Herpetologica Vol. 12 N. 2. December PRESS ISSN FIRENZE. Poste Italiane S.p.A. - Spedizione in Abbonamento Postale 70% DCB Firenze

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1 December 2017 Vol. 12 N. 2 Acta ISSN Herpetologica Iscritto al Tribunale di Firenze con il n 5450 del 03/11/2005 Poste Italiane S.p.A. - Spedizione in Abbonamento Postale 70% DCB Firenze FIRENZE UNIVERSITY PRESS

2 Acta Herpetologica Acta Herpetologica è la rivista ufficiale della Societas Herpetologica Italica (S.H.I.), un associazione scientifica che promuove la ricerca erpetologica di base e applicata, la divulgazione delle conoscenze e la protezione degli Anfibi e Rettili e dei loro habitat. Acta Herpetologica is the official journal of the Societas Herpetologica Italica (S.H.I.), a scientific association that promotes basic, applied, and conservation researches on Amphibians and Reptiles. Direttore responsabile (Editor): Marco Mangiacotti, DSTA, Università di Pavia, Via Taramelli 24, Pavia, Italia Redattori (Associate Editors): Aaron Matthew Bauer, Villanova University, United States Adriana Bellati, Department of Earth and Environmental Sciences University of Pavia, Italy Daniele Pellitteri-Rosa, Università degli Studi di Pavia, Italy Dario Ottonello, Centro Studi Bionaturalistici, Italy Ernesto Filippi, Sogesid-Ministero dell Ambiente, Roma, Italy Emilio Padoa-Schioppa, Università di Milano-Bicocca, Italy Emilio Sperone, Università della Calabria, Italy Fabio Maria Guarino, Università degli Studi di Napoli Federico II, Italy Gentile Francesco Ficetola, Université Grenoble-Alpes, France Giovanni Scillitani, Università degli Studi di Bari Aldo Moro, Italy Marcello Mezzasalma, Università degli Studi di Napoli Federico II, Italy Marco Sannolo, CIBIO-InBIO, Universidade do Porto, Vairão, Portugal Paolo Casale, Department of Biology University of Pisa, Italy Raoul Manenti, Dipartimento di Bioscienze, Università degli Studi di Milano, Milano Rocco Tiberti, Università di Pavia, Pavia, Italy Simon Baeckens, University of Antwerp, Belgium Stefano Scali, Museo Civico di Storia Naturale di Milano, Italy Uwe Fritz, Museum of Zoology, Senckenberg Dresden, Germany Consiglio direttivo S.H.I. (S.H.I. Council): Presidente (President): Roberto Sindaco Vice Presidente (Vice-President): Sandro Tripepi Segretario (Secretary): Dalila Giacobbe Tesoriere (Treasurer): Gulia Tessa Consiglieri (Council members): Gentile Francesco Ficetola, Lucio Bonato, Luciano di Tizio Sito ufficiale S.H.I. (Official S.H.I. website): Modalità di associazione Le nuove domande di associazione sono esaminate periodicamente dal Consiglio Direttivo; solo successivamente i nuovi soci riceveranno la comunicazione di accettazione con le modalità per regolarizzare l iscrizione (ulteriori informazioni sul sito: La quota annuale di iscrizione alla S.H.I. è di 35,00. I soci sono invitati a versare la quota di iscrizione sul conto corrente postale n intestato a: SHI Societas Herpetologica Italica. In alternativa è possibile effettuare un bonifico bancario sul Conto Corrente Postale: n. conto intestatario: SHI Societas Herpetologica Italica IBAN: IT-54-K Membership The S.H.I. Council will examine periodically new applications to S.H.I.: if accepted, new Members will receive confirmation and payment information (for more information contact the official website: Annual membership fee is (Euro). Payments are made on the postal account of SHI Societas Herpetologica Italica no , or by bank transfer on postal account no IBAN: IT-54-K to SHI Societas Herpetologica Italica. Versione on-line:

3 Acta Herpetologica Vol. 12, n. 2 - December 2017 Firenze University Press

4 Referee list. In alphabetical order the scientists that have accepted to act as editorial board members of Acta Herpetologica vol. 12 (2017). Elenco dei revisori. In ordine alfabetico gli studiosi che hanno fatto parte del comitato editoriale di Acta Herpetologica vol. 12 (2017). Abdala Virginia, Angelini Claudio, Antoniazzi Marta M., Avery Roger, Baeckens Simon, Beckmann Christa, Bellati Adriana, Bonato Lucio, Botto Valentina, Branch William, Busack Steven, Christoffersen Martin L., Conradie Werner, Correa Décio T., de Miranda Rogério, Díaz-Paniagua Carmen, Donihue Colin, Emmons Louise, Escoriza Daniel, Filippi Ernesto, Gazzola Andrea, Goldberg Stephen J., Gonçalves Sousa José G., Grosso Jimena, Hickerson Cari-Ann, Ibáñez Alejandro, Kelehear Crystal, Khannoon Eraqi, Köhler Jörn, Labra Antonieta, Leite Gabriela, Li Yi-ming, Luiselli Luca, Manenti Raoul, Mangiacotti Marco, Marin da Fonte Luis F., Marshall Kate, Mastrodonato Maria, Melotto Andrea, Mihalca Andrei D., Mikulíček Peter, Mockford Steve, Ogielska Maria, Ohler Annemarie, Pancharatna Katti, Pérez-Cembranos Ana, Pietersen Darren, Rato Catarina, Razzetti Edoardo, Rebelo Rui, Rebouças Raoni, Rheubert Justin L., Ribas Alexis, Rovatsos Michail, Sacchi Roberto, Salvidio Sebastiano, Santos Nago J. R., Saviola Anthony, Scali Stefano, Scillitani Giovanni, Scrocchi Gustavo, Sever David M., Silvano Débora L., Slavenko Alex, Steinfartz Sebastian, Stellatelli Oscar, Tiberti Rocco, Torres-Carvajl Omar, Vaglia Janet L., Valdecantos Soledad, Villa Andrea, Vinicius Caio, Vörös Judit, Watters Jessa, Žagar Anamarija, Zuffi Marco A.L.

5 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol Meristic and morphometric characters of Leptopelis natalensis tadpoles (Amphibia: Anura: Arthroleptidae) from Entumeni Forest reveal variation and inconsistencies with previous descriptions Susan Schweiger 1, James Harvey 2, Theresa S. Otremba 1, Janina Weber 1, Hendrik Müller 1, * 1 Institut für Spezielle Zoologie und Evolutionsbiologie mit Phyletischem Museum, Friedrich Schiller Universität Jena, Erbertstrasse 1, Jena, Germany. *Corresponding author. hendrik.mueller@uni-jena.de 2 41 Devonshire Avenue, Howick, 3290, South Africa Submitted on: 2017, 5 th June; revised on: 2017, 31 st August; accepted on: 2017, 3 rd October Editor: Rocco Tiberti Abstract. The tadpole of Leptopelis natalensis is described based on a series of 32 specimens from Entumeni Forest, KwaZulu-Natal, South Africa. Previous descriptions are brief, lack morphometric data, or are based on specimens of imprecise origin. The tadpole resembles other Leptopelis tadpoles and is generally in agreement with existing accounts, although some differences exist. Some of these differences seem to fall within the range of natural variation. Others, such as the presence of a fifth anterior row of keratodonts, might be indicative of variation at the population level and should be considered in future taxonomic revisions. Leptopelis natalensis tadpoles seem to be most readily distinguished by their more narrowly keratinized beaks from the geographically overlapping or adjacent L. mossambicus and L. xenodactylus. Keywords. South Africa, KwaZulu-Natal, Eastern Cape, taxonomy, buccal morphology, ontogenetic variation. INTRODUCTION The genus Leptopelis currently comprises 53 described species (Frost, 2017) of medium to large tree frogs that are distributed throughout most of Sub-Saharan Africa (Schiøtz, 1999). The most southerly distributed species of the genus is L. natalensis, which is found in a variety of habitats along the eastern region of the South African provinces of KwaZulu-Natal and part of Eastern Cape (Schiøtz, 1999; Bishop, 2004; Venter and Conradie, 2015). Although some, mostly brief, descriptions and illustrations of L. natalensis tadpoles have been published (Wager, 1930, 1965; van Dijk, 1966; Lambiris, 1988; Channing, 2001; du Preez and Carruthers, 2009; Channing et al., 2012), Channing (2001) considered none of the South African Leptopelis tadpoles to have been adequately described. The various descriptions furthermore differ in a number of diagnostic characters, such as labial keratodont formula. These differences in the existing descriptions could be the result of variation or population-specific differences that might be significant in future taxonomic revisions (Penske et al., 2015). To differentiate between these options, it is important to provide detailed locality information, which is lacking in some works providing information on tadpole morphology of L. natalensis (e.g., du Preez and Carruthers, 2009). While detailed, or at least limited, locality data are provided by Wager (1930, 1965, 1986), Pickersgill (2007) and Channing et al. (2012), these accounts provide mostly meristic data but few or no morphometric data that would help in assessing subtle differences that might exist between populations. We here provide a description and measurements of an ontogenetic series of tadpoles of L. natalensis based on a series collected at Entumeni Forest, ISSN (print) ISSN (online) Firenze University Press

6 126 Susan Schweiger et alii KwaZulu-Natal, assess variability and ontogenetic changes, and highlight differences between these and previous descriptions. MATERIALS AND METHODS A total of 32 tadpoles were collected at Entumeni Forest, KwaZulu-Natal, South Africa on 3 December The tadpoles were collected in a small forest stream ( S, E). Some tadpoles were preserved on the day of collection, while others were raised on a diet of commercial aquarium fish food and sampled in regular intervals to obtain different stages of development. Identification of the tadpoles was confirmed by raising some through metamorphosis. Specimens were euthanized in an aqueous solution of tricaine methanesulfonate (MS222; Fluka), fixed in 4% neutral buffered formalin, and transferred to 70% ethanol. Voucher specimens have been deposited in the herpetological collection of the Museum für Naturkunde Berlin, Germany (ZMB85717). Staging followed Gosner (1960). Standard measurements and labial tooth row formula followed Altig and McDiarmid (1999) and description of buccopharyngeal morphology Wassersug (1976). Drawings were prepared with the aid of a camera lucida attached to a Zeiss SV12 stereomicroscope. For inspection of the buccopharyngeal morphology, one tadpole of stage 36 was dissected, postfixed with 1% osmium tetroxide, dehydrated and critical point dried in an Emitech K850 critical point dryer, sputter coated with gold-palladium using an Emitech K500 and investigated using a Phillips XL30 ESEM scanning electron microscope with a digital image capture system. DESCRIPTION Tadpole. The description is based on 32 tadpoles from Gosner (1960) stages 25 to 42 (see Table 1 for measurements and detailed information). The tadpole is slender overall, with a moderately elongated, slightly dorsoventrally flattened body (wider than deep) and a relatively long tail with low dorsal and ventral fins. The widest point of the body is just behind the eyes (Fig. 1). No nares are visible until stage 34. From stage 35 to 37, the nares are indicated as light coloured spots, but do not seem to be open until stage 38. When fully formed, the nares are positioned dorsolaterally, about twice as far from the eye than the tip of the snout in lateral view. The eyes are positioned dorsally. A small anlage of the developing eyelid is first visible at stage 40, anterior of the eye. The spiracle is sinistral, about as far from the snout as from the body-tail junction. The spiracular opening is an upright oval, slightly slanted forward; its largest diameter almost as large as the diameter of the eye lens. The spiracular tube is angled upwards at about 45 ; the posterior end, including its inner wall, is free from the body. The tail is about 2.5 times as long as the body (see Table 1 for measurements) and very muscular. The myomeres are visible in the posterior half of the tail, but are otherwise indistinct or obscured by the dense pigmentation. The tailfins are very low, with the dorsal fin marginally deeper than the ventral fin. The dorsal fin has a low origin on the base of the tail, just behind the tail-body junction, and gradually rises towards the middle of the tail, where it reaches its maximum height. The ventral tailfin is very even, with the margin of the fin running more or less parallel to the ventral edge of the muscular tail. The overall deepest point of the tail is at about half its length. Tip of tail is pointed, with the muscular tail terminating some distance before the tail tip (Fig. 1B). The vent tube is attached to the right side of the ventral tailfin, with a very large opening forming a pointed arch. The coiled gut is well visible through the ventral body wall. Oral Disc. The oral disc is positioned subterminally and is not visible in dorsal view. The oral disc is lightly emarginated and has a broad rostral gap. One row of globular marginal papillae is present anterolaterally and laterally; posteriorly, two rows of papillae are present. Papillae are largest anterolaterally and laterally, and smaller posteriorly. A few submarginal papillae are present laterally. Keratodont formula is 4(2-4)/3(1) in the majority of the examined specimens (see below for variation). Moving inwards, supralabial keratodont rows are progressively smaller (Fig. 1A), infralabial rows are of nearly equal length, with P3 being slightly shorter than P1 and P2. Interruption of P1, if present, is very narrow in some specimens (Fig. 1A) but more pronounced in others. Keratodonts are about equally sized in most rows, except in P3 where they get progressively smaller laterally. The individual keratodonts are spoon-shaped and have eight or nine cusps along their margins, with the apical cusps being larger than the more laterally positioned ones (see inset in Fig. 2A). The jaw sheaths are serrated but only narrowly keratinised (indicated by the dark pigmentation). By stage 42, the lower jaw sheath and all keratodonts are absent and the papillation is much reduced in extent. Buccopharyngeal Morphology. The prenarial area of the buccal roof (Fig. 2A) is somewhat elongated and contains a few scattered pustules. In addition, a pair of short ridges is present and somewhat slanted laterally. The orientation of the choana is oblique to the midline (about 45 degrees) and both choanae form a forward-pointing angle of approximately 90 degrees. The jagged narial valves project deep into the buccal cavity and obscure the choanal openings. Anterolateral of each choana is a broad, flaplike papilla with a pustulate edge. Three to four thick, broad-based papillae are present posterolaterally to each

7 Tadpole of Leptopelis natalensis 127 Table 1. Measurements of Leptopelis natalensis. All measurements in millimeters (arithmetic mean ± SD). * indicates a damaged tail in one of the specimens of the series, which was omitted from the calculations. Gosner Stage 25 (n=3) 31 (n=1) 34 (n=1) 35 (n=2) 36 (n=14) 37 (n=2) 38 (n=2) 39 (n=1) 40 (n=1) 41 (n=1) 42 (n=4) Total length 32.1 ± 2.3* ± ± 2.7* 35.3* 37.9 ± ± 2.3 Body length 7.1 ± ± ± ± ± ± 0.3 Body width 3.8 ± ± ± ± ± ± 0.6 Body height 2.9 ± ± ± ± ± ± 0.2 Tail length 16.7 ± 1.6* ± ± 2.3* 24.5* 27.1 ± ± 1.8 Tail height 3.3 ± ± ± ± ± ± 0.5 Tail muscle height 1.9 ± ± ± ± ± ± 0.3 Width of oral disc 1.2 ± ± ± ± ± ± 0.2 Interorbital distance 2.7 ± ± ± ± ± ± 0.4 Internarial distance ± ± 0.2 Snout-naris distance ± ± 0.3 Snout-eye distance 2.5 ± ± ± ± ± ± 0.1 Snout-spiracle distance 4.9 ± ± ± ± ± Naris-eye distance ± ± 0.1 Eye diameter 0.6 ± ± ± ± ± ± 0.1 Fig. 1. Oral disc (A), lateral (B) and dorsal (C) view of a Gosner stage 36 tadpole of Leptopelis natalensis from Entumeni Forest, KwaZulu Natal, South Africa. Scale bar equals 0.5 mm in (A) and 5 mm in (B) and (C).

8 128 Susan Schweiger et alii Fig. 2. Scanning electron microscope images of the (A) buccal roof and (B) buccal floor of a Gosner stage 36 tadpole of Leptopelis natalensis. Inset in (A) shows a close-up of a keratodont. Scale bars in (A) and (B) equal 1 mm, and 5 µm in the inset.

9 Tadpole of Leptopelis natalensis 129 Table 2. Ontogenetic variation in labial keratodont formula. Number in brackets indicates number of specimens exhibiting the labial keratodont formula; asterisk (*) indicates asymmetry in the last (innermost) anterior keratodont row, with keratodonts present on one side only; keratodont data were only taken for 13 of the 14 investigated specimens of stage 36. Stage Labial keratodont formula 25 4(2-4)/3(1) [2]; 4(2-4*)/3 [1] 31 5(2-5)/3(1) [1] 34 5(2-5*)/3(1) [1] 35 4(2-4)/3(1) [2] 36 4(2-4)/3(1) [6]; 4(2-4)/3 [1]; 5(2-5*)/3(1) [1]; 5(2-5)/3(1) [5] 37 5(2-5)/3(1) [2] 38 4(2-4)/3(1) [2] 39 5(2-5)/3(1) [1] 40 5(2-5*)/3(1) [1] 41 5(2-5*)/3(1) [1] choana, and about six small pustules are present in the area between them. The median ridge separating postnarial arena and buccal roof arena is very prominent and forms an almost semi-circular flap. The buccal roof arena is fringed by eight pairs of medium to large papillae of similar sizes as in the buccal floor arena. An additional three to four pairs of smaller papillae are present in second row towards the posterior part of the buccal roof arena. There is furthermore a group of three to four short lateral roof papillae at each side of the arena. Within the buccal roof arena and posterior to it are ca. 100 pustules. A well-defined glandular zone with numerous secretory pits is present; it is broader laterally and has a relatively narrow medial gap. The dorsal velum has a broad medial gap and number of smaller papillae and pustules along its edge and sides. Additional pustules are present posterior to the dorsal velum and within its median gap. In the buccal floor (Fig. 2B), a pair of large, flaplike infralabial papillae, with smaller pustules along their margins, is present immediately inside the oral cavity on each side. An additional large, flap-like infralabial papilla is present medially just behind the lower jaw sheath. Four large lingual papillae are present on the tongue anlage. The area immediately behind the lingual papillae is marked by a transverse groove. To the left and right of this grove is a fairly large, bulbous structure. The buccal floor arena is fringed by nine to ten pairs of medium to large buccal floor papillae, all curved towards the buccal floor arena, except for the posteriormost pair of papillae, which point backwards (possibly an artefact of preservation). Around 60 pustules cover the posterior two thirds Table 3. Summary of published information on Leptopelis natalensis tadpoles. EC Eastern Cape Province, KZN KwaZulu-Natal Province, G Gosner (1960) stage. Reference Locality Keratodont formula Oral disc characters Maximum length /tail length as multiple of body length this study Entumeni Forest (KZN) 4(2-4)/3(1) or 5(2-5)/3(1), rarely 4(2-4)/3 double row of marginal papillae posteriorly, single row of slightly larger papillae laterally; jaw sheaths delicate and narrowly keratinized; disc emarginated 42mm (G39)/2.5x Wager (1930) Port St. Johns (EC) 4(2-4)/3 double row of marginal papillae posteriorly, single 51mm/2.5x row laterally; disc emarginated Wager (1965) Port St. Johns (EC) 4(2-4)/3 double row of marginal papillae posteriorly, single 49mm/2.75x row laterally; disc emarginated Durban (KZN) 4(2-4)/3 35mm/2.5x Nkandla (KZN) 5(2-5)/3 50mm/2.3x Lambiris (1988) - 4(2-4)/3 double row of marginal papillae posteriorly, single 50mm (G38)/- row laterally Pickersgill (2007) Hillcrest (KZN) 4(2-4)/3, sometimes 4(2-4)/3(1) double row of marginal papillae posteriorly, single row laterally; jaw sheaths narrowly keratinized; disc not emarginated 49mm (G37?)/2.5x du Preez and Carruthers (2009) - 4(2-4)/3, sometimes 4(2-4)/3(1) double row of marginal papillae posteriorly, single row laterally; jaw sheaths delicate Channing et al. (2012) KZN 4(2-4)/3 double row of marginal papillae posteriorly, single row of slightly larger papillae laterally; jaw sheaths delicate and keratinized along margins; disc emarginated 50mm/2.6x (figured tadpole) 35mm/2.2x

10 130 Susan Schweiger et alii of the buccal floor arena, as well as the area immediately posterior to it. In addition, ca. 15 pustules are present anterolaterally of the buccal floor arena, just in front of the buccal pockets. The buccal pockets are simple, narrow but deep, curved slits, with no associated papillae or pustules. It is unclear whether the buccal pockets are perforated to provide a bypass to the atrial chamber (Wassersug, 1976) or whether these end blind. The ventral velum is wide, with four marginal projections on each side, and a deep medial notch that exposes the glottis. The ventral velum contains secretory pits along its margin. Coloration in life. A nearly uniform dark olive, with a scatter of iridiophores across the entire dorsal and lateral sides of body and tail. Ventral side more sparsely pigmented anteriorly, but skin above the abdominal cavity completely unpigmented and translucent in younger stages but becoming somewhat more opaque in older specimens. Coloration in preservative. Dorsal body densely pigmented and overall homogenously dark brown in colour. Lateral line system very well visible as pigment-free spots in clearly defined lines along the body. Ventral body sparsely pigmented anteriorly but pigment-free above the abdominal cavity, with the coiled gut clearly visible. Pigmentation becomes somewhat less dense on tail and individual melanophores more easily discernible. Pigmentation on the dorsal tail-fin similar to the muscular tail, but distalmost edge pigment-free. Ventral fin free of pigment and translucent, with only some scattered melanophores present along the basal edge and towards the posterior end. Variation. Overall, little variation is present within the examined material. Specimens differ slightly in the distribution of melanophores on the tail-fins, with some specimens having a ventral tailfin that is almost entirely free of pigmentation except for the very tip of the tail, whereas others show scattered pigment to a various extent within the posterior half of the fin. Pigmentation of the dorsal fin also slightly less or more dense in some specimens. The most variation is seen in the oral disc, in particular the number and arrangement of keratodont rows. Slightly more than half of the specimens (14 of 27; see Table 2) have four anterior rows of keratodonts, with the first always undivided, and the remainder divided. The rest of the specimens have an additional, innermost fifth keratodont row (A5). In all specimens, the last anterior row is usually rather short and in a number of specimens present on one side only (Table 2). The first posterior row is usually divided by a small gap of variable width, but undivided in two of the 27 specimens examined that have an oral apparatus. DISCUSSION Overall, the tadpole of L. natalensis resembles other Leptopelis tadpoles in general shape and appearance (see Channing et al., 2012, and Barej et al., 2015, for most recent and comprehensive treatments of the group). In South Africa, the range of L. natalensis is close to or overlaps with the ranges of L. xenodactylus and L. mossambicus (Channing, 2001; Minter et al., 2004). Based on the available information, the tadpole of L. mossambicus appears to be somewhat larger and proportionally shorter-tailed than that of L. natalensis, and overall darker in colouration, being more brown than olive (du Preez and Carruthers, 2009). Leptopelis mossambicus further appears to differ from L. natalensis by having slightly higher tailfins, a very broad rostral gap in the papillation of the oral disc that is almost as broad as the disc itself, and somewhat more robust jaw sheaths that are keratinized for about half their width (du Preez and Carruthers, 2009; Channing et al., 2012). The tadpole of L. xenodactylus is very similar to L. natalensis and these species appear to be indistinguishable by overall shape and colouration alone. However, in contrast to L. natalensis, L. xenodactylus tadpoles do seem to have a more robustly keratinized jaw (Channing, 2008; du Preez and Carruthers, 2009; Channing et al., 2012), which should help facilitate a correct identification. In addition there are differences in papillation, with more submarginal papillae being present in L. xenodactyloides and the posterior papillae differing in size (inner row of shorter, more globular papillae and outer row of relatively long papillae; Channing 2008). The two species have so far not been found in sympatry, although they occur in close proximity to each other in central KwaZulu-Natal. A number of descriptions of the tadpole of L. natalensis have been provided before (summarized in Table 3), but most of them are brief, lack measurements, or do not provide precise locality information. All previous accounts and our observations agree on the overall shape and colouration of the tadpoles, but some differences especially in total length, oral disc morphology, and keratodont formula exist. While most accounts provide a maximum length of around 50 mm, Wager (1965) and Channing et al. (2012) reported 35 mm as total length, at least for some populations, but did not indicate what stage the examined specimens were at. Furthermore, there is some variation regarding the length of the tail, but all of this seems to be within the limit of normal variation, given that maximum length is largely dependent on condition. While most previous investigators reported or figured a slightly emarginated oral disc, which matches our own observations, Pickersgill (2007) illustrated a disc

11 Tadpole of Leptopelis natalensis 131 that is not emarginated. Assuming all observations to be correct, this would indicate a more substantial difference between that population and others. Both van Dijk (1966) and du Preez and Carruthers (2009) reported the presence of an elygium in the eye of L. natalensis tadpoles, although van Dijk (1966) indicated that an ocular elygium might not generally be present and is not easily detected. In our specimens, an ocular elygium is not present, but dorsally the pigmented skin seems to somewhat extend onto the eyeball, which may represent an epidermal elygium (see Kruger et al., 2013). Most previous descriptions gave the keratodont formula of L. natalensis tadpoles as 4(2-4)/3, indicating an undivided P1 (Wager, 1930, 1986; Lambiris, 1988; Pickersgill, 2007; du Preez and Carruthers, 2009; Channing et al., 2012), but Pickersgill (2007) and du Preez and Carruthers (2009) also stated that P1 can sometimes be divided. In our series, only two specimens had an undivided P1, the rest all had a divided P1 although the gap was very slight in some specimens. While this may be an indication of interpopulational variation, it seems possible that previous reports might have simply overlooked a narrow gap in P1. Similar variations in the presence of a divided vs. undivided P1 have been reported by Penske et al. (2015) for Leptopelis cf. grandiceps. Furthermore, only slightly more than half of the specimens (14, see Table 2) of our ontogenetic series of L. natalensis tadpoles had four anterior rows of keratodonts. Almost as many (13 specimens) had an additional, divided A5 and a resulting keratodont formula of 5(2-5)/3(1). Although also present in some younger specimens, the presence of an A5 seemed to be more pronounced in older tadpoles (Table 2). Variation in the number of keratodont rows has been reported for a number of species, including L. calcaratus, L. gramineus, L. vannutelli and L. yaldeni (see Channing et al., 2012). An ontogenetic increase has further been reported for L. aubryoides (Barej et al., 2015), L. calcaratus (Lamotte and Perret, 1961) and L. viridis (Rödel 2000), and specimens with an additional A5 have been reported for L. modestus, L. spiritusnoctis and L. rufus (Barej et al., 2015). In many anuran tadpoles, anterior keratodont rows are added during ontogeny and the presence of an A5 in some Leptopelis might be related to overall tadpole size. It is therefore possible that previous investigations did not examine specimens of a sufficient age for an A5 to be expressed. At the same time, the maximum length of 50 mm reported by several authors for L. natalensis (e.g., Wager, 1930; Lambiris, 1988; see Table 2 for full list), which substantially exceeds the 42 mm of the largest specimen in our sample, would argue against this. Only Wager (1965) reported tadpoles with an A5 from Nkandla Forest, KwaZulu-Natal. Entumeni Forest, the origin of the specimens examined here and only other reported population of L. natalensis tadpoles with a fifth anterior row of keratodonts, is less than 30 km away from Nkandla Forest. Given the current state of knowledge, these two populations seem diagnosably different from other L. natalensis populations at the level of tadpole morphology. Future studies of phylogeography of L. natalensis should include these populations and investigate their degree of differentiation compared to others. ACKNOWLEDGEMENTS We would like to thank Ezemvelo KZN for issuing the necessary permits (OP4976/2013, OP635/2014) to undertake our research and in particular Adrian Armstrong and Sharon Louw for their generous advice and help facilitating our work. For help in the field we are grateful to Lars Möckel and Katrin Friedemann. Funding was provided through a German Science Foundation (DFG) grant to HM (MU2914/2-1). REFERENCES Altig, R., Mc Diarmid, R.W. (1999): Body plan development and morphology. In: Tadpoles the biology of anuran larvae, pp Mc Diarmid, R.W., Altig, R., Eds, Chicago University Press, Chicago. Barej, M.F., Pfalzgraff, T., Hirschfeld, M., Liedtke, H.C., Gonwouo, N.L., Penner, J., Dahmen, M., Doherty- Bone, T., Schmitz, A., Rödel, M.O. (2015): The tadpoles of eight West and Central African Leptopelis species (Amphibia: Anura: Arthroleptidae). Amphib. Rept. Cons. 9: Bishop, P.J. (2004): Leptopelis natalensis (Smith, 1849). In: Atlas and red data book of the frogs of South Africa, Lesotho and Swasiland, pp Minter, L.R., Burger, M., Harrison, J.A., Braack, H.H., Bishop, P.J., Kloepfer, D., Eds, Smithsonian Institution, Washington, D.C. Channing, A. (2008): The mud dwelling tadpole of the long-toed tree frog, Leptopelis xenodactylus (Arthroleptidae). Herp. Rev. 39: Channing, A. (2001): Amphibians of Central and Southern Africa. Comstock Publishing Associates, Cornell University Press, Ithaca. Channing, A., Rödel, M.O., Channing, J. (2012): Tadpoles of Africa. Edition Chimaira, Frankfurt-am-Main. du Preez, L., Carruthers, V. (2009): A complete guide to the frogs of Southern Africa. Struik Nature, Cape Town.

12 132 Susan Schweiger et alii Frost, D.R. (2017): Amphibian Species of the World: an Online Reference. Version 6.0 (accessed 23 May 2017). Electronic Database accessible at research.amnh.org/herpetology/amphibia/index.html. American Museum of Natural History, New York. Gosner, K.L. (1960): A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16: Kruger, D.J.D., Weldon, C., Minter, L.R., du Preez, L.H. (2013): Morphology of the elygium and developing umbraculum in the eye of Amietia vertebralis tadpoles. J. Morphol. 274: Lambiris, A.J.L. (1988): A review of the amphibians of Natal. Lammergeyer 39: Lamotte, M., Perret, J.L. (1961): Contribution à l étude des batraciens de l Ouest africain XIII. Les formes larvaires de quelques espèces de Leptopelis: L. aubryi, L. viridis, L. anchietae, L. ocellatus et L. calcaratus. Bulletin de l Institut Fondamental d Afrique Noire, Serie A 3: Minter, L.R., Burger, M., Harrison, J.A., Braack, H.H., Bishop, P.J., Kloepfer, D., Eds. (2004): Atlas and Red Data Book of the Frogs of South Africa, Lesotho and Swasiland. SI/MAB Series 9. Smithsonian Institution, Washington, D.C. Penske, S., Gvoždík, V., Menegon, M., Loader, S.P., Müller, H. (2015): Description of the tadpole of Leptopelis cf. grandiceps (Amphibia: Anura: Arthroleptidae) from the Uluguru Mountains, Tanzania. Herpetol. J. 25: Pickersgill, M. (2007): Frog search: results of expeditions to Southern and Eastern Africa. Edition Chimaira, Frankfurt-am-Main. Rödel, M.O. (2000): Herpetofauna of West Africa. Volume I: Amphibians of the West African savanna. Edition Chimaira, Frankfurt-am-Main. Schiøtz, A. (1999): Treefrogs of Africa. Edition Chimaira, Frankfurt-am-Main. Van Dijk, D.E. (1966): Systematic and field keys to the families, genera and described species of South African anuran tadpoles. Ann. Natal. Mus. 18: Venter, A.J., Conradie, W. (2015): A checklist of the reptiles and amphibians found in the protected areas along the South African Wild Coast, with notes on conservation implications. Koedoe 57: 1-25 Wager, V.A. (1930): The breeding habits and life-histories of two rare South African Amphibia. I. Hylambates natalensis A. Smith. II. Natalobatrachus bonebergi Hewitt and Methuen (plates V-X). Trans. Roy. Soc. S. Afr. 19: Wager, V.A. (1965): The frogs of South Africa. Purnell and Sons, Cape Town. Wager, V.A. (1986): Frogs of South Africa: Their Fascinating Life Stories. Delta Books, Craighall. Wassersug, R.J. (1976): Oral morphology of anuran larvae: terminology and general description. Occ. Pub. Mus. Nat. Hist. Univ. Kansas 48: 1-23.

13 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol Brown anole (Anolis sagrei) adhesive forces remain unaffected by partial claw clipping Austin M. Garner*, Stephanie M. Lopez, Peter H. Niewiarowski Department of Biology, Program in Integrated Bioscience, University of Akron, Akron, OH , USA. *Corresponding author. Submitted on: 2017, 19 th April; Revised on: 2017, 28 th July; Accepted on: 2017, 31 th July Editor: Fabio M. Guarino Abstract. Morphological properties of animal locomotor systems should reflect costs and benefits associated with particular environments. Lizards that possess both claws and adhesive toe pads are specialized for environments that require movements in both the horizontal and vertical planes, and on both rough and smooth substrates. Although toe/claw clipping is a common technique for marking free-ranging lizards, this technique is disadvantageous to those lizards possessing adhesive toe pads. A previous study removed entire claws of Anolis carolinensis and observed a significant reduction in adhesive abilities, which was likely attributed to damage of underlying tendons that play critical roles in engaging the adhesive pads. Here, we report on the clinging ability of brown anoles (Anolis sagrei) with partial claw removal. We found that adhesive capacities were not affected by partial removal of the claw, suggesting that partial claw removal prevents damage to the underlying tendons and that it may be a safer alternative for short-term marking of adhesive pad-bearing lizards. Keywords. Adhesion, Anolis, claw clipping, clinging force, toe clipping. INTRODUCTION Substrate characteristics of the natural environments of most lizards are usually heterogeneous and complex. Presumably, particular morphological properties of the locomotor systems of lizards reflect evolutionary responses to the costs and benefits associated with specializations for particular substrates. For example, several clades of lizards have evolved elaborate adhesive toe pads (while at the same time retaining claws) allowing them to cling to smooth substrates (Ruibal and Ernst, 1965; Williams and Peterson, 1982; Peterson, 1983). Claws perform well on rough or irregularly patterned substrates, whereas subdigital adhesive pads excel on smooth substrates (Irschick et al., 1996; Zani, 2000). Lizards that encounter both types of substrates may benefit by having both claws and toepads, as do species in the genus Anolis, allowing them to travel effectively on both smooth and rough surfaces (Irschick et al., 1996; Zani, 2000; Crandell et al., 2014). Toe or claw clipping is a widely utilized technique for marking small reptiles and amphibians (Dunham et al., 1988). Although this may be a convenient marking technique for terrestrial species, few studies have investigated how toe or claw clipping affects adhesive toe pad function and whole organism performance in arboreal adhesive pad-bearing lizards. Bloch and Irschick (2005) examined the effects of complete claw clipping on clinging abilities of the adhesive pad-bearing lizard, Anolis carolinensis. After clipping toes at the distalmost portion of the adhesive toepad (effectively removing the entire claw; Fig. 1A), they found that claw removal significantly decreased clinging abilities. Several explanations were suggested including a change in the way the animal perceives its environment (a tactile response), and a morphological ISSN (print) ISSN (online) Firenze University Press

14 134 Austin M. Garner et alii effect on the function of the toepad after the claw was removed (i.e., tendon damage; Bloch and Irschick, 2005). Although these mechanisms are not mutually exclusive, we wanted to test the relative importance of the possibility that clinging performance would suffer due to tendon damage as a result of claw clipping. While Bloch and Irschick (2005) did not directly state a mechanism of tendon damage, anoles (like most tetrapods) control flexion of the digits via flexor tendons attached to the musculus flexor digitorum longus. These flexor tendons span across the entire toe, including the distal phalanges (Abdala et al., 2009). When Bloch and Irschick (2005) removed the distalmost portion of the toe, they likely severed flexor tendons as well. Given this, it is possible that the destruction of those tendons may have resulted in decreased adhesion because the anoles were not able to properly engage their adhesive system. In our study, we used partial claw clipping to leave those tendons intact and measured maximum clinging ability on smooth substrates. Partial claw clipping reduces claw length and changes claw shape, but also decreases the interference between the adhesive pad and the substrate (Fig. 1B). We hypothesized that partial claw clipping would not result in significantly reduced clinging abilities in Anolis sagrei on smooth substrates, likely because the less destructive marking technique would leave the underlying tendons preserved. The results of this study will not only increase our understanding of how claw clipping affects whole animal adhesive performance, but may also influence marking techniques utilized by researchers studying wild populations of adhesive pad-bearing lizards that also have claws. Animals MATERIALS AND METHODS A total of 19 captured brown anoles (Anolis sagrei) were used for experiments, including both adult males and females (Table 1). All lizards used for this study were collected at the Key West Botanical Gardens (Key West, FL) between 11 May 2008 and 16 May 2008, and then transported back to the Mote Marine Laboratory on Summerland Key, FL for clinging experiments. Anoles were released back to marked capture points in the gardens within 24 hours of capture. Experimental procedures Clinging experiments were completed using a custom-designed, motorized rig that measured adhesive force (Niewiarowski et al., 2008). The first set of trials were conducted on a sheet of glass covered with an acetate sheet (Bloch Fig. 1. Diagram of an anole toe indicating the differences in claw clipping locations between Bloch and Irschick (2005) and the present study. The clipping location is represented by the solid line and the removed portion of the claw is represented by the dashed line. Bloch and Irschick (2005) removed the entire claw at the distalmost portion of the adhesive toe pad (A), possibly compromising the underlying tendons that facilitate adhesive toe pad engagement. We partially removed the claw (B) in an effort to preserve the underlying tendons. Table 1. Sample size, mass, and mean clinging forces of brown anoles (Anolis sagrei) used in experimental trials. All values reported as mean ± SE. n Mass (g) Intact (N) Clipped (N) Acetate (N) Glass (N) ± ± ± ± ± 0.11 and Irschick, 2005). The two front feet of each anole were pulled three times in rapid succession along the substrate at a uniform speed of approximately 5 cm/s. For consistency, force measurements were done by a single individual. After the three pulls, the sheet of acetate was removed and the same anole was pulled along the sheet of glass three more times. Pulls on glass were used to test the possibility that claws (intact or clipped) contributed to clinging force by digging into the acetate sheet (Bloch and Irschick, 2005). Maximum clinging force was recorded for each set of pulls and was defined as the force recorded at the point when both front feet of the anole were visibly slipping on the substrate. Subsequent to pulls with intact claws, claws on both front feet were partially clipped using a small pair of nail scissors. Unlike Bloch and Irschick (2005), claws were only clipped halfway to avoid potential damage to the underlying tendons (Fig. 1B). After clipping, clinging ability was tested identically to tests withint act claws. Smooth substrates were chosen to isolate the effect of partial claw clipping on the adhesive capability of Anolis sagrei. Rough or irregular substrates generally increase clinging ability of lizards with claws because claws can generate friction or mechanically interlock with surface asperities, thus possibly resulting in a bias towards higher clinging ability with claws left intact (Zani, 2000; Crandell et al., 2014). The order in which anoles were tested was randomized.

15 Effect of claw clipping on brown anole adhesion 135 Statistical analysis We used a mixed model analysis of variance (ANOVA) to compare maximum clinging ability as a function of substrate, claw removal, and their interaction. Maximum clinging ability was the dependent variable. Substrate type (glass or acrylic), claw state (intact or clipped), and their interaction were the independent variables. Individual anole was modeled as a random effect. Before statistical analysis, maximum clinging ability data were log-transformed in order to meet the assumptions of an ANOVA. All statistical analyses were completed using JMP Pro 12 (SAS Institute, Inc., Cary, NC, USA). Table 2. Results of the mixed model analysis of variance (ANOVA). The table displays a significant difference in maximum clinging ability as a function of substrate (*P < 0.05). There is no significant difference in maximum clinging ability as a function of claw state or the interaction between substrate and claw state. Effect NumDF DenDF F P Substrate * Claw state Substrate * Claw state RESULTS The mixed model ANOVA using an assumption of compound symmetry (Littell et al., 2000) fit the data nearly as well as the model relaxing the assumption of compound symmetry and leads to qualitatively similar conclusions, but we report the latter as it is more conservative. The effect of claw removal on maximum clinging ability of Anolis sagrei revealed that neither claw state (F 1,18 = 0.683, P = 0.419) nor the substrate by claw state interaction (F 1,18 = 0.725, P = 0.406) had a significant effect on maximum clinging ability (Fig. 2 and Table 2). However, maximum clinging ability was significantly affected by substrate type, with glass producing higher maximum clinging forces than acetate (F 1,18 = 4.435, P = 0.049; Fig. 3 and Table 2). Fig. 3. Mean maximum clinging forces of Anolis sagrei on acetate and glass substrates. A mixed model ANOVA found that the mean maximum clinging force of A. sagrei was significantly higher on glass compared to acetate (F 1,18 = 4.435, P = 0.049). Error bars represent ± 1 SE. DISCUSSION Fig. 2. Mean maximum clinging forces of Anolis sagrei on acetate and glass substrates with fully intact or partially clipped claws. A mixed model ANOVA indicates no significant difference in the mean maximum clinging forces of A. sagrei as a function of claw state (F 1,18 = 0.683, P = 0.419) or the substrate by claw state interaction (F 1,18 = 0.725, P = 0.406). Error bars represent ± 1 SE. Bloch and Irschick (2005) reported a significant decrease in clinging forces when the claws of Anolis carolinensis were completely removed. Because several mechanisms were proposed to explain this effect, we examined the possibility that tendon damage may be responsible for the reduction in adhesion. To investigate this, we partially removed claws of Anolis sagrei and compared their clinging abilities to anoles with fully intact claws. Our results demonstrated that partial claw clipping had no significant effect on maximum clinging force, supporting the hypothesis that entire claw removal may actually damage the underlying tendons (Bloch and Irschick, 2005). The main difference between our study and Bloch and Irschick (2005) is that claws were only partially clipped, not removed, thus the underlying tendons were left intact. All claws on both front feet were clipped in our work, as opposed to remov-

16 136 Austin M. Garner et alii ing 2-4 claws on the front feet. Yet, our recorded clinging forces are similar to those observed by Bloch and Irschick (2005). Importantly, the difference in clinging forces between intact and partially clipped claws (~ 0.2 N or ~ 20 g) is about five times smaller in our study than what Bloch and Irschick (2005) found (~ 1 N or ~ 100 g). Although our samples sizes are smaller than Bloch and Irschick (2005), we used a mixed model ANOVA to account for the non-independence of observations attributable to individual lizards, and the small differences between treatments is likely to be biologically insignificant as well. Do claws interfere with toe pads? In lizards that possess both claws and subdigital adhesive pads, it has been demonstrated that increases in claw curvature, length, and height are correlated with increases in toe pad adhesive force on smooth substrates (Zani, 2000; Crandell et al., 2014). While morphological variation in claws appears to influence adhesion, the mere presence of claws should result in reduced adhesive capabilities. Clinging forces generated by adhesive toe pads are directly related to the numbers of individual setae that can make appropriate contact with the substrate (contact fraction) (Autumn et al., 2000; Autumn, 2006). Contact fraction depends on many factors, including pre-loads applied by the lizard, as well as the degree and nature of roughness of the substrate (Russell et al., 2007). On smooth substrates, it is intuitive to hypothesize that claws may result in decreased adhesive force because the toe pads may be more removed from the substrate resulting in decreased contact fraction. Thus, one would expect the partial removal of claws to result in an increase in adhesive force on smooth substrates. Surprisingly, our results reveal no significant difference in clinging ability between anoles with fully intact and partially clipped claws, at least on smooth substrates. Furthermore, Mahendra (1941) observed that the adhesive locomotion of Hemidactylus flaviviridis on smooth substrates appeared to be unaffected by claw removal. Thus, it may be that morphological or behavioral mechanisms minimize theoretical trade-offs between claws and toepads in their functional roles during adhesive locomotion. Lizards that possess both claws and subdigital adhesive pads are likely able to effectively cling to both smooth and rough substrates (Irschick et al., 1996; Zani, 2000). While it appears that partial claw clipping does not result in a reduction in adhesive performance on smooth substrates, it is possible that claw removal results in lower clinging abilities on rough substrates, although this has yet to be tested empirically. Therefore, it is interesting to consider the ecological implications of losing one of the two clinging mechanisms discussed (adhesion via van der Waals interactions or clinging via claws). However, considering the heterogeneity of substrates in the natural habitat of Anolis sagrei, it is difficult to gauge which mechanism of clinging would be more costly to A. sagrei if it was lost via toe/claw removal. Regardless, partial claw clipping is the least invasive of the two marking techniques discussed and does not result in a reduction of adhesive performance. Glass versus acetate We recorded a significantly higher clinging force on glass than on acetate when claws were left intact. Two aspects of this result are relevant. First, it suggests that claws do not enhance adhesion by digging into the acetate sheets (Bloch and Irschick, 2005). Second, and consistent with theory underlying the mechanics of van der Waals forces, substrates with higher surface energies should lead to higher clinging forces (glass is about 100 mj/m 2 and acetate is about mj/m 2 ) (Israelachvili, 1992). A potential confounding factor for future adhesion studies could be which of these substrates is more appropriate to use and which is most similar to a smooth substrate encountered by adhesive pad-bearing lizards in their natural environments. Marking techniques A non-destructive, permanent marking technique would be ideal for working with reptiles, yet it is very challenging to achieve, particularly with animals that rely on adhesive functions during locomotion (Paulissen and Meyer, 2000). Bloch and Irschick (2005) addressed this problem with complete claw clipping as a permanent, relatively reliable solution, but with an undesired effect: a reduction in adhesive performance. When comparing permanence of a marking technique, complete claw clipping/removal is more effective than partial claw clipping. However, our work here suggests that partial claw clipping may serve as an excellent alternative during shortterm mark and recapture studies because no significant change in adhesive capabilities was observed. ACKNOWLEDGEMENTS We thank D. Pund, T. Shultz, J. Flinn, and M. Sanchez for helping collect, measure, and weigh animals. We also thank Key West Botanical Gardens for allowing us to utilize their grounds for animal capture and to Mote

17 Effect of claw clipping on brown anole adhesion 137 Marine Laboratories for allowing us to use their Summerland Key facilities to conduct our research. All experimental procedures were in accordance with the ethical guidelines published by the American Society of Ichthyologists and Herpetologists (Beaupre, 2004). All protocols were in accordance with the regulations of The University of Akron IACUC, protocol #07-4G. REFERENCES Abdala, V., Manzano, A.S., Tulli, M.J., Herrel, A. (2009): The tendinous patterns in the palmar surface of the lizard manus: functional consequences for grasping ability. Anat. Rec. 292: Autumn, K. (2006): How Gecko Toes Stick. Am. Sci. 94: Autumn, K., Liang, Y.A., Hsieh, S.T., Zesch, W., Chan, W.P., Kenny, T.W., Fearing, R., Full, R.J. (2000): Adhesive force of a single gecko foot-hair. Nature 405: Beaupre, S.J., Ed. (2004): Guidelines for Use of Live Amphibians and Reptiles in Field and Laboratory Research, 2nd edition. American Society for Icthyologists and Herpetologists, USA. Bloch, N., Irschick, D.J. (2005): Toe-clipping dramatically reduces clinging performance in a pad-bearing lizard (Anolis carolinensis). J. Herpetol. 39: Crandell, K.E., Herrel, A., Sasa, M., Losos, J.B., Autumn, K. (2014): Stick or grip? Co-evolution of adhesive toepads and claws in Anolis lizards. Zoology 117: Dunham, A.E., Morin, P.J., Wilbur, H.M. (1988): Methods for the study of reptile populations. In: Biology of the Reptilia, pp Gans, C. Huey, R.B., Eds, Alan R. Liss, Inc., New York. Irschick, D.J., Austin, C.C., Petren, K., Fisher, R.N., Losos, J.B., Ellers, O. (1996): A comparative analysis of clinging ability among padbearing lizards. Biol. J. Linn. Soc. 59: Israelachvili, J. (1992): Intermolecular and Surface Forces, Academic Press Limited, San Diego, CA. Littell, R.C., Pendergast, J., Natarajan, R. (2000): Tutorial in biostatistics: modelling covariance structure in the analysis of repeated measures data. Stat. Med. 19: Mahendra, B.C. (1941): Contributions to the bionomics, anatomy, reproduction and development of the Indian house-gecko, Hemidactylus flaviviridis Rüppel. P. Indian Acad. Sci. B 13: Niewiarowski, P.H., Lopez, S., Ge, L., Hagan, E., Dhinojwala, A. (2008): Sticky gecko feet: the role of temperature and humidity. PLoS ONE 3: e2192. Paulissen, M.A., Meyer, H.A. (2000): The effect of toeclipping on the gecko Hemidactylus turcicus. J. Herpetol. 34: Peterson, J.A. (1983): The evolution of the subdigital pad in Anolis. I. Comparisons among the anoline genera. In: Advances in Herpetology and Evolutionary Biology: Essays in Honor of Ernest E. Williams, pp Rhodin, G.J., Miyata, K., Eds, Museum of Comparative Zoology, Harvard Univ., Cambridge, MA. Ruibal, R., Ernst, V. (1965): The structure of the digital setae of lizards. J. Morphol. 117: Russell, A.P., Johnson, M.K., Delannoy, S.M. (2007): Insights from studies of gecko-inspired adhesion and their impact on our understanding of the evolution of the gekkotan adhesive system. J. Adhes. Sci. Technol. 21: Williams, E., Peterson, J. (1982): Convergent and alternative designs in the digital adhesive pads of scincid lizards. Science 215: Zani, P. (2000): The comparative evolution of lizard claw and toe morphology and clinging performance. J. Evol. Biol. 13:

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19 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol Species and sex comparisons of karyotype and genome size in two Kurixalus tree frogs (Anura, Rhacophoridae) Shun-Ping Chang 1,2, Gwo-Chin Ma 2,3,4, Ming Chen 2,5,6,7,8, *, Sheng-Hai Wu 1, * 1 Department of Life Sciences, National Chung Hsing University, 145 Xingda Rd., Taichung, Taiwan; *Corresponding author. shwu@dragon.nchu.edu.tw 2 Department of Genomic Medicine, Changhua Christian Hospital, 176 Zhanghua Rd., Changhua, Taiwan; *Corresponding author. mingchenmd@gmail.com 3 Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, 110 Jianguo N. Rd. (Sec. 1), Taichung, Taiwan 4 Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, 666 Buzih Rd., Taichung, Taiwan 5 Department of Obstetrics and Gynecology, Changhua Christian Hospital, 135 Nanxiao St., Changhua, Taiwan 6 Department of Obstetrics and Gynecology, College of Medicine and Hospital, National Taiwan University, 1 Roosevelt Rd. (Sec. 4), Taipei, Taiwan 7 Department of Medical Genetics, National Taiwan University Hospital, 1 Changde St., Taipei, Taiwan 8 Department of Life Sciences, Tunghai University, 1727 Taiwan Boulevard (Sec. 4), Taichung, Taiwan Submitted on: 2017, 6 th June; revised on 2017, 22 nd September; accepted on 2017, 22 nd September Editor: Uwe Fritz Abstract. Kurixalus is a rhacophorid genus of tree frogs that are similar in morphology but vary in reproductive behavior. We investigated the cytogenetic features and genome size using conventional G-banding, C-banding and silver-staining techniques, fluorescence in situ hybridization (FISH), and flow cytometry in two representatives of Kurixalus (K. eiffingeri Boettger, 1895 and K. idiootocus Kuramoto and Wang, 1987) and compared the data between species and sex. The two Kurixalus species share a diploid chromosome number 2n = 26 and fundamental number FN = 52. Prominent differences between species were noted in the distribution of secondary constriction (SC)/nucleolus organizer region (NOR) and dense heterochromatin. Other interspecies differences including variations in the number of metacentric and submetacentric chromosomes and staining intensity of heterochromatin were also found. The cytogenetic results are consistent with the observed differences in their genome sizes. FISH with telomeric motif (TTAGGG)n for both species detected signals in the terminal regions. Intersex comparisons revealed no differences in terms of cytogenetic features and genome size in the two species. Despite the apparent highly conserved diploid chromosome number, data on the karyotype microstructure characterize the cytogenetic profile of the two Kurixalus species that contribute to clarification of the chromosomal homologies and the rearrangement mechanisms occurring during the karyotype evolution of Kurixalus. No heteromorphic chromosome pair in both species is consistent with the view that homomorphic sex chromosome is common in amphibians. Keywords. Kurixalus eiffingeri, K. idiootocus, karyotype, homomorphic chromosome, sex determination. ISSN (print) ISSN (online) INTRODUCTION Kurixalus is a rhacophorid genus of small tree frogs that occurs across East to Southeast Asia, including the Ryukyu Islands, Taiwan, China, India, Myanmar, Cambodia, Vietnam, Thailand, Malays, Sumatra, Borneo and the Philippines (Frost, 2017). Phylogenetic data support the Firenze University Press

20 140 Shun-Ping Chang et alii monophyletic origin of Kurixalus despite its broad distribution (Li et al., 2013; Biju et al., 2016; Jiang et al., 2016). However, since these frogs are similar in morphology and overlap in body size, taxonomy is difficult and mainly based on molecular analyses (Wilkinson et al., 2002; Li et al., 2008; Li et al., 2009; Hertwig et al., 2013; Yu et al., 2013; Nguyen et al., 2014a; Nguyen et al., 2014b; Wu et al., 2016). Currently, 14 species are recognized in Kurixalus (Frost, 2017). Despite the taxonomy of Kurixalus species continues to update, considerable differences in the reproductive behavior are noted among some known species. For example, K. eiffingeri Boettger, 1895, a species found in Taiwan and two southern islands of Japan (Maeda and Matsui, 1989), breeds in water-filled tree holes and bamboo stumps, and displays a complex parental care including paternal care for eggs and subsequent maternal care for larvae. The tadpoles are oophagous, feeding exclusively on unfertilized eggs laid by the female frogs which return to nests (Kam et al., 1997; Kam et al., 2001). On the contrary, K. idiootocus Kuramoto and Wang, 1987, a species endemic to Taiwan, breeds in soils and crevices that are frequently covered with dead leaves. Eggs hatch when heavy rainfall occurs and the hollow fills up, and the tadpoles get washed into nearby waterways or pools. The tadpoles are herbivorous, subsisting on algae and plants. While much attention has been put on subjects of the reproductive behavior in Kurixalus species (Lin and Kam, 2008; Cheng WC et al., 2013; Tung et al., 2015), little information is available on their genetic complements in terms of species difference and sex differentiation. It is thus interesting to investigate the cytogenetic and genomic features in members of this genus. Variations in chromosome number and structure among species and/or populations can be used to estimate phylogenetic relationships (Yates et al., 1979) and has been applied by various kinds of cytogenetic techniques for classifying species with similar morphology (Medeiros et al., 2003; Rosa et al., 2003; Veiga-Menoncello et al., 2003; Carvalho et al., 2009). Cytogenetic analyses can also reveal sex determination of targeted species. If heteromorphic sex chromosomes are present in species, genetic sex determination is inferred (Hanada, 2002). Even though heteromorphic chromosome pairs are not always recognized in species, different chromosome staining techniques, such as G-banding, C-banding and Ag-NOR staining are still widely accepted in detecting cryptic structural alterations of chromosomes (Hanada, 2002; Wu et al., 2007). Additionally, sophisticated painting techniques, such as fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH), are also useful tools in the studies of comparative cytogenetics. This can be instanced by the use of FISH with a highly conserved vertebrate telomeric motif (TTAGGG)n to identify chromosome rearrangements (e.g., fusion, fission and translocation events) if the interstitial telomeric sequences (ITSs) were detected. The important role of the telomeric sequences in karyotypic diversity, chromosome evolution, and chromosome instability has been highlighted (Bruschi et al., 2014). The CGH, a FISH-based method using total DNA from two taxa as probes, can differentiate chromosomes from different genomes and has been used in studies of genomewide comparison (Yu et al., 2012). Karyotypes in the genus Kurixalus are poorly understood. Available cytogenetic data are restricted to the common features, such as diploid chromosome number, in two species (Kuramoto, 1977; Kuramoto and Wang, 1987). In this study, with the aim of providing cytogenetic information of Kurixalus, the karyotypes of two representatives of Kurixalus (K. eiffingeri Boettger, 1895 and K. idiootocus Kuramoto and Wang, 1987) were characterized using G-banding, C-banding, and Ag-NOR staining. Because karyotypic variations may involve rearrangements that are not detected by conventional karyotyping, we also included here the telomeric FISH and CGH analyses in the karyotypes of both species. Moreover, the genomic sizes were also examined to yield a natural link to the cytogenetic data that provide a genetic basis for comparative studies of interspecies as well as intersex differentiation in Kurixalus. Sampling MATERIALS AND METHODS A total of 40 specimens of Kurixalus eiffingeri (male: n = 10, female: n = 10) and K. idiootocus (male: n = 10, female: n = 10) were collected from Xitou (elevation 1,092 m a.s.l.; N, E) and Yuchi (elevation 634 m; N, E), respectively, in Nantou County, Taiwan (Fig. 1). Both of the two Kurixalus species examined have been taxonomically identified, in addition to phenotypic classification, by phylogenetic analysis using the mitochondrial DNA cytochrome c oxidase subunit 1 (CO1) and the 16S rdna sequencing (Wu et al., 2016). The reference CO1 and 16S rdna sequences have been uploaded to GenBank with accession numbers of DQ and DQ for K. eiffingeri, and DQ and DQ for K. idiootocus. Tissues of gonad (from both sexes) and kidney were obtained and cultured according to Chinchar and Sinclair (1978). Cytogenetic analyses For chromosome preparation, mitotic metaphase arrest was induced by adding 0.1 μg/ml colcemid 4 h before cell harvest-

21 Comparisons of karyotype and genome size in two Kurixalus species 141 immersed the slide in 2x SSC for 2 min at RT without agitation, followed by a successive dehydration step with 70%, 85% and 100% ethanol respectively for 2 min and allowed to dry. The sample and probe were co-denatured by heating the slide at 75 C for 2 min, and then hybridizations were performed at 37 C for 12 h. The slide were immersed in 0.4 x SSC (ph 7.0) at 72 C for 2 min and then were immersed in 2 x SSC, 0.05% Tween-20 at RT (ph 7.0) for 1 min. Finally, the slides were drained and applied 10µl of µg/ml DAPI antifade onto hybridization areas, and were viewed with a fluorescence microscope. Comparative genomic hybridization (CGH) Fig. 1. Map showing sampling locations of the two Kurixalus species in Nantou County, Taiwan. 1. Yuchi ( N, E) for K. idiootocus; 2. Xitou ( N, E) for K. eiffingeri. ing. Hypotonic KCl solution (0.075 M) was applied for 20 min, followed by fixation with fresh Carnoy s fixative (3:1 of methanol to glacial acetic acid) at 4 C for 1 h. G-banding, C-banding and Ag-NOR staining were carried out according to published protocols (Wang et al., 2004; Wu et al., 2007). In short, for G-banding, the metaphase chromosomes were digested with trypsin at room temperature (RT) for 15 sec and subsequently stained with Wright s stain at RT for 2 min; for C-banding, the metaphase chromosomes were treated with an alkali barium hydroxide 5% Ba(OH) 2 at 50 C for 15 sec, incubated in 2 x SSC solution at 60 C for 1 h, and afterward stained with Wright s dye; for Ag-NOR staining, the working solution (mixture of 100 μl of the 2% gelatin colloid solution and 200 μl of the silver nitrate solution) was mixed immediately before use and pipetted onto the slides with the metaphase chromosomes at 70 C for 2 min. After rinsing off with deionized water, the metaphase chromosomes were stained with Wright s dye at RT for 2 min. Telomeric FISH The physical map of telomeric sequences was detected by fluorescence in situ hybridization (FISH) using All Human Telomeres Probe (TTAGGG)n (Qbiogene/MP Biomedicals LLC, Tucson, AZ, USA) and followed manufacturer s manual. Briefly, 10 µl of the fixed, pre-treated and homogenized samples were applied on a microscope slide and dried at 50 C for 15 min, Comparative genomic hybridization is often used for the evaluation of the differences between the chromosomal complements of different sexes (Badenhorst et al., 2013; Matsubara et al., 2013; Rovatsos et al., 2015). The CGH between sexes was performed as previously described (Yu et al., 2012), with some modifications. Briefly, the genomic DNA from male and female were labeled with fluorescein isothiocyanate (FITC)-12-dUTP and Texas Red (TxRed)-5-dUTP (Perkin Elmer Life Sciences, Boston, MA, USA) respectively by Nick Translation Kit (Roche Diagnostics, Basel, Switzerland) and co-precipitated with a 200- fold excess of Cot-1 DNA. Genomic probes were re-dissolved and kept in hybridization buffer (50% formamide, 10% dextran sulphate, and 2 x SSC). The hybridization mixture was denatured at 80 C for 10 min, followed by preannealing at 37 C for 60 min. The slides were incubated in a moist chamber at 37 C for 72 h. After standard washing procedure, chromosomes were counterstained with μg/ml DAPI added to antifade reagent (Abbott, Illinois, USA). Image capture and analysis Karyotype images, FISH and CGH results were analyzed for at least 10 well-spread metaphases in each sample by Cyto- Vision system (Applied Imaging, Carlsbad, CA, USA). For karyotyping, chromosome types were classified according to the nomenclature of Levan et al. (1964). Genome size estimation The genome sizes were estimated by flow cytometry based on the description of Matsuba and Merilä (2006). Blood samples from each sex of both species were collected in phosphatebuffered saline without divalent cations and stored at 4 C. As internal references, male blood cells from Lithobates catesbeianus (haploid genome size: C value = 7.37 pg) and Homo sapiens (C value = 3.5 pg) were analyzed simultaneously in 1:10 mixed with surveyed cells. Cells were suspended in 0.5 ml of a solution containing 25 μg propidium iodide (PI), 0.1% sodium citrate, 25 μg RNAase, and 0.1% Triton X-100. Samples were filtered through 30 μm mesh and kept at 4 C in the dark for over 2 h. Mean fluorescence of co-stained nuclei was quantified

22 142 Shun-Ping Chang et alii on a Beckman-Coulter EPICS XL-MCL flow cytometer with an argon laser (emission at 488 nm/15 mw power), and analyzed with WinMDI 2.9 software. The PI fluorescence and genome size of L. catesbeiana and H. sapiens were used as standards to calculate the unknown genome sizes in samples (Vinogradov, 1998). Student T-test was used to compare the difference of genome sizes between species/sexes by SPSS software (IBM Corp., Armonk, NY, USA). The Bonferroni correction was applied to set the significance cut-off at α/n, with α = 0.05 and n = number of samples. RESULTS A total of 138 and 125 metaphase spreads from K. eiffingeri (individual number = 10) and K. idiootocus (individual number = 10) respectively were subjected to chromosome analyses. Both species had the same haploid and diploid chromosome numbers (n = 13 and 2n = 26), and chromosomes are readily classified into two distinct size groups, large pairs of chromosomes (no. 1-5) and small pairs of chromosomes (no. 6-13). All chromosome pairs were either metacentric or submetacentric, giving a fundamental number (FN) of 52 (Table 1). Though karyotypes of the two species were similar, differences were noted in detailed chromosome morphology and banding pattern (Fig. 2). No chromosomal polymorphism (e.g., inversions, translocations, deletions and duplications) was noted among individuals examined in each of the two species. In K. eiffingeri the chromosome pairs 2, 3, and 9 were submetacentrics and the others metacentrics while in K. idiootocus chromosome pairs 2 and 4 were submetacentrics and the others metacentrics. The karyotype formulas of K. eiffingeri and K. idiootocus were thus 10 metacentric + 3 submetacentric and 11 metacentric + 2 submetacentric, respectively. An achromatic secondary constriction (SC) was found in the long arm near the centromere of chromosome pair 8 and pair 12 in K. eiffingeri and K. idiootocus respectively (Fig. 2). Additionally, significant differences (ANOVA test, P < ) in relative chromosome length were found in chromosome pairs 1-3 (longer in K. eiffingeri) and in chromosome pairs 6-9 and (longer in K. idiootocus) (Table 2). No heteromorphic chromosome pair (sex chromosomes) could be identified in each species. C-banding analysis showed that constitutive heterochromatin was located at the centromeres of all chromosomes of K. eiffingeri and K. idiootocus, but heterochromatins differed in banding intensity corresponding with blocks of constitutive heterochromatin. An additional dense heterochromatin was only found on proximal short arm of chromosome pair 8 of K. eiffingeri (Fig. 3). The nucleolus organizer region (NOR) was detected by silver staining on the long arm near the centromere of chromosome pair 8 in K. eiffingeri, but on the long arm near the centromere of chromosome pair 12 in K. idiootocus (Fig. 2). The active NORs are within the corresponding regions of SCs in both species (Fig. 2 and Table 1). FISH with telomeric probe (TTAGGG)n detected strong signals on the chromosome ends of all chromosomes of K. eiffingeri and K. idiootocus (Fig. 4). No (TTAGGG)n signal was found at interstitial region in either species. The CGH for K. eiffingeri and K. idiootocus revealed no notable chromosome difference between sexes. Most of stronger painting signals were observed in the centromeres with blocks of tandemly repeated satellite sequences, which are embedded in heterochromatic regions. A strong hybridization signal was noted in the telomeric region of the long arm of chromosome pair 3 of K. eiffingeri using either male or female probe (Fig. 5). The DNA C value of was estimated as 5.06 ± 0.13 pg (male: 5.02 ± 0.08 pg, female: 5.11 ± 0.16 pg) in K. eiffingeri (n = 20), and 4.18 ± 0.21 pg (male: 4.23 ± 0.22 pg, female: 4.13 ± 0.20 pg) in K. idiootocus (n = 17) (Table 1). Significant difference in genome size between species was evidenced (Student t-test, t 35 = 15.29, P < ). In contrast, no sexual difference in genome size was found in both species (Student t-test, t 18 = , P = in K. eiffingeri, 10 male vs 10 female; t 15 = 0.968, P = in K. idiootocus, 9 male vs 8 female). DISCUSSION The chromosomes of frogs in the family Rhacophoridae is considered to be conservative since almost all species studied so far show invariable 2n = 26 karyotypes (Li, 2007). Distinct diploid chromosome number in this family is extremely rare and only reported in few species (e.g., Chiromantis doriae 2n = 16, in Rao and Yang, 1996, but 2n = 16 or 26 in Tan, 1987). Rhacophorid karyotypes also share other characteristics including composition of two size groups of chromosomes (i.e., large and small chromosome pairs) and predominant presence of metacentric and submetacentric chromosomes (Kuramoto, 1977; Blommers-Schlösser, 1978; Kuramoto, 1985; Kuramoto and Wang, 1987; Tan, 1987; Rao and Yang, 1996; Joshy and Kuramoto, 2011). These features, together with the fact that very few acrocentric and telocentric chromosomes were found, have led to the suggestion that the karyotype of Rhacophoridae represent a derived condition in Anura (Rao and Yang, 1996) because the primary direction of Anuran karyotype evolution has been advocated to reduce chromosome number by fusions of acrocentrics to metacentrics (Morescalchi, 1980; Suarez

23 Comparisons of karyotype and genome size in two Kurixalus species 143 Table 1. Summary of karyotype and chromosomes features of the two species: 2n, diploid chromosome number; M, metacentric chromosomes; SM, submetacentric chromosomes; FN, fundamental number; SC, secondary constriction; NOR, nucleolus organizer region; NSF, no specific findings. 2n Karyotype formula FN Size group of chromosomes (large + small) SC NOR Heterochromatins (by C-banding) Telomeric motif (TTAGGG)n (by telomeric FISH) CGH C value (pg; mean ± SD) K. eiffingeri Male M + 3 SM q 8q Female M + 3 SM q 8q Overall M + 3 SM q 8q 1. Centromere of all chromosomes 2. Proximal 8p (dense) 1. Centromere of all chromosomes 2. Proximal 8p (dense) 1. Centromere of all chromosomes 2. Proximal 8p (dense) Telomeres of all chromosomes Telomeres of all chromosomes Telomeres of all chromosomes Stronger painting signal on 3q Stronger painting signal on 3q Stronger painting signal on 3q 5.02 ± ± ± 0.13 K. idiootocus Male M + 2 SM q 12q Centromere of all chromosomes Female M + 2 SM q 12q Centromere of all chromosomes Overall M + 2 SM q 12q Centromere of all chromosomes Telomeres of all chromosomes Telomeres of all chromosomes Telomeres of all chromosomes NSF 4.23 ± 0.22 NSF 4.13 ± 0.20 NSF 4.18 ± 0.21

24 144 Shun-Ping Chang et alii Fig. 2. G-banding karyotypes of K. eiffingeri for male (A), female (B); and proposed idiogram (C), and K. idiootocus for male (D), female (E) and proposed idiogram (F). The G-banding karyotypes of female and male are similar in each of the two species. Bars = 10 μm. An achromatic secondary constriction (SC; indicated by stars) was found in the long arm near the centromere of chromosome pair 8 of K. eiffingeri (G) and in the long arm near the centromere of chromosome pair 12 of K. idiootocus (H). The SCs were accompanied by positive Ag-NOR staining (indicated by arrows) in both species (G and H). et al., 2013). The two Kurixalus species analyzed here revealed conservative karyological features of Rhacophoridae, including the modal diploid number, 5 larger and 8 smaller chromosomal pairs, but no acrocentric (either metacentric or submetacentric) chromosomes. Although the diploid states of the two Kurixalus species are similar, evidence of karyotype divergence through chromosomal rearrangements was found. The two Kurixalus species are different in karyotype microstructure. A highly dense heterochromatin was only found in the proximal short arm of the chromosome pair 8 in K. eiffingeri, which could be a chromosome marker in karyotype of this species. Besides, the long arm of chromosome pair 3 of K. eiffingeri was painted with strong fluorescence signals in karyotypes of both sexes, suggesting the region is rich in repeated sequences in this species. In both species, NORs were only found in one pair of chromosomes and the NOR localizations were coincided with regions of SC. However, the NOR-bearing chromosome pairs differ between K. eiffingeri and K. idiootocus: one in pair 8 and the other in pair 12. Extensive studies have shown that NORs can differ in extent, localization and number among chromosomes from different species that make NORs a useful chromosome marker in comparative cytogenetic studies (Morescalchi, 1980; Mahony and Robinson, 1986; Bruschi et al., 2014). For instance, the NOR on chromosome pair 7 has been recognized as a structural distinction between Z and W chromosomes in Buergeria buergeri, which is the only known rhacophorid species with heteromorphic sex chromosomes (Schmid et al., 1993). Recently, the possible role of NORs as rearrangement hotspots during evolution is also discussed (Cazaux et al., 2011; Britton-Davidian et al., 2012). Chromosomal rearrangements can be traced by chromosomal mapping of telomeric sequences, which located at the ends of eukaryotic chromosomes with an

25 Comparisons of karyotype and genome size in two Kurixalus species 145 Table 2. Arm ratio (length of long arm/short arm) and relative length (percentage of total haploid chromosome length) of chromosomes in K. eiffingeri and K. idiootocus (mean ± SD). Abbreviation M and SM indicate metacentric and submetacentric respectively, and n is the number of metaphase spreads analyzed Pair no. Male (n = 19) Arm ratio Female (n = 17) K. eiffingeri K. idiootocus Relative length (%) Arm ratio Attribute Male Female Male Female (n = 19) (n = 17) (n = 21) (n = 19) Relative length (%) Attribute Male Female (n = 21) (n = 19) ANONA test F P ± ± 0.10 M ± ± ± ± 0.11 M ± ± < ± ± 0.11 SM ± ± ± ± 0.11 SM ± ± < ± ± 0.35 SM ± ± ± ± 0.09 M ± ± < ± ± 0.10 M ± ± ± ± 0.17 SM ± ± ± ± 0.11 M 9.61 ± ± ± ± 0.11 M 9.68 ± ± ± ± 0.15 M 5.48 ± ± ± ± 0.15 M 5.79 ± ± < ± ± 0.14 M 5.06 ± ± ± ± 0.07 M 5.46 ± ± < ± ± 0.13 M 4.85 ± ± ± ± 0.08 M 5.18 ± ± < ± ± 0.21 SM 4.67 ± ± ± ± 0.13 M 5.04 ± ± < ± ± 0.13 M 4.53 ± ± ± ± 0.06 M 4.45 ± ± ± ± 0.11 M 3.90 ± ± ± ± 0.09 M 4.40 ± ± < ± ± 0.13 M 3.77 ± ± ± ± 0.11 M 4.30 ± ± < ± ± 0.08 M 3.11 ± ± ± ± 0.09 M 4.09 ± ± < Levan et al. (1964). Statistical significance (α = 0.05/76, after Bonferroni correction). Fig. 3. C-banding karyotypes of K. eiffingeri for male (A) and female (B), and K. idiootocus for male (C) and female (D). Constitutive heterochromatins were regularly detected on the centromeres of all chromosomes. Particularly, a dense heterochromatin was also noted on the proximal region of short arm of chromosome pair 8 in K. eiffingeri. The C-banding karyotypes of female and male are similar in each of the two species. Bars = 10 μm.

26 146 Shun-Ping Chang et alii Fig. 4. Fluorescence in situ hybridization (FISH) with telomeric probe exclusively mapped (TTAGGG)n repeats to terminal regions of all chromosomes of K. eiffingeri (a) and K. idiootocus (b). No interstitial telomeric sequence (ITS) was detected in both species. important function of chromosomal stability by protecting them from end-to-end fusion, degradation, and recombination (Fagundes and Yonenaga-Yassuda, 1998; Bruschi et al., 2014). The presence of interstitial telomeric sequences (ITSs) in chromosomes has been considered as evidence of chromosome rearrangements that occurred during the evolution of karyotypes. Telomeric FISH for the karyotypes of the two Kurixalus species showed that the (TTAGGG)n motifs are restricted to the terminal regions of chromosomes. The absence of ITSs cannot be explained by the absence of chromosome rearrangements because evidence of karyotypic differences between K. eiffingeri and K. idiootocus were noted. We postulate that subtle chromosome changes irrelevant to ITSs (e.g., intrachromosomal rearrangements) may play a role in karyotype evolution. Recent genomic studies have shown that amphibians have a slow rate of interchromosomal rearrangements but intrachromosomal rearrangements of loci are not uncommon (Smith and Voss, 2006; Sun et al., 2015). Thus, the undetected chromosome changes Fig. 5. Comparative genomic hybridization (CGH) using male and female genomic probes (labeled by FITC and TxRed, respectively) for chromosomes from male and female of K. eiffingeri showed similar hybridization patterns between sexes (A). A large chromosomal fragment with stronger painting signal (indicated by an arrow) were observed on the terminal regions of long arm of chromosome pair 3, presumably rich in repeated sequences (B). Similar patterns between sexes without any stronger painting signal in chromosome pairs were observed in K. idiootocus (data not showed). p, short arm; q, long arm. in Kurixalus may be uncovered by advanced molecular analyses. The present data demonstrated that the karyotypes of the two Kurixalus species seem to be conserved, despite minor differences in chromosome morphology were observed. As a result, the interspecies differences at chromosomal level are unsatisfying to link directly with their behavioral difference in reproductive mode. In addition to cytogenetic characteristics, genome sizes of the two Kurixalus species are different. The estimated DNA C value of K. eiffingeri is about 1.21 times greater than that of K. idiootocus. Currently, no information is available about the C values of other rhacophorid species and the few data collected in Genome Size Database ( actually belonged species in the Mantellidae. In amphibians, the C values ranged from

27 Comparisons of karyotype and genome size in two Kurixalus species to 120 pg. The more than 130-fold variation in C value has rendered amphibian a taxon with the most variable genome size among vertebrates (Gregory, 2003). Genome size can be affected by various genetic events, such as duplication, insertion, recombination, deletion and polyploidization (Gregory, 2005). Difference in genome sizes thus reflects dissimilarity in genomic compositions. Identification of heteromorphic chromosome pairs has been generally acknowledged as the first step in recognition of sex determination mechanism (Valenzuela et al., 2003). Some heteromorphic chromosome pairs differ in chromosomal appearance and are easily observable, whereas others require advanced high-resolution tools to clarify. In this study, cytogenetic analyses for the two Kurixalus species revealed all chromosome pairs are homomorphism. Even the CGH was executed for both sexes in each species, no recognizable sex difference in karyotypes. This is not surprising because in amphibians most species lack morphologically distinguishable sex chromosomes despite exhibiting gonochorism (distinct male and female). So far, only a few of amphibian species with heteromorphic sex chromosomes were reported (Hayes, 1998; Berset-Brandli et al., 2006; Ezaz et al., 2006). It is a little strange that, despite the lack of heteromorphism in chromosome pairs of most amphibian species, genetic sex determination is generally considered the rule for this group (Nakamura, 2009). It was proposed that since sex chromosomes evolved from homomorphic autosomes through the acquisition of a sex determining gene (Ohno, 1967), the reason that heteromorphic sex chromosomes could not be highlighted in amphibians was not due to their nonexistence but because the genes involved in sex determination were located on poorly discriminated regions of the chromosomes (Hayes, 1998). For example, the gene for ADP/ATP translocase was described as a sex-linked marker in Rana rugosa (Miura et al., 1998) of XX/XY system and sex-specific loci in Xenopus laevis (Mawaribuchi et al., 2017) of ZZ/ZW system, but none of the species exhibited notable differences in karyotypes between sexes. However, in the two Kurixalus species analyzed the sexual differences in genomic size are too small to be detected, leading to inconclusive evidence if genetic differentiation exists between sexes. Traditionally, a good strategy to understand the possible sex determination mode in species is to examine the sex ratio in natural populations (Janzen and Paukstis, 1991). Balanced sex ratio is the most general explanation for species with genetic determination. The sex ratio of K. idiootocus in nature was not known, but K. eiffingeri had balanced adult sex ratio (51.2% proportion of males) (Kam et al., 2000). Although this provides a possible link to the genetic sex determination in K. eiffingeri, segregation distortion, such as temperature-induced differential mortality, differential fertilization, and embryo abortion, can result in biased primary (fertilization) or secondary (births) sex ratio (Valenzuela et al., 2003). Besides, in a few frog species the sex can be affected by temperature despite they are considered as genetic sex determination (Witschi, 1914; Piquet, 1930; Yoshikura, 1963). Basically, all above arguments must move ahead from a well-established base of sufficient cytogenetic information just like our efforts in the two Kurixalus species. We considered that the loss of cytogenetic differences between sexes of the two Kurixalus species does not refute the idea that these species possess genetic sex determination. However, the present data did not provide evidence to support the existence of sex-specific genetic markers in either species. It is possible that the sex chromosomes might be homomorphic, with the extensive pseudoautosomal region (Roco et al., 2015), and small sex specific regions that harbor key genes from the sexdetermination pathway, such as the cases with sex specific Dmrt1 gene in Rana temporaria (Ma et al. 2016; Rodriguez et al 2017), which would need more advanced molecular approaches (e.g., genotyping by length-polymorphic markers) to exploring the genetic sex-determination. At the moment, it remains unclear if all amphibian species are of genetic sex determination, but species with diverse reproductive behaviors like Kurixalus provides an interesting object to study this question. We demonstrated that the two Kurixalus tree frogs have distinct karyotypes and genome sizes. Cytogenetic characteristics, such as the distribution of SC/NOR and staining intensity of heterochromatins that distinguished the karyotypes of the two species, were not previously reported. In either species, the finding of no differences in cytogenetic and genomic features between sexes is in line with previous reports that showed no heteromorphic chromosome pair in most rhacophorid species. Moreover, our telomeric FISH demonstrated no ITS in chromosomes of both species, suggesting subtle chromosome changes irrelevant to ITSs (e.g., intrachromosomal rearrangements) may play a role in their karyotype evolution. The cytogenetic data and the genomic information described in this work contribute to our understanding of the genetic features in Kurixalus species and are potentially useful as taxonomic and reproductive traits for additional studies in this genus as well as in the family Rhacophoridae. ACKNOWLEDGEMENTS The authors are thankful to Yin-Ju Yang, Miin-Yu Horng, Yi Fu Lin, Chun-Hsin Tsai, Chia-Ming Kuo, and

28 148 Shun-Ping Chang et alii Hui-Shan Tsai for valuable help with sample collection, and to the staff of the Experimental Forest of the National Taiwan University at Xitou for a permit to collect samples in the experimental forest and providing accommodation. The wild animals were sampled according to the Taiwan Animal Protection Act with permits from Nantou County Government (permit no ) and Experimental Forest of the National Taiwan University (permit no ). The research was reviewed and approved by the Institutional Animal Care and Use Committee of Changhua Christian Hospital (CCH- AE ). The study was partly supported by Changhua Christian Hospital (project No: 104-CCH-IRP-101) and Ministry of Science and Technology, Taiwan (project No: B ). REFERENCES Badenhorst, D., Stanyon, R., Engstrom, T., Valenzuela, N. (2013): A ZZ/ZW microchromosome system in the spiny softshell turtle, Apalone spinifera, reveals an intriguing sex chromosome conservation in Trionychidae. Chromosome Res. 21: Berset-Brandli, L., Jaquiery, J., Dubey, S., Perrin, N. (2006): A sex-specific marker reveals male heterogamety in European tree frogs. Mol. Biol. Evol. 23: Biju, S.D., Senevirathne, G., Garg, S., Mahony, S., Kamei, R.G., Thomas, A., Shouche, Y., Raxworthy, C.J., Meegaskumbura, M., Van Bocxlaer, I. (2016): Frankixalus, a new rhacophorid genus of tree hole breeding frogs with Oophagous Tadpoles. PLoS One 11: e Blommers-Schlösser, R. (1978): Cytotaxonomy of the Ranidae, Rhacophoridae, Hyperoliidae (Anura) from Madagascar with a note on the karyotype of two amphibians of the Seychelles. Genetica 48: Britton-Davidian, J., Cazaux, B., Catalan, J. (2012): Chromosomal dynamics of nucleolar organizer regions (NORs) in the house mouse: microevolutionary insights. Heredity 108: Bruschi, D.P., Rivera, M., Lima, A.P., Zuniga, A.B., Recco- Pimentel, S.M. (2014): Interstitial Telomeric Sequences (ITS) and major rdna mapping reveal insights into the karyotypical evolution of Neotropical leaf frogs species (Phyllomedusa, Hylidae, Anura). Mol. Cytogenet. 7: 22. Carvalho, K.A., Garcia, P.C., Recco-Pimentel, S.M. (2009): Cytogenetic comparison of tree frogs of the genus Aplastodiscus and the Hypsiboas faber group (Anura, Hylidae). Genet. Mol. Res. 8: Cazaux, B., Catalan, J., Veyrunes, F., Douzery, E.J., Britton-Davidian, J. (2011): Are ribosomal DNA clusters rearrangement hotspots? A case study in the genus Mus (Rodentia, Muridae). BMC Evol. Biol. 11: 124. Cheng, W.C., Chen, Y.H., Yu, H.T., Roberts, J.D., Kam, Y.C. (2013): Sequential polygyny during egg attendance is rare in a tree frog and does not increase male fitness. Ethology 119: Ezaz, T., Stiglec, R., Veyrunes, F., Marshall Graves, J.A. (2006): Relationships between vertebrate ZW and XY sex chromosome systems. Curr. Biol. 16: R Fagundes, V., Yonenaga-Yassuda, Y. (1998): Evolutionary conservation of whole homeologous chromosome arms in Akodont rodents Bolomys and Akodon (Muridae, Sigmodontinae): maintenance of interstitial telomeric segments (ITS) in recent event of centric fusion. Chromosome Res. 6: Frost, D.R. (2017): Amphibian Species of the World: an Online Reference. Version org/vz/herpetology/amphibia/index.php (2017). Accessed 05 May Gregory, T.R. (2003): Variation across amphibian species in the size of the nuclear genome supports a pluralistic, hierarchical approach to the C-value enigma. Biol. J. Linn. Soc. Lond. 79: Gregory, T.R. (2005): Genome size evolution in animals. In: The Evolution of the Genome, pp Gregory T.R., Ed, Elsevier Academic Press, San Diego. Hanada, H. (2002): G and C banding show structural differences between the Z and W chromosomes in the frog Buergeria buergeri. Hereditas 136: Hayes, T.B. (1998): Sex determination and primary sex differentiation in amphibians: genetic and developmental mechanisms. J. Exp. Zool. 281: Hertwig, S.T., Schweizer, M., Das, I., Haas, A. (2013): Diversification in a biodiversity hotspot--the evolution of Southeast Asian rhacophorid tree frogs on Borneo (Amphibia: Anura: Rhacophoridae). Mol. Phylogenet. Evol. 68: Janzen, F.J., Paukstis, G.L. (1991): Environmental sex determination in reptiles: ecology, evolution, and experimental design. Q. Rev. Biol. 66: Jiang, K., Yan, F., Wang, K., Zou, D.H., Li, C., Che, J. (2016): A new genus and species of treefrog from Medog, southeastern Tibet, China (Anura, Rhacophoridae). Zool. Res. 37: Joshy, S.H., Kuramoto, M. (2011): Karyological studies on 5 anuran species (Rhacophoridae, Microhylidae) from the western ghats, southwest India. Cytologia 76: Kam, Y.C., Chen, Y.H., Chen, T.C., Tsai, I.R. (2000): Maternal brood care of an arboreal breeder, Chirix-

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31 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol Non-native turtles in a peri-urban park in northern Milan (Lombardy, Italy): species diversity and population structure Claudio Foglini 1, Roberta Salvi 2, * 1 Indipendent researcher. Via L. B. Alberti 8/A, I Cinisello Balsamo (MI), Italia 2 Indipendent researcher. Via A. Moro 1, I Carvico (BG), Italia. *Corresponding author. clafogli@libero.it Both authors contributed equally to this work. Submitted on: 2017, 30 th of May; revised on: 2017, 16 th of August; accepted on: 2017, 6 th of October Editor: Simon Baeckens Abstract. Trachemys scripta (slider turtle) and other freshwater turtle species have been traded worldwide as pets, but they are often released by owners in wild or in semi-natural habitats, especially in suburban and urbanized areas. In four artificial lakes and ponds of a Regional Park in the North outskirt of Milan (Italy, Lombardy), we trapped non native turtles using basking-traps and landing nets from April to August 2013.We captured 156 Trachemys scripta, 10 Graptemys pseudogeographica and four Pseudemys sp.; the capture-mark-recapture approach estimated an overall number of 224 individuals (95% C.I ). There was also a strong indirect support for a probable in situ reproduction, with small juveniles captured and a nest-digging female observed. Key-words. Invasive alien species, morphometrics, non-native-turtles, reproduction, slider turtle, Trachemys scripta. INTRODUCTION The global introduction of non-native amphibian and reptile species has increased exponentially trough the last 150 years (Çiçek and Ayaz, 2015). One animal group that is globally very popular in the pet trade is freshwater turtle, in particular subspecies and hybrids of the American slider turtle Trachemys scripta. Turtles belonging to this group are massively traded at the global scale (Ballasina, 1995; Kraus, 2009) and are usually sold when they are only few centimetres long. Due to their fast growing rate these turtles are then often released into the wild or in semi natural habitats (Crescente et al., 2014). Trachemys scripta compete for food and basking places in wetlands where they coexist with European Emys orbicularis and Mauremys leprosa (Cadi and Joly, 2003; Perez-Santigosa et al., 2008; Ficetola et al., 2009), and massive turtle presence can influence ecosystem functioning and aquatic communities (Lindsay et al., 2013). Free-living slider turtles populations occur in many countries of southern Europe (Spain: Martínez-Silvestre et al., 2011; France: Cadi et al., 2004; Italy: Agosta and Parolini, 1999; Ferri and Soccini, 2003; Ficetola et al., 2003; Monti, 2010) and the ongoing climate change will probably expand the areas of suitability in the future (Ficetola et al., 2009). In addition, following the European ban on import of T. s. elegans in the 1997 (European Commission, 1997), legal pet trade switched to other slider subspecies: mainly T. s. scripta, hybrids T. s. scripta x T. s. elegans and, but to lesser extent, T. s. troostii (Bringsøe, 2006). After a new European ban, extended to all Trachemys subspecies (European Commission, 2001 and subsequent amendments), different species of Graptemys and Pseudemys were also imported (Bringsøe, 2006). In this study we investigated: (1) the number and size structure of turtles; (2) the presence of different species and subspecies of several non-native turtles; (3) the possibil- ISSN (print) ISSN (online) Firenze University Press

32 152 Claudio Foglini, Roberta Salvi ity of reproduction in the artificial lakes and ponds of the Nord Milano Park (Italy, Lombardy, Province of Milan). MATERIALS AND METHODS The study was carried out in the Nord Milano Park (Fig. 1), a 640 ha peri-urban Regional Park located in the North outskirts of Milan (Italy, Lombardy). The park was created on industrial brownfield during later sixties, and first reforestation dates back to During the years, woods, artificial wetlands, small lakes and ditches were added, while many others environmental improvement measures are still in progress. The presence of several non-native turtles in the park is well known, but has never been deeply investigated. The counting sessions of this study were focused into four wetlands: three artificial lakes (Cinisello, Suzzani and Bresso Lakes) and one ditch-like site (Breda Ditch) (see Fig. 1 legend). To avoid problems with double-counts and with low detectability (i.e., water turbidity, lack of basking sites, aquatic macrophytes cover) turtles were captured with basking traps from April to August 2013: each trap was visited every two or three days between 11:00 AM and 03:00 PM, when the basking activity is greater (Cadi and Joly, 2000). During each visit additional turtles were captured with landing nets. Parameters such as the straight line plastron length (SPL), the minimum straight carapace length (SCLmin) and the straight carapace width (SCW) were measured with callipers (Bjorndal and Bolten, 1989) by the same operator. Each individual was weighted and sexed using secondary sexual characteristics such as foreclaws and tail length (Readel, 2008). Specimens with SPL less than 150 mm (Gibbons, 1990) and with doubtful elements were considered unsexed. Each animal was also unambiguously marked with a unique notches combination on the marginal carapace scutes. The release of invasive turtles after marking was an exceptional procedure, necessary to collect the basic knowledge for a pilot capture-recapture study; this was essential to develop and refine an effective control or eradication plan in the study area. Given the fact that capture and recapture invariably occurred for each individual within the same site, a close populations scenario was assumed. This assumption is supported by the fact that trapping sites are not connected and are separated by wooded areas, lawns, footpaths and bikeways. For this reason, in order to estimate the overall number of turtles, the capture-mark-recapture (CMR) model for close populations (JHE Closed Population Model Estimation) provided by the software Noremark (White, 1996) was used. Fig. 1. Study area (black box, not scaled). The position and a detailed view of each wetland is given by numbers: 1) Cinisello Lake; 2) Bresso Lake; 3) Breda Ditch; 4) Suzzani Lake. Wetland boundaries are marked in white (modified from and GeoPortale Regione Lombardia).

33 Non-native turtles population in peri-urban wetland 153 Table 1. Number and sex of non-native turtles captured in each pond, grouped by taxa. M=male; F=female; n.d.=unsexed. Wetland T. s. elegans T. s. scripta T.s. troostii T. s. hybrids Pseudemys sp. G. pseudogeographica M F n.d. M F n.d. M F n.d. M F n.d. M F n.d. M F n.d. Suzzani Lake Bresso Lake Breda Ditch Cinisello Lake Table 2. Morphometrics (mean ± SD and range) of captured specimens, grouped by taxa. SPL = Straight line Plastron Length; SCLmin = minimum straight carapace length; weight. N may vary, as some data is missing. T. s. elegans T. s. scripta T. s. troostii T. s. hybrids Pseudemys sp. G. pseudogeographica SPL (mm) ± SD ± ± ± ± ± ± n min. max. (mm) SCL min (mm) ± SD ± ± ± ± ± ± n min. max. (mm) weight (g) ± SD ± ± ± ± ± ± n min. max. (g) RESULTS Species richness and abundance We captured and individually marked, weighted and sexed 156 slider turtles: 16 red-eared slider Trachemys scripta elegans, 35 yellow-bellied slider T. scripta scripta, 7 Cumberland slider T. scripta troostii and 98 T. scripta hybrids. Specimens were classified as hybrids when they showed a mix of elements from parental species (Seidel et al., 1999; for details see supplementary Fig. S1). We also captured, measured and marked 10 false map turtle Graptemys pseudogeographica (supplementary Fig. S2) and cooter Pseudemys sp. (supplementary Fig. S3). Details about sex-ratio and species repartition into each sampling site are shown in Table 1, while details about morphometrics are shown in Table 2. Using the acquired data, the following overall population was estimated with Noremark software (95 % confidence interval in parentheses): 13 (13) turtles in the Cinisello Lake, 84 (73-102) in the Suzzani Lake, 106 (90-129) in the Bresso Lake and 21 (19-26) in the Breda Ditch (supplementary Table S4). Trachemys scripta population structure Due to the lack of data for the others turtles genera, only the population structure of Trachemys scripta subspecies was analysed. Among sexed specimens, in three sampled wetlands we found a shift towards a female-biased sex-ratio. In the Suzzani and Bresso lakes (Fig. 2a and 2b, respectively), we found mainly medium-small turtles; full-grown adults are present in small number, and we found also some juveniles. Small turtles were prevalent also in the Breda Ditch, in which we found also only two juveniles and one adult female (Fig. 2c). In the Cinisello Lake, the small number of individuals does not allow a population description (Fig. 2d). The presence of small specimens with SPL between 26 and 51 millimetres was noteworthy. Four were T. scripta hybrids, three of which trapped in the Bresso Lake and one in the Breda Ditch (SPL 26 to 51 mm, μ ± SD). One T. s. elegans (SPL 43 mm) was captured in the Suzzani Lake, and one T. s. scripta (SPL 42 mm) was captured in the Bresso Lake (supplementary Fig. S5a and S5b). Three more Trachemys juveniles have SPL

34 154 Claudio Foglini, Roberta Salvi Fig. 2. Number of slider turtles, classified after their Straight Plastron Length, hosted in each wetland (completion year shown in brackets). inside the same range, but their SCL exceed 60 mm. DISCUSSION Trachemys scripta has been included in the Top 100 World s Worst Invaders as a result of their formerly massive import by the pet trade (Lowe et al., 2000). It is now listed among the Invasive Alien Species (IAS) of European Union concern (European Commission, 2016). Pseudemys and Graptemys have partially replaced sliders as pets-turtle, but due to their higher price they are not so common as feral individuals (Bringsøe, 2006). Species composition and population status Captured slider turtles were mainly T. scripta hybrids and T. s. scripta, followed by red-eared slider and just few T. s. troostii. Few individuals of other species (i.e., false map turtle and cooter) were also captured. Among slider turtles, the female-biased sex ratio we found is common in non-native population, because slider turtles were reared with artificially high temperature: this accelerate their development (Prevot-Julliard et al., 2007), but increase female development in species with temperature-dependent sex determination (Godfrey et al., 2003). In suitable conditions, the unbalanced sex ratio could increase the number of recruits of non-native population (Ficetola et al., 2009). According to the number and the size of small turtles captured, Bresso Lake was the only wetland with apparently active recruiting: this could be due either to new release of relatively young individuals (uncommon with pet turtle) or to active reproduction (see below). Reproduction Several Mediterranean and southern-continental European areas currently have suitable climate conditions for slider turtles successful reproduction (Tzankov et al., 2015). Concerning breeding turtles in the park, before this study there was only an anecdotic report of one hatchling with eggshell remains, ran over on the side of a bikeway (Mariani G., pers. com.). Unfortunately, because of the advanced decomposition, it was possible to identify the specimen only as belonging to the Trachemys genus. The smallest slider turtles recorded (see results) can be considered clues of probably recent breeds, because their carapace lengths were similar to those reported for newly born juveniles in Ernst et al. (1994). Moreover, the juvenile T. s. elegans captured was almost certainly feral, because the trade of this specie in the EU was banned since 1997 (European Commission, 1997).

35 Non-native turtles population in peri-urban wetland 155 Although we did not record nests during this study, in July 2016 an adult female was observed during nestdigging (Gelso M.R., pers.com.; supplementary Fig. S6), and during the same month, in the Bresso Lake another juvenile of T. scripta was reported (Ghislandi A. and Ghislandi M., pers. com.; supplementary Fig. S7). Conclusions As the park is deeply engulfed in the urban matrix, no European pond turtle Emys orbicularis is present, but on the other end there is a small population of smooth newt (Lissotriton vulgaris), classified as near threatened (NT) in Italy (Rondinini et al., 2013). For all these reasons, we find unusual that the park has not yet adopted an invasive turtle control plan, even more after the ratification of the European Regulation 1143/2014 and the European Implementing Regulation 1141/2016 (European Commission, 2014; 2016). According to the evidences we found (i.e. reproductive population, number of potential breeding turtles), we hope that this work could represent a first step towards the adoption of a non-native turtles monitoring and control program in the area, even with eradication measures. Due to the high level of anthropization, and the easy accessibility of wetlands in the study area, we also urged the adoption of steady actions aimed at discouraging further releases by turtle owners. ACKNOWLEDGMENTS We would like to thank Nord Milano Park management for research opportunities, and all the employees (especially Gianmario Bernasconi, Francesco Carbone and Marco Siliprandi) for their generous support. We would like to thank Francesco Ficetola and Thania Manfredi for their valuable advice. Many thanks also to Anna and Maurizio Ghislandi and to Maria Rita Gelso for some of the pictures. SUPPLEMENTARY MATERIAL Supplementary material associated with this article can be found at < Manuscript number REFERENCES Agosta, F., Parolini, L. (1999): Autoecologia e rapporti sinecologici di popolazioni introdotte in Lombardia di Trachemys scripta elegans: cati preliminari. Riv. Idrobiol. 38: Ballasina, D. (1995): Salviamo Le Tartarughe! Edagricole, Padova. Bjorndal, K.A., Bolten, A.B. (1989): Comparison of straight-line and over-the-curve measurements for growth rates of green turtles, Chelonia mydas. B. Mar. Sci. 45: Bringsøe, H. (2006): NOBANIS Invasive Alien Species Fact Sheet Trachemys scripta. From: Online Database of the North European and Baltic Network on Invasive Alien Species NOBANIS accessed 25 June Cadi, A., Joly, P. (2000). The introduction of the slider turtle (Trachemys scripta elegans) in Europe: competition for basking sites with the European pond turtle (Emys orbicularis). Chelonii 2: Cadi, A., Joly, P. (2003): Competition for basking places between the endangered European pond turtle (Emys orbicularis galloitalica) and the introduced red-eared slider (Trachemys scripta elegans). Can. J. Zool. 81: Cadi, A., Delmas, V., Prévot-Julliard, A.C., Joly, P., Pieau, C., Girondot, M. (2004): Successful reproduction of the introduced slider turtle (Trachemys scripta elegans) in the South of France. Aquat. Conserv. 14: Çiçek, K., Ayaz, D. (2015): Does the red-eared slider (Trachemys scripta elegans) breed in Turkey? Hyla 1: Crescente, A., Sperone, E., Paolillo, G., Bernabò, I., Brunelli, E., Tripepi, S. (2014): Nesting ecology of the exotic Trachemys scripta elegans in an area of Southern Italy (Angitola Lake, Calabria). Amphibia-Reptilia 35: Ernst, C.H., Lovich, J.E., Barbour, R.W. (1994): Turtles of the United States and Canada. Smithsonian Institution Press, Washington. European Commission (1997): Council Regulation (EC) No 338/97 of 9 December 1996 on the protection of species of wild fauna and flora by regulating trade therein. Off. J. European Union 40: European Commission (2001): Commission Regulation (EC) No 2087/2001 of 24 October 2001 suspending the introduction into the Community of specimens of certain species of wild fauna and flora. Off. J. European Union 44: European Commission (2003): Commission Regulation (EC) No 349/2003 of 25 February 2003 suspending the introduction into the Community of specimens of certain species of wild fauna and flora. Off. J. European Union 46: 3-18.

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37 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol Species composition and richness of anurans in Cerrado urban forests from central Brazil Cláudia Márcia Marily Ferreira 1, *, Augusto Cesar de Aquino Ribas 2, Franco Leandro de Souza 3 1 Programa de Pós-Graduação em Ecologia e Conservação, Universidade Federal de Mato Grosso do Sul, Centro de Ciências Biológicas e da Saúde, Cidade Universitária, Caixa Postal 549, CEP , Campo Grande, MS, Brazil. *Corresponding author. claudiamarily@hotmail.com 2 Universidade Federal de Mato Grosso do Sul, Faculdade de Computação, Caixa Postal 549, CEP , Campo Grande, MS, Brazil 3 Universidade Federal de Mato Grosso do Sul, Centro de Ciências Biológicas e da Saúde, Cidade Universitária, Caixa Postal 549, CEP , Campo Grande, MS, Brazil Submitted on: 2016, 21 st April; revised on: 2017, 15 th April; accepted on: 2017, 25 th May Editor: Gentile Francesco Ficetola Abstract. Brazil harbors the greatest diversity of amphibians on the planet although there are few studies dealing with urban fauna. The objective of this study is to describe the species composition and richness of anurans in urban Cerrado fragments from Campo Grande municipality, Mato Grosso do Sul state, central Brazil. The specimens were sampled in three stages through pitfall traps and visual/acoustic surveys. Seventeen species were recorded (17.7% of anuran species registered in Mato Grosso do Sul), with Leptodactylidae and Hylidae being the most represented families. The existence of a high number of green areas and water bodies in the urban area likely favors anuran species in the region. The anuran communities in urban areas of Campo Grande were dominated by species which use a broad range of habitats. In this study there was the record of a new species of anuran, Proceratophrys dibernardoi, for the state of Mato Grosso do Sul. The forest fragments that had the highest similarity for species composition were those with similar environmental conditions. The knowledge of the fauna that occurs in urban areas is important because natural habitats suffer severe fragmentation and degradation and species present in these areas may disappear in a shorter period of time. Keywords. Anuran, city, urbanization, Cerrado, habitat fragmentation. INTRODUCTION There are approximately 7,600 amphibian species in the world (Frost, 2017), most of them occurring in tropical regions where natural landscapes have been altered by human activities (Ellis and Ramankutty, 2008). Brazil has 1,080 amphibian species (Segalla et al., 2016) and at least 209 are recorded for the Cerrado and adjoining biomes (Valdujo et al., 2012). The Cerrado is a biodiversity hotspot being currently affected by landscape changes (Soares-Filho et al., 2014) and so conservation strategies must be viewed as a priority (Overbeck et al., 2015). Threats to the Cerrado domain and associated biodiversity include land conversion for agriculture and pasture as well as urban expansion (Klink and Machado, 2005), which in turn can result in the isolation and reduction of the population size of several species and cause local extinctions. Amphibians are amongst the most endangered vertebrate groups in the world (Stuart et al., 2004; Hamer and McDonnell, 2008; Verdade et al., 2010). Some factors that affect amphibian populations are habitat destruction, introduced species, climate change, UV-B radiation, pollution and disease (Young et al., 2001; Verdade et al., 2010). ISSN (print) ISSN (online) Firenze University Press

38 158 Cláudia Márcia Marily Ferreira et alii Urbanization causes habitat fragmentation, loss, isolation and degradation (Hamer and McDonnell, 2008) and associated biotic and abiotic habitat changes (Aronson et al., 2014), being a process that promotes profound changes in the environment. Many amphibian species are threatened by the expansion of urban areas (Hamer and McDonnell, 2008). In urban habitats the major factors impacting amphibians are changes in natural vegetation and hydrological courses (Hamer and McDonnell, 2008), aquatic and terrestrial pollution (Paul and Meyer, 2001; Croteau et al., 2008), predation by domestic animals (Woods et al., 2003), roadkills (Andrews et al., 2008), and diseases (Croteau et al., 2008). Urban development may change the species composition and decrease the richness and diversity of amphibians (Rubbo and Kiesecker, 2005; Hamer and McDonnell, 2008; McKinney, 2008; Hamer and McDonnell, 2010). Many species recorded in the urban environment use multiple types of habitat, being habitat generalists (Hamer and McDonnell, 2008). However several species with specific habitat requirements, still poorly known to science or endangered are also recorded in this type of environment (Grandinetti and Jacobi, 2005; Knispel and Barros, 2009; Ferreira et al., 2010; Silva et al., 2011; Pereira et al., 2013). At least 84% of the population within Brazil resides within urban areas (IBGE, 2015) and the state of Mato Grosso do Sul (central Brazil) has one of the highest urbanization rates in the country (Almeida, 2009). In Mato Grosso do Sul the predominant Cerrado vegetation with its distinct physiognomies such as palm swamps (veredas), gallery forests, wet campos, and closed woodlands harbors 97 amphibian species (Souza et al., in press). Despite this considerable richness, there are few studies on urban species in this region (Ávila and Ferreira, 2004). The objectives of this study were to describe Fig. 1. Localization of the 11 urban Cerrado fragments in Campo Grande municipality, Mato Grosso do Sul state, central Brazil. 1. Itanhangá Square (IS); 2. Sóter Ecological Park (SEP); 3. Coqueiral Farm (CF); 4. Anhanduí Ecological Park (AEP); 5. Imbirussu Environmental Education Center (IEEC); 6. Mato Grosso do Sul Federal University Fish Center (UFMSF); 7. Dom Bosco University (DBU); 8. Mato Grosso do Sul Federal University Cerrado (UFMSC); 9. Military College (MC); 10. Prosa State Park (PSP); 11. Matas do Segredo State Park (MSSP).

39 Urban forest anurans in central Brazil 159 the species composition and richness of anurans in urban Cerrado fragments from Campo Grande municipality, Mato Grosso do Sul state, central Brazil. Study site MATERIAL AND METHODS Campo Grande municipality has 840,000 habitants living in an area of 8,100 km 2. The altitude is 532 m a.s.l. and climate is characterized by a dry (April to September) and wet period (October to March). Mean annual rainfall is 1,530 mm and mean annual temperature ranges between 18 and 28.8 o C. Around 21% of the area exhibits native Cerrado vegetation while the remainder is covered by pastures, agricultural fields and developed areas (PLANURB, 2015). The urban perimeter of Campo Grande has an area of km 2 and a population density of 104 inhabitants/km 2. The city has 28 headsprings and 18 green areas (parks and protected areas) covering about 864 hectares (PLANURB, 2015). Eleven urban forest fragments with size ranging from 1.8 to ha were selected for this study (Fig. 1): Itanhangá Square (IS; 1.8 ha), Sóter Ecological Park (SEP; 3.4 ha), Coqueiral Farm (CF; 10 ha), Anhanduí Ecological Park (AEP; 10.8 ha), Imbirussu Environmental Education Center (IEEC; 13.2 ha), Mato Grosso do Sul Federal University Fish Center (UFMSF; 15.4 ha), Dom Bosco University (DBU; 28.8 ha), Mato Grosso do Sul Federal University Cerrado (UFMSC; 35.2 ha), Military College (MC; 48.6 ha), Prosa State Park (PSP; 128 ha), and Matas do Segredo State Park (MSSP; ha). There are bodies of water in all forest fragments, except: DBU, UFMSC and MC. Data collection Anurans were sampled in three stages - from November 2012 to March 2013, November 2013 to April 2014, and November Sampling methodology included pitfall traps and visual/acoustic surveys (Heyer et al., 1994). Active sampling was made independent of the pitfall trap sampling. The pitfall traps were used in all periods of the study, while visual/ acoustic surveys were used during the second and third stage. Each pitfall trap line consisted of four 30 l plastic buckets connected by 50 cm height drift-fences. The number of lines varied from one to two according to fragment size (areas larger than 20 ha had two lines). Pitfall traps were kept opened during four consecutive days by month in the first stage of the study and seven consecutive days by month from the second and third stage (to increase the sampling effort), and checked every 48 hours. Visual/acoustic surveys were realized at night in one 30 x 30 m quadrat in each fragment. In forest fragments with water bodies, the active sampling was conducted in the wet areas or in their vicinity. In each forest fragment five nights of sampling were conducted, once a month (totaling five months of sampling), except in UFMSC and DBU where we made three and four nights of sampling, once a month, respectively. Visual/ acoustic surveys had an average duration of two hours, starting at dusk (18:30 h). Voucher specimens were deposited at the Coleção Zoológica de Referência of the Universidade Federal de Mato Grosso do Sul (ZUFMS, Campo Grande, Mato Grosso do Sul state). Nomenclature followed Frost (2017). Data analysis To evaluate the sampling effort, we used a species accumulation curve based on the samples, often known as Mao Tau estimate (Chiarucci et al., 2008; Colwell et al., 2012). These data were analysed with a presence-absence matrix, with taxa in rows and samples in columns; Mao Tau estimate is suitable when a number of samples are available (Hammer et al., 2001). Each forest fragment was considered a sampling unit and the sufficiency in sampling was considered when the slope of the curve approached zero. To estimate the similarity in anuran species composition among forest fragments sampled we used a cluster analysis (UPGMA) based on a similarity matrix constructed with the Sørensen index for the community. For this analysis, only presence and absence data of species on each site were used to be able to concatenate presence of species from the different methods used. It was also used the k-means algorithm to create several partitions forming a cascade from 2 to 6 groups. Calinski criteria (Calinski-Harabasz, 1974) was used to determine the optimum number of groups for the k-means. The groups assigned to each local were then used to determine the indicator value (Dufrene and Legendre, 1997) of each species to evaluate its importance in determining the local group. All analyses were performed with R (R Core Team, 2016), using vegan (Oksanen et al., 2016) and indicspecies (De Caceres and Legendre, 2009) packages. RESULTS Seventeen anuran species from four families were recorded (Table 1). Leptodactylidae was the richest family (11 species), followed by Hylidae (four species) and Bufonidae and Odontophrynidae (one species each). Dendropsophus nanus, Hypsiboas punctatus, Hypsiboas raniceps and Leptodactylus syphax were sampled exclusively by visual/acoustic search while Leptodactylus cf. elenae and Physalaemus nattereri were sampled only by pitfall traps. Richness among sites ranged from one to 10 (Table 1). Cumulative species curve reached the asymptote with eleven samples (Fig. 2), with an expected total richness of 17. Based on the Calinski criteria two major groups for the sampled locals (Fig. 3) were observed, with the group IS and SEP more similar (75%), followed by PSP and MSSP (67%) and AEP, CF, UFMSF, palm swamps areas, with 57% or more of similarity (Table 2). Hypsiboas punctatus and Leptodactylus podicipinus had the higher indi-

40 160 Cláudia Márcia Marily Ferreira et alii Table 1. Anuran species recorded at 11 Cerrado urban fragments in Campo Grande municipality, Mato Grosso do Sul state, central Brazil. See text for site abbreviation. Species/Family Sites IS SEP CF AEP IEEC UFMSF DBU UFMSC MC PSP MSSP Bufonidae Rhinella schneideri (Werner, 1894) x x x x x x x x Hylidae Dendropsophus nanus (Bounlenger, 1889) x x x x Hypsiboas punctatus (Schneider, 1799) x x x x x Hypsiboas raniceps Cope, 1862 x x Scinax fuscovarius (Lutz, 1925) x x x Leptodactylidae Adenomera diptyx (Boettger, 1885) x x x x x x x x Leptodactylus cf. elenae Heyer, 1978 x Leptodactylus fuscus (Schneider, 1799) x x Leptodactylus labyrinthicus (Spix, 1824) x x x Leptodactylus mystacinus (Burmeister, 1861) x x x x Leptodactylus podicipinus (Cope, 1862) x x x x Leptodactylus syphax Bokermann, 1969 x x x Physalaemus albonotatus (Steindachner, 1864) x x x x Physalaemus centralis Bokermann, 1962 x x Physalaemus cuvieri Fitzinger, 1826 x x x Physalaemus nattereri (Steindachner, 1863) x x x Odontophrynidae Proceratophrys dibernardoi Brandão, Caramaschi, Vaz-Silva and Campos, 2013 x x x Richness Fig. 2. Cumulative species curve (solid line) for 11 Cerrado urban fragments in Campo Grande municipality, Mato Grosso do Sul state, central Brazil. Gray space represents 95% confidence intervals. Fig. 3. Similarity cluster analysis (UPGMA) with the Sørensen index for 11 Cerrado urban fragments in Campo Grande municipality, Mato Grosso do Sul state, central Brazil. See text for abbreviations.

41 Urban forest anurans in central Brazil 161 Table 2. Similarity values of Sørensen index between 11 Cerrado urban fragments in Campo Grande municipality, Mato Grosso do Sul state, central Brazil. UFMSC CF MC IEEC IS AEP UFMSF PSP MSSP SEP CF 0.40 MC IEEC IS AEP UFMSF PSP MSSP SEP DBU Table 3. Group to which each species was classified, followed by the indicator value for the group 1 (CF, IEEC, AEP, UFMSF, PSP and MSSP) and group 2 (UFMSC, MC, IS, SEP and DBU) and the respective significance for use as an indicator species. Group IV for group 1 IV for group 2 P Adenomera diptyx Dendropsophus nanus Eupemphix nattereri Hypsiboas punctatus Hypsiboas raniceps Leptodactylus cf. elenae Leptodactylus fuscus Leptodactylus labyrinthicus Leptodactylus mystacinus Leptodactylus podicipinus Leptodactylus syphax Physalaemus albonotatus Physalaemus centralis Physalaemus cuvieri Proceratophrys dibernardoi Rhinella schneideri Scinax fuscovarius cator values for group 1, while no species were distinctiveness for group 2 (Table 3). DISCUSSION Seventeen species of anurans were recorded in the urban area of Campo Grande, representing 17.7% of the species reported for Mato Grosso do Sul state. The existence of a high number of green areas and water bodies in the urban environment can favor anuran species in the region. Most species detected in Campo Grande are able to tolerate environments altered by anthropogenic factors. Some of the species observed are endemic to the Cerrado and a new species (P. dibernardoi) was recorded in the state. Forest fragments with similar environmental conditions have similar anuran composition. The predominance of Leptodactylidae and Hylidae species is an expected result because of the large number of species of these families in the Neotropics (Duellman, 1988), also configuring the most specious families in Brazil (Segalla et al., 2016). On the other hand, the few species recorded for Bufonidae and Odontophrynidae in the present study can reflect the low diversity of these taxa in Cerrado areas (Valdujo et al., 2012). Although none of the species recorded in this study

42 162 Cláudia Márcia Marily Ferreira et alii was assigned to any threat status category (ICMbio, 2014), some points must be considered. The recently described Proceratophrys dibernardoi (Brandão et al., 2013) represents a new species record for Mato Grosso do Sul state and was found at least in three sampled urban areas. Furthermore, Physalaemus centralis, P. nattereri e P. dibernardoi are typical Cerrado species (Valdujo et al., 2012; Brandão et al., 2013). As a biodiversity hotspot (Myers et al., 2000), the Cerrado is suffering from intensive landscape changes resulting from land use (Ratter et al., 1997) which requires ongoing research on management, conservation and urban planning (Soares- Filho et al., 2014; Overbeck et al., 2015). Most (70.6%) of the anuran urban species found in Campo Grande municipality utilize a broad range of habitats and are found in anthropogenic environments, such as Dendropsophus nanus (Reichle et al., 2004). Anurans from urban areas are mainly represented by habitat generalist species (Hamer and McDonnell, 2008; Ferreira et al., 2010; Zocca et al., 2014) such as Scinax fuscovarius (Aquino et al., 2010), common species in the urban environment (Ávila and Ferreira, 2004; Grandinetti and Jacobi, 2005; Rodrigues et al., 2008; Torres, 2012; Pereira et al., 2013). Species that live in urban areas also have high fertility (Hamer and McDonnell, 2010), and reproduce in degraded aquatic environments (Lane and Burgin, 2008). Rhinella schneideri, for example, a species present in urban areas of Brazil (Ávila and Ferreira, 2004; Shibatta et al., 2009; Corrêa et al., 2014), uses a broad variety of habitats (Aquino et al., 2004), females produce more than eggs (Uetanabaro et al., 2008) and individuals of this species were found in forest fragments in Campo Grande whose water bodies are contaminated by sewage and garbage. Despite the predominance of habitat generalists in urban systems, species habitat specialists are also recorded (Ferreira et al., 2010) and species still little known to science (Santana et al., 2008). New anuran species are being discovered in the urban environment (Santana et al., 2012; Feinberg et al., 2014). This means that cities can harbor a considerable biological community that provides interesting appeal for management and environmental education programs (Feinberg et al., 2014). The species Physalaemus centralis and P. nattereri are more sensitive to anthropogenic habitat disturbances (Aquino et al., 2004; Colli et al., 2004) than others species recorded in this study; maybe for this reason there were low numbers recorded in the urban area (Melo et al., 2007; present study). Proceratophrys dibernardoi is a species with few biological data available because it was recently discovered (Brandão et al., 2013). The forest fragments with high similarity among species were those that presented similar environmental conditions, such as types of vegetation and levels of anthropogenic impact. Itanhangá Square and SEP have the highest similarity between species. They are smaller fragments and are much modified by human activities. The two green areas are surrounded by built-up areas and suffer great pressure of the surroundings. Matas do Segredo State Park and PSP are the largest fragments present in the urban area of Campo Grande and also the best preserved ones. The MSSP and PSP harbor headsprings and have similar Cerrado physiognomies (Imasul, 2009, 2011). The PSP is surrounded by roads and other human constructions, whereas the MSSP has part of its area facing rural area. The palm swamps of AEP, CF and UFMSF have a small area and suffer great anthropogenic surrounding pressures. Anhanduí Ecological Park is a palm swamp in regeneration and the entire fragment is surrounded by roadways and is affected by sporadic fires. Coqueiral Farm is a small privately owned area and harbor Bandeira s headspring. The site is completely surrounded by roads and is impacted with urban development in its vicinity. Mato Grosso do Sul Federal University Fish Center has a lot of garbage and the Cabaça stream is polluted by sewage (SEMADUR, 2015). The fragment is completely surrounded by human constructions. The knowledge of urban biodiversity is important since forest fragments in cities suffer high ecological impacts mainly associated to border effect with direct consequence for forest and litter dwelling species. Longterm and multidisciplinary studies are required to understand the dynamic connections between environmental and socioeconomic processes in the cities (Tanner et al., 2014), resulting in important tools for conservation and management programs for urban ecosystems. Preserving green areas inside an urban matrix is important for biodiversity protection (Cornelis and Hermy, 2004). For anurans, urban forest fragments are extremely important sites since they represent areas for shelter, feeding, dispersion and reproduction (Becker et al., 2008; Hamer and MacDonnell, 2008; Sabbag and Zina, 2011). In this way, some actions for conserving anurans in urban areas should include green areas restoration and management, including connecting corridors to facilitate the possibility of species dispersal. ACKNOWLEDGMENTS We thank Instituto de Meio Ambiente de Mato Grosso do Sul for authorization to carry out this study in state conservation units; Secretaria Municipal de Meio Ambiente e Desenvolvimento Urbano for authorizing this study in parks and other green areas of the

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47 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol The life-history traits in a breeding population of Darevskia valentini from Turkey Muammer Kurnaz, Alı İhsan Eroğlu, Ufuk Bülbül*, Halıme Koç, Bılal Kutrup Karadeniz Technical University, Faculty of Science, Department of Biology, 61080, Trabzon, Turkey * Corresponding author. ufukb@ktu.edu.tr Submitted on: 2015, 17 th December; revised on: 2017, 2 nd February; accepted on 2017, 16 th October Editor: Giovanni Scillitani Abstract. We investigated the age structure, body size, longevity and growth in a breeding population of Darevskia valentini inhabiting highland altitude in Balahor, Turkey. According to the skeletochronological analysis (n= 25; 14, 11 ), the estimated ages ranged from 3 to 9 years (from 4 to 7 in males and from 3 to 9 in females). The maximum life span was 7 years in males and 9 years in females. The age at maturity was found to be 3 years in both sexes. The mean age and SVL were not statistically different between sexes. For both sexes, we found a significant positive correlation between body size and the number of LAGs. The growth coefficient (k) was lower in females (0.30) than in males (0.76) while asymptotic SVL was higher in females (70.06) than in males (60.55). Growth rates were found to be significantly different between both sexes (females grew faster than males). However, a low level of female-biased sexual size dimorphism (SSD) was observed in the population. Keywords. Skeletochronology, longevity, von Bertalanffy model, growth rate, SSD. INTRODUCTION The Valentin s Lizard, Darevskia valentini (Boettger, 1892) inhabits highland areas above 1800 m (Baran and Atatür, 1998; Eiselt et al., 1992). It has three subspecies (D. v. valentini, D. v. lantzicyreni and D. v. spitzenbergerae) distributed in a large area including most of Anatolia. Many studies about systematic, distribution and ecology were performed on this species (Darevsky, 1972; Franzen, 1990; Eiselt et al., 1992; Mulder, 1995; Sindaco et al., 2000; Tarkhnishvili et al., 2013), but to date, there is no study related to age determination in Turkey. Age determination yields important data for ecological studies of the reptilian species. One of the most commonly used techniques to determine the individual age in reptiles is skeletochronology that many studies have proven to be a very successful and reliable method (Castilla and Castanet, 1986; Girons et al., 1989; Piantoni et al., 2006; Nayak et al., 2008; Kim et al., 2010; Guarino, 2010). Skeletochronology estimates the age using lines of arrested growth layers in the bone tissue (Castanet and Baez, 1991; Castanet, 1994). Due to its reliability and the fact that it is less time-consuming than other age-determination methods such as marking-recapture (Halliday and Verrell, 1988), the number of the studies using skeletochronology method has increased recently (Luís et al., 2003; Altunışık et al., 2013; Tok et al., 2013; Gül et al., 2014; Üzüm et al., 2014; Yakın and Tok, 2015; Kanat and Tok, 2015; Gül et al., 2015; Üzüm et al., 2015; Comas et al., 2016). Since there are no detailed studies based on age and growth parameters of Darevskia valentini, here we present data on life-history traits (age structure, body size and some growth parameters) of the species. In order to assess population dynamics of D. valentini living at altitudes between m a.s.l. (Tok et al., 2009), ISSN (print) ISSN (online) Firenze University Press

48 168 Muammer Kurnaz et alii we chose a mountain population (2400 m a.s.l) living at mean altitude regarded as typical for the species. Field Study MATERIALS AND METHODS A total of 25 individuals of Valentin s Lizard, Darevskia valentini (14, 11 ) were caught during breeding season from Balahor Plateau (near Gümüşhane), Turkey. The Balahor population is located in a highland area ( N, E) at an altitude of 2400 m a.s.l. The habitat consists of rocky areas and open ground. The active period for lizards varies from early May to the middle of September. During the sampling period (16-20 August 2015), the average air temperature in daytime was recorded as 27 C. Lizards were caught by hand. The sex of each individual was determined by direct examination of the sexual organs and secondary sex characters (e.g., dark blue spots on the margins of ventral plates and dorsal coloration of males). Snout-vent length (SVL) was measured to the nearest 0.01 mm using a digital caliper. We quantified Sexual Size Dimorphism (SSD) with the Lovich and Gibbons (1992) index according to the following formula: SDI = (mean length of the larger sex / mean length of the smaller sex) ± 1. In this formula, we used -1 because females were larger than males and defined as positive. For each lizard, the second phalange from the longest finger of the hind limb was clipped and preserved in 10% formalin solution for subsequent histologic analyses. After registration and toe-clipping, the lizards were released back into their natural habitats. The animals were treated in accordance with the guidelines of the local ethics committee. The procedure of skeletochronology is based on the calculation of the lines of arrested growth (LAGs) in transverse sections of the middle part of phalangeal diaphyses using a portion of the second phalanx from the third toe (Gül et al., 2014). After removing the skin, toes were treated for 2.5 hours with 5% nitric acid for decalcification of bone tissue. All samples were then loaded in a tissue processor (Leica Tissue Processor TP1020, Germany) and all tissue samples were embedded in paraffin by a tissue embedding device (Thermo Shandon B , USA). Crosssections (15 µm) were obtained with a rotary microtome and thereafter were stained using the haematoxylin procedure. The stained sections were mounted using Entellan and observed under a light microscope. Age determination was estimated using skeletochronology analysis (Castanet and Smirina, 1990; Smirina, 1994). The numbers of LAGs in each section were independently calculated by three observers (M. Kurnaz, A.İ. Eroğlu and U. Bülbül) and results were compared. Possible double lines were counted as a single LAG. A double line is two adjacent age rings formed during a winter season. As previously stated in the study of Özdemir et al. (2012), we assessed endosteal resorption of the first LAG by comparing the diameters of eroded marrow cavities with the diameters of non-eroded marrow cavities in sections from the youngest specimens. Endosteal resorption did not interfere with age estimation procedures because the resorption zone never reached the first LAG. The distance between two adjoining LAGs is a good indicator of individual growth in a given year (Kleinenberg and Smirina, 1969; Özdemir et al., 2012). Where we observed an obvious decrease in spacing between two subsequent LAGs, we took it to mark the age when sexual maturity was achieved (Ryser, 1998; Yılmaz et al., 2005; Özdemir et al., 2012). Uncountable cross section samples were not incorporated into our study. Statistical Analyses Normality of the SVL and age distribution for the males and females was tested with the One-Sample Kolmogorov-Smirnov test (P 0.05). Being that the variables normally distributed, we used parametric independent sample t-test to estimate significant differences (P < 0.05). Pearson s correlation was used to estimate the relationship between SVL and age (P < 0.05). All statistical tests were processed with IBM SPSS 21.0 for Windows. Skeletochronological Analyses Growth Pattern Estimating The growth patterns were estimated by using the von Bertalanffy equation model between body size and age according to previous studies (James, 1991; Wapstra et al., 2001; Roitberg and Smirina, 2006; Guarino et al., 2010). The general form of the von Bertalanffy equation used is L t = L (1 - e -k (t-t 0 ) ) where L t is length at age t, L is a parameter depicting asymptotic maximum length, e is the base of the natural logarithm, k is a growth coefficient, and t 0 is the age at hatching, which is the starting point of the growth interval under the present study. As

49 Age and growth of Darevskia valentini 169 applied in the study of Guarino et al. (2010), we assumed the mean value provided by Tayhan et al. (2011) as size at hatching (L t0 = 25.3 mm) because of the lack of incontrovertible data on the size at hatching of the populations studied, due to lack of young lizards collected during the field studies. The parameters L (asymptotic SVL) and k, and their asymptotic confidence intervals (CI) were estimated using the non-linear regression procedure by means of the IBM SPSS 21.0 software program. Then, the growth rates were calculated as R = k (L - L t ). Growth curves were considered to be significantly different if the 95% confidence intervals did not overlap (James, 1991; Wapstra et al., 2001). RESULTS Age ranged from 4-7 years in males and 3-9 years in females (Table 1). The mean age of the specimens was not different between the sexes (independent sample t-test: t = ; df = 23; P = 0.431). Intersexual differences in body size (length) was female-biased (SDI = 0.052). The mean SVL (t = ; df = 23; P = 0.431) did not differ between sexes. There was a significant positive correlation between SVL and age for both males (Pearson s correlation r = 0.797; P < 0.01) and females (r = 0.836; P < 0.01). The growth pattern estimated by von Bertalanffy s showed a best fit to the relation between age and SVL (Fig. 1). For males, the estimated asymptotic SVL was lower than the maximum SVL record (SVL asym ± CI: ± 5.80 mm) while for females, it was higher than the maximum SVL record (SVL asym ± CI, females: ± 5.30 mm). The growth coefficient was higher in males than in females (k ± CI, males: 0.27 ± 0.11; females: 0.19 ± 0.10). For all populations (males + females), the asymptotic SVL, growth coefficient (k) and mean growth rate were calculated as ± 4.33 mm, 0.24 ± 0.08 and 4.11 ± 0.79 mm per year, respectively. The growth curve of males was significantly different from that of females. In Table 1. Descriptive statistics of age and SVL of the Balahor population. For abbreviations, see text (n: number of samples; Range: maximum and minimum values and SE: standard error). Characters Sex n Mean Range SE Age SVL Age SVL Age SVL Fig. 1. The von Bertalanffy growth curves for males (open circle, solid line), females (solid circle, grey line) and all specimens (dot line) of D. valentini. Open square shows SVL mean of the lizards at hatching (25.3 mm) as reported by In den Bosch and Bouth (1998). Growth parameters are given in the text. Fig. 2. Relationships among the growth rates of age groups belonged to individuals of D. valentini from Balahor population. this population, females grow faster than males (the average growth rate was 0.97 ± 0.43 in males and 4.12 ± 0.98 in females) (Fig. 2). The mean growth rates of males and females were significantly different within the population (Independent Sample t-test: t = ; df = 9; P < 0.05). Descriptive statistics of the growth rates for the Balahor population are given in Table 2. A growth zone and thin hematoxylinophilic outer line most likely corresponding to a winter line of arrested growth were present in sections of the phalanges in 100% of both male (n = 14) and female (n = 11) adult specimens (Fig. 3). The resorption zone never reached the first LAG and did not interfere with age determination. We observed double lines in 18 (72%) specimens. The oldest females and males were 9 and 7 years old, respectively (Fig. 4). The age at maturation was 3 years for both sexes in the population.

50 170 Muammer Kurnaz et alii Table 2. Descriptive statistic of growth rate and growth coefficient (k) of the Balahor population. For abbreviations, see text (n: number of samples; Range: maximum and minimum values and SE: standard error). Characters Sex n Mean Range SE Growth rate k Growth rate k Growth rate k Fig. 4. Age distributions for both males and females of D. valentini from Balahor population. Fig. 3. A cross section (15 µm thick) through phalange of a fiveyear-old female (63.87 mm SVL) D. valentini from Balahor population. For abbreviations, see text (MC: Marrov Cavity; EB: Endosteal bone; RL: Resorption Line; DL: Double Line; ERL: Endosteal Resting Line; P: Periosteal Bone). DISCUSSION The present study provides data on the age structure and growth patterns of the Valentin s Lizard from a Turkish population. Life history traits (e.g., mean age, age at sexual maturity and maximum longevity) could depend on genetic characteristics and be species-specific. In the present study, the mean age of the Darevskia valentini was found to be 5.36 years in males and 5.82 years in females of the Balahor population located in a high elevation site (2400 m). However, Gül et al. (2014) reported a mean age about 1 year lower (4.3 in males and 4.8 in females) in a high altitude population (inhabiting 2137 m a.s.l.) of another rock lizard species (D. rudis). Conformably, Arakelyan et al. (2013) reported a lower mean age for the four parthenogenetic rock lizards (4.46 years for D. armeniaca, 4.44 years for D. unisexualis, 4.44 years for D. sapphirina and 4.06 years for D. uzzelli) living in highland habitats (between 1400 and 2000 m a.s.l.). Apart from species variation, age structure may also differ among different populations of the same species. The age structure of lizards may fluctuate according to environmental parameters (e.g., climate, altitude, latitude and predation) and other conditions such as proportion and energetic costs of reproduction, activity season and hibernation period (Roitberg and Smirina, 2006). A different climatic condition might affect population demography by generating differences in age structure and longevity (Özdemir et al., 2012). For instance, an increase in mean age with higher altitude has been reported for various lizard species in colder environments (Roitberg and Smirina, 2006). Apart climatic conditions, other factors such as different rates of predation might affect the demography of the lizards studied. Like mean age, longevity is dependent on the active period, which is in turn related to altitude, latitude and other climatic and environmental factors. Since the climatic conditions in Balahor were not favourable for lizard activity due to the presence of snow for at least 6.5 months, the active period of the studied population was found to be lower than other species inhabiting lowland habitats. In general, individuals from high-elevation sites have higher longevity than those from low-elevation sites (Wapstra et al. 2001; Roitberg and Smirina, 2006; Guarino et al., 2010). Congruently, we found a high longevity

51 Age and growth of Darevskia valentini 171 (7 years in males and 9 years in females) in the Balahor population. Similar to our results, the maximum longevity was found to be 8 years in D. armeniaca, D. unisexualis (Arakelyan et al., 2013) and D. rudis (Gül et. al., 2014) while it was found to be 6 years in D. sapphrina and D. uzzelli (Arakelyan et al., 2013). In the present study, the age of both sexes was significantly correlated with their body size (SVL). The adult body size depends on many factors including age at maturity and longevity (Özdemir et al., 2012). In some species, the male lizards mature earlier than females (Beebee and Griffiths, 2000; Olsson and Madsen, 2001). However, age at maturity and SVL were not found to be significantly different between both sexes in our study. A low level of female-biased sexual size dimorphism (SSD) was observed in the Balahor population. Longevity and age at first reproduction have been identified as the main determinants of SSD at an intra-specific level (Liao and Lu, 2010; Lyapkov et al., 2010; Liao et al., 2013; Liao et al. 2015). Congruently, age at maturity was found to be similar in both sexes. Moreover, SSD in many adult lizards arises due to sexual differences in the growth rates, and the larger sex grows faster than the smaller (John- Adler and Cox, 2007; Kolarov et al., 2010; Üzüm et al., 2014). Although there was not a high-level of femalebiased SSD, we found significant differences between the growth rates of both sexes. Our data show that females grew faster than males and that the growth trajectories were different between sexes. The lower value of k in the von Bertalanffy equation in females suggests that they attain the asymptotic body length slower than males. When considering the overall population, we found higher growth rates in our individuals. This is in accordance with the general prediction that lizards grow faster at high elevation sites. Double lines are found in higher percentages in some unsuitable ecological conditions (e.g., hot climate and dry period) (Jakob et al., 2002; Guarino and Erişmiş, 2008; Özdemir et al., 2012). On the other hand, food availability may negatively affect the number of double lines (Bülbül et al., 2016). Since the Balahor population has a cold climate and less food sources, the high percentage of double lines (72 %) observed in this steppe region suggests that the unfavorable food supply has a greater effect than climatic conditions. In conclusion, our data on the body size, age structure, longevity, and growth of D. valentini may contribute to the knowledge of the life-history traits of this species. Our results show different growth rates between sexes in the Balahor population, having different longevity, despite having a similar age at sexual maturity. However, long-term studies including different populations under various environmental conditions are neeeded to reveal a more comprehensive picture. ACKNOWLEDGEMENTS We are grateful to Zekeriya Azak for his help on field studies. The study was carried out by permissions of Ministry of Forest and Water Affairs ( ) and Karadeniz Technical University Animal Care and Ethics Committee (KTÜ /2015/23). English revision was accomplished by Duncan Gullick Lien who is an Instructor at Karadeniz Technical University, School of Foreign Languages in Turkey. REFERENCES Altunışık, A., Gül, Ç., Özdemir, N., Tosunoğlu, M., Ergül, T. (2013): Age structure and body size of the Strauch s racerunner, Eremias strauchi strauchi Kessler, Turk. J. Zool. 37: Arakelyan, M., Danielyan, F. (2000): Age and growth of some parthenogenetic and bisexual species of rock lizards (Lacerta), from Armenia. Zool. J. 79: Arakelyan, M., Petrosayan, R., Ilgaz, Ç., Kumlutaş, Y., Durmuş, S.H., Tayhan, Y., Danielyan, F. (2013): A skeletochronological study of parthenogenetic lizards of genus Darevskia from Turkey. Acta Herpetol. 8: Baran, İ., Atatür, M.K. (1998): Türkiye Herpetofaunası (Kurbağa ve Sürüngenler) (First Edition). TC Çevre Bakanlığı, Ankara. Beebee, T.J.C., Griffiths, R.A. (2000): Amphibians and Reptiles. A Natural History of the British Herpetofauna. Harper Collins New Naturalist, London. Bülbül, U., Kurnaz, M., Eroğlu, A. İ., Koç, H., Kutrup, B. (2016): Body size and age structure of the endangered Clark s lizard (Darevskia clarkorum) populations from two different altitudes in Turkey. Amphibia-Reptilia 37: Caetano, M.H., Castanet, J. (1993): Variability and microevolutionary patterns in Triturus marmoratus from Portugal: age, size, longevity and individual growth. Amphibia-Reptilia 14: Castanet, J., Smirina, E.M. (1990). Introduction to the skeletochronological method in amphibians and reptiles. Ann. Sci. Nat. Zool. 11: Castanet, J., Baez, M. (1991): Adaptation and evolution in Gallotia lizards from the Canary Islands: age, growth, maturity and longevity. Amphibia-Reptilia 12: Castanet, J. (1994): Age estimation and longevity in reptiles. Geront. 40:

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55 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol Influence of desiccation threat on the metamorphic traits of the Asian common toad, Duttaphrynus melanostictus (Anura) Santosh Mogali*, Srinivas Saidapur, Bhagyashri Shanbhag Department of Zoology, Karnatak University, Dharwad , Karnataka State, India *Corresponding author. Submitted on: 2017, 20 th July; revised on: 2017, 6 th August; accepted on 2017, 21 th August Editor: Raoul Manenti Abstract. Phenotypic plasticity of metamorphic traits, in response to desiccation threat, was studied in Duttaphrynus melanostictus under laboratory conditions. Newly hatched Gosner stage 19 tadpoles were exposed to decreasing water levels (gradually or rapidly) up to the beginning of metamorphic climax (MC, Gosner stage 42). The control group was reared in unchanging water levels. The tadpoles experiencing desiccation threat reached MC earlier than those reared in constant water levels and metamorphosed (Gosner stage 46) at smaller body sizes. Time to reach MC was comparable between the groups of tadpoles experiencing a gradual or rapid decrease in water levels but their size at the completion of metamorphosis varied. They emerged at a significantly smaller size under rapid desiccation threat compared to the gradual desiccation threat. Impact on size at emergence was in proportion to the level of desiccation threat and this accelerated development and led to an early metamorphosis. The study shows the ability of D. melanostictus for developmental plasticity under adverse ecological conditions like the desiccation threat. Keywords. Duttaphrynus melanostictus, desiccation threat, phenotypic plasticity, metamorphic climax, metamorphic traits, toad tadpoles. ISSN (print) ISSN (online) INTRODUCTION Phenotypic plasticity is widespread in nature; it allows exploitation of different habitats and, to face unpredictable ecological conditions (Relyea, 2002; Pigliucci, 2005; Miner et al., 2005; Fusco and Minelli, 2010). Phenotypic plasticity is also encountered during the development of anuran amphibians that generally have complex life cycles comprising of an aquatic larval stage, and transformation into an adult-body shape after completing metamorphosis (Newman, 1992; Denver et al., 1998; Relyea, 2002; Miner et al., 2005). Therefore, age and size at metamorphosis are important metamorphic traits in amphibians (Wilbur and Collins, 1973; Wilbur, 1980; Werner, 1986; Smith, 1987). These traits are subject to variation in response to changes in both biological and non-biological factors present in the habitat. The biological factors that influence amphibian development include availability of food resources (Travis, 1984; Newman, 1998; Laurila and Kujasalo, 1999; Enriquez-Urzelai et al., 2013), predatory pressures (Werner, 1986; Skelly and Werner, 1990; Benard, 2004; Mogali et al., 2011a, 2016), inter and intraspecific competitions, density (Semlitsch and Caldwell, 1982; Newman, 1987; Richter et al., 2009; Mogali et al., 2016), and association with kin or non-kin (Girish and Saidapur, 1999, 2003). Among non-biological factors, variations in water temperature (Newman, 1989; Hayes et al., 1993; Maciel and Juncá, 2009; Tejedo et al., 2010) and risk of pond drying/desiccation (Denver et al., 1998; Brady and Griffiths, 2000; Székely et al., 2010; Mogali et al., 2011b, 2016) are the two main factors influencing anuran metamorphosis. In transient water bodies, desiccation threat is serious and completion of metamorphosis before the ponds Firenze University Press

56 176 Santosh Mogali et alii dry is obligatory. Evidently, slow growth rates and/or prolonged larval periods in unpredictable hydroperiods of the ponds are sure to decrease the chances of tadpoles completing metamorphosis before the ponds dry (Altwegg and Reyer, 2003; Johansson et al., 2005; Wells, 2007). On the other hand, a hastened larval development can lower larval mortality, but it is invariably at the cost of growth resulting in a smaller size at metamorphosis that may have consequences in their later survival and reproductive success. Smaller metamorphs have lower locomotory capacity (Semlitsch et al., 1988; Richter- Boix et al., 2006), lower tolerance to dehydration (Newman and Dunham, 1994), reduced resistance to parasites (Goater, 1994), weaker immunity (Gervasi and Foufopoulos, 2008) lower juvenile survivorship (Reques and Tejedo, 1997; Altwegg and Reyer, 2003), and lower reproductive success (Smith, 1987; Scott, 1994). Yet, when larval mortality risk increases due to pond drying an early metamorphosis may be favoured despite the costs associated with a smaller size. Hence, a phenotypic plasticity involving the trade-off between certain life history traits (e.g., larval growth, duration of the larval period, size at transformation) is a useful strategy. The original models of amphibian metamorphosis that attempt to evaluate optimal size at transformation predict that in an aquatic environment when conditions are favourable for larval growth (i.e., in permanent or slowly desiccating ponds), tadpoles should delay metamorphosis and transform at a larger size (Wilbur and Collins, 1973; Werner, 1986). But when conditions of the temporary ponds become precarious a strategy to adjust the developmental processes so as to metamorphose early and emerge on land is useful. A developmental strategy of phenotypic plasticity can reduce the exposition to risky conditions and thereby enhance the survival rate. In Dharwad, many anuran species reproduce in rainfilled ephemeral water bodies formed during South-West monsoon. Tadpoles living in such ponds face the perennial threat of desiccation due to the failure of intermittent monsoon showers (Mogali et al., 2011b). The Asian common toad, Duttaphrynus melanostictus (earlier known as Bufo melanostictus) breeds both in rain-filled ephemeral ponds and in cement cisterns within the parks holding water round the year (Saidapur and Girish, 2001). Hence, D. melanostictus tadpoles offer an excellent model to study developmental plasticity in response to varying degrees of dropping levels of water (desiccation threat). Therefore, the present study was designed to determine, in a laboratory set-up, the influence of gradual or rapid water depletion on the two key metamorphic traits, the larval duration, and size at emergence. We hypothesized that tadpoles facing the rapid depletion of water (high desiccation threat) would metamorphose earlier and at a smaller size than those developing in a gradual decline in water levels (low desiccation threat). Importantly, the experimental design permitted us to exclude the influence of confounding factors such as food scarcity and predator pressure which generally interfere with the growth and development of these tadpoles in nature. MATERIAL AND METHODS Four egg clutches of D. melanostictus were collected on 29 May, 2014 from rain-filled ponds in and around (within 2 km distance) the Karnatak University Campus (latitude N, longitude E). Soon after collection, the eggs were brought to the laboratory and placed separately in plastic tubs (32 cm diameter and 14 cm deep) containing 5 L of aged (dechlorinated) tap water. All eggs hatched almost synchronously at Gosner stage 19 (Gosner, 1960) a day after their collection. Soon after hatching, the tadpoles from different clutches were mixed and used for the experiment. Tadpoles were picked randomly and were reared in the plastic tubs with 3 L of water until the onset of metamorphic climax stage (MC, Gosner stage 42). Fifteen such tubs with 20 tadpoles in each were maintained (in total 300 tadpoles, i.e., 100 tadpoles in each group). The experimental groups were as follows: I. Control: tadpoles were reared in constant water levels (3 L). II. Gradual desiccation: tadpoles were reared in 3 L of water for the first 4 days and then subjected to 0.5 L decrease in water at 4 day intervals. III. Rapid desiccation: tadpoles were reared in 3 L of water for a day and from the second day onwards 0.25 L of water was reduced each day. In receding water groups when water reached 0.5 L (day 20 in group II; day 10 in group III) no further reduction was made. The groups II and III thus provided low and high desiccation threat. All tadpoles were fed on the boiled spinach ad libitum. Water was changed on alternate days and fresh food was provided. The rearing tubs were placed on a flat surface in a room under natural photoperiod and temperature. The positions of tubs were randomized on an alternate day to avoid possible effects of position. The water temperature ( C) in tubs was recorded twice, daily at 10:00 h and 15:00 h. Following the onset of metamorphic climax (MC, emergence of forelimbs, Gosner stage 42), the subjects were transferred to small plastic tubs (19 cm diameter and 7 cm deep) covered with fine nylon mesh with a little water and, placed inclined to provide the semi-terrestrial environment to facilitate emergence. The days to reach MC were noted for each individual. After completion of metamorphosis (Gosner stage 46), snout-vent length (SVL in mm) and body mass (in mg) were recorded. Two tadpoles in group I, and one tadpole each in groups II and III died during the course of the experiment. After completion of experiments, the toadlets were released near natural water bodies. Data on days to reach MC, SVL, body mass of toadlets, and water tem-

57 Influence of desiccation threat on toad tadpoles 177 perature were analysed by one-way ANOVA followed by Tukey s post-hoc test. Data for each parameter were organized into frequency distribution to know the percentage of individuals falling within a particular dataset. RESULTS Time taken to reach MC, and size at metamorphosis (SVL, body mass) differed significantly between different groups (P < 0.001, Table 1). Tadpoles reared in declining water levels (groups II and III) reached MC earlier (P < 0.001) and metamorphosed at a smaller size (P < 0.001) than those reared in unchanging water levels (group I). Further, tadpoles experiencing rapid depletion of water (group III) metamorphosed at smaller sizes (P < 0.001) than those experiencing gradual depletion in water levels (group II). However, number of days required for the onset of MC was comparable in both these groups (P = 0.937). The daily water temperature of various tubs fluctuated between C and as such did not differ significantly throughout the course of the experiments (morning: F 2,86 = 0.298; P = 0.743; and afternoon hours: F 2,86 = 0.281; P = 0.756). Therefore, the effects of temperature, if any, were uniform across the control and experimental groups. The frequency distribution data showed that 78.78% of individuals from rapid desiccation group and 28.28% of individuals from gradual desiccation group metamorphosed (Gosner stage 46) at 7.99 mm SVL, but none subjected to constant water levels metamorphosed at comparable SVL (Fig. 1). Further, 96.96% individuals in rapid desiccation group and 83.83% individuals in gradual desiccation group metamorphosed at a smaller body mass ( 69 mg) while only 12.24% of tadpoles reared in unchanging water levels (group I) metamorphosed at this low body mass (Fig. 2). The data on Fig. 1. Percent metamorphs of Duttaphrynus melanostictus per snout-vent length class (mm) in different rearing groups Fig. 2. Percent metamorphs of Duttaphrynus melanostictus per body mass class (mg) in different rearing groups Table 1. Snout-vent length (SVL), body mass of metamorphs, and days required for the onset of metamorphic climax (MC; Gosner stage 42) in Duttaphrynus melanostictus reared in waters with different levels of desiccation. Data represent mean ± SE; n = 100 tadpoles for each group (300 tadpoles in total); dissimilar superscripts (a, b, c) indicate significant differences between the groups in the same column; significance level was set to Rearing groups SVL (mm) Body mass (mg) Onset of MC (in days) I. Control 9.42 ± 0.07 a ± 1.71 a ± 0.24 a II. Gradual desiccation 8.24 ± 0.05 b ± 1.14 b ± 0.18 b III. Rapid desiccation 7.62 ± 0.05 c ± 1.00 c ± 0.20 b F value F = F = F = P value P < P < P < Fig. 3. Percent tadpoles of Duttaphrynus melanostictus per class (days) to reach metamorphic climax (MC; Gosner stage 42) in different rearing groups

58 178 Santosh Mogali et alii days for onset of MC showed that 33.33% individuals reared in rapid desiccation group (with mean SVL, 7.10 ± 0.06 mm and mean body mass, ± 0.78 mg), 37.37% individuals from gradual desiccation group (with mean SVL, 7.73 ± 0.05 mm and mean body mass, ± 0.95 mg) took 23 days; while only 12.24% tadpoles reared in unchanging water levels initiated MC (with mean SVL, 8.58 ± 0.03 mm and mean body mass, ± 1.26 mg) by this same time (Fig. 3). DISCUSSION Amphibian metamorphosis is often characterized by the developmental phenotypic plasticity involving a trade-off between larval period and size at transformation. A risk of mortality in the larval environment increases due to two major factors like the predator pressure and desiccation threat as most anurans breed opportunistically in ephemeral ponds (Laurila and Kujasalo, 1999; Lardner, 2000). The present study deals with one such factor, the desiccation threat, imposed by decreasing water levels in the rearing tubs. In nature, the threat of pond drying leads to early metamorphosis and transformation at a smaller, vulnerable body size (Werner, 1986; Rowe and Ludwig, 1991; Brady and Griffiths, 2000; Rudolf and Rödel, 2007; Mogali et al., 2011b). The present empirical study shows that tadpoles of D. melanostictus when subjected to low or high desiccation threat, in fact, accelerate their development and emerge on the land earlier than the control group. Apparently, this is a strategy to escape mortality in the larval stage of development that occurs in water. In consistent with our predictions, the size of newly emerged toadlets was significantly small in response to the level of desiccation threat; those experiencing rapid desiccation threats emerged at a significantly smaller body size than those exposed to gradual water depletion. The former transformed at the smallest mean SVL and body mass (Table 1, Fig. 1 and 2). We had hypothesized that tadpoles facing rapid desiccation threat may advance the onset of MC over those facing gradual desiccation threats. Interestingly, there was no difference in the time taken for the onset of MC in both low or high desiccation risk groups; though these reached MC significantly early compared to the no desiccation risk group. The observations suggest that even though a relevant degree of plasticity can be adaptively present, a given species may need minimum threshold period to complete development. The present findings are however in good agreement with the view that anuran tadpoles from natural populations exhibit phenotypic plasticity in their growth and development when subjected to adverse ecological conditions (Lind et al., 2008). It may be noted that some individuals in all the groups took 23 days to reach MC; others took a variable amount of time, as much as 33 days, especially in the group having no desiccation threat. These observations suggest that phenotypic plasticity response in the developmental rate may depend upon the severity of the ecological conditions and, genotypic variations among the group members. The minimum time taken to reach MC in certain individuals of different groups was 20 days; this period appears to be the minimum threshold time for onset of MC in the toad. The minimum period required to reach MC may also be species-specific and therefore differ in different species. The mechanisms proposed to explain the acceleration of metamorphosis in anuran tadpoles facing the threat of desiccation differ. Elevated temperature (Newman, 1992; Tejedo and Reques, 1994) or a decrease in food (Alford and Harris, 1988; Newman, 1994) has been attributed to lowered growth rate with the accelerated developmental rate. Yet other studies indicated that the temperature has no influence on developmental rate (Loman, 1999; Laurila and Kujasalo, 1999; Márquez-García et al., 2009). In the present study, there was no difference in water temperature of the rearing tubs in all groups and food provided was in excess quantity. Hence, the major factor influencing accelerated metamorphosis of the toad tadpoles is the desiccation threat rather than temperature or scarcity of food availability. Interestingly, individuals reaching MC by 23 days differed in their size between the groups. Tadpoles in constant water level (group I) grew bigger than those reared in rapid and gradual depletion of water levels. Further, individuals from gradual desiccation group were bigger than those in rapid desiccation group. These findings suggest that plasticity in the developmental rate of D. melanostictus is a normal feature and allows adjusting the growth of individuals in relation to extrinsic factors operating in their habitat, in this case, desiccation risk. Attainment of a smaller size at the time of completion of metamorphosis in rapid desiccation group may be partly due to increased crowding and intraspecific competition among the group members. An earlier study on the toad tadpoles has shown that they metamorphose late and at a smaller size when raised in constant water (without any desiccation risk) but under crowded condition (Saidapur and Girish, 2001). The study also showed that under uncrowded conditions and in the absence of any desiccation threat the toad tadpoles extend the larval period and maximize their growth. It appears that the toad tadpoles are capable of assessing the level of desiccation risk and

59 Influence of desiccation threat on toad tadpoles 179 adjust their developmental and growth rates. Developmental plasticity is believed to be an attribute of the individual to generate different phenotypes depending upon the ecological conditions (Pigliucci, 2005). According to theoretical models of plastic responses, natural selection will favour reaction norms that balance cost avoidance with resource acquisition; for example, the cost of maintaining a plastic response is expected to trigger the evolution of reaction norms that increase adaptation to more frequently changing environmental conditions (Pigliucci, 2005). Indeed, the tadpoles of the toad and several other anuran species that breed with the onset of monsoon rains in southern India, and frequently encounter unpredictable ecological challenges like the desiccation risk. Therefore, observance of phenotypic plasticity in the development of the toad tadpoles in response to desiccation threat supports the above view. Further, it is believed that genetic variations among the populations also play a role in the expression of plasticity (Pigliucci, 2005). In our study, four parental lines were mixed creating more heterogeneity of the group members which may have actually lowered values of plastic response as the data represents an average response of pooled genotypes. Therefore, further studies are needed to clarify the relationship between variation in genotype and expression of phenotypic plasticity in the toad population. ACKNOWLEDGEMENTS SMM is thankful to UGC s DSKPDF, New Delhi and BAS is thankful to INSA, New Delhi for support. This research was conducted according to ethical guidelines laid down by CPCSEA, New Delhi under Registration No. 639/02/a/CPCSEA. REFERENCES Alford, R.A., Harris, R.N. (1988): Effects of larval growth history on anuran metamorphosis. Am. Nat. 131: Altwegg, R., Reyer, H.U. (2003): Patterns of natural selection on size at metamorphosis in water frogs. Evolution 57: Benard, M.F. (2004): Predator-induced phenotypic plasticity in organisms with complex life cycles. Annu. Rev. Ecol. Evol. Syst. 35: Brady, L.D., Griffiths, R.A. (2000): Developmental responses to pond desiccation in tadpoles of the British anuran amphibians (Bufo bufo, B. calamita and Rana temporaria). J. Zool. 252: Denver, R.J., Mirhadi, N., Phillips, M. (1998): Adaptive plasticity in amphibian metamorphosis: response of Scaphiopus hammondii tadpoles to desiccation. Ecology 79: Enriquez-Urzelai, U., San Sebastián, O., Garriga, N., Llorente, G.A. (2013): Food availability determines the response to pond desiccation in anuran tadpoles. Oecologia 173: Fusco, G., Minelli, A. (2010): Phenotypic plasticity in development and evolution: facts and concepts. Introduction. Phil. Trans. R. Soc. B. 365: Gervasi, S.S., Foufopoulos, J. (2008): Costs of plasticity responses to desiccation decrease post-metamorphic immune function in a pond-breeding amphibian. Funct. Ecol. 22: Girish, S., Saidapur, S.K. (1999): The effects of kinship and density on growth and metamorphosis of the bronze frog (Rana temporalis) tadpoles. Acta. Ethol. 2: Girish, S., Saidapur, S.K. (2003): Density-dependent growth and metamorphosis in the larval bronze frog Rana temporalis influenced by genetic relatedness of the cohort. J. Biosci. 28: Goater, C.P. (1994): Growth and survival of postmetamorphic toads: interactions among larval history, density and parasitism. Ecology 75: Gosner, K.L. (1960): A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16: Hayes, T., Chan, R., Licht, P. (1993): Interactions of temperature and steroids on larval growth, development, and metamorphosis in a toad (Bufo boreas). J. Exp. Zool. 266: Johansson, F., Hjelm, J., Giles, B.E. (2005): Life history and morphology of Rana temporaria in response to pool permanence. Evol. Ecol. Res. 7: Lardner, B. (2000): Morphological and life history responses to predators in larvae of seven anurans. Oikos 88: Laurila, A., Kujasalo, J. (1999): Habitat duration, predation and phenotypic plasticity in common frog (Rana temporaria) tadpoles. J. Am. Ecol. 68: Lind, M.I., Persbo, F., Johansson, F. (2008): Pool desiccation and developmental thresholds in the common frog, Rana temporaria. Proc. R. Soc. B. 275: Loman, J. (1999): Early metamorphosis in common frog Rana temporaria tadpoles at risk of drying: an experimental demonstration. Amphibia-Reptilia 20: Maciel, T.A., Juncá, F.A. (2009): Effects of temperature and volume of water on the growth and development

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61 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol Predation of common wall lizards: experiences from a study using scentless plasticine lizards Jenő J. Purger*, Zsófia Lanszki, Dávid Szép, Renáta Bocz Department of Ecology, Institute of Biology, University of Pécs, Ifjúság útja 6, 7624 Pécs, Hungary *Corresponding author. purger@gamma.ttk.pte.hu Submitted on: 2017, 19 th February; revised on: 2017, 18 th August; accepted on: 2017, 22 nd August Editor: Paolo Casale Abstract. The potential influence of predators on lacertid lizards has been studied by using models made of plasticine which shows the attack marks of predators and as such allows their identification and estimation of predation pressure. The general aim was to study predation on plasticine models of lizards and to improve methods, since the results depend on the number of plasticine models used, their spatial pattern and the duration of experiments. We estimated the density of the common wall lizard Podarcis muralis population on stone walls of a vineyard in the city of Pécs (Hungary) in August 2015 in order to imitate the real density in our experiment with plasticine models. The density of common wall lizards was 8.2 ind. /100 m 2 and accordingly we placed 25 scentless plasticine lizards on the stone walls on the first transect with 10 m distance between them, which imitates the real pattern. In the second transect 25 lizard models were placed more sparsely, the distance between them being 20 m. During four weeks the predation rate was 24% in densely spaced plasticine lizards and 40% in sparsely spaced plasticine lizards, but the difference was not significant. The daily survival rate of densely spaced lizards was 0.99 (=99.1%) and that of sparsely spaced lizard models was 0.98 (=98.25%), but this difference was not significant either. On the basis of marks left on plasticine lizards, mammal predators (e.g. beech marten) dominated, while the impact of bird predators was smaller than expected. Predators attacked the head of plasticine lizards more frequently than their trunk, tail or limbs, but a significant preference of body parts was not detected. From our experience it is important to study the distribution and density of real animals, to imitate their real pattern, instead of an arbitrarily designed experiment with models. The typical scent of plasticine also could influence the results, which can be avoided by using scentless plasticine models coated with liquid rubber. We suggest the calculation of daily survival rates in order to produce results that allow the comparison of different studies. Keywords. Abundance, density dependence, Hungary, Podarcis muralis, survival rates. INTRODUCTION Predator-prey interactions have been in the focus of ecological and evolutionary research (e.g., Cooper and Blumstein, 2015). There are many difficulties in studying predation events directly in the wild, especially on small vertebrates; therefore during the last decades models made of soft materials (e.g., plasticine) have been used (Bateman et al., 2016). The potential influence of predators on lacertid lizards has been also studied by using plasticine models which show the attack marks of predators and as such allow their identification and estimation of predation pressures (e.g., Castilla and Labra, 1998; Castilla et al., 1999; Diego-Rasilla, 2003a,b; Shepard, 2007; Vervust et al., 2007, 2011; Pérez-Mellado, 2014; Sato et al., 2014; Fresnillo et al., 2015; Stellatelli et al., 2015). The results of these studies mainly depend on the quality of materials and the number of plasticine models used, on ISSN (print) ISSN (online) Firenze University Press

62 182 Jenő J. Purger et alii the duration of experiment and also on how often they were checked for evidences of attack (Bateman et al., 2016). A further problem is that the unnatural smell of plasticine can influence the results, through modifying the behaviour of mammal predators which rely on olfactory cues (Bayne and Hobbson, 1999; Purger et al., 2012; Fresnillo et al., 2015). These models are immobile, while bird predators relying on visual cues mostly react to a moving prey (Rangen et al., 2000). The many lizard species are diurnal animals and their main predators are birds (e.g., Vervust et al., 2007; Pérez-Mellado, 2014; Fresnillo et al., 2015), therefore the unnatural attractiveness of models for nocturnal mammal predators should be avoided by using scentless models (Purger et al., 2012). The duration of experiments performed with plasticine lizards was mostly arbitrarily decided (e.g., Diego-Rasilla, 2003a,b) in dependence of the abundance of potential predators and their ability to discover and predate the models (e.g., Castilla and Labra, 1998). Since predation events depend on the density of prey, it is important to study the pattern (e.g., distribution, density) of real animals in the particular area before starting an experiment with models, to imitate real pattern, instead of an arbitrarily designed experiment. In some studies the distance between models was only 2 m (Vervust et al., 2007, 2011) or 5 m (e.g., Castilla et al., 1999; Diego-Rasilla, 2003a,b; Pérez-Mellado, 2014), which suggest a high density, while in other studies the distance was at least 100 m apart from one to another (Fresnillo et al., 2015) or the models were placed scattered (Pérez-Mellado, 2014). Predation rates are influenced by different circumstances therefore the results of different studies are difficult to compare. Our study was performed as an attempt to standardise predation experiments with lizard models. Our specific goals were: a) to find out whether predation is dependent on lizard density; b) to estimate predation rates and the daily survival rates of scentless plasticine lizards; c) to identify the predators; d) to find out if predators prefer any parts of the prey s body. MATERIAL AND METHODS The study was carried out at the St. Nicholas Hill Research Station (46 04'N, 18 09'E) of the Institute of Viticulture and Oenology, University of Pécs, on the southern slopes of Mecsek Mts., m a.s.l., 5 km to the west from the centre of the city of Pécs, Hungary. This area (14 ha) has been used as a vineyard since the 1750 s. Its surface is slightly undulating with stone walls between fields. The soil is Ramann-type brown forest soil formed on Pannonian red sandstone (Teszlák et al., 2013). From the north the area is bordered by manna ashdowny oak (Fraxino-Quercetum) dry forest. The common wall lizard Podarcis muralis (Laurenti, 1768) is the most common lizard species in Europe (Guillaume, 1997), occurring everywhere in Hungary with the exception of the Great Hungarian Plain (Puky et al., 2005). In suitable habitats such as open rocky hillsides, quarries and stone walls in urban environments with warm microclimate it can reach considerable densities (Trócsányi and Korsós, 2004). Active individuals of the species are observable even on warmer days of winter months in the southern region of the country (Trócsányi et al., 2007). It is an opportunistic species so habitat requirements are variable, it often lives in vineyards (Vogrin, 1998), but their densities could be influenced by shelter, food and the effects of predators (Gruschwitz and Böhme, 1986). Natural predators of this species are among mammals, birds and snakes which occur in their habitats (Gruschwitz and Böhme, 1986). We estimated the density of the wall lizard population on stone walls in the summer of 2015 (before the study) in order to imitate the real density in our experiment with plasticine models. The density was not estimated by capturing the animals. But instead lizards were counted by walking on the top of all six stonewalls ( m high and ca. 0.4 m width) which divided vineyard terraces. The average length of stonewalls was 310 m (SD = ) and the average of top surface area was 124 m 2 (SD = 59.97). Counting was performed by the same person, during six days from 24 August 2015, always beginning at 16:00 h, on one stonewall randomly selected each day. During counting the lizards escaped due to human appearance, and hid on the side of the walls, therefore they were counted only once. Before the experiment the workers in the vineyards were informed about our study and they tried to not cause disturbance. For our study lizard replicas were created using non-toxic natural colour plasticine (produced by KOH-I-NOOR Hardtmuth, Czech Republic). The lizard models were made of plasticine with a wire axis which was used also for hanging them on the stone wall. We used plasticine lizard models whose body size cca. 15 cm (±1 cm) and shape were similar to those of adult wall lizards (Diego-Rasilla, 2003a,b). The basic colour of male and female is similar (Arnold and Ovenden, 2002), therefore the sculpted plasticine lizards were painted uniformly in taupe colour (tempera, produced by Pannoncolor, Hungary) based on the colour of observed and photographed lizards in the study area. Then models were coated with uncoloured liquid rubber spray (PlastiDip, USA) and were aired for two weeks in order to eliminate the scent of plasticine and thus allow equal chances for avian and mammal predators in their visual search (Purger et al., 2012). In the morning of 7 September 2015 we placed 25 scentless plasticine lizards on the top of a stone wall in the first transect with 10 m spacing, imitating real density pattern. In the second transect 25 lizard models were sparsely spaced; the distance between them was 20 m. They were placed in an open area and were fully visible to avian predators (Pérez-Mellado, 2015). The study sites were homogeneous, very similar linear habitats, m apart from each other; therefore we assume that there were no differences in predator communities. On the south facing side of stone walls 50 plasticine lizards were placed (25 densely and 25 sparsely spaced). Unfortunately the majority of models placed on the vertical side of walls melted due high

63 Survival of plasticine lizards 183 solar radiation during the experiment, and these two transects were not included in the analysis. We checked the condition of lizard models after their placement, on the afternoon of days 1, 3, 7, 14, 17, 21 and 28 between 16:00 h and 18:00 h. Attacked models were removed during regular checking to avoid pseudoreplication. On the last checking day we gathered the remaining models. A lizard model was considered as being attacked by a predator when bill marks of birds, tooth marks of mammals were found, or if it had disappeared (e.g., Castilla and Labra, 1998; Castilla et al., 1999; Diego-Rasilla, 2003a,b). We recorded which body part of the lizards (head, trunk, tail or limbs) had been damaged by predators (Vervust et al., 2011). Based on the marks on plasticine models, mammal predators were identified by the help of our collection of mammal skulls (Fig. 1.). Predation rates on lizard models arranged in the two transect were calculated as percentage of damaged (predated) models. Daily survival rate is the probability that a lizard survives a single day. We used Mayfield s (1975) method (common in ornithological studies) for estimating the daily survival rate of a sample of plasticine wall lizards using exposure days (the cumulative number of days that the lizards in the sample were monitored) and the number of known losses. According to the Mayfield method, the estimated daily survival was calculated as 1 - [(number of lizard losses) / (total exposure days)]. In our study, for the comparison of daily survival rates the test proposed by Johnson (1979) was applied, calculating with the free software J-test developed by K. Halupka (2009). For comparing the proportions of predation causes and number of attacks on different body parts, chi-square goodness of fit for two and four categories was used (Zar, 1999). A minimum tail probability level of P < 0.05 was accepted for all the statistical tests, and all P-values were two-tailed. RESULTS AND DISCUSSION On the top of stone walls (total length 1860 m, surface area cca. 744 m 2 ) 61 common wall lizards (8.2 individuals/100 m 2 ) and five eastern green lizards Lacerta viridis (0.7 ind. /100 m 2 ) were counted, which means there was at least one lizard in every cca. 10 m 2. Our estimation of common wall lizard population density showed similarity to the results quoted by Puky et al. (2005). These authors summarised data from literature and concluded that the territory of common wall lizard ranges between 3 and 50 m 2. Such great variation in lizard density is affected by a complex variety of factors; e.g., habitat diversity, availability of resources, presence of predators, competitors and human disturbances (Pérez-Mellado et al., 2008). Trócsányi and Korsós (2004, 2007) suggest that in Mecsek Mts. near the city of Pécs the density of wall lizards on a brick wall was 36 individuals/100 m 2 while there was only 6.5 individuals/100 m 2 in a quarry. In comparison with these values the density of wall lizards estimated in the vineyard was low. Applying of some sampling methods often resulted in underestimation of lizard density (e.g., Smolensky and Fitzgerald, 2010; Ruiz de Infante Anton et al., 2013), however, these values may be useful in experiments with artificial models. During our study 24% of the densely spaced lizards and 40% of sparsely spaced lizards were damaged by predators, but based on the number of predation events the difference was not significant (χ 2 with Yates corrections = 0.56; df = 1; P = 0.546). Density-dependent predation was not detected by using plasticine lizards. With a view to the fact that a high variability in the density of common wall lizards in different habitats is shown (Trócsányi and Korsós, 2004, 2007), we can say that in our experiment the imitated density of the same habitat was low in both transects, and therefore we could not detect significant differences between predation rates. Despite this, we suggest taking into consideration the density of the studied species and placing the replicas accordingly in order to achieve more realistic results. The daily survival rate of densely spaced lizard models (total exposure days = 662, number of lizard losses = 6) was 0.99 (99.1%, 95% Confidence Intervals: ) and that of scarcely spaced lizards (total exposure days = 560.5, number of lizard losses = 10) was 0.98 (98.25%, 95% CI: ), but this difference was not significant (Z = 1.296, two tailed P = 0.195). The duration of our study (four weeks) was quite long because we had to wait for the first predation event to occur; scarce predation resulted in high daily survival rates. Similar studies took few days or a week since they used high density (2-5 m) of prey models (e.g., Castilla and Labra, 1998; Castilla et al., 1999; Diego-Rasilla, 2003a,b; Sato et al., 2014, Stellatelli et al., 2015) or even 20 days in the study with models 100 m apart from each other (Fresnillo et al., 2015). Our experience showed that in the case of few predators in the study area the studies should last longer, until the predation rate reach at least 30-40%, or the study should be repeated. We identified one mark of a bird, two marks of small mammals, two marks of weasel Mustela nivalis, three of red fox Vulpes vulpes (Fig. 1.) and six bites of beech marten Martes foina on the plasticine models. Two of the lizard models disappeared; we suppose that they were taken by large mammals. Predators did not damage two lizard models placed next to each other at the same occasion, which means that the liquid rubber obscured the plasticine smell, or maybe because the attacked lizard model was unpalatable and then the predator did not attack the other nearest. It is known that mammal predators use mainly olfactory cues during hunting (Rangen et al., 2000), but in our study it seems that the smell of plasticine did not attract the mammal predators. Noc-

64 184 Jenő J. Purger et alii Fig. 1. Tooth prints left on plasticine models were compared with skulls and in this case red fox was identified as predator. turnal mammals did not identify lizard models as prey; we found droppings of beech marten three times on the same lizard model, which means that it marked its revier (Seiler et al., 1994). Red foxes, beech martens, weasels and cats Felis catus were seen regularly in the study area and we found their traces and droppings. Most of them are known for preying upon lizards (e.g., Castilla et al., 1999; Diego-Rasilla, 2003a). We presume that in habitats with a lot of hiding places, preying upon small bodied fast moving lizards require big energy investment. According to ecological studies of mammal feeding, red fox, weasel and beech marten are reported to consume lizards periodically (occasionally) or rarely (e.g., Lanszki et al., 1999; Lanszki, 2003, 2012; Lanszki and Heltai, 2007). In our study the predation role of small mammals was not considerable, but it was not negligible either. Among small mammals shrew (Soricidae) species are often mentioned as wall lizard predators (Gruschwitz and Böhme, 1986), however in some predation studies replicas showing marks of rodents were considered as non-attacked (e.g., Castilla et al., 1999). In our study avian predation rate was also very low. Common kestrel Falco tinnunculus and common buzzard Buteo buteo, both being potential lizard predators (e.g., Castilla et al., 1999; Diego-Rasilla, 2003a; Vervust et al., 2011), were frequent in the study area. Also, there were hooded crows Corvus cornix and Eurasian jays Garrulus glandarius which, too, were identified as egg predators in our earlier study (Purger et al., 2004). There is evidence that birds are able to visually recognize lizards as prey, based on their shape and colour pattern, even if the animals remain immobile (e.g., Stuart-Fox et al., 2003; Shepard, 2007; Stellatelli et al., 2015). The possible reason for less attack by avian predators in our experiment may be that Fig. 2. Number of attacks by different predators on various body parts of plasticine wall lizard models (black bars head, grey bars trunk, white bars limbs, hatched bars tail). models did not resemble wall lizard coloration and pattern with sufficient precision, similarly to the study of Marshall et al. (2015). During our study we observed a smooth snake Coronella austriaca just when preying on a wall lizard, but tooth marks of this well-known lizard predator (Diego-Rasilla, 2003a; Amo et al., 2004) were not found on any of the lizard models. Based on the low number of predation events recorded (n = 30) the only fact we could determine was that tooth marks of large mammals were found on the head of plasticine lizards more frequently than on their trunk, tail or limbs (Fig. 2.), but significant preference of body parts was not detected (χ 2 = 1.2; df = 3; P = 0.753). Predators could grab different parts of the body with equal chance, since plasticine lizards were immobile. According to the results of experiments with lizard replicas (Castilla et al., 1999; Vervust et al., 2011) mammals tend to attack the head of a prey more often. From our experience in studies of daily active animals we suggest using scentless plasticine animal replicas coated with liquid rubber which eliminate unnatural plasticine smell and reduce the impact of nocturnal predators. It is important to study the recent distribution and density of real animals in the particular area, to imitate their real pattern, instead of an arbitrarily designed experiment with models. Since predation rate depends mainly on the pattern of prey, the activity of members of predator community, as well as on the duration of experiments, we suggest the calculation of daily survival rates in order to produce results that allow the comparison of different studies.

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67 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol Reproductive timing and fecundity in the Neotropical lizard Enyalius perditus (Squamata: Leiosauridae) Serena Najara Migliore 1,2, *, Henrique Bartolomeu Braz 2,3, André Felipe Barreto-Lima 4, Selma Maria Almeida-Santos 1,2 1 Setor de Anatomia, Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Orlando Marques de Paiva 87, Cidade Universitária, São Paulo, SP, Brazil Laboratório de Ecologia e Evolução, Instituto Butantan - Av. Dr. Vital Brazil, 1500 Butantã, São Paulo - SP, Brazil School of Life and Environmental Sciences, Heydon-Laurence Building, A08, University of Sydney, NSW, 2006, Australia 4 Laboratório de Herpetologia, Departamento de Zoologia, Instituto de Ciências Biológicas, Campus Darci Ribeiro, Universidade de Brasília - DF, Brazil *Corresponding author. serena_891@hotmail.com Submitted on: 2016, 16 th December; revised on: 2017, 24 th February; accepted on: 2017, 29 th July Editor: Giovanni Scillitani Abstract. Enyalius perditus is a semi-arboreal lizard species whose reproduction is poorly known. Here, we combine information obtained from preserved and live specimens to describe the reproductive timing (vitellogenesis, gravidity, and egg-laying) and fecundity (clutch size, egg size, and relative clutch mass) in females of E. perditus. Female reproduction is remarkably seasonal and occurs in the warmer and wetter periods of the year. Secondary vitellogenesis occurs from mid to late spring, whereas gravidity and egg-laying occur in early summer. Mating appears to be synchronized with secondary vitellogenesis, indicating an associated reproductive cycle. We suggest that E. perditus females produce only a single clutch per reproductive season. Clutch size ranged from three to 11 eggs and was positively correlated with female body size. Finally, the relative clutch mass was high, a recurrent feature to sit-and-wait foragers. Keywords. Clutch size, reproductive biology, reproductive cycle, seasonal reproduction. Detailed information on the reproductive biology of a number of species is critical for elaborating and testing ecological-evolutionary hypotheses and providing informed decisions on conservation strategies (Shine and Bonnet, 2009; Vitt, 2013). Despite the recent increase in the number of studies on the reproduction of Neotropical lizards (e.g., Balestrin et al., 2010; Ferreira et al., 2009; Vieira et al., 2001), knowledge about the reproductive biology of several species is still scarce. Enyalius is composed of insectivorous, diurnal, and semi-arboreal lizards (Jackson, 1978; Rautenberg and Laps, 2010; Sturaro and Silva, 2010; Barreto-Lima and Sousa, 2011; Barreto-Lima et al., 2013). Enyalius is distributed mostly in Atlantic Forest areas but some species may also occur in the Amazon Forest, forest galleries in Cerrado, and isolated forested areas in Caatinga (Barreto-Lima, 2012). Enyalius perditus is commonly found in Atlantic forest areas in southeastern Brazil, where it may occur in sympatry with E. brasiliensis, E. iheringii, and E. bilineatus (Barreto-Lima, 2012). Many studies have addressed several aspects of the natural history of the species, including feeding ecology, activity patterns, microhabitat use, sexual dimorphism, and behavior (Barreto-Lima and Sousa, 2006, 2011, Sturaro and Silva, 2010; Barreto-Lima et al., 2013; Migliore et al., 2014). Recently, Migliore et al. (2014) summarized the reproductive information available for E. perditus and E. iheringii and found that published data is ISSN (print) ISSN (online) Firenze University Press

68 188 Serena Najara Migliore et alii limited to punctual observations on clutch size, courtship and mating behavior, and timing of mating and gravidity. This limited information impairs both an overview on the reproduction of the species and broad comparisons across other species. To fill this gap, we combine information obtained from both museum and live specimens to describe the reproductive timing (vitellogenesis, gravidity, and egg-laying) and fecundity (clutch size, egg size, and relative clutch mass) in females of E. perditus. We analyzed 35 sexually mature females of E. perditus housed in six scientific collections from Brazil (Appendix 1). The specimens were mostly collected throughout the Atlantic forest domain. The climate in the area is seasonal and characterized by a distinct hot, rainy season from spring to summer (October-March) and a dry season from autumn to winter (April-September) associated with lower temperatures (Mendonça and Danni-Oliveira, 2007). Females were considered sexually mature if they contained vitellogenic follicles, oviductal eggs, or folded oviducts. For each individual, we recorded the: (1) snout-vent length (SVL) to the nearest 1 mm, (2) number of ovarian follicles or eggs, (3) diameter of the largest ovarian follicle, and (4) length and width of oviductal eggs. Additional observations on body sizes, egg-laying, egg size, and clutch size were obtained from two gravid females collected in the Biological Reserve of Boracéia, São Paulo state, on 8th December These females were kept in terrarium containing branches and leaf litter and allowed to oviposit naturally. Procedures for egg measurements and incubation were used according to Migliore et al. (2014). We determined the timing of secondary vitellogenesis using a scatterplot of the diameter of the largest ovarian follicle (Almeida-Santos et al., 2014). Relative clutch mass (RCM) was calculated by dividing the total clutch mass by maternal body mass after oviposition + total clutch mass (Vitt and Price, 1982). We used simple linear regression to determine the relationship between maternal SVL and clutch size (both log-transformed; King, 2000) and significance was assumed at P Mean values are always followed by ± standard deviation. Fig. 1. Reproductive timing in females of Enyalius perditus. The graph shows the seasonal variation in the diameter of the largest ovarian follicle or oviductal egg and the timing of egg-laying. Closed circle: ovarian follicles; open circle: oviductal eggs; arrows: egg-laying. Body sizes of E. perditus females ranged from 58 to 91 mm (mean = 74.7 ± 7.8 mm; n = 36). Females with follicles in primary vitellogenesis were found throughout the year (Fig. 1). Substantial increases in follicular size (and thus secondary vitellogenesis) were observed from mid to late spring (November-December; Fig. 1). Gravid females (n = 11) were observed in early-summer (January; Fig. 1). No female contained follicles in secondary vitellogenesis simultaneously with oviductal eggs. Egglaying was recorded in early summer (Table 1). The two wild-caught females laid eight and six eggs each on 26th December 2015 and 1st January 2016, respectively (Fig. 1). All eggs spoiled over incubation due to fungal contamination. Clutch size (including preserved and captive specimens) averaged 7.1 ± 2.4 eggs (range: 3-11 eggs; n = 13 clutches). Clutch size was positively correlated with maternal SVL (r = 0.70; n = 13; P = 0.008; Fig. 2). Egg length in all gravid females averaged ± 1.36 mm (range: mm; n = 92 eggs from 13 females) and egg width averaged 8.15 ± 0.56 mm (range: Table 1. Morphometrics of two clutches of Enyalius perditus from Biological Reserve of Boracéia, São Paulo state, Brazil. RCM: Relative clutch mass. Individual Date laid Clutch size Female mass 1 (g) Total clutch mass (g) RCM 2 Egg length (mm) Egg width (mm) Egg mass (g) Female 1 26 Dec ± ± ± 0.02 Female 2 1 Jan ± ± ± Post parition mass. 2 RCM was calculated by dividing the total clutch mass by maternal body mass after oviposition plus total clutch mass (Vitt and Price, 1982).

69 Reproduction in Enyalius perditus 189 Fig. 2. Relationship between maternal snout-vent length and clutch size (both log-transformed) in Enyalius perditus. Closed circles: data from preserved specimens; open circles: data from freshly laid clutches mm; n = 92 eggs from 13 females). RCM for the two wild-caught females that laid eggs in captivity was 0.27 and Reproductive timing in E. perditus females is remarkably seasonal, with secondary vitellogenesis, gravidity, and egg-laying occurring within three months, from November to January (see Sturaro and Silva, 2010; Barreto-Lima and Sousa, 2011 for additional records of vitellogenesis and gravidity). Therefore, reproductive timing in E. perditus females is associated with the warmer and wetter periods of the year. Reproductive information for other Enyalius species is rather limited. However, the reproductive timing in E. perditus appears to be concentrated within a shorter period of time than other Enyalius from the Atlantic forest (Marques and Sazima, 2004; Teixeira et al. 2005; Rautenberg and Laps, 2011; Migliore et al., 2014). This short reproductive season in females of E. perditus suggests that females produce only a single clutch per reproductive season. Indeed, this is corroborated by the absence of females containing follicles in secondary vitellogenesis simultaneously with oviductal eggs (Almeida-Santos et al., 2014). Associated reproductive cycles are common in lizards and consist of reproductive events in males and females (i.e., sperm production, mating, and ovulation) occurring at the same period (Crews and Gans, 1992; Méndez-de la Cruz et al., 2014). Courtship and mating in E. perditus have been reported in spring (November-December: Barreto-Lima and Sousa, 2006; Migliore et al., 2014) and thus are synchronized with the timing of secondary vitellogenesis and ovulation. This suggests that E. perditus exhibits associated reproductive cycles. However, histological investigations of the reproductive cycle of E. perditus males are required to confirm if the species exhibits associated reproductive cycles. Mean clutch size in E. perditus is low relative to at least three other congenerics (14.0 eggs in E. iheringii: Rautenberg and Laps, 2010; Migliore et al., 2014; 12.3 eggs in E. leechi: Vitt et al., 1996; and 11.5 eggs in E. brasiliensis: Teixeira et al., 2005) but high relative to another congeneric (4.4 eggs in E. bilineatus: Teixeira et al., 2005). These differences may be explained by interspecific differences in mean body size since all Enyalius species that showed higher clutch size than E. perditus also exhibited larger body sizes (see Rand, 1982; Vitt et al., 1996; Teixeira et al., 2005; Rautenberg and Laps, 2010). This idea is corroborated by our finding that the clutch size in E. perditus increased with maternal SVL, as observed in other Enyalius (Teixeira et al., 2005; Vitt et al., 1996) and in many lizard species with variable clutch size (Fitch, 1970; Tinkle et al., 1970). The RCM for E. perditus ( ) is similar to that reported for a congeneric (0.38 in E. iheringii; Migliore et al., 2014). In lizards, RCM tends to be relatively low in wide forager species and relatively high in sit-andwait foragers (Vitt and Price, 1982). Females of Enyalius species appear to be sit-and-wait foragers (Sousa and Cruz, 2008; Borges et al., 2013) and move shorter distances than males (Barreto-Lima et al., 2013). The RCM for Enyalius is higher than the upper limit reported for other oviparous lizards that forage widely (~ 0.21) and consistent with values reported for sit-and-wait foragers (Vitt and Price, 1982), thus in agreement with the association between RCM and foraging tactics. ACKNOWLEDGEMENTS We thank G. Puorto, P. R. Manzani, J. C. Moura- Leite, M. R. S. Pires, R. N. Feio, and V. Silva for allowing access to specimens under their care, M. T. Rodrigues for providing some specimens, M. Teixeira-Junior for assistance in field work. We also thank P. Monteiro, N. Torello-Vieira and K. Banci for criticism on an earlier draft. We thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for providing financial support (Master s scholarship to S. N. Migliore), SISBIO (Sistema de Autorização e Informação em Biodiversidade; permit number SISBIO ), and Butantan Institute Animal Ethics Committee (approval number ) for the permission to collect and maintain the lizard specimens at the Ecology and Evolution Lab Butantan Institute.

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71 Reproduction in Enyalius perditus 191 Vitt, L.J., Ávila-Pires, T.C.S., Zani, P.A. (1996): Observations on the ecology of the rare Amazonian lizard, Enyalius leechii (Polychrotidae). Herpetol. Nat. Hist. 4: Vitt, L.J., Price, H.J. (1982): Ecological and evolutionary determinants of relative clutch mass in lizards. Herpetologica 38: APPENDIX Appendix 1. List of museums and voucher specimens of Enyalius perditus examined. Collection (Abbreviation) Voucher number Instituto Butantan, São Paulo, Coleção de Referência (IBSPCR) SÃO PAULO: São José do Barreiro (IBSPCR 407). Museu de Zoologia, Universidade Estadual de Campinas (ZUEC) SÃO PAULO: Ilhabela (ZUEC 2934, 2935, 2936, 2938, 2942), Ubatuba (ZUEC 1887). Museu de Zoologia João Moojen (MZUFV) MINAS GERAIS: Lambari (MZUFV 633). MINAS GERAIS: Lambari (CHARW 94, 155, 158, 160, 161, 194, 289, Coleção Herpetológica Alfred Russel Wallace (CHARW) 293, 296, 321), Alfenas (CHARW 322), Boa Esperança (CHARW 317). Coleção Herpetológica da Universidade Federal de Ouro Preto (UFOP) MINAS GERAIS: Itatiaia, Serra de Ouro Branco (UFOP 939s, 968s, 976s, 978s, 993s, 995s, 996s, 997s, 1042s, 1062s, 1077s, 1087s). Museu de História Natural Capão da Imbuia (MHNCI) PARANÁ: Telêmaco Borba (MHNCI 3128, 12956, 12966).

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73 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol Observations on the intraspecific variation in tadpole morphology in natural ponds Eudald Pujol-Buxó 1,2, *, Albert Montori 1, Roser Campeny 3, Gustavo A. Llorente 1,2 1 Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals, Universitat de Barcelona, Barcelona, Spain. *Corresponding author. epujolbuxo@ub.edu 2 Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Barcelona, Spain 3 Minuartia Estudis Ambientals, Barcelona, Spain Submitted on: 2017, 10 th July; revised on: 2017, 21 st August; accepted on: 2017, 26 th August Editor: Marco Mangiacotti Abstract. Intraspecific morphological variation of anuran tadpoles occurs in response to several factors. Causes and consequences of this variation have been largely studied hitherto in controlled environments, but data from natural habitats is clearly less abundant. Here, we present a series of observations on the morphology mainly tail depth of three tadpole species from NE Iberian Peninsula across different pond typologies. According to experimental data on tadpole morphology and selective pressures along the pond permanency gradient, we should expect that tadpoles inhabiting ponds with a short hydroperiod mainly facing desiccation risk have shallower tail fins than tadpoles from ponds with longer hydroperiod mainly facing predation risk. Thus, we expected that the link between these complementary selective pressures predation risk, desiccation risk and hydroperiod could make possible to detect intraspecific variation in tadpole morphology among different typologies of natural ponds. Morphological differences were found in all studied species, and variation, when present, agreed with theory: tadpoles had deeper fin tails as they were collected in ponds with a longer hydroperiod. Interestingly, in most cases these morphological differences were more marked as tadpoles were larger in size. Although distances among the studied ponds were generally short posing phenotypic plasticity as the most plausible proximate mechanism specifically designed studies would be needed to disentangle the relative role of other processes like local adaptation. Keywords. Alytes obstetricans, Hyla meridionalis, Rana temporaria, predation risk, desiccation risk, phenotypic plasticity. INTRODUCTION Intraspecific morphological variation of anuran tadpoles occurs in response to several factors and is created through different mechanisms. Phenotypic plasticity and various processes creating population-level genetic changes (Van Buskirk and McCollum, 1999; Pfennig and Murphy, 2000; Relyea, 2004; 2005) have been listed as natural sources of this variation. Usually, a series of both biotic and abiotic stressors desiccation and predation risk, tadpole competition and density combined with the particular life history characteristics of each species, creates a set of predictable tadpole morphologies (Relyea, 2004; Richter-Boix et al., 2006a; 2006b; 2007; Touchon and Warkentin, 2008; Van Buskirk, 2009). Importantly, these morphologies have been proved to correlate with individual fitness during larval stages (Johnson et al., 2008; Dijk et al., 2016; Pujol-Buxó et al., 2017) and to influence also post-metamorphic morphology and fitness in turn (Tejedo et al., 2010; Johansson and Richter-Boix, 2013; Pujol-Buxó et al., 2013). Causes, effects and consequences of intraspecific morphological variation in tad- ISSN (print) ISSN (online) Firenze University Press

74 194 Eudald Pujol-Buxó et alii poles have been largely studied so far, but mainly using laboratory experimental procedures or controlled garden experiments (e.g., Relyea, 2004; 2005; Touchon and Warkentin, 2008). Hence, in this field of study, morphological data of tadpoles from natural ponds is clearly less abundant (but see Van Buskirk, 2009; 2014; Johnson et al., 2015). This data is crucial to confirm the trends observed in laboratory or garden experiments and to spur novel research questions and hypotheses. The pond permanency gradient ranging from ephemeral pools to permanent water bodies (Skelly, 1995; Schneider and Frost, 1996; Wellborn et al., 1996) correlates with most selective pressures acting on tadpoles in the Mediterranean area. Predation and pond desiccation are arguably the most important selective pressures acting on tadpole populations, and they tend to create a trade-off along the pond permanency gradient (Skelly, 1995): the mean time a pond contains water each year negatively correlates with its desiccation risk, but it is also commonly linked to an increasing number or diversity of predators (Smith, 1983; Pearman, 1995; Schneider and Frost, 1996; Richter-Boix et al. 2006b; 2007). Interestingly, as showed by laboratory experiments, both selective pressures also create opposite morphological outcomes in the tail shape of tadpoles. Thus, tadpoles under predation risk display deeper tail fins to lure predators away from lethal surfaces in case of attack (Van Buskirk et al., 2003; Johnson et al., 2008), while tadpoles under desiccation risk display shallower tails, investing more energy in the feeding and digesting structures located in the main body (Vences et al., 2002; Richter-Boix et al., 2006a). Therefore, assuming an inverse correlation between predation and desiccation risk along the pond permanency gradient, we can expect from experimental data that tadpoles inhabiting ponds with a long hydroperiod should usually display either by phenotypic plasticity or other mechanisms deeper tail fins than tadpoles from ponds with a short hydroperiod (Smith, 1983; Richter-Boix et al., 2006a; 2006b; 2007; Van Buskirk, 2009). Here, we explore this assumption re-analysing simple morphological data tail depth and total length of tadpoles on three European species inhabiting more than one pond typology. MATERIALS AND METHODS We gathered available morphological data of tadpoles of three anurans inhabiting different pond typologies in two Natural Parks (NP) located near Barcelona (Catalonia, Spain), namely Alytes obstetricans (Anura, Alytidae) and Hyla meridionalis (Anura, Hylidae) from Garraf NP; and Rana temporaria (Anura, Ranidae) from Montseny NP. Data from Garraf NP was initially collected as part of a monitoring of the parks anuran populations during spring of year 1991, and data from Montseny NP is from a PhD thesis by Campeny (2001) on tadpole trophic ecology made during years 1985 and In both cases, tadpoles had been dip-netted from natural ponds along several weeks or months of spring, being the ponds in Montseny NP the same for both years (Tables S1, S2 and S3). Since all tadpoles were euthanized for other purposes within each study, they could not be possibly sampled twice. Although tadpoles were measured differently in both studies using a caliper Garraf NP, and using a binocular microscope in Montseny NP we did not perform comparisons across species or parks, and therefore we can discard possible biases due to the measurement methods. In both cases, we assigned ponds to a certain category ephemeral, temporary or permanent according to criteria by Richter-Boix et al. (2006b) and each pond s usual hydroperiod during the years of sampling. According to these criteria, Alytes obstetricans in Garraf NP chooses mainly permanent water bodies as reproduction ponds, using temporary and even ephemeral ponds occasionally (Montori et al., 2015), while Hyla meridionalis mostly uses temporary ponds, breeding also in all pond typologies present in Garraf NP (Montori et al., 2015). On the other hand, Rana temporaria in Montseny NP breeds in most types of water bodies, from permanent streams to temporary or occasionally ephemeral ponds (Campeny, 2001). Since necessary assumptions for parametric tests were not met mainly due to important differences in the numbers of specimens measured in each pond, differences in tail depth (Fig. S1) were analysed using non-parametric randomization tests implemented in the package lmperm (Wheeler and Torchiano, 2016), using 1000 randomizations in each case. Tests were run separately for each species: tail depth as response variable, pond as factor and total length of the tadpole as a covariate, allowing for interactions. When there were multiple ponds to test for the same species, we used the same procedures in pairwise tests to detect statistically homogeneous groups if global differences were found. Experimental data for comparison using the same measurements (in this case on Discoglossus pictus and Pelodytes punctatus) was re-analysed from a study on inducible defences (Pujol-Buxó et al., 2013). In this case we used linear mixed models instead of permutation tests using the same model structure to account for lack of independence, by adding a random intercept depending on tank. All statistical analyses and figures were done in R v3.2.3 (R core team, 2015). RESULTS The relationship between tail depth and total length of A. obstetricans tadpoles significantly differed in slope (that is, effects of the interaction were significant: F 4,423 = 6.44, P < 0.001) and intercept (F 4,423 = 21.6, P < 0.001) when testing all five ponds together. However, there were clearly two types of ponds according to posterior pairwise analysis: on one hand, A. obstetricans tadpoles from permanent ponds displayed the steepest slopes, not differing in slope among them (F 1,391 = 0.01, P = 0.863) but having the pond G6 a higher intercept than pond G1

75 Intraspecific variation in tadpole morphology 195 Tail depth (mm) a) Alytes obstetricans permanent pond G1 permanent pond G6 temporary and ephemeral ponds Tail depth (mm) a) permanent pond M3 temporary pond M Total length (mm) Total length (mm) 8 7 b) b) Tail depth (mm) Hyla meridionalis permanent pond G6 temporary pond G2 ephemeral pond G3 Tail depth (mm) permanent pond M3 temporary pond M Total length (mm) Total length (mm) Fig. 1. Intraspecific morphological variation among different nearby natural ponds from Garraf NP (for pond information see supplementary material): a) Alytes obstetricans, b) Hyla meridionalis. (F 1,392 = 25.6, P < 0.001). On the other hand, tadpoles from temporary and ephemeral ponds showed more gentle slopes, not differing among them neither in slope (F 2,32 = 0.06, P = 0.883) nor in the intercept (F 2,34 = 2.65, P = 0.131) (Fig. 1, both pond typologies grouped together for clarity). Relationship between tail depth and total length of H. meridionalis tadpoles differed in slope (F 2,286 = 36.2, P < 0.001) and intercept (F 2,286 = 8.44, P = 0.039) among the three studied ponds when tested all together (Fig. 1). According to pairwise tests, the slope of the ephemeral pond is significantly more gentle than the ones of permanent (F 1,275 = 70.7, P < 0.001) and temporary (F 1,56 = 11.69, P = 0.001) ponds. Tadpoles from the permanent and temporary ponds did not differ in slope (F 1,241 = 0.71, P = 0.261). Differences in the intercept disappeared in pairwise analyses (all P > 0.05). Differences in morphology between R. temporaria tadpoles from the temporary and permanent ponds (Fig. 2) were significant in both studied years, being the slope Fig. 2. Intraspecific morphological variation in Rana temporaria from two nearby natural pools of Montseny NP in consecutive years (for pond information see supplementary material): a) year 1985, b) year between tail depth and total length of tadpoles always steeper in the permanent pond (F 1,266 = 6.48, P = for 1985, and F 1,189 = 29.84, P < for 1986). Differences in the intercept were also found in both cases (F 1,266 = 51.1, P < for 1985, and F 1,189 = 70.3, P < for 1986). Differences in experimental morphology between D. pictus tadpoles under or without predation risk from Anax sp. included as well a significant interaction (F 1,84 = 10.93, P = 0.001), being the slope between tail depth and total length of tadpoles steeper when a caged predator was present (Fig. S1). The same applies for experimental data on P. punctatus (F 1,85 = 6.29, P = 0.014), being again the slope steeper when a caged predator was present (Fig. S2). DISCUSSION Morphological differences among ponds were found in all studied species, and variation, when present,

76 196 Eudald Pujol-Buxó et alii agreed with theory: tadpoles had deeper fin tails as they were collected in ponds with a longer hydroperiod. Thus, observations coincide with theoretical predictions, arguably posing the trade-off among desiccation and predation risk (Skelly, 1995) as the possible underlying cause of the observed intraspecific morphological differences. Unluckily, given that these observations were not originally taken to explore this hypothesis, we lack data on predator density and diversity in the studied ponds among other potentially useful data, making impossible to assess if the observed morphological trends are in each case rather a consequence of desiccation risk, predation risk, or both. Interestingly, morphological differences among pond typologies were always expressed through a significant interaction between pond type and total length, that is, as changes in the relationship among both measures along growth (i.e., slope differences seen in Fig 1 and Fig 2). Thus, when morphological differences are found among pond typologies, these become more exaggerated as tadpoles are larger in size, coinciding with the re-analyzed experimental data on anti-predator morphology from Pujol-Buxó et al. (2013), and being consistent with similar studies examining tadpole morphology along wide size ranges (Relyea, 2003). Morphological differences between Alytes obstetricans tadpoles from the two permanent ponds, where differences were found in the intercept, represent the only exception to this pattern. The exaggeration of morphological differences with size might be consistent with previous works reporting that behavioural defences are, in relative terms, more used in the first stages of tadpole life, while morphological differences become more marked as tadpoles grow larger (Relyea, 2003; Pujol-Buxó et al., 2017). Which is the process creating the variation we observe in these ponds? The two ponds from Montseny NP are separated less than 1km, and the mean distance among studied ponds in the other study area (Garraf NP) is approximately 3.15 km (Tables S1 and S2). Given these distances, we cannot discard gene flow and therefore we suggest a role of phenotypic plasticity in shaping the observed morphological differences (DeWitt and Scheiner, 2004; Van Buskirk, 2009). However, another complementary option is that, even assuming moderate rates of gene flow (Lind et al., 2011), after several generations of natural selection the sub-populations breeding in the different ponds have also constitutively departed in their morphology (Ledón-Rettig et al., 2008; Lind et al., 2011; Van Buskirk, 2014). This could be expressed in a default production of or a greater tendency to produce deeptailed tadpoles in populations usually breeding in permanent ponds and shallow-tailed tadpoles in populations from temporary and ephemeral ponds. Interestingly, our data of R. temporaria in different consecutive years from the same two ponds shows that although general patterns may repeat year after year, exact results the degree of morphological divergence may vary across years (Fig. 2). Thus, in both areas, neither microevolutionary processes among nearby ponds mediated by processes like genetic accommodation (Ledón Rettig et al., 2008; Wund et al., 2008) nor a prominent role of phenotypic plasticity cannot be totally disregarded. Further studies specifically designed to disentangle the relative role of these mechanisms would be needed. Finally, it is necessary to highlight that, although results agreed with prediction and the number of tadpoles sampled was high in some cases, our observations are based on too few ponds to be conclusive, and other additional studies would be needed to confirm the observed pattern. ACKNOWLEDGEMENTS The study was carried according to the national and local laws in force at the time, and both natural parks gave permission to capture and to euthanize. We are grateful to several reviewers for useful discussions on the manuscript. SUPPLEMENTARY MATERIAL Supplementary material associated with this article can be found at < manuscript number REFERENCES Campeny, R. (2001): Ecologia de les larves d amfibis anurs al Montseny. Unpublished PhD dissertation. Universitat de Barcelona, Barcelona (ES). DeWitt, T.J., Scheiner, S.M. (2004): Phenotypic plasticity: functional and conceptual approaches. Oxford University Press, New York. Dijk, B., Laurila, A., Orizaola, G., Johansson, F. (2016): Is one defence enough? Disentangling the relative importance of morphological and behavioural predator-induced defences. Behav. Ecol. Sociobiol., 70: Johansson, F., Richter-Boix, A. (2013): Within-population developmental and morphological plasticity is mirrored in between-population differences: linking plasticity and diversity. Evol. Biol. 40:

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79 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol Reliable proxies for glandular secretion production in lacertid lizards Simon Baeckens Laboratory of Functional Morphology, Department of Biology, University of Antwerp, 2610 Wilrijk, Belgium Department of Organismic and Evolutionary Biology, Harvard University, Cambridge (MA), USA Submitted on: 2017, 21 st June; revised on: 2017, 26 th August; accepted on: 2017, 11 th October Editor: Marco Mangiacotti Abstract. The epidermal glands of lizards are considered an important source of semiochemicals involved in lizard communication. Many features of the lizard epidermal gland system vary among and within species (e.g., gland number, size, and shape), and some are believed to reflect the degree of intra- and interspecific variation in glandular secretion production, and by extension, the chemical signalling investment of lizards. Traditionally, herpetologists estimate secretion production based on the number of glands or the size of the glands, rather than quantifying the amount of secretion produced. Still, the reliability of these proxies for secretion production has never been validated. Here, I explored the relationship among secretion production (in mass), pore size (surface area, diameter), and gland number in three species of lacertid lizards (Acanthodactylus boskianus, Timon lepidus, Holaspis guentheri), and tested which proxies predicted secretion production variation best, and examined whether the same trend is true for all species. The findings of this study show that the total secretion production of lacertids is highly variable among and within species. Variation in secretion production among-species (but not within-species) could partly be explained by variation in body size. While both measures of pore size were positively related with secretion production, my tests revealed the model with only pore diameter as contributing variable explaining absolute secretion production variation (both within and across species) as the best one. Although gland number appeared a suboptimal estimate for secretion production in the three lacertids under study, only family-wide, multi-species comparative tests counting large within-species sample sizes can provide further insight on the matter. Keywords. Chemical signals; epidermal gland secretions; femoral pores, Lacertidae, secretion quantity. Chemical signals are essential for inter- and intrasexual communication in many animals, and lizards represent no exception (Mason and Parker, 2010). Yet, the extent to which lizards utilize their chemosensory system varies greatly among species (Baeckens et al., 2017a, b). This phenomenon seems also true for the signalling system of lizards, which is illustrated by the fact that merely half of all non-ophidian squamate species are equipped with epidermal glands (lizards leading source of socially relevant chemical signals; Mayerl et al., 2015). It is even so that the number of epidermal glands that lizards possess varies among (and sometimes even within) species (Martín and López, 2000; Pincheira-Donoso et al., 2008; Baeckens et al., 2015). In search for the constraints and selective pressures driving this variation in chemical signalling investment, researchers have focussed on various morphological characteristics of the lizard epidermal gland system to quantify chemical signalling investment, and ultimately, to compare among lizards. Based on the premise that overall secretion production (thus secretion quantity ) reflects how much a particular lizard invests in and relies upon chemical signalling, herpetologists traditionally use gland number and/or the size of the gland opening (i.e., pore) as proxies for secretion quantity (Alberts et al., 1992; Escobar et al., 2001; Pincheira- Donoso et al., 2008; Iraeta et al., 2011; Valdecantos et al., ISSN (print) ISSN (online) Firenze University Press

80 200 Simon Baeckens 2014; Baeckens et al., 2015, 2017c). However, whether these features are truly reliable measures for a lizard s total amount of secretion production (and reliable for interspecific comparisons) has never been validated. In this study, I quantify the overall secretion production (in mass) of lizards of three different lacertid species, and test which characteristics predict secretion production best (gland number, pore diameter, and/or pore surface area). In total, I obtained 32 adult male lizards (14 Acanthodactylus boskianus, 12 Holaspis guentheri, 6 Timon lepidus) from local reptile hobbyist or through the pet trade (Fantasia Reptiles, Belgium, license HK ). Lizards were accommodated at the University of Antwerp facility, and housed in glass terraria (100 x 40 x 50 cm). A 60-watt bulb suspended above one end of the terrarium provided light and heat so that lizards could maintain a body temperature within their preferred range. Lizards had access to freshwater at all times, and were fed up to three times a week. Snout-vent length (SVL) was measured using digital callipers (Mitutuyo, CD-15CPX, accuracy = 0.01 mm). Average pore size was estimated by digitising (ImageJ, Abramoff et al., 2004) the (1) diameter and (2) surface area of the two most proximal pores of the left femur on images obtained with a stereomicroscope (Leica M165 C), and by subsequently calculating mean pore diameter and mean pore surface area per individual (Fig. 1). Next, I collected gland secretions of all lizards, by gently pressing with forceps around the pores until each gland was completely emptied and all secretion yielded (following Baeckens et al., 2017c). Secretion collection occurred within the lizards reproductive period in June 2014 (Castilla and Bauwens, 1989; Schleich et al., Fig. 1. Measuring pore surface area and pore diameter of the epidermal pores of a Timon lepidus lizard. 1996; Pianka and Vitt, 2003; Khannoon, 2009; Grimm et al., 2014), when they were active (between 10:00 and 16:00 h). Secretions extracted from all glands of the left thigh were directly weighed on a microbalance (Mettler Toledo MT5, accuracy = 1 µg). Prior to statistical analyses in SPSS v. 24 (Chicago, IL, USA), variables were log 10 (SVL, pore surface area, pore diameter, secretion mass) or square root (gland number) transformed to meet assumptions of normality (Shapiro-Wilks test: W 0.95). The results of this study show significant intra- and interspecific variation in all aspects of the lizard epidermal gland system, including secretion production (Table 1). While interspecific variation in secretion production could be partially explained by among-species differences in body size (with the largest species in this study, T. lepidus, producing high amount of secretion in comparison to the other two smaller lizard species), the observed within-species variation could not (Table 1 and 2). Further, results show that none of the epidermal gland characteristics (gland number, pore area, pore diameter) of lizards belonging to A. boskianus and H. guentheri were affected by body size (all; Pearson correlation, r < 0.45, P > 0.15). The surface area and diameter of the pores of T. lepidus, however, were strongly linked with body size (resp., r = 0.94, P = 0.006; r = 0.95, P = 0.005). Similar to the other species, gland number did not correlate with SVL in T. lepidus (r = 0.08, P = 0.89). Overall, there was no significant relationship between a lizard s number of glands and its total secretion production (Table 2). It was even so that the lizard species with the least number of glands (i.e., T. lepidus) was equipped with the largest pores, which moreover produced the largest amount of glandular secretion (Table 1). Pore surface area and pore diameter, on the other hand, correlated strongly with secretion mass in H. guentheri and T. lepidus but not in A. boskianus (Table 2). In T. lepidus, where pore size is affected by SVL, a partial correlation test (controlling for SVL) revealed also a positive relationship between relative secretion mass and pore surface area, but not pore diameter. Multiple regression analyses (backward stepwise elimination with gland number, pore diameter, and pore surface area) indicated the model with only pore diameter as the significant contributing independent variable explaining secretion mass variation (A. boskianus, R 2 = 0.31, F 1,13 = 5.35, P = 0.039; H. guentheri, R 2 = 0.45, F 1,11 = 8.27, P = 0.017; T. lepidus, R 2 = 0.79, F 1,5 = 15.32, P = 0.017). The same was true for an analogous multiple regression, but then across species encompassing all individuals (R 2 = 0.74, F 1,31 = 83.43, P < 0.001, Fig. 2). Overall, the findings of this study reveal that the total glandular secretion production of lacertid lizards is highly variable among and within species. While body size

81 Proxies for lizard chemical signalling investment 201 Table 1. Descriptive statistics for the morphological variables measured on male lacertid lizards of the three species used in this study. The table also shows the results of the analyses of variance (ANOVA) testing for absolute among-species differences (followed by Tukey post-hoc tests), and the results of the analyses of covariance (ANCOVA, with SVL as covariate) testing for relative differences. Acanthodactylus boskianus (A) n = 14 Timon lepidus (T) n = 6 Holaspis guentheri (H) n = 12 Interspecific comparison Absolute differences Relative differences Mean SE Min Max Mean SE Min Max Mean SE Min Max F 2,31 P Post-hoc F 2,31 P SVL (mm) <0.001 T > A > H Gland number <0.001 A > H > T Pore surface area (mm 2 ) <0.001 T > A = H Pore diameter (mm) <0.001 T > A > H Secretion mass (mg) <0.001 T > A = H Abbreviations: A = Acanthodactylus; H = Holaspis; T = Timon. Table 2. Results of Pearson correlation tests (r), testing for correlations between (a) SVL and gland traits, and (b) secretion mass and gland traits. Also shown are the results of partial correlation tests (c), which accounted for differences in SVL. Variables were transformed prior to analyses to meet the assumptions of normality. Bold values indicate statistical significance (P < 0.05). Acanthodactylus boskianus (n = 14) Timon lepidus (n = 6) Holaspis guentheri (n = 12) r P r P r P (a) Correlation with SVL Secretion mass Gland number Pore surface area Pore diameter (b) Correlation with secretion mass Gland number Pore surface area Pore diameter (c) Partial correlation with secretion mass Gland number Pore surface area Pore diameter was unable to explain the observed intraspecific variation in secretion production in the species under study, a positive link between body size and secretion quantity has been observed in other lizard species, such as in the iguanid Iguana iguana (Alberts et al., 1992) and the lacertid Podarcis muralis (Baeckens et al., 2017c). Latter researchers argue that since secretion production is most probably an energetically costly affair (Martín and López, 2014; Mayerl et al., 2015), it is very likely that only individuals in a good condition (which are often the largest individuals; Jakob et al., 1996) can afford high secretory activity rates. These discordant findings among studies and species imply that the biosynthetic pathways that produce glandular secretion might be species-specific. An alternative explanation for not finding a link between body size and secretion production among individuals of the same species concerns the origin of the animals in this study. Since pet store animals generally receive plenty of nutritious food throughout their lives in the store, it is unlikely to find biological significant variation in body condition (linked with secretion production) among individuals. Notwithstanding, large-scale phylogenetical-informed comparative studies could shed light on how idiosyncratic or universal the link between body size and secretion production really is, and how they scale (allometric/isometric) among and within species. Aside from quantifying (variation in) chemical signalling investment in lacertid lizards, the ultimate aim of this

82 202 Simon Baeckens log10 (Secre on mass) Acanthodactylus boskianus Holaspis guentheri Timon lepidus log10 (Pore diameter) Fig. 2. A scatterplot showing the positive relationship (R 2 = 0.74, F 1,31 = 83.43, P < 0.001; slope = 1.85) between pore diameter and glandular secretion production in three species of lizards. Because the slopes of the three species did not significantly differ, only one regression line (covering all lizards of the three species) is shown. Shaded area represents 95% confidence intervals. study was to document reliable proxies, if any, for secretion production. While both measures for pore size were positively correlated with secretion mass, the findings here suggest that pore diameter was the best predictor of secretion production quantity, hence chemical signalling investment. This was true both within species and across species, advocating pore diameter as the most adequate estimate of gland productivity in interspecific comparisons. Alberts and colleagues (1992) established a similar relationship in I. iguana males (with n = 10, r = 0.81, P = 0.002), although Baeckens et al. (2017c) did not so in the species P. muralis. However, the latter investigators merely measured pore surface area, not pore diameter. Why diameter turned out to be a better predictor of secretion quantity than surface area might be partly ascribed to the shape of the pores. The lacertids under study bared pores of a long-stretched oval form (unlike, for example, those of the teiid Tupinambis merianae; Chamut et al. 2009), which varied among individuals largely in length (diameter) and less in area. The lengthy oval shape of the pores might allow lizards to maximize their scent-mark area by increasing the contact zone between pores and substrate (along the proximal-distal limb axis of the limb). More research on lizard scent-marking behaviour and the functional significance of pore shape is necessary to make well-founded predictions on the matter. Surprisingly, gland number came out as a poor predictor of secretion production in the three lacertids under study here. Yet, several intra- and interspecific comparative studies have assumed that gland number reflects species investment in and use of chemical communication (e.g., Escobar et al., 2001; Pincheira-Donoso et al., 2008; Iraeta et al., 2011; Baeckens et al., 2015). Notwithstanding, their theory cannot be considered as illogically, for a lizard with x number of glands will produce a lower amount of secretion than a hypothetical identical lizard, but with x+1 number of glands. Besides, since this study only comprises three different lizard species belonging to merely one lizard family (Lacertidae), it would be incorrect to generalize and label gland number as a poor proxy reflecting chemical signalling investment in lizards. Clearly, only a broad, multi-species study counting large within-species sample sizes can provide further insight on the matter. While the goal of this short note was to underscore the importance of choosing the appropriate proxy for lizard secretion production, I wish to note that the findings of this study should be interpreter cautiously due to the following. Firstly, I only used secretion quantity to estimate chemical signalling investment, whilst disregarding secretion quality. The chemical composition of the secretion of lizards is a mixture of proteins and lipids and is highly species-specific (Mayerl et al., 2015; Mangiacotti et al., 2017; Baeckens et al., 2017d). One can easily imagine a trade-off between secretion quantity and quality, with lizards producing low amounts of secretion, but investing highly in, for instance, a rich or diverse chemical design with high concentrations of certain key compounds (such as described for some invertebrates; Wyatt, 2014). Future studies on the chemical signalling investment of lizards should, ideally, integrate both the chemical architecture of the glandular secretion and the total amount of secretion produced. Secondly, this study concentrates solely on follicular epidermal gland secretions, while neglecting any other source of semiochemicals. Although it is generally believed that follicular gland secretions are the leading source of semiochemicals (Martín and López, 2014; Mayerl et al., 2015), there is plenty of evidence that generation glands, faeces, cloacal secretions, and skin lipids contain socially relevant chemical stimuli too (Cooper and Vitt, 1984; Mason and Gutzke, 1990; Cooper, 1995; Labra, 2008; Moreira et al., 2008; Mouton et al., 2010). Whether lizards that invest little in the production of gland secretions are investing more strongly in semiochemicals of other origins (and vice versa) is, however, uncertain, but certainly not improbable. Thirdly, the use of animals obtained through the commercial pet trade, rather than using life-caught animals from the wild,

83 Proxies for lizard chemical signalling investment 203 might bring along a series of uncertainties concerning pre-purchase animal stress, transport, and housing conditions. Yet, similar to previous experiments using pet trade lizards (obtained through the same commercial dealer as used in the current study; Herrel et al., 2007, Driessens et al., 2014), animals were in good condition at the onset of the experiments. Overall, I am confident that these limitations did not compromise the main objective of this work, which was: quantifying intra- and interspecific variation in secretion production in a small subset of lacertid lizards, and exploring the best possible morphological traits to estimate secretion production. Based on the findings of this study, I advise scholars, at times when assessing secretion mass seems unfeasible (e.g., in museum specimen), to be cautiously thorough and integrate pore area, diameter, and number in any future studies scoring secretion production quantity. Although gland number played out to be a suboptimal quantity-proxy in the three lacertid lizards under study, broad-scale comparative analyses should examine this in more detail. ACKNOWLEDGMENTS Author thanks Raoul Van Damme, Alison Heath, and Katleen Huyghe for co-designing this study. Jan Scholliers and Jorrit Mertens for animal care, and three anonymous reviewers for significantly improving drafts of this manuscript. All work was carried out in accordance with the University of Antwerp animal welfare standard and protocols (ECD ). This work was carried out at the University of Antwerp, and made possible through financial support from the University of Antwerp. REFERENCES Abràmof, M.D., Magalhães, P.J., Ram, S.J. (2005): Image processing with ImageJ Part II. Biophotonics Int. 11: Alberts, A., Pratt, N.C., Phillips, J.A. (1992): Seasonal productivity of lizard femoral glands: relationship to social dominance and androgen levels. Physiol. Behav. 51: Baeckens, S., Edwards, S., Huyghe, K., Van Damme, R. (2015): Chemical signalling in lizards: an interspecific comparison of femoral pore numbers in Lacertidae. Biol. J. Linn. Soc. 114: Baeckens, S., Van Damme, R., Cooper, W.E. (2017a): How phylogeny and foraging ecology drive the level of chemosensory exploration in lizards and snakes. J. Evol. Biol. 30: Baeckens, S., Herrel, A., Broeckhoven, C., Vasilopoulou- Kampitsi, M., Huyghe, K., Goyens, J., Van Damme, R. (2017c): Evolutionary morphology of the lizard chemosensory system. Sci. Rep. 7: Baeckens, S., Huyghe, K., Palme, R., Van Damme, R. (2017b): Chemical communication in the lacertid lizard Podarcis muralis: the functional significance of testosterone. Acta Zool. 98: Baeckens, S., Martín, J., Garcia-Roa, R., Pafilis, P., Huyghe, K., Van Damme, R. (2017d): Environmental conditions shape the chemical signal design of lizards. Funct. Ecol. DOI: / Castilla, A.M., Bauwens, D. (1989): Reproductive characteristics of the lacertid lizard Lacerta lepida. Amphibia-Reptilia 10: Chamut, S., Valdez, V.G. & Manes, M.E. (2009): Functional morphology of femoral glands in the Tegu lizard, Tupinambis merianae. Zool. Sci., 26: Escobar, C.A., Labra, A., Niemeyer, H.M. (2001): Chemical composition of precloacal secretions of Liolaemus lizards. J. Chem. Ecol. 27: Grimm, A., Prieto Ramírez, A.M., Moulherat, S., Reynaud, J. & Henle, K. (2014): Life-history trait database of European reptile species. Nat. Conserv. 9: Iraeta, P., Monasterio, C., Salvador, A., Díaz, J.A. (2011): Sexual dimorphism and interpopulation differences in lizard hind limb length: locomotor performance or chemical signalling? Biol. J. Linn. Soc. 104: Jakob, E.M., Marshall, S.D., Uetz, G.W. (1996): Estimating fitness: a comparison of body condition indices. Oikos 77: Khannoon, E.R.R. (2009): Comparative chemical ecology, behaviour, and evolutionary genetics of Acanthodactlylus boskianus (Squamata: Lacertidae). Unpublished doctoral dissertation, Hull University, Hull (UK). Mangiacotti, M., Fumagalli, M., Scali, S., Zuffi, M.A.L., Cagnone, M., Salvini, R., Sacchi, R. (2017): Inter- and intra-population variability of the protein content of femoral gland secretions from a lacertid lizard. Curr. Zool. 63: Martín, J., López, P. (2000): Chemoreception, symmetry and mate choice in lizards. Proc. Biol. Sci. 267: Martín, J., Lopez, P. (2014): Pheromones and Chemical Communication in Lizards. In: Reproductive Biology and Phylogeny of Lizards and Tuatara, pp Rheubert, J.L., Siegel, D.S., Trauth, S.E., Eds, CRC Press, London. Mason, R.T., Parker, M.R. (2010): Social behavior and pheromonal communication in reptiles. J. Comp. Physiol. A. Neuroethol. Sens. Neural. Behav. Physiol. 196:

84 204 Simon Baeckens Mayerl, C., Baeckens, S., Van Damme, R. (2015): Evolution and role of the follicular epidermal gland system in non-ophidian squamates. Amphibia-Reptilia 36: Mouton, P. Le F.N., Van Rensburg, Van Rensburg D. A J., Van Wyk, J.H. (2010): Epidermal glands in cordylid lizards, with special reference to generation glands. Zool. J. Linn. Soc. 158: Pianka, E. & Vitt, L. (2003): Lizards - Windows to the Evolution of Diversity. University of California Press, California. Pincheira-Donoso, D., Hodgson, D.J., Tregenza, T. (2008): Comparative evidence for strong phylogenetic inertia in precloacal signalling glands in a species-rich lizard clade. Evol. Ecol. Res. 10: Schleich, H.H., Kästle, W., & Kabisch, K. (1996): Amphibians and reptiles of North Africa. Königstein, Koeltz. Valdecantos, S., Martinez, V., Labra, A. (2014): Comparative Morphology of Liolaemus lizards Precloacal Glands. Acta Herpetol. 9: Wyatt, T.D. (2014): Pheromones and Animal Behaviour: Chemical Signals and Signatures. Cambridge University Press, Cambridge.

85 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol Diet of juveniles of the venomous frog Aparasphenodon brunoi (Amphibia: Hylidae) in southeastern Brazil Rogério L. Teixeira 1, Ricardo Lourenço-de-Moraes 2, Débora C. Medeiros 3, Charles Duca 3, Rogério C. Britto 4, Luiz C.P. Bissoli 5, Rodrigo B. Ferreira 3, * 1 In memoriam 2 Laboratório de Herpetologia e Comportamento Animal, Departamento de Ecologia, Universidade Federal de Goiás, Campus Samambaia, Goiânia, GO, Brazil 3 Laboratório de Ecologia de Populações e Conservação, Programa de Pós-graduação em Ecologia de Ecossistemas, Universidade Vila Velha. Rua Comissário José Dantas de Melo, Boa Vista II, Vila Velha, ES, Brazil. *Corresponding author. rodrigoecologia@yahoo.com.br 4 Associação Capixaba em Defesa da Água e da Mata Atlântica (ACADAMA). Rua Lourenço Roldi, São Roque do Canaã, ES, Brazil 5 Ello Ambiental. Av.Getúlio Vargas, Colatina, ES, Brazil Submitted on: 2017, 17 th April; revised on: 2017, 29 th May; accepted on: 2017, 27 st September Editor: Daniele Pellitteri-Rosa Abstract. Seventy juvenile individuals of Aparasphenodon brunoi were collected on the low parts of tree trunks in an Atlantic Forest remnant. Arthropods were the dominant prey found in their stomachs. Coleoptera (adult and larvae) was the most important prey regarding prey frequency, number, weight, and index of relative importance. Secondary preys included Hymenoptera that was important regarding number of prey and Hemiptera that was important regarding prey weight. Trophic ontogeny was detected. The diversity of prey suggests A. brunoi is an opportunistic sit-andwait predator. Keywords. Anura, ecology, Hylidae, predation, ontogeny, feeding pattern. Studies regarding dietary aspects of animals may provide important data for a better understanding of the fundamental niche, position in the food webs, feeding time strategy, and metabolic needs of a species (Christian et al., 2007; Miller et al., 2010). Anurans are usually considered opportunistic predators regarding their feeding habits and most of them are arthropod consumers (Wells, 2007). Generally, the diet of anurans is related to their snout-vent length, ability to detect and capture prey as well as prey availability in the environment (Giaretta et al., 1998; Ferreira et al., 2015). Aparasphenodon brunoi Miranda-Ribeiro, 1920 is a hylid treefrog that uses bromeliads and hollows of tree trunks as diurnal refuge (i.e., bromelicolous species, sensu Peixoto, 1995) and reproduce in temporary ponds (Wogel et al., 2006). It is endemic to the coastal plain of Brazil (i.e., restinga habitat) from São Paulo to Bahia states (Ruas et al., 2013; Frost, 2016). This species has a highly toxic secretion that may be injected by bony spines on the head (Jared et al., 2015). Furthermore, some studies have shown that the toxicity of a frog is determined by the compounds sequestered from the diet during early development stage (e.g., Daly et al., 1994; Sime et al., 2000). There is little information on the ecology of juveniles of the venomous treefrog A. brunoi, and diet studies on adults have shown that this treefrog is a generalist forager of arthropods (Teixeira et al., 2002; Mesquita et al., 2004). The aim of this study is to describe the diet of juveniles ISSN (print) ISSN (online) Firenze University Press

86 206 Rogério L. Teixeira et alii of A. brunoi in an Atlantic Forest remnant from southeastern Brazil. The study area is in the coastal plain of Pontal do Ipiranga district, municipality of Linhares, Espírito Santo state, Brazil (19 13 S, W). The region is characterized by pastures and remnants of Atlantic Forest. The treefrogs were manually captured at night between September and October According to Mesquita et al. (2004), individuals of this species smaller than 48 mm are considered juveniles. The individuals were anesthetized using lidocaine cream (5%) and fixed in a solution of formalin 10% for 72 hours and then washed and placed in a solution of alcohol 70%. In laboratory the specimens were measured to the snout-vent length (SVL in mm), weighted (0.01 g precision) and dissected to determine the sex and to analyze the stomach content. The stomach contents were removed, spread in Petri dish and identified to the lowest taxonomic level possible. Preys were dried on paper towels and the following parameters for each prey category were evaluated: occurrence frequency, number, wet weight ( g precision), and index of relative importance (IRI). The IRI was calculated according to Pinkas et al. (1971) and the percent IRI (%IRI) according to Cortés (1997). For intraspecific comparison, IRI was calculated for adults of A. brunoi using data available on Teixeira et al. (2002). Intact preys were measured to the total length with a caliper (0.1 mm precision). The voucher specimens were deposited at the zoological collection of the Instituto Nacional da Mata Atlântica/Museu de Biologia Professor Mello Leitão (MBML), Espírito Santo State, Brazil. The data were tested for normality using Kolmogorov-Smirnov test and for homogeneity using Bartlett test. SVL and weight were compared between sexes using the analysis of variance (ANOVA). In this analysis, sex was the independent variable while SVL and weight were the dependent variables (Neter et al., 1990). Cluster analysis based on Euclidean distance was used to evaluate trophic ontogeny based on size classes of A. brunoi. Only the weight value of the main prey was used in this analysis and the data were log transformed. The relationship between prey length and anuran SVL was tested by a regression analysis. Mean and standard deviation are provided. The significance level was set to Seventy juvenile individuals of A. brunoi were collected on the low parts of tree trunks (< 1m). Adults were on trees canopy and were not collected. Thirty-two individuals were juvenile males and 38 were juvenile females. Juvenile males ranged on SVL from 31.0 to 39.2 mm (mean = 34.3, SD = 2.0 mm) and in weight from 2.0 to 4.4 g (mean = 2.9, SD = 2.0 g). Juvenile females ranged on SVL from 32.1 to 43.6 mm (mean = 36.7, SD = 2.7 mm) and in weight from 2.1 to 6.7 g (mean = 3.6, SD = 2.7 g). The SVL and weight differed significantly between Table 1. Prey items of juveniles of Aparasphenodon brunoi from Espírito Santo state, southeastern Brazil. F = occurrence frequency; N = number of prey; W = weight; IRI = index of relative importance. Prey F %F N %N W %W IRI %IRI Insecta Blattodea Coleoptera (Adult) Coleoptera (Larvae) Hemiptera Homoptera Hymenoptera Isoptera Orthoptera Insect remnant Arachnida Araneae Crustacea Ostracoda Other Anuran skin Total

87 Diet of juveniles of Aparasphenodon brunoi 207 Fig. 1. Cluster analysis based on Euclidian distance shows the trophic ontogeny in juveniles of Aparasphenodon brunoi from Espírito Santo state, southeastern Brazil. Fig. 2. Prey weight percentage according to snout-vent length (SVL) classes of juveniles of Aparasphenodon brunoi from Espírito Santo state, southeastern Brazil. the sexes (F 1,68 = 17.3, P < 0.01; F 1,68 = 12.6, P < 0.01, respectively). Females were on average larger and heavier than males. Fifty-three (76%) individuals had food items in their stomach, representing 12 prey categories, mostly arthropods, (Table 1). Coleoptera (adult and larva) was the most important prey regarding prey frequency, number, weight, and IRI. Secondary preys included Hymenoptera that was important regarding number of prey and Hemiptera that was important regarding prey weight. It was detected trophic ontogeny in A. brunoi (Fig. 1). Specimens less than 36.9 mm had similar diet, possibly because they predated more Coleoptera (Fig. 2). Specimens larger than 36.9 mm had similar diet by feeding mostly upon Hemiptera. The length of intact prey ingested by A. brunoi varied from 2.8 to 15.9 mm (mean = 8.9, SD 3.7). There was no relationship between total length of prey and anuran SVL (R² = 0.003, P > 0.05). The variety of prey categories suggests that A. brunoi is an opportunistic sit-and-wait predator. The diet of A. brunoi from another two populations had also a variety of prey categories (Teixeira et al., 2002; Mesquita et al., 2004). In fact, the feeding upon small arthropods has been reported in several studies of anurans (Teixeira and Coutinho, 2002; Ferreira and Teixeira, 2009; Ferreira et al., 2012). The presence of ostracods, common on tank bromeliads (Oliveira et al., 1994), indicates bromelicolous habit of A. brunoi at the studied fragment. Apparently, juveniles of A. brunoi feed upon different prey categories compared to adults. In our study, Coleoptera was the most important prey (%IRI = 67.26). In Teixeira et al. (2002), Hymenoptera was the most important prey (%IRI = 20.1) for adults of A. brunoi. Coleoptera was the least important prey (%IRI = 0.7) for this population of adults. In our study, A. brunoi feeds on different prey categories according to their SVL. Surprisingly, A. brunoi SVL was not related to prey size. Changes on diet correlated to anuran SVL have been reported for other species (Giaretta et al., 1998; Teixeira and Vrcibradic, 2003; Ferreira et al., 2007). Trophic ontogeny may be an important mechanism to avoid intraspecific competition and predation. Probably the diet of the adults at tree canopy may strengthen the trophic ontogeny in our studied population. The studied remnant may provide necessary amount of prey for A. brunoi due to the high percentage of stomachs with prey. We recommend future studies to evaluate the relationship between prey items and A. brunoi toxicity. ACKNOWLEDGMENTS We thank Gladstone I. Almeida, Aílston Anastácio and José A. P. Schineider for fieldwork assistance. PETROBRAS funded this research. IBAMA provided the sampling permit (07/98, /97-21). RLM thanks CNPq for scholarship (process /2013-2; /2016-2). RBF and DCM thank CAPES for scholarships. REFERENCES Christian, K., Webb, J.K., Schultz, T., Green, B. (2007): Effects of seasonal variation in prey abundance on field metabolism, water flux, and activity of a tropical ambush foraging snake. Physiol. Biochem. Zool. 80:

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89 Acta Herpetologica 12(2): , 2017 DOI: /Acta_Herpetol Who are you? The genetic identity of some insular populations of Hierophis viridiflavus s.l. from the Tyrrhenian Sea Ignazio Avella, Riccardo Castiglia, Gabriele Senczuk* Dipartimento di Biologia e Biotecnologie Charles Darwin, Università di Roma La Sapienza, 00151, Roma, Italy. *Corresponding author. gabriele.senczuk@uniroma1.it Submitted on: 2017, 3 rd February; revised on: 2017, 13 th April; accepted on: 2017, 6 th June Editor: Adriana Bellati Abstract. This work investigates the genetic identity of Hierophis viridiflavus s.l. specimens from insular populations, to determine which of the two previously identified species is present on each island. Here, the authors hypothesise about times and modes of colonization and discuss the faunistic value of the obtained results. This follows the recent proposal to consider the two clades as two different species. Specimens from the islands of Favignana, Lipari and Vulcano belong to H. carbonarius and probably all belong to putative Sicilian source populations. Conversely, all individuals from the Pontine Islands (Ponza, Palmarola, Ventotene) should be considered to belong to H. viridiflavus. Even if genetically identical to the specimens from the Tyrrhenian Italian coast, these individuals show a darker colouration, very similar to the one usually shown by H. carbonarius specimens. Considering that the Pontine H. viridiflavus populations probably have a very recent origin, the dark livery of these individuals could be the result of a rapid morphological adaptation to insular environments. Keywords. Colour pattern, Hierophis viridiflavus, islands, nd4, phylogeography. The western whip snake Hierophis viridiflavus s.l. (Lacépède, 1789) is a colubrid snake with a wide distribution range. It can be found in Central Europe from Eastern Spain to Central France, Luxemburg, Switzerland, Slovenia and Croatia. Its range also includes all of the Italian Peninsula, Sicily, Sardinia and most of the smaller Italian islands, Corsica and some Croatian islands (Vanni and Nistri, 2006; Zuffi, 2007). The species is found from sea level to m a.s.l., although it is extremely rare above 1500 m in the Alps (Farinello and Bonato, 2000). Interestingly, individuals of this species show two main phenotypes, one named viridiflavus (usually brown/blackish with yellow stripes and spots) and the other named carbonarius, typically melanic (completely black with blackish/grey ventral colouration) or melanotic (almost completely black livery, but paler/ yellowish head scales and ventral surface). Individuals from some of the islands of the Tuscan Archipelago, ISSN (print) ISSN (online) Sardinia and Corsica show a third phenotype, a middle ground between carbonarius and viridiflavus, called abundistic (Zuffi, 2008). In the past, four H. viridiflavus s.l. subspecies have been described for Italy, mainly due to the chromatic pattern of the analysed individuals: H. v. viridiflavus (Rimpp, 1979), H. v. carbonarius (Bonaparte, 1833), H. v. kratzeri (Kramer, 1971) and H. v. antoniimanueli (Capolongo, 1984). However, based on mitochondrial and nuclear DNA evidences, they have all been recently rejected (Vanni and Zuffi, 2011). Indeed, Nagy et al. (2002) and Rato et al. (2009) showed the presence of two mitochondrial distinct haplogroups: the first, roughly corresponding to the subspecies H. v. viridiflavus (clade W) includes individuals from Spain, France, Corsica, Sardinia and central to north-western Italy on the West side of the Apennines; the second, in part matching with the subspecies H. v. carbonarius (clade E), occurs on the other side of the Apennines, from north-eastern Firenze University Press

90 210 Ignazio Avella et alii Italy to southern Italy (including Sicily). More recently, a study based on morphometric, genetic and karyological data, proposed the elevation of the two genetic groups to species status (Mezzasalma et al., 2015). In particular, the authors emphasized significant differences in sexual chromosomes, as while females from the Eastern group have a submetacentric W sex chromosome, females from the Western group have a telocentric W sex chromosome. Thus, individuals from the Eastern clade have been recognized as Hierophis carbonarius, while individuals from the Western clade have been recognized as Hierophis viridiflavus. Interestingly, the relationship between colour variation and genetic repartition does not match completely (Zuffi, 2008). The brown/blackish colouration with yellow stripes and spots pattern, generally almost exclusive of the H. viridiflavus range, can be found also in H. carbonarius specimens (Rato et al., 2009). Although the distribution of the two species in the Italian Peninsula and on the largest Mediterranean islands (Sardinia, Corsica and Sicily) has already been studied (Nagy et al., 2002; Rato et al., 2009; Mezzasalma et al., 2015), there is a lack of molecular data from smaller Italian archipelagos. The aim of the present work is to determine the genetic identity of individuals collected in some Tyrrhenian islands including: Ponza, Palmarola and Ventotene from the Pontine Archipelago; Vulcano and Lipari from the Aeolian Islands; and Favignana from the Aegadian Islands, in order to update the distribution of the two species of whip snakes. We sampled a total of seven individuals of H. viridiflavus s.l. from six different islands between March 2014 and July 2015 (geographic locations are reported in Table 1 and showed in Fig. 1). Snakes were caught and handled following standard protocols (Fowler, 1978), and some ventral scales were removed and preserved in pure ethanol. In one case (RS296 from Lipari), the tissue was obtained from a shedded skin. Genomic DNA was extracted following the protocol described in Aljanabi and Martinez (1997). A fragment including the terminal portion of the NADH dehydrogenase subunit 4 (nd4) was amplified by standard PCR protocols using primers published by Arèvalo et al. (1994). Amplification conditions were the same as described by Pinho et al. (2006). The PCR products were purified with a Sure Clean (Bioline ) purification kit and the sequencing reactions were run under Big-Dye TM Terminator cycling conditions by a commercial company, Macrogen ( The electropherograms were checked using the software FinchTV ( to ensure the absence of double peaks and ambiguous positions. The obtained sequences were deposited to GenBank (accession numbers: KY KY923287) and joined with additional 91 nd4 sequences of Hierophis viridiflavus s.l. retrieved from GenBank (accession numbers: FJ FJ430660, Rato et al., 2009; LN LN552095, Mezzasalma et al., 2015). Nucleotide sequences were translated into amino acids with MEGA 6.0 (Tamura et al., 2013) using the vertebrate mitochondrial genetic code in order to assess the absence of pseudogenes. One nd4 sequence of Hierophis gemonensis (Laurenti, 1768) was downloaded from GenBank (accession number: AY487044, Nagy et al., 2004) and included in the analysis as outgroup, as it is considered the closest related species to H. viridiflavus (Schätti, 1988). The software jmodeltest (Posada, 2008) was used to determine the most appropriate model of sequence evolution for the nd4 dataset. According to the Akaike information criterion (AIC), the most supported evolutionary model was the TrN + I, therefore applied in the subsequent analysis. To reconstruct phylogenetic relationships, we used a coalescent Bayesian approach as implemented in MrBayes (Ronquist et al., 2012). We run 2 million generations, with 4 Markov chains sampling every 1000 steps. After a burn-in of 10%, the remaining trees were used to compute a 50% majority rule consensus tree. In addition, a statistical parsimony network under 95% probability connection limits was constructed using TCS 1.21 (Clement et al., 2000). Number of haplotypes, nucleotide diversity (π) and haplotype diversity (H) were Table 1. individuals analysed including sampling location, group of islands, colour pattern, haplotype number and haplogroup. Sample code Locality Archipelago Colour pattern Haplotype Species RL22 Ponza Pontine abundistic H1 H. viridiflavus RL66 Ponza Pontine abundistic H1 H. viridiflavus RL79 Ventotene Pontine abundistic H1 H. viridiflavus RL80 Palmarola Pontine abundistic H1 H. viridiflavus RS85 Favignana Aegadian carbonarius H10 H. carbonarius RS276 Vulcano Aeolian carbonarius H9 H. carbonarius RS296 Lipari Aeolian carbonarius H9 H. carbonarius

91 Genetic identity from Tyrrhenian insular populations of Hierophis viridiflavus s.l. 211 A Hierophis viridiflavus 1 Hierophis carbonarius Hv34-49 Hv28-54 Hv43-47 V2-48 V32-75 Hv4-34 Hv19-35 Hv30-33 Hv35-37 V4-31 V6-31 V7-31 V8-31 V33-34 Hv45-67 V12-67 V22-67 V31-77 Hv6-29 RL22-2 RL66-2 H1 RL79-1 0,99 RL80-3 Hv8-30 Hv36-27 Hv31-38 Hv25-69 Hv26-74 Hv29-73 Hv37-78 Hv38-72 Hv39-71 Hv48-68 Hv49-70 V9-79 V10-79 V11-79 Hv23-63 Hv41-65 V28-64 Hv3-76 V29-77 V30-77 V41-66 H2 V42-43 H3 V38-26 H4 Hv2-23 Hv7-19 Hv16-21 Hv ,98 H11 V17-17 V35-16 V36-21 RS85-4 H10 Hv20-8 RS276-6 RS296-5 H9 Hv47-7 V37-7 Hv1-12 H8 0,8 Hv32-62 H7 V39-15 H6 Hv13-18 Hv17-52 Hv22-41 V1-46 V3-58 V18-50 V20-51 V25-45 V26-45 Hv24-11 Hv5-57 V13-57 V14-57 V15-57 V16-57 H5 Hv15-61 V ,99 Hv21-32 Hv40-36 Hv42-40 V23-39 V27-42 Hv10-28 Hv14-28 Hv50-25 V40-22 Hv11-20 Hv18-14 Hv33-56 V24-44 Hv9-10 Hv12-9 Hv46-13 V19-55 H12 Hv44-60 Hv27-59 H13 Hierophis gemonensis B Palmarola Ponza Ventotene Lipari 6 66 Favignana 4 Vulcano C H2 H11 H3 H9 H10 H1 H5 H13 H6 H4 H7 H12 H8 11 Fig. 1. Bayesian phylogenetic tree (A) based on nd4 sequences for 98 ingroup specimens of H. viridiflavus s.l. and one outgroup (H. gemonensis). The posterior probabilities are indicated at each node. Each label indicates the specimen code, the locality number and the relative haplotype. Insular individuals are shown in bold. Geographic distribution (B) of the two mitochondrial lineages corresponding to H. viridiflavus (blue) and H. carbonarius (red). Statistical parsimony network (C) connecting haplotypes. The circle size is proportional to the sequence frequencies and each filled rectangle represent one substitution. also calculated for each group using DnaSP 5.1 (Librado and Rozas, 2009). The final nd4 alignment (568 bp) of 99 sequences returned 67 polymorphic sites and 13 haplotypes. The phylogenetic analysis confirmed the presence of two well defined mitochondrial clades (Fig. 1A), as already stated in previous works and corresponding to the two species H. viridiflavus and H. carbonarius (Rato et al., 2009;

92 212 Ignazio Avella et alii Mezzasalma et al., 2015). Nei s standard genetic distance between the two species was 4.2%. Specimens from Favignana, Vulcano and Lipari, belong to the species H. carbonarius (Fig. 1A). This clade showed the presence of nine haplotypes (Fig. 1C, see Table A1 in supplementary materials for all the haplotype references) with H = ± 0.071, and π = ± (mean ± SD). The specimens from Vulcano (RS276) and Lipari (RS296) shared the same haplotype (H9) with individuals from localities seven and eight (Fig. 1B), corresponding to Iria and Lago Spartà (Sicily). These results may suggest a recent colonization, either human-mediated or by oversea dispersal, from Sicily to the Aeolian islands. On the other hand, the specimen from the island of Favignana (RS85) showed a new private haplotype (H10), separated by one mutational step from haplotype H9. In this case, the single fixed substitution may have occurred on the island through a vicariant mechanism. Indeed, during the last glacial phase this island was connected to Sicily and become separated following the Last Glacial Maximum because of the sea level drop. A similar scenario has also been suggested to explain the genetic differentiation observed in other reptiles from Favignana (Mizan, 2015; Senczuk et al., 2017). However, due to the small sample size from Sicily, we cannot completely rule out that insular distinctiveness may have derived from a recent dispersal process of a haplotype not yet sampled in Sicily. All the individuals from the Pontine Islands (RL22, RL66, RL79, RL80) shared one single haplotype (H1) and should therefore be recognized as belonging to H. viridiflavus (Fig. 1A). This clade is genetically less differentiated than H. carbonarius clade (H = ± 0.067; π = ± ; mean ± SD) and is composed by four haplotypes: a single highly represented haplotype (H1; with an allele frequency of 94%) and three derived and extremely localized haplotypes (H2, H3 and H4). This result may suggest an anthropic introduction in modern times or a recent colonization of the Pontine Islands from the Tyrrhenian coast of the Italian Peninsula. Interestingly, the four specimens from the Pontine Islands showed a colour pattern which resemble the abundistic morph, which is in the middle between the carbonarius (melanic/melanotic) and the viridiflavus (black and yellowish) colour patterns. The abundistic phenotype was previously reported only in Sardinia, Corsica and the Tuscan Archipelago. However, Schätti and Vanni (1986) reported similarities between specimens from the Pontine Islands and the dark coloured ones from Emilia Romagna now considered belonging to H. carbonarius. In particular, the individual sampled from Ventotene (RL79, Fig. 2) had a very dark dorsal with Fig. 2. The specimen from Ventotene island (RL79). The individual was found stuck in a mist net trap and later released. a yellowish ventral colouration, showing a phenotype which could easily be mistaken with the one observed in many populations of H. carbonarius. This observation confirms that colour pattern alone cannot help identifying the species to which a specimen belongs. The four H. viridiflavus specimens from the Pontine Islands are genetically indistinguishable from the usually brown-yellowish western whip snakes located on the Tyrrhenian coast of Italy, but show a darker phenotype. It has been reported in previous works that colour variation in reptiles can be associated to adaptive processes (Norris and Lowe, 1964; Rosenblum et al., 2004). For example, darker or melanotic colouration may give a benefit in terms of thermoregulation (Trullas et al., 2007; Broennimann et al., 2014) and reproduction (Capula and Luiselli, 1994), and similar conclusion had been already drawn by Rato et al. (2009) and Zuffi (2007), as they consider the colour types in Hierophis viridiflavus s.l. a by-product of different environmental conditions. Therefore, the dark colouration of the snakes from the Pontine Islands could be the result of adaptive morphological evolution which occurred in a very short time, a phenomenon already observed in other insular reptile populations (Losos et al., 1997; Herrel et al., 2008). Finally, despite changes in colour polymorphism might also be the outcome of nonadaptive processes (King, 1988; Lorioux et al., 2008), the independent recurrence of the abundistic chromatism in all the northern Tyrrhenian Islands suggests a prominent role of adaptive forces acting in similar insular environmental conditions, which would deserve further studies. ACKNOWLEDGEMENTS Thanks to Dario D Eustacchio, Emanuela De Simone, Marco Basile and Mattia Menchetti for providing samples, to Laura Gramolini for helping in laboratory and