Alain de Chambrier Æ Sandrine C. Coquille Æ Jean Mariaux Æ Vasyl Tkach

Syst Parasitol (2009) 73:49 64 DOI 10.1007/s10-009-9178-6 Redescription of Testudotaenia testudo (Magath, 1924) (Eucestoda: Proteocephalidea), a parasite of Apalone spinifera (Le Sueur) (Reptilia: Trionychidae) and Amia calva L. (Pisces: Amiidae) in North America and erection of the Testudotaeniinae n. subfam. Alain de Chambrier Æ Sandrine C. Coquille Æ Jean Mariaux Æ Vasyl Tkach Received: 29 July 2008 / Accepted: 20 October 2008 Ó Springer Science+Business Media B.V. 2009 Abstract Testudotaenia testudo (Magath, 1924) is redescribed from the intestine of the softshell turtle Apalone spinifera (Le Sueur) (Trionychidae) and the bowfin Amia calva Linnaeus (Amiidae) from Reelfoot Lake, Tennessee, United States. A new subfamily, the Testudotaeniinae, is erected. The new taxon differs from all proteocephalidean subfamilies in the position of the genital organs in relation to the longitudinal internal musculature, i.e. the testes are cortical, rarely medullary; the ovary is partly medullary, with cortical lobes; the vitelline follicles are mainly medullary, with some follicles in the cortex; and the uterus is cortical. A key to the subfamilies of the order Proteocephalidea Mola, 1928 is provided. The most characteristic A. de Chambrier (&) S. C. Coquille J. Mariaux Department of Invertebrates, Natural History Museum Geneva, PO Box 6434, 1211 Geneva 6, Switzerland e-mail: alain.dechambrier@ville-ge.ch S. C. Coquille Faculty of Sciences, Department of Zoology and Animal Biology, Geneva University, 30, quai Ernest-Ansermet, 1211 Geneva 4, Switzerland V. Tkach Department of Biology, University of North Dakota, 1, Campus Drive and Cornell Street, 101 Starcher Hall, Grand Forks, ND 58202-9019, USA V. Tkach Institute of Parasitology, Polish Academy of Sciences, 51/55 Twarda Street, 00-818 Warszawa, Poland features of T. testudo are the precocious uterine aperture, the presence of internal uterine pores (as previously described for Proteocephalus paraguayensis (Rudin, 1917)), the eggs laid unripe, the very long strobila (up to 970 mm), and the presence of an anterior circular musculature in the suckers, which is considered as a good differential character. Three other species were found in Amia calva: Proteocephalus perplexus La Rue, 1911, P. ambloplitis (Leidy, 1887) and a new, undescribed form. Sequences of the partial nuclear 28S rrna gene of specimens of T. testudo from Apalone spinulifera and Amia calva confirm the conspecificity of samples from these two very distinct hosts, which may represent a capture phenomenon. As the subfamily Adenobrechmoinae Bursey, Goldberg & Kraus, 2006 and the genus Adenobrechmos Bursey, Goldberg & Kraus, 2006 are based on the presence of an apical organ, a character which reflects a rather common convergence, we consider the Adenobrechmoinae to be a junior synonym of the Proteocephalinae La Rue, 1911 and Adenobrechmos a junior synonym of Ophiotaenia La Rue, 1911. Adenobrechmos greeri Bursey, Goldberg & Kraus, 2006 thus becomes Ophiotaenia greeri (Bursey, Goldberg & Kraus, 2006) n. comb. Introduction During the course of an investigation into the parasite fauna of fishes and reptiles in the United States,

50 Syst Parasitol (2009) 73:49 64 several species of proteocephalid tapeworms were found in spiny softshell turtles Apalone spinifera (Lesueur) and bowfins Amia calva Linnaeus collected from Reelfoot Lake, Tennessee. One of these tapeworm species, Testudotaenia testudo (Magath, 1924), was found in both hosts. Amia calva also hosted three additional proteocephalid species, namely Proteocephalus perplexus La Rue, 1911, P. ambloplitis (Leidy, 1887), both already known from this host (La Rue, 1911, 1914), and another, probably new, proteocephalidean cestode (hereafter new species 1 ) morphologically similar to members of the polyphyletic genus Proteocephalus (Weinland, 1858) (see de Chambrier et al., 2004). The morphological description of the latter taxon will be provided in a separate paper. T. testudo is redescribed here and a new subfamily is proposed to accommodate it. Materials and methods In June 2002, three eastern spiny softshells Apalone spinifera and four bowfins Amia calva were collected from Reelfoot Lake, Tennessee, N36 29 0 07 00, W89 22 0 44 00. Turtles were collected in baited traps under a permit from the Tennessee Wildlife Resources Agency; the fish were collected by angling. Tapeworms were fixed in 4% hot (almost boiling) formaldehyde solution, with pieces of several worms placed in 99% pure ethanol for DNA analysis. Specimens used for morphological studies were then stained with Mayer s hydrochloric carmine solution, dehydrated in an ethanol series, cleared with eugenol (clove oil) and mounted in Canada balsam. Eggs were studied in whole-mounts. For histology, fragments of strobila and several scoleces were embedded in paraffin wax, cross-sectioned at 12 15 lm, stained with Weigert s haematoxylin and counterstained with 1% eosin B (acidified with five drops of pure acetic acid per 100 ml of solution) following recently updated protocols (Scholz & Hanzelová, 1998; de Chambrier, 2001; de Chambrier et al., 2006). Specimens prepared for scanning electron microscopy (SEM) were dehydrated in a graded ethanol series, transferred to a graded amylacetate series, critical point dried in CO 2, sputtered with gold and examined with a Zeiss DSM 940A electron microscope at the Geneva Natural History Museum (MHNG). Microthrix terminology follows Thompson et al. (1980) and Hoberg et al. (1995). Acronyms of museum collections are as follows: MHNG INVE: Natural History Museum, Geneva, Switzerland, Invertebrate Collection; IPCAS: Institute of Parasitology, Academy of Sciences, České Budějovice, Czech Republic; USNPC: US National Parasite Collection, Beltsville, Maryland, USA. Measurements are given in micrometres unless otherwise stated. For two-dimensional measurements, length is given before width. Abbreviations: Ma, measurements according to Magath (1924); mean ± SD, mean ± standard deviation; N, number of measurements; CV, coefficient of variability (%); CI, consistency index; RC, rescaled consistency index. DNA was extracted from a few ethanol preserved proglottides with the DNeasy Tissue kit (Qiagen) according to the manufacturer instructions. About 1 kb long fragments of the 5 0 portion of the nuclear 28S rrna gene was amplified and sequenced as previously described by Zehnder & Mariaux (1999). New sequences were deposited with EMBL under accession numbers FM956082-90. The sequences of T. testudo from fishes were aligned with known T. testudo sequences from turtles and all available sequences of proteocephalidean cestodes from Nearctic Amia and Ictalurus. Two species of the basal group of the Proteocephalidea (Gangesia and Acanthotaenia), as well as a member of the true Proteocephalus [Proteocephalus aggregate, see de Chambrier et al. (2004a)] were used as outgroups. The matrix comprised 20 taxa: Gangesia parasiluri Yamaguti, 1934 ex Silurus asotus L., Lake Suwa, Nagano Prefecture, Japan, 28.vi.1996, MHNG INVE 22436, AJ388590. Acanthotaenia cf. shipleyi (Linstow, 1903) ex Varanus salvator Laurenti, Klang, Malaysia, 5.x. 2001, MHNG INVE 32837, AJ583453. Proteocephalus percae (Müller, 1780) ex Perca fluviatilis L., Neuchâtel Lake, Switzerland, 12.vi.1996, MHNG INVE 36744, AJ388594. Megathylacoides sp. ex Amia calva L., Reelfoot Lake, Tennessee, USA, 20.vi.2002, MHNG INVE 35373, FM956086. Megathylacoides sp. ex A. calva L., Reelfoot Lake, Tennessee, USA, 20.vi.2002, MHNG INVE 35384, FM956087. Proteocephalus ambloplitis (Leidy, 1887) ex Lepomis macrochirus Rafinesque, Pearl River, Mississippi, USA, AJ388633.

Syst Parasitol (2009) 73:49 64 51 P. ambloplitis (Leidy, 1887) ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 35374, FM956084. P. ambloplitis (Leidy, 1887) ex Ictalurus punctatus Rafinesque, Reelfoot Lake, Tennessee, USA, 20.vi.2002, MHNG INVE 36278, FM956085. P. perplexus La Rue, 1911 ex I. punctatus Rafinesque, Reelfoot Lake, Tennessee, USA, 20.vi.2002, MHNG INVE 36277, sequence identical to MHNG INVE 35366. P. perplexus La Rue, 1911 ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 35366, FM956089. P. perplexus La Rue, 1911 ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 36139, FM956090. P. perplexus La Rue, 1911 ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 35321, sequence identical to MHNG INVE 35366. New species 1 ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 35548, FM956088. Testudotaenia testudo, ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 36706, sequence identical to MHNG INVE 35320. T. testudo, ex A. calva L., Reelfoot Lake, Tennessee, USA, 30.vi.2002, MHNG INVE 36707, sequence identical to MHNG INVE 35320. T. testudo, ex Apalone spinifera L., Reelfoot Lake, Tennessee, USA, 26.vi.2002, MHNG INVE 35316, sequence identical to MHNG INVE 35320. T. testudo, exa. spinifera L., Reelfoot Lake, Tennessee, USA, 26.vi.2002, MHNG INVE 35317, FM956083. T. testudo, ex A. spinifera L., Reelfoot Lake, Tennessee, USA, 1.vii.2002, MHNG INVE 35319, sequence identical to MHNG INVE 35320. T. testudo, ex A. spinifera L., Reelfoot Lake, Tennessee, USA, 1.vii.2002, MHNG INVE 35320, FM956082. The alignment was generated with Sequencher TM v4.1.2 (Gene Codes Corp.) and minor corrections were made by hand; the final alignment was 1,051 bp long. A parsimony analysis was performed using PAUP* v4.0b10 (Swofford, 2002) with the following settings: characters unordered and equally weighted, gaps treated as missing characters. Node support was assessed by bootstrapping (5,000 repeats). Uncorrected distances between sequences (gapped positions removed) were also calculated with PAUP*. Testudotaeniinae n. subfam. Diagnosis Proteocephalidea, Proteocephalidae, with features of family (Rego, 1995). Scolex with four deep suckers. Testes cortical, rarely medullary, arranged in two fields, in one or several layers. Ovary partially medullary, with cortical lobes. Vitelline follicles mainly medullary, with some follicles in cortex. Uterus cortical. Precocious uterine aperture present. Parasitic in turtles (Trionychidae) and freshwater fishes (Actinopterygii). Type- and only genus: Testudotaenia Freze, 1965. Testudotaenia Freze, 1965 Diagnosis Testudotaeniinae, with feature of subfamily. Tapeworms up to one metre long, with segmented strobila. Scolex small, bearing four distinctly separated suckers opening antero-laterally. Testes in two fields. Vagina always anterior to cirrus-sac. Band of vitelline follicles wider at level of ovary. Uterus with precocious longitudinal openings. Eggs laid unripe. Parasitic in testudine reptiles, occasionally in amiiform fishes. Type-species: T. testudo (Magath, 1924). Testudotaenia testudo (Magath, 1924) Freze, 1965 Type-host: Apalone spinifera(le Sueur) (eastern spiny softshell turtle) (Testunides: Trionychidae). Other host: Amia calva Linnaeus (bowfin) (Amiiformes: Amiidae). Prevalence: 100% (3/3) in Apalone spinifera, 50% (2/4) in Amia calva. Site: Intestine. Distribution: Lake City, Minnesota (type-locality), summer 1921; Louisiana (Acholonu, 1970); Reelfoot Lake, Tennessee (this study), United States. Material studied Ex Amia calva 2 specimens (coll. nos USNPC 100650.00 and 100651.00, field number USA 06, 13 slides 3 whole-mounts and 10 serial sections; USA 12, 2 slides, serial sections, same specimen as MHNG INVE 36707); 2 specimens and fragments (coll. nos MHNG INVE 36707, 35369, 54153-7, field number USA 12, 39 slides; USA 11, 1 slide, fragments; USA

52 Syst Parasitol (2009) 73:49 64 6, 69 slides, fragments); fragments of strobila (coll. no. IPCAS C-482, field number USA 06, 7 slides). Ex Apalone spinifera 2 specimens (coll. nos. MHNG INVE 35317 and 35318, field numbers respectively USA 07, 3 whole-mounts, and USA 08, 4 whole-mounts). Redescription (Figs. 1 13) [Based on 5 specimens.] Cestodes flattened dorsoventrally. Strobila large, anapolytic, 470 970 mm long (Ma = 300 500 mm), up to 2.34 mm wide (Ma = 1.6 mm) (n = 2), consisting of 546 721 proglottides: 272 471 immature proglottides (up to appearance of spermatozoa in vas deferens), 70 84 mature proglottides (up to appearance of eggs in uterus), 33 77 pre-gravid proglottides (up to appearance of hooks in oncospheres), and 127 133 gravid proglottides. Strobila covered uniformly with filiform microtriches more than 2 long (Fig. 13). Immature proglottides (170 715 9 990 1,335; length/width ratio 1:0.16 0.54); mature proglottides (485 1,325 9 1930 2200; ratio 1:0.28 0.67); pregravid proglottides (860 1,305 9 1,980 2,200; ratio 1:0.41 0.67); gravid proglottides (1,170 1,930 9 1,960 2,345 (Ma = 2,100 9 1,600); ratio 1:0.52 0.89). Scolex aspinose, with slightly conical apical part, 585 840 (Ma = 420) long, 425 745 (Ma = 630) in diameter (n = 2), wider than neck which is 345 395 (Ma = 240) wide. Scolex formed by 4 deep suckers, each 200 315 (Ma = 120) wide (n = 8, mean = 250) (Figs. 1, 2). Anterior margin of suckers with internal circular musculature (Figs. 2, 3). Concentration of gland-cells at apex of scolex, c.50 in diameter (n = 2) (Fig. 1). Proliferation zone 3,380 4,765 long, 340 390 wide. Internal longitudinal musculature well developed, consisting of isolated bundles of fibres (Figs. 6 8, 10). Osmoregulatory canals almost impossible to observe in mature and gravid proglottides. In immature proglottides, ventral canals much wider than dorsal, situated 17 22% of proglottis width from lateral margins, crossing cirrus-sac in its distal region, longitudinally overlapping marginal testes. Testes spherical to oval, 73 120 9 68 115 (Ma = 50 9 63), numbering 130 178 (mean = 152 ± 18; n = 10; CV = 12%) (Ma = 125 200), in 2 fields, more numerous laterally, not crossing anterior margin of ovary (Figs. 4, 5), forming 1 2 incomplete layers medially (Figs. 5, 6). Testes sometimes reaching laterally to vitelline follicles and to anterior margin of proglottides, producing spermatozoa even in gravid proglottides. Cirrus-sac pyriform (Figs. 4, 5), 585 800 (mean = 685 ± 55; n = 41; CV = 8%) (Ma = 560) long, 200 285 (mean = 240 ± 25; n = 41; CV = 11%) (Ma = 270) wide (length/width ratio 2.9 ± 0.31; n = 41; CV = 11%), representing 26 34% (mean = 31 ± 2%; n = 41; CV = 8%) of proglottis width. Internal sperm duct (vas deferens) thin-walled, forming numerous loops; ejaculatory duct coiled, thick-walled; cirrus occupies more than half of cirrus-sac length (Fig. 5). Genital ducts passing between osmoregulatory canals. Vas deferens strongly coiled, reaching to mid-line of proglottis. Genital atrium present. Genital pore irregularly alternate, markedly pre-equatorial, situated at 8 15% (mean = 11 ± 2; n = 41; CV = 16%) (Ma = \17%) of proglottis length. Vagina always anterior (n = 690) (Ma = always anterior) to cirrus-sac, thin-walled. Vaginal sphincter elongate, near genital atrium, its length representing c.1/4 to 1/5 of cirrus-sac length (Fig. 5). Mehlis glands 150 260 in diameter (n = 30), representing 5 12% of proglottis width (Fig. 5). Ovary bilobed, with follicular lobes, occupying 50 63% (mean = 56 ± 3%; n = 41; CV = 5%) (Ma = c.50%) of proglottis width (Fig. 5). Ovary with isthmus in medulla and lobes in cortex (Fig. 7). Vitelline follicles arranged in 2 lateral bands, wider at level of ovary, with numerous follicles crossing longitudinal internal musculature, penetrating dorsal and ventral cortex, rarely present preporally (14%, n = 297), not overlapping vagina and cirrus-sac, representing porally 56 73% (mean = 66 ± 4%; n = 18; CV = 7%) and aporally 74 90% (mean = 82 ± 5%; n = 018; CV = 6%) of proglottis length, respectively (Figs. 4, 5). Formation of uterus. Uterus cortical, already present in immature proglottides, with development of type 2, according to de Chambrier et al. (2004a), i.e. in immature proglottides, uterine stem has concentration of numerous intensely-staining cells on both sides and occupies up to 22% of proglottis width. Lumen present in uterine stem in last immature proglottides, from which it develops dense lateral elongate digitations, which are terminally ramified or not, or develops into median field of undifferentiated

Syst Parasitol (2009) 73:49 64 53 Figs. 1 3 Testudotaenia testudo (Magath, 1924) from Amia calva Linnaeus. 1. Scolex, ventral view, USNPC 100650. 2. Frontal section of scolex, INVE 36707. 3. Detail of sucker, INVE 36707. Abbreviations: ao, concentration of gland-cells; cg, cells with finely granular cytoplasm; cm, circular musculature. Scale-bars: 1, 500 lm; 2, 3, 100 lm chromophilic cells (Fig. 4). In mature proglottides, lumen gradually appears into each digitiform diverticulum and extends from base to apex. Apex of diverticulum bordered by numerous chromophilic cells. Transverse uterine diverticula grow into cortical parenchyma. In pre-gravid proglottides, eggs completely fill uterine stem and thin-walled diverticula, occupying up to 36% of proglottis width (Fig. 5). Uterus in gravid proglottides with diverticula on each side comprising numerous chromophilic cells (Fig. 9), occupying most of gravid proglottis width (de Chambrier et al., 2004a). In gravid proglottides, diverticula occupy up to 47% of proglottis width, without eggs. Uterus with 17 28 (n = 10) (Ma = 15 20) lateral branches on each side. Uterus opens via single, ventral, longitudinal slit situated along almost whole length of proglottis (Figs. 9, 11, 12). Narrow internal uterine pores present, similar to those described for Proteocephalus paraguayensis (Rudin, 1917) (see Figs. 6c, d in de Chambrier, 1990), emerging from diverticula into uterine stem (Fig. 12). Eggs laid through longitudinal uterine opening before they are ripe, irregular in shape, with spherical embryophore of 23 25 (Ma = 29) in diameter; oncosphere spherical, 14 15 (Ma = 19 21) in diameter,

54 Syst Parasitol (2009) 73:49 64 Figs. 4, 5 Testudotaenia testudo (Magath, 1924) from Amia calva. 4. Immature proglottis, ventral view, INVE 54153. 5. Gravid proglottis, ventral view (eggs not present). Abbreviations: ci, cirrus; cs, cirrus-sac; vs, vaginal sphincter. Scale-bars: 500 lm with 3 pairs of hooks 7.5 9 long (measured in mounted slides). Remarks We were unable to locate Magath s (1924) typematerial of Ophiotaenia testudo (now Testudotaenia testudo), either in the USNPC (Beltsville, MD) or in the Harold W. Manter Collection (Lincoln, NE). According to Achonolu (1970), the Magath s specimens are believed lost. Instead we used the measurements of the original description for comparative purposes. Our material (from both Apalone spinifera and Amia calva) corresponds with the original description, although Magath s material is slightly smaller. Magath (1924, Figs. 7 9) figured a cross-section of O. testudo without pointing out the particular position of the genital organs in relation to the longitudinal internal musculature. Brooks (1978) redescribed Proteocephalus testudo (=T. testudo) from Trachemys scripta elegans. His material differs from that described by Magath (1924) in the absence of an apical organ, in the ratio of

Syst Parasitol (2009) 73:49 64 55 Figs. 6 10 Testudotaenia testudo (Magath, 1924) from Amia calva. 6, 7. Cross-sections of mature proglottides, INVE 36707. 6. At level of middle of proglottis; 7. At level of ovary. 8. Cross-sections of pre-gravid proglottis at level of uterus, INVE 36707. 9. Ventral view of a gravid uterus, INVE 36707 (diagrammatic). Note the shape of the lateral ramified diverticula and the uterine aperture. 10. Dorsal view of internal longitudinal musculature, INVE 54153 (diagrammatic). Abbreviations: du, uterine diverticula; lm, longitudinal internal musculature; ov, ovary; te, testes; ut, uterus; vi, vitelline follicles. Scale-bars: 6 9, 500 lm; 10, 250 lm the cirrus-sac length to the proglottis width (42 59 vs 26 34%), in the anterior and posterior position of the vagina in relation to the cirrus-sac versus always anterior in the present study, and in the diameter of oncospheres (18 27 vs 14 15 lm). It is likely that the material described by Brooks (1978) from Trachemys scripta elegans as Proteocephalus testudo may represent another species. Testudotaenia testudo has been reported from Minnesota and Louisiana by Achonolu (1970) and from Oklahoma by McKnight (1959). Tennessee is a new locality for this species.

56 Syst Parasitol (2009) 73:49 64 Figs. 11 13 Testudotaenia testudo (Magath, 1924) from Amia calva. INVE 36707, SEM photomicrographs of gravid proglottis. 11. Ventral view showing the uterine aperture (arrow). 12. Detail view of uterine aperture. 13. Microtriches at level of the ventral median surface (star in 11). Scale-bars: 11, 500 lm; 12, 100 lm; 13, 2 lm The most characteristic features of T. testudo are the presence of uterine apertures in pre-gravid proglottides, the presence of internal uterine pores, the eggs laid unripe, a large body size and the presence of an anterior circular musculature in the suckers. The precocious uterine aperture has previously been observed in Solenotaenia viperis Beddard, 1913 (syn. of Crepidobothrium viperis) by Beddard (1913). Other members of Crepidobothrium Monticelli, 1899 also possess a precocious uterine aperture (de Chambrier, 1989a, b). This structure seems to be functionally identical in Testudotaenia (see present paper) and Crepidobothrium (see de Chambrier, 1989a), although the two genera are not morphologically similar. In both genera, the eggs are laid unripe and precociously through a large longitudinal aperture, and the uterus is empty in pre-gravid proglottides. There is no clear explanation for this unusual egg dispersion mechanism and it may have evolved independently in Crepidobothrium and Testudotaenia. Although Proteocephalus paraguayensis (Rudin, 1917) exhibits a similar uterine aperture, as well as internal uterine pores (de Chambrier, 1990), its eggs remain in the uterus until the proglottides become gravid and the uterine aperture opens widely. An explanation for this difference could be in the diameter of the internal uterine pores; although the diameter of embryophores is similar in both groups (c.20 25 lm), the diameter of the internal uterine pore reaches only 15 40 lm in P. paraguayensis versus 40 60 lm in T. testudo. A large body size is rare within the Proteocephalidea. Most proteocephalideans are small and attain lengths of up to 30 70 mm (Freze, 1965). Some species, such as P. paraguayensis, Ophiotaenia calmettei (Barrois, 1898), O. phillipsi (Burt, 1937) and O. magna (Hannum, 1925), reach 600 mm (Barrois, 1898; Burt, 1937; de Chambrier, 1990; Hannum, 1925), and only Thaumasioscolex didelphidis Cañeda- Guzmán, de Chambrier & Scholz, 2001 (1,047 mm long) and Megathylacoides procerum Sneed, 1950 (1,113 mm long) are larger than T. testudo (see Cañeda-Guzmán et al., 2001; Freze, 1965). Although circular musculature is present in the suckers of several unrelated lineages of the Proteocephalidea, its appearance and the level of development vary in unrelated taxa. This musculature can be: only slightly developed [as in Brooksiella praeputialis (Rego, Santos & Silva, 1974) and Proteocephalus jarara (Fuhrmann, 1927) (Figs. 14, 15)]; hypertrophied, forming a double crescent [as in Mariauxiella pimelodi de Chambrier & Rego, 1995 (Fig. 16)]; horseshoe-shaped posteriorly [as in Mariauxiella piscatorum de Chambrier & Vaucher, 1999 and Manaosia bracodemoca (Woodland, 1935) de Chambrier, 2003 (Fig. 18)]; or horseshoe-shaped anteriorly [as in Megathylacoides lamothei García- Prieto, 1990) (Fig. 17) and Barsonella lafoni de Chambrier, Scholz, Beletew & Mariaux, 2009] (see de Chambrier et al., 1991, 2004b, 2009; de Chambrier & Rego, 1995; de Chambrier & Vaucher, 1999; de Chambrier, 2003; Scholz et al., 2003). This character is very constant within a species and may be used as a good discriminating feature.

Syst Parasitol (2009) 73:49 64 57 Figs. 14 18 14. Brooksiella praeputialis (Rego, Santos & Silva, 1974) de Chambrier et al., 2004, from Cetopsis coecutiens (Lichtenstein), slightly developed anterior circular musculature of suckers. 15. Proteocephalus jarara (Fuhrmann, 1927) de Chambrier et al., 1991, from Bothrops jararaca (Wied-Neuwied), slightly developed circular musculature. 16. Mariauxiella pimelodi de Chambrier & Rego, 1995, from Pimelodus ornatus Kner, hypertrophied circular musculature forming a double crescent. 17. Megathylacoides lamothei (Garcia- Prieto, 1990), from Ictalurus meridionalis (Günther), hypertrophied circular musculature horseshoe-shaped anteriorly. 18. Mariauxiella piscatorum de Chambrier & Vaucher, 1999, from Hemisorubim platyrhynchos (Valenciennes), hypertrophied circular musculature horseshoe-shaped posteriorly. Abbreviations: cm, circular musculature. Scale-bars: 14, 200 lm; 15 18, 250 lm Differential diagnosis The Testudotaeniinae differs from the 12 recognised proteocephalidean subfamilies (Ephedrocephalinae Mola, 1929; Gangesiinae Mola, 1929; Monticelliinae Mola, 1929; Proteocephalinae La Rue, 1911; Marsypocephalinae Woodland, 1933; Zygobothriinae Woodland, 1933; Endorchiinae Woodland, 1934; Peltidocotylinae, Woodland, 1934; Rudolphiellinae Woodland, 1935; Sandonellinae Khalil, 1960; Acanthotaeniinae Freze, 1963; and Corallobothriinae Freze, 1965) (Mola, 1929; Woodland, 1933a, 1934b, 1935b;

58 Syst Parasitol (2009) 73:49 64 Khalil, 1960; Freze, 1963, 1965) by a distinct arrangement of the genital organs in relation to the internal longitudinal musculature: cortical testes arranged in two fields, in one or several layers; an ovary with cortical lobes and a medullary isthmus; and vitelline follicles which are mainly medullary, with some follicles in the cortex; and a cortical uterus (Beddard, 1913; Schmidt, 1986; Rego, 1994, 1995;Regoetal.,1998;deChambrier & Vaucher, 1997, 1999). The relative position of these organs in various recognised subfamilies is summarised in Fig. 20. Molecular analysis A total of 128 characters were parsimony informative after the removal of gapped positions. A branch and bound parsimony analysis yielded a single shortest tree 223 steps long (CI = 0.68, RC = 0.54) (Fig. 19). All morphologically recognised species formed monophyletic assemblages with the exception of Proteocephalus perplexus La Rue, 1911 (intraspecific distance 0 1.9%), for which the sequence from a Canadian specimen branched separately from those of samples from the USA. The position of the Canadian sample was, however, poorly supported. All Testudotaenia (0 0.2%) from fishes and turtles formed an unambiguously monophyletic group. P. ambloplitis (0 1%) was found to be the sister taxon of P. perplexus and the new Proteocephalinae species (from Amia calva), the sister taxon of Testudotaenia. Megathylacoides Jones, Kerly & Sneed, 1956 (0.1%) was the most basal proteocephalidean from North American fishes included in the analyses. Discussion Although molecular tools have permitted significant progress in our understanding of the proteocephalidean systematics (Mariaux, 1998; Hoberg et al., 1999; Zehnder & Mariaux, 1999; Kodedová et al., 2000; Zehnder, 2000; Zehnder et al., 2000; Zehnder & de Chambrier, 2000; de Chambrier et al., 2004a; Hypša et al., 2005; Scholz et al., 2007), the current classification remains mainly based on the seminal series of Woodland s papers published almost a century ago (Woodland, 1925a, b, c, 1933a, b, 1934a, b, 1935a, b). His subfamily classification is essentially based on the position of the different genital organs in relation to the internal longitudinal musculature (Fig. 20). Based on this criterion, the new subfamily, Testudotaeniinae, is well defined because of a unique combination of key characters, i.e. medullary vitelline follicles associated with cortical testes, uterus and ovary. It must be emphasised that the current systematic structure of the Proteocephalidea remains provisional. We are aware that some of the subfamilies (and genera) will likely prove to be artificial grouping of unrelated taxa, especially when molecular tools are used. However, every attempt to reorganise the order (i.e. Zehnder & Mariaux, 1999; de Chambrier et al., 2004; Hypša et al., 2005) on a sound phylogenetic bases has failed for all but a few limited groups of species. On the other hand, as many as seven subfamilies defined by Woodland s characters (i.e. the Acanthotaeniinae, Corallobothriinae, Ephedrocephalinae, Gangesiinae, Peltidocotylinae, Rudolphiellinae and Sandonellinae) have not been invalidated by recent investigations (Zehnder & Mariaux, 1999; Scholz et al., 2003; Hypša et al., 2005; de Chambrier et al., 2008), and we believe that the cortical/medullary organisation of the reproductive systems will certainly remain an important systematic character. Nevertheless, Proteocephalus currently remains a polyphyletic assemblage (Zehnder & Mariaux, 1999; Scholz et al., 2003; Hypša et al., 2005). We anticipate that several species currently placed in this genus will need to be assigned to different genera and even subfamilies. Our molecular tree shows that new species 1 from Amia calva, which is clearly not a member of the new subfamily Testudotaeniinae, is the sister-group of Testudotaenia. It might, logically, belong to a new subfamily. The exact status of this taxon should be clarified together with its description, but, given the above considerations of the diagnoses of the subfamilies and the relatively weak sister-group relationship between Testudotaenia and the new species, this does not affect the erection of the Testudotaeniinae. The Adenobrechmoinae Bursey, Goldberg & Kraus, 2006 was not included in our comparison. Bursey et al. (2006) described a new genus and species, Adenobrechmos greeri Bursey, Goldberg & Kraus, 2006, a parasite of the scincid lizard Sphenomorphus aignanus in Papua New Guinea. They justified the erection of their new genus (and subfamily Adenobrechmoinae) by the presence of a glandular apical organ. Contrary to the opinion of

Syst Parasitol (2009) 73:49 64 59 Fig. 19 Phylogenetic tree showing the monophyly of Testudotaenia from fish and turtles and its relationship to the other proteocephalideans found in Amia calva. Parsimony analysis, single shortest tree, see text for details. Number on branches are bootstrap values [50% (5000 repeats)

60 Syst Parasitol (2009) 73:49 64 Fig. 20 Survey of subfamilies of the Proteocephalidea and position of their genital organs in relation to the internal longitudinal musculature (modified from Euzet, 1982). Abbreviations: lm, longitudinal internal musculature; ov, ovary; te, testes; ut, uterus; vi, vitelline follicles Bursey et al. (2006), the presence of such organs has already been described in numerous proteocephalidean species belonging to various subfamilies from a large variety of hosts and geographical regions (e.g. Proteocephalus glanduligerus (Janicki, 1928), P. sophiae de Chambrier & Rego, 1994, P. joanae de Chambrier & Paulino, 1997, P. vitellaris (Verma, 1928), P. tigrinus Woodland, 1925 and Ophiotaenia hylae Johnston, 1912) (Proteocephalinae), Nomimoscolex piraeeba Woodland, 1934, Vaucheriella bicheti de Chambrier, 1987, N. suspectus Zehnder, de Chambrier, Vaucher & Mariaux, 2000 (Zygobothriinae) (see Johnston, 1912; Woodland, 1925c; Janicki, 1928; Verma, 1928; Woodland, 1934b; de Chambrier, 1987, 2004; de Chambrier & Rego, 1994; de Chambrier & Paulino, 1997; Scholz et al., 1998; Zehnder et al., 2000). This character reflects a rather common convergence but certainly does not justify the erection of a new supraspecific taxon. Considering the medullary position of testes, ovary, uterus and vitelline follicles, coupled with a scolex with uniloculate suckers and testes in one field, this taxon could be placed in Proteocephalus Weinland, 1858. However, almost all proteocephalidean cestodes found in reptiles and amphibians are members of Ophiotaenia La Rue, 1911 (see Freze, 1965; de Chambrier et al., 2006; Ammann & de Chambrier, 2008; Coquille & de Chambrier, 2008), which differs from Proteocephalus essentially in the presence of two testicular fields and a preformed uterus (Freze, 1965). Both Proteocephalus and Ophiotaenia have been demonstrated to be polyphyletic in previous phylogenetic analyses (Zehnder & Mariaux, 1999; de Chambrier et al., 2004a; Hypša et al., 2005). Pending a complete revision of the group, we choose to provisionally place the concerned taxon in Ophiotaenia. Therefore, we consider the Adenobrechmoinae Bursey, Goldberg & Kraus, 2006 to be a junior synonym of the Proteocephalinae and Adenobrechmos Bursey, Goldberg & Kraus, 2006 a junior synonym of Ophiotaenia. Consequently, Adenobrechmos greeri Bursey, Goldberg & Kraus,

Syst Parasitol (2009) 73:49 64 61 2006 becomes Ophiotaenia greeri (Bursey, Goldberg & Kraus, 2006) n. comb. The finding of T. testudo in an evolutionary ancient fish, Amia calva, is rather surprising. There is no other example of a proteocephalidean species known to parasitise such different groups of hosts. Proteocephalidean species usually exhibit an oioxenic specificity, at least in the Neotropical region (de Chambrier & Vaucher, 1999), and rarely a stenoxenic specificity. As stenoxeny is uncommon in the New World proteocephalideans, it is interesting to note the other proteocephalideans of fishes from North America are often stenoxenic: both Proteocephalus ambloplitis and P. perplexus are found in Amia, Lepomis and Ictalurus. The situation is different for Palaearctic species of Proteocephalus, such as P. longicollis (Zeder, 1800), P. percae (Müller, 1780) and P. torulosus (Batsch, 1786), which may parasitise fish hosts belonging to different fish families or even orders (Scholz & Hanzelová, 1998). We assume that the presence of T. testudo in Amia calva represents an isolated local capture phenomenon, as both hosts were collected in the same place and habitat. They both are opportunist feeders and have a similar diet consisting of fishes, frogs, crayfishes, insects and shrimp. Key to subfamilies of the Proteocephalidea (modified after Khalil, 1960) 1 Genital organs entirely cortical... Monticelliinae Genital organs at least partly medullary... 2 2 Genital organs entirely medullary... 3 Genital organs partly medullary and partly cortical... 7 3 Vitelline follicles in two lateral compact bands... 4 Vitelline follicles in two compact bands posterior to ovary... Sandonellinae 4 Rostellum present... 5 Rostellum absent... 6 5 Rostellum without hooks; parasites of reptiles... Acanthotaeniinae Rostellum usually with hooks; parasites of siluriform fishes... Gangesiinae 6 Metascolex present... Corallobothriinae Metascolex absent... Proteocephalinae 7 Testes medullary... 8 Testes cortical... 9 8 Uterus medullary... Zygobothriinae Uterus cortical, with branches in medulla... Endorchiinae 9 Vitelline follicles medullary... 12 Vitelline follicles cortical... 10 10 Uterus cortical... Peltidocotylinae Uterus medullary... 11 11 Vitelline follicles in one entire dorsal field... Ephedrocephalinae Vitelline follicles in two lateral dorsal rows... Rudolphiellinae 12 Uterus cortical... Testudotaeniinae Uterus medullary... Marsypocephalinae Acknowledgements We are grateful to André Puiz for providing SEM photomicrographs, Janik Pralong and Florence Marteau for technical assistance, and to Gilles Roth for his help with drawings (all from Geneva). We thank Tomáš Scholz (České Budějovice) for fruitful suggestions and helpful comments. We also thank Susan Lim (University of Malaya, Kuala Lumpur) and Takeshi Shimazu (Nagano Prefectural College, Japan) for providing samples. Scott Snyder (University of Nebraska at Omaha) and Stephen Curran (Gulf Coast Research Laboratory, University of Southern Mississippi) kindly helped with the field collecting of turtles and fish. This project was supported in part by the National Science Foundation PBI award Nos 0818696 and 0818823. References Achonolu, A. D. (1970). On Proteocephalus testudo (Magath, 1924) (Cestoda: Proteocephalidae) from Trionyx spinifer (Chelonia) in Louisiana. Journal of Wildlife Diseases, 6, 171 172. Ammann, M., & de Chambrier, A. (2008). Ophiotaenia gilberti sp. n. (Cestoda: Proteocephalidea), a parasite of Thamnodynastes pallidus (Serpentes: Colubridae) from Paraguay. Revue Suisse de Zoologie, 115, 541 551. Barrois, T. (1898). Sur quelques Ichthyotaenia parasites de serpents. Bulletin de la Société des Sciences de l Agriculture et des Arts de Lille, 2, 6 9. Beddard, F. E. (1913). Contributions to the anatomy and systematic arrangement of the Cestoidea. IX. On a new genus of ichthyotaeniids. Proceedings of the Zoological Society of London, (1913), 243 261. Brooks, D. R. (1978). Systematic status of proteocephalid cestodes from reptiles and amphibians in North America with descriptions of three new species. Proceedings of the Helminthological Society of Washington, 45, 1 28. Bursey, C. R., Goldberg, S. R., & Kraus, F. (2006). New genus, new species of Cestoda (Proteocephalidae) from the lizard Sphenomorphus aignanus (Squamata: Scincidae) from

62 Syst Parasitol (2009) 73:49 64 Papua New Guinea. Comparative Parasitology, 73, 184 187. Burt, D. R. R. (1937). Two new reptilian cestodes of the genus Proteocephalus (Ophiotaenia). Ceylon Journal of Sciences, 20, 157 179. Cañeda-Guzmán, I., de Chambrier, A., & Scholz, T. (2001). Thaumasioscolex didelphidis n. gen. and n. sp. (Eucestoda: Proteocephalidae) from the black-eared opossum Didelphis marsupialis from Mexico, the first proteocephalidean tapeworm from a mammal. Journal of Parasitology, 87, 639 647. Coquille, S. C., & de Chambrier, A. (2008). Cairaella henrii gen. n. sp. n., a parasite of Norops trachyderma (Polychrotidae), and Ophiotaenia nicoleae sp. n. (Eucestoda: Proteocephalidea), a parasite of Thecadactylus rapicauda (Gekkonidae) in Ecuador. Folia Parasitologica, 55, 197 206. de Chambrier, A. (1987). Vaucheriella bicheti n. gen., n. sp. (Cestoda: Monticellidae, Zygobothriinae) parasite de Tropidophis cf. taczanowskyi (Steindachner, 1880) (Serpentes: Tropidophidae) des Andes Équatoriennes. Revue Suisse de Zoologie, 94, 829 840. de Chambrier, A. (1989a). Révision du genre Crepidobothrium Monticelli, 1900 (Cestoda: Proteocephalidae) parasite d Ophidiens néotropicaux. I. C. gerrardii (Baird, 1860 et C. viperis Beddard 1913. Revue Suisse de Zoologie, 96, 191 217. de Chambrier, A. (1989b). Révision du genre Crepidobothrium Monticelli, 1900 (Cestoda: Proteocephalidae) parasite d Ophidiens néotropicaux. II. C. dollfusi Freze, 1965, C. lachesidis (MacCallum, 1921) et conclusions. Revue Suisse de Zoologie, 96, 345 380. de Chambrier, A. (1990). Redescription de Proteocephalus paraguayensis (Rudin, 1917) (Cestoda: Proteocephalidae) parasite de Hydrodynastes gigas (Dum., Bibr. & Dum., 1854) du Paraguay. Systematic Parasitology, 16, 85 97. de Chambrier, A. (2001). A new tapeworm from the Amazon, Amazotaenia yvettae n. gen., n. sp. (Eucestoda: Proteocephalidea) from the siluriform fishes Brachyplatystoma filamentosum and B. vaillanti (Pimelodidae). Revue Suisse de Zoologie, 108, 303 316. de Chambrier, A. (2003). Systematic status of Manaosia bracodemoca Woodland, 1935 and Paramonticellia itaipuensis Pavanelli et Rego, 1991 (Eucestoda: Proteocephalidea), parasites of Sorubim lima (Siluriformes: Pimelodidae) from South America. Folia Parasitologica, 50, 121 127. de Chambrier, A. (2004). Redescription of Ophiotaenia hylae Johnston, 1912 (Eucestoda: Proteocephalidea), parasite of Litoria aurea (Amphibia: Hylidae) from Australia. Revue Suisse de Zoologie, 111, 371 380. de Chambrier, A., Coquille, S. C., & Brooks, D. R. (2006). Ophiotaenia bonneti sp. n. (Eucestoda: Proteocephalidea), a parasite of Rana vaillanti (Anura: Ranidae) in Costa Rica. Folia Parasitologica, 53, 125 133. de Chambrier, A., D Alessio, M. L., & de Azevedo Corrêa, F. M. (1991). Redescription de Proteocephalus jarara (Fuhrmann, 1927) (Cestoda: Proteocephalidae) parasite de Bothrops alternatus (Viperidae) au Brésil. Revue Suisse de Zoologie, 98, 15 32. de Chambrier, A., & Paulino, R. C. (1997). Proteocephalus joanae sp. n. (Eucestoda: Proteocephalidea), a parasite of Xenodon neuwiedi (Serpentes: Colubridae) from South America. Folia Parasitologica, 44, 289 296. de Chambrier, A., & Rego, A. A. (1994). Proteocephalus sophiae n. sp. (Cestoda: Proteocephalidae), a parasite of the siluroid fish Paulicea luetkeni (Pisces: Pimelodidae) from the Brazilian Amazon. Revue Suisse de Zoologie, 101, 361 368. de Chambrier, A., & Rego, A. A. (1995). Mariauxiella pimelodi n. g., n. sp. (Cestoda: Monticelliidae): A parasite of pimelodid siluroid fishes from South America. Systematic Parasitology, 30, 57 65. de Chambrier, A., Rego, A. A., & Mariaux, J. (2004a). Redescription of Brooksiella praeputialis and Goezeella siluri (Eucestoda: Proteocephalidea), parasites of Cetopsis coecutiens (Siluriformes) from the Amazon and proposition of Goezeella danbrooksi sp. n. Revue Suisse de Zoologie, 111, 111 120. de Chambrier, A., Scholz, T., Beletew, M., & Mariaux, J. (2009). Barsonella lafoni n. gen and n. sp. (Cestoda: Proteocephalidea) from Clarias catfish in Africa. Journal of Parasitology, 95, 160 168. de Chambrier, A., Sène, A., Mahmoud, Z. N., Mariaux, J., & Scholz, T. (2008). Sandonella sandoni (Lynsdale, 1960), an enigmatic and morphologically unique cestode parasitic in the osteoglossiform fish Heterotis niloticus in Africa. Journal of Parasitology, 94, 202 211. de Chambrier, A., & Vaucher, C. (1997). Révision des cestodes (Monticelliidae) décrits par Woodland (1934) chez Brachyplatystoma filamentosum avec redéfinition des genres Endorchis Woodland, 1934 et Nomimoscolex Woodland, 1934. Systematic Parasitology, 37, 219 233. de Chambrier, A., & Vaucher, C. (1999). Proteocephalidae et Monticelliidae (Eucestoda: Proteocephalidea) parasites de poissons d eau douce au Paraguay, avec descriptions d un genre nouveau et de dix espèces nouvelles. Revue Suisse de Zoologie, 106, 165 240. de Chambrier, A., Zehnder, M. P., Vaucher, C., & Mariaux, J. (2004b). The evolution of the Proteocephalidea (Platyhelminthes, Eucestoda) based on an enlarged molecular phylogeny, with comments on their uterine development. Systematic Parasitology, 57, 159 171. Euzet, L. (1982). Problèmes posés par la spécificité parasitaire des cestodes Proteocephalidea et Pseudophyllidea parasites de poissons. Mémoires du Muséum National d Histoire Naturelle, Paris (Zoologie),, 279 287. Freze, V. I. (1963). [Convergent formation of the rostellum in two branches of Proteocephalata; revision of subfamilies Gangesiinae Mola, 1929 and Acanthotaeniinae Freze, 1963 (Cestoda, Proteocephalata)]. Gel minty cheloveka, zhivotnykh i sel skokhozyaistvennykh rastenii (k 85-letiyu Akademika K.I. Skrjabina). Moscow: Izdatel tsvo Nauka, pp. 150 155 (in Russian). Freze, V. I. (1965). [Essentials of cestodology]. Vol. 5. [Proteocephalata in fish, amphibians and reptiles.]. Moskva: Izdatel stvo Nauka, 538 pp (in Russian; English translation, Israel Program of Scientific Translation, 1969, Jerusalem, v? 597 pp.). Hannum, C. A. (1925). A new species of cestode, Ophiotaenia magna n. sp. from the frog. Transactions of the American Microscopical Society, 64, 148 152.

Syst Parasitol (2009) 73:49 64 63 Hoberg, E. C., Brooks, D. R., & Mariaux, J. (1999). Phylogeny of the orders of the Eucestoda: Morphological and molecular evidence. Systematic Parasitology, 42, 12 37. Hypša, V., Skeríkova, A., & Scholz, T. (2005). Phylogeny, evolution and host-parasite relationships of the order Proteocephalidea (Eucestoda) as revealed by combined analysis and secondary structure characters. Parasitology, 130, 359 371. Janicki, C. (1928). Cestodes s. str. aus Fischen and Amphibiens. In L. A. Jägerskiöld (Ed.), Results of the Swedish zoological expedition to Egypt and White Nile, 1901 (Part 5, pp. 1 58). Uppsala. Johnston, T. H. (1912). Notes on some Entozoa. Proceedings of the Royal Society of Queensland, 24, 63 91. Khalil, L. F. (1960). On a new genus Sandonella, for Proteocephalus sandoni Lynsdale, 1960, (Proteocephalidae) and the erection of a new subfamily, Sandonellinae. Journal of Helminthology, 34, 47 54. Kodedová, I., Dolezel, D., Broucková, M., Jirku, M., Hypša, V., Lukes, J., et al. (2000). On the phylogenetic positions of the Caryophyllidea, Pseudophyllidea and Proteocephalidea (Eucestoda) inferred from 18S rrna. International Journal for Parasitology, 30, 1109 1113. La Rue, G. R. (1911). A revision of the cestode family Proteocephalidae. Zoologischer Anzeiger, 38, 473 482. La Rue, G. R. (1914). A revision of the cestode family Proteocephalidae. Illinois Biological Monographs, 1(1 2), 3 351. Magath, T. B. (1924). Ophiotaenia testudo, a new species from Amyda spinifera. Journal of Parasitology, 11, 44 49. Mariaux, J. (1998). A molecular phylogeny of the Eucestoda. Journal of Parasitology, 85, 114 124. McKnight, T. J. (1959). A taxonomic study of the helminth parasites of the turtles of Lake Texoma. PhD Dissertation, University of Oklahoma, 47 pp. Mola, P. (1929). Descriptio platodorum sine exstis. Zoologischer Anzeiger, 86, 101 113. Rego, A. A. (1994). The order Proteocephalidea. In L. F. Khalil, A. Jones, & R. A. Bray (Eds.), Keys to the cestode parasites of vertebrates (pp. 257 293). Wallingford, UK: CAB International. Rego, A. A. (1995). A new classification of the cestode order Proteocephalidea Mola. Revista Brasileira de Zoologia, 12, 791 814. Rego, A. A., de Chambrier, A., Hanzelová, V., Hoberg, E., Scholz, T., Weekes, P., et al. (1998). Preliminary phylogenetic analysis of subfamilies of the Proteocephalidea (Cestoda). Systematic Parasitology, 40, 1 19. Schmidt, G. D. (1986). Handbook of tapeworm identification. Boca Raton, FL: CRC Press, 675 pp. Scholz, T., Drábek, R., & Hanzelová, V. (1998). Scolex morphology of Proteocephalus tapeworms (Cestoda: Proteocephalidae), parasites of freshwater fish in the Palaearctic Region. Folia Parasitologica, 45, 27 43. Scholz, T., & Hanzelová, V. (1998). Tapeworms of the genus Proteocephalus Weinland, 1858 Cestoda: Proteocephalidae, parasites of fishes in Europe. Studie AV CR, (1998(2)) (p. 119 pp). Prague: Academia. Scholz, T., Hanzelová, V., Skeríkova, A., Shimazu, T., Rolbiecki, L., et al. (2007). An annotated list of species of the Proteocephalus Weinland, 1856 aggregate sensu de Chambrier et al. (2004) (Cestoda: Proteocephalidea), parasites of fishes in the Palaearctic region, the phylogenetic relationships and key to identification. Systematic Parasitology, 67, 139 156. Scholz, T., Rosas, R. R., Pérez-Ponce de León, G., Choudhury, A., & de Chambrier, A. (2003). Taxonomic status of Choanoscolex lamothei Garcia-Prieto, 1990 (Cestoda: Proteocephalidae) using morphological and molecular evidences. Journal of Parasitology, 89, 1212 1219. Swofford, D. L. (2002). PAUP 4.0: Phylogenetic analysis using parsimony (and other methods). Version 4.0 Beta. Sunderland, MA: Sinauer Associates. Thompson, R. C. A., Hayton, A. R., & Jue Sue, L. P. (1980). An ultrastructural study of the microtriches of adult Proteocephalus tidswelli (Cestoda: Proteocephalidea). Zeitschrift für Parasitenkunde, 64, 95 111. Verma, S. C. (1928). Some cestodes from Indian fishes, including four new species of Tetraphyllidea and revised keys to the genera Acanthobothrium and Gangesia. Allahabad University Studies, 4, 119 176. Woodland, W. N. F. (1925a). On some remarkable new Monticellia-like and other cestodes from Sudanese siluroids. Quarterly Journal of Microscopical Science, 69, 703 729. Woodland, W. N. F. (1925b). On Proteocephalus marenzelleri, P. naiae and P. viperis. Annals of Tropical Medicine and Parasitology, 19, 265 279. Woodland, W. N. F. (1925c). On three new proteocephalids (Cestoda) and a revision of the genera of the family. Parasitology, 17, 370 394. Woodland, W. N. F. (1933a). On a new subfamily of proteocephalid cestodes the Othinoscolecinae from the Amazon siluroid fish Platystomatichthys sturio (Kner). Parasitology, 25, 491 500. Woodland, W. N. F. (1933b). On the anatomy of some fish cestodes described by Diesing from the Amazon. Quarterly Journal of Microscopical Science, 76, 175 208. Woodland, W. N. F. (1934a). On the Amphilaphorchidinae, a new subfamily of proteocephalid cestodes, and Myzophorus admonticellia, gen. and sp. n., parasitic in Pirinampus spp. from the Amazon. Parasitology, 26, 141 149. Woodland, W. N. F. (1934b). On some remarkable new cestodes from the Amazon siluroid fish, Brachyplatystoma filamentosum (Lichtenstein). Parasitology, 26, 268 277. Woodland, W. N. F. (1935a). Additional cestodes from the Amazon siluroids pirarara, dorad, and sudobim. Proceedings of the Zoological Society London, 104, 851 862. Woodland, W. N. F. (1935b). Some more remarkable cestodes from Amazon siluroids fish. Parasitology, 27, 207 225. Zehnder, M. (2000). Molecular systematic analysis of the Proteocephalidea Mola, 1928 (Eucestoda) based on mitochondrial and nuclear ribosomal DNA sequences. PhD Thesis, Faculté des Sciences, Université de Neuchâtel, 139 pp. Zehnder, M., & de Chambrier, A. (2000). Morphological and molecular analyses of the genera Peltidocotyle Diesing, 1850 and Othinoscolex Woodland, 1933 and a morphological study of Woodlandiella Freze, 1965 (Proteocephalidea, Eucestoda), parasites of South American siluriform fishes (Pimelodidae). Systematic Parasitology, 46, 33 43.

64 Syst Parasitol (2009) 73:49 64 Zehnder, M., de Chambrier, A., Vaucher, C., & Mariaux, J. (2000). Nomimoscolex suspectus n. sp. (Eucestoda, Proteocephalidea) with morphological and molecular phylogenetic analyses of the genus. Systematic Parasitology, 47, 153 172. Zehnder, M., & Mariaux, J. (1999). Molecular systematic analysis of the order Proteocephalidea (Eucestoda) based on mitochondrial and nuclear rdna sequences. International Journal for Parasitology, 29, 1841 1852.