A preliminary study on the Mediterranean spur-thighed tortoise Testudo graeca Linnaeus, 1758 from northwestern Iran

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1 Herpetology Notes, volume 7: (2014) (published online on 11 April 2014) A preliminary study on the Mediterranean spur-thighed tortoise Testudo graeca Linnaeus, 1758 from northwestern Iran Elham Rezazadeh 1, Ramin Mohammadi Alucheh 1* and Haji Gholi Kami 2 Abstract. There are two species of Testudinidae, Testudo graeca Linnaeus 1758 and Testudo horsfieldi Gray 1844 in Iran. The ecological and biological status of Testudo graeca population in northwest of Iran is poorly known. Therefore in this study we provide morphological, coloration and distribution data on32 specimens (20 males, 12 females) from northwest of Iran. Additionally, new data on feeding, habitat features and threats of the species were studied. In this area some reproductive aspects including reproduction time, egg size and number were reported for the first time. Key words. Testudo graeca, Morphological characteristics, Biological status, Ardabil, Iran. Introduction Today, testudonine tortoises have a wide geographic distribution that includes the Palearctic, Oriental and Ethiopian biogeographic realms (Parham et al., 2006a). A large portion of this area is inhabited by the Mediterranean spur-thighed tortoise Testudo graeca Linnaeus These tortoises occur under very different climatic and environmental conditions, ranging from a Mediterranean climate with mild, frostfree winters to an extreme continental steppe climate with severe winter frost (Fritz et al., 2007). There are 11 known species of turtle and tortoise including six families and nine genera in Iran. Two species of Testudinidae, Testudo graeca and Testudo horsfieldi Gray 1844 are known from Iran (Rastegar- Pouyani et al., 2008). Anderson (1979) has described two subspecies of T. graeca on the Iranian Plateau: T. g. ibera and T. g. zarudnyi, whereas more recent molecular researches (Fritz et al., 2007; Parham et al., 2012) have introduced three subspecies of T.graeca that exist in Iran: T. g. perses, T. g. buxtoni, and T. g. zarudnyi. Several investigations have already been conducted on molecular phylogeny (Parham et al., 1 Young Researchers and elites Club, Ardabil Branch, Islamic Azad University, P. O. Box 196, Ardabil, Iran 2 Department of Biology, Faculty of Sciences, Golestan University, P.O. Box 49165, Gorgan, Iran *Corresponding author; ramin.p1363@gmail.com 2006a, Fritz et al., 2007; Parham et al., 2012; Mikulíček et al., 2013; Mashkaryan et al., 2013), population dynamics (Bayley and Highfield, 1996; El Mouden et al., 2001; Slimani et al., 2001), general ecology (Lambert, 1969, 1981, 1983), geographic variations (Highfield, 1990a, b), feeding ecology (El Mouden et al., 2006), morphological comparison (Turkozan and Olgun, 2005) and reproduction behavior (Sadeghi and Torki, 2012). However, T. graeca in Iran has received little scientific attention and its biology, ecology and habitat affinities are poorly known. Since this species is listed on the IUCN Red List as a Vulnerable species, such information is essential for planning conservation measures. The present study aims to present new data on morphological characteristics, habitat features, distribution, feeding, ectoparasite infection, reproduction and conservation status of T. graeca populations in the Ardabil province, NW Iran. Material and Methods Study area The study area was located in the northwestern part of Iran, Ardabil Province (38 15 E, to E, N to N). The region is surrounded by the Alborz Mountains and the Caspian Sea to the east, Aras River to the north, Arasbaran protected area and Gare-Dagh Mountain to the west and the Talesh Mountains to the south (Fig. 1A). Altitude ranges between 20 m in the Moghan steppe to 4811 m above sea level in the Sabalan Mountain. The prominent vegetation is alpine and semi alpine steppe.

2 128 Elham Rezazadeh et al. Figure 1. A: Map of the study area. B: Sampling stations in Ardabil province. Field studies and analysis In order to determine the occurrence of T. graeca, we visited different stations in Ardabil Province of northwestern Iran between 2009 and Distribution localities are shown in Fig. 1B. Habitat characteristics including vegetation and sympatric reptiles were also taken into consideration. Morphometric characteristics of 32 captured specimens (20 male, 12 female) were measured with calipers (to 0.02 mm). Captured animals were considered as juveniles when midline carapace length was less than 100 mm (Slimani et al., 2001); based on that, juveniles were not measured. Sexes were determined and the color pattern of specimens was recorded before releasing them. The tortoises were thoroughly checked for ectoparasites and the number of attached ticks was recorded. Nurtured plants by the species, in direct observation, were collected for identification; other plants in the habitat were also sampled for recognizing the habitat plant coverage. Reproduction behavior, nesting and egg laying were observed too. Eggs were measured (to 0.01 mm) and weighed (to 0.01 gr) before incubation. The morphometric characteristics that were measured are: Straight Carapace Length (SCL), Straight-line measurement from the outermost projection of the nuchal plate to the posterior end of the supracaudalia; Straight Carapace width (SCW), Greatest width in a straight line directly across turtle.; Carapace Height (CH), the vertical measurement between the highest point of carapace and the lowest point of plastron; Plastron Length (PL), the straight-line measurement from the outermost projection of the gular to the posterior end of the anal scute. For distinguish similarities and differences between males and females, some statistical analyses on morphometric variables were carried out. Data were examined for conformation to assumption of normality (the Shapiro-Wilk test) and homogeneity (Leven s test for equality of variance). The status of the sexual dimorphism was tested with an Independent t-test. Non normal data was analyzed by Mann-Withney U test. Regarding all statistical tests, significance level was set at p<0.05. Statistical analyses were carried out using the program SPSS 16.0.

3 A preliminary study on the Mediterranean spur-thighed tortoise Testudo graeca 129 Table 1. Descriptive statistics of morphometric characters obtained from Testudo graeca specimens collected from northwestern Iran (Ardabil province). For abbreviations, see text (N: number of specimens; min: minimum value; Table1. Descriptive statistics of morphometric characters obtained from Testudo graeca specimens max: collected maximum from value; northwestern S.E: standard Iran (Ardabil error province). of the mean; For abbreviations, S. see text (N: D: number standard of specimens; deviation) min: minimum value; max: maximum value; SE: standard error of the mean; SD: standard deviation) Characters Overall Characters N Min Overall Max Mean S.E S. D N Min Max Mean S.E S. D SCL SCW SCL CH SCW CH PLmax PLmax PLmin PLmin Table 2. Results of Independent t-test comparing males Table 2. Results of Independent t-test comparing males and females in terms of morphometric and females characteristics. in terms of morphometric characteristics. Reproduction length of males and femles was mm and 300 mm, respectively. Sexual dimorphism was found in SCL (t = 2.234, df = 30, p = 033), SCW (t = 2.497, df = 30, p = 018), CH (t = 2.67, df = 30, p = 012), PLmax (t = 1.99, df = 30, p = 0.049), and PLmin (t = 2.109, df = 30, p = 0.043) measured characters (Table 2). In all metric measurements males have larger body size than females; SCL in males (222.5 mm) has higher mean value than that in females (171.2 mm). Mean value of SCW in males (155.9 mm) was higher than that in females (124.6 mm). Also males (mean = 93 mm) have higher carapace than females (mean = 70.6 mm). In addition, males PLmax and PLmin (mean = mm, 179 mm respectively) have higher mean value than those in females (mean = mm, mm respectively). Characters t df Sig. Characters t df Sig. In the study area, courtship occurred in mid-april. The SCL largest number of matings took place in shady places SCW SCL SCW between 2 pm and 4 pm. In one case, mating lasted CH CH twelve minutes at 16:30 pm (Fig. 2). Approximately PLmax PLmax 1.99 PLmin one month later, females started to lay eggs. Egg laying PLmin in one of the females was observed on 26 May and nine mature eggs were numbered. This process started at 16:20 pm and lasted minutes. At first, the animal Table 3. Table Egg measurements 3. Egg measurements for female Testudo for female graeca Testudo from northwestern graeca Iran dug (Ardabil up soil with her claws to a depth of cm. Province). from northwestern Iran (Ardabil Province). Entrance of burrow was narrow and deep seated part was broad. After egg laying the nest was filled up to Characters N Min Max Mean S.E S. D 2-3 cm higher than the soil surface. While finishing this Egg length(mm) process, shakes were observed in fore- and hindlimbs Egg width(mm) of the animal, after minutes it began feeding. Collected Egg weight(gr) data from the eggs of this specimen are provided in Table 3. The length of the largest egg was 39 mm, 33 mm width and gr weights. Results Morphology The ground color of the carapace is uniformly dark olive with some pale yellow-brown spots, especially on the lateral margins. Head is black with small brown spots, limbs and forelimbs are brown with black scales. Descriptive statistics of morphometric measurements of all specimens of T. graeca and separately by gender are provided in Table 1. SCL ranges between mm (mean = mm) were found for mature male specimens, whereas the mean value of this character in females was mm (range: mm). Among our specimens, maximum straight carapace Feeding, habitat affinities and threats This species is completely herbivorous and in study area it was observed while feeding on Astragalus sp., and Agrpyron sp. The habitats of this species are stony foothills, hillsides covered with meadow, and foothill vegetation is alpine steppe with plant species such as Stachys sp., Eremostachys sp.,nepeta sp., Tris sp., Verbascum sp., Alyssum sp., Sencio sp., Agrpyron sp. and some other species of Astragalus. Additionally, T. graeca also occurs in plains and farmlands. In its habitat, T. graeca was sympatric with some reptiles, Ophisops elegans, Laudakia caucasia and Ophisaurus apodus.

4 130 Elham Rezazadeh et al. Figure 2. Testudo graeca while mating. Development of modern farming in the study area in one hand and live stock overgrazing on the other hand, have caused to destruct the habitat of species. However, there are some additional threat factors in the study area; one of the main predators of this species is Buteo buteo (common buzzard). As we observed, this bird collected an adult tortoise and dropped it from 25 meters elevation, due to hitting a stone its carapace was broken (Fig. 3A). Also in some study sites skeletons of the species were observed (Fig. 3B). Likewise, ectoparasites pose another threat; multiple taxa of ticks infect the species. Most of these parasites attach to soft parts of animal body where hands and limbs join the body and under marginals as well. In our specimens prevalence of infection in males was 100% and in females was 66% (overall = 88% of specimens). Also, average numbers of ticks in males and females was six and two parasites per specimen, respectively. Hence, males are more vulnerable than females in this case. Discussion Some researchers have asserted that Middle Eastern T. graeca should be split into more than nine species, but Parham et al. (2006b) by genetic evidence have considered that this species in the Middle East is a single species, pending further study. However, according to this molecular study and recent research (Parham et al., 2012), the subspecies occurring in study area is T. g. buxtoni. In the measured characters there were significant differences between the two sexes. Males have a larger and wider carapace than females. Also, they have a higher carapace and larger plastron than females. The results of straight carapace measurements revealed that

5 A preliminary study on the Mediterranean spur-thighed tortoise Testudo graeca 131 Figure 3. A: Testudo graeca hit by a common buzzard in Ardabil. B: Skeleton of T. graeca in study area. males (mean = mm) are significantly larger than females (mean = mm). Disregarding subspecies, there are clear differences between Iranian and Turkish populations. Turkozan, et al. (2003) studied a population of this species from Mardin, southeastern Turkey. Males in this study were larger than females. Males in the Iranian population (mean = mm) are larger than the Turkish ones (mean = mm), but females of the Turkish population (mean = mm) are larger than Iranian ones (mean = mm). The maximum SCL recorded from the Mediterranean region is 295 mm, Aegean region is 242 mm and Mardin population is 239 mm respectively (Turkozan, et al., 2005), while the maximum SCL of the Iranian population in this study is mm. Anderson (1979) has indicated that most Iranian specimen s SCL are under 300 mm. According to this, and to author s knowledge, the represented SCL measurement is a new size record for Iran. It shows that the Iranian population has a larger body size in comparison with a Turkish population. Comparison between two populations, south-east Anatolia and northwest Iran, shows significant morphometric differences (Table 4). This difference may be due to geographical barriers and mountain chains like Ararat and Sabalan Table 4. Morphometric (Turkozan comparison et al., 2005). for males and females between two population of Testudo graeca from Northwestern Iran (this study) and Southeastern Anatolia (Turkozan et al., 2005). Males Northwest Iran South-east Anatolia Characters N Min Max Mean S.E S. D N Min Max Mean S.E S. D SCL SCW CH PL Females Northwest Iran South-east Anatolia Characters N Min Max Mean S.E S. D N Min Max Mean S.E S. D SCL SCW CH PL

6 132 Elham Rezazadeh et al. Table 5. Egg Size and weight comparison of Testudo graeca between two populations of Testudo graeca ibera from Northern Greece and Western Iran (Zagros), with one population of Testudo graeca buxtoni from Northwestern Iran (Transcaucasia). Hiley and Loumbourdis, 1988 Sadeghi and Torki, 2012 Present study Year assessment Location Northern Greece Western Iran (Zagros) Northwestern Iran (Transcaucasia) Egg length (mm) 35.4± ± ± 1.4 Egg width (mm) 29.2± ± ± 0.6 Egg weight (gr) 17.5± ± ± 1.1 crests with 5000 m and 4811 m above sea level respectively. These two populations may tend to differ from each other and additional measurement features should be examined closely in subsequent studies to clarify other differences. Nikolosky (1915) recorded courtship in Transcaucasia in April-May, our observations confirm this study. Sadeghi and Torki (2012) have studied the reproduction phases of Testudo graeca ibera in Zagros Mountains, Iran. They compared their results with Hiley and Loumbourdis study (1988) on northern Greek population of the species. Sadeghi and Torki (2012) showed that eggs of the Iranian population have greater length (mean: 44.8±0.5 mm), width, and especially mass. But interestingly, egg size (length) of northwestern Iran population (this study; mean = 36.8± 1.4 mm) is more similar to the Greek population (mean = 35.4± 2.0 mm) rather than the Zagros ones (Table 5). Sadeghi and Torki (2012) reported four eggs for a female while egg laying whereas we observed nine eggs for one female. Clearly, more studies are needed to explore why it varies. Although T. graeca is widespread, it is classified as Vulnerable on the IUCN Red List. This group is often threatened by human activity: pet-trade, hunting for food, or habitat destruction (Van Abbema, 1997; Parham et al., 2006a). In Iran, the meat of this tortoise is generally not consumed and it seems that the major threat for the Iranian population in the study site is habitat destruction because of obvious development of farming and excessive live stock pasturage. In addition, bird as a predator and high intensity of the infection can be of the threatening factors. To protect the population of T. graeca, some conservation measures should be taken: protection of nesting area, prevention of overgrazing and disturbing pastures, and education programs for local people. This study is presenting preliminary information regarding morphological and biological status of T. graeca. Therefore, further investigations are necessary to better clarify other detailed biological and ecological aspects, habitat status and define a strategy for conservation of the species in NW Iran. Acknowledgements. The authors are indebted to Steven. C. Anderson for reviewing manuscript. We are grateful to Hadi Rabti for helping in field work, Mehdi Asadi, head of the Department of the Environment of Pars Abad for his advice and collaboration. Also thanks to Ms. Madani for her support on the manuscript. References Anderson, S.C. (1979): Synopsis of the turtles, Crocodiles and amphibians of Iran. Proceedings of the California Academy of Sciences, Series 4, 41(22): Bayley, J.R., Highfield, A.C. (1996): Observations on ecological changes threatening a population of Testudo graeca graeca in the Souss Valley, Southern Morocco. Chelonian Conservation and Biology 2: El Mouden, E.H., Slimani, T., Ben Kaddour, K. (2001): Croissance et dimorphisme sexuel chez la Tortue Mauresque (Testudo graeca graeca L. 1758). Chelonii 3, Proceeding International Congress on Testudo Genus, March El Mouden, E.H., Slimani, T., Ben Kaddour, K., Lagarde, F., Ouhammou, A., Bonnet, X. (2006): Testudo graeca graeca feeding ecology in an arid and overgrazed zone in Morocco. Journal of Arid Environments 64: Fritz, U., Hundsorfer, A.K., Siroky, P., Auer, M., Kami, H.G., Lehmann, J., Mazanaeva, L.F., Turkozan, O., Wink, M. (2007): Phenotypic plasticity leads to incongruence between morphologybased taxonomy and genetic differentiation in western Palaearctic tortoises (Testudo graeca complex;testudines, Testudinidae). Amphibia-Reptilia 28: Highfield, A.C. (1990a): Preliminary Report on the Taxonomic, Biotypic and Conservation Status of the Land Tortoises of Tunisia. Tortoise Trust, London, 14pp.

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