ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah Acta Herpetologica 8(2): 99-104, 2013 A skeletochronological study of parthenogenetic lizards of genus Darevskia from Turkey Marine Arakelyan1,*, Ruzanna Petrosyan1, Çetin Ilgaz2, Yusuf Kumlutaş2, Salih Hakan Durmuş3, Yahya Tayhan4, Felix Danielyan1 1 Yerevan State University, Alek Manoogian, 1, Yerevan 0025, Armenia, *Corresponding author. E-mail: arakelyanmarine@yahoo.com 2 Dokuz Eylül University, Faculty of Sciences, Department of Biology, 35160, Buca-İzmir, Turkey 3 Dokuz Eylül University, Faculty of Education, Department of Biology, 35160, Buca-İzmir, Turkey 4 Hakkari University, Healty Vocational College, Hakkari, Turkey Submitted on 2012, 20th February; revised on 2013, 13th May; accepted on 2013, 10th December. Abstract. The skeletochronological method has been used to assess age distribution and age-related differences in body size among populations of the parthenogenetic lizards Darevskia sapphirina, D. uzzelli, D. armeniaca and D. uni- sexualis from Turkey and Armenia. The age distribution between D. armeniaca and D. unisexualis did not significantly differ and ranged from 1 to 8 years. Maximum age for both D. uzzelli and D. sapphirina was 6 years, and 8 years for both D. unisexualis and D. armeniaca. In all the studied species, individuals reached sexual maturity after third hiber- nation. According to patterns of growth marks resorption, D. sapphirina is distinguished from all other rock lizards of genus Darevskia by a narrowest periosteal bone as result of high rate of endosteal resorption resulting in complete destruction of hatchling line and line of the first hibernation. Keywords. Darevskia, parthenogenesis, age, SVL, skeletochronology. INTRODUCTION The genus Darevskia includes seven parthenoge- netic species of Caucasian rock lizards widely distributed throughout the inner part of the Armenian Plateau on terri- tories of central, northwestern and northern Armenia, adja- cent region of southern Georgia, western Azerbaijan and northeastern Turkey. Among parthenogenetic species, D. armeniaca, D. dahli, D. unisexualis and partly D. rostombe- kowi have comparatively wide areas of distribution, while D. uzzelli, D. sapphirina and D. bendimahiensis are endem- ics of eastern and northeastern Turkey with limited distri- bution ranges. The habitats of the cited species are similar and occur primarily in steppe zone, where they prefer rocks and conglomerates of stones at elevation above 1500 m with drastic seasonal climatic variations (Darevsky, 1967; Darevs- ky and Danielyan, 1977; Schmidtler et al., 1994; Ananjeva et al., 2006; Ilgaz, 2006; Arakelyan et al., 2011). Previous studies of parthenogenetic Darevskia spe- cies revealed low genetic variability in comparison with their bisexual parental species, in which genome diversity may arise as a result of mutation or multiple hybridiza- tion events (Fu et al., 2000, Moritz et al., 1992, Marti- rosyan et al, 2002). According to results obtained with mitochondrial and allozyme markers (Fu et al., 2000; Murphy et al., 2000), parthenogenetic species D. uni- sexualis, D. uzzelli, D. sapphirina and D. bendimahiensis originated from hybridization events of the same bipa- rental species D. raddei and D. valentini during reticu- late evolution. Thus, comparative studies about age and growth of parthenogenetic lizards may provide additional information about the origin of parthenogenetic species and possibility to study the influence of enviroment on phenotypic diversity in the clonal populations of lizards. However, the data on growth, longevity and age composi- tion of populations of Darevskia parthenogenetic species 100 Marine Arakelyan et al. are scarce. Skeletochronological data for D. armeniaca, D. dahli, D. unisexualis and D. rostombekowi from Arme- nia show that parthenogenesis does not influence growth rate, longevity and maturity and are correlated with body size rather than reproduction mode (Arakelyan and Dan- ielyan, 2000; Arakelyan, 2001; Arakelyan, 2002). Despite their conservation importance due to restricted ranges (Akarsu et al., 2009, Kaska et al., 2009), no data are cur- rently available on the life history of endemic partheno- genetic species of genus Darevskia from Turkey. The purpose of this study is to determine and com- pare the age and body size of parthenogenetic species of genus Darevskia from Turkey and analyze differences in body size for each age group among populations of uni- sexual lizards. MATERIALS AND METHODS A total of 255 individuals of partenogenetic species D. armeniaca (Mehely, 1909), D. sapphirina (Schmidtler et al, 1994), D. unisexualis (Darevsky, 1966) and D. uzzelli (Darevsky and Danielyan, 1977) were collected from different localities from northeastern and eastern part of Turkey and compared with populations of D. armeniaca and D. unisexualis from Armenia (Table 1 and Figure 1). We used 70% ethanol pre- served specimens that had already been collected and incor- porated into the collections of ZDEU (Zoology Department Ege University, Turkey) and Yerevan State University, Armenia. Lizard snout-vent length (SVL) was measured from the tip of snout to anal cleft by using a dial caliper with an accuracy of 0.01 mm. The age of individuals was determined by skeletochro- nological method (Smirina, 1974; Castanet and Smirina, 1990) which is based on counting of numbers of arrested growth lines (LAG) formed in the bones of reptiles as a result of seasonal growth processes. Cross-sections of femur bones were used for counting of concentric arrested growth lines. The bones were decalcified in 5% nitric acid solution to one hour and rinsed in tap water for over 12 hours. The cross sections (10 mm thick- ness) of diaphysis of femur were prepared using a freezing microtome and then were stained with Erlich’s haematoxylin for 20 min. The sections were mounted on the slide with a drop of glycerin and a cover slip was placed over the slide. The cross sections were examined under a light microscope and record- ed with a digital Canon A100 camera. LAGs were counted and verified independently by two authors. The cross-sections were processed by image analysis. For each individual at least three selected cross-sections from the middle of diaphysis were used for measurement. Measurements were made on images of bone sections using the morphometric software tpsDig (Rohlf, 2004). The square roots of mean values of longest and shortest axes of diameters of bone sections and diameters of marrow cav- ity were calculated. The periosteal bone width was expressed as difference between periosteal diameter (bone width) and the marrow diameters of bones. The hatchling line (neonatal line) and the first hibernation resting line suggests that the first Table 1. Data on the specimens of parthenogenetic rock lizard used for skeletochronological analysis (N – number of samples) Species N Locality Date Altitude Coordinates D. armeniaca 76 Ardahan, Turkey Artavazd vil., Kotayk Province, Armenia 5 km of Gyulagarak vil., Lori Province, Armenia 09.07.2003 20.07.1999 30.06.1999 1750 1870 1400 41°04’N, 42°50’E 40°37’N, 44°33’E 40.59’N, 44°77’E D. uzzelli 52 28 km SE of Horasan, Erzurum, Turkey 7 km W of Selim, Kars, Turkey 03.09.2002 29.06.2001 2000 1950 39°08’N, 43°06’E 39°08’N, 43°06’E D. unisexualis 112 28 km SE of Horasan, Erzurum, Turkey 7 km W of Selim, Kars, Turkey 5 km of Noratuz vil., Gegharkunik Province, Armenia 2 km of Tcovinar vil., Gegharkunik Province, Armenia Artavazd vil., Kotayk Province, Armenia 03.09.2002 29.06.2001 07.06.2004 08.07.2000 20.07.1999 2000 1950 1930 1920 1870 39°08’N, 43°06’E 39°08’N, 43°06’E 40°23’N, 45°12’E 40°09’N, 45°27’E 40°37’N, 44°33’E D. sapphirina 15 26 km NW of Erciş, Van, Turkey 19.06.2002 1950 39°08’N, 43°06’E Fig. 1. Sampling localities of parthenogenetic species (square: Darevskia sapphirina, triangle: Darevskia uzzelli, circle: Darevskia unisexualis, star: Darevskia armeniaca). 101A skeletochronological study of parthenogenetic lizards of genus Darevskia from Turkey LAGs deposited in juveniles are retained into later life time of lizards (Smirina, 1974; Castanet, 1994). Both of these closely spaced lines were completely or partly present in all samples of D. armeniaca, D. unisexualis and D. uzzelli, which allowed a correct assessment of the number of their LAGs (Fig. 2). In order to account for the number of resorbed LAGs in process of endosteal resorption in some samples of D. sapphirina, “back calculation” was performed by comparing the diameter of the marrow cavity of the adult lizards with that of the first LAG and outer edge of a bone of the subadult lizards (Smirina, 1974; Castanet and Smirina, 1990). All data were tested for normality (Shapiro-Wilk) and for homogeneity of variances (Levene test). We used one-way analysis of variance (ANOVA) and Scheffé’s post hoc test to test for differences in snout-vent length (SVL) or bone width within each age class in species or populations. Significance was defined as P < 0.05. Statistica software version 7.0 was used for data treatment (StatSoft, 2004). RESULTS Age structure of all studied species is characterized by a maximum age of 6-8 year and predominance of 4-5 year-olds among adult individuals (Fig. 3). The maximum age estimated by skeletochronological method for D. armeniaca and D. unisexualis was 8 years, while the old- A B C D Fig. 2. Femur bone cross–sections of four-year old parthenogenetic lizards of four species (A - D. armeniaca, B - D. unisexualis, C - D. uzzelli, and D - D. sapphirina). Arrows indicate LAGs, Mc - marrow cavity, Eb - endosteal bone. 102 Marine Arakelyan et al. est lizards of D. uzzelli, D. sapphirina in our samples were 6 years. According to the presence of vitellogenic eggs, individuals reach sexual maturity after third hibernation in all studied species. The average age of adults in our samples was similar among species (Mean ± SE - D. armeniaca: 4.46 ± 0.11 n = 73; D. unisexualis: 4.44 ± 0.11 n = 95; D. sapphirina: 4.44 ± 0.33 n = 10; D. uzzelli: 4.06 ± 0.25 n = 17) and not differed significantly (F3, 195= 1.02, P = 0.39) from one another. Significant differences in SVL between four parthe- nogenetic species in each age class (Table 2) were found only between 4 years-old D. uzzelli and D. unisexualis (P < 0.05) and between 4 years-old D. uzzelli and D. armeniaca (P < 0.01) according to Scheffé’s post-hoc test for one-way ANOVA. The maximum SVL of 71 mm was recorded for a 6 years-old D. armeniaca, 70 mm in a 6 years-old D. unisexualis, 67 mm in a 6 years-old D. uzzel- li, 59 mm in a 5 years-old D. sapphirina. No differences in body size (SVL) for each age class of D. unisexualis parthenogenetic lizards were detected among seven localities while among three populations of D. armeniaca significant differences were found only between populations from Stepanavan in Armenia and Ardahan in Turkey (P < 0.05) in 4-years old lizards (Fig. 4). D. unisex ualis Horasan, Turkey Noratuz, Armenia Hankavan, Armenia T covinar, Armenia 3 4 5 6 Age 40 45 50 55 60 65 70 75 80 S V L D. arm eniaca Ardahan, Turkey Stepanavan, Armenia Hankavan, Armenia 3 4 5 6 Age 45 50 55 60 65 70 75 SV L Fig. 4. Differences in body size (SVL) between populations of D. unisexualis and D. armeniaca within each age group (yr.). Vertical bars show0 .95 confidence intervals.   Age N um be r o f l iz ar ds D.armeniaca D.unisexualis D.sapphirina D.uzzelli 3 4 5 6 7 8 0% 3% 5% 8% 10% 13% 15% 18% 21% Fig. 3. Distribution of adult age groups of parthenogenetic species. Table 2. Snout-vent length of adults of parthenogenetic species in each age group (N: Number of samples, SE: Standard error of the mean, SD: Standard deviation) Species Age group 3 4 5 6 N Mean ± SE SD N Mean ± SE SD N Mean ± SE SD N Mean ± SE SD D. armeniaca 9 55.25 ± 1.24 3.45 31 56.60 ± 0.67 3.42 26 61.84 ± 0.73 3.33 5 64.54 ± 1.67 4.13 D. unisexualis 16 53.35 ± 0.93 4.54 38 57.92 ± 0.61 3.85 29 61.27 ± 0.69 2.73 8 63.91 ± 1.32 2.86 D. uzzelli 6 58.66 ± 1.52 4.98 6 59.24 ± 1.52 7.01 3 60.41 ± 2.15 1.78 2 63.73 ± 2.64 3.90 D. sapphirina 2 48.64 ± 2.64 0.22 4 54.62 ± 2.64 2.67 4 57.85 ± 1.87 1.20 1 59.47 103A skeletochronological study of parthenogenetic lizards of genus Darevskia from Turkey We found differences between patterns of growth marks resorption among studied species. Examination of stained cross sections of femur bones of D. unisexu- alis and D. uzzelli revealed the low endosteal resorption which did not destroy any part of first resting lines in 53% and 34% of cases respectively, whereas in others cas- es it locally destroyed up to 3/4 of the innermost rings. D. armeniaca had intermediate rate of resorption where the sections with intact first lines comprised 8%, and there were no sections with completely destroyed hatchling and first hibernation lines. D. sapphirina had considerably high resorption with 98% of cases completely destroyed hatching and line of first hibernation (Fig. 2, 5). Conse- quently, among compared four species, D. sapphirina had a significantly narrower periosteal bone (F3, 115 = 41.94, P < 0.0001) in comparison with D. unisexualis (Scheffé’s post-hoc test P < 0.001), D. uzzelli (P < 0.001), and D. armeniaca (P < 0.001) (Fig. 5). DISCUSSION Although skeletochronology has been applied to four parthenogenetic Darevskia species in Armenia (Arakely- an and Danielyan, 2000), no studies have been conducted in D. sapphirina and D. uzzelli in Turkey. The range of ages of D. uzzelli and D. sapphirina in our samples did not differ from other species of Darevskia, where maxi- mum age was 6 years, similar to D. rostombekowi and D. dahli (Arakelyan and Danielyan, 2000) and lower than in D. armeniaca and D. unisexualis. However, the oldest liz- ards of D. armeniaca (8 years) identified by skeletochro- nological methods in different populations of Armenia and Turkey did not represent the oldest individuals docu- mented under natural conditions estimated by mark-and- recapture method. The long term observation of D. arme- niaca in Armenia showed that the maximum longevity is up to 10 year (Galoyan, 2011). Thus, the actual longev- ity of parthenogenetic lizards may exceed the maximum recorded by skeletochronological estimated age, especially for larger species, probably due to outer LAGs distances in older individuals that are very narrow or missing. There were no significant differences in the snout- vent length (SVL) of parthenogenetic species among age groups, however D. rostombekowi had the smallest SVL in each age group while D. uzzelli, D. unisexualis and D. armeniaca had larger sizes. Among studied species D. unisexualis, D. uzzelli and D. sapphirina apparently origi- nated from hybridization of the same parent species: D. raddei and D. valentini (Murphy et al., 2000). The com- parison of their SVL indicated that the species D. uni- sexualis and D. uzzelli are close to each other, whereas D. sapphirina has a smaller size. It is possible that body size between parthenogenetic species originating from same parents does not change. The comparisons of body size of the same unisexual species will allow studies of influence of the environment on clonal organisms. We did not find significant differences in body size between populations of parthenogenetic species, with the exception of 4 years- old D. armeniaca. However, we found some population- specific variation in body size in each age group of D. armeniaca and D. unisexualis from Armenia and Turkey, where body size of lizards from Turkey was larger than that of Armenian lizards. The climatic and habitat char- acteristics of sampling localities in Armenia and Turkey were mostly similar. Observed differences among popu- lations could result from the temperature and the length of active periods at lower and higher elevations. Previ- ous studies showed that body length, growth rates, age of maturity, and longevity can vary considerably among populations of the same species, where lizards inhab- iting high-elevation sites and northern latitudes live longer than those from low-elevation sites and southern latitudes in the Northern Hemisphere (Arakelyan, 2000; Wapstra and Swain, 2001; Roitberg and Smirina, 2006). Surprisingly, the periosteal bones of D. sapphirina were notably thinner than those of other species appar- ently as the result of intensive endosteal resorption. The rate of resorption in Darevskia rock lizards is quite slow (Arakelyan and Danielyan 2000; Arakelyan, 2002) and the first growth arrested lines are always locally present with with the exception of D. sapphirina. The apparently high rate of resorption may result from either a specific mutation or new allele combination following a hybridi- zation event among bisexual species which gave rise to the parthenogenetic species.   D. sapphirina D. unisex ualis D. uzzelli D. arm eniaca3 4 5 6 Age 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 B on e w id th Fig. 5. The periosteal bone width (in µm) of four parthenogenetic species within each age group (yr.). Vertical bars show 0.95 confi- dence intervals. 104 Marine Arakelyan et al. ACKNOWLEDGMENTS The authors wish to express their gratitude to Dr. Marco A.L. Zuffi, Museo di Storia Naturale e del Territorio, Univer- sità di Pisa, and anonymous reviewers for helpful comments on earlier drafts of the manuscript. We thank Project No. 2009. KB.FEN.003 Scientific Research Project Coordination Unit, Dokuz Eylül University, Turkey for their support. REFERENCES Akarsu, F., Tuniyev, B., Ananjeva, N., Agasyan, A., Orlov, N., Tuniyev, S. (2009): D. uzzelli. In: IUCN 2011. IUCN Red List of Threatened Species. Version 2013.2. . Downloaded on 22 May 2013. Ananjeva, N.B., Orlov, N.L., Khalikov, R.G., Darevsky, I.S., Ryabov, S.A., Barabanov, A.V. (2006): The Rep- tiles of Northern Eurasia. Taxonomic Diversity, Distri- bution, Conservation Status. Pensoft Series Faunistica, Sofia. Arakelyan, M., Danielyan, F. (2000): Age and growth of some parthenogenetic and bisexual species of rock lizards (Lacerta), from Armenia. Zool. J. 79: 585-590. Arakelyan, M. (2001): Development of ovaries in bisexual and parthenogenetic rock lizards of genus Darevskia. In: The Problem of Herpetology. Proceedings of the 1st Meeting of the Nikolsky Herpetological Society, p. 21-22. Ananjeva, N.B. et al., Ed., Pushchino-Moscow (in Russian). Arakelyan, M. (2002): The study of age, growth, and longevity in triploid hybrids of rock lizards of genus Darevskia in Armenia. Russian J. Herpetol. 9: 63-68. Arakelyan, M., Danielyan, F., Corti, C., Sindaco, R., Leviton, A. (2011): Herpetofauna of Armenia and Nagorno-Karabakh. SSAR, Salt Lake City. Castanet, J., Smirina, E. (1990): Introduction to the skel- etochronological method in amphibians and reptiles. Ann. Sci. Nat. Zool. 11: 191-196. Darevsky, I.S. (1967): Rock lizards of the Caucasus: sys- tematics, ecology and phylogenesis of the polymor- phic groups of Caucasian rock lizards of the subgenus Archeolacerta. Nauka, Leningrad (in Russian). Darevsky, I.S., Danielyan, F.D. (1977): Lacerta uzzeli sp. nov. (Sauria, Lacertidae) - a new parthenogenetic spe- cies of rock lizard from eastern Turkey. Trudy Zool. Inst. 76: 55-59 (in Russian). Fu, J., Murphy, R.W., Darevsky, I.S. (2000): Divergence of the cytochrome b gene in the Lacerta raddei complex and its parthenogenetic daughter species: evidence for recent multiple origins. Copeia 2: 432-440. Galoyan, E.A. (2011). The role of social relations in the formation of space structure of the settlements of bisexual and parthenogenetic species of rock lizards. Unpublished PhD thesis. Moscow State University, Moscow (in Russian). Ilgaz, Ç. (2006): On specimens of Darevskia armeniaca (Sauria: Lacertidae: Darevskia) collected from Arda- han. Turk. J. Zool. 30: 47-54. Kaska, Y., Kumlutaş, Y., Avcı, A., Üzüm, N., Yeniyurt, C., Akarsu, F. (2009): D. sapphirina. In: IUCN 2011. IUCN Red List of Threatened Species. Version 2013.2. . Downloaded on 22 May 2013. Martirosyan, I.A., Ryskov, A.P., Petrosian, V.G., Arake- lian, M.S., Aslanian, A.V., Danielian, F.D., Darevskiĭ, I.S., Tokarskaia, O.N. (2002): Variation of mini- and microsatellite DNA markers in populations of parthe- nogenetic rock lizard Darevskia rostombekovi. Rus. J. Gen. 38: 691-698. Moritz, C., Uzzel, T., Spolsky, C., Hotz, H., Darevsky, I., Kupriyanova, L., Danielyan, F. (1992): The maternal ancestry and approximate age of parthenogenetic spe- cies of Caucasian rock lizards (Lacerta: Lacertidae). Genetica 87: 53-62. Murphy, R.W, Fu, J., Macculloch, R.D., Darevsky, I.S., Kupri- yanova, L.A. (2000): A five line between sex and unisex- uality: the phylogenetic constraints on parthenogenesis in lacertid lizards. Zool. J. Linn. Soc. 130: 527-549. Rohlf, F.J., 2004. tpsDig. Version 1.40. SUNY, Stony Brook. Roitberg, E.S., Smirina, E.M. (2006): Adult body length and sexual size dimorphism in Lacerta agilis boemica (Reptilia, Lacertidae): between-year and interlocality variation. In: Mainland and Insular Lizards: a Medi- terranean Perspective, pp. 175-187. Corti, C., Lo Cas- cio, P., Biaggini, M., Florence University Press, Flor- ence: 175-187. Schmidtler, J.F., Eiselt, J., Darevsky, I.S. (1994): Untersu- chungen an Feldeidechsen (Lacerta-saxicola-Gruppe) in der östlichen Türkei: 3. Zwei neue parthogenetische Arten. Salamandra 30: 55-70. Smirina, E.M. (1974): Prospects of age determination by bone layers in Reptilia. Zool. Zhur. 53: 111-117 (in Russian). Wapstra, E., Swain, R. (2001): Geographic and annu- al variation in life history traits in a temperate zone Australian skink. J. Herpetol. 35: 194-203. Acta Herpetologica Vol. 8, n. 1 - June 2013 Firenze University Press Journal of the Societas Herpetologica Italica ACTA HERPETOLOGICA