Testudo Species Research Articles

Symmetry-Breaking Stabilities in Carapace Curvature on Testudo (Reptilia, Testudinidae).

Simple SummaryWhen turned upside down, terrestrial turtles have no active control on self-righting if the animal has been flipped over. Turtles belonging to Testudo genus present high-domed carapaces and short neck and legs. Using geometric morphometric techniques, we studied left-right frontal symmetry among some Testudo species to study if carapaces’ geometry may serve as the tool to roll right side up. A clear right directionality was detected in the studied sample. This fact. more that easing the self-righting potential (“kinematic instability”, understood as the ability to self-right without effort), would make the stable ventral turning difficult (“static stability”, understood as the ability to resist passively turning of the body produced by destabilizing forces).The aim of this research was to contribute to the study of the doming geometry of Testudo carapace as an unstable point of equilibrium when animals are overturned. We performed this research using geometric morphometric using a sample of 64 Testudo individuals belonging to different species (T. hermanni n = 30, T. graeca n = 3, T. marginata n = 13 and T. horsfieldii n = 18), sexes and ages. A set of four sagittal landmarks (discrete homologous points) and 15 pairs of semi-landmarks, on the frontal doming of the carapace, were digitized on individual carapace pictures. Significative fluctuating asymmetry was detected, defined as small, completely random departures from bilateral symmetry, but much less than directional asymmetry, which appeared highly significative. Anti-symmetry did not appear. Carapace asymmetry was dominated by a clear right directionality. A possible biological speculation could be that this asymmetry more that easing the self-righting potential (“kinematic instability”, understood as the ability to self-right without effort), makes stable ventral turning difficult (“static stability”, understood as the ability to resist passively turning the body produced by destabilizing forces). This asymmetry is present among both sexes but more marked among males. An explanation for this sexually differentiated pattern could be the higher locomotion and the fight for mating in males, making them consequently more prone to losing their balance and falling on their back. These data may be useful in studying adaptative traits in Testudo species as well as establishing a seminal base for future studies. This research is the first attempt to explore a suitable method to assess doming asymmetry which could be useful in future, more extensive investigations, on a larger interspecific sample.

Risk factors influencing brumation success in captive tortoises in the United Kingdom.

This survey of tortoise owners was performed to investigate the factors contributing to morbidity and mortality during and post-brumation in captive Testudo species in the United Kingdom. Information regarding a total of 270 tortoises was included in the study, from 252 completed surveys. Binary logistic regression and multivariate modelling were used to evaluate the potential predictors associated with brumation mortality and those associated with post-brumation problems. A 7.78% mortality rate was found, with 7.22% of surviving tortoises reported to have post-brumation health concerns. Brumation in a garden and an uncontrolled reduction of temperature prior to brumation were the main risk factors for morbidity and mortality in the study. Veterinarians should be aware of these risk factors to ensure they target husbandry and brumation advice to owners of tortoises to reduce these risks.

Divergent scute asymmetry among pure and crossed individuals of Testudo hermanni (Gmelin, 1789)

Fluctuating asymmetry (FA) refers to subtle differences between left and right sides in bilaterally symmetrical organisms or their parts. Both genetic and environmental changes can increase FA, reflecting deterioration in developmental homeostasis of adult morphology due to a loss of developmental stability. In this study, we used geometric morphometric techniques to examine plastral scute asymmetries in a sample of 31 pure and crossed Testudo species (T. hermanni hermanni n = 23 and crosses with T. hermanni boettgeri n = 8) only females by means of 19 anatomical landmarks. Procrustes ANOVA indicated that FA in crossed individuals was significantly higher than that in pure individuals. Crossed individuals also showed a greater degree of phenotypic plasticity than T. hermanni hermanni. We conclude that crosses among T. hermanni hermanni and T. hermanni boettgeri can increase homozygosity and are responsible for greater developmental instabilities. Nonetheless, more information on crossed phenotypes could be of great interest to raise pure Hermann’s tortoises for reintroduction programmes. Key words: Carapace, Geometric morphometrics, Hybridization, Plastron, Testudines

A case of massive infestation of a female Spur-thighed tortoise Testudo graeca by blood-sucking ticks Hyalomma aegyptium (Acari: Ixodidae)

ABSTRACTInfestation by ticks affects several vertebrate groups, including tortoises. Testudo species are among chelonian fauna that are infested in their natural habitat by several tick species, with Hyalomma aegyptium as the dominant one. We report a heavy tick infestation found in female T. graeca in May 2018 in Had Draa (Essaouira region, Morocco). The female, with carapace length of 175 mm and body weight of 779.3 g was found to be infested by 288 H. aegyptium in different stages. It was more infested by female ticks than males, and the hind limbs (59.6%) and forelimbs (30.25%) were the most frequent predilection sites of ticks. Our data provide the first record of massive parasitism by H. aegyptium in this vulnerable and threatened tortoise.

The utility of plastron shape for uncovering cryptic diversity in Hermann's tortoise

AbstractAlthough a significant unexploited potential of the geometric morphometrics in taxonomy and systematics has been documented, only a few studies so far have focused on the utility of this method in the detection of population genetic structure in turtles. For the first time, we employed geometric morphometrics to explicitly analyse whether populations possessing divergent haplotypes evolved different patterns of plastral shape in any Testudo species using the Greek populations of Hermann's tortoise as a case study. To molecularly characterize the Greek populations of Testudo hermanni boettgeri used for plastron shape analyses, 12s rRNA gene of tortoises sampled from seven localities in Greece was sequenced. Three divergent haplotypes were observed, regionally corresponding to the north‐western (HI), south‐western (HII) and Peloponnese (HIII) areas of Greece. In both females and males, haplotypes HI and HII showed the lack of plastral shape divergence, but were both statistically significantly different relative to HIII found in Peloponnese. The Peloponnesian populations (HIII) had longer bridges and the wider and shorter anterior part of the plastron, relative to populations from the mainland Greece (HI and HII). Hence, both morphological and molecular markers suggested Peloponnese as a distinct lineage in relation to mainland populations. Finally, we consider geometric morphometrics as an efficient method to detect and characterize subtle interpopulation differences. Findings in this study have conservation applications as well, implying the presence of cryptic genetic and taxonomic diversity, and, thus, filling the crucial gap in the assessment of the conservation status of T. hermanni.

Intraspecific variation in digit reduction in Testudo: the case of the Hermann’s tortoise

Phalangeal reduction is a common and widespread phenomenon among tortoises that has been associated with the adaptation to terrestrial life. While reduced manual digit 1 appears characteristic in almost all Testudo species, it is uncertain why the metacarpal I and distal carpal of the same digit are completely missing in some individuals of Hermann’s tortoise (Testudo hermanni hermanni). To clarify this issue, we investigated the number of manual claws in six populations of Hermann’s tortoise (one from the Ebro Delta in the Iberian Peninsula and five from Minorca Island), their age, sex, genetic lineage, and the substrate type that they inhabit. The number of claws was ascertained based on direct counts (n > 1500 individuals) and by X-rays (n = 32 individuals), obtaining three different phalangeal formulae: (1-2-2-2-1, D-2-2-2-1, 0-2-2-2-1). Thus, claw counts through both methodologies (direct count and X-ray) further confirm that the observed claws serve as a good proxy to assess the actual number of digits. Our results show no loss of phalanges, metacarpal and carpal bones in digit 1 associated with age, sex, or substrate, contrary to some previous authors who hypothesized a relationship between this loss and sexual dimorphism. Therefore, variations in the number of manual digits and the loss of metacarpal I and distal carpal in digit 1 in Hermann’s tortoise are related to population and genetic lineage. More detailed comparisons with other Testudo hermanni populations from elsewhere in Europe would be required to understand the evolutionary significance concerning the intrapopulation variability in the number of digits remaining.

The role of Virus "X" (Tortoise Picornavirus) in kidney disease and shell weakness syndrome in European tortoise species determined by experimental infection.

Tortoise Picornavirus (ToPV) commonly known as Virus "X" was recently discovered in juvenile European tortoises suffering from soft carapace and plastron as well as kidney disease. Therefore, this virus was a potential candidate to be a causative agent for these disease patterns. Spur thighed tortoises (Testudo graeca) seemed to be more susceptible to establish clinical symptoms than other European species like T. hermanni. Thus this trial investigated the role of ToPV in the described syndrome. Two groups of juvenile European tortoises (T. graeca and T.hermanni) each of 10 animals, were cloacally, oronasally and intracoelomically inoculated with an infectious dose (~ 2000 TICD) of a ToPV strain isolated from a diseased T. graeca. A control group of two animals of each species received non-infected cell culture supernatant. The tortoises were examined daily and pharyngeal and cloacal swabs for detection of ToPV-RNA by RT-PCR were taken from each animal every six days for a period of 6 months. At the end of the study the remaining animals were euthanised and dissected. Bacteriological and parasitological tests were performed and organ samples of all tortoises were investigated by RT-PCR for the presence of ToPV and histopathology. Animals that were euthanised at the end of the experiment, were examined for presence of specific anti-ToPV antibodies. Several animals in both inoculated groups showed retarded growth and a light shell weakness, in comparison to the control animals. Three animals were euthanised during the trial, showing reduced weight gain, retarded growth, severe shell weakness and apathy, in parallel to clinical observations in naturally infected animals. In all inoculated animals of both species an intermittent virus shedding, starting from 18 days post inoculation (d.p.i.), till 164 d.p.i. was detected, while the control animals remained negative. The virus was successfully reisolated in terrapene heart cell culture in 16 of 20 inoculated animals of both species. Histopathology of most inoculated animals revealed a lack of bone remodeling and vacuolisation in kidney tubuli which supports the described pathogenesis of nephropathy and osteodystrophy. Anti- ToPV antibody titres ranged from 1:2 to >1:256 in 13 of 20 animals, whereas all control animals were seronegative. The study proofed the Henle Koch`s postulates of ToPV as causative agent for shell dystrophy and kidney disease in both testudo species. The proposed species specific sensitivity towards clinical disease was not observed.

Nutrition and husbandry conditions of Palearctic tortoises (Testudo spp.) in captivity

ABSTRACTMediterranean and Russian tortoises (Testudo spp.) are popular companion animals (pets), despite ongoing controversy concerning privately keeping reptiles. The arguments used during these controversial discussions have often been based on outdated facts. Therefore, a survey was developed to evaluate the current population structure, husbandry conditions, diet regime, and health status of Testudo species in captivity. More than 75% of the 1075 respondents housed their tortoises in an outdoor enclosure containing a greenhouse or cold frame, which is considered the most species-appropriate way of husbandry. Of the respondents, 67.7% fed their tortoises with the optimum diet of more than 80% grasses and weeds during the summer vegetation period. Only 8.2% of respondents owned a tortoise with a diagnosed disease. According to the results, the likelihood of tortoises developing pyramidal growth syndrome, which can be used as an indicator of the quality of tortoise husbandry, was high in tortoises kept in a terrarium and/or fed a diet of less than 80% grasses and weeds in summer. This likelihood varied among species, with a higher incidence in Hermann’s tortoises (Testudo hermanni).

Four new species of the genus Caccothryptus (Coleoptera, Limnichidae)

Four new species of the genus Caccothryptus (Coleoptera: Limnichidae) are described: C. taiwanus from Taiwan; C. orion from Okinawa; C. tibetanus and C. chayuensis from Tibet. All the species belong to the testudo species group (sensu Hernando & Ribera 2014). Additional specimen data and an updated species list are also given, and C. testudo Champion, 1923 is newly recorded from Thailand.

Geometry of self righting: the case of Hermann’s tortoises

Armoured animals can easily lose their balance and fall on their back. When overturned, they can be exposed to suboptimal environment conditions, starvation or predation, thus, self-righting has a vital importance. Chelonians’ stiff shells prevent torsion of the torso, thus influence of the dorsum shape on self-righting can be significant. We explored the relationship between duration of active self-righting attempts and shell geometry in Hermann’s tortoises. We used geometric morphometrics and shell shape indices (R index, sphericity and flatness indices) to explore this effect. Our results showed a negative relationship between size and righting performance in both females and males. Enlarged females’ body, and thus reduced agility, could be favoured by fecundity selection. On the other hand, the body size of males is probably affected by sexual selection. Additionally, enlarged flared rear marginal scutes in males negatively affect self-righting. This part of males’ shells could be shaped by the trade-off between self-righting and stability during courtship and mating. However, the relationship between carapace shape and righting performance is not straightforward, as different results were obtained when various shape indices were applied in the analyses. The indices imply that greater curvature of the carapace shape facilitates self-righting in Hermann’s tortoises. Comparison of available literature data and results of this study imply that shell shape across the Testudo genus, except for T. marginata, largely overlaps with shell shape of T. hermanni. Thus, a similar effect of shell shape on self-righting performance could be expected for most of Testudo species.

Comparison of total calcium, ionised calcium and albumin concentrations in the plasma of captive tortoises (Testudo species)

Plasma total calcium, ionised calcium and albumin levels in Hermann's tortoises (Testudo hermanni), spur-thighed tortoises (Testudo graeca), marginated tortoises (Testudo marginata) and Horsfield's tortoises (Testudo horsfieldi) were investigated. The animals...

Variations in the concentration of ionised calcium in the plasma of captive tortoises (Testudo species)

The concentration of ionised calcium was measured in the plasma of 13 Hermann's tortoises (Testudo hermanni boettgeri), nine spur-thighed tortoises (Testudo graeca ibera), two marginated tortoises (Testudo marginata) and six...

Phalangeal formulae and ontogenetic variation of carpal morphology in Testudo horsfieldii and T. hermanni

AbstractWe compared variation in phalangeal and carpal morphology of the Central Asian Testudo horsfieldii, a burrow-digging tortoise species, with its sister taxon T. hermanni, a Mediterranean species without extensive digging behaviour. Thirty-two Testudo horsfieldii kazachstanica shared the same phalangeal formula (0-2-2-1-1). The distal carpal 1 and metacarpal I as well as the pisiform were consistently lacking, sometimes also the medial centrale. Phalangeal morphology was more variable in Testudo hermanni hermanni. In 29 specimens four phalangeal formulae were found that differed with respect to the reduction of digit 1 (1-2-2-2-1, M-2-2-2-1, D-2-2-2-1, 0-2-2-2-1). The pisiform develops late in ontogeny and is lacking in very most juveniles and subadults. In contrast to T. h. kazachstanica, the medial centrale is always present. In both T. h. kazachstanica and T. h. hermanni carpalia increasingly fuse with age and size. The underlying morphological patterns differ however. The extreme character state in aged T. h. kazachstanica is one large solid bone element, formed by the fused intermedium, ulnare and both centralia. Aged T. h. hermanni have, in contrast, two separate larger carpal elements, one formed by the fused lateral and medial centralia and the other by the fused distal carpalia 1 and 2; the intermedium and ulnare never fuse with one another or with other carpalia. While a partial or complete loss of digit 1 seems to be characteristic for all Testudo species, we propose that the extensive fusion of carpal elements in T. horsfieldii is correlated with its natural history because a rigid manus could be advantageous for burrow-digging. Also the reduction of digit 4 (one phalanx present), a rare character among testudinids and not occurring in other Testudo species, could be linked with its mode of life.

When genes meet nomenclature: Tortoise phylogeny and the shifting generic concepts of Testudo and Geochelone

We used a five-gene data set (mtDNA: 12S rRNA, 16S rRNA, cyt- b; nDNA: Cmos, Rag2) comprising approximately two-thirds of all extant testudinid species and, for the first time, including all five Testudo species to investigate the question of whether all western Palaearctic testudinids are monophyletic. Further, we examined whether the recently suggested allocation of the African Geochelone pardalis in the otherwise exclusively South African genus Psammobates and of the Malagasy G. yniphora in the monotypic genus Angonoka is justified in the face of considerable morphological evidence against such placements. Our phylogenetic analyses do not support the paraphyly and generic break-up of Testudo, as suggested by previous papers using a smaller taxon sampling and mtDNA data only. We propose a continued usage of the generic name Testudo for all five western Palaearctic tortoise species. Within Testudo, two monophyletic subclades are present, one containing T. hermanni+ T. horsfieldii, and the other comprising ( T. kleinmanni+ T. marginata)+ T. graeca. Nomenclaturally, we demonstrate that Eurotestudo Lapparent de Broin et al., 2006, which was recently erected with the type species T. hermanni, is an objective junior synonym of Chersine Merrem, 1820 and Medaestia Wussow, 1916. Recognition of a monotypic genus Angonoka for G. yniphora is unwarranted according to both our re-analysis of sequence data and morphological data. Acknowledging the strong morphological similarity between G. yniphora and G. radiata, we suggest placing both species into the genus Astrochelys. Although sequence data for only one of the three Psammobates species was available for analysis, there is currently no cause to challenge the monophyly of this genus as established on the basis of morphological evidence. Thus, we hypothesize that G. pardalis is sister to a monophyletic Psammobates. In light of the clear morphological gap between G. pardalis and Psammobates species, the recognition of a distinct genus Stigmochelys for the former seems justified.

Is Testudo werneri a distinct species?

AbstractSequence variation of a 1066 bp long mtDNA fragment (cytochrome b gene, adjacent part of tRNA-Thr gene) of four known-locality samples of Testudo kleinmanni (Tripolitania, Libya) and of four samples of T. werneri (Negev, Israel) is compared with additional five sequences of pet trade tortoises allegedly representing T. kleinmanni. Four haplotypes, differing in one to four mutation steps occur. The most common haplotype was shared by all known-locality samples of T. kleinmanni and three T. werneri. Sequence variation within each nominal species and in the pooled sample of T. kleinmanni, T. werneri and pet trade tortoises is the lowest known for any Testudo species. We conclude there is no support for the validity of T. werneri Perälä, 2001.

Phenotypic plasticity leads to incongruence between morphology-based taxonomy and genetic differentiation in western Palaearctic tortoises (Testudo graeca complex; Testudines, Testudinidae)

Abstract Tortoises of the Testudo graeca complex inhabit a patchy range that covers part of three continents (Africa, Europe, Asia). It extends approximately 6500 km in an east-west direction from eastern Iran to the Moroccan Atlantic coast and about 1600 km in a north-south direction from the Danube Delta to the Libyan Cyrenaica Peninsula. Recent years have seen a rapid increase of recognized taxa. Based on morphological investigations, it was suggested that this group consists of as many as 20 distinct species and is paraphyletic with respect to T. kleinmanni sensu lato and T. marginata. Based on samples from representative localities of the entire range, we sequenced the mitochondrial cytochrome b gene and conducted nuclear genomic fingerprinting with ISSR PCR. The T. graeca complex is monophyletic and sister to a taxon consisting of T. kleinmanni sensu lato and T. marginata. The T. graeca complex comprises six well-supported mtDNA clades (A-F). Highest diversity is found in the Caucasian Region, where four clades occur in close neighbourhood. This suggests, in agreement with the fossil record, the Caucasian Region as a radiation centre. Clade A corresponds to haplotypes from the East Caucasus. It is the sister group of another clade (B) from North Africa and western Mediterranean islands. Clade C includes haplotypes from western Asia Minor, the southeastern Balkans and the western and central Caucasus Region. Its sister group is a fourth, widely distributed clade (D) from southern and eastern Asia Minor and the Levantine Region (Near East). Two further clades are distributed in Iran (E, northwestern and central Iran; F, eastern Iran). Distinctness of these six clades and sister group relationships of (A + B) and (C + D) are well-supported; however, the phylogeny of the resulting four clades (A + B), (C + D), E and F is poorly resolved. While in a previous study (Fritz et al., 2005a) all traditionally recognized Testudo species were highly distinct using mtDNA sequences and ISSR fingerprints, we detected within the T. graeca complex no nuclear genomic differentiation paralleling mtDNA clades. We conclude that all studied populations of the T. graeca complex are conspecific under the Biological Species Concept. There is major incongruence between mtDNA clades and morphologically defined taxa. Morphologically well-defined taxa, like T. g. armeniaca or T. g. floweri, nest within clades comprising also geographically neighbouring, but morphologically distinctive populations of other taxa (clade A: T. g. armeniaca, T. g. ibera, T. g. pallasi ; clade D: T. g. anamurensis, T. g. antakyensis, T. g. floweri, T. g. ibera, T. g. terrestris), while sequences of morphologically similar tortoises of the same subspecies (T. g. ibera sensu stricto or T. g. ibera sensu lato) scatter over two or three genetically distinct clades (A, C or A, C, D, respectively). This implies that pronounced morphological plasticity, resulting in phenotypes shaped by environmental pressure, masks genetic differentiation. To achieve a more realistic taxonomic arrangement reflecting mtDNA clades, we propose reducing the number of T. graeca subspecies considerably and regard in the eastern part of the range five subspecies as valid (T. g. armeniaca, T. g. buxtoni, T. g. ibera, T. g. terrestris, T. g. zarudnyi). As not all North African taxa were included in the present study, we refrain from synonymizing North African taxa with T. g. graeca (mtDNA clade B) that represents a further valid subspecies.

Isolation and characterization of eight microsatellite loci for the study of gene flow between Testudo marginata and Testudo weissingeri (Testudines: Testudinidae)

AbstractThe taxonomic status of the Greek Testudo species, marginated tortoise (Testudo marginata Schoepff, 1793) and dwarf marginated tortoise (Testudo weissingeri Bour, 1995), is controversed. In order to study the gene flow among these Greek Testudo species, eight polymorphic microsatellite markers were characterized and screened for 32 individuals. The number of alleles observed per locus ranged from two to 19. Observed heterozygosities varied from 0.235 to 0.830. These microsatellite loci were also successfully tested on seven closely related species of Testudinidae. This set of microsatellites offers an efficient tool to investigate genetic differentiation among testudinid species.

The phylogeny of Mediterranean tortoises and their close relatives based on complete mitochondrial genome sequences from museum specimens

As part of an ongoing project to generate a mitochondrial database for terrestrial tortoises based on museum specimens, the complete mitochondrial genome sequences of 10 species and a ∼14 kb sequence from an eleventh species are reported. The sampling of the present study emphasizes Mediterranean tortoises (genus Testudo and their close relatives). Our new sequences are aligned, along with those of two testudinoid turtles from GenBank, Chrysemys picta and Mauremys reevesii, yielding an alignment of 14,858 positions, of which 3238 are parsimony informative. We develop a phylogenetic taxonomy for Testudo and related species based on well-supported, diagnosable clades. Several well-supported nodes are recovered, including the monophyly of a restricted Testudo, T. kleinmanni + T. marginata (the Chersus clade), and the placement of the enigmatic African pancake tortoise ( Malacochersus tornieri) within the predominantly Palearctic greater Testudo group ( Testudona tax. nov.). Despite the large amount of sequence reported, there is low statistical support for some nodes within Testudona and so we do not propose names for those groups. A preliminary and conservative estimation of divergence times implies a late Miocene diversification for the testudonan clade (6–10 million years ago), matching their first appearance in the fossil record. The multi-continental distribution of testudonan turtles can be explained by the establishment of permanent connections between Europe, Africa, and Asia at this time. The arrival of testudonan turtles to Africa occurred after one or more initial tortoise invasions gave rise to the diverse (>25 species) ‘ Geochelone complex.’ Two unusual genomic features are reported for the mtDNA of one tortoise, M. tornieri: (1) nad4 has a shift of reading frame that we suggest is resolved by translational frameshifting of the mRNA on the ribosome during protein synthesis and (2) there are two copies of the control region and trnF, with the latter having experienced multiple-nucleotide substitutions in a pattern suggesting that each is being maintained by selection.

Environmentally caused dwarfism or a valid species—Is Testudo weissingeri Bour, 1996 a distinct evolutionary lineage? New evidence from mitochondrial and nuclear genomic markers

We examine the evolutionary relationships of the five traditionally recognized species of the western Palearctic tortoise genus Testudo ( T. graeca, T. hermanni, T. horsfieldii, T. kleinmanni, and T. marginata) and the newly described dwarfed species T. weissingeri by using sequence data of the mitochondrial cytochrome b gene and nuclear genomic fingerprints with inter-simple sequence repeats (ISSR). Testudo weissingeri differs from T. marginata mainly by its smaller size and some color-pattern characteristics. T. weissingeri lives in the driest, poorest and hottest part of the distributional range of T. marginata. While both data sets demonstrated phylogenetic distinctness of the five traditionally recognized species of Testudo, some subspecies and even some local populations, we detected no differentiation between T. marginata and T. weissingeri. We conclude that T. weissingeri is not a distinct evolutionary unit. We suggest that its small size is the result of suboptimal environmental conditions with limited resources and synonymize it with T. marginata. T. marginata and T. kleinmanni form a clade that is supported both by our mtDNA and nuclear genomic data sets. According to mtDNA data, this clade is the sister taxon to the T. graeca complex. A sister group relationship of T. hermanni and (( T. marginata + T. kleinmanni) + T. graeca) is moderately to weakly supported by mtDNA data; T. horsfieldii is the sister taxon to a clade comprising all other Testudo species.

Thermostable lichenase as a translational reporter

The polymerase chain reaction with arbitrary primers (RAPD-PCR) was used to study intraspecific variation in Mediterranean turtle Testudo graeca, which is represented by the Dagestan (T. g. pallasi) and Nikolskii (T. g. nikolskii) subspecies in Russia. To study the phylogenetic relationships, the RAPD variation was also compared in two other T. graeca subspecies (T. g. ibera and T. g. terrestris), two closely related Testudo species (T. kleinmanni and T. marginata), and Central Asian turtle Agrionemys horsfieldii. Parameters of RAPD variation showed that the sample from different geographical regions of Dagestan was more polymorphic and heterogeneous than that from Central Asia. The two samples differed in the mean number of RAPD fragments N (48.761 vs. 40.400), number of polymorphic fragments P (78.7 vs. 32.3), and within-group similarity index APS (0.607 vs. 0.784). In T. g. pallasi, no significant difference in N, P, or APS was observed between samples from different localities of Dagestan or between groups of turtles with four- or five-clawed forelegs. A dendrogram of genetic similarity between the species and subspecies under study contained two clusters, one comprising all A. horsfieldii individuals and the other, all turtles of the genus Testudo. In the latter, T. marginata and T. kleinmanni showed higher similarity to each other than to T. graeca. The four T. graeca subspecies clustered separately from each other with a high reliability, T. g. nikolskii and T. g. ibera (Turkey) being more similar to each other than to T. g. terrestris or T. g. pallasi. The possible causes of the presence of four claws on a foreleg and the relationships among members of the genus Testudo were discussed.