Phylogenetic relationships among the species of the genus Testudo (Testudines: Testudinidae) inferred from mitochondrial 12S rRNA gene sequences

To test phylogenetic relationships within the genus Testudo (Testudines: Testudinidae), we have sequenced a fragment of the mitochondrial (mt) 12S rRNA gene of 98 tortoise specimens belonging to the genera Testudo, Indotestudo, and Geochelone. Maximum likelihood and neighbor-joining methods identify two main clades of Mediterranean tortoises, one composed of the species Testudo graeca, Testudo marginata, and Testudo kleinmanni and a second of Testudo hermanni, Testudo horsfieldii, and Indotestudo elongata. The first clade, but not the second, was also supported by maximum parsimony analysis. Together with the genus Geochelone, a star-like radiation of these clades was suggested, as a sister-group relationship between the two Testudo clades could not be confirmed. The intraspecies genetic variation was examined by sequencing the mt 12S rRNA fragment from 28 specimens of T. graeca and 49 specimens of T. hermanni from various geographic locations. Haplotype diversity was found to be significantly larger in T. graeca compared with T. hermanni, suggestive of reduced genetic diversity in the latter species, perhaps due to Pleistocene glaciations affecting northern and middle Europe or other sources of lineage reduction. No ancient mt 12S rRNA gene haplotypes were identified in T. graeca and/or T. hermanni originating from islands in the Mediterranean Sea, suggesting that these islands harbor tortoise populations introduced from the European and African mainland.     

Redescription of four species of Mehdiella from Testudinidae, with a key to the species and discussion on the relationships among the species of this genus

Four species of the genus Mehdiella Seurat, 1918 are redescribed: M. cristata Petter, 1966 and M. stylosa dollfusi Petter, 1966, parasite of Pyxix arachnoides Bell, 1827 from Madagascar, M. s. stylosa (Thapar, 1925) and M. uncinata (Drasche, 1884), parasite of Testudo graeca Linneaus, 1758, Testudo hermanni Gmelin, 1789 and Testudo horsfieldii (Gray, 1844) from Palaearctic region. Light microscopy and scanning electron microscopy (SEM) studies revealed new informations on the morphology of these species. On the basis of this morphological study, the sub-species Mehdiella stylosa dollfusi and M. s. stylosa are raised to level of species. The position of Mehdiella cristata among the species of the genus Mehdiella and the relationships among the species of the genus Mehdiella are discussed. A key to the eight valid species Mehdiella is given.     

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.     

Sexual dimorphism of body size and shell shape in European tortoises

Adult body size and shape were examined in almost 1400 individuals of the tortoises Testudo graeca , T. hermanni and T. marginata from Greece. The size at maturity was greater in females than in males in all three species. Maximum and mean adult sizes were also greater in females than in males in T. graeca and T. hermanni . Males grew to a larger size than females in T. marginata , and mean adult size was similar in the sexes in this species. Sexual dimorphism of shape (adjusted for size covariate) was shown in most of the characters examined, and the degree of this dimorphism differed significantly among the three species. Differences were related to their contrasting courtship behaviours: horizontal head movements and severe biting in T. marginata , vertical head bobs and carapace butting in T. graeca , and mounting and tail thrusting in T. hermanni . There was no difference in the frequency of observations of courtship or fighting among the three species, but courtship was about 10 times more common than combat in males. All species showed greatest courtship activity in autumn; copulation was rarely observed in T. hermanni (only 0.36% of courting males) and not seen in the other species in the field. Observations made throughout the activity season indicated that feeding was equally common in males and females in all three species. Differences in shape were more likely to be the result of sexual selection than of natural selection for fecundity. Detailed predictions are made for sexual dimorphism of other characters in these species.     

Detection and characterization of Mycoplasma spp. and Salmonella spp. in free-living European tortoises (Testudo hermanni, Testudo graeca, and Testudo marginata)

Free-living and captive chelonians might suffer from upper respiratory tract disease (URTD), a pathology primarily caused by Mycoplasma agassizii. Wild tortoises can also be an important reservoir of Salmonella spp., which are commensal in the host reptile but are potential zoonotic agents. Between July 2009 and June 2010, we screened free-living European tortoises (spur-thighed tortoises Testudo graeca, Hermann's tortoises Testudo hermanni, marginated tortoises Testudo marginata) temporarily housed in a wildlife center in Italy. We molecularly characterized 13 Mycoplasma isolates detected in all Testudo spp. studied, and three PCR-positive animals showed typical URTD clinical signs at the time of sampling. Three Salmonella enterica serotypes (Abony, Potsdam, Granlo), already related to reptile-associated human infections, were also identified. These results highlight the potential role played by wildlife recovery centers in the spread and transmission of pathogens among wild chelonians and to humans.     

RAPD variation in Mediterranean turtle Testudo graeca L. (Testudinidae)

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.     

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.     

Co-distribution pattern of a haemogregarine Hemolivia mauritanica (Apicomplexa: Haemogregarinidae) and its vector Hyalomma aegyptium (Metastigmata: Ixodidae)

Hyalomma aegyptium ticks were collected from tortoises, Testudo graeca, at localities in northern Africa, the Balkans, and the Near and Middle East. The intensity of infestation ranged from 1-37 ticks per tortoise. The sex ratio of feeding ticks was male-biased in all tested populations. Larger tortoises carried more ticks than did the smaller tortoises. The juveniles were either not infested, or carried only a poor tick load. Hyalomma aegyptium was absent in the western Souss Valley and Ourika Valley in Morocco, the Cyrenaica Peninsula in Libya, Jordan, and the Antilebanon Mountains in Syria. Hemolivia mauritanica, a heteroxenous apicomplexan cycling between T. graeca and H. aegyptium, was confirmed in Algeria, Romania, Turkey, Syria, Lebanon, and Iran. Its prevalence ranged from 84% in Romania (n = 45), 82% in eastern Turkey (n = 28), and 82% in the area of northwestern Syria with adjacent Turkish borderland (n = 90), to 38% in Lebanon (n = 8) and in only 1 of 16 sampled tortoises in Algeria. The intensity of parasitemia in the studied areas ranged from 0.01% up to 28.17%. The percentage of Hemolivia-infected erythrocytes was significantly higher in adults. All tortoises from Hyalomma-free areas were Hemolivia-negative. Remarkably, all 29 T. graeca from Jabal Duruz (southwestern Syria) and 36 T. graeca from the area north of Middle Atlas (Morocco) were Hemolivia-negative, despite the fact that ticks parasitized all adult tortoises in these localities. Identical host preferences of H. aegyptium and H. mauritanica suggest the occurrence of co-evolution within the Testudo-Hyalomma-Hemolivia host-parasite complex.     

Mitochondrial haplotype diversity in the tortoise species <Emphasis Type="Italic">Testudo graeca</Emphasis> from North Africa and the Middle East

Background  

To help conservation programs of the endangered spur-thighed tortoise and to gain better insight into its systematics, genetic variation and evolution in the tortoise species Testudo graeca (Testudines: Testudinidae) was investigated by sequence analysis of a 394-nucleotide fragment of the mitochondrial 12S rRNA gene for 158 tortoise specimens belonging to the subspecies Testudo graeca graeca, Testudo graeca ibera, Testudo graeca terrestris, and a newly recognized subspecies Testudo graeca whitei. A 411-nucleotide fragment of the mitochondrial D-loop was additionally sequenced for a subset of 22 T. graeca, chosen because of their 12S gene haplotype and/or geographical origin.     

Identification and characterization of microsatellite markers in Hermann's tortoise (Testudo hermanni,Testudinidae)

The isolation and characterization of six polymorphic loci from a Testudo hermanni genomic library is reported. Enrichment was performed for AC but four of the characterized microsatellites present also an additional motif. Variability was tested on populations of the two recognized subspecies, Testudo hermanni hermanni and Testudo hermanni boettgeri. For one locus, a size range specific for the subspecies T. h. hermanni was observed. These are the first primers identified directly in the genome of T. hermanni     

Descriptions of Tachygonetria africana n. sp. and T. pretoriensis n. sp. and redescriptions of two other species of Tachygonetria Wedl, 1862 (Nematoda: Pharyngodonidae), all parasitic in Geochelone pardalis (Testudinidae) from South Africa

Two new species of Tachygonetria, T. africana n. sp. and T. pretoriensis n. sp. are described. Both are parasites of Geochelone pardalis (Bell) from South Africa. In addition, two subspecies, T. longicollis fitzsimmonsi Petter, 1966 and T. macrolaimus macrolaimus (Linstow, 1899), also parasites of Geochelone pardalis (from Pretoria Zoo, South Africa), are redescribed and new information on their morphology is presented. On the basis of their morphology, these subspecies are raised to full specific level.     

Carapace and plastron sensitivity to touch and vibration in the tortoise (Testudo hermanni and T. graeca)

Neural impulses in response to tactile stimulation of the shell were recorded from afferent nerve fibres in tortoises ( T. graeca and T. hermanni). It was found that there is a mechanoreceptive innervation in the superficial layers of the shell which is sensitive to transient stimuli, particularly to vibration at frequencies up to 100 Hz. Receptive fields pertaining to single and small groups of individual afferent fibres were mapped: the fields were sharply circumscribed and distributed in relation to the scutes of the shell. The tactile innervation that was found would be consistent with a capacity for recognition and accurate localization of innocuous stimuli and may play a central role in courtship and mating behaviour.     

Studies on the growth, structure and abundance of the Mediterranean spur-thighed tortoise, Tesudo graeca in field populations

The blackness and physical appearance of Mediterranean spur-thighed tortoises ( Testudo graeca L.) are recorded in field populations. The inter-relationships between carapace and plastron length, fourth vertebral scute width and other morphometric characters are related to growth. The rear fontanelle is larger in males and the plastron length correspondingly smaller. The relationship between carapace length ( y ) in mm and body mass ( x ) in g is strongly allometric and given by y = 16.63 x ^0–34. The maximum carapace lengths recorded for male and female T. graeca graeca L. in NW. Africa are 151 and 198 mm, and for larger T. graeca ibera Pallas in W. Turkey and NE. Greece 208 and 218 mm, respectively. The growth annuli of epidermal scutes are deposited yearly until external growth ceases. The number is used to assess the age of wild animals. Tortoises can be full-grown by 19 years but some, especially slower growing ones, deposit further annuli. The biggest mean growth increments occur up to 7 years with the largest in individuals over the 5–12 year period. The proportion of immature tortoises, adult sex ratios, size and body mass structure, age distribution, annual recruitment and survivorship until full-grown, and abundance (sighting frequency) are compared in northern and southern range populations of NW. African T. g. graeca and S. Anatolian and SE. European (Thrace and Macedonia) ones of T. g. ibera. These are discussed in relation to other tortoise species.     

Pharyngodonidae (Oxyuroidea; Nematoda) of Agama yemenensis in Saudi Arabia: hypothesis on the origin of pharyngodonids of herbivorous reptiles

Summary Five new species of pharyngodonid (Oxyuroidea) nematodes are described from the posterior gut of Agama yemenensis (Agamidae) in Saudi Arabia: Tachygonetria paradentata n.sp., a member of the T. dentata complex, most closely resembles T. quentini Petter, 1966 from Testudo tentoria verreauxi from South Africa in the form and disposition of cephalic sense organs; it is distinguished by having caudal alae, an anterior anal lip in the male formed by two sharply-elbowed lobes and by the fact that the terminal spine on the caudal appendage of the male is much shorter than that in T. quentini. Alaeuris asirensis n.sp. is most similar to a species described as Thelandros sexlabiata Ortlepp, 1933 from Testudo tentoria verreauxi from South Africa: the last pair of caudal papillae in the male is subterminal, and there are six lips and a slight prevulvar swelling in the female. Males of the two species are easily distinguished by the presence of caudal alae and a longer caudal appendage in A. asirensis females are distinguished by the fact that lips are less developed in the new species. The fact that two such similar species cannot be included in the same genus reflects the need for a redefinition of Alaeuris. Thelandros agama n.sp. resembles T. alatus Wedl, 1862 from Uromastix sp. in North Africa in the cephalic structure of the female and in the shape of the peduncles supporting adanal papillae in males; the new species is distinguished by its longer spicule and by the presence of caudal alae and the absence of lips in males. T. masaae n.sp. resembles T. taylori Chatterji, 1935 and T. baylisi Chatterji, 1935 from Agama sp. and Uromastix sp. in India in that the peduncles supporting the caudal papillae in the male are elongate; it differs from both and from all other members of the genus in that the vulva is located at the end of a long tube-like exvagination of the body wall and the anterior anal lip of the male is simple rather than fringed. T. petterae n.sp. resembles T. taylori and T. baylisi in having well-developed caudal alae in males and six prominent lips in females; it is distinguished from both by its flask-shaped adanal peduncles in the male. Thelandros is redefined to include only those species in which the pre- and adanal papillae are pedunculate and the opening to the spicular pouch is markedly postanal. The nine genera of the Pharyngodonidae in herbivorous and omnivorous reptiles form an evolutionary line distinct from that in insectivorous reptiles. It is suggested that this line arose in tortoises in Laurasia in the Eocene, spread with these hosts to Africa, Madagascar and South America, and subsequently underwent evolutionary radiations in each of these regions. Pharyngodonids of A. yemenensis have a double origin: the Thelandros spp. probably evolved with agamids whereas species of Tachygonetria and Alaeuris are likely captures from tortoises. ac]19831111     

Mitochondrial DNA haplotyping of Testudo graeca on both continental sides of the Straits of Gibraltar

Testudo graeca is an endangered species of tortoise that inhabits Mediterranean areas of Africa, Asia, and Europe. Western populations are found on both sides of the Straits of Gibraltar. The effects of geographical isolation on genetic divergence were assessed by the sequence analysis of two mitochondrial DNA regions of the 12S rRNA and cytochrome b genes. Four different haplotypes were identified. A single haplotype was shared by all Spanish and some east Moroccan specimens. Two haplotypes were unique to the west Moroccan T. graeca populations and allowed the clear discrimination between individual specimens found west of the Moulouya River. Phylogenetic analysis based on the estimation of nucleotide sequence distances of the haplotypes suggests an African origin for the Spanish populations and a subspecies status for the west Moroccan pool.     

Genetic assessment,illegal trafficking and management of the Mediterranean spur-thighed tortoise in Southern Spain and Northern Africa

Wild populations of many species are declining as a result of habitat destruction and climate change but also through the over-collection for wild meat and the pet trade. With a long history of trade around the Mediterranean, populations of the spur-thighed tortoise (Testudo graeca graeca) have become highly disturbed. In this study we utilise a molecular approach to investigate the diversity, population admixture and structure of T. g. graeca populations in northern Africa and southern Spain, as well as to obtain an insight into the origin of newly established populations in the south of Europe. We infer this from the sequencing of two partial regions of the mitochondria (12s rRNA + cyt b) and genotyping at 16 microsatellite markers in 448 tortoises. Our results are consistent with the hypothesis that Spanish populations were founded from North Africa, the consequence of multiple introductions or exchanges in genetic material as a result of trans-oceanic dispersal. Despite the trade of individuals between both sides of the Gibraltar Strait, our analysis of population structure showed clear differences between both the African and European populations, suggesting an incipient evolutionary lineage in southeast of Spain. As such, these populations possess unique genetic identities and should be treated as different management units. Surprisingly, the genetic data identified a great deal of diversity contained within pet (captive) stock and also allowed us to infer hybrids among individuals with another species of terrestrial tortoise from northern Spain (T. hermanni hermanni). Additionally, our results provide insight into the local movement and trade of individuals that has occurred around the Mediterranean basin (between northern Africa and southern Spain) and as such provides guidance for the effective management of T. g. graeca captive stock and the illegal trafficking.