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.     

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.     

Mitochondrial sequence data expose the putative cosmopolitan polychaete Scoloplos armiger (Annelida, Orbiniidae) as a species complex

Background  

Polychaetes assigned as Scoloplos armiger (Orbiniidae) show a cosmopolitan distribution and have been encountered in all zoogeographic regions. Sibling S. armiger -like species have been revealed by recent studies using RAPDs and AFLP genetic data. We sequenced a ~12 kb fragment of the Scoloplos cf. armiger mitochondrial genome and developed primers for variable regions including the 3' end of the cox3 gene, trnQ, and most of nad6. A phylogenetic analysis of this 528-nucleotide fragment was carried out for S. armiger -like individuals from the Eastern North Atlantic as well as Pacific regions. The aim of this study is to test the cosmopolitan status, as well as to clarify the systematics of this species complex in the Eastern North Atlantic, while using a few specimens from the Pacific Ocean for comparision.     

Differentiation of tortoises of the genera Testudo and Agrionemys (Testudinidae) based on the polymorphism of nuclear and mitochondrial markers

Based on polymorphism of the 12S rRNA gene and RAPD markers, differentiation of 122 tortoise individuals belonging to the three species of genus Testudo (T. kleinmanni, T. marginata, and T. graeca), six subspecies of T. graeca (T. g. nikolskii, T. g. pallasi, T. g. armeniaca, T. g. zarudnyi, T. g. terrestris, T. g. ibera), and two subspecies of the Central Asian tortoise Agrionenemys horsfieldii (A. h. horsfieldii, A. h. kazakhstanica) was performed. For comparison, 32 known sequences of 12S rRNA gene (392 bp) from tortoises of the two genera inhabiting the territories of Europe, Asia, and Africa were used. In the populations of A. horsfieldii, a total of six haplotypes, including three newly described variants, were identified. In the examined tortoises of the genus Testudo, eleven 12S rRNA haplotypes were identified. One new haplotype was detected in T. kleinmanni. Among the eight subspecies of T. graeca, eight haplotypes were identified, with four newly described ones. The reported RAPD markers generally supported the reconstructions obtained with the use of the mitochondrial marker. Similarly to the 12S rRNA-based reconstructions, two independent clusters included representatives of the two genera, Agrionemys and Testudio. Among the latter, representatives of T. marginata and T. kleinmanni, as well as T. graeca, with high statistical support values, formed two reciprocally monophyletic groups. Compared to the mitochondrial markers, RAPDs more statistically significantly discriminated the sample of T. g. terrestris and the four subspecies, T. g. ibera, T. g. armeniaca, T. g. pallasi, and T. g. nikolskii. In almost all cases except the representatives of T. g. ibera, the representatives of each of four subspecies formed individual subclusters. The geographical haplotype distribution patterns and possible evolutionary scenario of the origin and dispersal of tortoises of the two genera are discussed.     

Mitochondrial phylogeography and subspecies of the wide‐ranging sub‐Saharan leopard tortoise Stigmochelys pardalis (Testudines: Testudinidae) – a case study for the pitfalls of pseudogenes and GenBank sequences

The leopard tortoise (Stigmochelys pardalis) is the most widely distributed sub‐Saharan tortoise species, with a range extending from the Horn of Africa all over eastern Africa to the Republic of South Africa, Namibia and southernmost Angola. Using 1938 bp of mitochondrial DNA (cyt b gene, partial ND4 gene plus adjacent tRNA genes) from a nearly range‐wide sampling, we examined its phylogeographic structure and compared our findings with previously published GenBank sequences. We identified seven major clades that are largely parapatrically distributed. A few records of distinct haplotypes at the same locality or in close proximity could be the result of translocation of tortoises by man. The greatest diversity occurs in the south of the species’ range, with five out of the seven clades. Testing for isolation‐by‐distance suggests that the observed phylogeographic structure is the result of restricted geographical gene flow and not of historical vicariance. This is in sharp contrast to wide‐ranging thermophilic reptiles from the western Palaearctic, whose phylogeographic structure was significantly shaped by Pleistocene range interruptions, but also by earlier dispersal and vicariant events. Most cyt b sequences of S. pardalis from GenBank turned out to be nuclear pseudogenes, or to be of chimerical origin from such pseudogenes and authentic mitochondrial sequences, which argues for caution regarding uncritical usage of GenBank sequences. The recent revalidation of the two subspecies of S. pardalis was based on such a chimerical sequence that was erroneously identified with the subspecies S. p. babcocki. Furthermore, according to our data, the distribution of mitochondrial clades does match neither the traditional subspecies ranges nor the pronounced geographical size variation of leopard tortoises. We conclude that there is no rationale for recognizing subspecies within S. pardalis.     

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.     

Relative role of life-history traits and historical factors in shaping genetic population structure of sardines (<Emphasis Type="Italic">Sardina pilchardus</Emphasis>)

Background  

Marine pelagic fishes exhibit rather complex patterns of genetic differentiation, which are the result of both historical processes and present day gene flow. Comparative multi-locus analyses based on both nuclear and mitochondrial genetic markers are probably the most efficient and informative approach to discerning the relative role of historical events and life-history traits in shaping genetic heterogeneity. The European sardine (Sardina pilchardus) is a small pelagic fish with a relatively high migratory capability that is expected to show low levels of genetic differentiation among populations. Previous genetic studies based on meristic and mitochondrial control region haplotype frequency data supported the existence of two sardine subspecies (S. p. pilchardus and S. p. sardina).     

Phylogeny,Diversity, Distribution,and Host Specificity of Haemoproteus spp. (Apicomplexa: Haemosporida: Haemoproteidae) of Palaearctic Tortoises

A complex wide‐range study on the haemoproteid parasites of chelonians was carried out for the first time. Altogether, 811 samples from four tortoise species from an extensive area between western Morocco and eastern Afghanistan and between Romania and southern Syria were studied by a combination of microscopic and molecular‐genetic methods. Altogether 160 Haemoproteus‐positive samples were gathered in the area between central Anatolia and eastern Afghanistan. According to variability in the cytochrome b gene, two monophyletic evolutionary lineages were distinguished; by means of microscopic analysis it was revealed that they corresponded to two previously described species—Haemoproteus anatolicum and Haemoproteus caucasica. Their distribution areas overlap only in a narrow strip along the Zagros Mts. range in Iran. This fact suggests the involvement of two different vector species with separated distribution. Nevertheless, no vectors were confirmed. According to phylogenetic analyses, H. caucasica represented a sister group to H. anatolicum, and both of them were most closely related to H. pacayae and H. peltocephali, described from South American river turtles. Four unique haplotypes were revealed in the population of H. caucasica, compared with seven haplotypes in H. anatolicum. Furthermore, H. caucasica was detected in two tortoise species, Testudo graeca and Testudo horsfieldii, providing evidence that Haemoproteus is not strictly host‐specific to the tortoise host species.     

RAPD variation in Mediterranean turtle <Emphasis Type="Italic">Testudo graeca</Emphasis> 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 (48.76 vs. 40.40), 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 forelegs of turtles from Dagestan and the relationships among members of the genus Testudo were discussed.Translated from Genetika, Vol. 40, No. 12, 2004, pp. 1628–1636.Original Russian Text Copyright © 2004 by Semyenova, Korsunenko, Vasilyev, Pereschkolnik, Mazanaeva, Bannikova, Ryskov.     

The type IV pilin,PilA, is required for full virulence of <Emphasis Type="Italic">Francisella tularensis</Emphasis> subspecies <Emphasis Type="Italic">tularensis</Emphasis>

Background  

All four Francisella tularensis subspecies possess gene clusters with potential to express type IV pili (Tfp). These clusters include putative pilin genes, as well as pilB, pilC and pilQ, required for secretion and assembly of Tfp. A hallmark of Tfp is the ability to retract the pilus upon surface contact, a property mediated by the ATPase PilT. Interestingly, out of the two major human pathogenic subspecies only the highly virulent type A strains have a functional pilT gene.     

Individualistic response to past climate changes: niche differentiation promotes diverging Quaternary range dynamics in the subspecies of Testudo graeca

Understanding how species have responded to past climate changes might inform how current climate change may affect species. However, current perspectives on species’ response to Quaternary cycles mainly stem from studies on mid and high latitudes and limited information is available at lower latitudes. In these systems a greater variation in individual species’ responses to glacial cycles has been suggested, but we lack of clear case studies of such variations. Here, by means of ecological niche modelling, we characterized the niche differentiation of the five subspecies of the tortoise Testudo graeca in north Africa and described the range dynamics suffered by each of the subspecies since the Last Glacial Maxima. Molecular data was employed to infer past population dynamics. The five subspecies present a clear niche differentiation, particularly in relation to rainfall, covering a range from semiarid to humid species. The ecological differentiation among subspecies promotes very different, even opposite, post‐glacial range dynamics. Since the Last Glacial Maxima, the ranges have either expanded (T. g. graeca), contracted (T. g. marokkensis), shifted northwards (T. g. soussensis) or remained stable (T. g. cyrenaica and T. g. nabeulensis). Molecular data supported the majority of these range dynamics. Our work exemplifies how, in a climate change scenario, phylogenetically very close taxa (i.e. at the subspecies level) might experience strikingly different biogeographical dynamics due to former niche differentiation. Our work supports an individualistic response to glacial cycles, which can be particularly strong in lower latitudes. In these areas precipitation changes during glacial periods may have strongly affected distribution ranges in idiosyncratic ways. We hypothesize that in those species limited by precipitation the response to glacial cycles (and climate changes) is more unpredictable than in those limited by temperature.     

Histochemical and ultrastructural study of the digestive tract of the tortoise Testudo graeca (Testudines)

The digestive tract of the tortoise Testudo graeca (Testudines) was investigated by means of light and electron microscopy. The esophagus of T. graeca was lined by two types of epithelium: non-keratinized stratified squamous in the upper portion and stratified columnar in the lower. The lamina propria of the esophagus contained tubular or tubuloacinar glands. The mucosa of the stomach showed similar characteristics to those of other reptiles. The small intestine exhibited longitudinal folds lined by absorptive and goblet cells. The large intestine was lined by columnar mucous cells. Within the lamina propria of the large intestine, masses of 10–15 epithelial cells connecting with the surface epithelium by means of slender cytoplasmic processes were observed. A battery of six lectins (Con-A, PNA, WGA, DBA, SBA, and LTA) was used to identify the epithelial mucins. WGA and DBA reacted with all types of mucous cells throughout the alimentary canal. PNA was only unreactive in the intestine, LTA in the esophagus, and SBA in the large intestine. These results indicate a similar lectin-binding pattern throughout the gut of T. graeca.     

Integrating phylogeographic patterns of microsatellite and mtDNA divergence to infer the evolutionary history of chamois (genus <Emphasis Type="Italic">Rupicapra</Emphasis>)

Background  

The chamois, distributed over most of the medium to high altitude mountain ranges of southern Eurasia, provides an excellent model for exploring the effects of historical and evolutionary events on diversification. Populations have been grouped into two species, Rupicapra pyrenaica from southwestern Europe and R. rupicapra from eastern Europe. However, a previous study of cytochrome b revealed that the two proposed species were non-monophyletic. The reconstruction of phylogenetic relationships between animal species often depends on the markers studied. To further elucidate the evolutionary history of chamois, we extended earlier studies by analysing DNA sequences of four mitochondrial regions (ND1, 12S, tRNApro and Control Region) and microsatellites (20 loci) to include all subspecies and cover its entire distribution range.     

Hybridization and recurrent evolution of left–right reversal in the land snail genus Schileykula (Orculidae,Pulmonata)

The land snail genus Schileykula Gittenberger, 1983 is distributed in arid limestone areas from western Turkey to north-western Iran. It comprises eight species, which display high variation in shell size and morphology. The cylindrical shells are 5–12 mm in height and the last shell whorls bear several inner lamellae and plicae. Two taxa differ in their chirality having sinistral shells, while all the others are dextrals such as the vast majority of orculids. The aim of this study was to establish a molecular genetic phylogeny of Schileykula and to test whether it conforms to the current morphology-based classification. Furthermore, we were interested in the phylogenetic position of the two sinistral forms in order to assess whether one or two reversals happened in the evolution of the genus. Nine out of ten species, including all four subspecies of Schileykula trapezensis and three of six subspecies of Schileykula scyphus, were investigated. A section of the mitochondrial cytochrome c oxidase subunit I gene was analyzed in 54 specimens of Schileykula and from a subsample, partial sequences of the mitochondrial genes for the 12S rRNA and the 16S rRNA, and a section of the nuclear H4/H3 histone gene cluster were obtained. The phylogenetic trees based on the mitochondrial sequences feature high support values for most nodes, and the species appear well differentiated from each other. The two chiral forms evolved independently and are not sister lineages. However, some groupings disagree with the present morphology-based classification and taxonomical conclusions are drawn. Schileykula trapezensis is polyphyletic in the molecular genetic trees; therefore, three of its subspecies are elevated to species level: Schileykula acampsis Hausdorf, 1996 comb. nov., Schileykula neuberti Hausdorf, 1996 comb. nov., and Schileykula contraria Neubert, 1993 comb. nov. Furthermore, Schileykula sigma is grouped within S. scyphus in the mitochondrial and nuclear trees and consequently treated as a subspecies of the latter (Schileykula scyphus sigma Hausdorf, 1996 comb. nov.). Schileykula nordsiecki, whose shell morphology is indistinguishable from that of the neighboring Schileykula scyphus lycaonica, but who differs in its genital anatomy, was confirmed to represent a distinct lineage. The phylogenies produced by the mitochondrial and nuclear data sets are to some extent conflicting. The patterns differ concerning the grouping of some specimens, suggesting at least two independent hybridization events involving S. contraria, S. scyphus and S. trapezensis. The results exemplify the importance of integrating both mitochondrial and nuclear sequence data in order to complement morphology-based taxonomy, and they provide further evidence for hybridization across distantly related lineages in land snails.     

From speciation to introgressive hybridization: the phylogeographic structure of an island subspecies of termite, <Emphasis Type="Italic">Reticulitermes lucifugus corsicus</Emphasis>

Background  

Although much research has been carried out into European Reticulitermes taxonomy in recent years, there is still much discussion about phylogenetic relationships. This study investigated the evolution from intra- to interspecific phylogeny in the island subspecies Reticulitermes lucifugus corsicus and threw new light on this phenomenon. An integrative approach based on microsatellites and mitochondrial and nuclear DNA sequences was used to analyze samples taken from a wide area around the Tyrrhenian sea and showed how the subspecies evolved from its origins to its most recent form on continental coasts.     

Detection of a novel Chlamydia species in captive spur-thighed tortoises (Testudo graeca) in southeastern Spain and proposal of Candidatus Chlamydia testudinis

The spur-thighed tortoise (Testudo graeca) is an endangered Mediterranean tortoise that lives in North Africa, Southern Europe and Southwest Asia. In the wake of recent legislation making their keeping as domestic animals illegal, many of these animals have been returned to wildlife recovery centers in Spain. In the present study, a population of such tortoises showing signs of ocular disease and nasal discharge was examined for the presence of Chlamydia spp. Cloacal, conjunctival and/or choanal swabs were collected from 58 animals. Using a real-time PCR specific for the family Chlamydiaceae, 57/58 animals tested positive in at least one sample. While only a few samples proved positive for C. pecorum, sequencing of the 16S rRNA gene revealed a sequence identical to previously published sequences from specimens of German and Polish tortoises. Whole-genome sequences obtained from two conjunctival swab samples, as well as ANIb, TETRA values and a scheme based on 9 taxonomic marker genes revealed that the strain present in the Spanish tortoises represented a new yet non-classified species, with C. pecorum being its closest relative. We propose to designate the new species Candidatus Chlamydia testudinis.     

Speciation in the stonechat (Saxicola torquata) inferred from nucleotide sequences of the cytochrome-b gene

The geographical differentiation and speciation in the stonechat (Saxicola torquata; Aves: Turdidae) was studied by sequencing a 300-bp fragment of the mitochondrial cytochrome-b gene. Stonechats from three different subspecies inhabiting three continents (S. t. rubicola, the European stonechat; S. t. maura, the Siberian stonechat; and S. t. axillaris, an African stonechat) and stonechats from seven European populations were examined. While variation within the populations of the European subspecies was less than 0.3%, the genetic distances between the subspecies were substantial (2.7-5.7%) in comparison with published data for subspecies of other birds. If speciation is indeed reflected in the cytochrome-b gene of stonechats, species status for the African stonechat, but also for the Siberian taxon, should be reconsidered, especially in the light of differences in distributions, morphology and habitat preference.     

Body size development of captive and free‐ranging Leopard tortoises (Geochelone pardalis)

The growth and weight development of Leopard tortoise hatchings (Geochelone pardalis) kept at the Al Wabra Wildlife Preservation (AWWP), Qatar, was observed for more than four years, and compared to data in literature for free‐ranging animals on body weight or carapace measurements. The results document a distinctively faster growth in the captive animals. Indications for the same phenomenon in other tortoise species (Galapagos giant tortoises, G. nigra; Spur‐thighed tortoises, Testudo graeca; Desert tortoises, Gopherus agassizi) were found in the literature. The cause of the high growth rate most likely is the constant provision with highly digestible food of low fiber content. Increased growth rates are suspected to have negative consequences such as obesity, high mortality, gastrointestinal illnesses, renal diseases, “pyramiding,” fibrous osteodystrophy or metabolic bone disease. The apparently widespread occurrence of high growth rates in intensively managed tortoises underlines how easily ectothermic animals can be oversupplemented with nutrients. Zoo Biol 29:517–525, 2010. © 2009 Wiley‐Liss, Inc.