Diversity and biogeography of South American mud turtles elucidated by multilocus DNA sequencing (Testudines: Kinosternidae).

Kinosternon is the most speciose genus of extant turtles, with 22 currently recognized species, distributed across large parts of the Americas. Most species have small distributions, but K. leucostomum and K. scorpioides range from Mexico to South America. Previous studies have found discordance between mitochondrial and nuclear phylogenies in some kinosternid groups, with the current taxonomy following the nuclear-based results. Herein, based on extended molecular, geographic, and taxonomic sampling, we explore the phylogeographic structure and taxonomic limits for K. leucostomum and the K. scorpioides group and present a fossil-calibrated nuclear time tree for Kinosternon. Our results reveal contrasting differentiation patterns for the K. scorpioides group and K. leucostomum, despite overlapping distributions. Kinosternon leucostomum shows only shallow geographic divergence, whereas the K. scorpioides group is polyphyletic with up to 10 distinct taxa, some of them undescribed. We support the elevation of K. s. albogulare and K. s. cruentatum to species level. Given the deep divergence within the genus Kinosternon, we propose the recognition of three subgenera, Kinosternon, Cryptochelys and Thyrosternum, and the abandonment of the group-based classification, at least for the K. leucostomum and K. scorpioides groups. Our results show an initial split in Kinosternon that gave rise to two main radiations, one Nearctic and one mainly Neotropical. Most speciation events in Kinosternon occurred during the Quaternary and we hypothesize that they were mediated by both climatic and geological events. Additionally, our data imply that at least three South American colonizations occurred, two in the K. leucostomum group, and one in the K. scorpioides group. Additionally, we hypothesize that discordance between mitochondrial and nuclear phylogenetic signal is due to mitochondrial capture from an extinct kinosternine lineage.

Integrating morphological, molecular and cytogenetic data for F2 sea turtle hybrids diagnosis revealed balanced chromosomal sets.

Hybridization could be considered part of the evolutionary history of many species. The hybridization among sea turtle species on the Brazilian coast is atypical and occurs where nesting areas and reproductive seasons overlap. Integrated analysis of morphology and genetics is still scarce, and there is no evidence of the parental chromosome set distribution in sea turtle interspecific hybrids. In this study, chromosome markers previously established for pure sea turtle species were combined with morphological and molecular analyses aiming to recognize genetic composition and chromosome sets in possible interspecific hybrids initially identified by mixed morphology. The data showed that one hybrid could be an F2 individual among Caretta caretta × Eretmochelys imbricata × Chelonia mydas, and another is resulting from backcross between C. caretta × Lepidochelys olivacea. Native alleles of different parental lineages were reported in the hybrids, and, despite this, it was verified that the hybrid chromosome sets were still balanced. Thus, how sea turtle hybridism can affect genetic features in the long term is a concern, as the implications of the crossing-over in hybrid chromosomal sets and the effects on genetic function are still unpredictable.

How do maternal androgens and estrogens affect sex determination in reptiles with temperature-dependent sex?

Temperature sex determination (TSD) in reptiles has been studied to elucidate the mechanisms by which temperature is transformed into a biological signal that determines the sex of the embryo. Temperature is thought to trigger signals that alter gene expression and hormone metabolism, which will determine the development of female or male gonads. In this review, we focus on collecting and discussing important and recent information on the role of maternal steroid hormones in sex determination in oviparous reptiles such as crocodiles, turtles, and lizards that possess TSD. In particular, we focus on maternal androgens and estrogens deposited in the egg yolk and their metabolites that could also influence the sex of offspring. Finally, we suggest guidelines for future research to help clarify the link between maternal steroid hormones and offspring sex.

Detection of Hemopathogens in Chelonoidis carbonaria: Microscopic, Molecular, Hematological, and Clinical Biochemistry Aspects.

Background: The growing contact between men and wild animals, caused by the increase in the population in urban centers and the destruction of the habitat of these animals, has been leading to a greater circulation of pathogens between humans and wildlife. Chelonoidis carbonaria, a tortoise found throughout South America, is one of the animals most rescued from animal trafficking and illegal breeding. Considering this situation, this study aimed to verify the occurrence of hemoparasites in C. carbonaria. Materials and Methods: Blood samples from 73 C. carbonaria were collected from animals located in (1) a rural commercial breeding unit, (2) an urban zoo, and (3) a center of rescued animal screening. Genomic DNA was extracted from these animals and used in PCRs to detect specific genomic fragments of haemogregarines (i.e., Hepatozoon and Hemolivia), and members of the Anaplasmataceae Family (i.e., Ehrlichia sp. and Anaplasma sp.). Blood samples were screened for hemopathogens by direct microscopy and were used for hematological assays, and serum samples were analyzed to determine the concentration of serum components. Results: It was found that 34.2% of the tortoises presented Sauroplasma sp. in their blood samples; these animals showed clinical biochemistry changes that indicate altered liver function. Two zoo animals were positive for Ehrlichia sp. in PCR, and also presented clinical biochemistry and hematological changes. Conclusion: The present project is pioneer in the detection of Ehrlichia sp. in C. carbonaria, and was able to identify changes in clinical biochemistry that can be a result of the infection by hemopathogens in this species.

The importance of cryptic diversity in the conservation of wide-ranging species: The red-footed tortoise Chelonoidis carbonarius in Colombia.

Wide-ranging species are seldom considered conservation priorities, yet they have the potential to harbour genetically deeply differentiated units across environments or ecological barriers, including some that warrant taxonomic recognition. Documenting such cryptic genetic diversity is especially important for wide-ranging species that are in decline, as they may comprise a set of even more endangered lineages or species with small distributions. However, studies of wide-ranging species, particularly when they cross political borders, are extremely challenging. One approach to overcoming these challenges is to conduct detailed local analyses in combination with less detailed, range-wide studies. We used this approach with the red-footed tortoise (Chelonoidis carbonarius), a threatened species likely to contain cryptic diversity given its vast range and the distinctive ecoregions that it inhabits. Previous single-gene molecular studies indicated the presence of at least five lineages, two of which occur in different ecoregions separated by the Andes within Colombia. We used a comprehensive genomic analysis to test the hypothesis of cryptic diversity within the single jurisdiction of Colombia. We used a combination of restriction-site-associated DNA sequencing and environmental niche modelling to provide three independent lines of evidence that support the presence of important cryptic diversity that may deserve taxonomic recognition: allopatric reproductive isolation, local adaptation and ecological divergence. We also provide a fine-scale genetic map with the distribution of conservation units in Colombia. As we complete ongoing range-wide analyses and make taxonomic adjustments, we recommend that the two lineages in Colombia be treated as separate units for conservation purposes.

Las especies con distribuciones amplias rara vez son consideradas prioridades de conservación, sin embargo, tienen el potencial de albergar unidades genéticamente diferenciadas que en algunos casos justifican reconocimiento taxonómico. Documentar dicha diversidad genética críptica es especialmente importante para las especies de rangos amplios que ya están en peligro de extinción, pues pueden comprender un conjunto de linajes o especies aún más amenazadas y con distribuciones más pequeñas. Sin embargo, los estudios de especies de rangos amplios, particularmente cuando cruzan fronteras políticas, son extremadamente desafiantes. Un enfoque para superar estos desafíos es realizar análisis locales detallados en combinación con estudios en todo el rango de distribución menos detallados. Nosotros usamos este enfoque con la tortuga de patas rojas (Chelonoidis carbonarius), una especie amenazada que probablemente contiene diversidad genética críptica dada su amplia distribución y las distintas ecorregiones en las que habita. Estudios moleculares previos de un solo gen indicaron la presencia de al menos cinco linajes, dos de los cuales ocurren en diferentes ecorregiones separadas por los Andes en Colombia. En este estudio utilizamos una combinación de secuenciación de ADN asociada a sitios de restricción (RADseq) y modelamiento de nicho ecológico para proporcionar tres líneas independientes de evidencia que respaldan la presencia de diversidad críptica importante que puede merecer reconocimiento taxonómico: aislamiento reproductivo alopátrico, adaptación local y divergencia ecológica. También proporcionamos un mapa genético a escala fina con la distribución de unidades de conservación en Colombia. Mientras completamos análisis genómicos en todo el rango de distribución y hacemos ajustes taxonómicos, recomendamos que los dos linajes en Colombia se traten como unidades independientes para fines de conservación.

Mitochondrial DNA and local ecological knowledge reveal two lineages of leatherback turtle on the beaches of Oaxaca, Mexico.

Despite multiple conservation efforts of the Mexican government, the leatherback turtle is at serious risk of extinction. In this study, we investigated the possible presence of a genetic bottleneck that could prevent the recovery of this species and compared these findings with those of the olive ridley turtle, which is in true recovery. Our results confirmed that a demographic change occurred in the past and the presence of two different leatherback turtle lineages that diverged approximately 13.5 million years ago. Local ecological knowledge (LEK) also described the presence of these two lineages and warned that one is at higher risk of extinction than the other. Genetic analysis confirmed 124 mutations between the two lineages, and much lower genetic diversity in one lineage than the other. Our study highlights and substantiates the power of mixing LEK, environmental history, and genetics to better understand conservation challenges of highly threatened species such as the leatherback turtle. Moreover, we report a new lineage of the leatherback turtle which may represent a distinct species. Future studies should focus on morphological, ecological, biogeographical, evolutionary and conservation perspectives for the analysis of the new lineage.

Matamatas Chelus spp. (Testudines, Chelidae) have a remarkable evolutionary history of sex chromosomes with a long-term stable XY microchromosome system.

The genus Chelus, commonly known as Matamata is one of the most emblematic and remarkable species among the Neotropical chelids. It is an Amazonian species with an extensive distribution throughout Negro/Orinoco and Amazonas River basins. Currently, two species are formally recognized: Chelus orinocensis and Chelus fimbriata and although it is still classified as "Least Concern" in the IUCN, the Matamatas are very appreciated and illegally sold in the international pet trade. Regardless, little is known regarding many aspects of its natural history. Chromosomal features for Chelus, for instance, are meagre and practically restricted to the description of the diploid number (2n = 50) for Chelus fimbriata, and its sex determining strategies are yet to be fully investigated. Here, we examined the karyotype of Chelus fimbriata and the newly described Chelus orinocensis, applying an extensive conventional and molecular cytogenetic approach. This allowed us to identify a genetic sex determining mechanism with a micro XY sex chromosome system in both species, a system that was likely present in their most common recent ancestor Chelus colombiana. Furthermore, the XY system found in Chelus orinocensis and Chelus fimbriata, as seen in other chelid species, recruited several repeat motifs, possibly prior to the split of South America and Australasian lineages, indicating that such system indeed dates back to the earliest lineages of Chelid species.

A new lineage of Galapagos giant tortoises identified from museum samples.

The Galapagos Archipelago is recognized as a natural laboratory for studying evolutionary processes. San Cristóbal was one of the first islands colonized by tortoises, which radiated from there across the archipelago to inhabit 10 islands. Here, we sequenced the mitochondrial control region from six historical giant tortoises from San Cristóbal (five long deceased individuals found in a cave and one found alive during an expedition in 1906) and discovered that the five from the cave are from a clade that is distinct among known Galapagos giant tortoises but closely related to the species from Española and Pinta Islands. The haplotype of the individual collected alive in 1906 is in the same clade as the haplotype in the contemporary population. To search for traces of a second lineage in the contemporary population on San Cristóbal, we closely examined the population by sequencing the mitochondrial control region for 129 individuals and genotyping 70 of these for both 21 microsatellite loci and >12,000 genome-wide single nucleotide polymorphisms [SNPs]. Only a single mitochondrial haplotype was found, with no evidence to suggest substructure based on the nuclear markers. Given the geographic and temporal proximity of the two deeply divergent mitochondrial lineages in the historical samples, they were likely sympatric, raising the possibility that the lineages coexisted. Without the museum samples, this important discovery of an additional lineage of Galapagos giant tortoise would not have been possible, underscoring the value of such collections and providing insights into the early evolution of this iconic radiation.

Complex genetic patterns and distribution limits mediated by native congeners of the worldwide invasive red-eared slider turtle.

Non-native (invasive) species offer a unique opportunity to study the geographical distribution and range limits of species, wherein the evolutionary change driven by interspecific interactions between native and non-native closely related species is a key component. The red-eared slider turtle, Trachemys scripta elegans (TSE), has been introduced and successfully established worldwide. It can coexist with its native congeners T. cataspila, T. venusta and T. taylori in Mexico. We performed comprehensive fieldwork, executed a battery of genetic analyses and applied a novel species distribution modelling approach to evaluate their historical lineage relationships and contemporary population genetic patterns. Our findings support the historical common ancestry between native TSE and non-native (TSEalien ), while also highlighting the genetic differentiation of the exotic lineage. Genetic patterns are associated with their range size/endemism gradient; the microendemic T. taylori showed significant reduced genetic diversity and high differentiation, whereas TSEalien showed the highest diversity and signals of population size expansion. Counter to our expectations, lower naturally occurring distribution overlap and little admixture patterns were found between TSE and its congeners, exhibiting reduced gene flow and clear genetic separation across neighbouring species despite having zones of contact. We demonstrate that these native Trachemys species have distinct climatic niche suitability, probably preventing establishment of and displacement by the TSEalien . Moreover, we found major niche overlap between TSEalien and native species worldwide, supporting our prediction that sites with closer ecological optima to the invasive species have higher establishment risk than those that are closer to the niche-centre of the native species.

Mercury bioaccumulation, genotoxic and biochemical biomarkers reveal the health status of yellow-spotted Amazon River turtles (Podocnemis unifilis) in an environmental protection area in the Amazon / Bioacumulação de mercúrio, biomarcadores genotóxicos e bioquímicos revelam o estado de saúde de tracajás (Podocnemis unifilis) em uma área de proteção ambiental na Amazônia

Chelonians are considered good bioindicators of environmental quality. The assessment of the health status of chelonian populations in the Amazon is important because they are traditionally consumed in large numbers in riverine communities and sustainable use reserves. The present study aimed to evaluate the health of Podocnemis unifilis (Testudines, Podocnemididae) in an environmental protection area in the Amazon region in Brazil. We analyzed the biomarkers lipoperoxidation, carbonylation of proteins, occurrence of micronuclei and erythrocytic nuclear abnormalities, quantified metallothioneins, and evaluated mercury bioaccumulation. We generated pioneering data regarding biomarkers in wild Amazonian freshwater turtles. All biomarker responses did not vary significantly between the sexes. The occurrence of oxidative and genotoxic damage, as well as concentrations of metallothioneins was low compared to other studies. In addition, the bioaccumulation of mercury in the muscle of the animals was below the limit recommended for human consumption by the World Health Organization. Our results provide baseline data on mercury bioaccumulation and biomarker responses that can be useful for future comparisons with other freshwater turtles. The data also provide evidence that the sustainable exploitation of these turtles in the study area (Piagaçu-Purus Sustainable Development Reserve) does not pose a risk to local riverine communities, as the detected mercury concentrations are safe for human consumption. In this sense, our results highlight the importance of protected areas for the conservation of healthy turtle populations, at the same time ensuring the health of the human populations that use them as a food resource.(AU)

Quelônios são considerados bons bioindicadores da qualidade ambiental. A avaliação do estado de saúde de populações de quelônios na Amazônia também é importante porque estes animais são tradicionalmente consumidos em grandes quantidades em comunidades ribeirinhas e em reservas de uso sustentável. O presente estudo avaliou a saúde de Podocnemis unifilis (Testudines, Podocnemididae) em uma área de proteção ambiental da Amazônia no Brasil. Analisamos lipoperoxidação, carbonilação de proteínas, ocorrência de micronúcleos e anormalidades nucleares eritrocíticas, quantificamos metalotioneínas, e avaliamos a bioacumulação de mercúrio. Geramos dados pioneiros sobre biomarcadores em quelônios de água doce amazônicos silvestres. Todas as respostas aos biomarcadores não variaram significativamente entre os sexos. A ocorrência de danos oxidativos e genotóxicos, bem como as concentrações de metalotioneínas foram baixas em comparação a outros estudos. Adicionalmente, a bioacumulação de mercúrio no músculo dos animais ficou abaixo dos limites recomendados para consumo humano pela Organização Mundial de Saúde. Nossos resultados constituem um conjunto de dados de referência para bioacumulação de mercúrio e respostas de biomarcadores que podem ser úteis para futuras comparações com outros quelônios de água doce. Os dados também fornecem evidências de que a exploração sustentável desses quelônios na área de estudo (Reserva de Desenvolvimento Sustentável Piagaçu-Purus) não apresenta risco às comunidades ribeirinhas locais, já que as concentrações de mercúrio detectadas são seguras para o consumo humano. Nesse sentido, nossos resultados evidenciam a importância das áreas protegidas para a conservação de populações saudáveis de quelônios, concomitantemente assegurando a saúde das populações humanas que os utilizam como fonte alimentar.(AU)

"Ancient DNA" reveals that the scientific name for an extinct tortoise from Cape Verde refers to an extant South American species.

We examined the type material of the extinct tortoise species Geochelone atlantica López-Jurado, Mateo and García-Márquez, 1998 from Sal Island, Cape Verde, using aDNA approaches and AMS radiocarbon dating. High-quality mitochondrial genomes obtained from the three type specimens support that all type material belongs to the same individual. In phylogenetic analyses using mitochondrial genomes of all species groups and genera of extant and some recently extinct tortoises, the type material clusters within the extant South American red-footed tortoise Chelonoidis carbonarius (Spix, 1824). Our radiocarbon date indicates that the tortoise from which the type series of G. atlantica originates was still alive during 1962 and 1974. These results provide firm evidence that the type material of G. atlantica does not belong to the Quaternary tortoise bones excavated on Sal Island in the 1930s, as originally thought. Thus, the extinct tortoise species remains unstudied and lacks a scientific name, and the name G. atlantica has to be relegated into the synonymy of C. carbonarius. The circumstances that led to this confusion currently cannot be disentangled.

Lipid profiling suggests species specificity and minimal seasonal variation in Pacific Green and Hawksbill Turtle plasma.

In this study, we applied multiple reaction monitoring (MRM)-profiling to explore the relative ion intensity of lipid classes in plasma samples from sea turtles in order to profile lipids relevant to sea turtle physiology and investigate how dynamic ocean environments affect these profiles. We collected plasma samples from foraging green (Chelonia mydas, n = 28) and hawksbill (Eretmochelys imbricata, n = 16) turtles live captured in North Pacific Costa Rica in 2017. From these samples, we identified 623 MRMs belonging to 10 lipid classes (sphingomyelin, phosphatidylcholine, free fatty acid, cholesteryl ester, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, ceramide, and triacylglyceride) and one metabolite group (acyl-carnitine) present in sea turtle plasma. The relative ion intensities of most lipids (80%) were consistent between species, across seasons, and were not correlated to body size or estimated sex. Of the differences we observed, the most pronounced was the differences in relative ion intensity between species. We identified 123 lipids that had species-specific relative ion intensities. While some of this variability is likely due to green and hawksbill turtles consuming different food items, we found indications of a phylogenetic component as well. Of these, we identified 47 lipids that varied by season, most belonging to the structural phospholipid classes. Overall, more lipids (n = 39) had higher relative ion intensity in the upwelling (colder) season compared to the non-upwelling season (n = 8). Further, we found more variability in hawksbill turtles than green turtles. Here, we provide the framework in which to apply future lipid profiling in the assessment of health, physiology, and behavior in endangered sea turtles.

Green, yellow or black? Genetic differentiation and adaptation signatures in a highly migratory marine turtle.

Marine species may exhibit genetic structure accompanied by phenotypic differentiation related to adaptation despite their high mobility. Two shape-based morphotypes have been identified for the green turtle (Chelonia mydas) in the Pacific Ocean: the south-central/western or yellow turtle and north-central/eastern or black turtle. The genetic differentiation between these morphotypes and the adaptation of the black turtle to environmentally contrasting conditions of the eastern Pacific region has remained a mystery for decades. Here we addressed both questions using a reduced-representation genome approach (Dartseq; 9473 neutral SNPs) and identifying candidate outlier loci (67 outlier SNPs) of biological relevance between shape-based morphotypes from eight Pacific foraging grounds (n = 158). Our results support genetic divergence between morphotypes, probably arising from strong natal homing behaviour. Genes and enriched biological functions linked to thermoregulation, hypoxia, melanism, morphogenesis, osmoregulation, diet and reproduction were found to be outliers for differentiation, providing evidence for adaptation of C. mydas to the eastern Pacific region and suggesting independent evolutionary trajectories of the shape-based morphotypes. Our findings support the evolutionary distinctness of the enigmatic black turtle and contribute to the adaptive research and conservation genomics of a long-lived and highly mobile vertebrate.

Population genetics of wild and captive Trachemysvenusta (Gray, 1856) (Reptilia: Emydidae) in the Usumacinta river basin in Mexico.

The Mesoamerican slider Trachemysvenusta is endemic to Central America and Southern Mexico. Several human-mediated disturbances, including habitat degradation and illegal hunting for food, have impacted its populations along the Usumacinta river basin. The extent to which these disturbances have affected the genetic diversity and population structure of T. venusta inhabiting the basin remains unresolved. To this end, we analyzed eight microsatellite markers in five wild populations of T. venusta from the middle and lower reaches of the basin as well as one captive population. Our results show high levels of genetic diversity for all analyzed populations, low F ST values, high gene flow and no genetic structure, indicating an absence of genetic differentiation across sites and, thus, a single panmictic population for the basin. Evidence of a genetic bottleneck was observed in two of the wild populations (and the captive one), indicating some impact from disturbances, whether from poaching or habitat fragmentation, despite the seemingly high connectivity of most populations. Results are discussed in terms of the relative importance of genetic parameters for the conservation of T. venusta, particularly in light of the importance of demographic stochasticity in local conditions undergoing rapid changes.

Ancient mitogenomics elucidates diversity of extinct West Indian tortoises.

We present 10 nearly complete mitochondrial genomes of the extinct tortoise Chelonoidis alburyorum from the Bahamas. While our samples represent morphologically distinct populations from six islands, their genetic divergences were shallow and resembled those among Galápagos tortoises. Our molecular clock estimates revealed that divergence among Bahamian tortoises began ~ 1.5 mya, whereas divergence among the Galápagos tortoises (C. niger complex) began ~ 2 mya. The inter-island divergences of tortoises from within the Bahamas and within the Galápagos Islands are much younger (0.09-0.59 mya, and 0.08-1.43 mya, respectively) than the genetic differentiation between any other congeneric pair of tortoise species. The shallow mitochondrial divergences of the two radiations on the Bahamas and the Galápagos Islands suggest that each archipelago sustained only one species of tortoise, and that the taxa currently regarded as distinct species in the Galápagos should be returned to subspecies status. The extinct tortoises from the Bahamas have two well-supported clades: the first includes one sample from Great Abaco and two from Crooked Island; the second clade includes tortoises from Great Abaco, Eleuthera, Crooked Island, Mayaguana, Middle Caicos, and Grand Turk. Tortoises belonging to both clades on Great Abaco and Crooked Island suggest late Holocene inter-island transport by prehistoric humans.

DNA barcode analysis of the endangered green turtle (Chelonia mydas) in Mexico1.

Technological and analytical advances to study evolutionary biology, ecology, and conservation of green turtles (Chelonia mydas) are realized through molecular approaches including DNA barcoding. We characterized the usefulness of COI DNA barcodes in green turtles in Mexico to better understand genetic divergence and other genetic parameters of this species. We analyzed 63 sequences, including 25 from green turtle field specimens collected from the Gulf of Mexico and from the Mexican Pacific and 38 already present in the Barcode of Life Data Systems (BOLD). A total of 13 haplotypes were identified with four novel haplotypes from the Pacific Ocean and three novel haplotypes from the Atlantic Ocean. Intraspecific distance values among COI gene sequences by two different models were 0.01, demonstrating that there is not a subdivision for green turtle species. Otherwise, the interspecific distance interval ranged from 0.07 to 0.13, supporting a clear subdivision among all sea turtle species. Haplotype and total nucleotide diversity values of the COI gene reflect a medium genetic diversity average. Green turtles of the Mexican Pacific showed common haplotypes to some Australian and Chinese turtles, but different from the haplotypes of the Mexican Atlantic. COI analysis revealed new haplotypes and confirmed that DNA barcodes were useful for evaluation of the population diversity of green turtles in Mexico.

Ecology and genetic identification of freshwater turtles in Pakistan

The turtle population plays an important role in sustaining the water ecosystem by minimizing pollution from water. The identification and molecular investigation of freshwater fauna is essential for conservation of the species that are near to extinction. The quality of water, type of flora, fauna, and environmental condition are the major factors that directly affect the distribution of freshwater turtles. Two families including eight species of freshwater turtles are found in Pakistan. The Geoemydidae (Geoclemys hamiltonii, Hardella thurjii, Pangshura smithii, and Pangshura tecta) and Trionychidae (Chitra indica, Nilssonia gangetica, Nilssonia hurum, and Lissemys punctata andersoni). Studies on the species diversity and habitat of freshwater turtle have not been focused previously in the region. The present study was the first conducted to estimate the habitat and genetic diversity of freshwater turtles using 12S rRNA (ribosomal RNA) gene in Pakistan. A total of 26 samples were collected from various localities using hand net, cast net, gills net, steel hooks, thick chemical wire, using chicken intestine and small fishes. The collected turtle specimens were morpho-taxonomically categorized into two genera, Lissemys punctata andersoni (n=13, 50%) and Nilssonia gangetica (n=13, 50%). The collected species showed an aggressive and active behavior in captivity during summer. Genomic DNA was extracted from collected specimens and used in PCR reaction by using specific primers for the amplification of short fragments of 12S rRNA gene. Analysis of generated sequences confirmed the existence of L. p. andersoni in the region. The generated sequences of L. p. andersoni correspond to Clad A and showed a close resemblance among different species of the genus Lissemys. The climatic change such as temperature and rainfall have great effects on the occurrence of turtles. Habitat degradation occurred due to various factors such as draining wetlands, deforestation, converting clear water rivers to stagnant multi-purpose reservoirs and mortality on roads when turtles move around to feed. Current study concluded that the freshwater turtles L. p. andersoni and N. gangetica are interested in natural feeds. The analysis of 359 bp of 12S rRNA gene of the genus Lissemys turtles showed relationships of these turtles with cyclanorbines flap shell turtles, which agrees with previous reports. The African taxa are paraphyletic with respect to the Asian Lissemys. The ancestors of the extant genus cyclanorbines spread from North America to Asia [26]. It should be expected that each of the 3 taxa, L. p. andersoni, L. p. punctata and L. scutata represents a distinct genetic lineage. Present molecular investigation concluded that Clad A comprising L. p. punctata, L. scutata, L. cylonensis also include L. p. andersoni species. Clad B also contains one sequence from India, identified as L. p. andersoni. Their classification as conspecific evolutionary lineages are suggested by similar genetic divergences, the observation of mismatches between morphology (spotted vs. unspotted) and mitochondrial haplotypes in clades A and B. The clades A and B provides evidence for gene flow between the spotted subspecies L. p. andersoni and adjacent populations with unspotted flap shell turtles. This study is the first investigation about the habitat and of the endemic turtle species L. p. andersoni and N. gangetica in Pakistan. The genetic identification followed by phylogenetic analysis based on 12S rRNA partial genes revealed a closest similarity with the sequences generated for the same species from the neighboring countries. This study provided information to conduct further molecular studies that are essential to provide significant genetic data about turtle species.(AU)

Distribution of genetic diversity reveals colonization patterns and philopatry of the loggerhead sea turtles across geographic scales.

Understanding the processes that underlie the current distribution of genetic diversity in endangered species is a goal of modern conservation biology. Specifically, the role of colonization and dispersal events throughout a species' evolutionary history often remains elusive. The loggerhead sea turtle (Caretta caretta) faces multiple conservation challenges due to its migratory nature and philopatric behaviour. Here, using 4207 mtDNA sequences, we analysed the colonisation patterns and distribution of genetic diversity within a major ocean basin (the Atlantic), a regional rookery (Cabo Verde Archipelago) and a local island (Island of Boa Vista, Cabo Verde). Data analysis using hypothesis-driven population genetic models suggests the colonization of the Atlantic has occurred in two distinct waves, each corresponding to a major mtDNA lineage. We propose the oldest lineage entered the basin via the isthmus of Panama and sequentially established aggregations in Brazil, Cabo Verde and in the area of USA and Mexico. The second lineage entered the Atlantic via the Cape of Good Hope, establishing colonies in the Mediterranean Sea, and from then on, re-colonized the already existing rookeries of the Atlantic. At the Cabo Verde level, we reveal an asymmetric gene flow maintaining links across island-specific nesting groups, despite significant genetic structure. This structure stems from female philopatric behaviours, which could further be detected by weak but significant differentiation amongst beaches separated by only a few kilometres on the island of Boa Vista. Exploring biogeographic processes at diverse geographic scales improves our understanding of the complex evolutionary history of highly migratory philopatric species. Unveiling the past facilitates the design of conservation programmes targeting the right management scale to maintain a species' evolutionary potential.

The role of selection in the evolution of marine turtles mitogenomes.

Sea turtles are the only extant chelonian representatives that inhabit the marine environment. One key to successful colonization of this habitat is the adaptation to different energetic demands. Such energetic requirement is intrinsically related to the mitochondrial ability to generate energy through oxidative phosphorylation (OXPHOS) process. Here, we estimated Testudines phylogenetic relationships from 90 complete chelonian mitochondrial genomes and tested the adaptive evolution of 13 mitochondrial protein-coding genes of sea turtles to determine how natural selection shaped mitochondrial genes of the Chelonioidea clade. Complete mitogenomes showed strong support and resolution, differing at the position of the Chelonioidea clade in comparison to the turtle phylogeny based on nuclear genomic data. Codon models retrieved a relatively increased dN/dS (ω) on three OXPHOS genes for sea turtle lineages. Also, we found evidence of positive selection on at least three codon positions, encoded by NADH dehydrogenase genes (ND4 and ND5). The accelerated evolutionary rates found for sea turtles on COX2, ND1 and CYTB and the molecular footprints of positive selection found on ND4 and ND5 genes may be related to mitochondrial molecular adaptation to stress likely resulted from a more active lifestyle in sea turtles. Our study provides insight into the adaptive evolution of the mtDNA genome in sea turtles and its implications for the molecular mechanism of oxidative phosphorylation.

Comparative Cytogenetics of Four Sea Turtle Species (Cheloniidae): G-Banding Pattern and in situ Localization of Repetitive DNA Units.

Sea turtles are considered flagship species for marine biodiversity conservation and are considered to be at varying risk of extinction globally. Cases of hybridism have been reported in sea turtles, but chromosomal analyses are limited to classical karyotype descriptions and a few molecular cytogenetic studies. In order to compare karyotypes and understand evolutive mechanisms related to chromosome dif-ferentiation in this group, Chelonia mydas, Caretta caretta, Eretmochelys imbricata, and Lepidochelys olivacea were cytogenetically characterized in the present study. When the obtained cytogenetic data were compared with the putative ancestral Cryptodira karyotype, the studied species showed the same diploid number (2n) of 56 chromosomes, with some variations in chromosomal morphology (karyotypic formula) and minor changes in longitudinal band locations. In situ localization using a 18S ribosomal DNA probe indicated a homeologous microchromosome pair bearing a 45S ribosomal DNA locus and size heteromorphism in all 4 species. Interstitial telomeric sites were identified in a microchromosome pair in C. mydas and C. caretta. The data showed that interspecific variations occurred in chromosomal sets among the Cheloniidae species, in addition to other Cryptodira karyotypes. These variations generated lineage-specific karyotypic diversification in sea turtles, which will have considerable implications for hybrid recognition and for the study, the biology, ecology, and evolutionary history of regional and global populations. Furthermore, we demonstrated that some chromosome rearrangements occurred in sea turtle species, which is in conflict with the hypothesis of conserved karyotypes in this group.