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.

Genomics reveals broad hybridization in deeply divergent Palearctic grass and water snakes (Natrix spp.).

Understanding speciation is one of the cornerstones of biological diversity research. Currently, speciation is often understood as a continuous process of divergence that continues until genetic or other incompatibilities minimize or prevent interbreeding. The Palearctic snake genus Natrix is an ideal group to study speciation, as it comprises taxa representing distinct stages of the speciation process, ranging from widely interbreeding parapatric taxa through parapatric species with very limited gene flow in narrow hybrid zones to widely sympatric species. To understand the evolution of reproductive isolation through time, we have sequenced the genomes of all five species within this genus and two additional subspecies. We used both long-read and short-read methods to sequence and de-novo-assemble two high-quality genomes (Natrix h. helvetica, Natrix n. natrix) to their 1.7 Gb length with a contig N50 of 4.6 Mbp and 1.5 Mbp, respectively, and used these as references to assemble the remaining short-read-based genomes. Our phylogenomic analyses yielded a well-supported dated phylogeny and evidence for a surprisingly complex history of interspecific gene flow, including between widely sympatric species. Furthermore, evidence for gene flow was also found for currently allopatric species pairs. Genetic exchange among these well-defined, distinct, and several million-year-old reptile species emphasizes that speciation and maintenance of species distinctness can occur despite continued genetic exchange.

Socioeconomic differences in the utilization of diagnostic imaging and non-pharmaceutical conservative therapies for spinal diseases.

BACKGROUND: A different utilization of health care services due to socioeconomic status on the same health plan contradicts the principle of equal treatment. We investigated the presence and magnitude of socioeconomic differences in utilization of diagnostic imaging and non-pharmaceutical conservative therapies for patients with spinal diseases. METHODS: The cohort study based on routine healthcare data from Germany with 11.7 million patient-years between 2012 and 2016 for patients with physician-confirmed spinal diseases (ICD-10: M40-M54), occupation and age 20 to 64 years. A Poisson model estimated the effects of the socioeconomic status (school education, professional education and occupational position) for the risk ratio of receiving diagnostic imaging (radiography, computed tomography, magnetic resonance imaging) and non-pharmaceutical conservative therapies (physical therapy including exercise therapy, manual therapy and massage, spinal manipulative therapy, acupuncture). RESULTS: Patients received diagnostic imaging in 26%, physical therapy in 32%, spinal manipulative therapy in 25%, and acupuncture in 4% of all patient-years. Similar to previous survey-based studies higher rates of utilization were associated with higher socioeconomic status. These differences were most pronounced for manual therapy, exercise therapy, and magnetic resonance imaging. CONCLUSIONS: The observed differences in health care utilization were highly related to socioeconomic status. Socioeconomic differences were higher for more expensive health services. Further research is necessary to identify barriers to equitable access to health services and to take appropriate action to decrease existing social disparities.

Indication for spinal surgery: associated factors and regional differences in Germany.

BACKGROUND: Rising surgery rates have raised questions about the indications for spinal surgery. The study investigated patient-level and regional factors associated with spinal surgery for patients with spinal diseases. METHODS: We undertook a cohort study based on routine healthcare data from Germany of 18.4 million patients within 60.9 million episodes of two patient-years before a possible spinal surgery in the time period 2008 to 2016. Using a Poisson model, the effects of a broad range of patient-related (sociodemographic, morbidity, social status), disease- and healthcare-related (physicians' specialty, conservative treatments) and regional variables were analyzed. RESULTS: There was substantial regional heterogeneity in the occurrence of spinal surgery which decreased by only one quarter when controlling for the various determinants assessed. Previous musculoskeletal and mental health disorders as well as physical therapy were associated with a lower probability of surgery in the fully-adjusted model. Prescriptions for pain medication and consultations of specialists were associated with a higher probability of surgery. However, the specific severity of the vertebral diseases could not be taken into account in the analysis. Furthermore, a substantial proportion of patients with surgery did not receive a consultation with an outpatient specialist (29.5%), preoperative diagnostics (37.0%) or physical therapy (48.3%) before hospital admission. CONCLUSION: This large study on spinal diseases in Germany highlights important patterns in medical care of spinal diseases and their association with the probability of spinal surgery. However, only a relatively small proportion of the regional heterogeneity in spinal surgery could be explained by the extensive consideration of confounders, which suggests the relevance of other unmeasured factors like physicians' preferences.

Biogeography of Italy revisited: genetic lineages confirm major phylogeographic patterns and a pre-Pleistocene origin of its biota.

Warm-adapted species survived the cold stages of the past glacial-interglacial cycles in southern European peninsulas and recolonized major parts of Central and Northern Europe in the wake of postglacial warming. However, many of the genetic lineages which differentiated within these refugia predate even the Pleistocene. One of these refugia is the Italian Peninsula with its surrounding islands. In this meta-analysis, we compiled phylogeographic patterns of multiple species across this region. We transformed existing geographic information on 78 animal and plant species (with a total of 471 genetic lineages) within 17 predefined areas into presence/absence matrices. We elaborated three matrices: (i) only old lineages exclusively distinguished by deep splits, (ii) only young lineages distinguished by shallow (i.e. recent) splits, and (iii) presence/absence of the respective species. To infer biogeographic relationships between the predefined areas, we performed bootstrapped neighbour joining cluster analyses on these three matrices. In addition, we reviewed the geological history of Italy to identify causes of the observed biogeographic patterns. We found Sardinia and Corsica to be biogeographically closely linked with each other, and that they diverge strongly from all other regions. Sicily also diverges strongly from all other regions, while the intra-island differentiation was comparatively low. On the Italian mainland, Calabria exhibited the most pronounced biogeographic differentiation, often with several lineages present, resulting from old vicariance events within the region. Furthermore, southern Apulia and the Po Plain with adjoining areas of northern peninsular Italy displayed considerable distinctiveness. Admixture prevailed in the areas between these three regions. The ancient isolation of Sicily, as well as Sardinia plus Corsica, resulted in endemic lineages with only moderate recent exchange with adjacent mainland regions. Pronounced diversification occurs within the Italian Peninsula. The complex tectonic activities, such as shifting (micro)plates, submergence of major parts of peninsular Italy with the genesis of numerous Pliocene islands, in combination with the climatic cycles during the Pleistocene have most likely generated the current biogeographic pattern of species. Immigrations from the Balkan Peninsula into northern Italy partly accounted for the distinctiveness of this region.

Population structure and gene flow of the syntopic turtles Emys and Mauremys from coastal and inland regions of Anatolia (Turkey): results from mitochondrial and microsatellite data.

Revealing the genetic basis of the existence of different species living together in different geographic regions provides clarification of this phylogeographic differentiation. In this study, we investigated the population genetics and evaluated the level of genetic variation of inland and coastal populations of Mauremys and Emys in Turkey. Tissue samples of 196 terrapins were studied which were collected from syntopic coastal (Gölbent-Söke/Aydin; M. rivulata and E. orbicularis) and inland populations (Bahçesaray/Aksaray; M. caspica and E. orbicularis). DNA was isolated using the InnuPREP DNA Mini Kit. Mitochondrial DNA sequences and allelic variation at 13 microsatellite loci for Mauremys and 12 microsatellite loci for Emys were examined.  Three haplotypes were found for Emys orbicularis (Im, Ip and Iw) collected from the coastal region and two haplotypes for Emys orbicularis (Ig and Im) collected from inland. Two haplotypes were identified for M. caspica (Cmt8 and Cmt9) and three haplotypes were identified for M. rivulata (Rmt3, Rmt24 and Rmt26). Using microsatellites and the software STRUCTURE the most probable value for K was revealed as two 2 for both species. The FST value between M. rivulata and M. caspica was 0.39, and between the coastal and inland populations of E. orbicularis 0.09. It can be concluded that Emys populations tend to evolve by somehow preserving the allelic richness they have and Mauremys populations continue to differentiate so that new species emerge in the evolutionary process to reach the ideal allelic structure.

Genomic analyses reveal two species of the matamata (Testudines: Chelidae: Chelus spp.) and clarify their phylogeography.

The matamata is one of the most charismatic turtles on earth, widely distributed in northern South America. Debates have occurred over whether or not there should be two subspecies or species recognized due to its geographic variation in morphology. Even though the matamata is universally known, its natural history, conservation status and biogeography are largely unexplored. In this study we examined the phylogeographic differentiation of the matamata based on three mitochondrial DNA fragments (2168 bp of the control region, cytochrome oxidase subunit I, and the cytochrome b gene), one nuclear genomic DNA fragment (1068 bp of the R35 intron) and 1661 Single Nucleotide Polymorphisms (SNPs). Our molecular and morphological analyses revealed the existence of two distinct, genetically deeply divergent evolutionary lineages of matamatas that separated in the late Miocene (approximately 12.7 million years ago), corresponding well to the time when the Orinoco Basin was established. As a result of our analyses, we describe the genetically and morphologically highly distinct matamata from the Orinoco and Río Negro Basins and the Essequibo drainage as a species new to science (Chelus orinocensis sp. nov.). Chelus fimbriata sensu stricto is distributed in the Amazon Basin and the Mahury drainage. Additionally, the analyses revealed that each species displays phylogeographic differentiation. For C. orinocensis, there is moderate mitochondrial differentiation between the Orinoco and the Río Negro. For C. fimbriata, there is more pronounced differentiation matching different river systems. One mitochondrial clade was identified from the Amazon, Ucayali, and Mahury Rivers, and another one from the Madeira and Jaci Paraná Rivers. The C. orinocensis in the Essequibo and Branco Rivers have haplotypes that constitute a third clade clustering with C. fimbriata. Phylogenetic analyses of the R35 intron and SNP data link the matamatas from the Essequibo and Branco with the new species, suggesting past gene flow and old mitochondrial introgression. Chelus orinocensis is collected for the pet trade in Colombia and Venezuela. However, neither the extent of the harvest nor its impact are known. Hence, it is crucial to gather more information and to assess its exploitation throughout its distribution range to obtain a better understanding of its conservation status and to design appropriate conservation and management procedures. RESUMEN: La matamata es una de las tortugas más carismáticas del mundo, ampliamente distribuida en el norte de Sudamérica. Debido a su variación morfológica geográfica, se debate sobre el reconocimiento de dos subespecies o especies. A pesar de que la matamata es universalmente conocida, su historia natural, estado de conservación y biogeografía han sido muy poco estudiados. En este estudio examinamos la diferenciación filogeográfica de las matamatas en base ​​a tres fragmentos de ADN mitocondrial (2168 pb de la región de control, la subunidad I del citocromo oxidasa y el gen del citocromo b), un fragmento de ADN genómico nuclear (1068 pb del intrón R35) y 1661 polimorfismos de nucleótido único (SNPs). Nuestros análisis moleculares y morfológicos revelaron la existencia de dos linajes evolutivos distintos de matamatas, genéticamente divergentes que se separaron en el Mioceno tardio (hace aproximadamente 12.7 millones de años), correspondiendo al tiempo en que se estableció la cuenca del Orinoco. Como resultado de nuestros análisis, describimos las genéticamente y morfológicamente distintas matamatas de las cuencas del Orinoco, Río Negro y Essequibo como una especie nueva para la ciencia (Chelus orinocensis sp. nov.). Chelus fimbriata sensu stricto se distribuye en la cuenca del Amazonas y en el drenaje del Mahury. Adicionalmente, los análisis revelaron que cada especie muestra diferenciación filogeográfica. Para C. orinocensis, hay una moderada diferenciación mitocondrial entre el Orinoco y el Río Negro. Para C. fimbriata, hay una diferenciación más pronunciada, concordando con los diferentes sistemas fluviales. Se identificó un clado de los ríos Amazonas, Ucayali y Mahury y otro de los ríos Madeira y Jaci Paraná. Las C. orinocensis de los ríos Essequibo y Branco tienen haplotipos que constituyen un tercer clado que se agrupa con C. fimbriata. Los análisis filogenéticos del intrón R35 y los datos de SNP asocian las matamatas de Essequibo y Branco con la nueva especie, sugiriendo flujo de genes pasado ​​e introgresión mitocondrial antigua. Chelus orinocensis se colecta para el comercio de mascotas en Colombia y Venezuela. Sin embargo, ni se conoce el alcance de las colectas ni su impacto. Por lo tanto, es crucial recopilar más información y evaluar su explotación en todo su rango de distribución, comprender mejor su estado de conservación y para diseñar acciones apropiadas de conservación y manejo.

Low Hospital Volume Increases Revision Rate and Mortality Following Revision Total Hip Arthroplasty: An Analysis of 17,773 Cases.

BACKGROUND: With the number of primary total hip arthroplasty (THA), the amount of revision THA (R-THA) increases. R-THA is a complex procedure requiring special instruments, implants, and surgical skills. Therefore it is likely that hospitals performing a higher number of R-THAs have more experience with this type of surgery and therefore fewer complications. The purpose of this study was to evaluate the relationship between hospital volume and risk of postoperative complications following R-THA. METHODS: Using nationwide healthcare insurance data for inpatient hospital treatment, 17,773 aseptic R-THAs in 16,376 patients treated between January 2014 and December 2016 were included. Outcomes were 90-day mortality, 1-year revision procedures, and in-house adverse events. The effect of hospital volume on outcome was analyzed by means of multivariate logistic regression. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. RESULTS: Hospital volume had a significant effect on 90-day mortality (≤12 cases per year: OR 2.13, CI 1.53-2.96; 13-24: OR 1.79, CI 1.29-2.50; 25-52: OR 1.53, CI 1.11-2.10; ≥53: reference) and 1-year revision procedures (≤12: OR 1.26, CI 1.09-1.47; 13-24: OR 1.18, CI 1.02-1.37; 25-52: OR 1.03, CI 0.90-1.19; ≥53: reference). There was no significant effect on risk-adjusted major in-house adverse events. CONCLUSION: We found evidence of higher risk for revision surgery and mortality in hospitals with fewer than 25 and 53 R-THA per year, respectively. To improve patient care, complex elective procedures like R-THA which require experience and a specific logistic background should be performed in specialized centers.

Millennium-old farm breeding of Chinese softshell turtles (Pelodiscus spp.) results in massive erosion of biodiversity.

Chinese softshell turtles (Pelodiscus spp.) are widely distributed, ranging from the Amur and Ussuri Rivers in the Russian Far East through the Korean Peninsula, Japan, and eastern, central, and southern China to southern Vietnam. In East and Southeast Asia, Chinese softshell turtles are traditionally exploited for food and have been farm-bred in China since the Spring and Autumn Period, more than 2400 years ago. Currently, the annual production of Pelodiscus amounts to 340,000 t in China alone. Using mitochondrial DNA (2428 bp) and five nuclear loci (3704 bp), we examined broad sampling of wild and farm-bred Pelodiscus to infer genetic and taxonomic differentiation. We discovered four previously unknown mitochondrial lineages, all from China. One lineage from Jiangxi is deeply divergent and sister to the mitochondrial lineage of Pelodiscus axenaria. The nuclear loci supported species status for P. axenaria and the new lineage from Jiangxi. Pelodiscus maackii and P. parviformis, both harboring distinct mitochondrial lineages, were not differentiated from P. sinensis in the studied nuclear markers. The same is true for two new mitochondrial lineages from Zhejiang, China, represented by only one individual each, and another new lineage from Anhui, Guangdong, Jiangxi and Zhejiang, China. However, Vietnamese turtles yielding a mitochondrial lineage clustering within P. sinensis were distinct in nuclear markers, suggesting that these populations could represent another unknown species with introgressed mitochondria. Its species status is also supported by the syntopic occurrence with P. sinensis in northern Vietnam and by morphology. In addition, we confirmed sympatry of P. axenaria and P. parviformis in Guangxi, China, and found evidence for sympatry of P. sinensis and the new putative species from Jiangxi, China. We also discovered evidence for hybridization in turtle farms and for the occurrence of alien lineages in the wild (Zhejiang, China), highlighting the risk of genetic pollution of native stock. In the face of the large-scale breeding of Pelodiscus, we claim that the long-term survival of distinct genetic lineages and species can only be assured when an upscale market segment for pure-bred softshell turtles is established, making the breeding of pure lineages lucrative for turtle farms. Our findings underline that the diversity of Pelodiscus is currently underestimated and threatened by anthropogenic admixture. We recommend mass screening of genetic and morphological variation of Chinese softshell turtles as a first step to understand and preserve their diversity.

Tropical ancient DNA reveals relationships of the extinct Bahamian giant tortoise Chelonoidis alburyorum.

Ancient DNA of extinct species from the Pleistocene and Holocene has provided valuable evolutionary insights. However, these are largely restricted to mammals and high latitudes because DNA preservation in warm climates is typically poor. In the tropics and subtropics, non-avian reptiles constitute a significant part of the fauna and little is known about the genetics of the many extinct reptiles from tropical islands. We have reconstructed the near-complete mitochondrial genome of an extinct giant tortoise from the Bahamas (Chelonoidis alburyorum) using an approximately 1 000-year-old humerus from a water-filled sinkhole (blue hole) on Great Abaco Island. Phylogenetic and molecular clock analyses place this extinct species as closely related to Galápagos (C. niger complex) and Chaco tortoises (C. chilensis), and provide evidence for repeated overseas dispersal in this tortoise group. The ancestors of extant Chelonoidis species arrived in South America from Africa only after the opening of the Atlantic Ocean and dispersed from there to the Caribbean and the Galápagos Islands. Our results also suggest that the anoxic, thermally buffered environment of blue holes may enhance DNA preservation, and thus are opening a window for better understanding evolution and population history of extinct tropical species, which would likely still exist without human impact.

Are TKAs Performed in High-volume Hospitals Less Likely to Undergo Revision Than TKAs Performed in Low-volume Hospitals?

BACKGROUND: High-volume hospitals have achieved better outcomes for THAs and unicompartmental knee arthroplasties (UKAs). However, few studies have analyzed implant survival after primary TKA in high-volume centers. QUESTIONS/PURPOSES: Is the risk of revision surgery higher when receiving a TKA in a low-volume hospital than in a high-volume hospital? METHODS: Using nationwide billing data of the largest German healthcare insurer for inpatient hospital treatment, we identified 45,165 TKAs in 44,465 patients insured by Allgemeine Ortskrankenkasse who had undergone knee replacement surgery between January 2012 and December 2012. Revision rates were calculated at 1 and 2 years in all knees. The hospital volume was calculated using volume quintiles of the number of all knee arthroplasties performed in each center. We used multiple logistic regression to model the odds of revision surgery as a function of hospital volume. Age, sex, 31 comorbidities, and variables for socioeconomic status were included as independent variables in the model. RESULTS: After controlling for socioeconomic factors, patient age, sex, and comorbidities, we found that having surgery in a high-volume hospital was associated with a decreased risk of having revision TKA within 2 years of the index procedure. The odds ratio for the 2-year revision was 1.6 (95% CI, 1.4-2.0; p < 0.001) for an annual hospital volume of 56 or fewer cases, 1.5 (95% CI, 1.3-1.7; p < 0.001) for 57 to 93 cases, 1.2 (95% CI, 1.0-1.3; p = 0.039) for 94 to 144 cases, and 1.1 (95% CI, 0.9-1.2; p = 0.319) for 145 to 251 cases compared with a hospital volume of 252 or more cases. CONCLUSIONS: We found a clear association of higher risk for revision surgery when undergoing a TKA in a hospital where less than 145 arthroplasties per year were performed. The study results could help practitioners to guide potential patients in hospitals that perform more TKAs to reduce the overall revision and complication rates. Furthermore, this study underscores the importance of a minimum hospital threshold of arthroplasty cases per year to get permission to perform an arthroplasty. LEVEL OF EVIDENCE: Level III, therapeutic study.

Early mesozoic coexistence of amniotes and hepadnaviridae.

Hepadnaviridae are double-stranded DNA viruses that infect some species of birds and mammals. This includes humans, where hepatitis B viruses (HBVs) are prevalent pathogens in considerable parts of the global population. Recently, endogenized sequences of HBVs (eHBVs) have been discovered in bird genomes where they constitute direct evidence for the coexistence of these viruses and their hosts from the late Mesozoic until present. Nevertheless, virtually nothing is known about the ancient host range of this virus family in other animals. Here we report the first eHBVs from crocodilian, snake, and turtle genomes, including a turtle eHBV that endogenized >207 million years ago. This genomic "fossil" is >125 million years older than the oldest avian eHBV and provides the first direct evidence that Hepadnaviridae already existed during the Early Mesozoic. This implies that the Mesozoic fossil record of HBV infection spans three of the five major groups of land vertebrates, namely birds, crocodilians, and turtles. We show that the deep phylogenetic relationships of HBVs are largely congruent with the deep phylogeny of their amniote hosts, which suggests an ancient amniote-HBV coexistence and codivergence, at least since the Early Mesozoic. Notably, the organization of overlapping genes as well as the structure of elements involved in viral replication has remained highly conserved among HBVs along that time span, except for the presence of the X gene. We provide multiple lines of evidence that the tumor-promoting X protein of mammalian HBVs lacks a homolog in all other hepadnaviruses and propose a novel scenario for the emergence of X via segmental duplication and overprinting of pre-existing reading frames in the ancestor of mammalian HBVs. Our study reveals an unforeseen host range of prehistoric HBVs and provides novel insights into the genome evolution of hepadnaviruses throughout their long-lasting association with amniote hosts.

Disentangling the Pelomedusa complex using type specimens and historical DNA (Testudines: Pelomedusidae).

Recent research has shown that the helmeted terrapin (Pelomedusa subrufa), a species that occurs throughout sub-Saharan Africa, in Madagascar and the southwestern Arabian Peninsula, consists of several deeply divergent genetic lineages. Here we examine all nominal taxa currently synonymized with Pelomedusa subrufa (Bonnaterre, 1789) and provide mitochondrial DNA sequences of type specimens or topotypic material for most taxa. Lectotypes are designated for Testudo galeata Schoepff, 1792, Pentonyx capensis Duméril & Bibron, 1835, Pelomedusa nigra Gray, 1863, Pelomedusa galeata var. disjuncta Vaillant & Grandidier, 1910, and Pelomedusa galeata damarensis Hewitt, 1935. For Pelomedusa gasconi Rochebrune, 1884, a taxon without preserved type material, a neotype is designated. Type material of Pentonix americana Cornalia, 1849, a nominal species without credible type locality, is lost and its identity remains questionable. Also the holotype of Pelomedusa galeata orangensis Hewitt, 1935 is lost, but its allocation to the only genetic lineage occurring in South Africa is unambiguous. Phylogenetic analyses of type sequences or topotypic material reveal that the remaining nominal taxa represent three of the nine previously identified lineages of Pelomedusa. Among these three lineages is the South African one. Type specimens of Pentonyx gehafie Rüppell, 1835 correspond to an additional distinct lineage. The present study provides a sound basis for a subsequent integrative taxonomic revision of the Pelomedusa complex.

A revision of African helmeted terrapins (Testudines: Pelomedusidae: Pelomedusa), with descriptions of six new species.

Using nearly range-wide sampling, we analyze up to 1848 bp of mitochondrial DNA of 183             helmeted terrapins and identify a minimum of 12 deeply divergent species-level clades. Uncorrected p distances of these clades equal or clearly exceed those between the currently recognized species of Pelusios, the genus most closely related to Pelomedusa. We correlate genetic discontinuities of Pelomedusa with data on morphology and endoparasites and describe six new Pelomedusa species. Moreover, we restrict the name Pelomedusa subrufa (Bonnaterre, 1789) to one genetic lineage and resurrect three further species from its synonymy, namely P. galeata (Schoepff, 1792), P. gehafie (Rüppell, 1835), and P. olivacea (Schweigger, 1812). In addition to these ten Pelomedusa species, we identify two further clades from Cameroon and Sudan with similar levels of genetic divergence that remain unnamed candidate species. We also note that some problematical terrapins from South Africa and Somalia may represent two additional candidate species. Some of the Pelomedusa species are morphologically distinctive, whilst others can only be identified by molecular markers and are therefore morphologically cryptic taxa.

Genetic identification, morphology and distribution of Natrixhelvetica subspecies in southern and western Switzerland (Reptilia, Squamata, Serpentes).

Most of Switzerland is inhabited by the nominotypical subspecies of the barred grass snake (Natrixhelveticahelvetica), which is characterized by mitochondrial DNA lineage E. Only in the northeast of the country, the common grass snake (N.natrix) occurs and hybridizes with N.h.helvetica in a narrow contact zone. However, we discovered that in southern and western Switzerland barred grass snakes representing another mtDNA lineage (lineage C) are widely distributed. Lineage C is typical for Alpine populations of the southern subspecies N.h.sicula. Our microsatellite analyses of the Swiss samples revealed differences between the two subspecies and also a substructure with two clusters in each subspecies. Furthermore, we discovered a contact and hybrid zone of N.h.helvetica and N.h.sicula along the northern shore of Lake Geneva and also confirm that interbreeding with alien common grass snakes (N.n.moreotica, mtDNA lineage 7) occurs there. This finding is of concern for nature conservation and measures should be taken to prevent further genetic pollution. Using morphometrics, we found no differences between the two subspecies of N.helvetica, while N.natrix was slightly distinct from N.helvetica.

Taking advantage of reference-guided assembly in a slowly-evolving lineage: Application to Testudo graeca.

BACKGROUND: Obtaining de novo chromosome-level genome assemblies greatly enhances conservation and evolutionary biology studies. For many research teams, long-read sequencing technologies (that produce highly contiguous assemblies) remain unaffordable or unpractical. For the groups that display high synteny conservation, these limitations can be overcome by a reference-guided assembly using a close relative genome. Among chelonians, tortoises (Testudinidae) are considered one of the most endangered taxa, which calls for more genomic resources. Here we make the most of high synteny conservation in chelonians to produce the first chromosome-level genome assembly of the genus Testudo with one of the most iconic tortoise species in the Mediterranean basin: Testudo graeca. RESULTS: We used high-quality, paired-end Illumina sequences to build a reference-guided assembly with the chromosome-level reference of Gopherus evgoodei. We reconstructed a 2.29 Gb haploid genome with a scaffold N50 of 107.598 Mb and 5.37% gaps. We sequenced 25,998 protein-coding genes, and identified 41.2% of the assembly as repeats. Demographic history reconstruction based on the genome revealed two events (population decline and recovery) that were consistent with previously suggested phylogeographic patterns for the species. This outlines the value of such reference-guided assemblies for phylogeographic studies. CONCLUSIONS: Our results highlight the value of using close relatives to produce de novo draft assemblies in species where such resources are unavailable. Our annotated genome of T. graeca paves the way to delve deeper into the species' evolutionary history and provides a valuable resource to enhance direct conservation efforts on their threatened populations.

Complex phylogeography in Rhinoclemmys melanosterna: conflicting mitochondrial and nuclear evidence suggests past hybridization (Testudines: Geoemydidae).

We examined differentiation within the Colombian wood turtle Rhinoclemmys melanosterna, and among R. melanosterna and the closely allied species R. diademata, R.funerea and R. punctularia, based on 1060 base pairs of the mitochondrial cyt b gene. We also assessed the phylogenetic relationships among these species using 2050 bp of mtDNA (partial cyt b, 12S and 16S genes) and 3620 bp of nuclear DNA (partial Rag 1, Rag 2, C-mos, R35 and ODC genes). There is considerable phylogeographic structuring within R. melanosterna, with seven distinct clades distributed across the species' range. These clades correlate to some extent with previously described differences in the dorsal pattern of head coloration. Individual and combined analyses of mitochondrial and nuclear DNA indicated contradictory relationships among R. melanosterna, R. diademata, R. funerea and R. punctularia. Mitochondrial DNA sequences revealed R. melanosterna to be non-monophyletic with respect to R. diademata, R. funerea and R. punctularia. In contrast, R. melanosterna constituted a well-supported monophyletic clade using nuclear DNA. This conflict between mitochondrial and nuclear data suggests past gene flow among the allopatrically and parapatrically distributed species R. melanosterna, R. diademata, R. funerea and R. punctularia. Compared to the other Rhinoclemmys species, the taxa under study are weakly differentiated. To assess their taxonomic status, further research is warranted using additional nuclear markers and additional samples of R. diademata, R. funerea and R. punctularia. For the time being, a continued classification of R. melanosterna, R. diademata, R. funerea and R. punctularia as distinct species is justified owing to their allopatric and parapatric distributions, and to conserve the established usage of names that is based on morphological and karyotypic differentiation.

Skeletal repatterning enhances the protective capacity of the shell in African hinge-back tortoises (Kinixys).

Changes in the structural association of skeletal traits are crucial to the evolution of novel forms and functions. In vertebrates, such rearrangements often occur gradually and may precede or coincide with the functional activation of skeletal traits. To illustrate this process, we examined the ontogeny of African hinge-back tortoises (Kinixys spp.). Kinixys species feature a moveable "hinge" on the dorsal shell (carapace) that enables shell closure (kinesis) when the hind limbs are withdrawn. This hinge, however, is absent in juveniles. Herein, we describe how this unusual phenotype arises via alterations in the tissue configuration and shape of the carapace. The ontogenetic repatterning of osseous and keratinous tissue coincided with shifts in morphological integration and the establishment of anterior (static) and posterior (kinetic) carapacial modules. Based on ex vivo skeletal movement and raw anatomy, we propose that Kinixys employs a "sliding hinge" shell-closing system that overcomes thoracic rigidity and enhances the protective capacity of the carapace. Universal properties of the vertebrate skeleton, such as plasticity, modularity, and secondary maturation processes, contributed to adaptive evolutionary change in Kinixys. We discuss a hypothetical model to explain the delayed emergence of skeletal traits and its relevance to the origins of novel form-to-function relationships.

Genotyping the phenotypic diversity in Aegean Natrixnatrixmoreotica (Bedriaga, 1882) (Reptilia, Serpentes, Natricidae).

We examined the mitochondrial identity of Aegean Natrixnatrixmoreotica representing different morphotypes, with a focus on new material from Milos and Skyros. We found no correlation between distinct morphotypes and mitochondrial identity. Our results support that grass snake populations are polyphenetic and that southern subspecies, including island populations, show a higher variability than northern ones.

Ancient DNA elucidates the lost world of western Indian Ocean giant tortoises and reveals a new extinct species from Madagascar.

Before humans arrived, giant tortoises occurred on many western Indian Ocean islands. We combined ancient DNA, phylogenetic, ancestral range, and molecular clock analyses with radiocarbon and paleogeographic evidence to decipher their diversity and biogeography. Using a mitogenomic time tree, we propose that the ancestor of the extinct Mascarene tortoises spread from Africa in the Eocene to now-sunken islands northeast of Madagascar. From these islands, the Mascarenes were repeatedly colonized. Another out-of-Africa dispersal (latest Eocene/Oligocene) produced on Madagascar giant, large, and small tortoise species. Two giant and one large species disappeared c. 1000 to 600 years ago, the latter described here as new to science using nuclear and mitochondrial DNA. From Madagascar, the Granitic Seychelles were colonized (Early Pliocene) and from there, repeatedly Aldabra (Late Pleistocene). The Granitic Seychelles populations were eradicated and later reintroduced from Aldabra. Our results underline that integrating ancient DNA data into a multi-evidence framework substantially enhances the knowledge of the past diversity of island faunas.