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Acoustic communication, broadly distributed along the vertebrate phylogeny, plays a fundamental role in parental care, mate attraction and various other behaviours. Despite its importance, comparatively less is known about the evolutionary roots of acoustic communication. Phylogenetic comparative analyses can provide insights into the deep time evolutionary origin of acoustic communication, but they are often plagued by missing data from key species. Here we present evidence for 53 species of four major clades (turtles, tuatara, caecilian and lungfish) in the form of vocal recordings and contextual behavioural information accompanying sound production. This and a broad literature-based dataset evidence acoustic abilities in several groups previously considered non-vocal. Critically, phylogenetic analyses encompassing 1800 species of choanate vertebrates reconstructs acoustic communication as a homologous trait, and suggests that it is at least as old as the last common ancestor of all choanate vertebrates, that lived approx. 407 million years before present.

Despite the relevance of chemical communication in vertebrates, comparative examinations of macroevolutionary trends in chemical signaling systems are scarce. Many turtle and tortoise species are reliant on chemical signals to communicate in aquatic and terrestrial macrohabitats, and many of these species possess specialized integumentary organs, termed mental glands (MGs), involved in the production of chemosignals. We inferred the evolutionary history of MGs and tested the impact of macrohabitat on their evolution. Inference of ancestral states along a time-calibrated phylogeny revealed a single origin in the ancestor of the subclade Testudinoidea. Thus, MGs represent homologous structures in all descending lineages. We also inferred multiple independent losses of MGs in both terrestrial and aquatic clades. Although MGs first appeared in an aquatic turtle (the testudinoid ancestor), macrohabitat seems to have had little effect on MG presence or absence in descendants. Instead, we find clade-specific evolutionary trends, with some clades showing increased gland size and morphological complexity, whereas others exhibiting reduction or MG loss. In sister clades inhabiting similar ecological niches, contrasting patterns (loss vs. maintenance) may occur. We conclude that the multiple losses of MGs in turtle clades have not been influenced by macrohabitat and that other factors have affected MG evolution.

Telomeres are nucleoprotein complexes protecting chromosome ends in most eukaryotic organisms. In addition to chromosome ends, telomeric-like motifs can be accumulated in centromeric, pericentromeric and intermediate (i.e., between centromeres and telomeres) positions as so-called interstitial telomeric repeats (ITRs). We mapped the distribution of (TTAGGG)n repeats in the karyotypes of 30 species from nine families of turtles using fluorescence in situ hybridization. All examined species showed the expected terminal topology of telomeric motifs at the edges of chromosomes. We detected ITRs in only five species from three families. Combining our and literature data, we inferred seven independent origins of ITRs among turtles. ITRs occurred in turtles in centromeric positions, often in several chromosomal pairs, in a given species. Their distribution does not correspond directly to interchromosomal rearrangements. Our findings support that centromeres and non-recombining parts of sex chromosomes are very dynamic genomic regions, even in turtles, a group generally thought to be slowly evolving. However, in contrast to squamate reptiles (lizards and snakes), where ITRs were found in more than half of the examined species, and birds, the presence of ITRs is generally rare in turtles, which agrees with the expected low rates of chromosomal rearrangements and rather slow karyotype evolution in this group.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Map turtles of the genus Graptemys are native to North America, where a high degree of drainage endemism is believed to have shaped current diversity. With 14 species and one additional subspecies, Graptemys represents the most diverse genus in the family Emydidae. While some Graptemys species are characterized by pronounced morphological differences, previous phylogenetic analyses have failed yet to confirm significant levels of genetic divergence for many taxa. As a consequence, it has been debated whether Graptemys is taxonomically inflated or whether the low genetic divergence observed reflects recent radiations or ancient hybridization. In this study, we analysed three mtDNA blocks (3228 bp) as well as 12 nuclear loci (7844 bp) of 89 specimens covering all species and subspecies of Graptemys. Our analyses of the concatenated mtDNA sequences reveal that the widespread G. geographica constitutes the sister taxon of all other Graptemys species. These correspond to two clades, one comprised of all broad-headed Graptemys species and another clade containing the narrow-headed species. Most species of the broad-headed clade are reciprocally monophyletic, except for G. gibbonsi and G. pearlensis, which are not differentiated. By contrast, in the narrow-headed clade, many currently recognized species are not monophyletic and divergence is significantly less pronounced. Haplotype networks of phased nuclear loci show low genetic divergence among taxa and many shared haplotypes. Principal component analyses using coded phased nuclear DNA sequences revealed eight distinct clusters within Graptemys that partially conflict with the terminal mtDNA clades. This might be explained by male-mediated gene flow across drainage basins and female philopatry within drainage basins. Our results support that Graptemys is taxonomically oversplit and needs to be revised.

Turtles demonstrate variability in sex determination and, hence, constitute an excellent model for the evolution of sex chromosomes. Notably, the sex determination of the freshwater turtles from the family Chelidae, a species-rich group with wide geographical distribution in the southern hemisphere, is still poorly explored. Here we documented the presence of an XX/XY sex determination system in seven species of the Australasian chelid genera Chelodina, Emydura, and Elseya by conventional (karyogram reconstruction, C-banding) and molecular cytogenetic methods (comparative genome hybridization, in situ hybridization with probes specific for GATA microsatellite motif, the rDNA loci, and the telomeric repeats). The sex chromosomes are microchromosomes in all examined species of the genus Chelodina. In contrast, the sex chromosomes are the 4th largest pair of macrochromosomes in the genera Emydura and Elseya. Their X chromosomes are submetacentric, while their Y chromosomes are metacentric. The chelid Y chromosomes contain a substantial male-specific genomic region with an accumulation of the GATA microsatellite motif, and occasionally, of the rDNA loci and telomeric repeats. Despite morphological differences between sex chromosomes, we conclude that male heterogamety was likely already present in the common ancestor of Chelodina, Emydura and Elseya in the Mesozoic period.

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.

Using up to 2456 bp mtDNA and up to 2716 bp nDNA of fresh samples and short sequences of three mitochondrial genes of historical museum material, we examine the phylogeography of Amyda cartilaginea. This data set provides evidence for the existence of deeply divergent genetic lineages which we interpret as three distinct species, two of which are polytypic. On the Great Sunda Islands, the distribution ranges of the two subspecies of Amyda cartilaginea (Boddaert, 1770) sensu stricto and of an undescribed species match palaeodrainage systems. Amyda cartilaginea cartilaginea occurs in the East Sunda palaeodrainage, with records in eastern Borneo and Java. Also a record from Sulawesi, most probably not representing a native population, refers to A. c. cartilaginea. In the North Sunda palaeodrainage (Sumatra, western Borneo) lives Amyda cartilaginea maculosa subsp. nov., which is described herein. One sample from the Baram river (Sarawak, Malaysia) is genetically highly distinct and represents a new species. We refrain from naming this taxon until more material becomes available for morphological characterization. For the continental populations, we resurrect the species Amyda ornata (Gray, 1861). We identify Asian softshell turtles from the Mekong drainage with the nominotypical subspecies, while the genetically distinct populations from Thailand and Myanmar are assigned to Amyda ornata phayrei (Theobald, 1868). Samples from Bangladesh are also genetically distinct and represent an undescribed subspecies and the first country record for Amyda.

Fritz, U., Alcalde, L., Vargas-Ramirez, M., Goode, E.V., Fabius-Turoblin, D.U. & Praschag, P. (2012). Northern genetic richness and southern purity, but just one species in the Chelonoidis chilensis complex. —Zoologica Scripta, 41, 220–232. The Chelonoidis chilensis complex, the sister group of the famous Galapagos tortoises, is a widely distributed group of South American land tortoises, ranging from the dry Chaco of Bolivia, Paraguay and northern Argentina to northern Patagonia. Within this complex, up to three distinct species have been recognized. Using sequence data of the mitochondrial cytochrome b gene and length polymorphisms of 10 microsatellite loci, we investigate genetic differentiation among all three nominal species. We find only negligible differentiation, with decreasing genetic diversity from north to south. We conclude that only one species, Chelonoidis chilensis (Gray, 1870), is valid, with C. donosobarrosi (Freiberg, 1973) and C. petersi (Freiberg, 1973) as its junior synonyms. Morphological variation within C. chilensis sensu lato is in accord with the observation that size variation in chelonians follows Bergmann’s rule, with body size increasing with latitude. The observed phylogeographic differentiation inverses the well-known pattern of southern genetic richness and northern purity from the northern hemisphere, resulting from dispersal from glacial refugia. This implies that in higher latitudes of both hemispheres genetic diversity may decrease with increasing distance from the refugium. For C. chilensis sensu lato, it seems likely that long-distance dispersal via rafting on the Desaguadero River led to the foundation of the southernmost populations in northern Patagonia during the Holocene.

For a long time, turtles of the family Geoemydidae have been considered exceptional because representatives of this family were thought to possess a wide variety of sex determination systems. In the present study, we cytogenetically studiedGeoemyda spengleriandG. japonicaand re-examined the putative presence of sex chromosomes inPangshura smithii. Karyotypes were examined by assessing the occurrence of constitutive heterochromatin, by comparative genome hybridization andin situhybridization with repetitive motifs, which are often accumulated on differentiated sex chromosomes in reptiles. We found similar karyotypes, similar distributions of constitutive heterochromatin and a similar topology of tested repetitive motifs for all three species. We did not detect differentiated sex chromosomes in any of the species. ForP. smithii, a ZZ/ZW sex determination system, with differentiated sex chromosomes, was described more than 40 years ago, but this finding has never been re-examined and was cited in all reviews of sex determination in reptiles. Here, we show that the identification of sex chromosomes in the original report was based on the erroneous pairing of chromosomes in the karyogram, causing over decades an error cascade regarding the inferences derived from the putative existence of female heterogamety in geoemydid turtles.

Using DNA sequences of the mitochondrial cytochrome b gene, we investigated phylogeny and taxonomy of South and South-east Asian turtles of all species and subspecies of the genera Batagur, Callagur, Hardella, Kachuga and Pangshura. We found three major clades: (i) a moderately to well-supported clade containing all large riverine species assigned so far to Batagur, Callagur and Kachuga; (ii) a well-supported monophylum comprising the four Pangshura species; and (iii) Hardella that could constitute either the sister-taxon of Pangshura or of a clade comprising Batagur, Callagur, Kachuga and Pangshura. The genus Kachuga is clearly polyphyletic. Therefore, we recommend placing all Batagur, Callagur and Kachuga species in one genus. According to the International Code of Zoological Nomenclature Batagur Gray, 1856, being originally erected at higher rank, takes precedence over the simultaneously published name Kachuga Gray, 1856, and the younger name Callagur Gray, 1870, resulting in an expanded genus Batagur. Indonesian and Malaysian Batagur baska proved to be highly distinct from our sequences of this species from the Sundarbans (Bangladesh, adjacent India), suggesting that a previously unidentified species is involved. This finding is of high conservation relevance in the critically endangered B. baska. The currently recognized subspecies within Hardella thurjii, Pangshura smithii and P. tentoria do not correspond well with mtDNA clades. Considering that the two subspecies of H. thurjii are likely to be based only on individual ontogenetic differences, we propose abandoning the usage of subspecies within H. thurjii. In the Ghaghra River, Uttar Pradesh (India) we detected shared haplotypes in P. smithii and P. tentoria, implying that the unusual morphological characters of the Ghaghra River population of P. tentoria could be the result of interspecific hybridization.

Based on 2354 bp of mitochondrial DNA (12S rRNA, ND4, cyt b) and 2573 bp of nuclear DNA (C-mos, ODC, R35), we re-examine the phylogenetic relationships of Nilssonia species. Individual and combined analyses of mitochondrial and nuclear DNA using Maximum Likelihood and Bayesian approaches confirm the monophyly of the genus. While mitochondrial data alone could not resolve the phylogenetic position of N. formosa, nuclear data support a sister group relationship of N. formosa and the remaining Nilssonia species. Combined analyses of mitochondrial and nuclear DNA suggest the following branching pattern, with N. formosa as the sister taxon of the remaining species: N. formosa + ((N. gangetica + N. leithii) + (N. hurum + N. nigricans)). Among the samples we studied is the first record of N. formosa for Yunnan, China, and the first record of wild-living N. nigricans for Bangladesh. In N. gangetica, each of the studied major river basins harbours a genetically distinct population, suggesting that at least three distinct management units should be distinguished: (1) Brahmaputra River; (2) Indus and Ganges Rivers plus Ganges Delta; and (3) Mahanadi River.

We investigated relationships among Asian fl apshell turtles by using 2286 bp of mitochondrial DNA for phylogenetic reconstructions and relaxed molecular clock calculations. Currently three taxa are recognized, the unspotted species Lissemys scutata and L. punctata, with the unspotted subspecies L. p. punctata and the spotted subspecies L. p. andersoni. However, we found fi ve deeply divergent clades, two of which correspond to L. scutata (Myanmar; perhaps also adjacent Thailand and Yunnan, China) and L. p. andersoni (Indus, Ganges and Brahmaputra drainages; western Myanmar), respectively. Within L. p. punctata from peninsular India and Sri Lanka three distinct clades were identifi ed, two from peninsular India and one from Sri Lanka. The two clades from peninsular India are more closely related to L. p. andersoni than to fl apshell turtles from Sri Lanka. Due to a genetic divergence resembling L. scutata, we propose to separate Sri Lankan populations as the distinct species L. ceylonensis (Gray, 1856) from L. punctata. Furthermore, we suggest to restrict the name L. p. punctata (Lacepède, 1788) = L. p. punctata (Bonnaterre, 1789) to populations from southern peninsular India, whereas the name L. p. vittata (Peters, 1854) should be applied to unspotted fl apshell turtles from northern peninsular India. We classify all three taxa from the Indian subcontinent as subspecies because (1) there is morphological and genetic evidence that L. p. andersoni intergrades with L. p. vittata, and (2) the genetic divergence among L. p. punctata, L. p. andersoni and L. p. vittata resembles the degree of differentiation as observed between the latter two subspecies, whereas the differences between L. ceylonensis and L. scutata and among these species and the subspecies of L. punctata are about twice the values as observed among the subspecies of L. punctata. The formation of the subspecies of L. punctata was dated to have occurred between the uppermost Miocene and the Early Pleistocene (mean split ages of approx. 4.5 and 4.2 million years); the origin of L. ceylonensis and L. scutata, to a range between the Early Miocene and the Lower Pliocene (mean split ages of approx. 8 and 11 million years, respectively).

We describe external morphology and habitat of ten specimens of Cyclemys gemeli, a recently discovered leaf turtle species from north-eastern India, previously known only from its incomplete holotype and photos of a live female. Further, we assess the phylogenetic position of C. gemeli using sequence data of the mitochondrial cytochrome b gene as well as of three nuclear DNA fragments (C-mos, Rag2 genes, intron 1 of R35 gene) and confirm its genetic distinctiveness. Mitochondrial data strongly suggest a sister group relationship of C. gemeli and C. fusca, another species occurring in Myanmar. According to our new records, the Naga Hills and the Arakan Mts could constitute the geographical divide between C. gemeli and C. fusca. Morphologically, C. gemeli resembles other dark-bellied Cyclemys species and determination by external morphology alone is quite difficult.

Using mtDNA sequences of historical museum specimens, including the herein designated lectotype of Tetraonyx affinis Cantor 1847 and topotypic specimens of Trionyx (Tetraonyx) cuvieri Gray 1831 and Tetronyx longicollis Lesson 1834, we demonstrate that the name Batagur affinis (Cantor 1847) has to be used for a recently identified critically endangered terrapin species from Southeast Asia. Further, we provide evidence that Batagur baska (Gray 1830) historically was distributed from north-easternmost India and Bangladesh to at least the Ayeyarwady and Bago estuaries in Myanmar while B. affinis occurs in the southern Malay Peninsula and Sumatra. The taxonomic allocation of the extant and extirpated Batagur populations in the northern Malay Peninsula, Cambodia and southern Vietnam remains unclear. A museum specimen from the mid-19 th century suggests that B. baska once also occurred in the Indus Delta of southern Pakistan.

The systematic position of the rare Indian turtle Geoemyda silvatica Henderson is examined by a phylogenetic analysis of mtDNA sequence data (cytochrome-b gene) of most species of Geoemydidae. Geoemyda silvatica represents a basal and isolated taxon within Geoemydidae, definitely not a close relative of any species of Geoemyda or Heosemys, the genera in which G. silvatica has been placed in the past. Therefore, the new genus Vijayachelys is proposed for G. silvatica. Cranial morphology and some other osteological characters of Vijayachelys silvatica are described and illustrated. Differential diagnoses are given for the type species of Melanochelys and the respective type species of the superficially similar genera Geoemyda, Heosemys, and Leucocephalon. According to Bayesian analysis of mtDNA data, Melanochelys trijuga could be distantly related to V. silvatica, whereas the morphological similarity of the other species probably is the result of a similar mode of life. The discovery of the phylogenetically isolated position of V. silvatica highlights the importance of the Western Ghats as a biodiversity hotspot rich in higher-level endemics.

Estuarine Batagur are among the most critically endangered chelonian species. We assess the taxonomic status of the recently discovered Cambodian relic population of Batagur by phylogenetic analyses of three mitochondrial (2096 bp) and three nuclear DNA fragments (1909 bp) using sequences from all other Batagur species and selected allied geoemydids. Furthermore, we calculated haplotype networks of the mitochondrial cytochrome b gene for Cambodian terrapins, B. affinis, B. baska, and B. kachuga and compare external morphology of estuarine Batagur populations. Genetically, Cambodian Batagur are closely related with, but distinct from B. affinis from Sumatra and the west coast of the Malay Peninsula. Morphologically, Cambodian Batagur resemble the distinctive B. affinis populations from the eastern Malay Peninsula that were not available for genetic study. We suggest that the Batagur populations from the eastern Malay Peninsula and Cambodia represent a new subspecies of B. affinis that once was distributed in estuaries surrounding the Gulf of Thailand (Batagur affinis edwardmolli subsp. nov.). Its patchy extant distribution is most probably the result of large-scale habitat alteration and century-long overexploitation. In addition, our phylogenetic analyses suggest repeated switches between riverine and estuarine habitats during the evolution of the extant Batagur species.