Hidden hotspots of amphibian biodiversity in China.

Identifying and protecting hotspots of endemism and species richness is crucial for mitigating the global biodiversity crisis. However, our understanding of spatial diversity patterns is far from complete, which severely limits our ability to conserve biodiversity hotspots. Here, we report a comprehensive analysis of amphibian species diversity in China, one of the most species-rich countries on Earth. Our study combines 20 y of field surveys with new molecular analyses of 521 described species and also identifies 100 potential cryptic species. We identify 10 hotspots of amphibian diversity in China, each with exceptional species richness and endemism and with exceptional phylogenetic diversity and phylogenetic endemism (based on a new time-calibrated, species-level phylogeny for Chinese amphibians). These 10 hotspots encompass 59.6% of China's described amphibian species, 49.0% of cryptic species, and 55.6% of species endemic to China. Only four of these 10 hotspots correspond to previously recognized biodiversity hotspots. The six new hotspots include the Nanling Mountains and other mountain ranges in South China. Among the 186 species in the six new hotspots, only 9.7% are well covered by protected areas and most (88.2%) are exposed to high human impacts. Five of the six new hotspots are under very high human pressure and are in urgent need of protection. We also find that patterns of richness in cryptic species are significantly related to those in described species but are not identical.

A reinvestigation of phylogeny and divergence times of Hynobiidae (Amphibia, Caudata) based on 29 nuclear genes.

Although several recent studies have investigated the major phylogenetic relationships within Hynobiidae, their evolutionary history remains partially resolved and the phylogenetic positions of some genera, particularly Pachyhynobius and Salamandrella are still disputed. Notably, previous studies relied primarily on mitochondrial DNA data and concatenated analyses; thus, a new investigation based on multiple nuclear genes and species-tree inference is needed. Here, we provide an in-depth phylogenetic analysis, based on 29 nuclear genes comprising 29,232bp of data from a comprehensive taxonomic sampling (24 hynobiids and 7 outgroups), using both concatenated and species-tree methods. Our results robustly resolved most genus-level relationships within Hynobiidae, including the placement of Salamandrella as the sister group to a clade containing Batrachuperus, Liua and Pseudohynobius, and the placement of Pachyhynobius as the sister group to a clade containing all hynobiids excluding Onychodactylus, Paradactylodon and Ranodon. Time estimates based on our data suggest that the major group of living hynobiids (excluding Onychodactylus) originated approximately 40Ma, ∼50% younger than estimates from mtDNA data (62.5Ma) but 10% older than estimates from three nuclear genes (36Ma). Our results highlight the benefits of using a large number of nuclear loci to infer both phylogeny and time for relatively old lineages.

Rediscovery of Protohynobius puxiongensis (Caudata: Hynobiidae) and its phylogenetic position based on complete mitochondrial genomes.

The mysterious Asian hynobiid salamander, Protohynobius puxiongensis, was described based on a single specimen collected in 1965 and never found again since then. Because the specimen had an internasal bone, Pr. puxiongensis was thought to retain a primitive character lost by a common ancestor of all other hynobiid salamanders, and it was thus considered to be not only a new genus, but also a new subfamily. This conclusion bothered herpetologists for decades because it was based on only one specimen and one character without other living specimens being rediscovered. After years of field effort, we rediscovered living individuals of Pr. puxiongensis at its type locality. All characters observed in rediscovered specimens are identical to the original description of the holotype except the internasal bone, implying that the internasal bone observed in the holotype may be just an individual variation. To examine the phylogenetic position of Pr. puxiongensis, we sequenced complete mitochondrial genomes for this species, together with two Pseudohynobius species. By combining 18 published hynobiid mitochondrial genomes and our new sequences, we reconstructed a comprehensive phylogenetic relationship of Hynobiidae at the genus level. Our results indicate that Pr. puxiongensis is deeply nested within the hynobiid phylogeny. It is the sister group of the Pseudohynobius species, and the validity of subfamily Protohynobiinae is not supported.

A new species of the genus Batrachuperus (Urodela: Hynobiidae) from Southwestern China.

A new stream salamander species, Batrachuperus daochengensis sp. nov., from southwestern China, is described herein based on morphological and molecular evidence. Molecular phylogeny derived from the mitochondrial gene together with previous nuclear data revealed that B. daochengensis sp. nov. is sister to B. yenyuanensis. The new species differs from all other species of the genus by the following combination of characters: brown horny epidermis on tips of fingers and toes absent; tubercles on palms and soles absent; costal grooves 12; dorsal brown, mottled with blackish spots; fingers 2-3-4-1 in order of decreasing length; tips of longest digits of fore- and hindlimbs largely separated by one to two costal spaces when adpressed towards each other along sides of body. The new species is currently known in the central and southern Shaluli Mountains in southwestern China.

ITS non-concerted evolution and rampant hybridization in the legume genus Lespedeza (Fabaceae).

The internal transcribed spacer (ITS) as one part of nuclear ribosomal DNA is one of the most extensively sequenced molecular markers in plant systematics. The ITS repeats generally exhibit high-level within-individual homogeneity, while relatively small-scale polymorphism of ITS copies within individuals has often been reported in literature. Here, we identified large-scale polymorphism of ITS copies within individuals in the legume genus Lespedeza (Fabaceae). Divergent paralogs of ITS sequences, including putative pseudogenes, recombinants, and multiple functional ITS copies were sometimes detected in the same individual. Thirty-seven ITS pseudogenes could be easily detected according to nucleotide changes in conserved 5.8S motives, the significantly lower GC contents in at least one of three regions, and the lost ability of 5.8S rDNA sequence to fold into a conserved secondary structure. The distribution patterns of the putative functional clones were highly different between the traditionally recognized two subgenera, suggesting different rates of concerted evolution in two subgenera which could be attributable to their different extents/frequencies of hybridization, confirmed by our analysis of the single-copy nuclear gene PGK. These findings have significant implications in using ITS marker for reconstructing phylogeny and studying hybridization.

Complete mitochondrial genome of Amolops mantzorum (Anura: Ranidae).

Sichuan torrent frog, Amolops mantzorum (family Ranidae, suborder Neobatrachia), possesses heteromorphic sex chromosomes unusual characteristics among amphibians. We determined the complete nucleotide sequence of the A. mantzorum mitogenome. This genome is 17,744 bp in length and contains 37 genes, 1 control region, and 1 light strand replication origin typically found in vertebrate mtDNAs. In the A. mantzorum mitogenome, a novel gene arrangement is observed within the WANCY tRNA gene cluster region. This mt gene arrangement seems to be usable as a molecular maker to distinguish to this species from other species in the genus Amolops.

[The research on the karyotypes of six species in the genus Eremias from China].

Based on the Giemsa-dyeing karyotypes and silver-staining bands of 15 populations from different localities in China belonging to 6 species of the genus Eremias , We found all species studied have 19 pairs of chromosomes, the size of chromosomes reduces gradually and there are no marked differences between the arranged pairs of macrochromosomes except the last pair of microchromosome. There are the same karyotype formula as 2n=38=36I+2m with NF=38 in E. argus, E. multiocellata, E. velox, E. arguta and E. grammica; but the karyotype formula of E. vermiculata is different as 2n=38=12V+2sI+22I+2m with NF=50. The NOR are all located on one small pair in female of E. velox, and E. arguta , in male of E. grammica and E. vermiculata ,and in both male and female of E. multiocellata. We have not found two or more than two pairs of NOR. Having one pair of NOR may be common in Genus Eremias and also the trait of Eremias. We speculate that the derivation of the karyotype of E. vermiculata had two possible way: one experienced the stage of triploid, and later the Robertsonian transposal of chromosomes; the other way was through the inversions between the arms on the chromosome and the phenomenon of inversions might occur during or subsequently after the upheaval of the Tibet and Qinghai plateau and the founding of the Tianshan . With regard to the trend of the evolution of chromosomes in the lizards ([1]), the karyotype of E. vermiculata is more advanced. Making specialties of E. vermiculata will help in building the phylogenic tree of Eremias. In both male and female of the species studied, the heteromorphic sex-chromosomes were not found.

The complete mitochondrial genome of Quasipaa boulengeri (Anura: Dicroglossidae).

Quasipaa boulengeri, an Asian spiny frog that belongs to Neobatrachia, has been reported as a de novo case of chromosomal rearrangements. In this paper, we determined the whole mitochondrial DNA (mtDNA) sequence of Q. boulengeri. The complete mtDNA sequence of Q. boulengeri is 17,741 bp in length, which contains 13 protein-coding genes (PCGs), two rRNAs, 23 tRNAs and one control region. The overall nucleotides base composition of the complete mtDNA is A (28.86%), G (14.43%), C (26.09%), T (30.62%), with a much higher A + T content. Compared to other Neobatrachia species, the mtDNA genome of Q. boulengeri shows a novel gene order at WANCY region.

[Application of ND-FISH in amphibians].

The recently popularized non-denaturing fluorescence in situ hybridization (ND-FISH) is a new technique that is both quick and efficient, in part because denaturing of both of the probes and the chromosomes is unnecessary. Synthetic simple sequence repeats (SSRs) labeled with fluorescein are used as probes to detect SSR-enriched chromosome regions and provide markers to identify the chromosomes. To date this method has not been applied to amphibians, even though the polymorphism of the distribution of SSRs may help to advance genetic polymorphism research. This paper also improved the double-colour FISH method by simultaneously using probes labelled with fluorescein and probes labelled with DIG to get double-color signals. This study found 5 SSRs markers that may be useful in the polymorphism research, and that the amphibian chromosomes must be denatured in ND-FISH.

COI is better than 16S rRNA for DNA barcoding Asiatic salamanders (Amphibia: Caudata: Hynobiidae).

The 5' region of the mitochondrial DNA (mtDNA) gene cytochrome c oxidase I (COI) is the standard marker for DNA barcoding. However, because COI tends to be highly variable in amphibians, sequencing is often challenging. Consequently, another mtDNA gene, 16S rRNA gene, is often advocated for amphibian barcoding. Herein, we directly compare the usefulness of COI and 16S in discriminating species of hynobiid salamanders using 130 individuals. Species identification and classification of these animals, which are endemic to Asia, are often based on morphology only. Analysis of Kimura 2-parameter genetic distances (K2P) documents the mean intraspecific variation for COI and 16S rRNA genes to be 1.4% and 0.3%, respectively. Whereas COI can always identify species, sometimes 16S cannot. Intra- and interspecific genetic divergences occasionally overlap in both markers, thus reducing the value of a barcoding gap to identify genera. Regardless, COI is the better DNA barcoding marker for hynobiids. In addition to the comparison of two potential markers, high levels of intraspecific divergence in COI (>5%) suggest that both Onychodactylus fischeri and Salamandrella keyserlingii might be composites of cryptic species.