Yuchi Zheng

Exploring patterns and extent of bias in estimating divergence time from mitochondrial DNA sequence data in a particular lineage: A case study of salamanders (Order Caudata)

In the practice of molecular dating, substitution saturation will bias the results if not properly modeled. Date estimates based on commonly used mitochondrial DNA sequences likely suffer from this problem because of their high substitution rate. Nevertheless, the patterns and extent of such expected bias remain unknown for many major evolutionary lineages, which often differ in ages, available calibrations, and substitution rates of their mitochondrial genome. In this case study of salamanders, we used estimates based on multiple nuclear exons to assess the effects of saturation on dating divergences using mitochondrial genome sequences on a timescale of ~200-300 My. The results indicated that, due to saturation for older divergences and in the absence of younger effective calibration points, dates derived from the mitochondrial data were considerably overestimated and systematically biased toward the calibration point for the ingroup root. The overestimate might be as great as 3-10 times (about 20 My) older than actual divergence dates for recent splitting events and 40 My older for events that are more ancient. For deep divergences, dates estimated were strongly compressed together. Furthermore, excluding the third codon positions of protein-coding genes or only using the RNA genes or second codon positions did not considerably improve the performance. In the order Caudata, slowly evolving markers such as nuclear exons are preferred for dating a phylogeny covering a relatively wide time span. Dates estimated from these markers can be used as secondary calibrations for dating recent events based on rapidly evolving markers for which mitochondrial DNA sequences are attractive candidates due to their short coalescent time. In other groups, similar evaluation should be performed to facilitate the choice of markers for molecular dating and making inferences from the results.

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.

The evolution of mitochondrial genomes in modern frogs (Neobatrachia): nonadaptive evolution of mitochondrial genome reorganization

BackgroundAlthough mitochondrial (mt) gene order is highly conserved among vertebrates, widespread gene rearrangements occur in anurans, especially in neobatrachians. Protein coding genes in the mitogenome experience adaptive or purifying selection, yet the role that selection plays on genomic reorganization remains unclear. We sequence the mitogenomes of three species of Glandirana and hot spots of gene rearrangements of 20 frog species to investigate the diversity of mitogenomic reorganization in the Neobatrachia. By combing these data with other mitogenomes in GenBank, we evaluate if selective pressures or functional constraints act on mitogenomic reorganization in the Neobatrachia. We also look for correlations between tRNA positions and codon usage.ResultsGene organization in Glandirana was typical of neobatrachian mitogenomes except for the presence of pseudogene trnS (AGY). Surveyed ranids largely exhibited gene arrangements typical of neobatrachian mtDNA although some gene rearrangements occurred. The correlation between codon usage and tRNA positions in neobatrachians was weak, and did not increase after identifying recurrent rearrangements as revealed by basal neobatrachians. Codon usage and tRNA positions were not significantly correlated when considering tRNA gene duplications or losses. Change in number of tRNA gene copies, which was driven by genomic reorganization, did not influence codon usage bias. Nucleotide substitution rates and dN/dS ratios were higher in neobatrachian mitogenomes than in archaeobatrachians, but the rates of mitogenomic reorganization and mt nucleotide diversity were not significantly correlated.ConclusionsNo evidence suggests that adaptive selection drove the reorganization of neobatrachian mitogenomes. In contrast, protein-coding genes that function in metabolism showed evidence for purifying selection, and some functional constraints appear to act on the organization of rRNA and tRNA genes. As important nonadaptive forces, genetic drift and mutation pressure may drive the fixation and evolution of mitogenomic reorganizations.

Coalescence patterns of endemic Tibetan species of stream salamanders (Hynobiidae: Batrachuperus)

Orogenesis of topographically diverse montane regions often drives complex evolutionary histories of species. The extensive biodiversity of the eastern edge of the Tibetan Plateau, which gradually decreases eastwardly, facilitates a comparison of historical patterns. We use coalescence methods to compare species of stream salamanders (Batrachuperus) that occur at high and low elevations. Coalescent simulations reveal that closely related species are likely to have been influenced by different drivers of diversification. Species living in the western high‐elevation region with its northsouth extending mountains appear to have experienced colonization via dispersal followed by isolation and divergence. In contrast, species on the eastern low‐elevation region, which has many discontinuous mountain ranges, appear to have experienced fragmentation, sometimes staged, of wide‐ranging ancestral populations. The two groups of species appear to have been affected differently by glaciation. High‐elevation species, which are more resistant to cooler temperatures, appear to have experienced population declines as recently as the last glaciation (0.016–0.032 Ma). In contrast, salamanders dwelling in the warmer and wetter habitats at low‐elevation environs appear to have been affected less by the relatively recent, milder glaciation, and more so by harsher, extensive glaciations (0.5–0.175 Ma). Thus, elevation, topography and cold tolerance appear to drive evolutionary patterns of diversification and demography even among closely related taxa. The comparison of multiple species in genealogical analyses can lead to an understanding of the evolutionary drivers.

A De Novo Case of Floating Chromosomal Polymorphisms by Translocation in Quasipaa boulengeri (Anura, Dicroglossidae)

Very few natural polymorphisms involving interchromosomal reciprocal translocations are known in amphibians even in vertebrates. In this study, thirty three populations, including 471 individuals of the spiny frog Quasipaa boulengeri, were karyotypically examined using Giemsa stain or FISH. Five different karyomorphs were observed. The observed heteromorphism was autosomal but not sex-related, as the same heteromorphic chromosomes were found both in males and females. Our results indicated that the variant karyotypes resulted from a mutual interchange occurring between chromosomes 1 and 6. The occurrence of a nearly whole-arm translocation between chromosome no. 1 and no. 6 gave rise to a high frequency of alternate segregation and probably resulted in the maintenance of the translocation polymorphisms in a few populations. The translocation polymorphism is explained by different frequencies of segregation modes of the translocation heterozygote during meiosis. Theoretically, nine karyomorphs should be investigated, however, four expected karyotypes were not found. The absent karyomorphs may result from recessive lethal mutations, position effects, duplications and deficiencies. The phylogenetic inference proved that all populations of Q. boulengeri grouped into a monophyletic clade. The mutual translocation likely evolved just once in this species and the dispersal of the one karyomorph (type IV) can explain the chromosomal variations among populations.

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

Abstract 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.

Intraspecific rearrangement of mitochondrial genome suggests the prevalence of the tandem duplication- random loss (TDLR) mechanism in Quasipaa boulengeri

BackgroundTandem duplication followed by random loss (TDRL) is the most frequently invoked model to explain the diversity of gene rearrangements in metazoan mitogenomes. The initial stages of gene rearrangement are difficult to observe in nature, which limits our understanding of incipient duplication events and the subsequent process of random loss. Intraspecific gene reorganizations may represent intermediate states, and if so they potentially shed light on the evolutionary dynamics of TDRL.ResultsNucleotide sequences in a hotspot of gene-rearrangement in 28 populations of a single species of frog, Quasipaa boulengeri, provide such predicted intermediate states. Gene order and phylogenetic analyses support a single tandem duplication event and a step-by-step process of random loss. Intraspecific gene rearrangements are not commonly found through comparison of all mitochondrial DNA records of amphibians and squamate reptiles in GenBank.ConclusionsThe intraspecific variation in Q. boulengeri provides insights into the rate of partial duplications and deletions within a mitogenome, and reveals that fixation and gene-distribution in mitogenomic reorganization is likely non-adaptive.

Isolation and characterization of eleven polymorphic tetranucleotide microsatellite loci for Quasipaa boulengeri (Anura: Dicroglossidae)

We isolated and characterized eleven polymorphic tetranucleotide microsatellite loci in the spiny-bellied frog (Quasipaa boulengeri). The loci were screened in 62 individuals from two natural populations. All loci were variable, with the number of alleles ranging from 8 to 26. The average observed and expected heterozygosity per locus ranged from 0.500 to 0.947 and from 0.735 to 0.932, respectively. Two loci were found to be significant departure from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was detected after sequential Bonferroni corrections. These polymorphic loci will be useful for conservation and population genetics studies of this threated species.

Isolation and characterization of ten microsatellite loci for the Chong’an moustache toad Leptobrachium liui (Anura: Megophryidae)

We isolated and characterized ten polymorphic microsatellite loci in the Chong’an moustache toad Leptobrachium liui, nine novel loci designed for this species and one locus from cross-species amplification. Loci were screened in 43 individuals of a single population. The number of alleles per locus ranged from 3 to 17, on average of 10. The average observed and expected heterozygosity per locus ranged from 0.512 to 0.884 and from 0.580 to 0.898, respectively. No significant departures from Hardy–Weinberg equilibrium were found after sequential Bonferroni corrections. Three loci, CHA8, CHA12, and VIB-D5, were detected to be linked. These polymorphic loci will be useful for conservation genetics and phylogeography studies of this endangered species.

Development of microsatellite markers for the spiny-bellied frog Quasipaa boulengeri (Anura: Dicroglossidae) through transcriptome sequencing

We detected and isolated thirty-two microsatellite loci from transcriptome-derived microsatellite DNA for Quasipaa boulengeri. The isolated loci were all polymorphic and the number of alleles per locus ranged from 2 to 10. The observed and expected heterozygosity per locus varied from 0.1493 to 0.9375 and from 0.1563 to 0.9688, respectively. By multiple comparisons with Bonferroni correction, seven loci were deviated from Hardy–Weinberg equilibrium and significant linkage disequilibrium was detected among twenty-five pairs of loci. Those variable microsatellite sites will be applied to assess gene flow between populations and investigate the evolutionary process of chromosomal polymorphism. Moreover, microsatellite loci were served as a valuable resource for drawing up criterion for the conservation management of this threatened species.