Phylogeny of the Asian spiny frog tribe Paini (Family Dicroglossidae) sensu Dubois

The anuran tribe Paini, family Dicroglossidae, is known in this group only from Asia. The phylogenetic relationships and often the taxonomic recognition of species are controversial. In order to stabilize the classification, we used approximately 2100 bp of nuclear (rhodopsin, tyrosinase) and mitochondrial (12S, 16S rRNA) DNA sequence data to infer the phylogenetic relationships of these frogs. Phylogenetic trees reconstructed using Bayesian inference and maximum parsimony methods supported a monophyletic tribe Paini. Two distinct groups (I,II) were recovered with the mtDNA alone and the total concatenated data (mtDNA+nuDNA). The recognition of two genera, Quasipaa and Nanorana, was supported. Group I, Quasipaa, is widespread east of the Hengduan Mountain Ranges and consists of taxa from relatively low elevations in southern China, Vietnam and Laos. Group II, Nanorana, contains a mix of species occurring from high to low elevation predominantly in the Qinghai-Tibetan Plateau and Hengduan Mountain Ranges. The occurrence of frogs at high elevations appears to be a derived ecological condition. The composition of some major species groups based on morphological characteristics strongly conflicts with the molecular analysis. Some possible cryptic species are indicated by the molecular analyses. The incorporation of genetic data from type localities helped to resolve some of the taxonomic problems, although further combined analyses of morphological data from type specimens are required. The two nuDNA gene segments proved to be very informative for resolving higher phylogenetic relationships and more nuclear data should be explored to be more confident in the relationships.     

Nuclear counterparts of the cytoplasmic mitochondrial 12S rRNA gene: A problem of ancient DNA and molecular phylogenies

Monkey mummy bones and teeth originating from the North Saqqara Baboon Galleries (Egypt), soft tissue from a mummified baboon in a museum collection, and nineteenth/twentieth-century skin fragments from mangabeys were used for DNA extraction and PCR amplification of part of the mitochondrial 12S rRNA gene. Sequences aligning with the 12S rRNA gene were recovered but were only distantly related to contemporary monkey mitochondrial 12S rRNA sequences. However, many of these sequences were identical or closely related to human nuclear DNA sequences resembling mitochondrial 12S rRNA (isolated from a cell line depleted in mitochondria) and therefore have to be considered contamination. Subsequently in a separate study we were able to recover genuine mitochondrial 12S rRNA sequences from many extant species of nonhuman Old World primates and sequences closely resembling the human nuclear integrations. Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters. One cluster contained all temporary cytoplasmic mitochondrial DNA sequences and approximately half of the monkey nuclear mitochondriallike sequences. A second cluster contained most human nuclear sequences and the other half of monkey nuclear sequences with a separate branch leading to human and gorilla mitochondrial and nuclear sequences. Sequences recovered from ancient materials were equally divided between the two clusters. These results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence: Nuclear counterparts of mitochondrial genes may lead to faulty interpretation of results.Correspondence to: A.C. van der Kuyl     

Molecular insights into the taxonomy of Hypanis (Bivalvia, Cardiidae, Lymnocardiinae) in the Black Sea lagoons

In this study, we used data from morphology and three DNA markers to assess the taxonomic status of the putative bivalve species Hypanis colorata and Hypanis angusticostata in a Black Sea lagoon, the Razelm Lake in Romania. The morphological data (the shape of shell ribs and the multivariate analysis of morphometric variance of three variables constructed as the ratios between the main dimensions of the shell) confirmed that the two analyzed species are distinct morphological entities. Three molecular markers, one from the nuclear genome (18S rRNA) and two from the mitochondrial genome (16S rRNA and COI), showed extremely reduced sequence divergence (0?C0.1%) between the two putative species. Based on these results, we suggest that H. angusticostata and H. colorata are morphotypes of a single species.     

Genetic Characterization of Clinical Acanthamoeba Isolates from Japan using Nuclear and Mitochondrial Small Subunit Ribosomal RNA

Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype ({"type":"entrez-nucleotide","attrs":{"text":"AF479533","term_id":"28194589","term_text":"AF479533"}}AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.     

Burmoniscus kitadaitoensis Nunomura, 2009 (Crustacea,Isopoda, Oniscidea) from southern Japan,a junior synonym of B. meeusei (Holthuis, 1947)

Re-examination of the holotype of Burmoniscus kitadaitoensis Nunomura, 2009 from Kitadaitojima Island, southern Japan reveals that this species is a junior synonym of B. meeusei (Holthuis, 1947). Partial regions of mitochondrial COI, 12S and 16S rRNA genes, and nuclear 18S and 28S rRNA genes were detected for species identification in the future.     

Evaluation of a novel 16S rRNA/tRNA mitochondrial marker for the identification and phylogenetic analysis of shrimp species belonging to the superfamily Penaeoidea

In this study, we infer the phylogenetic relationships within commercial shrimp using sequence data from a novel mitochondrial marker consisting of an approximately 530-bp region of the 16S ribosomal RNA (rRNA)/transfer RNA (tRNA)^Val genes compared with two other mitochondrial genes: 16S rRNA and cytochrome c oxidase I (COI). All three mitochondrial markers were considerably AT rich, exhibiting values up to 78.2% for the species Penaeus monodon in the 16S rRNA/tRNA^Val genes, notably higher than the average among other Malacostracan mitochondrial genomes. Unlike the 16S rRNA and COI genes, the 16S rRNA/tRNA^Val marker evidenced that Parapenaeus is more closely related to Metapenaeus than to Solenocera, a result that seems to be more in agreement with the taxonomic status of these genera. To our knowledge, our study using the 16S rRNA/tRNA^Val gene as a marker for phylogenetic analysis offers the first genetic evidence to confirm that Pleoticus muelleri and Solenocera agassizi constitute a separate group and that they are more related to each other than to genera belonging to the family Penaeidae. The 16S rRNA/tRNA^Val region was also found to contain more variable sites (56%) than the other two regions studied (33.4% for the 16S rRNA region and 42.7% for the COI region). The presence of more variable sites in the 16S rRNA/tRNA^Val marker allowed the interspecific differentiation of all 19 species examined. This is especially useful at the commercial level for the identification of a large number of shrimp species, particularly when the lack of morphological characteristics prevents their differentiation.     

First genetic record of the non-native muzzled blenny Omobranchus punctatus (Teleostei: Blenniidae) in the Atlantic Coast of Central and South America

In this study we sequenced two mitochondrial (COI and 16S rRNA) and one nuclear (18S rRNA) gene fragment of an introduced muzzled blenny (Omobranchus punctatus) specimen collected from the Orinoco Delta (Gulf of Paria estuary) in Venezuela. This is the first genetic data generated for this species' introduced range in Central and South America, suggesting an introduction from the Indian Ocean.     

Comparative Sequence Analysis of the Non-Protein-Coding Mitochondrial DNA of Inbred Rat Strains

The proper function of mammalian mitochondria necessitates a coordinated expression of both nuclear and mitochondrial genes, most likely due to the co-evolution of nuclear and mitochondrial genomes. The non-protein coding regions of mitochondrial DNA (mtDNA) including the D-loop, tRNA and rRNA genes form a major component of this regulated expression unit. Here we present comparative analyses of the non-protein-coding regions from 27 Rattus norvegicus mtDNA sequences. There were two variable positions in 12S rRNA, 20 in 16S rRNA, eight within the tRNA genes and 13 in the D-loop. Only one of the three neutrality tests used demonstrated statistically significant evidence for selection in 16S rRNA and tRNA-Cys. Based on our analyses of conserved sequences, we propose that some of the variable nucleotide positions identified in 16S rRNA and tRNA-Cys, and the D-loop might be important for mitochondrial function and its regulation.     

MRM2 encodes a novel yeast mitochondrial 21S rRNA methyltransferase

Mitochondria of the yeast Saccharomyces cerevisiae assemble their ribosomes from ribosomal proteins, encoded by the nuclear genome (with one exception), and rRNAs of 15S and 21S, encoded by the mitochondrial genome. Unlike cytoplasmic rRNA, which is highly modified, mitochondrial rRNA contains only three modified nucleotides: a pseudouridine (Psi(2918)) and two 2'-O-methylated riboses (Gm(2270) and Um(2791)) located at the peptidyl transferase centre of 21S rRNA. We demonstrate here that the yeast nuclear genome encodes a mitochondrial protein, named Mrm2, which is required for methylating U(2791) of 21S rRNA, both in vivo and in vitro. Deletion of the MRM2 gene causes thermosensitive respiration and leads to rapid loss of mitochondrial DNA. We propose that Mrm2p belongs to a new class of three eukaryotic RNA-modifying enzymes and is the orthologue of FtsJ/RrmJ, which methylates a nucleotide of the peptidyl transferase centre of Escherichia coli 23S rRNA that is homologous to U(2791) of 21S rRNA. Our data suggest that this universally conserved modified nucleotide plays an important function in vivo, possibly by inducing conformational rearrangement of the peptidyl transferase centre.     

Molecular Phylogeny of Weakfish Species of the Stellifer Group (Sciaenidae,Perciformes) of the Western South Atlantic Based on Mitochondrial and Nuclear Data

The phylogenetic relationships within the Stellifer group of weakfishes (Stellifer, Odontoscion, Ophioscion, and Bairdiella) were evaluated using 2723 base pairs comprising sequences of nuclear (rhodopsin, TMO-4C4, RAG-1) and mitochondrial (16S rRNA and COI) markers obtained from specimens of nine species. Our results indicate a close relationship between Bairdiella and Odontoscion, and also that the genus Stellifer is not monophyletic, but rather that it consists of two distinct lineages, one clade containing S. microps/S. naso/S. brasiliensis and the other, S. rastrifer/S. stellifer/Stellifer sp. B, which is closer to Ophioscion than the former clade. The O. punctatissimus populations from the northern and southern Brazilian coast were also highly divergent in both nuclear (0.8% for rhodopsin and 0.9% for RAG-1) and mitochondrial sequences (2.2% for 16S rRNA and 7.3% for COI), which we conclude is consistent with the presence of two distinct species. The morphological similarities of the members of the Stellifer group is reinforced by the molecular data from both the present study and previous analyses, which have questioned the taxonomic status of the Stellifer group. If, on the one hand, the group is in fact composed of four genera (Stellifer, Ophioscion, Odontoscion, and Bairdiella), one of the two Stellifer clades should be reclassified as a new genus. However, if the close relationship and the reduced genetic divergence found within the group is confirmed in a more extensive study, including representatives of additional taxa, this, together with the morphological evidence, would support downgrading the whole group to a single genus. Obviously, these contradictory findings reinforce the need for a more systematic taxonomic revision of the Stellifer group as a whole.     

Additional support for Afrotheria and Paenungulata,the performance of mitochondrial versus nuclear genes,and the impact of data partitions with heterogeneous base composition

We concatenated sequences for four mitochondrial genes (12S rRNA, tRNA valine, 16S rRNA, cytochrome b) and four nuclear genes [aquaporin, alpha 2B adrenergic receptor (A2AB), interphotoreceptor retinoid-binding protein (IRBP), von Willebrand factor (vWF)] into a multigene data set representing 11 eutherian orders (Artiodactyla, Hyracoidea, Insectivora, Lagomorpha, Macroscelidea, Perissodactyla, Primates, Proboscidea, Rodentia, Sirenia, Tubulidentata). Within this data set, we recognized nine mitochondrial partitions (both stems and loops, for each of 12S rRNA, tRNA valine, and 16S rRNA; and first, second, and third codon positions of cytochrome b) and 12 nuclear partitions (first, second, and third codon positions, respectively, of each of the four nuclear genes). Four of the 21 partitions (third positions of cytochrome b, A2AB, IRBP, and vWF) showed significant heterogeneity in base composition across taxa. Phylogenetic analyses (parsimony, minimum evolution, maximum likelihood) based on sequences for all 21 partitions provide 99-100% bootstrap support for Afrotheria and Paenungulata. With the elimination of the four partitions exhibiting heterogeneity in base composition, there is also high bootstrap support (89-100%) for cow + horse. Statistical tests reject Altungulata, Anagalida, and Ungulata. Data set heterogeneity between mitochondrial and nuclear genes is most evident when all partitions are included in the phylogenetic analyses. Mitochondrial-gene trees associate cow with horse, whereas nuclear-gene trees associate cow with hedgehog and these two with horse. However, after eliminating third positions of A2AB, IRBP, and vWF, nuclear data agree with mitochondrial data in supporting cow + horse. Nuclear genes provide stronger support for both Afrotheria and Paenungulata. Removal of third positions of cytochrome b results in improved performance for the mitochondrial genes in recovering these clades.     

Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules

5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator.     

Phylogenetic relationships and altered genome structures among Tetrahymena mitochondrial DNAs.

Tetrahymena thermophila mitochondrial DNA is a linear molecule with two tRNAs, large subunit beta (LSU beta) rRNA (21S rRNA) and LSU alpha rRNA (5.8S-like RNA) encoded near each terminus. The DNA sequence of approximately 550 bp of this region was determined in six species of Tetrahymena. In three species the LSU beta rRNA and tRNA(leu) genes were not present on one end of the DNA, demonstrating a mitochondrial genome organization different from that of T. thermophila. The DNA sequence of the LSU alpha rRNA was used to construct a mitochondrial phylogenetic tree, which was found to be topologically equivalent to a phylogenetic tree based on nuclear small subunit rRNA sequences (Sogin et al. (1986) EMBO J. 5, 3625-3630). The mitochondrial rRNA gene was found to accumulate base-pair substitutions considerably faster than the nuclear rRNA gene, the rate difference being similar to that observed for mammals.     

Molecular systematics of sclerosomatid harvestmen (Opiliones, Phalangioidea, Sclerosomatidae): geography is better than taxonomy in predicting phylogeny

Phylogenetic relationships within the Sclerosomatidae, the largest family of harvestmen, are explored using molecular data from four nuclear genes (28S and 18S rRNA, Histone 3 and Elongation factor-1α) and two mitochondrial gene regions (COI-COII, 16S and 12S rRNA). The taxon sample includes representative species from all families in Phalangioidea and all subfamilies of Sclerosomatidae (Gagrellinae, Gyinae, Leiobuninae, Sclerosomatinae). Our results solve several major taxonomic problems, including placement of Gyinae sensu stricto in Phalangiidae, the monophyly of the Metopilio group and its exclusion from Sclerosomatidae, and reaffirmation of the familial rank of Protolophidae. However, most major groups of sclerosomatids (Leiobuninae, Gagrellinae, Leiobunum, Nelima) are recovered as polyphyletic, although with a phylogenetic structure suggesting a strong association between geography and monophyly as well as notable morphological convergence in traditional diagnostic characters. Phylogenetic affinities between biotas of the New World and Asian tropics, as well as between temperate North American and East Asia, suggest that sclerosmatid historical biogeography may conform with the Boreotropic Concept. Finally, we discuss how the many problems that remain in sclerosomatid systematics might be addressed.     

Characterization of a novel small RNA encoded by Dictyostelium discoideum mitochondrial DNA

In this study, we analyzed a mitochondrial small (ms) RNA in Dictyostelium discoideum, which is 129 nucleotides long and has a GC content of only 22.5%. In the mitochondrial DNA, a single-copy gene (msr) for the ms RNA was located downstream of the gene for large-subunit rRNA. The location of msr was similar to that of the 5S rRNA gene in prokaryotes and chloroplasts, but clearly different from that in mitochondria of plants, liverwort and the chlorophycean alga Prototheca wikerhamii, in which small-subunit rRNA and 5S rRNA genes are closely linked. The primary sequence of ms RNA showed low homology with mitochondrial 5S rRNA from plants, liverwort and the chlorophycean alga, but the proposed secondary structure of ms RNA was similar to that of cytoplasmic 5S rRNA. In addition, ms RNA showed a highly conserved GAAC sequence in the same loop as in common 5S rRNA. However, ms RNA was detected mainly in the mitochondrial 25?000?×?g supernatant fraction which was devoid of ribosomes. It is possible that ms RNA is an evolutionary derivative of mitochondrial 5S rRNA.     

Ancient DNA analysis reveals woolly rhino evolutionary relationships

With ancient DNA technology, DNA sequences have been added to the list of characters available to infer the phyletic position of extinct species in evolutionary trees. We have sequenced the entire 12S rRNA and partial cytochrome b (cyt b) genes of one 60-70,000-year-old sample, and partial 12S rRNA and cyt b sequences of two 40-45,000-year-old samples of the extinct woolly rhinoceros (Coelodonta antiquitatis). Based on these two mitochondrial markers, phylogenetic analyses show that C. antiquitatis is most closely related to one of the three extant Asian rhinoceros species, Dicerorhinus sumatrensis. Calculations based on a molecular clock suggest that the lineage leading to C. antiquitatis and D. sumatrensis diverged in the Oligocene, 21-26 MYA. Both results agree with morphological models deduced from palaeontological data. Nuclear inserts of mitochondrial DNA were identified in the ancient specimens. These data should encourage the use of nuclear DNA in future ancient DNA studies. It also further establishes that the degraded nature of ancient DNA does not completely protect ancient DNA studies based on mitochondrial data from the problems associated with nuclear inserts.     

Nuclear gene sequences resolve species phylogeny and mitochondrial introgression in Leptocarabus beetles showing trans-species polymorphisms

We studied the phylogenetic relationships among Japanese Leptocarabus ground beetles, which show extensive trans-species polymorphisms in mitochondrial gene genealogies. Simultaneous analysis of combined nuclear data with partial sequences from the long-wavelength rhodopsin, wingless, phosphoenolpyruvate carboxykinase, and 28S rRNA genes resolved the relationships among the five species, although separate analyses of these genes provided topologies with low resolution. For both the nuclear gene tree resulting from the combined data from four genes and a mitochondrial cytochrome oxidase subunit I (COI) gene tree, we applied a Bayesian divergence time estimation using a common calibration method to identify mitochondrial introgression events that occurred after speciation. Three mitochondrial lineages shared by two or three species were likely subject to introgression due to interspecific hybridization because the coalescent times for these lineages were much shorter than the corresponding speciation times estimated from nuclear gene sequences. We demonstrated that when species phylogeny is fully resolved with nuclear gene sequence data, comparative analysis of nuclear and mitochondrial gene trees can be used to infer introgressive hybridization events that might cause trans-species polymorphisms in mitochondrial gene trees.     

Studying sources of incongruence in arthropod molecular phylogenies: Sea spiders (Pycnogonida) as a case study

In this report, we analyze the phylogeny of Pycnogonida using the three nuclear and three mitochondrial markers currently sequenced for studying inter- and intrafamilial relationships within Arthropoda: 18S and 28S rRNA genes, Histone H3, cytochrome c oxidase subunit 1 (CO1), 12S and 16S rRNA genes. We identify several problems in previous studies, due to the use of inappropriate sequences (taxonomic misidentification, DNA contamination, sequencing errors, missing data) or taxa (outgroup choice). Our analyses show that most markers are not powerful to study the phylogeny of sea spiders. The results suggest however a recent diversification of the group (Mesozoic rather than Paleozoic) and the early divergence of Austrodecidae, followed by Colossendeidae, Pycnogonidae and Rhynchothoracidae. Except Ammotheidae and Callipallenidae, all other families were recovered as monophyletic. Analyses of synonymous sites in CO1 sequences reveal an extreme heterogeneity of nucleotide composition within sea spiders, as six unrelated species show a reverse strand-specific bias. We therefore suggest that several independent reversals of asymmetric mutational constraints occurred during the evolution of Pycnogonida, as a consequence of genomic inversions involving either the control region or a fragment containing the CO1 gene. These hypotheses are supported by the comparison of two complete mitochondrial genomes of sea spiders (Achelia bituberculata and Nymphon gracile) with that of Limulus.     

Multigene Barcoding and Phylogeny of Geographically Widespread Muricids (Gastropoda: Neogastropoda) Along the Coast of China

The identification and phylogeny of muricids have been in a state of confusion for a long time due to the morphological convergence and plasticity. DNA-based identification and phylogeny methods often offer an analytically powerful addition or even an alternative. In this study, we employ a DNA barcoding method to identify 17 known and easily confused muricid species (120 individuals) from the whole China coast based on mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA sequences, and nuclear ITS-1 and 28S rRNA sequences. The phylogeny of muricid subfamilies is also analysed based on all mitochondrial and nuclear sequences. The universal COI and 16S rRNA primers did not work broadly across the study group, necessitating the redesign of muricid specific COI and 16S rRNA primers in this paper. Our study demonstrates that COI gene is a suitable marker for barcoding muricids, which can distinguish all muricid species studied. Phylogenetic analysis of 16S rRNA, ITS-1 and 28S rRNA data also provide good support for the species resolution observed in COI data. The relationships of muricid subfamilies are resolved based on the separate and combined gene data that showed the monophyly of each the subfamilies Ergalataxinae, Rapaninae, Ocenebrinae and Muricinae, especially that Ergalataxinae did not fall within Rapaninae.     

Molecular phylogeny of the family Dicroglossidae (Amphibia: Anura) inferred from complete mitochondrial genomes

The fork-tongued frogs, members of the amphibian Order Anura, belong to the family Dicroglossidae and are one of the most diverse groups of Anuran frogs; however, their taxonomy and phylogeny remain controversial. In the present study, sixteen dicroglossine mitochondrial genomes representing nine dicroglossine genera and 23 other neobatrachian taxa, were used to reconstruct the phylogenetic relationships of the family Dicroglossidae using different partitioned maximum likelihood and partitioned Bayesian inference methods at both the nucleotide and amino acid levels. The sampled fork-tongued frogs form a strongly supported monophyletic group that is the sister taxon to another well-supported clade that includes representatives of the families Ranidae, Rhacophoridae, and Mantellidae. The monophyly of the subfamily Occidozyginae and Dicroglossinae was revealed with strong supports, and two major clades were supported within Dicroglossinae. The sister-group relationship between the genera Limnonectes and the tribe Paini was supported. In addition, a sister-group relationships between Fejervarya and Euphlyctis + Hoplobatrachus, between Quasipaa and Yerana, and between Feirana and Nanorana are well supported. Estimates of divergence times revealed the divergence of Dicroglossidae during the Late Upper Cretaceous to the Early Eocene, and diversification of the major dicroglossine genera from the Early Eocene to the Middle Miocene.