Molecular Evidence for the Early History of Living Amphibians

The evolutionary relationships of the three orders of living amphibians (lissamphibians) has been difficult to resolve, partly because of their specialized morphologies. Traditionally, frogs and salamanders are considered to be closest relatives, and all three orders are thought to have arisen in the Paleozoic (>250 myr). Here, we present evidence from the DNA sequences of four mitochondrial genes (2.7 kilobases) that challenges the conventional hypothesis and supports a salamander–caecilian relationship. This, in light of the fossil record and distribution of the families, suggests a more recent (Mesozoic) origin for salamanders and caecilians directly linked to the initial breakup of the supercontinent Pangaea. We propose that this single geologic event isolated salamanders and archaeobatrachian frogs on the northern continents (Laurasia) and the caecilians and neobatrachian frogs on the southern continents (Gondwana). Among the neobatrachian frog families, molecular evidence supports a South American clade and an African clade, inferred here to be the result of mid-Cretaceous vicariance.     

Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the bell-ring frog, Buergeria buergeri (family Rhacophoridae)

In this study we determined the complete nucleotide sequence (19,959 bp) of the mitochondrial DNA of the rhacophorid frog Buergeria buergeri. The gene content, nucleotide composition, and codon usage of B. buergeri conformed to those of typical vertebrate patterns. However, due to an accumulation of lengthy repetitive sequences in the D-loop region, this species possesses the largest mitochondrial genome among all the vertebrates examined so far. Comparison of the gene organizations among amphibian species (Rana, Xenopus, salamanders and caecilians) revealed that the positioning of four tRNA genes and the ND5 gene in the mtDNA of B. buergeri diverged from the common vertebrate gene arrangement shared by Xenopus, salamanders and caecilians. The unique positions of the tRNA genes in B. buergeri are shared by ranid frogs, indicating that the rearrangements of the tRNA genes occurred in a common ancestral lineage of ranids and rhacophorids. On the other hand, the novel position of the ND5 gene seems to have arisen in a lineage leading to rhacophorids (and other closely related taxa) after ranid divergence. Phylogenetic analysis based on nucleotide sequence data of all mitochondrial genes also supported the gene rearrangement pathway.     

大头蛙和脆皮大头蛙线粒体3个基因的测定及两栖类亲缘关系研究

现存的两栖类系统发生关系一直存在争议,特别是3个目间的亲缘关系。本文设计了5对引物,扩增和测定了大头蛙和脆皮大头蛙线粒体12S和16S rRNA基因和Cytb基因的全序列。在对所测序列进行分析的同时,基于3个基因全序列的相加数据,运用MEGA 3.1和PHYLIP 3.64软件中的NJ法、MP法和ML法,对两爬类17个物种,以鱼类非洲肺鱼为外群,重建出3个树形完全一致的分子系统树。研究结果显示:现存两栖类中无尾目和有尾目为姐妹群关系,并推断有尾目内小鲵科和隐鳃鲵科亲缘关系较近。此外,在研究两栖类系统发生关系方面,作者分析前人研究中产生两种不同观点的可能原因,同时总结了在此类研究中产生偏差的几种影响因素。     

用线粒体tRNA基因探讨现存两栖动物三个目间系统发生关系

现存两栖类3个目的系统发生关系仍然没有统一意见,最广泛被接受的假说是单系起源,并且无尾类和有尾类为姐妹群关系而排斥蚓螈类(蛙类假说)。然而,这一假说一直存在争议。我们在测定了泽蛙线粒体基因组全序列的基础上,与已知其他的6种两栖类进行详细的比较分析,同时选择了11种高等脊椎动物的线粒体全基因序列,以硬骨鱼类作外群,用22个tRNA基因合并数据进行系统发生重建分析,结果表明MP、ML树都强力地支持现生两栖类动物为单系群,并且有尾目和蚓螈目为姐妹群关系。这个结果与蛙类假说是相矛盾的,与Bolt(1991)在形态学基础上提出的有尾类和蚓螈类为姐妹群关系的假说相一致,并得到建立在线粒体和核rRNA基因数据基础上的许多分子研究的支持。另外还探讨了本结果与前人的研究不一致的原因,以及利用线粒体全基因序列进行系统发生分析可能存在的偏差。     

Phylogeny and comparative substitution rates of frogs inferred from sequences of three nuclear genes

Phylogenetic relationships among major clades of anuran amphibians were studied using partial sequences of three nuclear protein coding genes, Rag-1, Rag-2, and rhodopsin in 26 frog species from 18 families. The concatenated nuclear data set comprised 2,616 nucleotides and was complemented by sequences of the mitochondrial 12S and 16S rRNA genes for analyses of evolutionary rates. Separate and combined analyses of the nuclear markers supported the monophyly of modern frogs (Neobatrachia), whereas they did not provide support for the monophyly of archaic frog lineages (Archaeobatrachia), contrary to previous studies based on mitochondrial data. The Neobatrachia contain two well supported clades that correspond to the subfamilies Ranoidea (Hyperoliidae, Mantellidae, Microhylidae, Ranidae, and Rhacophoridae) and Hyloidea (Bufonidae, Hylidae, Leptodactylidae, and Pseudidae). Two other families (Heleophrynidae and Sooglossidae) occupied basal positions and probably represent ancient relicts within the Neobatrachia, which had been less clearly indicated by previous mitochondrial analyses. Branch lengths of archaeobatrachians were consistently shorter in all separate analyses, and nonparametric rate smoothing indicated accelerated substitution rates in neobatrachians. However, relative rate tests confirmed this tendency only for mitochondrial genes. In contrast, nuclear gene sequences from our study and from an additional GenBank survey showed no clear phylogenetic trends in terms of differences in rates of molecular evolution. Maximum likelihood trees based on Rag-1 and using only one neobatrachian and one archaeobatrachian sequence, respectively, even had longer archaeobatrachian branches averaged over all pairwise comparisons. More data are necessary to understand the significance of a possibly general assignation of short branches to basal and species-poor taxa by tree-reconstruction algorithms.     

Complete nucleotide sequence of the mitochondrial genome of a salamander, Mertensiella luschani

The complete nucleotide sequence (16,650 bp) of the mitochondrial genome of the salamander Mertensiella luschani (Caudata, Amphibia) was determined. This molecule conforms to the consensus vertebrate mitochondrial gene order. However, it is characterized by a long non-coding intervening sequence with two 124-bp repeats between the tRNA(Thr) and tRNA(Pro) genes. The new sequence data were used to reconstruct a phylogeny of jawed vertebrates. Phylogenetic analyses of all mitochondrial protein-coding genes at the amino acid level recovered a robust vertebrate tree in which lungfishes are the closest living relatives of tetrapods, salamanders and frogs are grouped together to the exclusion of caecilians (the Batrachia hypothesis) in a monophyletic amphibian clade, turtles show diapsid affinities and are placed as sister group of crocodiles+birds, and the marsupials are grouped together with monotremes and basal to placental mammals. The deduced phylogeny was used to characterize the molecular evolution of vertebrate mitochondrial proteins. Amino acid frequencies were analyzed across the main lineages of jawed vertebrates, and leucine and cysteine were found to be the most and least abundant amino acids in mitochondrial proteins, respectively. Patterns of amino acid replacements were conserved among vertebrates. Overall, cartilaginous fishes showed the least variation in amino acid frequencies and replacements. Constancy of rates of evolution among the main lineages of jawed vertebrates was rejected.     

Sexual size dimorphism in caecilian amphibians: analysis, review and directions for future research

Sexual dimorphism, widespread in the animal kingdom, describes differences between the sexes in size, shape and many other traits. Sexual size dimorphism (SSD) plays a significant role in understanding life history evolution and mating systems. The snakelike morphology of limbless caecilian amphibians lacking obvious secondary sexual characters (in contrast to frogs and salamanders) impedes accurate intrasexual comparisons. In this study, sexual size dimorphism in the oviparous caecilian Ichthyophis cf. kohtaoensis, a phylogenetically basal caecilian, was analysed. Females were larger in all body and head characters tested. However, when adjusted to body size (total length), females differed only in their cloacal shape. Clutch volume was positively correlated to female body size, thus female fecundity increased with body size supporting the hypothesis of a fecundity-selected SSD in the oviparous Ichthyophis cf. kohtaoensis. A review of the present SSD data for caecilians shows that many species are monomorphic for body size but show dimorphism in head size, while other species demonstrate female-biased SSD. Male-biased SSD has not been reported for caecilians. To understand life history evolution in caecilians, further studies on the reproductive biology of other taxa are urgently needed, in particular for rhinatrematids and uraeotyphlids. New data will allow phylogenetically controlled comparative analyses to fully explore the pattern of SSD among caecilian lineages.     

Development of the tectum in Gymnophiones,with comparison to other amphibians

Tectal development in a number of caecilian (Gymnophiona: Amphibia) species was examined and compared with that in frogs and salamanders. The caecilian optic tectum develops along the same rostrocaudal and lateromedial gradients as those of frogs and salamanders. However, differences exist in the time course of development. Our data suggest that, as in salamanders, simplification of morphological complexity in caecilians is due to a retardation or loss of late developmental stages. Differences in the time course of development (heterochrony) among different caecilian species are correlated with phylogenetic history as well as with variation in life histories. The most pronounced differences in development occur between the directly developing Hypogeophis rostratus and all other species examined. In this species, the increase in the degree of morphological complexity is greatly accelerated. J. Morphol. 236:233–246, 1998. © 1998 Wiley-Liss, Inc.     

Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the Japanese pond frog Rana nigromaculata

In this study, we determined the complete nucleotide sequence of the mitochondrial genome of the Japanese pond frog Rana nigromaculata. The length of the sequence of the frog was 17,804 bp, though this was not absolute due to length variation caused by differing numbers of repetitive units in the control regions of individual frogs. The gene content, base composition, and codon usage of the Japanese pond frog conformed to those of typical vertebrate patterns. However, the comparison of gene organization between three amphibian species (Rana, Xenopus and caecilian) provided evidence that the gene arrangement of Rana differs by four tRNA gene positions from that of Xenopus or caecilian, a common gene arrangement in vertebrates. These gene rearrangements are presumed to have occurred by the tandem duplication of a gene region followed by multiple deletions of redundant genes. It is probable that the rearrangements start and end at tRNA genes involved in the initial production of a tandemly duplicated gene region. Putative secondary structures for the 22 tRNAs and the origin of the L-strand replication (OL) are described. Evolutionary relationships were estimated from the concatenated sequences of the 12 proteins encoded in the H-strand of mtDNA among 37 vertebrate species. A quartet-puzzling tree showed that three amphibian species form a monophyletic clade and that the caecilian is a sister group of the monophyletic Anura.     

Ontogenetic evidence for the Paleozoic ancestry of salamanders

The phylogenetic positions of frogs, salamanders, and caecilians have been difficult to establish. Data matrices based primarily on Paleozoic taxa support a monophyletic origin of all Lissamphibia but have resulted in widely divergent hypotheses of the nature of their common ancestor. Analysis that concentrates on the character states of the stem taxa of the extant orders, in contrast, suggests a polyphyletic origin from divergent Paleozoic clades. Comparison of patterns of larval development in Paleozoic and modern amphibians provides a means to test previous phylogenies based primarily on adult characteristics. This proves to be highly informative in the case of the origin of salamanders. Putative ancestors of salamanders are recognized from the Permo-Carboniferous boundary of Germany on the basis of ontogenetic changes observed in fossil remains of larval growth series. The entire developmental sequence from hatching to metamorphosis is revealed in an assemblage of over 600 specimens from a single locality, all belonging to the genus Apateon. Apateon forms the most speciose genus of the neotenic temnospondyl family Branchiosauridae. The sequence of ossification of individual bones and the changing configuration of the skull closely parallel those observed in the development of primitive living salamanders. These fossils provide a model of how derived features of the salamander skull may have evolved in the context of feeding specializations that appeared in early larval stages of members of the Branchiosauridae. Larvae of Apateon share many unique derived characters with salamanders of the families Hynobiidae, Salamandridae, and Ambystomatidae, which have not been recognized in any other group of Paleozoic amphibians.     

Universal COI primers for DNA barcoding amphibians

DNA barcoding is a proven tool for the rapid and unambiguous identification of species, which is essential for many activities including the vouchering tissue samples in the genome 10K initiative, genealogical reconstructions, forensics and biodiversity surveys, among many other applications. A large‐scale effort is underway to barcode all amphibian species using the universally sequenced DNA region, a partial fragment of mitochondrial cytochrome oxidase subunit I COI. This fragment is desirable because it appears to be superior to 16S for barcoding, at least for some groups of salamanders. The barcoding of amphibians is essential in part because many species are now endangered. Unfortunately, existing primers for COI often fail to achieve this goal. Herein, we report two new pairs of primers (?, ?) that in combination serve to universally amplify and sequence all three orders of Chinese amphibians as represented by 36 genera. This taxonomic diversity, which includes caecilians, salamanders and frogs, suggests that the new primer pairs will universally amplify COI for the vast majority species of amphibians.     

Higher-level salamander relationships and divergence dates inferred from complete mitochondrial genomes

Phylogenetic relationships among the salamander families have been difficult to resolve, largely because the window of time in which major lineages diverged was very short relative to the subsequently long evolutionary history of each family. We present seven new complete mitochondrial genomes representing five salamander families that have no or few mitogenome records in GenBank in order to assess the phylogenetic relationships of all salamander families from a mitogenomic perspective. Phylogenetic analyses of two data sets—one combining the entire mitogenome sequence except for the D-loop, and the other combining the deduced amino acid sequences of all 13 mitochondrial protein-coding genes—produce nearly identical well-resolved topologies. The monophyly of each family is supported, including the controversial Proteidae. The internally fertilizing salamanders are demonstrated to be a clade, concordant with recent results using nuclear genes. The internally fertilizing salamanders include two well-supported clades: one is composed of Ambystomatidae, Dicamptodontidae, and Salamandridae, the other Proteidae, Rhyacotritonidae, Amphiumidae, and Plethodontidae. In contrast to results from nuclear loci, our results support the conventional morphological hypothesis that Sirenidae is the sister-group to all other salamanders and they statistically reject the hypothesis from nuclear genes that the suborder Cryptobranchoidea (Cryptobranchidae + Hynobiidae) branched earlier than the Sirenidae. Using recently recommended fossil calibration points and a “soft bound” calibration strategy, we recalculated evolutionary timescales for tetrapods with an emphasis on living salamanders, under a Bayesian framework with and without a rate-autocorrelation assumption. Our dating results indicate: (i) the widely used rate-autocorrelation assumption in relaxed clock analyses is problematic and the accuracy of molecular dating for early lissamphibian evolution is questionable; (ii) the initial diversification of living amphibians occurred later than recent estimates would suggest, from the Late Carboniferous to the Early Permian (294 MYA); (iii) living salamanders originated during the Early Jurassic (183 MYA), and (iv) most salamander families had diverged from each other by Late Cretaceous. A likelihood-based ancestral area reconstruction analysis favors a distribution throughout Laurasia in the Early Jurassic for the common ancestor of all living salamanders.     

Frequency of independent origins of viviparity among caecilians (Gymnophiona): evidence from the first 'live-bearing' Asian amphibian

Viviparity is reported for Gegeneophis seshachari (Gymnophiona: Caeciliidae) from a gravid female containing four oviductal foetuses. The oviducts are highly vascularized and contain patches of thickened, layered tissue similar to foetal gut contents. Gegeneophis seshachari probably resemble other viviparous caecilians in having foetuses that ingest thickened oviduct lining using specialized deciduous teeth. This is the first report of viviparity in Asian amphibians and Indo-Seychellean caeciliids. Gegeneophis is the only caecilian genus known to include oviparous and viviparous species, and G. seshachari is the smallest known viviparous caecilian. Phylogenetic analysis of mitochondrial DNA sequences supports assignment of G. seshachari to a monophyletic Gegeneophis. Character optimization indicates that viviparity has evolved independently at least four times within Gymnophiona--a rate of incidence relative to the number of extant species that is higher than for other vertebrate groups except squamate reptiles. Our findings strengthen the proposal that caecilian reproduction demands further attention.     

Mitogenomic perspectives on the origin and phylogeny of living amphibians

Establishing the relationships among modern amphibians (lissamphibians) and their ancient relatives is necessary for our understanding of early tetrapod evolution. However, the phylogeny is still intractable because of the highly specialized anatomy and poor fossil record of lissamphibians. Paleobiologists are still not sure whether lissamphibians are monophyletic or polyphyletic, and which ancient group (temnospondyls or lepospondyls) is most closely related to them. In an attempt to address these problems, eight mitochondrial genomes of living amphibians were determined and compared with previously published amphibian sequences. A comprehensive molecular phylogenetic analysis of nucleotide sequences yields a highly resolved tree congruent with the traditional hypotheses (Batrachia). By using a molecular clock-independent approach for inferring dating information from molecular phylogenies, we present here the first molecular timescale for lissamphibian evolution, which suggests that lissamphibians first emerged about 330 million years ago. By observing the fit between molecular and fossil times, we suggest that the temnospondyl-origin hypothesis for lissamphibians is more credible than other hypotheses. Moreover, under this timescale, the potential geographic origins of the main living amphibian groups are discussed: (i) advanced frogs (neobatrachians) may possess an Africa-India origin; (ii) salamanders may have originated in east Asia; (iii) the tropic forest of the Triassic Pangaea may be the place of origin for the ancient caecilians. An accurate phylogeny with divergence times can be also helpful to direct the search for "missing" fossils, and can benefit comparative studies of amphibian evolution.     

Evolutionary relationships of the lungless caecilian Atretochoana eiselti (Amphibia: Gymnophiona: Typhlonectidae)

Appreciation of the diversity of caecilian amphibians has recently been enhanced by the discovery of a radically divergent aquatic caecilian of the Neotropical Typhlonectidae. Atretochoana eiselti is the largest lungless tetrapod and the only lungless caecilian, and it possesses a suite of remarkable cranial modifications that set it apart from all other caecilians. Numerical phylogenetic analyses, using 141 morphological characters, were performed in order to resolve the evolutionary relationships of Atretochoana and representatives of all other typhlonectid genera. These analyses yield a single most parsimonious tree, (Chthonerpeton (Nectocaecilia (Typhlonectes natans, Typhlonectes compressicauda) (Potomotyphlus, Atretochoana)))) , that is both well resolved and, as judged by Bremer support and by bootstrapping, is well supported. This tree is used as a basis for interpreting ecological shifts and associated morphological evolution within the Typhlonectidae. The available data suggest that the rate of morphological evolution in the Atretochoana lineage is significandy greater than that in other typhlonectid lineages.     

Fetal adaptations for viviparity in amphibians

Live‐bearing has evolved in all three orders of amphibians—frogs, salamanders, and caecilians. Developing young may be either yolk dependent, or maternal nutrients may be supplied after yolk is resorbed, depending on the species. Among frogs, embryos in two distantly related lineages develop in the skin of the maternal parents' backs; they are born either as advanced larvae or fully metamorphosed froglets, depending on the species. In other frogs, and in salamanders and caecilians, viviparity is intraoviductal; one lineage of salamanders includes species that are yolk dependent and born either as larvae or metamorphs, or that practice cannibalism and are born as metamorphs. Live‐bearing caecilians all, so far as is known, exhaust yolk before hatching and mothers provide nutrients during the rest of the relatively long gestation period. The developing young that have maternal nutrition have a number of heterochronic changes, such as precocious development of the feeding apparatus and the gut. Furthermore, several of the fetal adaptations, such as a specialized dentition and a prolonged metamorphosis, are homoplasious and present in members of two or all three of the amphibian orders. At the same time, we know little about the developmental and functional bases for fetal adaptations, and less about the factors that drive their evolution and facilitate their maintenance. J. Morphol. 276:941–960, 2015. © 2014 Wiley Periodicals, Inc.     

Molecular phylogenetics of western North American frogs of the Rana boylii species group

Phylogenetic relationships among frogs of the genus Rana from western North America are investigated using 2013 aligned bases of mitochondrial DNA sequence from the genes encoding ND1 (subunit one of NADH dehydrogenase), tRNA(Ile), tRNA(Gln), tRNA(Met), ND2, tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), tRNA(Tyr), and COI (subunit I of cytochrome c oxidase), plus the origin for light-strand replication (O(L)) between the tRNA(Asn) and tRNA(Cys) genes. The aligned sequences contain 401 phylogenetically informative characters. A well-resolved phylogenetic hypothesis in which the Rana boylii species group (R. aurora, R. boylii, R. cascadae, R. muscosa, and R. pretiosa) is monophyletic is obtained. Molecular sequence divergence suggests that the R. boylii species group is approximately 8 million years old. The traditional hypothesis showing monophyly of the yellow-legged frogs (R. boylii and R. muscosa) is statistically rejected in favor of a hypothesis in which R. aurora, R. cascadae, and R. muscosa form a clade. Reanalyses of published nuclear ribosomal DNA restriction-site data and allozymic data support a monophyletic R. boylii group, but do not effectively resolve relationships among species within this group. Eight populations of R. muscosa form two major clades separated by a biogeographic break in the Sierra Nevada of California. This biogeographic break is broadly concordant with breaks found in four other amphibian and reptilian taxa. The two major clades within R. muscosa are estimated to have diverged approximately 2.2 million years before present. Each of these major clades contains two subgroups showing approximately 1.5 million years divergence, implicating climatic effects of Pleistocene glaciation in vicariance. The four distinct subgroups of R. muscosa separated by at least 1.4 million years of evolutionary divergence are suggested as potential units for conservation.