Evolutionary relationships and reproductive isolating mechanisms in the rice frog (Fejervarya limnocharis) species complex from Sri Lanka, Thailand, Taiwan and Japan, inferred from mtDNA gene sequences, allozymes, and crossing experiments

The rice frog (Fejervarya limnocharis) species complex is widely distributed, from India to Japan, and most prevalently in Southeast Asia. Conspicuous morphological variation has been reported for this species complex throughout its distribution range. In the present study, we used mtDNA gene sequence and allozyme analyses to infer evolutionary affinities within this species complex using eight populations (Sri Lanka; Bangkok and Ranong in Thailand; Taiwan; and Hiroshima, Okinawa, Ishigaki and Iriomote in Japan). We also conducted crossing experiments among four populations from Japan, Thailand, and Sri Lanka in order to find out more about the reproductive isolating mechanisms that might exist among the East, Southeast, and South Asian populations of this species complex. The crossing experiments revealed that the Sri Lanka population is reproductively isolated from the Hiroshima, Bangkok, and Ranong populations by complete hybrid inviability, and that the Bangkok population may be reproductively isolated from the Hiroshima population by partial hybrid inviability. Thus, it is not unreasonable to regard the Sri Lanka population as a species separated from F. limnocharis. The mtDNA and allozyme data showed that the Ranong population is most closely related to the Bangkok population in nuclear genome, but more similar to the Okinawa and Taiwan populations in mtDNA genome. The present, preliminary survey may raise questions about the species status of these particular populations and also about the nature of the biological species concept.     

Genetic differentiation of the Fejervarya limnocharis complex from Bangladesh and other Asian countries elucidated by allozyme analyses

The present study was conducted to elucidate the genetic divergence and the phylogenetic relationships in the F. limnocharis complex from Bangladesh and other Asian countries such as Sri Lanka, Thailand, Malaysia, Taiwan and Japan by allozyme analyses. We used a total of 95 frogs of the F. limnocharis complex from these countries and F. cancrivora from the Philippines as an outgroup. Based on body size, the F. limnocharis complex from Bangladesh was divided into three distinct groups: large, medium and small types. Allozyme analyses were carried out with 28 loci encoding 20 enzymes and two blood proteins by horizontal starch-gel electrophoresis. When genetic distance was calculated, distinct divergence was found among the three types: mean genetic distance was 0.782 between the small and medium types, 1.458 between the large and medium types, and 1.520 between the large and small types. Phylogenetic trees based on genetic distance showed that all populations of Bangladesh small type strongly formed a cluster and were found to be most closely related to the Sri Lanka population; that all populations of Bangladesh large type formed a very strong cluster and were grouped with several populations from Thailand, Malaysia, Japan, and Taiwan; and that the medium type was segregated from all other groups. This may imply that each of the three types is a different species, and that the medium type is possibly an undescribed taxon.     

Reproductive isolating mechanisms and molecular phylogenetic relationships among Palearctic and Oriental brown frogs

Crossing experiments were made among various brown frog species and populations collected from Japan, China, Russia and Taiwan. The main purpose of these experiments was to confirm the existence of reproductive isolating mechanisms among Rana pirica from Japan, R. chensinensis from China and R. chensinensis from Russia, and between these three taxa and the other brown frogs distributed in the Palearctic and Oriental regions. It was found that there was no or a slight gametic isolation among the three taxa. While there was a nearly equal number of male and female offspring in the control groups, the hybrid frogs were all males, and completely sterile upon attaining sexual maturity. Thus, each of the Japanese R. pirica and the Russian R. chensinensis is a valid species, distinct from the Chinese R. chensinensis. The phylogenetic tree based on nucleotide sequence data from the mitochondrial 12S and 16S rRNA genes of the Palearctic and Oriental brown frogs showed that the three taxa are included in a cluster together with the other species with 2n=24 chromosomes. The present crossing experiments and molecular data support the hypothesis that each of them is a separate but closely related species.     

Multiple pathways for invasion of anurans on a Pacific island

Since 1937, thirteen species of non-indigenous anurans have made their way to Guam. Of these, at least six have established breeding populations. Various pathways led to the introduction of these species to the island. The only anuran intentionally introduced was Chaunus marinus (formerly Bufo marinus ), which was brought to Guam as a biocontrol agent. Kaloula picta, K. pulchra, Polypedates leucomystax , and probably Litoria fallax arrived as stowaways via maritime or air-transport vessels. Eleutherodactylus coqui and Euhyas (formerly Eleutherodactylus ) planirostris appear to have entered Guam through the horticultural trade. Specimens of Pseudacris regilla were found among agricultural products and Christmas trees. Five species have been transported to Guam via the aquacultural trade. The importation of tilapia, milkfish, and white shrimp from China, Hong Kong, Taiwan, and the Philippines was associated with the introduction to Guam of Fejervarya cancrivora, F. limnocharis sensu lato, Microhyla pulchra, Polypedates megacephalus , and Sylvirana guentheri (formerly Rana guentheri ). Presently, no quarantine or containment guidelines have been established for Guam's aquacultural industry.     

用RAPD分析四种无尾类的系统演化关系

利用RAPD技术检测了分属无尾目3个科（雨蛙科、蟾蜍科、蛙科）的黑眶蟾蜍（Bufomelanostictus)、中国雨蛙（Hyla chinensis）、泽陆蛙（Rana limnocharis）、沼水蛙（R.guentheri)的系统发生关系。经19个随机引物对4个物种基因组DNA进行扩增,选择其中扩增谱带清晰的16个引物进行分析,计算不同科间及同一科内不同种间的遗传距离,结果表明：16个引物获得的RAPD谱带均表现出不同程度的多态性;泽陆蛙与沼水蛙间的亲缘关系最近,而黑眶蟾蜍与中国雨蛙之间的亲缘关系较黑眶蟾蜍与蛙科的泽陆蛙、沼水蛙之间以及中国雨蛙与泽陆蛙、沼水蛙之间的亲缘关系近,从基因组DNA水平上也说明雨蛙科与蟾蜍科间的亲缘关系更近,与蛙科的亲缘关系更远,这与形态学、染色体和线粒体DNA多态性研究的分析结果一致,从而进一步从分子水平上为无属目这3科的系统演化提供了新的证据。     

Multiple invasions of the Ryukyu Archipelago by Oriental frogs of the subgenus Odorrana with phylogenetic reassessment of the related subgenera of the genus Rana

The genus Rana, notably diversified in Oriental regions from China to Southeast Asia, includes a group of cascade frogs assigned to subgenera Odorrana and Eburana. Among them, R. ishikawae and the R. narina complex represent the northernmost members occurring from Taiwan to the Ryukyu Archipelago of Japan. Relationships of these frogs with the continental members, as well as the history of their invasions to islands, have been unclear. The taxonomic status of Odorrana and related genera varies among authors and no phylogenetic reassessment has been done. Using partial sequences of mitochondrial 12S and 16S rRNA genes, we estimated phylogenetic relationships among 17 species of the section Hylarana including Odorrana and Eburana, and related species from the Ryukyus, Taiwan, China, Thailand, Malaysia, and Indonesia. We estimate that (1) Odorrana is monophyletic and encompasses species of Eburana and R. hosii, which is now placed in Chalcorana, (2) the ancestor of R. ishikawae separated from other Rana in the middle to late Miocene prior to its entry to the Ryukyu Archipelago, (3) the ancestor of the R. narina complex later diversified in continental Asia, and invaded the Ryukyu Archipelago through Taiwan, (4) the R. narina complex attained its current distribution within the Ryukyus through niche segregations, and (5) vicariance of R. hosii between Malay Peninsula and Borneo occurred much later than the divergence events in the R. narina complex. Current subgeneric classification of Rana, at least of Southeast Asian members, requires full reassessment in the light of phylogenetic relationships.     

A taxonomic study of the Rana narina complex, with description of three new species (Amphibia: Ranidae)

A morphometric and electrophoretic survey was conducted to examine taxonomic relationships among eight population samples of the Rana narina complex from the Ryukyu Archipelago of Japan and Taiwan. Five discrete morphotypes are differentiated, and these showed considerable genetic differentiation from each other. From the available information, each of the five morphotypes is judged to represent a species, and three are described as new. Rana utsunomiyaorum sp. nov. and R. supranarina sp. nov. are sympatric in Ishigakijima and Iriomotejima islands of the Yaeyama Group, and differ from the other members by having a shorter hindlimb. Rana utsunomiyaorum is the smallest in the complex, while R. supranarina is the largest. Rana amamiensis sp. nov . occurs on Amamioshima and Tokunoshima islands of the Amami Group and, like R. narina from Okinawajima of the Okinawa Group, has a long hindlimb; it differs from the latter species by having a larger body and relatively small tympanum. These two species differ from R. swinhoana from Taiwan by having a narrower disk on the third finger. A key to known species of the complex is given. Further, syslematics of the R. narina complex within Rana , body size in the two sympatric species, and sexual dimorphism found in this complex are discussed.     

从12S rRNA基因序列研究中国蛙科24种的进化关系

测定了中国蛙科动物24种和蟾蜍科的中华大蟾蜍线粒体12S rRNA基因长约400bp片段的序列。采用邻接法,对其数据的系统发生分析表明24种可分成3个支系：第一个支系包括棘腹蛙、合江棘蛙、棘胸蛙、大头蛙、虎纹蛙、倭蛙、泽陆蛙共７种,其中３种棘蛙组成一个单系;第二个支系由３种湍蛙,即戴云湍蛙、华南湍蛙和武夷湍蛙组成,应属于湍蛙亚科;第三支系包含余下的１４种蛙,其中５种林蛙和４种臭蛙分别组成一个单系;２种侧褶蛙先聚合后,再与弹琴水蛙聚合,２种粗上先聚合在一起,再与由林蛙组成的单系群聚合。第三支系首先与第二支系相聚,然后它们与第一支系形成姐妹群,即由蛙亚科的７个物种组成的第一支系,不与蛙亚科另１４个物种组成的第三支系直接聚合,表明第一支系的物种可能不属于蛙亚科。此外,分子证据提示把陆蛙属、大头蛙属、虎纹蛙属、棘棘属、臭蛙属、粗皮蛙属和侧褶蛙属从原来的蛙属分出有其合理性。、     

Phylogenetic relationships of the Chinese brown frogs (genus Rana) inferred from partial mitochondrial 12S and 16S rRNA gene sequences

Based on partial sequences of the 12S and 16S ribosomal RNA genes, we estimated phylogenetic relationships among brown frogs of the Rana temporaria group from China. From the phylogenetic trees obtained, we propose to include Rana zhengi in the brown frogs. Monophyly of the brown frogs was not unambiguously supported, but four well-supported clades (A, B, C, and D) always emerged, although relationships among them remained unresolved. Clade A contained brown frogs with 24 chromosomes and was split into two distinct subclades (Subclade A-1: R. chensinensis and R. huanrenensis; Subclade A-2: R. dybowskii). Polytomous relationships among populations of R. chensinensis and R. huanrenensis suggested the necessity of further taxonomic assessment. Rana kunyuensis proved to be the sister group to R. amurensis, and these two species formed Clade B. Clade C was composed of R. omeimontis and R. chaochiaoensis, and Clade D included R. sauteri, which has been placed in other ranid genera. These relationships did not change after adding published data, and monophyly of Subclade A-1, A-2, and other East Asian brown frogs with 24 chromosomes (R. pirica and R. ornativentris) was ascertained, though their relationships were unresolved. Clade C, together with R. japonica and R. longicrus, also formed a monophyletic group. Brown frogs related to Clades A and C were estimated to have dispersed from continental Asia to adjacent regions through multiple events.     

中国林蛙的分子系统关系

测定了 6种林蛙和用作外群的 2种侧褶蛙和 1种陆蛙的线粒体 12SrRNA基因序列 393bp。序列两两对位比较表明内外群间的位点替换率是 7 3%到 2 3 1% ,内群中物种间则为 0 0 %到 9 2 %。依据上述DNA序列 ,用距离法和最大简约法的系统发育分析表明 :①研究的 6种林蛙聚为一支 ,构成单系群 ,并有高的BPs值(90 %以上 )支持 ;② 6种林蛙可以分成 2个姐妹群 ,即中国林蛙、黑龙江林蛙和桓仁林蛙为一组 (BPs >94% ) ,峨眉林蛙、昭觉林蛙和镇海林蛙为一组 (BPs >5 0 % ) ;③昭觉林蛙与镇海林蛙有较近的亲缘关系 ;④中国林蛙的榆中种群与牡丹江种群间的遗传分化似乎达到了种级分化水平。     

A new species of the Fejervarya limnocharis complex from Japan (Anura, Dicroglossidae)

We describe a new species of dicroglossid frog of the Fejervarya limnocharis complex from western Honshu, Japan Mainland. The new species, Fejervarya kawamurai, is genetically closer to F. sakishimensis than to F. limnocharis. It differs from F. sakishimensis by smaller tympanum, head, forelimb, hindlimb, foot, and tibia lengths, all relative to snout-vent length, and from F. multistriata by relatively shorter forelimb, hindlimb, foot, and tibia. From F. limnocharis and F. iskandari, it is differentiated by relatively smaller forelimb, hindlimb, foot, and tibia lengths. Taxonomic problems of Fejervarya populations occurring in Central Ryukyus, continental China, and Taiwan are discussed.     

Survey of Spirometra erinaceieuropaei in frogs in Taiwan and its experimental infection in cats

Eighteen of 56 (32.1%) wild Rana limnocharis from central and south Taiwan were found to contain plerocercoids of Spirometra erinaceieuropaei. This is the first report of S. erinaceieuropaei infections in frogs in Taiwan, with the plerocercoids being recovered from the thigh and back muscles or under the skin. Other species of frogs examined, including nine wild R. latouchii, one wild Buergeria robustus and 110 cultured R. rugulosa were free of infection. The plerocercoids were orally inoculated into four cats; three of which were each given a single plerocercoid and one a dose of three plerocercoids. Daily faecal examination showed that two cats started shedding eggs of S. erinaceieuropaei on day 8 postinfection (PI) and the other two on day 10 PI. The highest eggs per gram and eggs per day for a single worm was found to be 428,000 and 14,416,000 respectively. Only the cat inoculated with three plerocercoids shed proglottids in its faeces during the 2 month observation period.     

Morphological divergence, reproductive isolating mechanism, and molecular phylogenetic relationships among Indonesia, Malaysia, and Japan populations of the Fejervarya limnocharis complex (Anura, Ranidae)

In order to elucidate the taxonomic status of the Fejervarya limnocharis complex relative to Malaysia and Japan populations, morphological observations and molecular phylogenetic analysis were carried out using three populations from Indonesia (type locality), Malaysia, and Japan. In addition, we conducted histological and spermatogenic observations using hybrids among these populations. Principal component and cluster analyses demonstrated that these populations could be clearly separated from one another. Abnormal testes were found in the hybrids between the Japan and Indonesia populations and between the Japan and Malaysia populations, but testes of the controls and hybrids between the Malaysia and Indonesia populations were quite normal. The mean number of univalents per cell was 5.42, 4.58, and 0.20 in hybrids between the Indonesia and Japan populations, Malaysia and Japan populations, and Indonesia and Malaysia populations, respectively. Sequence divergences in 16S rRNA and Cyt b genes were 0-0.4% (xbar=0.2%) and 0.3-1.5% (xbar=1.0%), respectively, between the Malaysia and Indonesia populations, and 2.4-2.6% (xbar=2.5%) and 11.0-12.0% (xbar=11.5%) between the Japan population and F. limnocharis complex, including the Malaysia and Indonesia populations and F. multistriata from China. This study indicated that the Malaysia population and F. multistriata from China should be designated as a subspecies of topotypic F. limnocharis, and that the Japan population should be regarded as a distinct species.     

Genetic relationships and reproductive-isolation mechanisms among the Fejervarya limnocharis complex from Indonesia (Java) and other Asian countries

In order to elucidate the genetic relationships and reproductive-isolation mechanisms among the Fejervarya limnocharis complex from Indonesia and other Asian countries, allozyme analyses and crossing experiments were carried out using 208 individuals from 21 localities in eight Asian countries. The allozyme analyses revealed that 17 enzymes examined were controlled by genes at 27 loci, and that 7.9 phenotypes were produced by 5.2 alleles on average. The two species recognized in F. limnocharis sensu lato from Southeast Asia (i.e., F. limnocharis sensu stricto and F. iskandari) were found to occur sympatrically at three localities (Bogor, Cianjur and Malingping), all on Java, Indonesia. Fejervaya iskandari was dominant at each of these localities and showed substantial geographic genetic variation. Laboratory-produced hybrids between F. limnocharis and F. iskandari from Java became underdeveloped and died at the tadpole stage, suggesting that these species are completely isolated by hybrid inviability. Hybrids between topotypic F. limnocharis and the Malaysian and Japanese conspecific populations developed normally to metamorphosis. Likewise, hybrids between topotypic F. iskandari and the Thailand and Bangladesh conspecific populations also showed normal viability throughout larval development. The present allozyme analyses and crossing experiments strongly suggested the presence of two distinct forms, the large type and the small type, in the F. limnocharis complex from Asia, and further subdivision of the large type into the F. limnocharis assemblage and the F. iskandari assemblage. The small type was found in samples from India, Thailand, Bangladesh and Sri Lanka, and included at least three different species. The sample from Pilok, Thailand, was considered to represent an undescribed species.     

虎纹蛙及其它几种蛙科动物线粒体16S rRNA的空间结构分析

测定了虎纹蛙16S rRNA基因全序列,并从GeneBank上下载了5种蛙科动物同源序列,利用CLUSTAL与MEGA3.1软件比较了种间的序列差异,用RNAdraw软件推测分析二级结构的空间构象,并根据空间结构对这6种蛙科动物的分类地位进行讨论分析。结果表明：虎纹蛙与黑斑蛙的二级结构最为相似,与泽蛙结构大体一致,而与黑斑蛙同属的金线蛙则表现出较大的差异;凹耳蛙与上述几种差别较大;福建大头娃与这几种蛙科动物差距最为明显。空间结构分析与序列比较结果存在一定的差异。     

Evidence of Segregated Spawning in a Single Marine Fish Stock: Sympatric Divergence of Ecotypes in Icelandic Cod?

There is increasing recognition of intraspecific diversity and population structure within marine fish species, yet there is little direct evidence of the isolating mechanisms that maintain it or documentation of its ecological extent. We analyzed depth and temperature histories collected by electronic data storage tags retrieved from 104 Atlantic cod at liberty ≥1 year to evaluate a possible isolating mechanisms maintaining population structure within the Icelandic cod stock. This stock consists of two distinct behavioral types, resident coastal cod and migratory frontal cod, each occurring within two geographically distinct populations. Despite being captured together on the same spawning grounds, we show the behavioral types seem reproductively isolated by fine-scale differences in spawning habitat selection, primarily depth. Additionally, the different groups occupied distinct seasonal thermal and bathymetric niches that generally demonstrated low levels of overlap throughout the year. Our results indicate that isolating mechanisms, such as differential habitat selection during spawning, might contribute to maintaining diversity and fine-scale population structure in broadcast-spawning marine fishes.     

Molecular phylogenetics and reproductive incompatibility in a complex of cryptic species of aphid parasitoids

We infer the phylogeny of a complex of cryptic species and populations of parasitic wasps and examine how reproductive incompatibility maps onto the molecular phylogeny. We used four nuclear (28S-D2, ITS1, ITS2, ArgK) and two mitochondrial (COI, COII) gene regions to analyze relationships among populations in the Aphelinus varipes species complex (Hymenoptera: Aphelinidae) from throughout Eurasia (France, Georgia, Israel, China, Korea and Japan) and from three aphid hosts (Aphis glycines, Diuraphis noxia and Rhopalosiphum padi; Hemiptera: Aphididae). A combined analysis of 21 genotypes of Aphelinus resulted in six most-parsimonious trees, and successive approximations character-weighting selected two of these as best supported by the data. All six gene regions were necessary to fully resolve the relationships among taxa. Four clades within the A. varipes complex were distinguished: (1) Aphelinus kurdjumovi, (2) Aphelinus hordei, (3) Aphelinus atriplicis, Aphelinus varipes, and Aphelinus albipodus, and (4) Aphelinus certus (populations from China, Korea, and Japan). Based on rates of nucleotide substitutions, these clades diverged between 78 and 526 thousand years ago during a period of repeated glaciations in Eurasia. In laboratory crosses, A. kurdjumovi, A. hordei, and A. varipes were reproductively incompatible with one another and all other populations. A. atriplicis was incompatible with these three species, but not with A. certus. The populations of A. certus from China, Japan, and Korea were reproductively compatible with one another but not with the other populations. Thus, with one exception, entities that were phylogenetically distinct were also reproductively incompatible with one another. Our evidence on molecular differentiation and reproductive incompatibility supports recognition of at least five cryptic species in the A. varipes complex. We discuss likely reasons for the high rate of speciation in this complex.     

从线粒体基因探讨中国大头蛙群的分类及其属内地位

Partial sequences of the mitochondrial 12S rRNA and 16S rRNA gene were determined for 8 populations of three species of Chinese Limnonectes, and aligned with the published sequences of Limnonectes from other parts of the world. When Nanorana parker, Paa boulengeri, Fejervarya limnocharis and Hoplobatrachus rugulosus was used as outgroup taxa (Accession Nos. AY158705, AY313685, AF206111, AF206491, AY322311). The sequences of the 12S rRNA and 16S rRNA genes totaled 950 nueleotide positions with gaps including 510 variable sites. We reconstructed phylogenetie trees using Clustal X 1.8, Mega 2.1 and PHYLIP 3.5e software, and using the maximum parsimony and maximum likelihood methods, respectively. Our analyses suggest that these fanged frogs from China are another monophyletie group in addition to the four monophyletie groups identified by previous studies. The Chinese Limnonectes were grouped into three elades (BCL 55% ). The first elade contains one species (BCL 100% ), from a population of Limnoneetes fragilis from Hainan Province. The second contains four individuals (BCL 100% ), i. e. two populations of Limnonectes kuhlii from Yunnan Province. The third contains one species (BCL 100% ), i. e. five populations of Limnonectes fujianensis from Fujian Province and 1 from Taiwan Province. The resulted phylogenetie trees indicate L. fragilis is basal to L. kuhlii L. fujianensis 。     

Molecular evidence of the existence of two sibling species within the echinothurioid echinoid Asthenosoma ijimai from Japanese waters

The echinoid, Asthenosoma ijimai belonging to the order Echinothurioida from Japanese waters shows the geographical variation in morphological and ecological characters. The echinothurioid from Ryukyu Islands in southern Japan is cleary different from that of Sagami Bay and Suruga Bay in the middle part of Japan at non-molecular level.Their phylogenetic and taxonomic relationships were studied at the molecular level by allozyme analysis. The results demonstrated that the two echinothurioids from Ryukyu Islands and Sagami Bay do not share gene pools with each other, and they were fixed for different alleles at five genetic loci (Mdh, G6pd, Po, Alk-3 and Est-7) in a total of 23 enzyme genetic loci scored. This indicates no gene flow between the two echinothurioids, and is a molecular evidence for that they are reproductively isolated and genetically distinct species. The Nei's genetic distance (D=0.181) between the two were significantly higher than those between conspecific local populations, and comparable to those between closely related species in many other animals containing echinoderms. The present molecular data are well consistent with the non-molecular evidence from morphology, developmental biology and ecology. Putting these data together, we propose that the two echinothurioids should be classified as two sibling species of the genus Asthenosoma and would like to give the following scientific names: the echinothurioid species from Sagami Bay is Asthenosoma ijimai and that from Ryukyu Islands is A. ijimai R.     

Complete nucleotide sequence and gene arrangement of the mitochondrial genome of the crab-eating frog Fejervarya cancrivora and evolutionary implications

The complete nucleotide sequence of the mitochondrial (mt) genome of the crab-eating frog, Fejervarya cancrivora Gravenhorst (Amphibia: Anura: Ranidae), was determined. The mt genome is 17,843 bp long and contains 13 protein-coding (ATP6, ATP8, COI-III, ND1-6 and 4L, and Cyt b) and two ribosomal RNA (12S and 16SrRNA) genes. Although metazoan mt genomes typically encode 22 transfer RNA genes (tRNAs), the F. cancrivora mtDNA contains 23 tRNAs due to the presence of an extra copy of tRNA(Met). A major noncoding region and a prominent intergenic spacer corresponding to the control region and light-strand replication origin were also found. To confirm the phylogenetic position of F. cancrivora, we compared the gene arrangement with that of other anurans and performed phylogenetic analyses based on mt genomic data. The genome organization of F. cancrivora mtDNA differs from that of typical vertebrates and neobatrachian frogs but is identical with that of F. limnocharis, suggesting that the unique gene arrangement occurred in the common ancestor of the genus. Phylogenetic analyses supported the monophyly of the Fejervarya species used here as well as the dicroglossini clade. Although the family Ranidae as previously recognized (= Ranidae, Discoglossidae, and some other natatanuran families; sensu Frost et al., 2006) is shown as a clade in the maximum parsimony analysis, the maximum likelihood and the Bayesian analyses suggest the paraphyly of the Ranidae with respect to the families, Mantellidae and Rhacophoridae. Three-tandem duplications of gene regions followed by subsequent deletions of supernumerary genes were proposed to explain the evolution of the extra tRNA(Met) and translocation of ND5 from the original neobatrachian gene order.