Higher-level snake phylogeny inferred from mitochondrial DNA sequences of 12S rRNA and 16S rRNA genes

Portions of two mitochondrial genes (12S and 16S ribosomal RNA) were sequenced to determine the phylogenetic relationships among the major clades of snakes. Thirty-six species, representing nearly all extant families, were examined and compared with sequences of a tuatara and three families of lizards. Snakes were found to constitute a monophyletic group (confidence probability [CP] = 96%), with the scolecophidians (blind snakes) as the most basal lineages (CP = 99%). This finding supports the hypothesis that snakes underwent a subterranean period early in their evolution. Caenophidians (advanced snakes), excluding Acrochordus, were found to be monophyletic (CP = 99%). Among the caenophidians, viperids were monophyletic (CP = 98%) and formed the sister group to the elapids plus colubrids (CP = 94%). Within the viperids, two monophyletic groups were identified: true vipers (CP = 98%) and pit vipers plus Azemiops (CP = 99%). The elapids plus Atractaspis formed a monophyletic clade (CP = 99%). Within the paraphyletic Colubridae, the largely Holarctic Colubrinae was found to be a monophyletic assemblage (CP = 98%), and the Xenodontinae was found to be polyphyletic (CP = 91%). Monophyly of the henophidians (primitive snakes) was neither supported nor rejected because of the weak resolution of relationships among those taxa, except for the clustering of Calabaria with a uropeltid, Rhinophis (CP = 94%).      

Phylogenetic relationships of African microhylid frogs inferred from DNA sequences of mitochondrial 12S and 16S rRNA genes

The phylogenetic relationships of microhylid frogs are poorly understood. The first molecular phylogeny for continental African microhylids is presented, including representatives of all subfamilies, six of the eight genera, and the enigmatic hemisotid Hemisus. Mitochondrial 12S and 16S rRNA sequence data were analysed using parsimony, likelihood and Bayesian methods. Analyses of the data are consistent with the monophyly of all sampled subfamilies and genera. Hemisus does not nest within either brevicipitines or non-brevicipitines. It is possibly the sister group to brevicipitines, in which case brevicipitines might not be microhylids. Phrynomantis and Hoplophryne potentially group with non-African, non-brevicipitine microhylids, in agreement with recent morphological and molecular data. Within brevicipitines, Breviceps is recovered as the sister group to a clade of Callulina+Spelaeophryne+Probreviceps. The relationships among the genera within this latter clade are unclear, being sensitive to the method of analysis. Optimal trees suggest the Probreviceps macrodactylus subspecies complex might be paraphyletic with respect to P. uluguruensis, corroborating preliminary morphological studies indicating that P. m. rungwensis may be a distinct species. P. m. loveridgei may be paraphyletic with respect to P. m. macrodactylus, though this is not strongly supported. Some biogeographic hypotheses are examined in light of these findings.     

Novel relationships among hyloid frogs inferred from 12S and 16S mitochondrial DNA sequences

Advanced frogs (Neobatrachia) are usually divided into two taxa, Ranoidea (the firmisternal frogs) and Hyloidea (all other neobatrachians). We investigated phylogenetic relationships among several groups of Hyloidea using 12S and 16S rRNA mitochondrial gene sequences and tested explicit relationships of certain problematic hyloid taxa using a sample of 93 neobatrachians. Parsimony, maximum likelihood, and Bayesian inference methods suggest that both the Ranoidea and Hyloidea are well-supported monophyletic groups. We reject three hypotheses using parametric bootstrap simulation: (1) Dendrobatidae lies within the Ranoidea; (2) The group containing Hylidae, Pseudidae, and Centrolenidae is monophyletic; and (3) Brachycephalus is part of Bufonidae.     

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.     

Inter- and intraspecific evolutionary relationships of the rice frog Rana limnocharis and the allied species R. cancrivora inferred from crossing experiments and mitochondrial DNA sequences of the 12S and 16S rRNA genes

The rice frog Rana limnocharis is widely distributed in Southeast Asia and the rest of the Asian region extending from India to Japan. In Japan, the Sakishima-island populations of this species were regarded as a distinct species based on morphological and genetic divergences. The main purposes of this study were to confirm the presence of intraspecific reproductively isolating mechanisms in the Sakishima-island populations of R. limnocharis, and to clarify molecular inter- and intraspecific relationships of R. limnocharis and an allied species, Rana cancrivora. The hybridization experiments revealed that there were no reproductively isolating mechanisms between the Sakishima-island populations and other populations of R. limnocharis. The molecular evolutionary relationships were investigated by analyzing nucleotide sequences of the mitochondrial 12S and 16S rRNA genes using 12 populations of R. limnocharis from Japan and Taiwan, and two populations of R. cancrivora from Thailand and the Philippines. The phylogenetic trees constructed by the NJ method showed that the two populations of R. cancrivora were clearly separated from the 12 populations of R. limnocharis, and that the 12 populations of R. limnocharis were broadly divided into three clades; the first comprising eight populations from the main islands of Japan, the second comprising the Sakishima-island populations, and the third comprising the Okinawa-island and Taiwan populations. Interestingly, the Okinawa-island and Taiwan populations of R. limnocharis showed a close relationship that possibly reflected a secondary contact between the two populations. Based on the present crossing experiments and molecular data, it seems reasonable to regard the Sakishima-island populations as a single subspecies of R. limnocharis.     

Phylogenetic relationships of the Japanese minnows,Pseudorasbora (Cyprinidae), as inferred from mitochondrial 16S rRNA gene sequences

The phylogenetic relationships among threePseudorasbora fishes (Cyprinidae, Sarcocheiichthyinae) occurring in Japan (P. parva, P. pumila pumila andP. pumila subsp. sensu Nakamura [1963]) were inferred from nucleotide sequences of the mitochondrial 16S rRNA gene. The sequences. of 1240 bp, were determined and compared for 22 specimens from 2–8 populations for each taxon, with a singlePungtungia herzi specimen as an outgroup. A total of 171 sites (13.8%) were variable among the specimens, but only 0–2 sites within each population. The phylogenetic relationships estimated by neighbor-joining, maximum-parsimony and maximum-likelihood methods confirmed a sister relationship between the twoP. pumila subspecies, with a high level of confidence. However, their genetic distinction from each other (4.1±0.4SD % sequence difference on average) was at a level similar to that between them andP. parva (5.9±0.5%). The geographic distribution of the twoP. pumila subspecies, which are separated by the Fossa Magna region, suggests that the genetic divergence of the two subspecies originated from a vicariant process separating the freshwater ichthyofaunas of eastern and western Honshu.Pseudorasbora parva populations were divided into two genetic groups (1.8±0.2% sequence difference), one group comprising continental and part of the Japanese populations, and the other the remaining Japanese populations. This suggests that at least two genetically divergent lineages had been originally distributed in Japan, but a strong possibility remains that the present situation has resulted from artificial transplantation.     

Phylogenetic analysis of sequences of the 12S and 16S rRNA mitochondrial genes in the family Bovidae: new evidence

The phylogeny of the family Bovidae has been inferred from our data on the 12S and 16S rRNA gene sequences and from the results of other authors. A considerable (2,460 bp) length of the analyzed fragments of these conserved genes and the use of different methods of cladogram construction allowed us to verify the systematic position of the genera Saiga, Pantholops, Procarpa, and Oreamnos. Saigas were shown to be phylogenetically far closer to gazelles than black-tailed gazelles and pygmy antelopes. In general, the genetic analysis data are in agreement with the results of morphological studies.     

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 。     

The phylogeny of Paralabrax (Perciformes: Serranidae) and allied taxa inferred from partial 16S and 12S mitochondrial ribosomal DNA sequences

Partial sequences of 16S and 12S mitochondrial ribosomal DNA were used to examine the phylogenetic relationships of the primarily eastern Pacific genus Paralabrax (Perciformes: Serranidae) and allied taxa. Paralabrax is considered a basal serranine, which is itself considered the basal subfamily in the Serranidae. Multiple serranines reported closely related to Paralabrax from the genera Serranus, Hypoplectrus, Cratinus, and Centropristis were used as outgroups. Species from the remaining two subfamilies, Epinephilinae and Anthiinae, of the Serranidae were also used in the analyses. The tree of the Serranidae was rooted with the families Polyprionidae and Priacanthidae. Paralabrax, the Serranidae, and the Serraninae were monophyletic in this study. Serranus was found to be paraphyletic. Centropristis, formerly considered the sister taxon to Paralabrax, was not closely related in these analyses. Cratinus agassizii, a monotypic genus from the eastern Pacific, was found to be the sister taxon to Paralabrax. There is greater resolution for intergeneric and subfamily relations than interspecific relationships. A single most parsimonious tree for the interspecific relationships of Paralabrax and allied taxa is proposed. This proposed molecular phylogeny is consistent with known biogeographic processes in the eastern Pacific.     

Molecular phylogeny of the speciose vole genus Microtus (Arvicolinae, Rodentia) inferred from mitochondrial DNA sequences

Voles of the genus Microtus represent one of the most speciose mammalian genera in the Holarctic. We established a molecular phylogeny for Microtus to resolve contentious issues of systematic relationships and evolutionary history in this genus. A total of 81 specimens representing ten Microtus species endemic to Europe as well as eight Eurasian, six Asian and one Holarctic species were sequenced for the entire cytochrome b gene (1140 bp). A further 25 sequences were retrieved from GenBank, providing data on an additional 23, mainly Nearctic, Microtus species. Phylogenetic analysis of these 48 species generated four well-supported monophyletic lineages. The genus Chionomys, snow voles, formed a distinct and well-supported lineage separate from the genus Microtus. The subgenus Microtus formed the strongest supported lineage with two sublineages displaying a close relationship between the arvalis species group (common voles) and the socialis species group (social voles). Monophyly of the Palearctic pitymyid voles, subgenus Terricola, was supported, and this subgenus was also subdivided into two monophyletic species groups. Together, these groupings clarify long-standing taxonomic uncertainties in Microtus. In addition, the Asian and the Nearctic lineages reported previously were identified although the latter group was not supported. However, relationships among the main Microtus branches were not resolved, suggesting a rapid and potentially simultaneous radiation of a widespread ancestor early in the history of the genus. This and subsequent radiations discernible in the cytochrome b phylogeny, show the considerable potential of Microtus for analysis of historical and ecological determinants of speciation in small mammals. It is evident that speciation is an ongoing process in the genus and that the molecular data provides a vital insight into current species limits as well as cladogenic events of the past.     

Phylogeny and biogeography of paradoxical frogs (Anura, Hylidae, Pseudae) inferred from 12S and 16S mitochondrial DNA

We used mitochondrial DNA sequences of 12S and 16S ribosomal RNA genes to reconstruct phylogenetic relationships of the nine species of South American aquatic hylids known as paradoxical frogs. Pseudis minuta and P. cardosoi form the sister-group to a clade comprising Lysapsus and the remaining Pseudis. We suggest the resurrection of Podonectes, including P. minutus and P. cardosoi, to avoid the nonmonophyly of Pseudis. Some doubt is cast on the species status of P. cardosoi. Lysapsus limellum, P. bolbodactyla, and P. paradoxa each may comprise more than one species, but lack of comprehensive geographic and morphological appraisals precludes taxonomic changes. Biogeographic implications of the phylogeny are discussed. The correlation between hydrographic basins and Pseudis species is not fully supported, and a recent contact between Amazon populations in eastern Bolivia and western Brazil (Rond?nia) and Paraná basin populations in the Pantanal is suggested. A dispersal-vicariance analysis showed that major diversification of Pseudis and Lysapsus species occurred in the Brazilian Shield, and that the presence of P. paradoxa and L. limellum in the Amazon and Paraná basins is due to recent dispersals. Evidence suggests a vicariant event, most likely caused by Miocene marine introgressions, as the cause for the restricted distribution of L. laevis in the Guiana Shield.     

Phylogenetic relationship among cockroach families inferred from mitochondrial12S rRNA gene sequence

A number of phylogenies exist for cockroaches that differ in the postulated relationships among families and genera. The relationship of the wood-feeding genus, Cryptocercus, to other cockroach families and to termites, has generated considerable debate. Grandcolas (1994), based on morphological analysis, synonymized the family Cryptocercidae with Polyphagidae and placed the genus Cryptocercus in the subfamily Polyphaginae. To determine if an independent set of characters supports the placement of Cryptocercus in Polyphaginae, a phylogenetic analysis of relationships among representative genera of the five cockroach families was undertaken. DNA sequence of a -430 base pair portion of the mitochondrial small ribosomal subunit gene from representatives of Blattidae, Blattellidae, Blaberidae and Cryptocercus, previously published by Kambhampati (1995) and Kambhampati et al. (1996), and the homologous sequence from representatives of Polyphagidae were used in the analysis. A total of twenty cockroach taxa and three termite genera were included in the study. Because a recent study showed that Cryptocercus punctulatus consists of a species complex, DNA sequence from four individuals collected in different parts of the U.S.A. was included in the study. The trees estimated from parsimony and neighbour-joining analyses indicated that Cryptocercus is a monophyletic clade which is most closely related to members of Blattidae. Polyphagidae is indicated as a sister group to the Blattidae + Cryptocercus complex, suggesting that Polyphagidae may belong to the superfamily Blattoidea rather than to Blaberoidea as proposed by McKittrick (1964). Blaberidae and Blattellidae were sister groups as previously proposed. Based on the present analysis, I propose that the genus Cryptocercus be retained in the family Cryptocercidae. Cockroaches     

Phylogeny of bradyrhizobia from Chinese cowpea miscellany inferred from 16S rRNA, atpD, glnII, and 16S-23S intergenic spacer sequences

The cowpea (Vigna unguiculata L.), peanut (Arachis hypogaea L.), and mung bean (Vigna radiata L.) belong to a group of plants known as the "cowpea miscellany" plants, which are widely cultivated throughout the tropic and subtropical zones of Africa and Asia. However, the phylogeny of the rhizobial strains that nodulate these plants is poorly understood. Previous studies have isolated a diversity of rhizobial strains from cowpea miscellany hosts and have suggested that, phylogenetically, they are from different species. In this work, the phylogeny of 42 slow-growing rhizobial strains, isolated from root nodules of cowpea, peanut, and mung bean from different geographical regions of China, was investigated using sequences from the 16S rRNA, atpD and glnII genes, and the 16S-23S rRNA intergenic spacer. The indigenous rhizobial strains from the cowpea miscellany could all be placed in the genus Bradyrhizobium , and Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense were the main species. Phylogenies derived from housekeeping genes were consistent with phylogenies generated from the ribosomal gene. Mung bean rhizobia clustered only into B. liaoningense and B. yuanmingense and were phylogenetically less diverse than cowpea and peanut rhizobia. Geographical origin was significantly reflected in the phylogeny of mung bean rhizobia. Most cowpea rhizobia were more closely related to the 3 major groups B. liaoningense, B. yuanmingense, and Bradyrhizobium elkanii than to the minor groups Bradyrhizobium japonicum or Bradyrhizobium canariense . However, most peanut rhizobia were more closely related to the 2 major groups B. liaoningense and B. yuanmingense than to the minor group B. elkanii.     

Phylogenetic analysis of gamma-proteobacteria inferred from nucleotide sequence comparisons of the house-keeping genes adk, aroE and gdh: comparisons with phylogeny inferred from 16S rRNA gene sequences

Nucleotide sequence comparisons of three house-keeping genes, adenylate kinase (adk), shikimate dehydrogenase (aroE), and glucose-6-phosphate dehydrogenase (gdh), were used to infer the phylogeny of 33 gamma-proteobacteria. Phylogenetic trees inferred from each gene, and from the concatenated sequences of all three genes, are, in general, similar to a 16S rRNA gene-inferred tree. Similar grouping of bacteria are revealed at the family, genus, species and strain levels in all five trees. The house-keeping genes, however, show a higher rate of nucleotide sequence substitutions. Consequently, they can possibly probe deeper branches of a phylogenetic tree than the 16S rRNA gene. However, because their nucleotide sequences are not as highly conserved among gamma-proteobacteria, family- or genus-specific primers would need to be designed for the amplification of any of these three house-keeping genes. Since these genes are used in multilocus sequence typing, it is expected that the number of sequences publicly available for many taxa will increase over time proving them very useful either at complementing 16S rRNA-inferred phylogenies or for specific, targeted, phylogenetic analysis.     

Higher-level systematics of rodents (Mammalia,Rodentia): Evidence from the mitochondrial 12S rRNA gene

Phylogenetic relationships among major rodent superfamilies traditionally have been difficult to establish because of the apparent high level of convergence and parallelism seen among morphological characters and/or rapid differentiation of rodent groups in the Paleocene/Eocene. Nucleotide sequence data from the mitochondrial 12S rRNA gene were used to clarify phylogenetic relationships among the major groups of rodents as defined by Brandt (1855) and Tullberg (1899). Based on the approximately 800 bp analyzed for the 12S rRNA gene in 59 mammalian species, including 25 of the 32 extant rodent families, the major rodent groups that could be defined as monophyletic clades were the Hystricognathi, the Muroidea, and the Geomyoidea. In addition, support for superfamilial sister-group relationships was found for Aplodontoidea with Sciuroidea and Dipodoidea with Muroidea.     

Phylogeny and biogeography of ratite birds inferred from DNA sequences of the mitochondrial ribosomal genes

The origin of the flightless ratite birds of the southern continents hasbeen debated for over a century. Whether dispersal or vicariance(continental breakup) best explains their origin depends largely on theirphylogenetic relationships. No consensus has been reached on this issuedespite many morphological and molecular studies. To address this questionfurther we sequenced a 2.8-kb region of mitochondrial DNA containing theribosomal genes in representative ratites and a tinamou. Phylogeneticanalyses indicate that Struthio (Africa) is basal and Rhea (South America)clusters with living Australasian ratites. This phylogeny agrees withtransferrin and DNA hybridization studies but not with sequence analyses ofsome protein-coding genes. These results also require reevaluation of thephylogenetic position of the extinct moas of New Zealand. We propose a newhypothesis for the origin of ratites that combines elements of dispersaland vicariance.     

Molecular phylogeny of the nasuta subgroup of Drosophila based on 12S rRNA, 16S rRNA and CoI mitochondrial genes, RAPD and ISSR polymorphisms

The nasuta subgroup is a cluster of morphologically almost similar forms with a wide range of geographic distribution. During the last three decades nature of inter-relationship among the members has been investigated at different levels of organization. The phylogenetic relationships of the members of the nasuta subgroup of the immigrans species group of Drosophila was made by employing Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats-PCR (ISSR-PCR) polymorphisms, mitochondrial 12S rRNA, 16S rRNA and Cytochrome C Oxidase subunit I (CoI) gene sequences. The phylogenetic tree generated by RAPD analysis is in nearly complete congruence with the classification based on morphophenotypic characters. The 12S and 16S rRNA genes were highly conserved across the nasuta subgroup and revealed only 3 and 4 variable sites respectively, of which only one site was informative. The CoI gene, on the other hand, revealed 57 variable sites of which 25 sites were informative. All the three species of orbital sheen complex were included in a major cluster in the phylogenetic trees derived from mitochondrial gene sequence data consistent with the morphophenotypic classification. The CoI analysis placed two species of frontal sheen complex, D. n. nasuta and D. n. albomicans in two different clades and this is inconsistent with morphological classification. The molecular clock suggested that divergence between the kohkoa complex and the albomicans complex occurred approximately 2.2 MYA, indicating recent evolution of the nasuta subgroup. The higher transition bias in the mitochondrial genes reported in the present study also suggested recent evolution of the nasuta subgroup.     

Angiosperm phylogeny inferred from sequences of four mitochondrial genes

An angiosperm phylogeny was reconstructed in a maximum likelihood analysis of sequences of four mitochondrial genes, atpl, matR, had5, and rps3, from 380 species that represent 376 genera and 296 families of seed plants. It is largely congruent with the phylogeny of angiosperms reconstructed from chloroplast genes atpB, matK, and rbcL, and nuclear 18S rDNA. The basalmost lineage consists of Amborella and Nymphaeales (including Hydatellaceae). Austrobaileyales follow this clade and are sister to the mesangiosperms, which include Chloranthaceae, Ceratophyllum, magnoliids, monocots, and eudicots. With the exception of Chloranthaceae being sister to Ceratophyllum, relationships among these five lineages are not well supported. In eudicots, Ranunculales, Sabiales, Proteales, Trochodendrales, Buxales, Gunnerales, Saxifragales, Vitales, Berberidopsidales, and Dilleniales form a basal grade of lines that diverged before the diversification of rosids and asterids. Within rosids, the COM (Celastrales-Oxalidales-Malpighiales) clade is sister to malvids (or rosid Ⅱ), instead of to the nitrogen-fixing clade as found in all previous large-scale molecular analyses of angiosperms. Santalales and Caryophyllales are members of an expanded asterid clade. This study shows that the mitochondrial genes are informative markers for resolving relationships among genera, families, or higher rank taxa across angiosperms. The low substitution rates and low homoplasy levels of the mitochondrial genes relative to the chloroplast genes, as found in this study, make them particularly useful for reconstructing ancient phylogenetic relationships. A mitochondrial gene-based angiosperm phylogeny provides an independent and essential reference for comparison with hypotheses of angiosperm phylogeny based on chloroplast genes, nuclear genes, and non-molecular data to reconstruct the underlying organismal phylogeny.     

Phylogenetic relationships among Pneumocystis from Asian macaques inferred from mitochondrial rRNA sequences

The presence of Pneumocystis organisms was detected by nested-PCR at mitochondrial large subunit (mtLSU) rRNA gene in 23 respiratory samples from Asian macaques representing two species: Macaca mulatta and M. fascicularis. A very high level of sequence heterogeneity was detected with 18 original sequence types. Two genetic groups of Pneumocystis could be distinguished from the samples. Within each group, the extent of genetic divergence was low (2.5+/-1.4% in group 1 and 2.3+/-1.7% in group 2). Genetic divergences were systematically higher when macaque-derived sequence types were compared with Pneumocystis mtLSU sequences from other primate species (from 5.3+/-2.7% to 19.3+/-3.0%). The two macaque-derived groups may be considered as distinct Pneumocystis species. Surprisingly, these Pneumocystis species were recovered from both M. mulatta and M. fascicularis suggesting that host-species restriction may not systematically occur in the genus Pneumocystis. Alternatively, these observations question about the species concept in macaques.     

Molecular phylogenetic relationships of pond frogs distributed in the Palearctic region inferred from DNA sequences of mitochondrial 12S ribosomal RNA and cytochrome b genes

The evolutionary relationships of pond frogs distributed in the Far East and Europe were investigated by analyses of nucleotide sequences of mitochondrial 12S ribosomal RNA (12S rRNA) and cytochrome b (cyt b) genes. The nucleotide sequences of a 412-bp segment of the 12S rRNA gene and a 534-bp segment of the cyt b gene were determined by the PCR-direct sequencing method using 19 frogs belonging to six species and one subspecies distributed in the Palearctic region. Phylogenetic trees were constructed by the neighbor-joining and maximum-likelihood methods using Rana catesbeiana or Xenopus laevis as an outgroup. The 412-bp segment of the 12S rRNA gene contained 65 variable sites including gap sites, and the 534-bp segment of the cyt b gene contained 160 variable sites. The nucleotide sequence divergences of the 12S rRNA gene were 0.25-4.83% within the Far Eastern frogs, 0.25-6.22% within the European frogs, and 8.74-11.24% between the Far Eastern and the European frogs, whereas those of the cyt b gene were 3.64-14.73% within the Far Eastern frogs, 0.38-14.42% within the European frogs, and 16.53-23.58% between the Far Eastern and the European frogs. Although most nucleotide substitutions were at the third codon position of the cyt b gene and were silent mutations, 4 amino acid replacements occurred within the Far Eastern frogs, 4 within the European frogs, and 11 between the Far Eastern and the European frogs. The phylogenetic trees constructed from the nucleotide sequence divergences showed slightly different topologies for the 12S rRNA and cyt b genes. R. esculenta from Ukraine was closely related to R. lessonae from Luxembourg in both the 12S rRNA and the cyt b gene sequences.