Phylogenetic relationships of bitterlings based on mitochondrial 12S ribosomal DNA sequences

Analysis of the nucleotide sequence of the mitochondrial 12S ribosomal RNA gene of 27 species or sub-species of bitterlings showed that bitterlings comprise an Acheilognathus clade and a Tanakia-Rhodeus clade, partially supporting an earlier classification based on morphology and karyology. The monophyly of Acheilognathus is confirmed, but that of Tanakia and Rhodeus remains poorly resolved. Within the Tanakia–Rhodeus clade, all species or sub-species having a diploid chromosome number of 46 form a monophyletic group. Results support the hypothesis that evolutionary trends of bitterling karyotypes involve reduction from 2 n =48 to either 2 n =44 (by Robertsonian translocation) or 2 n =46 (by non-Robertsonian translocation).     

Molecular phylogeny of elopomorph fishes inferred from mitochondrial 12S ribosomal RNA sequences

Wang, C. H., Kuo, C. H., Mok, H. K. & Lee, S. C. (2003). Molecular phylogeny of elopomorph fishes inferred from mitochondrial 12S ribosomal RNA sequences. — Zoologica Scripta , 32 , 231–241.
Fishes of the superorder Elopomorpha include tenpounders ( Elops ), tarpon ( Megalops ), bonefishes ( Albula ), spiny eels ( Notacanthus ), apodes, and gulper eels; despite highly diversified morphological features, all undergo a leptocephalus larval stage and are thus treated (although with some dissenting views) as monophyletic. Following analysis of 12S rRNA sequences we present results that confirm a monophyletic Elopomorpha clearly separated from Clupeomorpha. Elops and Megalops share a common ancestor and are clustered in a subclade at the bottom of Elopomorpha. Albula and Notacanthus share a common ancestor forming the sister group to Anguilliformes. Saccopharyngiformes is not a sister group of Anguilliformes, as the single species sequenced here is nested deeply within the latter. Neither the suborder Congroidei nor the superfamily Congroidea within Anguilliformes are monophyletic.     

Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting

Denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA (rDNA) fragments has frequently been applied to the fingerprinting of natural bacterial populations (PCR/DGGE). In this study, sequences of bacterial universal primers frequently used in PCR/DGGE were compared with 16S rDNA sequences that represent recently proposed divisions in the domain Bacteria. We found mismatches in 16S rDNA sequences from some groups of bacteria. Inosine residues were then introduced into the bacterial universal primers to reduce amplification biases caused by these mismatches. Using the improved primers, phylotypes affiliated with Verrucomicrobia and candidate division OP11, were detected in DGGE fingerprints of groundwater populations, which have not been detected by PCR/DGGE with conventional universal primers.     

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.     

PCR primers and amplification methods for 12S ribosomal DNA, the control region, cytochrome oxidase I, and cytochrome b in bufonids and other frogs, and an overview of PCR primers which have amplified DNA in amphibians successfully

New primers (N = 24) for the amplification and sequencing of the complete or near complete 12S ribosomal DNA, about 1000 bp of the control region, 390 bp of cytochrome oxidase I, and the near complete cytochrome b are described. The 12S ribosomal DNA primers successfully amplify DNA in tetrapods; other primers successfully amplify DNA in bufonoids and other anurans. An overview of published literature and sequence data banks identified 170 mitochondrial and 96 nuclear DNA primers that have been used or are highly likely to be useful in amphibians. Primer sequences, their locations within genes, and sequence location and identity in Xenopus and human and/or mouse are presented for each primer. The utility of each primer was estimated by identifying the smallest, yet most inclusive, taxonomic category within which each primer has been successful. Primers from all published sources are mapped together. We hope that these new primers, as well as the list of primers that have been useful in amphibians, will encourage further systematic and population genetic studies of amphibians.     

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.     

Evidence for homologous recombination between repeated sequences containing 18S and 5S ribosomal RNA genes in wheat mitochondrial DNA

Closely linked genes for 18S and 5S rRNAs have been located on four different cloned SalI restriction fragments of wheat (Triticum aestivum L.) mitochondrial DNA. Restriction analysis has revealed that in each of the cloned fragments, the 18S and 5S rRNA genes are contained within the same basic structural unit, R, which is at least 4 kbp long. This unit is flanked by sequences designated u (0.8 kbp), v (13.7 kbp), w (0.7 kbp), and y (1.4 kbp), in the orientations v-R-w, v-R-y, u-R-w, and u-R-y in the four different SalI fragments. We conclude that 18S + 5S rRNA genes are located at several distinct sites in the wheat mitochondrial genome, and suggest that reciprocal intra- and/or intermolecular recombination between such repeated sequences could promote extensive genomic rearrangement and thus contribute to the physical heterogeneity that is a hallmark of most plant mitochondrial DNAs.     

Unravel the hidden protistan diversity: application of blocking primers to suppress PCR amplification of metazoan DNA

Applied Microbiology and Biotechnology - Planktonic protists, including both autotroph and heterotroph, have been recognized as a major contributor to primary production and consumers of bacteria,...     

16S ribosomal DNA-directed PCR primers for ruminal methanogens and identification of methanogens colonising young lambs

The population densities and identities of methanogens colonising new-born lambs in a grazing flock were determined from rumen samples collected at regular intervals after birth. Methanogen colonisation was found at the first sampling (1-3 days after birth) and population densities reached around 10(4) methanogens per gram at 1 week of age. Population densities increased in an exponential manner to a maximum of 10(8)-10(9) per gram at 3 weeks of age. To identify methanogens, PCR primers specific for each of the Archaea; a grouping of the orders Methanomicrobiales, Methanosarcinales and Methanococcales; the order Methanobacteriales; the order Methanococcales; the order Methanosarcinales; the genus Methanobacterium; and the genus Methanobrevibacter were designed. Primer-pair specificities were confirmed in tests with target and non-target micro-organisms. PCR analysis of DNA extracts revealed that all the detectable ruminal methanogens belonged to the order Methanobacteriales, with no methanogens belonging to the Methanomicrobiales, the Methanosarcinales, or the Methanococcales being detected. In 3 lambs, the initial colonising methanogens were Methanobrevibacter spp. and in 2 lambs were a mixture of Methanobrevibacter and Methanobacterium spp. In the latter case, the initial colonising Methanobacterium spp. subsequently disappeared and were not detectable 12-19 days after birth. Seven weeks after birth, lambs contained only Methanobrevibacter spp. This study, the first to provide information on the identities of methanogens colonising pre-ruminants, suggests that the predominant methanogens found in the mature rumen establish very soon after birth and well before a functioning rumen develops.     

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 elopomorph fishes inferred from mitochondrial ribosomal DNA sequences

The superorder Elopomorpha, a grouping which includes all teleost fishes that possess a specialized leptocephalous larva [true eels (Anguilliformes), gulpers and bobtail snipe eels (Saccopharyngiformes), bonefishes, spiny eels, and halosaurs (Albuliformes, including Notacanthiformes), ladyfishes and tarpons (Elopiformes, including Megalopiformes)] comprises >800 species for which phylogenetic relationships are poorly understood. In the present study, we analyzed mitochondrial DNA sequences in segments of the 12S and 16S rRNA genes in 33 elopomorph taxa encompassing all of the previously proposed orders, and 9 of the 15 currently recognized families of the Anguilliformes, as well as outgroup representatives from the superorders Osteoglossomorpha (nine species) and Clupeomorpha (three species), to develop phylogenetic hypotheses based on distance and parsimony methods. Both methods failed to support the monophyly of the Elopomorpha, casting doubt on the validity of the leptocephalus as an elopomorph synapomorphy. The orders Elopiformes, Albuliformes, and Anguilliformes, however, were resolved as monophyletic assemblages. Parsimony analysis supported the separation of the Anguilliformes into two groups (primitive and advanced) based on the presence of divided versus fused frontal bones. In addition, the molecular data indicated a close affinity of the anguilliform Thalassenchelys coheni (incertae sedis), known only from the leptocephalus, with the family Serrivomeridae. The implications of these data as regards the evolution of the elopomorph assemblage are discussed.     

Species-specific PCR primers for Pythium developed from ribosomal ITS1 region

AIMS: To develop a specific method for distinguishing and detecting Pythium species. METHODS AND RESULTS: Twenty PCR primers were designed from the sequences of the rDNA internal transcribed spacer 1 (ITS1) region from 34 Pythium species. The specificity of these forward primers paired with ITS2 or ITS4 and reverse universal primers was tested. Five species-specific primers were obtained, other primers showed different specificity to Pythium species. The specific amplifications enabled nine Pythium species to be differentiated. Specific detection of Pythium aphanidermatum from infested plants and P. dimorphum from soil was demonstrated. CONCLUSIONS: A method for identifying nine Pythium species using specific PCR amplification was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its rapidness and ease, the results of PCR amplified with different primers can be a powerful method for identifying Pythium species and detecting or monitoring the target fungus directly from plant material, soil and water samples.     

Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies

16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of ‘best available’ primer pairs for Bacteria and Archaea for three amplicon size classes (100–400, 400–1000, ≥1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.     

Phylogenetic relationships ofPolemoniaceae inferred from 18S ribosomal DNA sequences

Cladistic analyses of chloroplast DNA disagree with current classifications by placingPolemoniaceae near sympetalous families with two staminal whorls, includingFouquieriaceae andDiapensiaceae, rather than near sympetalous families with a single staminal whorl, such asHydrophyllaceae andConvolvulaceae. To explore further the affinities ofPolemoniaceae, we sequenced 18S ribosomal DNA for eight genera ofPolemoniaceae and 31 families representing a broadly definedAsteridae. The distribution of variation in these sequences suggest some sites are hypervariable and multiple hits at these sites have obscured much of the hierarchical structure present in the data. Nevertheless, parsimony, least-squares minimum evolution, and maximum likelihood methods all support a monophyleticPolemoniaceae that is placed nearFouquieriaceae, Diapensiaceae and related ericalean families.     

Phylogenetic relationships among cirrate octopods (Mollusca: Cephalopoda) resolved using mitochondrial 16S ribosomal DNA sequences

PHYLOGENETIC RELATIONSHIPS AMONG THE CIRRATE OCTOPODS (MOLLUSCA: Cephalopoda) were investigated using partial sequences of the 16S rRNA mitochondrial gene. The derived phylogeny supports the traditional separation of cirrate families based on web form. Genera with a single web (Opisthoteuthis, Grimpoteuthis, Luteuthis, and Cirroctopus) are clearly distinct from those with an intermediate or secondary web (Cirroteuthis, Cirrothauma, and Stauroteuthis). The cirrates with a single web are separated into three groups. The first group is represented by Opisthoteuthis species, the second by Grimpoteuthis and Luteuthis, and the third by members of the genus Cirroctopus. There is no support for the isolation of Luteuthis in a separate family (Luteuthidae). There is, however, evidence of two groupings within the genus Opisthoteuthis. The data suggest the following revisions in the systematic classification of the cirrates: (1) Cirrothauma, Cirroteuthis, and Stauroteuthis be united in the Cirroteuthidae; (2) Grimpoteuthis and Luteuthis be placed in the Grimpoteuthidae; (3) Opisthoteuthis in the Opisthoteuthidae, and; (4) Cirroctopus be considered sufficiently distinct from both Opisthoteuthidae and Grimpoteuthidae to warrant placement in a new family.     

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.