Haplotype diversity and phylogenetic relationships among the Iberian barbels (Barbus,Cyprinidae) reveal two evolutionary lineages

The phylogenetic relationships and haplotype diversity of all Iberian barbels were examined by analyzing the complete mitochondrial cytochrome b gene sequence (1141 bp) of 72 specimens from 59 Iberian localities. Phylogenetic findings demonstrated a clear distinction between two mitochondrial lineages and confirmed the existence of two previously considered subgenera: Barbus and Luciobarbus: The first subgenus, Barbus, is represented on the Iberian Peninsula by Barbus haasi and Barbus meridionalis. The second subgenus, Luciobarbus, includes the remaining endemic Iberian species: Barbus comizo, Barbus bocagei, Barbus microcephalus, Barbus sclateri, Barbus guiraonis, and Barbus graellsii. Mean haplotype divergence between these subgenera was 10.40%, providing evidence of a clear subdivision within the Iberian barbels. Our results conflict with those reported in a recent study, based on 307 cytochrome b base pairs, that failed to identify any division within the genus Barbus in the Iberian Peninsula. The inclusion of nine further species belonging to this genus (used as outgroups) allowed us to establish a closer relationship of the Iberian species of the subgenus Barbus with other European taxa than with the Iberian Luciobarbus, which was found to cluster with North African, Caucasian, and Greek species. At the population level, no biogeographic structure was shown by specimens of each species (only 5.98% of the variation was attributable to differences among populations of each species). Given the discrete amount of divergence found among the Luciobarbus species, the formation of current hydrographic basins during the Plio-Pleistocene seems to have played a major role in their isolation and evolution.     

Evolutionary history and speciation modes in the cyprinid genus Barbus.

Phylogenetic relationships and evolutionary patterns in the genus Barbus were examined through the analysis of the complete sequences of three mitochondrial genes: ATPases 8 and 6, which overlap slightly, and cytochrome b. This complex genus includes diploid as well as tetraploid and hexaploid species that are distributed throughout the Palaearctic, Ethiopian and Asiatic biogeographical regions. Given that genome duplication is an important evolutionary mechanism in eukaryotes, in the present report we attempt to describe its role in the evolution of the genus Barbus, as well as drawing systematic and phylogenetic conclusions. The phylogenetic results indicated the splitting of the current Barbus genus into five main mitochondrial lineages corresponding to (i) the genus Barbus sensu stricto (tetraploid, which is subdivided into the subgenera Barbus and Luciobarbus), (ii) the hexaploid species, (iii) the Ethiopian tetraploid species, (iv) the African diploid species, and (v) the Asian diploid species. The branching of 'foreign' genera as sister groups of some of these monophyletic assemblages (such as Aulopyge is to Barbus sensu stricto or Varicorhinus is to the hexaploid barbels) demonstrates the polyphyly of the group. Moreover, the relationships between the proposed lineages also show that genome duplication may be considered as a homoplasic character since it must have occurred over at least three independent periods and/or in three independent areas. In relation to the possible saltational evolutionary model for the polyploid species examined here, it was found that, although feasible at the nuclear level, the mitochondrial markers looked at do not appear to have undergone this type of evolution. Rather, they seem to have experienced more or less constant change through time.     

The phylogenetic relationships of flies in the family Drosophilidae deduced from mtDNA sequences.

The phylogenetic relationships of several taxa from representative genera, subgenera, groups, and subgroups in the Drosophilidae were examined using sequences from a 905-bp mtDNA fragment. Conventional cloning and sequencing techniques were used to obtain nucleotide sequences. In addition, polymerase chain reaction primers were designed for the rapid amplification and sequencing of this region for the species examined in the Drosophilidae. Phylogenetic analysis was done by cladistic techniques. Because of the coding nature of the 905-bp mtDNA fragment, several separate analyses of these sequences were performed. The genera Scaptomyza and Hirtodrosophila occupy ancestral branching positions in the molecular phylogeny. The genera Chymomyza and Zaprionus have intermediate branching positions, while the subgenera Drosophila and Sophophora are in the most derived position in the molecular phylogeny. Within the subgenus Sophophora, there is little resolution using these sequences, while within the subgenus Drosophila, D. melanica, D. funebris, and D. pinicola form a clade in a derived part of the phylogeny, with D. robusta and D. immigrans branching in an intermediate position in the phylogeny. D. mercatorum, a member of the repleta species group, occupies an ancestral position in the molecular phylogeny.     

Rapid radiation of the Mediterranean Luciobarbus species (Cyprinidae) after the Messinian salinity crisis of the Mediterranean Sea, inferred from mitochondrial phylogenetic analysis

Phylogenetic relationships of 64 freshwater Barbus s.s. species distributed in basins around the Mediterranean Sea were assessed using cytochrome b sequences. Our results are in concordance with previous morphological and genetic studies, which proposed that these species belong to two major lineages (or subgenera): Barbus and Luciobarbus . We were particularly interested in phylogenetic relationships among species of the Luciobarbus lineage that are primarily found in the southern Mediterranean region from the Iberian Peninsula to the Middle East. In the Luciobarbus lineage, species that were previously attributed to the Capoeta genus were clustered. In this study, we observed short internodes between monophyletic groups having a geographical agreement around the Mediterranean. However, groups from the opposite sides of the Mediterranean Sea (Iberian Peninsula– Capoeta , north-western Africa–Middle East) seem to be phylogenetically close. We therefore infer that rapid radiation of Luciobarbus species in the Late Miocene better fits our data rather than gradual founder events in the southern Mediterranean. We propose that the biogeographical event along an east–west route, responsible for the present distribution of Luciobarbus species, was the 'Lago Mare' phase of the Mediterranean Sea that provided a rapid dispersal route over extensive distances. This provides new insights into the speciation pattern of this group, and may be of general use in the study of freshwater species in these regions.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 207–222.     

Development of primer sets for PCR amplification of the PgiC gene in ferns

Polymerase chain reaction (PCR)-based nuclear DNA markers were developed for fern species. We first determined the partial nucleotide sequence of cDNA of the pgiC gene encoding cytosolic phosphoglucose isomerase from Dryopteris caudipinna, and then PCR primers for exon-primed, intron-crossing (EPIC) amplifications were designed. The EPIC primers are universally applicable to the most derived indusiate fern families such as Dryopteridaceae, Thelypteridaceae, and Woodsiaceae. The PCR products of primers 14F/16R containing two introns are moderate in size (534 bp-ca.1000 bp) and are possibly of value in phylogenetic reconstruction at specific and generic levels. Codominant nuclear DNA markers applicable to the estimation of mating systems and other population genetic studies were also developed by a combination of single-strand conformation polymorphism (SSCP) and EPIC amplification using primers 14F/15R and 15F/16R. In order to provide a case study using these markers, allelic variation of PCR products using 15F/16R was examined in populations of Arachniodes standishii (Dryopteridaceae).     

PCR Primers for Metazoan Nuclear 18S and 28S Ribosomal DNA Sequences

Background

Metagenetic analyses, which amplify and sequence target marker DNA regions from environmental samples, are increasingly employed to assess the biodiversity of communities of small organisms. Using this approach, our understanding of microbial diversity has expanded greatly. In contrast, only a few studies using this approach to characterize metazoan diversity have been reported, despite the fact that many metazoan species are small and difficult to identify or are undescribed. One of the reasons for this discrepancy is the availability of universal primers for the target taxa. In microbial studies, analysis of the 16S ribosomal DNA is standard. In contrast, the best gene for metazoan metagenetics is less clear. In the present study, we have designed primers that amplify the nuclear 18S and 28S ribosomal DNA sequences of most metazoan species with the goal of providing effective approaches for metagenetic analyses of metazoan diversity in environmental samples, with a particular emphasis on marine biodiversity.

Methodology/Principal Findings

Conserved regions suitable for designing PCR primers were identified using 14,503 and 1,072 metazoan sequences of the nuclear 18S and 28S rDNA regions, respectively. The sequence similarity of both these newly designed and the previously reported primers to the target regions of these primers were compared for each phylum to determine the expected amplification efficacy. The nucleotide diversity of the flanking regions of the primers was also estimated for genera or higher taxonomic groups of 11 phyla to determine the variable regions within the genes.

Conclusions/Significance

The identified nuclear ribosomal DNA primers (five primer pairs for 18S and eleven for 28S) and the results of the nucleotide diversity analyses provide options for primer combinations for metazoan metagenetic analyses. Additionally, advantages and disadvantages of not only the 18S and 28S ribosomal DNA, but also other marker regions as targets for metazoan metagenetic analyses, are discussed.     

Development of primer sets for PCR amplification of the PgiC gene in ferns

Polymerase chain reaction (PCR)-based nuclear DNA markers were developed for fern species. We first determined the partial nucleotide sequence of cDNA of the pgiC gene encoding cytosolic phosphoglucose isomerase from Dryopteris caudipinna, and then PCR primers for exon-primed, intron-crossing (EPIC) amplifications were designed. The EPIC primers are universally applicable to the most derived indusiate fern families such as Dryopteridaceae, Thelypteridaceae, and Woodsiaceae. The PCR products of primers 14F/16R containing two introns are moderate in size (534 bp–ca.1000 bp) and are possibly of value in phylogenetic reconstruction at specific and generic levels. Codominant nuclear DNA markers applicable to the estimation of mating systems and other population genetic studies were also developed by a combination of single-strand conformation polymorphism (SSCP) and EPIC amplification using primers 14F/15R and 15F/16R. In order to provide a case study using these markers, allelic variation of PCR products using 15F/16R was examined in populations of Arachniodes standishii (Dryopteridaceae). Received: July 4, 2001 / Accepted: September 12, 2001     

CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design

We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 3' degenerate core. A longer 5' non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).     

Recent radiation of Iberian barbel fish (Teleostei, Cyprinidae) inferred from cytochrome b genes

Nucleotide sequence fragments of the mitochondrial DNA gene encoding cytochrome b were examined in 26 individuals belonging to the seven species of Barbus endemic to the Iberian Peninsula: Barbus haasi, B. bocagei, B. graellsii, B. sclateri, B. comiza, B. guiraonis, and B. microcephalus. Six of the seven currently recognized species can be distinguished on the basis of their cytochrome b nucleotide sequences. Sequence divergence estimates for Spanish species of Barbus (0-6.5%) are, in general, low in comparison to those reported for other fish species, and hybrid individuals were found. All of these observations suggest a recent radiation. The inferred phylogenetic tree has two main clades, one including B. graellsii, B. guiraonis, and B. microcephalus, and the other the remaining species groups.     

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini)

Accurate species-level identifications underpin many aspects of basic and applied biology;however,identifications can be hampered by a lack of discriminating morphological characters,taxonomic expertise or time.Molecular approaches,such as DNA"barcoding"of the cytochrome c oxidase(COI)gene,are argued to overcome these issues.However,nuclear encoding of mitochondrial genes(numts)and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications.One insect group for which these molecular and morphological problems are significant are the dacine fruit flies(Diptera:Tephritidae:Dacini).We addressed these issues associated with COI barcoding in the dacines by first assessing several"universal"COI primers against public mitochondrial genome and numt sequences for dacine taxa.We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region.Our new primers were tested alongside universal primers on a selection of dacine species,including both fresh preserved and decades-old dry specimens.Additionally,Bactrocera tiyoni mitochondrial and nuclear genomes were compared to identify putative numts.Four numt clades were identified,three of which were amplified using existing universal primers.In contrast,our new primers preferentially amplified the"true"mitochondrial COI barcode in all dacine species tested.The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable.Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.     

Microsatellite primers for the Western Pebble‐mound Mouse (Pseudomys chapmani) that show cross amplification for other species of Australian rodent

We describe 12 dinucleotide and one tetranucleotide microsatellite loci for the Western Pebble‐mound Mouse (Pseudomys chapmani) that can be amplified with the polymerase chain reaction. All primers produced clear and highly polymorphic amplification patterns containing between seven and 14 alleles and with high expected heterozygosities. The amplification of these primers across seven related conspecifics makes them useful for population genetic studies and conservation work in several of these species.     

Molecular phylogeny and biogeography of African diploid barbs, ‘Barbus’, and allies in Africa and Asia (Teleostei: Cypriniformes)

African diploid barbs (‘Barbus’, Clypeobarbus, Barboides, etc.) are a group of small cyprinids with a body size less than 20 cm and widely distributed in drainages across Africa. These species constitute a significant component of African freshwater fish fauna. This study is the first to focus on the molecular systematics and biogeography of African diploid barbs ‘Barbus’ and its African and Asian allies using both mitochondrial and nuclear genes. We test for monophyly of groups, determine interspecific relationships and estimate the time of divergence of 52 species of ‘Barbus’ and allies using two mitochondrial and four nuclear genes. Resulting trees demonstrate that ‘Barbus’ and allies (Systomus, Barboides, Clypeobarbus and African tetraploid barbs) form a strongly supported clade; however, ‘Barbus’ is not resolved as monophyletic. Divergence time analyses identify the separation between Systomus and ‘Barbus’ plus African allies may have occurred around 26 MYA. In addition to the phylogenetic results, these findings highlight the need for more thorough taxonomic and systematic studies on ‘Barbus’ and allies using morphological and additional molecular data and greater taxon sampling, including the type species of the genus Enteromius, ‘Barbus’ potamogalis.     

Evolutionary conservation and versatility of a new set of primers for amplifying the ribosomal internal transcribed spacer regions in insects and other invertebrates

The evolutionary conservation and versatility of a new set of nuclear primers for the amplification of the ribosomal internal transcribed spacer regions in insects and other invertebrates have been studied. These primers, conserved across Insecta and other invertebrates, are based on a comprehensive taxonomic survey of the current DNA sequence databases. Their versatility was demonstrated by extensive polymerase chain reaction assays in 16 species from two arachnid orders, eight insect orders, three invertebrate and vertebrate chordate orders and by direct sequencing of the amplified products. The availability of these primers to the insect research community should facilitate the use of internal transcribed spacer regions in intraspecific studies as well as phylogenetic analysis of closely related taxa.     

Variability in nuclear DNA among nonhuman primates: Application of molecular genetic techniques to intra- and inter-species genetic analyses

Nuclear DNA clones and sequence information derived from human genetic analyses were used to detect and characterize intra- and inter-species DNA variation at several nuclear loci in hominoids and cercopithecoids. Restriction fragment length polymorphisms were found at five loci among captive rhesus monkeys. Cross-species polymerase chain reaction (PCR) amplification detected an insertion within the beta-globin gene cluster in hylobatids. The combined use of cross-species PCR and denaturing gradient gel electrophoresis detected both species differences and intra-species polymorphism in the homeobox cluster 2 of hominoids. These results a) demonstrate that DNA clones and nucleotide sequence information from human molecular genetics can be used to facilitate studies of the molecular genetics of nonhuman primates, and b) document specific examples of intra- and inter-species molecular variability at several loci. © 1992 Wiley-Liss, Inc.     

Noncoding chloroplast DNA variation in Mexican pines

Universal primers were used for PCR amplification of three noncoding regions of chloroplast DNA in order to study restriction site variation in 12 Mexican pine species. Two length mutations were identified that are of diagnostic value for two subgenera or sections of the genus. Phylogenetic analysis of the restriction site and length variation showed patterns of variation largely consistent with previous arrangements of these pines, except for the position of Pinus nelsonii, indicating that Pinus section Parraya Mayr, as circumscribed by Little and Critchfield (1969) and later authors, is not a monophyletic group.     

Microfluidic PCR-based target enrichment: A case study in two rapid radiations of Commiphora (Burseraceae) from Madagascar

Developing effective and cost-efficient multilocus nuclear datasets for angiosperm species is a continuing challenge to the systematics community. Here we describe the development and validation of a novel set of 91 nuclear markers for PCR-based target enrichment. Using microfluidic PCR and Illumina MiSeq, we generated nuclear, subgenomic libraries for 96 species simultaneously and sequenced them for a total cost of ca. $6000 USD. Approximately half of these costs include reusable reagents (primers, barcodes, and custom sequencing primers) and taxon sampling could be increased by an order of magnitude to maximize sequencing depth efficiency. The principle benefit of microfluidic PCR over alternative target enrichment strategies is that it bypasses costly library preparation. After sequencing, we evaluated the ability of the loci to resolve species level relationships within two recently radiated lineages of endemic Madagascan Commiphora Jacq. (Burseraceae) species. Our results demonstrate that (i) effective nuclear markers can be designed for non-model angiosperm taxa from these publicly available datasets; (ii) that microfluidic PCR amplification followed by high throughput sequencing can produce highly complete taxon by locus sequence data matrices with minimal resource investment; and (iii) that these numerous nuclear phylogenomic markers can improve our understanding of phylogenetic relationships withinCommiphora. We provide a synopsis of ongoing activities to enhance this microfluidic PCR-based target enrichment strategy through broader primer assays, multiplexing, and increased efficiency of sequencing depth.     

Identification of Spanish barbel species using the RAPD technique

The RAPD-PCR technique was employed to identify three endemic Spanish species of Barbus: Barbus bocagei, B. graellsii and B. sclateri , that present very similar morphologies. Using seven primers, six diagnostic bands were found in B. bocagei , 11 in B. graellsii and nine in B. sclateri . Cluster analysis of the genetic similarity values obtained from RAPD data indicated that the species B. bocagei and B. graellsii are more related to each other than to B. sclateri .     

Age estimates of Frullania (Frullaniaceae,Porellales) main lineages: another example of rapid and recent diversification in liverwort evolution

Frullania Raddi is an extant genus of liverworts (Bryophytes) widespread around the world. It belongs to the family Frullaniaceae Lorch., with a large number of species distributed into several subgenera, sections and subsections according with different morphological classifications. As shown in previous studies, most Frullania sub-generic classifications are supported by molecular data, indicating that morphological characters appear well suitable to discriminate species. However, deep among-clade relationships remain unclear, probably due to the rapid diversification of main clades, paralleling other plant lineages. In this study, we reconstruct Frullania phylogeny based on available molecular data used in previous studies, and we present time estimates for the origin of its main branches. Results supported the monophyly of most subgenera as demonstrated in previous studies and supported a rapid diversification of these main lineages. Time estimates under a relaxed molecular clock and with integrated fossil evidence and nucleotide mutation rates further suggested a Jurassic origin of the genus and a rapid diversification during Palaeogene and Neogene. This may have been influenced by geographical and climate changes during these periods as predicted for most leafy liverworts.     

Development of a molecular diagnostic system to discriminate Dreissena polymorpha (zebra mussel) and Dreissena bugensis (quagga mussel)

A 3-primer PCR system was developed to discriminate invasive zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel. The system is based on: 1) universal primers that amplifies a region of the nuclear 28s rDNA gene from both species and 2) a species-specific primer complementary to either zebra or quagga mussel. The species-specific primers bind to sequences between the binding sites for the universal primers resulting in the amplification of two products from the target species and one product from the nontarget species. Therefore, nontarget products are positive amplification controls. The 3-primer system accurately discriminated zebra and quagga mussels from seven geographically distinct populations.     

Unusually limited nucleotide sequence variation of the expressed major histocompatibility complex class I genes of a New World primate species (Saguinus oedipus)

Although major histocompatibility complex (MHC) class I molecules are, as a rule, highly polymorphic in mammalian species, those of the New World primate Saguinus oedipus (cotton-top tamarin) exhibit limited polymorphism. We have cloned and sequenced twelve MHC class I cDNAs from this species. Since cloned cotton-top tamarin cell lines express three to six MHC class I molecules, this species must have at least three functional MHC class I loci. There was, however, no evidence of locus-specific substitutions in the tamarin cDNAs. Unlike all other species studied, tamarin MHC class I cDNAs displayed limited nucleotide sequence variation. The sequence similarity between the two most divergent tamarin cDNAs was 95%. To ensure that the polymerase chain reaction (PCR) primers employed in these studies had amplified all of the tamarins' expressed MHC class I genes, we used another set of primers to amplify only exons 2 and 3 from RNA and DNA. PCR of genomic DNA resulted in the amplification of six distinct clones, of which only three were well expressed. Two of these nonexpressed genes were pseudogenes and the other was a nonclassical gene. Southern blot analysis demonstrated that the tamarin has 8–11 MHC class I genes, suggesting we had indeed cloned the majority of these genes. Cotton-top tamarins are, therefore, unique among mammalian species studied to date in that they express MHC class I molecules with limited nucleotide sequence variation.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M38403-15.