Molecular identification of dolphinfish species (genus Coryphaena) using multiplex haplotype-specific PCR of mitochondrial DNA

A rapid and reliable method of dolphinfish species identification was designed based on PCR amplification of diagnostic DNA fragments from the mitochondrial cytochrome b gene. It consisted in a tetraplex reaction producing a positive control amplicon and species-specific fragments in Coryphaena hippurus and C. equiselis. It was successfully tested in specimens of known identity and in nominal C. hippurus samples among which two C. equiselis were discovered. This approach has significant advantages over other molecular species identification methods and may help in determining species composition of mixed catches, and in forensic and food control applications of dolphinfish specimens or products.     

A molecular phylogeny of the Sylvia cantillans complex: cryptic species within the Mediterranean basin

The subalpine warbler Sylvia cantillans is formally considered a polytypic species, with four subspecies, European S. c. cantillans, albistriata, moltonii (recently resumed name: subalpina) and North African S. c. inornata. They are very similar in external morphology but clearly differ in their vocalizations. We evaluated their uncertain taxonomic status reconstructing the phylogenetic and phylogeographic relationships among populations sampled across major biogeographical areas in the European species’ range, using nucleotide sequences of the mitochondrial cytochrome b gene (mtDNA cyt b). A variety of phylogenetic analyses concordantly led to identify four major groups, only partially corresponding to the three European nominal subspecies. Phylogenetic trees showed a monophyletic group including all moltonii individuals, well diverged from all other taxa. Populations taxonomically assigned to cantillans were polyphyletic being split into two distinct clades (western and southern cantillans), with monophyletic albistriata closely related to southern cantillans. Individuals of moltonii and southern cantillans sampled in sites of sympatry in central Italy were assigned to their respective groups, with perfect concordance between phenotypic and genetic identifications. All findings indicate that moltonii should be ranked as a distinct species. Former subspecies cantillans is polyphyletic, but additional data are needed to define the taxonomic status of its two clades. Albistriata is phylogenetically related to southern cantillans and should be provisionally kept as a subspecies of S. cantillans. The cantillans complex thus provides an interesting case-study illustrating geographical structuring across small geographical ranges, and it exemplifies speciation through differentiation in allopatry leading to reproductive isolation after a secondary contact.     

Molecular phylogenetics and diversification of the genus Sporophila (Aves: Passeriformes)

The evolutionary affinities within and among many groups of nine-primaried oscines remain unresolved. One such group is Sporophila, a large genus of New World tanager-finches. Our study focused particularly on clarifying the relationship between this genus and a closely related one, Oryzoborus, and on examining the phylogenetic affinities of the "capuchinos," a group of 11 Sporophila species that share a similar male plumage coloration pattern. Our phylogenetic analyses, based on 498 bp of mitochondrial DNA sequence, indicated that: (1) Oryzoborus is embedded within a well-supported clade containing all Sporophila species, which strongly suggests that both genera should be merged, (2) the species of capuchinos comprise a monophyletic group, implying that the plumage patterns common to all probably arose only once, and (3) the capuchinos clade is comprised of two sub-clades, one including two species that are distributed in northern South America and the other one containing eight species that are present south of the Amazon River. Mean sequence divergence among the southern capuchinos species was extremely low, suggesting a rapid radiation within the last half-million years that may be related to the high level of sexual selection present in the genus and might have been promoted by marine ingressions and egressions that occurred in some southern coastal regions of South America in the Late Pleistocene.     

Phylogenetic relationships of glyptosternoid fishes (Siluriformes: Sisoridae) inferred from mitochondrial cytochrome b gene sequences

To explore phylogenetic relationships among glyptosternoid fishes, we determined nucleotide sequences of the complete mitochondrial cytochrome b gene region (1138 base pair). Thirteen species of glyptosternoid fishes and six species of non-glyptosternoids represent 10 sisorid genera were examined. Molecular phylogenetic trees were constructed using the maximum parsimony, minimum evolution, maximum likelihood, and Bayesian methods. Bayesian and maximum likelihood analyses support the monophyly of glyptosternoids, but our hypothesis of internal relationships differs from previous hypothesis. Results indicated that glyptosternoid is a monophyletic group and genera Glyptosternum and Exostoma are two basal species having a primitive position among it. Genera Euchiloglanis and Pareuchiloglanis form a sister-group. Then they form a sister-group with Pseudexostoma plus Oreoglanis. Our result also found that Pareuchiloglanis anteanalis might be considered as the synonyms of Parechiloglanis sinensis, and genus Euchiloglanis might have only one valid species, Euchiloglanis davidi.     

Molecular architecture of Pipistrellus pipistrellus/Pipistrellus pygmaeus complex (Chiroptera: Vespertilionidae): further cryptic species and Mediterranean origin of the divergence

Previous genetic analyses have demonstrated that two phonic types of one of the most common European bats, the Common pipistrelle, belong to distinct species, although they are almost identical morphologically (45 kHz Pipistrellus pipistrellus and 55 kHz Pipistrellus pygmaeus). To reconstruct the history of the species complex and explain the codistribution of both forms in Europe and the Mediterranean, we performed phylogenetic analysis based on a 402-bp portion of the cytochrome b gene. Particular attention was paid to the eastern and southern parts of the range where no data were available. We found further distinctive allopatric haplotypes from Libya and Morocco. The difference of about 6-7% described in the Libyan population suggests the occurrence of a new species in the southern Mediterranean. The species status of Moroccan population is also discussed. The phylogeographic patterns obtained and analysis of fossil records support the hypothesis of expansion of both species into Europe from the Mediterranean region during the Holocene. The allopatric speciation model fits our data best. The paleobiographic scenario envisaged is corroborated also by molecular clock estimations and correlations with Late Neogene environmental changes in the Mediterranean region which ended with the Messinian salinity crisis.     

Extensive mtDNA variation within the yellow-pine chipmunk,Tamias amoenus (Rodentia: Sciuridae), and phylogeographic inferences for northwest North America

The yellow-pine chipmunk, Tamias amoenus, is common in xerophytic forests throughout much of northwest North America. We analyzed cytochrome b sequence variation from 155 individuals representing 57 localities across the distribution of T. amoenus including 10 additional species of Tamias. Maximum likelihood and parsimony tree estimation methods were used in conjunction with nested clade analysis to infer both deep and population-level processes. Our results indicate that two currently recognized subspecies of T. amoenus (T. a. canicaudus and T. a. cratericus) are not nested within other samples of T. amoenus. Maximum uncorrected levels of intraspecific sequence divergence within remaining samples of T. amoenus are >7%. Substantial geographic variation is characterized by 12 well-supported clades that correspond to distinct mountain ranges, but do not necessarily follow existing subspecific taxonomy. Significant association between geography and genealogy was detected within many of these clades and can be attributed to different population-level processes including past fragmentation, recent range expansion, and isolation by distance.     

Phylogeny and phylogeography of critically endangered Gyps species based on nuclear and mitochondrial markers

Populations of Oriental White-backed Vulture (Gyps bengalensis) and Long-billed Vulture (Gyps indicus) dramatically declined by 95–100% on the Indian subcontinent in mid-1990s. The present study was conducted to discover the phylogeny and phylogeography of Gyps species based on nuclear (recombination activating gene, RAG-1) and mitochondrial (cytochrome b, cytb) markers. Gyps species showed monophyly and no geographic partition was observed within the three groups of Gyps species (G. bengalensis, G. indicus and G. fulvus) despite the large sample size available (n = 149). Our study supports the treatment of G. indicus and G. tenuirostris as separate species. In all analyses, the earliest divergence separated G. bengalensis from all other Gyps taxa while a sister relationship was supported between G. fulvus and G. rueppellii, and these two taxa together were sister group to a clade consisting of G. indicus, G. tenuirostris and G. coprotheres. Molecular clock estimates of both nuclear and mitochondrial (RAG-1, cytb) genes indicated a rapid and recent diversification within the Gyps species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular phylogeny and biogeography of woolly flying squirrel (Rodentia: Sciuridae), inferred from mitochondrial cytochrome b gene sequences

To investigate the genetic diversity between the populations of woolly flying squirrels (Eupetaurus) from the eastern and western extremes of the Himalayas, partial mitochondrial cytochrome b gene sequences (390-810 bp) that were determined from the museum specimens were analyzed using maximum parsimony (MP) and maximum likelihood (ML) methods. The molecular data reveal that the two specimens that were collected in northwestern Yunnan (China) are members of the genus Eupetaurus. Reconstructed phylogenetic relationships show that the populations of Eupetaurus in the eastern and western extremes of the Himalayas are two distinct species with significant genetic differences (12%) and diverged about 10.8 million years ago. Eupetaurus is significantly different from Petaurista and Pteromys. The level of estimated pairwise-sequence divergence observed between Eupetaurus and Petaurista or Pteromys is greater than that observed between Eupetaurus and Trogopterus, Belomys, Glaucomys, or Hylopetes. Considering the divergence time of the two Eupetaurus groups, the glaciations and the uplift of the Himalayas and Qinghai-Tibet plateau during the Pliocene-Pleistocene period might be the major factors affecting the present distribution of Eupetaurus along the Himalayas.     

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.     

Molecular phylogenetic perspective on speciation in the genus <Emphasis Type="Italic">Sebastes</Emphasis> (Scorpaenidae) from the Northwest Pacific and the position of <Emphasis Type="Italic">Sebastes</Emphasis> within the subfamily Sebastinae

In the Northeast and the Northwest Pacific, 26 and 68 species of Sebastes occur, respectively, the similar appearance of some pairs of shallow coastal water species in the two regions having been interpreted as evidence either for migration of ancestral species or of morphological convergence. To distinguish between these competing hypotheses, a portion of the mitochondrial DNA (mtDNA) cytochrome b gene was sequenced from 23 species of Sebastes from the Northwest Pacific and phylogenetic trees constructed so as to include the existing data from Northeastern species of Sebastes. The results clearly indicated separate origins for external morphology in Northeast and Northwest Pacific species. The shallow coastal water species in the Northwest Pacific are thought to have diverged from about 9.8MYA. Furthermore, the phylogenetic position of Sebastes was inferred among the subfamily Sebastinae, Helicolenus, Hozukius, Sebastes, and Sebastiscus, the results suggesting that Helicolenus and Hozukius are more closely related to Sebastes than Sebastiscus.     

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.     

Molecular and morphological data suggest that Spinibarbus caldwelli (Nichols) (Teleostei: Cyprinidae) is a valid species

The taxonomic problem of the cyprinid species of genus Spinibarbus, occurring in southern China and northern Vietnam, was resolved on the basis of molecular and morphological analyses. Spinibarbus caldwelli and Spinibarbus hollandi have a smooth posterior edge of the last unbranched dorsal fin ray among species in the genus. Spinibarbus caldwelli is currently regarded as a junior synonym of S. hollandi because of ambiguities in diagnostic characters. In this article, 11 mtDNA cytochrome b sequences of Spinibarbus specimens were analyzed together with Barbodes gonionotus and Puntius conchonius as outgroups. Our results showed that specimens identified as S. hollandi from Taiwan were different from those from the Asian mainland at a high level of genetic divergence (0.097–0.112), which is higher than that between the two valid species, S. sinensis and S. yunnanensis (0.089), and suggested that Taiwan specimens should be considered as a different species from the Asian mainland one. In a molecular phylogenetic analysis, the sister-group relationship between Taiwan specimens and the Asian mainland specimens was supported strongly by a high confidence level (100% in bootstrap value). Further analysis of morphological characters showed that overlap of diagnostic characters is much weaker than previously suggested. Taiwan specimens had 8 branched rays in the dorsal fin, whereas those from the mainland had almost 9–10. The molecular and morphological differences suggest S. caldwelli to be valid. The molecular divergence shows the genetic speciation of S. hollandi and S. caldwelli might have occurred 5.6–4.9 million years ago; the former could be a relict species in Taiwan, and the latter dispersed in the Asian mainland.     

Molecular phylogeny of musk deer: a genomic view with mitochondrial 16S rRNA and cytochrome b gene

The phylogenetic status of the infra order Pecora is controversial, even though it is supported by paleontological, morphological, and molecular evidence. We analyzed two mitochondrial genes (i.e., 16S rRNA and cytochrome b) to resolve the phylogenetic position of pecoran species, i.e., the Bovidae, Cervidae, and Moschidae endemic to the Indian subcontinent. We used phylogenetic analysis based on different algorithms, including neighbor joining, maximum parsimony, Bayesian inference, maximum likelihood, minimum evolution, median joining network, along with multidimensional scaling, and DNA word analysis. Our results established the basal position of Tragulidae and the monophyly of the infra order Pecora within the Suborder Ruminantia. Our results also demonstrated that Bovidae, Cervidae, and Moschidae are allied with the placement of musk deer as more closely related to bovids than to cervids. Molecular dating based on sequence analysis shows that the radiation of Pecora occurred during the early Oligocene and that the majority of the pecoran families radiated and dispersed rapidly during the Oligocene/Miocene transition.     

The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.     

Molecular approaches for identification and construction of novel insecticidal genes for crop protection

Summary The insecticidal cry (crystal) genes from Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. Discovery of new insecticidal genes is of importance for delaying the development of resistance in target insects. The diversity of Bt strains facilitates isolation of new types of cry and vip (vegetative insecticidal protein) genes. PCR is a useful technique for quick and simultaneous screening of Bt strains for classification and prediction of insecticidal activities. PCR together with other methods of analysis such as RFLP, gene sequence determination, electrophoretic, immunological and chromatographic analysis of Cry proteins and insect bioassays for evaluation of toxicity have been employed for identification of new insecticidal proteins. Some other new approaches have also been devised. Many Bt strains with novel insecticidal genes have been found. A desired combination of Cry proteins can be assembled via site-specific recombination vectors into a recipient Bt strain to create a genetically improved biopesticide. For better pest control, the cry genes have been transferred to plants. Stacking of more than one insecticidal gene is required for resistance management in transgenic crops. Modification of Cry proteins through protein engineering for increasing the toxicity and/or the insecticidal spectrum is also a promising approach, but requires detailed understanding of the structure and function of these proteins and analysis of toxin-receptor interactions. More research into this area will provide useful insights for the design of toxins for management of insect resistance. Insecticidal genes from other bacteria and plants are also being examined for their potential for deployment in transgenic crops. Stringent implementation of resistance management is needed for maintaining the efficacy of Bt transgenic crops and deriving maximum economic and environmental benefit.     

Molecular genetics of Rhabdomys pumilio subspecies boundaries: mtDNA phylogeography and karyotypic analysis by fluorescence in situ hybridization

The phylogeography of the African four-striped mouse, Rhabdomys pumilio, was investigated using complete sequences of the mtDNA cytochrome b gene (1140 bp) and a combination of fluorescence in situ hybridization (FISH) and conventional cytogenetic banding techniques (G- and C-banding). Two cytotypes (2n=46 and 2n=48) were identified by cytogenetic analysis. There is no evidence of diploid number variation within populations, difference in gross chromosome morphology or of subtle interchromosomal rearrangements at levels detected by ZOO-FISH. Analysis of the mtDNA cytochrome b resulted in two major lineages that correspond roughly to the xeric and mesic biotic zones of southern Africa. One mtDNA clade comprises specimens with 2n=48 and the other representatives of two cytotypes (2n=48 and 2n=46). The mean sequence divergence (12%, range 8.3–15.6%) separating the two mtDNA clades is comparable to among-species variation within murid genera suggesting their recognition as distinct species, the prior names for which would be R. dilectus and R. pumilio. Low sequence divergences and the diploid number dichotomy within the mesic lineage support the recognition of two subspecies corresponding to R. d. dilectus (2n=46) and R. d. chakae (2n=48). Our data do not support subspecific delimitation within the nominate, R. pumilio. Molecular dating places cladogenesis of the two putative species at less than five million years, a period characterised by extensive climatic oscillations which are thought to have resulted in habitat fragmentation throughout much of the species range.     

Evidence from DNA nucleotide sequences and ISSR profiles indicates paraphyly in subspecies of the Southern Grey Shrike (Lanius meridionalis)

We obtained DNA sequence data from mitochondrial (cytochrome b) and nuclear genes (myoglobin and ornithine decarboxylase) to reconstruct the phylogenetic relationships among eight species of shrikes (Lanius). Phylogenetic analyses based on maximum parsimony, maximum likelihood and Bayesian inference all converged into a congruent topology and defined several well-supported clades. Our multi-gene approach based on nucleotide sequences from fast-evolving and conserved genes strongly supported the paraphyly of Southern Grey Shrikes (Lanius meridionalis). The Canary Islands subspecies (L. m. koenigi) differed significantly from its European counterpart (L. m. meridionalis). Furthermore, the genetic distinctiveness of L. m. koenigi was confirmed by ISSR genomic fingerprinting. By contrast, we did not find evidence to distinguish the Canarian Southern Grey Shrike from L. m. algeriensis on the African mainland (Tunisia), and therefore these two taxa may be considered as synonymous. Together, they correspond to a separate species. The origin of the taxa investigated in this study might have originated about 6 Mya at the Miocene/Pliocene boundary when a remarkable worldwide faunal turnover and global vegetation change occurred. The Lanius genus represents a complex and taxonomically challenging group that requires additional research.     

Evaluation of random amplified polymorphic DNA for species identification and phylogenetic analysis inStylosanthes (Fabaceae)

Random amplified polymorphic DNA (RAPD) was assessed for its suitability as a tool to be used in the identification of taxa from the genusStylosanthes (Fabaceae, Papilionoideae, Aeschynomeneae). Five random primers were used to fingerprint accessions from seven species in the genus, and generated RAPD profiles that were species-specific. Data were used to examine evolutionary relationships between taxa, employing both clustering and ordination techniques, and the results were compared with those from a previous cladistic analysis of chloroplast DNA (cpDNA) restriction fragments. Both multivariate approaches indicated relationships that were generally similar to those obtained by RFLP analysis of cpDNA. However, while cluster analysis grouped together all accessions within species, ordination placed certain accessions ofS. humilis, S. macrocephala andS. capitata into separate groups. Experiments to test the assumed homology of comigrating RAPDs estimated 85.7% homology for accessions within species, and 53.8% homology for accessions between species. The value of RAPD data in systematics is discussed.     

Testing the phylogeny of swordtail fishes using split decomposition and spectral analysis

We examine ways of testing for the reliability of inference from biological sequence data using sequences from Xiphophorus fishes and newly implemented methodology for sequence analysis. The approach we take provides one means to examine the fit between model and data for different sequences and hence to evaluate heterogeneity between data sets. In the case of the present study we show D-loop sequences to be a better molecule for studying the phylogeny of Xiphophorus fishes than cytochrome b sequences. The results of the split decomposition and spectral analysis confirm an earlier phylogenetic hypothesis which had been based on maximum parsimony, neighbor-joining, maximum likelihood analyses. Correspondence to: A. Meyer     

Phylogenetic relationships among Palearctic and Nearctic burbot (Lota lota): Pleistocene extinctions and recolonization

The burbot (Lota lota Linnaeus, 1758) is the only freshwater species from the cod family. Various taxonomic hypotheses were tested against molecular data by sequencing the mitochondrial cytochrome b locus of 120 burbot from 41 populations together with the related species Molva molva (ling) and Brosme brosme (tusk), which represented the other Lotinae genera. Within the genus Lota two distinct phylogroups were observed: one in North America south of the Great Slave Lakes (Lota lota maculosa) and one in Eurasia and the remainder of the Nearctic region (Lota lota lota). The burbot lineage separated 10 Myr BP from the other Lotinae, while the genetic variation within burbot appeared to be approximately 1 Myr old. However, fossil evidence suggested that burbot already existed in the Early Pliocene in Europe, from were it probably colonized North America in the Early Pleistocene. While Nearctic burbot survived climatic oscillations and diverged in several refugia, the Eurasian form became extinct or was reduced to a very small population. In the Late Pleistocene the species recolonized the Palearctic region to establish its present distribution range.