Molecular phylogenetics of the family Cyprinidae (Actinopterygii : Cypriniformes) as evidenced by sequence variation in the first intron of S7 ribosomal protein-coding gene: Further evidence from a nuclear gene of the systematic chaos in the family

The family Cyprinidae is the largest freshwater fish group in the world, including over 200 genera and 2100 species. The phylogenetic relationships of major clades within this family are simply poorly understood, largely because of the overwhelming diversity of the group; however, several investigators have advanced different hypotheses of relationships that pre- and post-date the use of shared-derived characters as advocated through phylogenetic systematics. As expected, most previous investigations used morphological characters. Recently, mitochondrial DNA (mtDNA) sequences and combined morphological and mtDNA investigations have been used to explore and advance our understanding of species relationships and test monophyletic groupings. Limitations of these studies include limited taxon sampling and a strict reliance upon maternally inherited mtDNA variation. The present study is the first endeavor to recover the phylogenetic relationships of the 12 previously recognized monophyletic subfamilies within the Cyprinidae using newly sequenced nuclear DNA (nDNA) for over 50 species representing members of the different previously hypothesized subfamily and family groupings within the Cyprinidae and from other cypriniform families as outgroup taxa. Hypothesized phylogenetic relationships are constructed using maximum parsimony and Basyesian analyses of 1042 sites, of which 971 sites were variable and 790 were phylogenetically informative. Using other appropriate cypriniform taxa of the families Catostomidae (Myxocyprinus asiaticus), Gyrinocheilidae (Gyrinocheilus aymonieri), and Balitoridae (Nemacheilus sp. and Beaufortia kweichotvensis) as outgroups, the Cyprinidae is resolved as a monophyletic group. Within the family the genera Raiamas, Barilius, Danio, and Rasbora, representing many of the tropical cyprinids, represent basal members of the family. All other species can be classified into variably supported and resolved monophyletic lineages, depending upon analysis, that are consistent with or correspond to Barbini and Leuciscini. The Barbini includes taxa traditionally aligned with the subfamily Cyprininae sensu previous morphological revisionary studies by Howes (Barbinae, Labeoninae, Cyprininae and Schizothoracinae). The Leuciscini includes six other subfamilies that are mainly divided into three separate lineages. The relationships among genera and subfamilies are discussed as well as the possible origins of major lineages. (c) 2008 Published by Elsevier Inc.

Molecular phylogeography of endangered sharp-snouted pitviper (Deinagkistrodon acutus; Reptilia, Viperidae) in Mainland China

Using phylogenetic and population genetic approaches, the present study reports the phylogeographic structure of the sharp-snouted pitviper (Deinagkistrodon acutus), a threatened snake species with commercial and medicinal importance in China. The entire mitochondrial ND2 gene (NADH dehydrogenase subunit 2) sequences of 86 individuals of D. acutus from 14 localities across its range in China were determined. Based on the results of phylogenetic analyses, distribution of diagnostic sites, haplotype network, and AMOVA hierarchical analysis, an cast-west division of the whole D. acutus population could be observed. Geographically, a line formed by a lake, river, and mountain chain (the Poyang Lake, Gan River to the southern end of the Wuyi Mountains), results in vicariance and approximately vertically splits the range into two and the whole population into two main lineages (western and eastern). The bifurcating tree suggested generally west to east dispersal trend. The data fit the isolation by distance (IBD) model well. Star-like clusters in haplotype network, significantly negative values of Fs statistics, and unimodal mismatch distributions all suggest recent demographic expansions in four areas. The results show that isolation, dispersal, bottleneck, and expansion jointly constitute the history of D. acutus. In a haplotype network, the excessive predominance of central haplotypes, few medium-frequency haplotypes, predominance (73.1 %) of the singletons among the derived haplotypes, most of which are connected to the central haplotype by only one mutational step, unsymmetrical campanulate unimodal curve of mismatch distributions and leftwards shift of the peaks, all suggest that the whole D. acutus population is a young population with low genetic diversity. Based on the data, the first priority for conservation action should be given to the Huangshan unit. (c) 2007 Elsevier Inc. All rights reserved.

Phylogeny of Oedogoniales (Chlorophyceae, Chlorophyta) inferred from 18S rDNA sequences with emphasis on the relationships in the genus Oedogonium based on ITS-2 sequences

The phylogeny of Oedogoniales was investigated by using nuclear 18S rDNA sequences. Results showed that the genus Oedocladium, as a separated clade, was clustered within the clade of Oedogonium; whereas the genus Bulbochaete was in a comparatively divergent position to the other two genera. The relationship among the species of Oedogonium was discussed, focusing on ITS-2 phylogeny analyzed combining with some morphological characteristics. Our results showed that all the dioecious nannandrous taxa involved in this study were resolved into one clade, while all the monocious taxa were clustered into another clade as a sister group to the former. The report also suggests that the dioecious macrandrous taxa form a paraphyly and could be more basally situated than the dioecious nannandrous and the monoecious taxa by means of molecular phylogeny and morphotype investigations.

PCR survey of Hox genes in the goldfish Carassius auratus auratus

A tetraploidization event took place in the cyprinid lineage leading to goldfishes about 15 million years ago. A PCR survey for Hox genes in the goldfish Carassius auratus auratus (Actinopterygii: Cyprinidae) was performed to assess the consequences of this genome duplication. Not surprisingly, the genomic organization of the Hox gene clusters of goldfish is similar to that of the closely related zebrafish (Danio rerio). However, the goldfish exhibits a much larger number of recent pseudogenes, which are characterized by indels. These findings are consistent with the hypothesis that dosage effects cause selection pressure to rapidly silence crucial developmental regulators after a tetraploidization event.

Molecular characterization of common carp (Cyprinus carpio) Sonic Hedgehog and discovery of its maternal expression

Sonic hedgehog (Shh), one of important homologous members of the hedgehog (Hh) family in vertebrates, encodes a signaling molecule that is involved in short- or long-range patterning processes during embryogenesis. In zebrafish, maternal activity of Hh was found to be contributing to the formation of primary motoneurons. However, we found that all of the known Hh members were not maternally expressed in zebrafish. In the present study, full-length cDNA of common carp (Cyprinus carpio) Shh (cShh) was gained by degenerate reverse-transcription PCR (RT-PCR) and rapid amplification of cDNA ends. Sequence comparison shows that cShh coding sequence shares 93.4% identity with zebrafish Shh coding sequence, and their corresponding protein sequences have 91.9% similarity. Comparative analysis of Shh genomic sequences and Hh protein sequences from different species revealed that the genomic structures of Hh are conserved from invertebrate to vertebrate. In contrast to zebrafish Shh, cShh transcripts were detectable from one-cell stage by RT-PCR analysis. Whole mount in situ hybridization verified the maternal expression of Shh in common carp, which is, to our knowledge, the first report of that in vertebrates, suggesting that Shh might be responsible for the maternal Hh activity in common carp.

Molecular evidence for the monophyly of East Asian groups of Cyprinidae (Teleostei : Cypriniformes) derived from the nuclear recombination activating gene 2 sequences

The family Cyprinidae is one of the largest families of fishes in the world and a well-known component of the East Asian freshwater fish fauna. However, the phylogenetic relationships among cyprinids are still poorly understood despite much effort paid on the cyprinid molecular phylogenetics. Original nucleotide sequence data of the nuclear recombination activating gene 2 were collected from 109 cyprinid species and four non-cyprinid cypriniform outgroup taxa and used to infer the cyprinid phylogenetic relationships and to estimate node divergence times. Phylogenetic reconstructions using maximum parsimony, maximum likelihood, and Bayesian analysis retrieved the same clades, only branching order within these clades varied slightly between trees. Although the morphological diversity is remarkable, the endemic cyprinid taxa in East Asia emerged as a monophyletic clade referred to as Xenocypridini. The monophyly for the subfamilies including Cyprininae and Leuciscinae, as well as the tribes including Labeonini, Gobionini, Acheilognathini, and Leuciscini, was also well resolved with high nodal support. Analysis of the RAG2 gene supported the following cyprinid molecular phylogeny: the Danioninae is the most basal subfamily within the family Cyprinidae and the Cyprininae is the sister group of the Leuciscinae. The divergence times were estimated for the nodes corresponding to the principal clades within the Cyprinidae. The family Cyprinidae appears to have originated in the mid-Eocene in Asia, with the cladogenic event of the key basal group Danioninae occurring in the early Oligocene (about 31-30 MYA), and the origins of the two subfamilies, Cyprininae and Leuciscinae, occurring in the mid-Oligocene (around 26 MYA). (c) 2006 Elsevier Inc. All rights reserved.

Phylogeny and biogeography of Chinese sisorid catfishes re-examined using mitochondrial cytochrome b and 16S rRNA gene sequences

The family Sisoridae is one of the largest and most diverse Asiatic catfish families, most species occurring in the water systems of the Qinhai-Tibetan Plateau and East Himalayas. To date published morphological and molecular phylogenetics hypotheses of sisorid catfishes are part congruent, and there are some areas of significant disagreement with respect to intergeneric relationships. We used mitochondrial cytochrome b and 16S rRNA gene sequences to clarify existing gaps in phylogenetics and to test conflicting vicariant and dispersal biogeographical hypotheses of Chinese sisorids using dispersal-vicariance analysis and weighted ancestral area analysis in combination with palaeogeographical data as well as molecular clock calibration. Our results suggest that: (1) Chinese sisorid catfishes form a monophyletic group with two distinct clades, one represented by (Gagata (Bagarius, Glyptothorax)) and the other by (glyptosternoids, Pseudecheneis); (2) the glyptosternoid is a monophyletic group and Glyptosternum, Glaridoglanis, and Exostoma are three basal species having a primitive position among it; (3) a hypothesis referring to Pseudecheneis as the sister group of the glyptosternoids, based on morphological evidence, is supported; (4) the genus Pareuchiloglanis, as presently defined, is not monophyletic; (5) congruent with previous hypotheses, the uplift of Qinghai-Tibetan Plateau played a primary role in the speciation and radiation of the Chinese sisorids; and (6) an evolutionary scenario combining aspects of both vicariance and dispersal theory is necessary to explain the distribution pattern of the glyptosternoids. In addition, using a cytochrome b substitution rate of 0.91% per million years and 0.23% for 16S rRNA, we tentatively date that the glyptosternoids most possibly originated in Oligocene-Miocene boundary (19-24Myr), and radiated from Miocene to Pleistocene, along with a center of origin in the Irrawaddy-Tsangpo drainages and several rapid speciation in a relatively short time. (c) 2005 Elsevier Inc. All rights reserved.

Molecular phylogeny of three subspecies of common carp Cyprinus carpio, based on sequence analysis of cytochrome b and control region of mtDNA

The complete cytochrome b and the control region of mtDNA (about 2070 bp in total) of 10 strains belonging to three subspecies of the common carp, including three wild subspecies (the Yangtze River wild common carp - Cyprinus carpio haematopterus, Yuanjiang River wild common carp Cyprinus carpio rubrofuscus and Volga River wild common carp - Cyprinus carpio carpio) and seven domestic strains (Xingguo red carp, Russian scattered scaled mirror carp, Qingtian carp, Japanese Koi carp, purse red carp, Big-belly carp, German mirror carp) were sequenced. Phylogenetic analysis indicated that the 10 strains form three distinct clades, corresponding to C. c. haematopterus, C. c. rubrofuscus and C. c. carpio respectively. Purse red carp, an endemic domestic strain in Jiangxi province of China, showed a higher evolution rate in comparison with the other strains of C. c. haematopterus, most probably because of intensive selection and a long history of domestication. Base variation ratios among the three subspecies varied from 0.78% (between C. c. haematopterus and C. c. rubrofuscus) to 1.47%(between C. c. carpio and C. c. rubrofuscus). The topography of the phylogenetic tree and the geographic distribution of three subspecies closely resemble each other. The divergence time between C. c. carpio and the other two subspecies was estimated to be about 0.9 Myr and about 0.5 Myr between C. c. haematopterus and C. c. rubrofuscus. Based on phylogenetic analysis, C. c. rubrofuscus might have diverged from C. c. haematopterus.

Positive selection on multiple antique allelic lineages of transferrin in the polyploid Carassius auratus

Transferrin polymorphism has been studied in the polyploid Carassius auratus by cloning and sequence analysis of cDNAs from its three subspecies C. auratus gibelio, C. auratus auratus, and C. auratus cuvieri. DNA polymorphism of extremely high extent was shown for the transferrin gene by the 248 segregation sites among coding region sequences of its alleles. The deduced amino acid sequences of the transferrin alleles showed variable theoretical physicochemical parameters, which might constitute molecular basis for their electrophoretic heterogeneity. Positive selection was inferred by the replacement/synonymous ratios larger than 1 in partial allelic lineages which was subsequently confirmed by likelihood simulation under neutral or selection models. Furthermore, the correspondent sites to these selected codons were collectively located at two planes in the crystallographic structure of rabbit transferrin, which suggested that the rapid evolution of C. auratus transferrin might correlate to its adaptation to variable environmental elements such as oxygen pressure. The minimal 26 recombination events were detected among coding sequences of C. auratus transferrin, with partial mosaic sequences and breakpoints identified by identity scanning and information site analyses. Phylogenetic analyses revealed multiple antique allelic lineages of transferrin, which was estimated to diverge fifteen to twenty MYA. All these features strongly suggested the role of balancing selection in long persistence of high transferrin polymorphism in C. auratus. Furthermore, owing to its particular evolutionary backgrounds, the silver crucian carp might possess a distinctive balancing selection mechanism.

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. (C) 2003 Elsevier Inc. All rights reserved.

Molecular systematics of Xenocyprinae (Teleostei : Cyprinidae): Taxonomy, biogeography, and coevolution of a special group restricted in east Asia

We surveyed mitochondrial DNA (mtDNA) sequence variation in the subfamily Xenocyprinae from China and used these data to estimate intraspecific, interspecific, and intergeneric phylogeny and assess biogeographic scenarios underlying the geographic structure of lineages. We sequenced 1140 bp of cytochrome b from 30 individuals of Xenocyprinae and one putative outgroup (Myxocypris asiaticus) and also sequenced 297 bp of ND4L, 1380 bp of ND4, 68 bp of tRNA(His), and 69 bp of tRNA(Ser) from 17 individuals of Xenocyprinae and the outgroup (M. asiaticus). We detected high levels of nucleotide variation among populations, species, and genera. The phylogenetic analysis suggested that Distoechodon hupeinensis might be transferred to the genus Xenocypris, the taxonomic status of the genus Plagiognathops might be preserved, and species of Xenocypris and Plagiognathops form a monophyletic group that is sister to the genus Distoechodon and Pseudobrama. The introgressive hybridization might occur among the populations of X. argentea and X. davidi, causing the two species to not be separated by mtDNA patterns according to their species identification, and the process and direction of hybridization are discussed. The spatial distributions of mtDNA lineages among populations of Xenocypris were compatible with the major geographic region, which indicated that the relationship between Hubei + Hunan and Fujian is closer than that between Hubei + Hunan and Sichuan, From a perspective of parasite investigation, our data suggested that the fauna of Hexamita in Xenocyprinae could be used to infer the phylogeny of their hosts. (C) 2001 Academic Press.

Genetic evidence for gonochoristic reproduction in gynogenetic silver crucian carp (Carassius auratus gibelio bloch) as revealed by RAPD assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp.