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.

Investigations into the perplexing interrelationship of the Genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodontidae)

The phylogenetic relationships within the genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodontidae) remain unresolved. Because of the use of Takifugu as model organisms, the resolution of these relationships is crucial for the interpretation of evolutionary trends in biology. Pufferfishes of this genus are comprised of a comparatively small number of species and are mainly distributed along the coastal region of the western part of the Sea of Japan and the coastline of China. Mitochondrial gene sequences were employed to test the phylogenetic hypotheses within the genus. Seventeen species of the genus were examined. Molecular phylogenetic trees were constructed using the maximum parsimony, neighbor-joining, maximum likelihood and Bayesian methods. Our hypothesis of internal relationships within the genus differs from previous hypotheses. Our results indicate that (1) the genus Takifugu is a monophyletic assemblage; (2) the genus is divided into 6 subgroups based on the molecular data; and (3) there is low genetic diversity among the species within this genus. In addition, speciation within Takifugu appears to be driven by hybridization and isolation by distribution. Our results also suggested that the taxonomy in the genus should be clarified based on both molecular and morphological data.

Phylogenetic relationships among the genera of the Penaeidae (Crustacea : Decapoda) revealed by mitochondrial 16S rRNA gene sequences

The phylogenetic relationships within the family Penaeidae are examined based on mitochondrial 16S rRNA gene sequence analysis of 30 species from 20 genera. The analysis generally supports the three- tribe scheme proposed by Burkenroad ( 1983) but it is not consistent with the five- group classification of Kubo ( 1949). Three clades are resolved: ( Penaeus sensu stricto + Fenneropenaeus + Litopenaeus + Farfantepenaeus + Marsupenaeus + Melicertus + Funchalia + Heteropenaeus), ( Metapenaeus + Parapenaeopsis + Xiphopenaeus + Rimapenaeus + Megokris + Trachysalambria) and ( Metapenaeopsis + Penaeopsis + Parapenaeus), corresponding to the Penaeini, Trachypenaeini and Parapenaeini respectively, while the affinities of Atypopenaeus and Trachypenaeopsis are obscure. The molecular data support that Miyadiella represents the juvenile stage of Atypopenaeus. Within the Trachypenaeini, Trachypenaeus sensu lato is clearly paraphyletic, while the monophyly of Penaeus sensu lato in the Penaeini is questionable.

Polyphasic characterization of water bloom forming Raphidiopsis species (cyanobacteria) from central China

Two strains of Raphidiopsis Fritsch et Rich were isolated from a fishpond in Wuhan city, China and rendered axenic, and characterized by a combination of morphological, physiological, biochemical and genetic methods. Morphologically the strains were identified as Raphidiopsis mediterranea Skuja (straight trichomes) and R. curvata Fritsch et Rich (coiled trichomes). These two strains demonstrated slight differences in optimal temperature range and GC content, while sharing some common characteristics including inability to grow hetertrophically, similar salinity tolerance (up to 0.78%) and an identical fatty acid composition. Cyanotoxins were not found in the strain of R. mediterranea, however, the strain of R. curvata contained both deoxycylindrospermopsin and cylindrospermopsin. Phylogenetic affiliations inferred from 16S rRNA gene sequences demonstrated that both Raphidiopsis strains clustered with Cylindrospermopsis, demonstrating their phylogenetic ties to Nostocaceae. (c) 2007 Elsevier B.V. All rights reserved.

Phylogenetic relationships of Danio within the order cypriniformes: A framework for comparative and evolutionary studies of a model species

The evolutionary relationships of species of Danio and the monophyly and phylogenetic placement of the genus within the family Cyprinidae and subfamily Rasborinae provide fundamentally important phyloinformatics necessary for direct evaluations of an array of pertinent questions in modern comparative biology. Although the genus Danio is not one of the most diverse within the family, Danio rerio is one of the most important model species in biology. Many investigations have used this species or presumed close relatives to address specific questions that have lasting impact on the hypothesis and theory of development in vertebrates. Largely lacking from this approach has been a holistic picture of the exact phylogenetic or evolutionary relationships of this species and its close relatives. One thing that has been learned over the previous century is that many organismal attributes (e.g., developmental pathways, ecologies, behaviors, speciation) are historically constrained and their origins and functions are best explained via a phylogenetic approach. Herein, we provide a molecular evaluation of the phylogenetic placement of the model species Danio rerio within the genus Danio and among hypothesized closely related species and genera. Our analysis is derived from data using two nuclear genes (RAG1, rhodopsin) and five mitochondrial genes (ND4, ND4L, ND5, COI, cyt b) evaluated using parsimony, maximum likelihood, and Bayesian analyses. The family Cyprinidae is resolved as monophyletic but the subfamily Rasborinae (priority over Danioinae) is an unnatural assemblage. Danio is identified as a monophyletic group sister to a clade inclusive of the genera Chela, Microrasbora, Devario, and Inlecypris, not Devario nor Esomus as hypothesized in previous studies. Danio rerio is sister to D. kyathit among the species of Danio evaluated in this analysis. Microrasbora and Rasbora are non-monophyletic assemblages; however, Boraras is monophyletic.

First human-caused extinction of a cetacean species?

The Yangtze River dolphin or baiji ( Lipotes vexillifer), an obligate freshwater odontocete known only from the middle-lower Yangtze River system and neighbouring Qiantang River in eastern China, has long been recognized as one of the world's rarest and most threatened mammal species. The status of the baiji has not been investigated since the late 1990s, when the surviving population was estimated to be as low as 13 individuals. An intensive six-week multivessel visual and acoustic survey carried out in November-December 2006, covering the entire historical range of the baiji in the main Yangtze channel, failed to find any evidence that the species survives. We are forced to conclude that the baiji is now likely to be extinct, probably due to unsustainable by-catch in local fisheries. This represents the first global extinction of a large vertebrate for over 50 years, only the fourth disappearance of an entire mammal family since AD 1500, and the first cetacean species to be driven to extinction by human activity. Immediate and extreme measures may be necessary to prevent the extinction of other endangered cetaceans, including the sympatric Yangtze finless porpoise ( Neophocaena phocaenoides asiaeorientalis).

The complete mitochondrial genome of the Chinese hook snout carp Opsariichthys bidens (Actinopterygii : Cyprinifonnes) and an altemative pattem of mitogenomic evolution in vertebrate

The complete mitochondrial genome sequence of the Chinese hook snout carp, Opsariichthys bidens, was newly determined using the long and accurate polymerase chain reaction method. The 16,611-nucleotide mitogenome contains 13 protein-coding genes, two rRNA genes (12S, 16S) 22 tRNA genes, and a noncoding control region. We use these data and homologous sequence data from multiple other ostariophysan fishes in a phylogenetic evaluation to test hypothesis pertaining to codon usage pattern of O. bidens mitochondrial protein genes as well as to re-examine the ostariophysan phylogeny. The mitochondrial genome of O. bidens reveals an alternative pattern of vertebrate mitochondrial evolution. For the mitochondrial protein genes of O. bidens, the most frequently used codon generally ends with either A or C, with C preferred over A for most fourfold degenerate codon families; the relative synonymous codon usage of G-ending codons is greatly elevated in all categories. The codon usage pattern of O. bidens mitochondrial protein genes is remarkably different from the general pattern found previously in the relatively closely 9 related zebrafish and most other vertebrate mitochondria. Nucleotide bias at third codon positions is the main cause of codon bias in the mitochondrial protein genes of O. bidens, as it is biased particularly in favor of C over A. Bayesian analysis of 12 concatenated mitochondrial protein sequences for O. bidens and 46 other teleostean taxa supports the monophyly of Cypriniformes and Otophysi and results in a robust estimate of the otophysan phylogeny. (C) 2007 Published by Elsevier B.V.

Morphological and genetic differences of Trypanosoma in some Chinese freshwater fishes: difficulties of species identification

Blood smears and purified trypanosome from freshwater fishes yellow catfish (Pseudobagras fulvidraco) and common carp (Cyprinus carpio) captured from Niushan Lake, Hubei Province were examined to determine whether all of their trypanosomes were Trypanosoma pseudobagri, a species of supposed host specificity and widespread existence across China. Trypanosomes occurred in 16/16 blood smears, and morphometric character analysis of trypanosomes from these smears showed that there were three morphospecies, Trypanosoma sp Carpio, T. sp Pseudobagri, and T. sp. 18S rDNA sequences of trypanosomes from 16 samples revealed three genetic groups among these fish trypanosomes. Group 1 was from C. carpio containing T. sp Carpio; groups 2 and 3 were from P. fulvidraco containing T. sp Pseudobagri and T. sp, respectively. The high similarity of morphometric characters and 18S rDNA sequences showed that T. sp Carpio and T. siniperca probably were the same species. T. sp Pseudobagri was the first occurrence in China. Sequence comparison showed that T. sp Pseudobagri sequence was most similar to that of clone Marv, whereas T. sp sequence differ from those of T. sp Carpio and T. sp Pseudobagri by 5.4 and 5.8%, respectively, and tentatively identified as T. pseudobagri. It was concluded that three species of trypanosomes, at least three genotypes occur in Niushan Lake fishes, and P. fulvidraco in this region appear to contain both types, although the identification of T. pseudobagri remains a problem.

Utility of ITS1-5.8S-ITS2 sequences for species discrimination and phylogenetic inference of two closely related bucephalid digeneans (Digenea : Bucephalidae): Dollfustrema vaneyi and Dollfustrema hefeiensis

The complete internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA, and ITS2 region of the ribosomal DNA from 60 specimens belonging to two closely related bucephalid digeneans (Dollfustrema vaneyi and Dollfustrema hefeiensis) from different localities, hosts, and microhabitat sites were cloned to examine the level of sequence variation and the taxonomic levels to show utility in species identification and phylogeny estimation. Our data show that these molecular markers can help to discriminate the two species, which are morphologically very close and difficult to separate by classical methods. We found 21 haplotypes defined by 44 polymorphic positions in 38 individuals of D. vaneyi, and 16 haplotypes defined by 43 polymorphic positions in 22 individuals of D. hefeiensis. There is no shared haplotypes between the two species. Haplotype rather than nucleotide diversity is similar between the two species. Phylogenetic analyses reveal two robustly supported clades, one corresponding to D. vaneyi and the other corresponding to D. hefeiensis. However, the population structures between the two species seem to be incongruent and show no geographic and host-specific structure among them, further indicating that the two species may have had a more complex evolutionary history than expected.

Origin of Gymnocypris przewalskii and phylogenetic history of Gymnocypris eckloni (Teleostei : Cyprinidae)

The origins and phylogenetic patterns were assessed for G. przewalskii and G. eckloni by analyzing the complete mtDNA cytochrome b gene sequence (1140bp). Phylogenetic analyses further supported that there were three mtDNA lineages (A-C) identified in G. przewalskii and G. eckloni, demonstrating that outer rakers of the first gill have little significance in the phylogeny of the Gymnocypris fishes. The network established showed that G. eckloni of the Yellow River specific haplotype A1 was a founder and it radiated all haplotypes of G. przewalskii which suggested G. przewalskii might only originate from one of two maternals of G. eckloni from the Yellow River. Fs test and mismatch analysis showed at least two expansion events in the population of G. przewalskii about 0.2734 Ma and 0.0658 Ma, while G. eckloni from Qaidam Basin could have experienced severe bottleneck effect about 0.0693 Ma. The population expansion was detected in subclades A1 and A21 with the most recent common ancestor (TMRCA) about 0.2308 +/- 0.01 Ma and 0.1319 +/- 0.015 Ma, respectively, which were within the geological age range of "Gonghe Movement" event that caused the separation of Lake Qinghai from the upper Yellow River. These results suggested the effect of the fish diversification by rapid uplift of the Qinghai-Tibetan Plateau in the Late Pleistocene.

Non-monophyly of fish in the Sinipercidae (Perciformes) as inferred from cytochrome b gene

The sinipercids represent a group of 12 species of freshwater percoid fish, including nine in Siniperca and three species in Coreoperca. Despite several classification attempts and a preliminary molecular phylogeny, the phylogenetic relationships and systematic position of sinipercids remained still unsolved. The complete cytochrome b gene sequences from nine sinipercid species four non-sinipercid fish species were cloned, and a total of 12 cyt b sequences from 10 species of sinipercids and 11 cyt b sequences from 10 species of non-sinipercid fish also in Perciformes were included in the phylogenetic analysis. As expected, the two genera Siniperca and Coreoperca within sinipercids are recovered as monophyletic. However, nine species representing Moronidae, Serranidae, Centropomidae, Acropomatidae, Emmelichtyidae, Siganidae and Centrarchidae included in the present study are all nested between Coreoperca and Siniperca, which provides marked evidence for a non-monophyly of sinipercid fishes. Coreoperca appears to be closest to Centrachus representing the family Centrarchidae. Coreoperca whiteheadi and C. herzi are sibling species, which together are closely related to C. kawamebari. In the Siniperca, the node between S. roulei and the remaining species is the most ancestral, followed by that of S. fortis. S. chuatsi and S. kneri are sibling species, sister to S. obscura. However, the sinipercids do not seem to have a very clear phylogenetic history, for different methods of phylogenetic reconstruction result in different tree topologies, and the only conclusive result in favor of a paraphyletic origin of the two sinipercid genera is the parametric bootstrap test. The paraphyly of Sinipercidae may suggest that the "synapomorphs" such as cycloid scales, upon which this family is based, were independently derived at least twice within sinipercid fishes, and further study should be carried out to include the other two Siniperca species and to incorporate other genes.

Phylogenetic relationships of the Chinese sisorid catfishes: a nuclear intron versus mitochondrial gene approach

Several recent molecular phylogenetic studies of the sisorid catfishes (Sisoridae) have challenged some aspects of their traditional taxonomy and cladistic hypotheses of their phylogeny. However, disagreement with respect to relationships within this family in these studies highlights the need for additional data and analyses. Here we subjected 15 taxa representing 12 sisorids genera to comprehensive phylogenetic analyses using the second intron of low-copy nuclear S7 ribosomal protein (rpS7) gene and the mitochondrial 16S rRNA gene segments both individually and in combination. The competing sisorid topologies were then tested by using the approximately unbiased (AU) test and the Shimodaira-Hasegawa (SH) test. Our results support previously suggested polyphyly of Pareuchiloglanis. The genus Pseudecheneis is likely to be nested in the glyptosternoids and Glaridoglanis might be basal to the tribe Glyptosternini. However, justified by AU and SH test, the sister-group relationship between Pseudecheneis and the monophyletic glyptosternoids cannot be rejected based on the second intron of rpS7 gene and combined data analyses. It follows that both gene segments are not suitable for resolving the phylogenetic relationships within the sisorid catfishes. Overall, the second intron of rpS7 gene yielded poor phylogenetic performance when compared to 16S rRNA gene, the evolutionary hypothesis of which virtually agreed with the combined data analyses tree. This phenomenon can be explained by the insufficient length and fast saturation of substitutions in the second intron of rpS7 gene, due to substitution patterns such as frequent indels (insertion/deletion events) of bases in the sequences during the evolution.

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.

Evidence of host specificity and congruence between phylogenies of bitterling and freshwater mussels

Evidence of host specificity and congruence between phylogenies of bitterling and freshwater mussels. Zoological Studies 45(3): 428-434. Bitterling (Cyprinidae: Acheilognathinae) are freshwater fishes with a unique spawning relationship with freshwater mussels on whose gills they lay their eggs. During the breeding season of bitterling fishes, we collected 843 mussels belonging to 16 species from Lake Qinglan, central China and examined their gill chambers for the presence of bitterling larvae. Three species of bitterling larvae were identified; Acheilognathus tonkinensis, Ach. cf. meridianus, and Ach. barbatulus, in 3 species of mussel: Unio douglasiae, Lamprotula caveata, and L. tortuosa, suggesting host specialization. Using our own and other published data, we compared the respective phylogenies of bitterling and mussels, but failed to show clear congruence. However, broad specializations are evident, with Acheilognathus and Tanakia showing preferences for mussels with a relatively simple gill structure (Ableminae), and Rhodeus spp. showing preferences for mussels of the Anodontinae and Unioninae, which have more-complex gill structures.

Reevaluation of the phylogenetic relationship between Mobilid and Sessilid peritrichs (Ciliophora, Oligohymenophorea) based on small subunit rRNA genes sequences

Based on morphological characters, peritrich ciliates (Class Olygohymenophorea, Subclass Peritrichia) have been subdivided into the Orders Sessilida and Mobilida. Molecular phylogenetic studies on peritrichs have been restricted to members of the Order Sessilida. In order to shed more light into the evolutionary relationships within peritrichs, the complete small subunit rRNA (SSU rRNA) sequences of four mobilid species, Trichodina nobilis, Trichodina heterodentata, Trichodina reticulata, and Trichodinella myakkae were used to construct phylogenetic trees using maximum parsimony, neighbor joining, and Bayesian analyses. Whatever phylogenetic method used, the peritrichs did not constitute a monophyletic group: mobilid and sessilid species did not cluster together. Similarity in morphology but difference in molecular data led us to suggest that the oral structures of peritrichs are the result of evolutionary convergence. In addition, Trichodina reticulata, a Trichodina species with granules in the center of the adhesive disc, branched separately from its congeners, Trichodina nobilis and Trichodina heterodentata, trichodinids without such granules. This indicates that granules in the adhesive disc might be a phylogenetic character of high importance within the Family Trichodinidae.