Phylogenetic relationships of Cyprinidae (Teleostei : Cypriniformes) inferred from the partial S6K1 gene sequences and implication of indel sites in intron 1

The family Cyprinidae is widely distributed in East Asia, and has the important phylogenetic significance in the fish evolution. In this study, the 5' end partial sequences (containing exon 1, exon 2 and indel 1) of S6K1 gene were obtained from 30 representative species in Cyprinidae and outgroup using PCR amplification and sequencing. The phylogenetic relationships of Cyprinidae were reconstructed with neighbor joining (NJ), maximum parsimony (MP), maximum likelihood (ML), and Bayesian methods. Myxocyprinus asiaticus (Catostomidae) was assigned to the outgroup taxon. Similar phylogenetic relationships within the family Cyprinidae were achieved with the four analyses. Leuciscini and Barbini were monophyletic lineages respectively with the high nodal supports. Leuciscini comprises Hypophthalmichthyinae, Xenocyprinae, Cultrinae, Gobioninae, Acheilognathinae and East Asian species of Leuciscinae and Danioninae. Monophyly of East Asian clade was supported with high nodal support. Barbini comprises Schizothoracinae, Barbinae, Cyprininae and Labeoninae. The monophyletic lineage consisting of Danio rerio, D. myersi, and Rasbora trilineata was basal in the tree. In addition, the large fragment indels in intron 1 were analyzed to improve the understanding of Cyprinidae relationships. The results showed that the large fragment indels were correlated with the relations among species. Some conserved regions in intron 1 were thought to be involved in the functional regulation. However, no correlation was found between sequence variations and species characteristic size.

Development of novel EST-SSR markers in common carp by data mining from public EST sequences

Expressed sequence tags (ESTs) are a source for microsatellite development. In the present study, EST-derived microsatelltes (EST-SSRs) were generated and characterized in the common carp (Cyprinus carpio) by data mining from updated public EST databases and by subsequent testing for polymorphism. About 5.5% (555) of 10,088 ESTs contain repeat motifs of various types and lengths with CA being the most abundant dinucleotide one. Out of the 60 EST-SSRs for which PCR primers were designed, 25 loci showed polymorphism in a common carp population with the alleles per locus ranging from 3 to 17 (mean 7). The observed (H-O) and expected (HE) heterozygosities of these EST-SSRs were 0.13-1.00 and 0.12-0.91, respectively. Six EST-SSR loci significantly deviated from the Hardy-Weinberg equilibrium (HWE) expectation, and the remaining 19 loci were in HWE. Of the 60 primer sets, the rates of polymorphic EST-SSRs were 42% in common carp, 17% in crucian carp (Carassius auratus), and 5% in silver carp (Hypophthalmichthys molitrix), respectively. These new EST-SSR markers would provide sufficient polymorphism for population genetic studies and genome mapping of the common carp and its closely related fishes. (c) 2007 Published by Elsevier B.V.

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.

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.

The c-myc coding DNA sequences of cyprinids (Teleostei : Cypriniformes): Implications for phylogeny

The family Cyprinidae is one of the largest fish families in the world, which is widely distributed in East Asian, with obvious difference in characteristic size among species. The phylogenetic analysis of cyprinid taxa based on the functionally important genes can help to understand the speciation and functional divergench of the Cyprinidae. The c-myc gene is an important gene regulating individual growth. In the present study, the sequence variations of the cyprinid c-myc gene and their phylogenetic significance were analyzed. The 41 complete sequences of the c-myc gene were obtained from cyprinids and outgroups through PCR amplification and clone. The coding DNA sequences of the c-myc gene were used to infer molecular phylogenetic relationships within the Cyprinidae. Myxocyprinus asiaticus (Catostomidae), Misgurnus anguillicaudatus (Cobitidae) and Hemimyzon sinensis (Homalopteridae) were assigned to the outgroup taxa. Phylogenetic analyses using maximum parsimony (MP), maximum likelihood (ML), and Bayesian retrieved similar topology. Within the Cyprinidae, Leuciscini and Barbini formed the monophyletic lineage respectively with high nodal supports. Leuciscini comprises Xeno-cyprinae, Cultrinae, East Asian species of Leuciscinae and Danioninae, Gobioninae and Acheilognathinae, and Barbini contains Schizothoracinae, Barbinae, Cyprininae and Labeoninae. Danio rerio, D. myersi and Rasbora trilineata were supposed to separate from Leuciscinae and Barbini and to form another lineage, The positions of some Danioninae species were still unresolved. Analyses of both amino acid variation with parsimony information and two high variation regions indicated that there is no correlation between variations of single amino acid or high variation regions and characteristic size of cyprinids. In,addition, the species with smaller size were usually found to be basal within clades in the tree, which might be the results of the adaptation to the primitive ecology and survival pressure.

Phylogeny of diplozoids in five genera of the subfamily diplozoinae palombi, 1949 as inferred from ITS-2 rDNA sequences

The phylogenetic relationship of 5 genera, i.e. Diplozoon Nordmann, 1832, Paradiplozoon Achmerov, 1974, Inustiatus Khotenovsky, 1978, Sindiplozoon Khotenovsky, 1981, and Eudiplozoon Khotenovsky, 1985 in the subfamily Diplozoinae Palombi, 1949 (Monogenea, Polyopisthocotylea) was inferred from rDNA ITS-2 region using neighbour-joining (NJ), maximum likelihood (ML) and Bayesian methods. The phylogenetic trees produced by using NJ, ML and Bayesian methods exhibit essentially the same topology. Surprisingly, freshwater species of Paradiplozoon from Europe clustered together with species of Diplozoon, but separated from Chinese Paradiplozoon species. The results of molecular phylogeny and lower level of divergence (4(.)1-15(.)7%) in ITS-2 rDNA among Paradiplozoon from Europe and Diplozoon and, on the other hand, high level of divergence (45(.)3-53(.)7%) among Paradiplozoon species from Europe and China might indicate the non-monophyletic origin of the genus Paradiplozoon. Also, the generic status of European Paradiplozoon needs to be revised. The species of Paradiplozoon in China is a basal group in Diplozoinae as revealed by NJ and Bayesian methods, and Sindiplozoon appears to be closely related to European Paradiplozoon and Diplozoon. with their relationship to Eudiplozoon and Inustiatus being unresolved.

Molecular phylogeny and biogeography of the highly specialized grade schizothoracine fishes (Teleostei : Cyprinidae) inferred from cytochrome b sequences

We recovered the phylogenetic relationships among 23 species and subspecies of the highly specialized grade schizothoracine fishes distributing at 36 geographical sites in the Tibetan Plateau and its Surrounding regions by analyzing sequences of cytochrome b genes. Furthermore, we estimated the possible divergent times among lineages based on a historical geological isolation event in the Tibetan Plateau. The molecular data revealed that the highly specialized grade schizothoracine fishes were not a monophyletic group, but were the same as genera Gymnocypris and Schizogypsis. Our results indicated that the molecular phylogenetic relationships apparently reflected their geographical and historical associations with drainages, namely species from the same and adjacent drainages clustered together and had close relationships. The divergence times of different lineages were well consistent with the rapid uplift phases of the Tibetan Plateau in the late Cenozoic, suggesting that the origin and evolution of schizothoracine fishes were strongly influenced by environment changes resulting from the upheaval of the Tibetan Plateau.

Identification of novel SINEs from Cyprinidae and their evolutionary significance

Short interspersed nuclear elements (SINEs) are widespread among eukaryotic genomes. They are repetitive DNA sequences that have been amplified by retrotransposition. In this study, a class of SINEs were isolated from the Opsariichthys bidens genome, and named Opsar. Sequence analysis confirmed that Opsar is a new class of typical SINEs derived from tRNA molecules. With the tRNA-derived region of Opsar and through BLASTN search, we further identified Zb-SINEs from the zebrafish genome, which includes two groups: Zb-SINE-A and Zb-SINE-B. The Zb-SINE-A group comprises subfamilies of -Al--A5, and the Zb-SINE-B group is a dimer of the tRNA(Ala)-derived region and shares a similar dimeric composition to Alu. Zb-SINEs are composed of three distinct regions: a 5 end tRNA-derived region, a tRNA-unrelated region and a 3 end AT-rich region. The flanking regions are AT rich. The average length of Zb-SINEs elements is about 340 6p. Zb-SINEs account for as much as 0.1% of the whole zebrafish genome. About 70% of the Zb-SINEs are on chromosomes 11, 18, and 19. These Zb-SINEs were characterized by PCR and dot hybridization. The distribution pattern of Zb-SINEs in genome strongly supports the master genes model. The tRNA-derived regions of Opsar and Zb-SINEs were compared with the tRNA(Ala) gene, and they showed 76% similarity, indicating that Opsar and Zb-SINEs originated from an inactive tRNA(Ala) sequence or a tRNA(Ala)-like sequence. In view of the evolutionary status of zebrafish in the Cyprinidae, we deduced that Zb-SINEs were a very old class of interspersed sequences.

Molecular phylogeny of Stentor (Ciliophora : Heterotrichea) based on small subunit ribosomal RNA sequences

To determine the phylogenetic position of Stentor within the Class Heterotrichea, the complete small subunit rRNA genes of three Stentor species, namely Stentor polymorphus, Stentor coeruleus, and Stentor roeseli, were sequenced and used to construct phylogenetic trees using the maximum parsimony, neighbor joining, and Bayesian analysis. With all phylogenetic methods, the genus Stentor was monophyletic, with S. roeseli branching basally.

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.

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.

Biogeography and molecular phylogeny of the genus Schizothorax (Teleostei : Cyprinidae) in China inferred from cytochrome b sequences

Aim: To test a vicariant speciation hypothesis derived from geological evidence of large-scale changes in drainage patterns in the late Miocene that affected the drainages in the south-eastern Tibetan Plateau. Location: The Tibetan Plateau and adjacent areas. Methods: The cytochrome b DNA sequences of 30 species of the genus Schizothorax from nine different river systems were analysed. These DNA sequences were analysed using parsimony, maximum likelihood and Bayesian methods. The approximately unbiased and Shimodaira-Hasegawa tests were applied to evaluate the statistical significance of the shortest trees relative to alternative hypotheses. Dates of divergences between lineages were estimated using the nonparametric rate smoothing method, and confidence intervals of dates were obtained by parametric bootstrapping. Results: The phylogenetic relationships recovered from molecular data were inconsistent with traditional taxonomy, but apparently reflected geographical associations with rivers. Within the genus Schizothorax, we observed a divergence between the lineages from the Irrawaddy-Lhuit and Tsangpo-Parlung rivers, and tentatively dated this vicariant event back to the late Miocene (7.3-6.8 Ma). We also observed approximately simultaneous geographical splits within drainages of the south-eastern Tibetan Plateau, the Irrawaddy, the Yangtze and the Mekong-Salween rivers in the late Miocene (7.1-6.2 Ma). Main conclusions: Our molecular evidence tentatively highlights the importance of palaeoriver connections and the uplift of the Tibetan Plateau in understanding the evolution of the genus Schizothorax. Molecular estimates of divergence times allowed us to date these vicariant scenarios back to the late Miocene, which agrees with geological suggestions for the separation of these drainages caused by tectonic uplift in south-eastern Tibet. Our results indicated the substantial role of vicariant-based speciation in shaping the current distribution pattern of the genus Schizothorax.

The phylogenetic relationships of the gobioninae (Teleostei : Cyprinidae) inferred from mitochondrial cytochrome b gene sequences

The Gobioninae are a group of morphologically and ecologically diverse Eurasian freshwater cyprinid fishes. The intergeneric relationships of this group are unresolved and the possible monophyly of this subfamily remains to be established. We used complete mitochondrial cytochrome b gene sequences from most genera within the gobionine group, in addition to a selection of cyprinid outgroups, to investigate the possible monophyly of this group and resolve the interrelationships within the group. Our results support the monophyly of the Gobioninae and identify four monophyletic groups within the subfamily; the Hemibarbus group, the Sarcocheilichthys group, the Gobio group, and the Pseudogobio group. The morphologically aberrant genera Gobiobotia, Xenophysogobio and Gobiocypris are included in the Gobioninae, with the latter a sister group of Gnathopogon.

Phylogeny of the East Asian botiine loaches (Cypriniformes, Botiidae) inferred from mitochondrial cytochrome b gene sequences

The Botiinae have traditionally represented a subfamily of the Cobitidae. At present, the classification and phylogenetic relationships of the Botiinae are controversial. To address systematic and phylogenetic questions concerning this group, we sequenced the complete cytochrome b gene from 34 samples, of which 24 represented 13 species of the East Asian botiine fishes, while the other 10 were non-botiine loach species. For the 1140 bp sequences determined, 494 sites were variable ones, of which 424 were parsimony informative. With Myxocyprinus asiaticus as an outgroup, molecular phylogenetic trees were constructed using the neighbor-joining, maximum parsimony, maximum likelihood and Bayesian methods. All molecular phylogenetic trees revealed that botiine fishes form a monophyletic group and are distantly related to other loaches, suggesting that the Botiinae should be placed in their own family. Within the Botiinae, there are three genera; Botia, Parabotia, and Leptobotia, each genus forming a monophyletic group, with the genus Botia as the most ancestral split. Our molecular results are in agreement with morphological analyses of botiines, suggesting that Botia is the ancestral genus, while Leptobotia and Parabotia were resolved as more derived sister groups.