Complete mitochondrial genome of the grass carp (Ctenopharyngodon idella, Teleostei): insight into its phylogenic position within Cyprinidae

Genome comparison has shed light on many fields of both basic and applied research, including the study of species phylogeny. Grass carp (Ctenopharyngodon idella) belongs to Cyprinidae, the largest freshwater fish family; but which subfamily it belongs to remains a controversial issue. In this study, the complete mitochondrial genome (mitogenome) sequence of grass carp was determined and phylogenetic analyses of all mitochondrial protein-coding genes and a nuclear gene (RAG 2) were conducted to explore the evolutionary relationship of grass carp with other cyprinid species. The mitogenome of grass carp is 16,609 bp in length. As with most other vertebrates, it contains the same gene order and an identical number of genes or regions, including 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one putative control region. Phylogenetic analyses using two different datasets (mitochondrial and nuclear) and three different computational algorithms (Bayesian, MP and ML) all revealed two distinct groups with high statistical support, indicating that Cyprininae and Leuciscinae are two separate, valid subfamilies. Importantly, our phylogenetic result provides strong molecular evidence in support of the placement of Ctenopharyngodon in Leuciscinae rather than in Cyprininae.     

Male primer endocrine responses to preovulatory female cyprinids under natural conditions in Sweden

This study investigated two related aspects of male-female reproductive interactions in the family Cyprinidae: (1) whether ovulating female rudd Scardinius erythrophthalmus (subfamily Leuciscinae) induce endocrine and gonadal priming responses in conspecific males, a phenomenon which has been described only in species from the subfamily Cyprininae such as goldfish, Carassius auratus, crucian carp Carassius carassius and common carp, Cyprinus carpio and (2) whether the stimuli mediating these responses are species-specific. Field studies of three sympatric European cyprinids, two leuciscins (S. erythrophthalmus and white bream Blicca bjoerkna) and one cyprinin (C. carassius), were conducted on fishes captured in Sweden in the spawning season and held in net pens under natural conditions. As previously reported in C. carassius, male S. erythrophthalmus increased milt (sperm and seminal fluid) volume and plasma concentrations of the sperm maturation hormone 4-pregnen-17,20β-diol-3-one (17,20β-P) when they were held with female S. erythrophthalmus induced to ovulate by injection of Ovaprim (GnRH analogue plus dopamine antagonist). Male S. erythrophthalmus had larger milt volumes than male C. carassius prior to and following exposure to ovulatory conspecifics, but exhibited a smaller proportional milt increase in response to stimulation, suggesting species differences in sperm allocation at spawning. The presence of female S. erythrophthalmus and B. bjoerkna did not affect milt volumes of C. carassius under two experimental conditions: (1) ovulating S. erythrophthalmus and B. bjoerkna did not increase the milt volumes of C. carassius and (2) S. erythrophthalmus and B. bjoerkna did not interfere with the milt volume increase induced in male C. carassius by ovulating conspecifics. These results suggest that, as in C. auratus, C. carassius and C. carpio (subfamily Cyprininae), female S. erythrophthalmus (subfamily Leuciscinae) release a preovulatory pheromone that exerts priming effects on male hormones and sperm allocation. The findings also indicate that C. carassius discriminate between the reproductive odours of conspecifics and heterospecifics.     

Cyprininae phylogeny revealed independent origins of the Tibetan Plateau endemic polyploid cyprinids and their diversifications related to the Neogene uplift of the plateau

Origin and diversification of the Tibetan polyploid cyprinids (schizothoracins) may help us to explore relationships between diversification of the cyprinids and the Tibetan Plateau uplift. Cyprininae phylogeny was analyzed using mitochondrial and nuclear DNA sequences to trace origins of polyploidy and diversifications of schizothoracins. Ancestral states reconstruction for ploidy levels indicated that the Cyprininae was diploid origin and the schizothoracin clades tetraploid origins. There were two diversification rate shifts along with diversification of the cyprinine fishes in response to the Tibetan uplift. The unusual diversification shifts were located to branches subtending the clades of Tibetan polyploid cyprinids. Our analyses suggested that (i) phylogeny of Cyprininae recovered two independent origins of the Tibetan polyploidy schizothoracins; (ii) diversifications of the schizothoracins were closely related to the Neogene uplift of the Tibetan plateau in the following ways: the relatively ancient Late Oligocene-Middle Miocene adaptive radiation may be associated with the uplift of the southern Tibet and Himalaya; the Middle Miocene-Early Pleistocene lineage-specific diversification broadly coincident with major phase of the Neogene Tibetan uplift; and the most recent Pleistocene diversification shift in Schizothorax closely coincident with the successive Kunlun-Huanghe and Gonghe movements of the Tibetan uplift and the glaciation-induced climate oscillations on the plateau.     

The formation of the polyploid hybrids from different subfamily fish crossings and its evolutionary significance

This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp x blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (female symbol) x blunt snout bream (BSB) (male symbol) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.     

鲤亚科鱼类的系统发育

鲤亚科是由原始的鲃亚科派生的一个单源群,因此,把鲤亚科各属筛选出的性状与纪亚科的相应性状比较,以确定其性质,即该性状为祖征,还是为离征。经比较,发现25项性状在鲤亚科的5个属中呈镶嵌分布。根据性状的镶嵌分布情况和简约性原则,推导出鲤亚科的属间系统发育分支图。以往的观点认为,鲃鲤属与原鲤属的关系较近。现在系统发育关系分析的结果与此全然不同,原鲤属与鲤属的关系较近,而纪鲤属在鲤亚科内是较早分化出的一支,与亚科内其他4个属的关系相对较远。最后,依化石资料分析了鲤亚科的分化时间。     

Cross-priming of microsatellite loci in subfamily cyprininae (family Cyprinidae): their utility in finding markers for population genetic analysis in three Indian major carps

This study is aimed to identify polymorphic microsatellite markers and establish their potential for population genetics studies in three carp (family cyprinidae; subfamily cyprininae) species, Labeo rohita, Catla catla and Cirrhinus mrigala through use of cyprinid primers. These species have high commercial value and knowledge of genetic variation is important for management of farmed and wild populations. We tested 108 microsatellite primers from 11 species belonging to three different cyprinid subfamilies, Cyprininae, Barbinae and Leuciscinae out of which 63 primers (58.33 %) successfully amplified orthologous loci in three focal species. Forty-two loci generated from 29 primers were polymorphic in these three carp species. Sequencing of amplified product confirmed the presence of SSRs in these 42 loci and orthologous nature of the loci. To validate potential of these 42 polymorphic loci in determining the genetic variation, we analyzed 486 samples of three focal species collected from Indus, Ganges and Brahmaputra river systems. Results indicated significant genetic variation, with mean number of alleles per locus ranging from 6.80 to 14.40 and observed heterozygosity ranging from 0.50 to 0.74 in the three focal species. Highly significant (P < 0.00001) allelic homogeneity values revealed that the identified loci can be efficiently used in population genetics analysis of these carp species. Further, thirty-two loci from 19 primers were useful for genotyping in more than one species. The data from the present study was compiled with cross-species amplification data from previous results on eight species of subfamily cyprininae to compare cross-transferability of microsatellite loci. It was revealed that out of 226 heterologous loci amplified, 152 loci that originated from 77 loci exhibited polymorphism and 45 primers were of multispecies utility, common for 2–7 species.     

Phylogeny and biogeography of the family Cyprinidae in the Middle East inferred from cytochrome b DNA- evolutionary significance of this region.

The phylogenetic relationships of cyprinid species from the Middle East and neighboring biogeographical areas were investigated using cytochrome b sequence variation in order to test hypotheses that consider the Middle Eastern area as an important interchange area or a center of speciation for the freshwater fauna. A total of 62 cyprinid species were analyzed over the complete cytochrome b fragment (1140 bp); 28 belong to the Leuciscinae subfamily and 34 to the Cyprininae. All the Leuciscinae lineage fish recorded in the Middle East are also found in Europe, which was interpreted as an important Palearctic influence in the Middle Eastern ichthyofauna consistent with the Lago Mare dispersion. However, it has also been suggested that several Danube species have their origins in the Middle East. In contrast, the Cyprininae subfamily showed three highly divergent lineages, one shared with the Euro-Mediterranean area (Barbus/Luciobarbus genus) relict of the Lago Mare dispersion, one shared with Africa (Carasobarbus/Varicorhinus subgenus), and the third shared with Asia (Garra genus). Furthermore, clades observed in the phylogenetic reconstructions are not consistent with morphometric or karyological data and disagree with previous taxonomic assumptions. Lastly, the dispersion history in the Middle East of this subfamily appears much more complicated and ancient than that of the Leuciscinae. However, taking into account Cyprininae and Leuciscinae distribution, the Middle East appears more like an important interchange area for the freshwater ichtyofauna than a center of speciation.     

指环虫属的早期辐射及其与宿主鲤科鱼类的协同进化关系

本研究利用28SrDNAC1-D2区序列分析采自鲤科鱼类中6亚科宿主和寄生在花鲈、梅花鲈上的共17种指环虫的系统发育关系。同时,通过比较宿主鲤科鱼类与指环虫的系统发育树,检验指环虫与其宿主是否存在协同进化关系。结果表明:17种指环虫形成5个进化支(Clade),其中寄生在团头鲂(亚科)和鲢、鳙(鲢亚科)上的6种指环虫聚为一支(Clade1),而它们的宿主鱼类在系统发育分析中也表现为近缘关系;寄生在鲮鱼(野鲮亚科)上的D.quanfami(Clade5)位于系统树最基部,鲫鱼和鲤鱼(鲤亚科)的寄生指环虫处在系统树的次基部位置,而鲤亚科与野鲮亚科组成的姐妹群在宿主系统树上同样处在基部位置,寄生虫和宿主在进化上较为原始的地位得到了很好地相互印证。因而,本研究首次利用分子系统学手段分析指环虫属远缘物种间的系统关系,揭示了指环虫属与宿主鱼类之间存在协同进化关系。另外,本研究首次发现,野鲮亚科鱼类也可能是指环虫类的早期宿主,这与先前认为鲤亚科鱼类为指环虫类的祖先宿主的推测有所不同。     

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).     

A study of the systematics of cyprinid fishes by two-dimensional gel electrophoresis

This study was carried out to shed light on confused subfamilial groupings in the Cyprinidae from the biochemical viewpoint at the molecular level, specifically by using two-dimensional gel electrophoresis of liver proteins. Six pairs of cypriniform fishes, which are different from one another at familial, subfamilial, generic, specific, subspecific, and individual levels, were compared. The genetic distances between pairs of fishes increased as taxonomic ranks of the pairs became higher, confirming the reliable usefulness of this technique. Four species representing the different subfamilies, Cyprininae, Gobioninae, Acheilognathinae, and Leuciscinae, were compared to give new insight into relationships at the subfamilial level. Cyprinus carpio (Cyprininae) and Pseudogobio esocinus esocinus (Gobioninae) gave the smallest genetic distance and the largest values were obtained between either one of the above species and Acheilognathus melanogaster (Acheilognathinae), suggesting that the former two subfamilies compose the most closely related group that is in turn distantly related to Acheilognathinae. Tribolodon hakonensis (Leuciscinae) had almost equal genetic distances to the three other species.     

Molecular phylogeny of congeneric monogenean parasites (Dactylogyrus): a case of intrahost speciation

Dactylogyrus species (Dactylogyridae: Monogenea) are a group of monogenean gill parasites that are highly specific to freshwater fish of the family Cyprinidae. Dactylogyrus species were sampled from 19 cyprinids and one percid collected in Europe. Using partial 18S rDNA and ITS1 sequences, a phylogeny of 51 Dactylogyrus species was reconstructed to investigate the patterns of parasite speciation and diversification. Three main Dactylogyrus lineages were recognized from all phylogenetic trees, that is, analysis of 18S rDNA alone and combined 18SrDNA and ITS1. The first lineage associates the Dactylogyrus species of Cyprinus carpio and Carassius auratus of the Cyprininae; the second associates Dactylogyrus species of the Gobioninae, Pseudorasbora parva of the Rasborinae, and Ctenopharyngodon idella of the Cyprininae; and the third associates Dactylogyrus species of the Leuciscinae and Alburninae and Barbus barbus of the Cyprininae. Our results suggest that the genus Dactylogyrus is of quite recent origin and that these three lineages separated from each other in a very short period of time. Host subfamily mapping onto the parasite tree inferred from analysis of the combined dataset showed that the Cyprininae could be plesiomorphic hosts for Dactylogyrus. Dactylogyrus parasites would have secondarily colonized the Percidae and representatives of the Leuciscinae, Alburninae, Gobioninae, and Rasborinae. Comparison of host and parasite phylogenetic relationships indicated that a very high number of parasite duplications occurred within two of the three Dactylogyrus lineages. Dactylogyrus diversification can be mainly explained by sympatric intrahost speciation events that seem to be correlated to strict host specificity. Moreover, the present study shows that the congeneric parasites speciating within one host tend to occupy niches within hosts differing at least in one niche parameter.     

Evolution and trans-species polymorphism of MHC class IIbeta genes in cyprinid fish

The polymorphism of DAB genes encoding MHC IIbeta was investigated in 11 cyprinid species from central Europe. The species belonged to four subfamilies: Cyprininae, Tincinae, Gobioninae and Leuciscinae. Two paralogous groups of sequences, DAB1 and DAB3, were recognised according to the similarity of their nucleotide and amino-acid sequences and from phylogenetic analyses using either partial exon 2 or partial exon 3 sequences. A high allelic variability among species was found for exon 2, indicating extensive MHC polymorphism. Time divergence estimation supports the separation of DAB1 and DAB3 groups predating the separation into fish subfamilies, and a cyprinid origin of the DAB genes. Phylogenetic trees using exon 2 support the hypothesis of trans-species polymorphism, which appears to be limited to the subfamily level, i.e. the presence of sequences from different species in the same allelic group was more often recognised within subfamilies Cyprininae and Leuciscinae than between them. Phylogenetic trees using exon 3 reflect the phylogenetic patterns previously found for Cyprinidae systematics. Specific nucleotides and amino-acids in exon 3 that separate both subfamilies, as well as the species within the Cyprininae subfamily were observed. A lack of segregation in leuciscin species was recognised and the alleles of different leuciscin species tend to share similar motifs in exon 3. This could be explained by the ancient and complicated dispersion history of Cyprininae and the radiation of Leuciscinae. The effects of selective pressures were investigated: (1) within species, (2) among lineages, and (3) among sites. From intraspecific analyses, exon 2 sequences were identified as the targets of diversifying selection, whilst the evolution of exon 3 seems to be under the influence of purifying selection. The analyses among lineages indicate positive selection in many branches when using exon 2, therefore confirming trans-species polymorphism, whilst the DAB lineages of exon 3 are potentially submitted to purifying selection to some extent. Moreover, our results suggest the secondary acquisition of function of DAB1 group after duplication. The analyses among sites reveal that exon 2 exhibits sites under positive selection mostly corresponding to the putative PBR sites involved in the alpha-helix structure of the protein.     

中国鲤亚科鱼类的分类、分布、起源及演化

本文报道中国鲤亚科鱼类5属23种,内有1新种——尖鳍鲤Cyprinus acutidorsalis sp.nov.,该种的主要鉴别特征为:(1)背鳍前部呈三角形突出,后缘具一很深的缺刻;(2)腹鳍起点前于背鳍;(3)尾鳍下叶不呈桔红色。中国鲤亚科鱼类区系,远较其他国家丰富,地方种有乌原鲤Procypris merus、岩原鲤P.rabaudi、尖鳍鲤、龙州鲤Cyprinus longzhouensis、短鳍鲤C.micristius、抚仙鲤C.fuxianensis、异龙鲤C.yilongensis、大眼鲤C.megalophthalmus、厚唇鲤C.crassilabris、杞麓鲤C.chilia、春鲤C.longipectoralis、大头鲤C.pellegrini、洱海鲤C.barbatus、云南鲤C.yunnanensis、大理鲤C.daliensis和翘嘴鲤C.ilishaestomus,共16种。云南高原鲤亚科种类特多,仅见于云南的中国地方种多达12种。在云南各湖泊,鲤属鱼类种的分化十分明显,尤其在洱海,所产6种除杞麓鲤外,均为该湖所特有,同域种如此之多,殊属罕见,大概洱海一带即为鲤亚科鱼类的分布中心。云南高原特有种如此丰富,可能与云南地形地貌几经变迁,并具备着使该亚科鱼类区系得以繁荣和分化的良好生态条件密切有关。本文还对鲤亚科鱼类的起源和演化,进行了初步的讨论。       

Genetic identification of larvae and juveniles reveals the difference in the spawning site among Cyprininae fish species/subspecies in Lake Biwa

To understand the differences in the spawning sites among Cyprininae fishes in Lake Biwa, we conducted periodic sampling of larvae and juveniles at three sites (irrigation ditch, St. 1; river, St. 2; and satellite lake, St. 3). On the basis of species/subspecies identification by using RAPD analysis, we examined the species composition of the larvae and juveniles at these three sampling sites. The number of specimens was 616, 68, and 117 at St. 1, St. 2, and St. 3, respectively. Based on morphological and genetic identification, the specimens were found to include nine fish species/subspecies, namely, Carassius auratus grandoculis, Carassius cuvieri, Carassius auratus langsdorfii, Cyprinus carpio, Sarcocheilichthys sp., Silurus asotus, Oryzias latipes, Odontobutis obscura obscura, and Rhinogobius sp. The species composition at the three sites also differed. Among the Cyprininae fishes, C. auratus grandoculis, C. auratus langsdorfii, and Cyprinus carpio were found in abundance at St. 1; C. cuvieri was not collected from St. 1 but was found at the other two sites, particularly St. 3. Among the other fishes, Rhinogobius sp. was collected at St. 1 and St. 3, whereas the other four occurred only at St. 1. These results suggest that the selection of spawning sites by C. cuvieri differs to a certain extent from that of the other Cyprininae fishes, and the irrigation ditch in the lake is an important habitat for the larvae and juveniles of native fish species.     

Partial combination applied to phylogeny of European cyprinids using the mitochondrial control region

Previous molecular phylogenies of European cyprinids led to some solid facts and some uncertainties. This study is based on a stretch of more than 1 kb in the mitochondrial control region newly sequenced for 35 European cyprinids and on previous cytochrome b and 16S rDNA data. The trees based on the control region are more accurate and robust than those obtained from the two other genes. Character incongruence among the three genes was tested using the incongruence length difference (ILD) test. Iterative removals of individual sequences followed by new ILD tests identified two sequences responsible for statistically significant incongruence. A partial combination was conducted, that is, a combination of the three data sets, removing the two sequences previously identified. The phylogenetic analysis of this partial combination gives a more robust and resolved picture of subfamilial interrelationships. The Rasborinae are the sister group of all other cyprinids. The monophyletic Cyprininae emerges next. Tinca tinca first and then Rhodeus are the sister groups of all the remaining nonrasborine and noncyprinine species. Gobio is the sister group of the Leuciscinae, in which the Phoxinini are the sister group of the Leuciscini. Within the Leuciscini, the genus Leuciscus and the subfamily Alburninae are both paraphyletic. The Rasborinae are the most basal cyprinid subfamily and the Tincinae are not the sister group of the Cyprininae. These two results challenge only two anatomical characters, which need to be reinterpreted or considered as homoplastic in cyprinid evolution: the modification of the first pleural rib and its parapophysis and the bony composition of the interorbital septum.     

异源四倍体鲫鲤、三倍体湘云鲫和它们父母本线粒体DNA 12S rRNA基因遗传变异的分析

采用质粒克隆测序方法,获得了异源四倍体鲫鲤5个个体、异源四倍体鲫鲤雌核发育二倍体后代2个个体、三倍体湘云鲫2个个体及红鲫、湘江野鲤和日本白鲫各1个个体的线粒体DNA 12S rRNA基因的全序列。经对比发现,异源四倍体5个个体共享2种单元型,异源四倍体鲫鲤雌核发育二倍体后代2个个体、三倍体湘云鲫2个个体以及红鲫、湘江野鲤和日本白鲫各1个个体分别共享1种单元型。用MEGA 1.0 软件分析了它们的碱基组成和核苷酸序列差异,用邻接法构建系统进化树。它们间的序列同源性在95%~99%之间,异源四倍体鲫鲤、三倍体湘云鲫和它们母本（分别为红鲫和日本白鲫）之间的序列同源性大于异源四倍体鲫鲤、三倍体湘云鲫和它们父本（分别为湘江野鲤和异源四倍体鲫鲤）之间的序列同源性,结果表明：异源四倍体鲫鲤和三倍体湘云鲫在线粒体DNA 12S rRNA基因上具有母性遗传特征。本研究另一值得注意地方的是异源四倍体鲫鲤经过9代(F3-F11)繁殖后,在5个个体中发现了2种单元型,说明在四倍体基因库中存在遗传多样性,为四倍体基因库的繁殖、保护和种群复壮提供了一些有价值的信息。     

环境毒物对金黄色葡萄球菌毒性影响的研究

采用毒物在营养琼脂中垂直扩散方法,通过测定毒物在营养球脂中抑制金黄色葡萄球菌生长产生蓝色抑菌带的长度,研究Hg、Cr^6 、Pb、CN^—、As、NO2^—、F^—及苯酚对金黄色葡萄球菌的毒性影响。结果表明：受试毒物的浓度与抑菌带有相关性,相关系数具显著意义;对毒物的敏感性为Cr^6 ＞Hg＞As＞CN^—＞Pb＞NO2^—＞苯酚＞F—;多种毒物共同作用其毒性影响增加。     

Molecular phylogeny of the fishes traditionally referred to Cyprinini sensu stricto (Teleostei: Cypriniformes)

Yang, L., Mayden, R. L., Sado, T., He, S., Saitoh, K. & Miya, M. (2010). Molecular phylogeny of the fishes traditionally referred to Cyprinini sensu stricto (Teleostei: Cypriniformes). —Zoologica Scripta, 39, 527–550. Carps (e.g. Koi) of the genus Cyprinus and Crucian carps (e.g. Goldfish) of the genus Carassius are among the most popular freshwater fishes around the world. However, their phylogenetic positions within the subfamily Cyprininae, relationships with their allies (e.g. Procypris, Carassioides), and the monophyly of the group formed by them and their allies, which is referred as the tribe Cyprinini sensu stricto, are far from clear. Historically, the Cyprinini was defined by different people according to whether a cyprinine fish possessed a spinous anal‐fin ray (or anal spine), the spine was serrated or not, and occasionally, the number of branched dorsal‐fin rays. Some definitions were established without providing any diagnostic characters. In this study, we investigated the monophyly of the tribe Cyprinini sensu stricto, based on four different historical definitions, and explored the phylogenetic relationships of these members in the subfamily Cyprininae. Using five mitochondrial genes as markers, both maximum‐likelihood and Bayesian trees were constructed using the optimal partitioning strategy. Both analyses successfully resolved a monophyletic Cyprininae and recovered seven major clades from this subfamily. The diagnosis limiting the tribe Cyprinini sensu stricto to four genera, Cyprinus, Carassius, Carassioides and Procypris, received most support. We propose that only those cyprinines that possess a serrated anal spine and have no <10 branched dorsal‐fin rays should be considered members of this tribe. Cyprinini is sister to the Sinocyclocheilus clade, a group traditionally considered a barbin, and together they form the ‘Cyprinini‐Sinocyclocheilus’ clade. Procypris forms the basal clade of the Cyprinini, whereas species of Carassius and Carassioides locate at the top.     

Variation patterns of the mitochondrial 16S rRNA gene with secondary structure constraints and their application to phylogeny of cyprinine fishes (Teleostei: Cypriniformes)

The mitochondrial 16S ribosomal RNA (rRNA) gene sequences from 93 cyprinid fishes were examined to reconstruct the phylogenetic relationships within the diverse and economically important subfamily Cyprininae. Within the subfamily a biased nucleotide composition (A>T, C>G) was observed in the loop regions of the gene, and in stem regions apparent selective pressures of base pairing showed a bias in favor of G over C and T over A. The bias may be associated with transition-transversion bias. Rates of nucleotide substitution were lower in stems than in loops. Analysis of compensatory substitutions across these taxa demonstrates 68% covariation in the gene and a logical weighting factor to account for dependence in mutations for phylogenetic inference should be 0.66. Comparisons of varied stem-loop weighting schemes indicate that the down-weightings for stem regions could improve the phylogenetic analysis and the degree of non-independence of stem substitutions was not as important as expected. Bayesian inference under four models of nucleotide substitution indicated that likelihood-based phylogenetic analyses were more effective in improving the phylogenetic performance than was weighted parsimony analysis. In Bayesian analyses, the resolution of phylogenies under the 16-state models for paired regions, incorporating GTR + G + I models for unpaired regions was better than those under other models. The subfamily Cyprininae was resolved as a monophyletic group, as well as tribe Labein and several genera. However, the monophyly of the currently recognized tribes, such as Schizothoracin, Barbin, Cyprinion + Onychostoma lineages, and some genera was rejected. Furthermore, comparisons of the parsimony and Bayesian analyses and results of variable length bootstrap analysis indicates that the mitochondrial 16S rRNA gene should contain important character variation to recover well-supported phylogeny of cyprinid taxa whose divergences occurred within the recent 8 MY, but could not provide resolution power for deep phylogenies spanning 10-19 MYA.