Genomic analysis and phylogenetic characterization of Himalayan snow trout, Schizothorax esocinus based on mitochondrial protein-coding genes.

BACKGROUND: Mitochondrial DNA (mtDNA) has become a significant tool for exploring genetic diversity and delineating evolutionary links across diverse taxa. Within the group of cold-water fish species that are native to the Indian Himalayan region, Schizothorax esocinus holds particular importance due to its ecological significance and is potentially vulnerable to environmental changes. This research aims to clarify the phylogenetic relationships within the Schizothorax genus by utilizing mitochondrial protein-coding genes. METHODS: Standard protocols were followed for the isolation of DNA from S. esocinus. For the amplification of mtDNA, overlapping primers were used, and then subsequent sequencing was performed. The genetic features were investigated by the application of bioinformatic approaches. These approaches covered the evaluation of nucleotide composition, codon usage, selective pressure using nonsynonymous substitution /synonymous substitution (Ka/Ks) ratios, and phylogenetic analysis. RESULTS: The study specifically examined the 13 protein-coding genes of Schizothorax species which belongs to the Schizothoracinae subfamily. Nucleotide composition analysis showed a bias towards A + T content, consistent with other cyprinid fish species, suggesting evolutionary conservation. Relative Synonymous Codon Usage highlighted leucine as the most frequent (5.18%) and cysteine as the least frequent (0.78%) codon. The positive AT-skew and the predominantly negative GC-skew indicated the abundance of A and C. Comparative analysis revealed significant conservation of amino acids in multiple genes. The majority of amino acids were hydrophobic rather than polar. The purifying selection was revealed by the genetic distance and Ka/Ks ratios. Phylogenetic study revealed a significant genetic divergence between S. esocinus and other Schizothorax species with interspecific K2P distances ranging from 0.00 to 8.87%, with an average of 5.76%. CONCLUSION: The present study provides significant contributions to the understanding of mitochondrial genome diversity and genetic evolution mechanisms in Schizothoracinae, hence offering vital insights for the development of conservation initiatives aimed at protecting freshwater fish species.

An overview of the taxonomic instability of endangered Golden and Zhobi mahseer in Pakistan / Uma visão geral da instabilidade taxonômica do golden em perigo e do Zhobi mahseer no Paquistão

In South and South East Asia three genera of fish species i.e. Tor, Neolissochilus and Naziritor are commonly known as Mahseer with at least 47 species. Among these 23 belongs to genus Tor, 22 to Neolissochilus and one to Naziritor i.e. Naziritor zhobensis. Recently another species added to genus Naziritor is Naziritor chelynoides in India. Among Tor species Tor putitora (Hamilton) is the most widely distributed Mahseer in Pakistan and other countries of the Indian subcontinent. However, based alone on morphological characters some authors identify the Pakistani counterparts as Tor macrolepis (Heckel), (a species presumed to be found exclusively in the Indus River system) distinct from Tor putitora (a species found in Ganga Brahmaputra River system). In order to resolve this taxonomic ambiguity, present study carried out meristic and morphometric measurements of Mahseer collected from a total of 11 water bodies of Pakistan. Ratios between the morphometric characters were calculated and statistically analyzed using t-test and correlation coefficient. Two species identified as Tor putitora and Naziritor zhobensis were the sole Mahseer inhabitants of Indus system in Pakistan. Tor putitora occurred at all surveyed sites while Nazirtor zhobensis had a distribution range from river Zhob to tributaries of river Gomal the right bank tributaries of River Indus. The study corroborates that there are no unequivocal morphological synapomorphies in any existing populations of both species. The study further demonstrates that head length, a character frequently used in Mahseer taxonomy, is not a good measure for species identification. Finally the present study establishes that Naziritor zhobensis still exists in the water bodies of Pakistan and that golden Mahseer occurring in Indus riverine system of Pakistan is Tor putitora.

No sul e sudeste da Ásia, três gêneros de espécies de peixes, ou seja, Tor, Neolissochilus e Naziritor, são comumente conhecidos como Mahseer com pelo menos 47 espécies. Entre estas, 23 pertencem ao gênero Tor, 22 a Neolissochilus e um a Naziritor, ou seja, Naziritor zhobensis. Recentemente, outra espécie adicionada ao gênero Naziritor é Naziritor chelynoides na Índia. Entre as espécies de Tor, Tor putitora (Hamilton) é o Mahseer mais amplamente distribuído no Paquistão e em outros países do subcontinente indiano. No entanto, com base apenas em caracteres morfológicos, alguns autores identificam as contrapartes paquistanesas como Tor macrolepis (Heckel) (uma espécie que se presume ser encontrada exclusivamente no sistema do rio Indo), distinta de Tor putitora (uma espécie encontrada no sistema do rio Ganga Brahmaputra). A fim de resolver essas ambiguidades taxonômicas, o presente estudo realizou medidas merísticas e morfométricas de Mahseer coletadas em um total de 11 corpos d’água do Paquistão. As razões entre os caracteres morfométricos foram calculadas e analisadas estatisticamente usando o teste t e o coeficiente de correlação. Duas espécies identificadas como Tor putitora e Naziritor zhobensis foram os únicos habitantes Mahseer do sistema fluvial Indo no Paquistão. Tor putitora ocorreu em todos os locais pesquisados, enquanto Nazirtor zhobensis tinha uma faixa de distribuição do rio Zhob aos afluentes do rio Gomal, afluentes da margem direita do rio Indo. O estudo corrobora que não há sinapomorfias morfológicas inequívocas em nenhuma das populações existentes de ambas as espécies. O estudo demonstra ainda que o comprimento da cabeça, um caractere frequentemente usado na taxonomia de Mahseer, não é boa medida para identificação das espécies. Finalmente, o presente estudo estabelece que Naziritor zhobensis ainda existe nos corpos d'água do Paquistão e que o Mahseer dourado ocorrendo no sistema fluvial Indo do Paquistão é Tor putitora.

Phylogenetic analysis and expression differences of eye-related genes in cavefish genus Sinocyclocheilus.

The adaptive evolution of visual systems has been observed in many cavefish. However, little is known about the molecular mechanisms underlying these adaptations, which include regressive changes such as eye degeneration. Here, we analyzed phylogenetic and expression patterns of 6 eye-related genes (crx, foxg1b, opn1sw2, otx2, rho and sox2) in 12 Sinocyclocheilus species from China, including 8 stygobionts and 4 stygophiles, and examined photoreceptor cell morphology of these species. Those eye-degenerated species of Sinocyclocheilus were polyphyletic and showed different degrees of photoreceptor defects in responses to cave environments. The eye loss and degeneration are the result of convergent evolution. Although S. anophthalmus grouped with the eye-normal species, it displayed not only a high degree of eye degeneration but also significant expression differences in eye-related genes compared with the eye-normal species. The gene foxg1b, which was determined to be under positive selection, might play an important role in the process of eye degeneration in S. anophthalmus based on differential expression. Eye-related gene expression and selection may have contributed to the polyphyly of the cave species. We examined gene expression and duplication in 6 eye-related genes and revealed that these genes displayed considerable diversity in relative expression in Sinocyclocheilus fishes. Otx2 and sox2 were significantly up-regulated in individual cave species, while the other 4 genes (crx, foxg1b, opn1sw2 and rho) were significantly down-regulated. These findings provide a valuable resource for elucidating molecular mechanisms associated with visual system evolution in cavefish.

Differential regulation of select osmoregulatory genes and Na+/K+-ATPase paralogs may contribute to population differences in salinity tolerance in a semi-anadromous fish.

The Sacramento splittail (Pogonichthys macrolepidotus) is a species of special concern that is native to the San Francisco Estuary, USA. Two genetically distinct populations exist and differ in maximal salinity tolerances. We examined the expression of 12 genes representative of osmoregulatory functions in the gill over a 14  day time course at two different salinities [11 or 14 PSU (Practical Salinity Units)] and revealed that each population showed distinct patterns of gene expression consistent with population differences in response to osmotic regimes. The relatively more salinity-tolerant San Pablo population significantly upregulated nine out of the 12 transcripts investigated on day 1 of 11 PSU salinity exposure in comparison to the day zero freshwater control. Three transcripts (nka1a, nka1b, and mmp13) were differentially expressed between the populations at 7 and 14 days of salinity exposure, suggesting a reduced ability of the relatively salinity-intolerant Central Valley population to recover. Additionally, a phylogenetic analysis of several Sacramento splittail Na+/K+-ATPase α1 sequences resulted in grouping by proposed paralog rather than species, suggesting that different paralogs of this gene may exist. These findings, together with prior research conducted on the Sacramento splittail, suggest that the San Pablo population may be able to preferentially regulate select osmoregulatory genes, including different Na+/K+-ATPase α1 paralogs, to better cope with salinity challenges.

Deep evolutionary origin of limb and fin regeneration.

Salamanders and lungfishes are the only sarcopterygians (lobe-finned vertebrates) capable of paired appendage regeneration, regardless of the amputation level. Among actinopterygians (ray-finned fishes), regeneration after amputation at the fin endoskeleton has only been demonstrated in polypterid fishes (Cladistia). Whether this ability evolved independently in sarcopterygians and actinopterygians or has a common origin remains unknown. Here we combine fin regeneration assays and comparative RNA-sequencing (RNA-seq) analysis of Polypterus and axolotl blastemas to provide support for a common origin of paired appendage regeneration in Osteichthyes (bony vertebrates). We show that, in addition to polypterids, regeneration after fin endoskeleton amputation occurs in extant representatives of 2 other nonteleost actinopterygians: the American paddlefish (Chondrostei) and the spotted gar (Holostei). Furthermore, we assessed regeneration in 4 teleost species and show that, with the exception of the blue gourami (Anabantidae), 3 species were capable of regenerating fins after endoskeleton amputation: the white convict and the oscar (Cichlidae), and the goldfish (Cyprinidae). Our comparative RNA-seq analysis of regenerating blastemas of axolotl and Polypterus reveals the activation of common genetic pathways and expression profiles, consistent with a shared genetic program of appendage regeneration. Comparison of RNA-seq data from early Polypterus blastema to single-cell RNA-seq data from axolotl limb bud and limb regeneration stages shows that Polypterus and axolotl share a regeneration-specific genetic program. Collectively, our findings support a deep evolutionary origin of paired appendage regeneration in Osteichthyes and provide an evolutionary framework for studies on the genetic basis of appendage regeneration.

Phylogenetic relationships of Cypriniformes and plasticity of pharyngeal teeth in the adaptive radiation of cyprinids.

The Cypriniformes comprise approximately 4,200 species accounting for 25% of the diversity of all freshwater fish, which is widely distributed across the world's continents except Antarctica, South America, and Australia. The highest species diversity is found in Southeastern Asia. Despite its remarkable species diversity and broad-scale geographic patterns of distribution, the evolutionary history of this major freshwater fish group remains largely unresolved. To gain insight of the evolutionary history of Cypriniformes, we present a phylogeny of this group using 1 mitochondrial gene and 15 nuclear genes comprising a total of 14,061 bp. Bayesian inference using all gene fragments yielded a well resolved phylogeny, which is mostly consistent with topologies obtained from Maximum Likelihood analyses. Our results further confirmed the monophyly of Cypriniformes and seven constituent subclades including Cyprinidae, Catostomidae, Gyrinocheilidae, Balitoridae, Cobitidae, Nemacheilidae, and Botiidae. Bayesian divergence time analysis indicated that the origin of the Cypriniformes was about 193 Mya during the early Jurassic, coinciding with the onset of the Pangaea breakup. The basal divergence of Cypriniformes is 154 Mya during the late Jurassic. Our findings from molecular divergence and biogeographical analysis indicate the most likely initial geographical range of the ancient Cypriniformes was both East and South Asia (Southeastern area of Mesozoic Laurasia). Moreover, the burst in species diversity in Cyprinidae afforded by the nearly worldwide colonization is possibly in response to the plasticity of pharyngeal dentition. The present study demonstrates that the Cypriniformes was about 193 Mya during the early Jurassic, coinciding with the onset of the Pangaea breakup. The plasticity of pharyngeal dentition of cyprinids might contribute to the burst and radiation of this lineage. The phylogenetic and biogeographic analyses in this study help to improve our understanding of the evolutionary history of this diverse and important freshwater fish group.

Finding stories in noise: Mitochondrial portraits from RAD data.

Mitochondrial DNA (mtDNA) has formed the backbone of phylogeographic research for many years; however, recent trends focus on genome-wide analyses. One method proposed for calibrating inferences from noisy next-generation data, such as RAD sequencing, is to compare these results with analyses of mitochondrial sequences. Most researchers using this approach appear to be unaware that many single nucleotide polymorphisms (SNPs) identified from genome-wide sequence data are themselves mitochondrial, or assume that these are too few to bias analyses. Here, we demonstrate two methods for mining mitochondrial markers using RAD sequence data from three South African species of yellowfish, Labeobarbus. First, we use a rigorous SNP discovery pipeline using the program stacks, to identify variant sites in mtDNA, which we then combine into haplotypes. Second, we directly map sequence reads against a mitochondrial genome reference. This method allowed us to reconstruct up to 98% of the Labeobarbus mitogenome. We validated these mitogenome reconstructions through blast database searches and by comparison with cytochrome b gene sequences obtained through Sanger sequencing. Finally, we investigate the organismal consequences of these data including ancient genetic exchange and a recent translocation among populations of L. natalensis, as well as interspecific hybridization between L. aeneus and L. kimberleyensis.

Wider geographic distribution and higher diversity of hexaploids than tetraploids in Carassius species complex reveal recurrent polyploidy effects on adaptive evolution.

Polyploidy roles on adaptive evolution and ecological novelty have been extensively studied in plants but remained unclear in vertebrates owing to the rare polyploidy incidences. Here, a huge number of 3105 specimens in Carassius species complex including 2211 hexaploids and 894 tetraploids were sampled from 34 locations through mainland China. And hexaploids had wider geographic distribution than tetraploids especially in the areas with high altitude, high latitude and low annual precipitation. Then, an approximate 1050 bp transferrin (tf) fragments were amplified from all the samples, and 526 tf alleles were identified from a total of 37260 sequences at last. Intriguingly, higher nucleotide diversity of tf alleles in hexaploids than in tetraploids was revealed. Moreover, via phylogenetic analysis of tf alleles, potential origin center of Carassius species complex was deduced to be Yangtze River basin and hexaploids should undergo multiple independent polyploidy origins from sympatric tetraploids. These findings indicate that the hexaploids might possess stronger environmental adaptation and ecological novelty than the tetraploids, which provide an association paradigm of recurrent polyploidy and ecological context in polyploid vertebrates.

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.

Molecular identification and functional characterisation of the interferon regulatory factor 1 in the blunt snout bream (Megalobrama amblycephala).

Interferon regulatory factors (IRFs) play a key role in mediating the host response against pathogen infection and other important biological processes. This is the first report of an IRF family member in blunt snout bream Megalobrama amblycephala. The complete cDNA of M. amblycephala (Ma) IRF1 gene has 1422 nucleotides (nt.), with an open reading frame of 858 nt, encoding a polypeptide of 285 amino acids. The putative MaIRF1 polypeptide shared significant structural homology with known IRF1 homologs: a conserved IRF domain was found at the N-terminal and an IRF association domain 2 at the C-terminal. Phylogenetic analysis showed that MaIRF1 amino acid sequence clustered with other teleost IRF1s, with a grass carp ortholog exhibiting the highest similarity. MaIRF1 mRNA expression patterns were studied using quantitative real-time PCR in healthy fish tissues and after a challenge with Aeromonas hydrophila bacterium. It was constitutively expressed in all examined tissues: the highest in blood, the lowest in muscle. The expression after A. hydrophila challenge was up-regulated in liver, spleen and kidney, but down-regulated in intestine and gills. At the protein level, similar expression patterns were observed in liver and gills. Patterns differed in intestine (up-regulation), spleen (down-regulation) and kidney (expression mostly unchanged). This study indicates that MaIRF1 gene plays an important role in the blunt snout bream immune system, hence providing an important base for further studies.

Cloning, characterization and mRNA expression of interleukin-6 in blunt snout bream (Megalobrama amblycephala).

In the present study, the interleukin-6 gene (IL-6) cDNA in blunt snout bream (Megalobrama amblycephala) was identified and its expression profiles under ammonia stress and bacterial challenge were investigated. The IL-6 sequence consisted of 1045 bp, including a 696 bp ORF which translated into a 232 amino acid (AA) protein. The protein contained a putative signal peptide of 24 AA in length. IL-6 expression analysis showed that the it is differentially expressed in various tissues under normal conditions and the highest IL-6 level was observed in the intestine tissue, followed by the liver, and then in the gills. Under ammonia stress, the IL-6 mRNA level both in spleens and intestine increased significantly (P < 0.05), with the maximum levels attained at 6 h, 12 h (72, 10-fold, respectively). Thereafter, they all significantly decreased (P < 0.01) and returned to the basal value within 48 h. Whereas, in livers it slightly decreased at 3 h firstly (0.5-fold), and then significantly (P < 0.05) increased with the maximum level attained 12 h (3-fold). Further expression analysis showed that the mRNA level of IL-6 in spleens, intestine and livers of blunt snout bream all increased significantly (P < 0.05), with maximum values attained at 6 h, 3 h, 6 h (10, 6, 18-fold, respectively) after Aeromonas hydrophila (A. hydrophila) injection, and then decreased to the basal value within 24 h which suggested that IL-6 was involved in the immune response to A. hydrophila. The cloning and expression analysis of the IL-6 provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions in blunt snout bream.

Inheritance of the complete mitochondrial genomes in three different ploidy fishes.

Diploid natural gynogenetic goldfish (2nGRCG), triploid hybrids (3nRB) and tetraploid hybrids (4nRB) are generated by distant hybridization of red common goldfish (RCG, Carassius auratus red var.) and blunt snout bream (BSB, Megalobrama amblycephala). In the present study, we obtained the complete mitochondrial DNA (mtDNA) sequences of the hybrid offspring and compared them with the homologous sequences of RCG and BSB. All mtDNA sequences of these hybrids were 16,580bp in length, and the genes number, size, and order were quite similar to that of RCG. Genetic analysis revealed that the mtDNA sequences of these hybrids had high similarity (>99%) and low divergence (<2%) to their maternal RCG, yet lower similarities (84%) and higher divergences (16%) to their paternal BSB. The phylogenetic analysis also showed that the sequences of 2nGRCG, 3nRB and 4nRB were clustered with RCG rather than with BSB. These results indicate that the mitochondrial genomes of 2nGRCG, 3nRB and 4nRB remain maternally inherited after hybridization and polyploidization. Moreover, clade separation of hybrid offspring from their paternal BSB in the phylogenetic tree implies that phylogenetic analysis of mtDNA is incomplete for elucidating the true relationships between different species, particularly when they have undergone hybridization or allopolyploidization. Our study provides significant information for both evolution and genetic studies of mtDNA for hybrid species and allopolyploidization species.

Cambios morfológicos en gametos del barbo tigre Puntius tetrazona (Cypriniformes: Cyprinidae) e implementación de la fertilización in vitro / Morphological changes in gametes of tiger barb Puntius tetrazona (Cypriniformes: Cyprinidae) and the implementation of in vitro fertilization

The production of ornamental fishes represents an economic activity of a growing number of Mexican families. Nevertheless, the reproduction of fish in captivity is one of the complications faced by farmers. This study was set up to: (i) evaluate the morphological and functional changes induced by hydration in the gametes of fish tiger barb (Puntius tetrazona; 240 samples) at tree times after hydration (10, 20 and 30s) with classic spermograms (volume, sperm concentration, viability, motility, and normal morphology); and (ii) evaluate the implementation of in vitro fertilization based on the ovulation rate, the percentage of fertilization and hatching, and the larval numbers obtained after 72 hours. The average volume of milt was 3.0±0.7μL, and the minimum, maximum and average concentration of sperm was 44.4x10(6) spz/mL, 52.3x10(6)spz/mL, and 48.1±5.9x10(6)spz/mL, respectively. The viability and motility of the sperm was 84.6±3.2% and 81.5±2.2%, respectively. The diameter of the sperm with/without water contact was 2.1±0.6μm and 3.8±1.0μm (p<0.05); the largest diameter was recorded 30 seconds after the contact with water. For oocytes, the smaller and larger diameters were recorded at 10 and 30s, respectively (both with/without water contact); the oocytes diameters after 10 and 30 seconds of contact with water were 1.11 and 1.55mm, respectively. A higher ovulation rate was recorded using the in vitro fertilization: 250±50 oocytes versus 28±09 oocytes (during natural fertilization; p<0.05). Nevertheless, fertilization and hatching rates were higher for the natural fertilization (80 and 60%, respectively). Considering the number of larvae obtained after 72 hours, our results showed a higher value for the in vitro fertilization (75±18 compared to 13.4±12 of the natural fertilization; p<0.05). We propose this fish as a model for other ornamental fishes of commercial interest. Our results demonstrate that the in vitro fertilization is a very high viable option to optimize and maximize resources; besides, the reproduction management optimization under controlled conditions may enhance wild fish stocks preservation. Rev. Biol. Trop. 62 (4): 1353-1363. Epub 2014 December 01.

El objetivo del presente estudio fue conocer las características de los gametos de Puntius tetrazona (n=240), los cambios morfológicos a partir de su activación mediante espermogramas cásicos y por otro lado, se evaluó la implementación de la fertilización in vitro a partir de la tasa ovulatoria, el % de fertilización y eclosión y el número de larvas vivas a las 72h. El volumen promedio de semen fue de 3.0±0.7µL. La concentración espermática mínima, máxima y promedio fue 44.48x10(6)spz/mL, 52.3x10(6)spz/mL y 48.1±5.9x10(6)spz/mL, respectivamente. La viabilidad promedio fue de 84.68±3.27%. La motilidad promedio fue 81.53±2.28%. El diámetro de los espermatozoides fluctuó entre 2.16±0.2 y 2.79±0.3µm; 3.84±0.3 y 4.86±0.31µm sin y con contacto con el agua respectivamente, con diferencias significativas. El diámetro mayor fue a los 30s en contacto con el agua. Los ovocitos de menor y mayor diámetro se registraron a los diez y 30s sin y con contacto con el agua respectivamente. Los diámetros de los ovocitos en diez y 30s en contacto con el agua fluctuaron entre 1.11 y 1.55mm respectivamente. La mayor tasa ovulatoria fue en la fertilización in vitro con 250±50 ovocitos frente a 28±09 de la natural, con diferencias significativas. Los porcentajes de fertilización y eclosión fueron más elevados en la fertilización natural con 80% y 60% respectivamente. Se registraron 75±18 larvas a las 72 horas en el grupo in vitro comparado con 13.4±12 larvas de la fertilización natural. Con lo anterior, la técnica que permitió mayor cantidad de larvas fue la de fertilización in vitro.

Detection of morphometric differentiation in Sattar snowtrout, Schizothorax curvifrons (Cypriniformes: Cyprinidae) from Kashmir Himalaya using a truss network system

Schizothorax curvifrons is a morphometrically and meristically most variable and economically valuable promising fish food from Kashmir Valley. Since there is a lack of information on stock structure of wild populations on this species, this study was aimed to investigate the intraspecific variation of this important snowtrout. For this, two rivers and one lake in Kashmir Himalaya were sampled from January 2011 to October 2012. Fish body measurements were taken and morphometric characters using the truss network system was constructed. Altogether, 506 fish specimens were collected. Data were subjected to principal component analysis, discriminant function analysis and univariate analysis of variance. The first principal component explained 63.44% of total variation, while second and third components explained 8.34% and 5.31%, respectively. The step-wise discriminant function analysis retained two variables that significantly discriminated the populations. Using these variables 83.4% of the original specimens were classified into their correct groups and 81.1% of the cross-validated (leave one out procedure) specimens were classified into their correct groups. All of the total 31 transformed truss measurements exhibited highly significant (p<0.001) differences between the populations. This represents the first attempt on stock structure of S. curvifrons; therefore, this study will hopefully guide fisheries taxonomists about its current stock structure and would help in its management and conservation programme across Kashmir Himalaya area.

Schizothorax curvifrons es un alimento para peces, morfométricamente y merísticamente más variable y de mayor valor económico del Valle de Kashmir. Dado que existe una falta de información sobre la estructura de las poblaciones silvestres de esta especie, este estudio tuvo como objetivo investigar la variación intraespecífica de esta importante trucha de nieve. Para ello, se tomaron muestras de dos ríos y un lago en Kashmir Himalaya, de enero 2011 a octubre 2012. Con las mediciones de los peces se construyó una red truss. En total, se recolectaron 506 muestras de peces. Los datos se sometieron a análisis de componentes principales, análisis de función discriminante y el análisis univariante de la varianza. El primer componente principal explicó 63.44% de la variación total, mientras que los componentes segundo y tercero explicaron el 8.34% y 5.31%, respectivamente. El análisis discriminante por etapas, retuvo dos variables que discriminaron significativamente las poblaciones. El uso de estas variables de 83.4% de los especímenes originales se clasificaron en sus grupos correctos y 81.1% de la validación cruzada (deja fuera un procedimiento) especímenes fueron clasificados en sus grupos correctos. Todas las mediciones para entramados transformados, del total de 31, mostraron diferencias altamente significativas (p<0.001) entre las poblaciones. Esto representa el primer intento de obtener la estructura de la población de S. curvifrons, por lo tanto, este estudio espera orientar a los taxonomistas de peces acerca de la estructura de la población actual y ayudar en su manejo y poder establecer un programa de conservación en el área de Kashmir Himalaya.

Molecular characterization and expression analysis of a peroxiredoxin 1 cDNA from Korean rose bitterling (Rhodeus uyekii).

Peroxiredoxins (Prxs), also known as natural killer cell enhancing factors in fish, role as antioxidant proteins and participate in a variety of biological processes, including H2O2-mediated cell signaling, molecular chaperoning, and mitochondrial function. In this study, we isolated and characterized a Prx 1 cDNA from the Korean rose bitterling Rhodeus uyekii, and designated it RuPrx 1. The RuPrx 1 cDNA encodes a 197-amino-acid polypeptide that belongs to the class of typical 2-Cys Prxs that contain peroxidatic and resolving cysteines. The deduced RuPrx 1 protein shows strong homology (77.38-92.89 %) with Prx 1 proteins from other species, including fish, amphibians, and mammals, and it is most closely related to rainbow smelt Prx 1. RuPrx 1 mRNA was ubiquitously detected in all tested tissues and its expression was comparatively high in the brain, intestine, kidney, liver, ovary, stomach, and testis. Expression of RuPrx 1 mRNA in liver peaked 3 h post-infection with Aeromonas hydrophila and decreased 24 h post-infection while the expression in intestine decreased 24 h post-infection. These results suggest that RuPrx 1 is conserved through evolution and may play roles similar to its mammalian counterparts.

Polyphyletic origins of schizothoracine fish (Cyprinidae, Osteichthyes) and adaptive evolution in their mitochondrial genomes.

The schizothoracine fish, also called snow trout, are members of the Cyprinidae, and are the most diversified teleost fish in the Qinghai-Tibetan Plateau (QTP). Clarifying the evolutionary history of the schizothoracine fish is therefore important for better understanding the biodiversity of the QTP. Although morphological and molecular phylogenetic studies have supported the monophyly of the Schizothoracinae, a recent molecular phylogenetic study based on the mitochondrial genome questioned the monophyly of this taxon. However, the phylogenetic analysis of that study was on the basis of only three schizothoracine species, and the support values were low. In this report, we inferred the phylogenetic tree on the basis of mitochondrial genome data including 21 schizothoracine species and five closely related species, and the polyphyletic origins of the Schizothoracinae were strongly supported. The tree further suggests that the Schizothoracinae consists of two clades, namely the "morphologically specialized clade" and the "morphologically primitive clade", and that these two clades migrated independently of each other to the QTP and adapted to high altitude. We also detected in their mitochondrial genomes strong signals of positive selection, which probably represent evidence of high-altitude adaptation. In the case of the morphologically specialized clade, positive selection mainly occurred during the Late Paleocene to the Early Oligocene. Its migration also seems to have occurred in the Early Eocene, and this timing is consistent with the drastic uplifting of the QTP. On the other hand, positive selection in the morphologically primitive clade has mainly occurred since the Late Miocene. Because its members are thought to have migrated to the QTP recently, it is possible that they are now undergoing high-altitude adaptation.

The lateral line receptor array of cyprinids from different habitats.

The lateral line system of teleost fishes consists of an array of superficial and canal neuromasts (CN). Number and distribution of neuromasts and the morphology of the lateral line canals vary across species. We investigated the morphology of the lateral line system in four diurnal European cyprinids, the limnophilic bitterling (Rhodeus sericeus), the indifferent gudgeon (Gobio gobio), and ide (Leuciscus idus), and the rheophilic minnow (Phoxinus phoxinus). All fish had lateral line canals on head and trunk. The total number of both, CN and superficial neuromasts (SN), was comparable in minnow and ide but was greater than in gudgeon and bitterling. The ratio of SNs to CNs for the head was comparable in minnow and bitterling but was greater in gudgeon and ide. The SN-to-CN ratio for the trunk was greatest in bitterling. Polarization of hair cells in CNs was in the direction of the canal. Polarization of hair cells in SNs depended on body area. In cephalic SNs, hair cell polarization was dorso-ventral or rostro-caudal. In trunk SNs, it was rostro-caudal on lateral line scales and dorso-ventral on other trunk scales. On the caudal fin, hair cell polarization was rostro-caudal. The data show that, in the four species studied here, number, distribution, and orientation of CNs and SNs cannot be unequivocally related to habitat.

High altitude adaptation of the schizothoracine fishes (Cyprinidae) revealed by the mitochondrial genome analyses.

The schizothoracine fishes, also known as "mountain carps" are widely distributed in the Qinghai-Tibetan Plateau and its peripheral regions. Although they provide a prime example of high altitude adaptation, the phylogenetic relationships and the divergence times among these carp lineages are still controversial. Moreover, the genetic basis for high altitude adaptation is also poorly understood. In this study, we determined the mitochondrial genomes from two species of the schizothoracine fishes, representing a "morphologically primitive" clade and "morphologically specialized" clade, respectively. The phylogenetic tree and the divergence times were estimated within the evolutionary framework of the entire order Cypriniformes. Our results indicate a polyphylyetic relationship of the schizothoracine fishes and suggest two independent migration events into the Qinghai-Tibetan Plateau: one by the "morphologically primitive" clade in the Late Miocene and another by the "morphologically specialized" clade in the Eocene. Rapid speciation events of each clade from the Late Miocene to the Pliocene correspond to the timing of the geologic acceleration of the Qinghai-Tibetan Plateau. Interestingly, we found evidence for positive selection acting on the protein coding genes in the mitochondrial genomes of the "morphologically specialized" clade, implying a possible genetic basis for high altitude adaptation in this derived lineage of cypriniform fishes.

Levels, distribution, and health risks of polycyclic aromatic hydrocarbons in four freshwater edible fish species from the Beijing market.

We first estimated the content of polycyclic aromatic hydrocarbons (PAHs) in the brain, liver, bladder, roe, and muscle of four species of edible freshwater fish from the Beijing market. The distribution characteristics of PAHs in these tissues and organs were analyzed to determine their health risks to humans. The results showed that the residual levels of wet weight and lipid-normalized weight ∑PAHs in various tissues of these fish ranged from 0.51 ng·g(-1) to 28.78 ng·g(-1) and from 93.62 ng·g(-1) to 8203.43 ng·g(-1), respectively. The wet weight contents of ∑PAHs were relatively higher in the brain and lower in the liver and muscle. But the differences were not significant. And the differences of lipid-normalized weight PAHs were significant, which in the bighead carp were found significantly the highest, followed in crucian carp, and the lowest in grass carp and carp. The contents of ∑PAHs were the highest in the liver and the lowest in the brain. In the tissues with a higher lipid content, higher residual levels of PAHs were found. The carcinogenic risks for humans from residual ∑PAHs in the various fish tissues were far below 10(-5).

The evolutionary history of the allopolyploid Squalius alburnoides (Cyprinidae) complex in the northern Iberian Peninsula.

Understanding the population structure, population dynamics and processes that give rise to polyploidy and helps to maintain it is central to our knowledge of the evolution of asexual vertebrates. Previous studies revealed high genetic diversity and several reproductive pathways in the southern populations of the Squalius alburnoides hybrid complex. In contrast, lower genetic variability and the associated limited chance of introducing new genetic combinations may threaten the survival of the northern Mondego populations. We analysed the genetic diversity and structure of nine populations of S. alburnoides in the Iberian Peninsula using microsatellite loci to provide further insights on the evolutionary history of this complex. Special attention was given to the less-studied northern populations (Mondego and Douro basins). Marked population structure, a high frequency of private alleles and a high diversity of some biotypes in the Douro basin indicate that some northern populations may not be at high risk of extinction, contrary to what was expected. The genetic diversity found in the northern Douro populations contradicts the general trend of remarkable genetic impoverishment northwards that occurs in other species and regions. The results indicate the possible existence of a glacial refugium in the Rabaçal River, corroborating findings in other species of this region. Historical events seem to have affected the geographical patterns of genetic variability found among and within the northern and southern populations of this complex and contributed to different patterns of genome composition. Therefore, historical events might have a major role in the long-term persistence of some polyploid hybrid taxa.