川陕哲罗鲑Cyt b基因克隆及其在鲑亚科中的系统发育关系

采用聚合酶链式反应克隆川陕哲罗鲑Hucho bleekeri线粒体细胞色素b基因 (Cyt b),首次报道该基因的全长序列(1140 bp,GenBank 登录号FJ597623),并与鲑亚科中其他5属14个物种的Cyt b基因进行同源性比较,分析了碱基组成和变异情况,以白鲑亚科的白鲑Coregonus lavaretus作为外群构建了ML、NJ和MP系统发育树.遗传距离和分子系统学分析表明,川陕哲罗鲑与细鳞鲑属的细鳞鱼Brachymystax lenok之间的遗传距离相对较小(8.47%),在系统发育树上两者聚在一起,提示川陕哲罗鲑与细鳞鱼存在较近的亲缘关系,该结果与以往的形态学分类不相符.     

哲罗鲑性别特异性标记筛选

研究通过比对哲罗鲑Hucho taimen (Pallas)基因组草图与虹鳟(Oncorhynchus mykiss)Y染色体序列,获得哲罗鲑性别相关的候选序列,并设计3对PCR扩增引物,以此筛选哲罗鲑性别特异性标记。初步筛选结果显示,在设计的3对引物中,引物ST2在雌鱼中无扩增条带,在雄鱼中有153 bp的扩增条带,可作为哲罗鲑雄性特异性候选标记。为了消除样本降解及失误等因素导致的条带缺失,研究以12S rRNA为参照,采用双重PCR法,在12S rRNA引物扩增出条带的前提下,用ST2引物条带的有无来判断性别,雌鱼为单带,无ST2引物条带;雄鱼为双带,有ST2引物条带。同时为了验证本方法的可靠性,对已知性别的哲罗鲑48尾雌、雄样本进行了检测,结果显示该方法遗传性别鉴别准确率为100%。用此标记筛选哲罗鲑雌、雄鱼简单易行,为哲罗鲑遗传学研究、单性养殖和性别控制育种等研究奠定了基础。     

Phylogenetic relationships of Sakhalin taimen Parahucho perryi inferred from PCR-RFLP analysis of mitochondrial DNA

RFLP analysis of three amplified mtDNA fragments (Cytb/D-loop, ND1/ND2, and ND3/ND4L/ND4) was performed in the following taxa: Parahucho perryi, Hucho taimen, Brachymystax lenok, B. tumensis, Salmo salar, Salvelinus leucomaenis, and S. levanidovi. For mtDNA of P. perryi, a substantial decrease in the haplotype and nucleotide diversity was observed as a result of random genetic drift, caused by a reduction in the effective population size. Nucleotide divergence estimates between the mtDNA haplotypes were determined. Sakhalin taimen P. perryi was found to be approximately equally diverged from S. salar and from the charrs of the genus Salvelinus, by 11.0 and 10.0%, respectively. The divergence between P. perryi and H. taimen constituted 14.6%, between P. perryi and lenoks of the genus Brachymystax, 14.2%, and between H. taimen and Brachymystax, 7.7%. The analysis of possible phylogenetic relationships of the mtDNA from P. perryi among the group of taxa examined confirmed validity of the genus Parahucho. Phylogenetic reconstructions performed showed that robustness of the trees constructed for the complex of phylogenetically informative characters over three mtDNA fragments was considerably higher than that of the trees constructed for individual genes.     

Spawning behaviour of Sakhalin taimen, <Emphasis Type="Italic">Parahucho perryi</Emphasis>, from northern Hokkaido,Japan

A video camera mounted in an underwater housing and remotely operated was used to monitor the behaviour of five different Sakhalin taimen (Parahucho perryi), females and attendant males spawning in three coastal tributary streams in Northern Hokkaido, Japan. Based on three complete and two incomplete spawnings, we describe in detail for the first time the complete spawning behavioural repertoire of this species. The Sakhalin taimen was originally placed within Hucho, then removed from that genus based on morphological, life history and molecular data. Our study supports that removal—none of the behavioural traits we recorded clustered Parahucho with Hucho uniquely. Similarities between the two genera were all plesiomorphic traits that are widespread throughout the salmonines. The immediate behaviour right after spawning was found to be a major difference between Hucho and Parahucho. Like female Oncorhynchus and Salmo, Sakhalin taimen females cover their eggs by beats of their tails immediately after spawning. This is different from the “rest, then cover” behaviour shown by Siberian taimen (Hucho taimen) as well as lenok (Brachymystax lenok), supporting again that the Sakhalin taimen be removed from Hucho and placed in its own genus.     

额尔齐斯河流域不同来源哲罗鲑形态及COⅠ基因比较研究

为对哲罗鲑(Hucho)的引种放流和土著物种的保护提供科学依据, 研究采集了数尾来源于天然水域的哲罗鲑(Hucho BHB和Hucho BEJ), 与引进的哲罗鲑(Hucho HLJ)一起进行了形态特点观察描述, 同时对COⅠ基因扩增测序并从GenBank下载哲罗鲑属COⅠ基因构建系统发育树、计算遗传距离进行分子生物学比较研究。结果显示, Hucho BEJ和Hucho HLJ形态特点相一致, Hucho BHB与其他两种来源的哲罗鲑在体色、斑点大小等均有差异。基于COⅠ基因的系统发育树显示几种不同来源的哲罗鲑均与太门哲罗鲑(H. taimen)聚为一个大的分枝, 但在这个分枝下, Hucho BEJ和Hucho HLJ与俄罗斯阿穆尔河(中国称黑龙江)的哲罗鲑聚为一枝, 而Hucho BHB单独形成一支。基于COⅠ基因的遗传矩阵显示, Hucho BEJ和Hucho HLJ与俄罗斯阿穆尔河的哲罗鲑遗传距离较近(0—0.0044), 而Hucho BHB与Hucho BEJ、Hucho HLJ及俄罗斯阿穆尔河的哲罗鲑遗传距离较远(0.0057—0.0082)。这说明Hucho BHB在形态和基因序列上与Hucho HLJ及俄罗斯阿穆尔河的哲罗鲑具有较大差异, 可能是不同的生态地理类型, Hucho BEJ和Hucho HLJ在形态和基因序列上基本一致, 我们推测Hucho BEJ是放流或养殖场逃逸的哲罗鲑。     

Comparative mtDNA sequence (control region, ATPase 6 and NADH‐1) divergence in Hucho taimen(Pallas) across four Siberian river basins

Hucho taimen from eight populations spanning four drainage basins (Amur, Lena, Enisei and Khatanga) were analysed for nucleotide sequence variation across three mitochondrial genes (ATP6, NADH‐1 and control region). Samples of H. hucho , Brachymystax lenok (sharp‐snouted and blunt‐snouted forms) and Parahucho perryi were also included for comparison. Nucleotide variation across a total of 1826 base pairs in H. taimen revealed shared haplotypes between the Amur and Lena basins, further supporting a previous hypothesis of late to post‐Pleistocene hydrological exchange between these now disjunct basins. In contrast to an earlier study using the control region alone, clear phylogeographic structure was seen at a large geographic scale, reflected by two phylogroups, one corresponding to the Amur and Lena basins, and the other to the Enisei and Khatanga basins. Comparative rates of divergence revealed considerably faster and less heterogeneous substitution rates for the two coding genes, especially at interspecific levels compared to the mtDNA control region.     

Mitochondrial DNA Variation and Introgression in Siberian Taimen Hucho taimen

Siberian taimen Hucho taimen is the largest representative of the family Salmonidae inhabiting rivers of northern Eurasia. The species is under intensive aquaculture activity. To monitor natural taimen populations we have sequenced a portion (8,141 bp) of the mitochondrial (mt) genome in 28 specimens of H. taimen from six localities in the Amur River basin. Nucleotide variability is low (π = 0.0010), but structured in two divergent haplotype groups. A comparison of the data with the GenBank H. taimen mt genome ({"type":"entrez-nucleotide","attrs":{"text":"HQ897271","term_id":"325127000","term_text":"HQ897271"}}HQ897271) reveals significant differences between them in spite of the fact that the fish specimens come from neighboring geographical areas. The distribution of divergence is non-uniform with two highly pronounced divergent regions centered on two genes, ND3 and ND6. To clarify the pattern of divergence we sequenced the corresponding portion of the mt genome of lenok Brachymystax tumensis and analyzed the GenBank complete mt genomes of related species. We have found that the first and second divergent regions are identical between the GenBank H. taimen and two lenok subspecies, B. lenok and B. lenok tsinlingensis, respectively. Consequently, both divergent regions represent introgressed mtDNA resulting from intergeneric hybridization between the two lenok subspecies and H. taimen. Introgression is, however, not detected in our specimens. This plus the precise identity of the introgressed fragments between the donor and the recipient GenBank sequence suggests that the introgression is local and very recent, probably due to artificial manipulations involving taimen – lenok intergeneric hybridization. Human-mediated hybridization may become a major threat to aquatic biodiversity. Consequently we suggest that due attention needs to be given to this threat by means of responsible breeding program management, so as to prevent a potential spread of hybrid fishes that could jeopardize the resilience of locally adapted gene pools of the native H. taimen populations.     

Genetc divergence and phylogeny of the Salmoninae based on allozyme data

The data on 31–37 allozyme loci in 21 species of nine salmonid genera are used for phylogenetic analysis by seven distance methods and several variants of cladistic analysis. Monophyletic origin for all genera and three sub-families of the Salmonidae is corroborated. The closest phylogenetic relationships are characteristic of Parasalmo and Oncorhynchus (bootstrap support is 88–99%), Brachymystax and Hucho (68–97%), and the clade ( Brachymystax + Hucho )+ Salmo (up to 85%). The patterns of phylogenetic relationships in the group Salmo-Parasalmo-Oncorhynchus are analogous to those in the group Parahucho-Hucho-Brachymystax. The position of Parahucho in phylogenetic trees of the Salmoninae is extremely unstable, although it is most likely associated with the clade ( Brachymystax + Hucho)+Salmo ) or Salvelinus. When using the out group analysis, Salvelinus appears as the earliest branch of the Salmoninae tree, whereas if the molecular clock is assumed, the basal position is occupied by Oncorhynchus. However, the latter genus is probably characterized by a substantially increased rate of molecular evolution.     

Variability of Siberian Taimen Hucho taimen (Salmonidae) of The Amur River

Journal of Ichthyology - The morphological variability of nine characters in six samples of Siberian taimen Hucho taimen from the Amur River was studied. The differences in the level of fluctuating...     

Serum levels of precursors to vitelline envelope proteins (choriogenins) in Sakhalin taimen after treatment with oestrogen and during oocyte growth

In Sakhalin taimen Hucho perryi two choriogenins have been identified in serum, designated high- and low-molecular-weight vitelline envelope-related protein ( h VERP and l VERP). Two immunoassays were developed using specific antibodies; single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA), for measurement of the precursors to vitelline envelope proteins. ELISA measures h VERP and l VERP in serum at concentrations as low as 16 and 47 ng ml⁻¹, respectively. Using these immunoassays, changes in serum levels of VERP were measured after treatment with E₂ and during oocyte growth. When immature 2-year-old taimen were injected with 2 mg E₂ per kg body weight, both VERPs were induced in serum within 24 h. In the course of the first ovulation, serum VERPs levels increased in September (8 months before ovulation), reached maximum in January, and decreased sharply prior to ovulation in May. These changes generally paralleled that of vitellogenin except for the drastic decrease of VERPs in May.     

Evolutionary and taxonomic relationships among Far-Eastern salmonid fishes inferred from mitochondrial DNA divergence

Mitochondrial DNA (mtDNA) restriction analysis was used to examine the evolutionary and taxonomic relationships among 11 taxa of the subfamily Salmoninae. The genera Brachymystax and Hucho were closely related, diverging by sequence divergence estimates of 3.1%. Because the mtDNA sequence divergence between blunt- and sharp-snouted forms of Brachymystax (2.24%) was similar to divergence level of Brachymystax and Hucho , then taking into account the distinct morphological, ecological and allozyme differences between them, it is possible to recognize these forms as two separate species. The subgenus Parahucho formed a very distinct group differing by 6.35–7.08% (sequence divergence estimate) from both Brachymystax and Hucho and must be considered as a valid genus. The UPGMA and neighbour-joined phenograms showed that the five genera studied are divided into two main groupings: (1) Hucho, Brachymystax and Salvelinus ; and (2) Oncorhynchus and Parahucho species. The mtDNA sequence divergence estimates between these groupings were about 8.1%. However, the subsequent bootstrap analysis of mtDNA RFLP data did not support the monophyly of the latter grouping. The concordance of morphological and mtDNA phylogenetic patterns is discussed.     

Spawning behaviour of taimen (<Emphasis Type="Italic">Hucho taimen</Emphasis>) from the Uur River,Northern Mongolia

This study presents the first observations of Hucho taimen spawning in the wild based on underwater video recordings. One pair of taimen was monitored during a 19 h period, supplemented with visual observations from two other spawning pairs. We recorded two full spawning events performed in two different locations separated by approximately 30 m. The absence of an established male hierarchy along a nesting female was the most important difference between taimen and other salmonine breeding biology. Taimen spawning, based on our observations, is a single pair event. The male prevented the approach of other males by launching intense attacks that extended for several meters away from the redd. Our data suggests that taimen females, differently from other salmonines, do not cover their eggs immediately after having spawned but rest for a variable number of minutes before covering them.     

The complete mitochondrial genome of the Sichuan taimen (Hucho bleekeri): repetitive sequences in the control region and phylogenetic implications for Salmonidae

The complete mitochondrial DNA genome of the Sichuan taimen (Hucho bleekeri) was determined by the long and accurate polymerase chain reaction (LA-PCR) and primer walking sequence method. The entire mitochondrial genome of this species is 16,997 bp in length, making it the longest among the completely sequenced Salmonidae mitochondrial genomes. It consists of two ribosomal RNA (rRNA) genes, 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and one control region (CR). The gene arrangement, nucleotide composition, and codon usage pattern of the mitochondrial genome are similar to those of other teleosts. A T-type mononucleotide microsatellite and an 82 bp tandem repeat were identified in the control region, which were almost identical among the three H. bleekeri individuals examined. Both phylogenetic analyses based on 12 concatenated protein-coding genes of the heavy strand and on just the control region show that H. bleekeri is a basal species in Salmoninae. In addition, Salmo, Salvelinus and Oncorhynchus all represent monophyletic groups, respectively. All freshwater species occupied basal phylogenetic positions, and also possessed various tandem repeats in their mitochondrial control regions. These results support established phylogenetic relationships among genera in Salmonidae based on morphological and molecular analyses, and are consistent with the hypothesis that Salmonidae evolved from freshwater species.     

Morphology,stomach contents and growth of the endangered salmonid,Sakhalin taimen <Emphasis Type="Italic">Hucho perryi</Emphasis>, captured in the Sea of Okhotsk,northern Japan: evidence of an anadromous form

Synopsis We describe a total of 25 anadromous Sakhalin taimen Hucho perryi collected on the coast of Sarufutsu, northern Hokkaido, Japan in June 1997 and 1999. We examined morphology, stomach contents and growth of three anadromous taimen (one male and two females) in detail, which were preserved in good condition. The three taimen were aged and one female still had some eggs retained in her abdomen. The stomach contents of the three taimen consisted of sand lance Ammodytes personatus Girard and sculpin Triglops sp. On the basis of scale analyses, the growth rate of the three taimen was estimated by using the back-calculation method, and the highest rates were observed at young ages. Guanine pigmentation was present at the base of the caudal fin of each taimen and is considered as a potential morphological trait to differentiate anadromous from fluvial specimens. Another anadromous taimen, of which some individuals had reared for more than 3 years in seawater are also reported. For the conservation of this rare and endangered species, their migration route between rivers and sea should be protected     

哲罗鱼稚鱼最佳投喂策略

为探讨哲罗鱼稚鱼的最佳投喂策略,设置了饥饿再投喂试验、饥饿再投喂恢复试验以及日投喂频率试验.结果表明: 饥饿再投喂试验中,各饥饿组未表现出补偿生长现象.但在饥饿再投喂恢复试验中,各饥饿组表现出不同程度的补偿生长,其中S_1/2组(饥饿1/2 d投喂1/2 d)体质量的增加量与对照组接近,表现出完全补偿生长.表明在哲罗鱼早期稚鱼阶段(体质量0～2 g,水温9～15.3 ℃),S_1/2是可以考虑使用的投喂方法.日投喂频率试验中,T₃组(日投喂3次)体长、体质量的增加量以及特定生长率均最高,饵料转化率也相对较高.表明在哲罗鱼后期稚鱼阶段(体质量2～21 g,水温8.8～15.5 ℃),以日投喂3次为宜.     

Analysis of genetic diversity on 9 wild stocks of taimen (Hucho taimen) by microsatellite markers

Taimen (Hucho taimen) is a native fish species in China and it is in the state of endangerment. To explain clearly the genetic diversity and genetic structure, 9 wild populations of taimen were investigated using 20 microsatellite markers. The results showed that their observed heterozygosity ranged from 0.0994 to 0.8882, the expected heterozygosity varied from 0.2005 to 0.8759, and the range of PIC index was from 0.3432 to 0.5261 while population from Huma River had low genetic diversity. Fst of matching group ranged from 0.0246 to 0.2333 (P <0.0001)and Nm varied among 0.8216 to 9.9292, which indicated that the genetic differentiation was remarkable among populations.The half/full-sib family tests detected a proportion of half/full-sib family groups varying among 27.78% to 90.91%, showing a high inbred pressure and a risk of bottlenecks experienced by most groups. The AMOVA results showed that the global Fst was 0.1081; the clustering result showed that individuals from Beiji tributary of Heilongjiang River clustered as one clade, all individuals from Huma River and Wusuli River clustered as one clade and all individuals from the upper reaches of the Heilongjiang River clustered as another clade. All these results indicated that the decrease of taimen resource has affected the gene exchange among their populations. In order to achieve full protection of taimen germplasm resources, we should put an end to the destructive fishing for taimen and promotegene exchange among their populations.     

The complete mitochondrial genome of the taimen, Hucho taimen, and its unusual features in the control region

The whole mitochondrial genome of Hucho taimen was firstly sequenced and characterized. The genome is 16,833 bp in length and contains 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a noncoding control region. Twelve protein-coding genes on the heavy strand showed that the content of A+T was higher than that of G+C, whereas the nd6 protein-coding gene on the light strand displayed an opposite pattern. We described the secondary structure of the origin of light strand (oriL) replication and found that the conserved 5'-GCCGG-3' sequence motif is variable in H. taimen and some other salmonids. We conclude that the control region is variable in length and represents the high A+T content, compared with other mitochondrial control regions available in Salmonidae and other non-salmonids. Additionally, another interesting feature of H. taimen mitogenome is that a T-type mononucleotide microsatellite and an 82 bp tandem repeat were identified in the control region.     

First mtDNA sequencing of Volga and Ob basin taimen Hucho taimen: European populations stem from a late Pleistocene expansion of H. taimen out of western Siberia and are not intermediate to Hucho hucho

New concatenated mtDNA sequences (three genes; n = 22) of Siberian taimen Hucho taimen primarily from west Siberian and European regions of the species' range were added to 12 previously published sequences to provide a phylogeographic overview of the species. European samples show only very minor divergence from west Siberian populations, supporting a late Pleistocene expansion from Siberia into the Urals, with no particular relation to the Danube River basin huchen Hucho hucho as once hypothesized. The disjunct distribution of the genus is most likely based on an early Pleistocene vicariant event.     

Spawning migration and redd construction of Sakhalin taimen, Hucho perryi (Salmonidae) on northern Hokkaido Island, Japan

Spatial and temporal patterns of redd construction by Sakhalin taimen, Hucho perryi , were studied in small streams on northern Hokkaido, Japan. Spawning took place in late April with close relation to discharge patterns. Observed redd size (mean ± 95% C.I.) was 145 ± 11·8 cm in length and 84 ± 5·8 cm in width. The number of egg pockets per redd was either one or two, which is fewer than that of other salmonids reported in the literature. Spawning ground distributions in 1991 and 1992 were similar to each other in terms of the distribution mean and dispersion of the redds. Unlike other salmonids Sakhalin taimen probably constructed multiple redds during one spawning season.