利用Cyt b基因序列探讨柴达木裸裂尻鱼的分类学地位

长期以来,柴达木裸裂尻鱼(Schizopygopsis kessleri)的分类学地位存在争议。为此,测定了柴达木裸裂尻鱼格尔木河及托素湖种群24个个体及黄河裸裂尻鱼(S.pylzovi)黄河及大通北川河种群12个个体的Cytb基因全序列。通过遗传距离和系统发育分析,探讨了柴达木裸裂尻鱼的分类学地位。结果显示,柴达木裸裂尻鱼与黄河裸裂尻鱼在系统进化树上并未形成相应的单系群;柴达木裸裂尻鱼与黄河裸裂尻鱼种间平均遗传距离为0.54%,明显低于裂腹鱼亚科其他属鱼类的种间遗传距离及本属其他种间遗传距离,表明来自柴达木盆地格尔木河和托素湖的裸裂尻鱼与黄河裸裂尻鱼之间没有达到种级水平的显著分化。综合地质学资料和柴达木水系与黄河水系现今的隔离格局,建议将柴达木裸裂尻鱼作为黄河裸裂尻鱼的一个亚种,即S.pylzovi kessleri。     

青藏高原裸裂尻属鱼类两个疑难种的分子系统学

裸裂尻鱼属[Schizopygopsis（Steindachner）]鱼类广泛分布于青藏高原各河流上游干支流及其湖泊之中,由于其广泛的分布域和特殊的生态环境而被生物学家视为青藏高原隆升过程中淡水鱼类物种起源、进化的模式物种。然而,该属鱼类种间具有非常相似的形态特征,致使大渡软刺裸裂尻鱼（Sp．malacanthus chengi）和前腹裸裂尻鱼（Sp．anteroventris）的分类学地位一直存在争议。本文采用聚合酶链式反应（PCR）和直接测序的方法,获得了裸裂尻鱼属两个分类疑难种和5个指名种（n=17）以及5个近缘种（n=5）共23个个体的细胞色素b（Cytb）基因的全序列（1140bp）,并以同亚科的齐口裂腹鱼（Schizothorax cprenanti）作为外群构建了ML、MP和Bayesian进化树。序列差异和分子系统学分析表明,Sp．malacanthus schengi和软刺裸裂尻鱼（Sp．malacanthus）间存在较大的差异（2．06％）,两者并不聚在同一枝上;Sp．anteroventris独立成一枝,与其它指名裸裂尻鱼间显示出较大差异（4．38％-5．53％）,综合其它形态差异共同提示,Sp．mnlacanthus chengi应提升为独立种,沿用Sp．chengi的种名,而Sp．anteroventris应该是裸裂尻鱼属的一个独立种。     

裂腹鱼亚科鱼类Hif-α基因的分子进化及低氧诱导表达

目的 探究低氧诱导因子Hif-α在裂腹鱼亚科鱼类适应低氧环境中的作用及表达调控。方法 利用RT-PCR技术获得了裂腹鱼亚科鱼类13个代表物种的Hif-1αΑ、Hif-1αB、Hif-2αΑ和Hif-2αB基因编码区序列,基于裂腹鱼亚科鱼类系统进化关系开展了基因选择压力分析,并选取花斑裸鲤进行低氧诱导表达研究。结果 Hif-1αΑ、Hif-1αB、Hif-2αΑ和Hif-2αB基因编码区长度分别为2196、2325、2544和2511 bp。通过序列比对发现裂腹鱼亚科鱼类HIF1αΑNODD结构域中保守脯氨酸羟基化基序LxxLAP发生缺失,特化及高度特化的裂腹鱼的HIF1αBCODD结构域中脯氨酸羟基化基序突变为PxxLAP。与常氧组相比,在重度缺氧条件下花斑裸鲤的脑和心脏组织中部分Hif-α基因表达量显著下降,在中度低氧中心脏组织中的Hif-1αA和Hif-2αA基因表达量上调。结论 Hif-α基因在13个物种中受到正向选择作用,但具体物种分支有待进一步研究,同时裂腹鱼亚科鱼类在适应慢性低氧和急性低氧环境可能存在两种不同的表达调控机制。     

黄河裸裂尻鱼群体遗传结构和Cyt b序列变异

测定了来自黄河上游和柴达木盆地托索湖的裸裂尻鱼共16个个体的Cytb基因全序列(1141bp),探讨了种群结构和遗传多样性。用MEGA2.1软件分析了碱基组成和序列变异;以青海湖裸鲤、花斑裸鲤和极边扁咽齿鱼为外类群,用PAUP*4.0b10程序构建了单倍型NJ树;用Arlequin Ver.2000程序计算了群体间遗传变异值(Fst)和Nm值以及群体分化概率值。结果显示,来自柴达木水系托索湖的裸裂尻鱼没有形成单系群,Fst=0.204(P0.05),Nm=1.95。初步判断,黄河和柴达木水系托索湖的裸裂尻鱼未显著分化,支持将柴达木裸裂尻鱼(Schizopygopsis kessleri)归并入黄河裸裂尻鱼(Schizopygopsis pylzovi)的形态学结果。两种群核苷酸多样度()分别为0.0012和0.0026,表现为较低水平。根据校正的分子钟推测,黄河和托索湖裸裂尻鱼群体分歧时间为距今7万年左右的更新世末期,结合地理分布的资料和古地质事件,对黄河裸裂尻鱼群体分布水系间的历史联系进行了分析。       

青海湖裸鲤和黄河裸裂尻鱼感染多子小瓜虫的病理学比较研究

为探讨不同裂腹鱼类感染多子小瓜虫后的病理学差异, 利用多子小瓜虫对青海湖裸鲤指名亚种(Gymnocypris przewalskii przewalskii)和黄河裸裂尻鱼(Schizopygopsis pylzovi Kessler)实施感染实验, 并对2种鱼进行了深入的病理学研究。研究结果显示: (1) 2种鱼的死亡量均呈现先激增后明显回落的趋势, 青海湖裸鲤死亡高峰在感染后第3至第4天, 黄河裸裂尻鱼死亡高峰在第3至第5天, 青海湖裸鲤的死亡比黄河裸裂尻鱼急剧。(2)感染后2种鱼体表均出现大量肉眼可见的白点。青海湖裸鲤皮肤黏液分泌量明显增加, 体表形成胶状黏液层, 黏液层中见不同细胞期小瓜虫包囊。黄河裸裂尻鱼鳍出现蛀鳍现象, 皮肤出现细菌感染样溃烂。(3)解剖发现, 感染组青海湖裸鲤和黄河裸裂尻鱼肝脏发生病理改变呈淡黄色, 胆有不同程度肿大。(4)组织切片和电镜观察显示, 小瓜虫在鳃部位的寄生导致青海湖裸鲤和黄河裸裂尻鱼鳃丝黏连, 鳃小片和鳃丝上皮细胞萎缩、脱落, 鳃丝结构被严重破坏。小瓜虫在2种鱼皮肤的寄生使寄生部位组织突起, 周围组织塌陷。青海湖裸鲤表皮下层出现空隙, 表皮结构被严重破坏。黄河裸裂尻鱼皮肤表皮细胞出现空泡化病理改变, 失去原有紧密结构, 表皮层和固有层间界限变模糊。综上所述, 小瓜虫的感染对青海湖裸鲤和黄河裸裂尻鱼的鳃和皮肤组织造成严重损伤, 但2种鱼表现的症状和造成的组织损伤类型有明显差异, 这与2种鱼长期适应不同水体环境密切相关。     

雅鲁藏布江中游裂腹鱼类的分布及栖息地特征

分别于2015年4月和10月对雅鲁藏布江中游裂腹鱼类的分布与栖息地环境特征进行了调查。结果表明:雅鲁藏布江中游现分布有裂腹鱼类6种,即异齿裂腹鱼(Schizothorax oconnori)、双须叶须鱼(Ptychobarbus dipogon)、拉萨裸裂尻鱼(Schizothorax younghusbandi)、拉萨裂腹鱼(Schizothorax waltoni)、巨须裂腹鱼(Schizothorax macropogon)和尖裸鲤(Oxygymnocypris stewarti);尖裸鲤仅在雅鲁藏布江仁布以上江段出现,异齿裂腹鱼和拉萨裸裂尻鱼在所有调查断面均有分布。RDA分析显示,河宽、流速和pH是影响裂腹鱼类栖息地选择的主要因子。针对当前雅鲁藏布江中游存在的过度捕捞、外来物种入侵和梯级水电开发等问题,建议管理部门采取划定保护区、禁捕、宣传放生土著鱼类等措施来维持裂腹鱼类栖息地的有效性。     

黄河裸裂尻鱼肌酸激酶M-CK cDNA的克隆及组织表达分析

肌酸激酶(CK)能够催化磷酸基在二磷酸腺苷(ADP)和磷酸肌酸之间的可逆性转移,在细胞能量代谢过程中发挥重要作用。本研究利用RT-PCR和RACE方法克隆了黄河裸裂尻鱼(Schizopygopsis pylzovi)肌酸激酶基因全长c DNA序列,并进行了生物信息学分析和系统发育关系研究。黄河裸裂尻鱼肌酸激酶c DNA全长1 599 bp,开放阅读框(ORF)1 143 bp,编码380个氨基酸,具有典型的N-端结构域和C-端催化结构域。氨基酸序列同源性分析表明,黄河裸裂尻鱼肌酸激酶与斑马鱼(Danio rerio)和鲤(Cyprinus carpio)M3-CK有较高的同源性,其序列一致度达94%以上。系统发育分析显示,黄河裸裂尻鱼肌酸激酶与斑马鱼和鲤的M3-CK聚在一支,形成一个单系群,初步判定克隆获得的黄河裸裂尻鱼肌酸激酶基因可能与鲤和斑马鱼M3-CK是直系同源基因。利用Real-time PCR方法检测和分析了黄河裸裂尻鱼主要组织肌酸激酶m RNA表达水平,肌肉、肠、眼、心中肌酸激酶转录本表达较高,在肝胰脏和脑中表达微弱。肌酸激酶在肌肉、肠、和心中高表达与其能量代谢功能相适应,而在眼组织中的高表达是否与肌酸激酶的其他功能相关,有待于进一步研究。     

怒江上游三种裂腹鱼类摄食及消化器官比较研究

于2017年5和6月以及9和10月在怒江西藏段收集了怒江裂腹鱼(Schizothorax nukiangensis)194尾、裸腹叶须鱼(Ptychobarbus kaznakovi)152尾,热裸裂尻鱼(Schizopygopsis thermalis)117尾。综合运用3种多元分析方法分析3种鱼类摄食及消化器官形态的种间差异,结果显示,主要种间差异部位为头部及肠道;种间形态指标均为显著性差异(P <0.05);3种裂腹鱼的头部形态已有了一定程度的分化。鱼类食物组成及食物竞争情况研究表明,怒江裂腹鱼和裸腹叶须鱼属杂食性偏动物食性鱼类,两者食物重叠指数较高(0.91);热裸裂尻鱼属杂食性偏植物食性鱼类。食物多样性指数的种间差异明显。3种裂腹鱼营养及空间生态位均有分化,摄食与水温、流速和海拔等环境因素密切相关。     

拉萨河鱼类调查及保护

拉萨河是雅鲁藏布江最大的支流, 其鱼类区系为隶属于裂腹鱼亚科、条鳅亚科和科的13 种鱼类以及裂腹鱼属的一个自然杂交种。最近十几年, 由于人类活动的影响, 导致了大量的外来鱼类进入拉萨河水体。文章介绍了2004 年至2006 年对拉萨河鱼类进行采样调查的情况。目前在拉萨河, 鱼类组成包括以往记录的14 种鱼类: 拉萨裂腹鱼Schizothorax waltoni (Regan)、异齿裂鱼 S. o’connori (Lloyd)、巨须裂腹鱼S.macropogon (Regan)、双须叶须鱼 Ptychobarbus dipogon (Regan)、尖裸鲤 Oxygymnocypris stewartii (Lloyd)、拉萨裸裂尻鱼Schizopygopsisyounghusbandi younghusbandi Regan、异齿裂腹鱼和拉萨裂腹鱼的一个自然杂交种、异尾高原鳅Triplophysa stewartii (Hora)、细尾高原鳅T. stenura (Herzenstein)、西藏高原鳅T. tibetana(Regan)、东方高原鳅T. orientalis (Herzenstein)、斯氏高原鳅T. stoliczkae (Steindachner)、短尾高原鳅T.brevicauda (Herzenstein)、黑斑原Glyptosternum maculatum (Regan); 8 种外来鱼类: 鲫Carassius auratus(Linnaeus)、麦穗鱼Pseudorasbora parva (Temminck et Schlegel)、泥鳅Misgurnus anguillicaudatus (Cantor)、黄黝鱼Hypseleotris swinhonis (Gunther)、鲤Cyprinus (C.) caupio Linnaeus、草鱼Ctenopharyngodon idellus(Cuvier et Valenciennes)、银鲫Carassius auratus gibelio (Bloch)、鲇Silurus asotus Linnaeus; 2 个未定种。调查结果表明拉萨河鱼类资源面临的两个主要问题是过度捕捞导致鱼类资源的减少和小型化以及外来鱼类入侵。文章针对这些情况作了分析和探讨, 并对土著鱼类的保护与合理利用提出了一些建议: 合理捕捞、严格禁渔期、控制外来鱼类、保护栖息地等。       

青海湖裸鲤类胰岛素生长因子IGF-Ⅱ的原核表达

青海湖裸鲤(Gymnocypris przewalskii)俗称湟鱼,属鲤科裂腹鱼亚科裸鲤属,是我国最大内陆咸水湖青海湖中唯一的经济鱼类,其生长极其缓慢,平均每生长到500g需要近10年[1]。青海湖裸鲤具有明显的生殖洄游,每年     

Mitochondrial cytochrome b Sequence Variation and Phylogenetics of the Highly Specialized Schizothoracine Fishes (Teleostei: Cyprinidae) in the Qinghai-Tibet Plateau

The complete 1140 bp mitochondrial cytochrome b sequences were obtained from 39 individuals representing five species of all four genera of highly specialized schizothoracine fishes distributed in the Qinghai-Tibet plateau. Sequence variation of the cytochrome b gene was surveyed among the 39 individuals as well as three primitive schizothoracines and one outgroup. Phylogenetic analysis suggested that the group assignment based on 1140 bp of the cytochrome b sequence is obviously different from previous assignments, and the highly specialized schizothoracine fishes (Schizopygopsis pylzovi, Gymnocypris przewalskii, G. eckloni, Chuanchia labiosa, and Platypharodon extremus) form a monophyletic group that is sister to the clade formed by the primitive schizothoracine fishes (Schizothorax prenanti, S. pseudaksaiensis, and S. argentatus). The haplotypes of Schizopygopsis pylzovi and G. przewalskii were paraphyletic based on cytochrome b data, which most likely reflected incomplete sorting of mitochondrial DNA lineages. The diploid chromosome numbers of Schizothoracinae were considered in phylogenetic analysis and provided a clear pattern of relationships. Molecular dating estimated for highly specialized schizothoracine fishes suggested that the highly specialized schizothoracine fishes diverged in the late Miocene Pliocene to Pleistocene (4.5 × 10⁴–4.05 × 10⁶ years BP). The relationship between the cladogenesis of highly specialized schizothoracine fishes and geographical events of the Qinghai-Tibet plateau is discussed.     

Molecular phylogeny of the subfamily Schizothoracinae (Teleostei: Cypriniformes: Cyprinidae) inferred from complete mitochondrial genomes

The schizothoracine fishes, members of the Teleost order Cypriniformes, are one of the most diverse group of cyprinids in the Qinghai–Tibetan Plateau and surrounding regions. However, taxonomy and phylogeny of these species remain unclear. In this study, we determined the complete mitochondrial genome of Schizopygopsis malacanthus. We also used the newly obtained sequence, together with 31 published schizothoracine mitochondrial genomes that represent eight schizothoracine genera and six outgroup taxa to reconstruct the phylogenetic relationships of the subfamily Schizothoracinae by different partitioned maximum likelihood and partitioned Bayesian inference at nucleotide and amino acid levels. The schizothoracine fishes sampled form a strongly supported monophyletic group that is the sister taxon to Barbus barbus. A sister group relationship between the primitive schizothoracine group and the specialized schizothoracine group + the highly specialized schizothoracine group was supported. Moreover, members of the specialized schizothoracine group and the genera Schizothorax, Schizopygopsis, and Gymnocypris were found to be paraphyletic.     

The youngest split in sympatric schizothoracine fish (Cyprinidae) is shaped by ecological adaptations in a Tibetan Plateau glacier lake

Although new empirical evidence shows that sympatric speciation has occurred in some species, there are few indisputable model organisms for this process of speciation. The two subspecies ( Gymnocypris eckloni eckloni and G . e . scoliostomus ) of the schizothoracine Gymnocypris fish species complex from a small glacier lake in the Tibetan Plateau, Lake Sunmcuo, fit several of the key characteristics of the sympatric speciation model. We used combined mitochondrial control region sequences and the cytochrome b gene (1894 bp) to address the phylogenetics and population genetics of 232 specimens of G . e . eckloni and G . e . scoliostomus , as well as all of its closely related sister species. We found that: (i) a total of four old lineages were uncovered in the widespread G . e . eckloni , of which only one was shown to be shared with all G . e . scoliostomus individuals and (ii) the new subspecies ( G . e . scoliostomus ) evolved in Lake Sunmcuo from the ancestral G . e . eckloni population within approximately 0.057 Ma. These two taxa of the species complex are morphologically distinct, and reproductive isolation is further suggested. Ecological disruptive selection based on morphological traits (e.g. mouth cleft characters) and food utilization may be a mechanism of incipient speciation of two sympatric populations within Lake Sunmcuo. This study provides the first genetic evidence for sympatric speciation in the schizothoracine fish.     

几何形态测量法结合Micro CT扫描对两水系花斑裸鲤的形态分析

研究采用现代几何形态测量法并结合Micro CT扫描技术对两水系(黄河上游与柴达木水系格尔木河)花斑裸鲤(Gymnocypris eckloni Herzenstein)的形态进行定量分析, 讨论两水系花斑裸鲤是否存在先前研究未能发现的形态差异。结果显示: 花斑裸鲤的两个地理种群在基于其头部形态轮廓二维坐标的主成分分析和判别分析的结果均聚在一起, 这与之前文献记载的两水系花斑裸鲤的形态学特征相一致, 并且对两水系花斑裸鲤进行的典型变量分析也同样支持这一结果; 对花斑裸鲤两个地理种群背鳍支鳍骨插入椎骨间的相对位置这一形态特征进行卡方检验, 结果表明在骨骼这一更具分类学意义的形态特征上表现出显著性差异。通过对花斑裸鲤两个地理种群的几何形态与Micro CT扫描, 准确定量地分析了两水系花斑裸鲤形态上的异同, 为正确评价两水系花斑裸鲤真实的演化关系奠定了形态学基础。     

黄河裸裂尻鱼五种群mtDNA控制区的遗传结构

黄河裸裂尻鱼（Schizopygopsis pylzovi）主要分布于青藏高原东北部兰州以上黄河干支流水系和高原北部柴达木内流水系,由于其对高原特殊生境表现出独特的适应性而受研究者关注。本文采用聚合酶链式反应（PCR）和直接测序的方法获得了一个分布于柴达木内流水系种群和四个黄河支流种群共99条个体的线粒体DNA（mtDNA）控制区（D-100p）821bp核苷酸的序列,探讨了其种群遗传结构和分化。99条个体的序列经比对后,发现77个（9．37％）多态性位点,共定义了53个单倍型,其中有一个单倍型（DT12）为两个种群（大通种群和互助种群）所共享,其余52个单倍型均为某个种群所特有。遗传多样性分析表明,柴达木种群的单倍型多样度（h=0．79&#177;0．06）和核苷酸多样度（π=0．0027&#177;0．0017）均略低于黄河支流各种群。AMOVA分析结果显示,遗传差异主要发生在种群之内,占75．39％,而小同水系的种群间只有24．61％,各种群间成对的Fst及遗传距离均表明种群间出现了一定程度的种群分化,但系统进化树最示出一种混杂的单倍型分布格局,提示青藏高原东北部地质事件所造成的水系间的隔离形成较晚。单倍型歧点分布呈现为单峰以及中性检验Tajima的D（-1．497,P=0．058）和Fu的Fs（-24．741,P=0．001）结果综合表明,黄河裸裂尻鱼在青藏高原隆升过程中曾经历过近期的种群扩张事件[动物学报54（6）：972—980,2008]。     

Mitochondrial DNA phylogeny of two morphologically enigmatic fishes in the subfamily Schizothoracinae (Teleostei: Cyprinidae) in the Qinghai-Tibetan Plateau

The complete mitochondrial DNA (mtDNA) cytochrome b gene (1140 bp) was sequenced in Herzenstein macrocephalus and Gymnocypris namensis and in 13 other species and sub-species ( n = 22), representing four closely related genera in the subfamily Schizothoracinae. Conflicting taxonomies of H. macrocephalus and G. namensis have been proposed because of the character instability among individuals. Parsimony, maximum likelihood and Bayesian methods produced phylogenetic trees with the same topology and resolved several distinctive clades. Previous taxonomic treatments, which variously placed these two species of separate genera or as sub-species, are inconsistent with the mtDNA phylogeny. Both H. macrocephalus and G. namensis appear in a well-supported clade, which also includes nine species of Schizopygopsis , and hence should be transferred to the genus Schizopygopsis . Morphological changes are further illustrated, and their adaptive evolution in response to the local habitat shifts during the speciation process appears to be responsible for conflicting views on the systematics of these two species and hence the contrasting taxonomic treatments. These species are endemic to the Qinghai-Tibetan Plateau, a region with a history of geological activity and a rich diversity of habitats that may have result in the parallel and reversal evolution of some morphological characters used in their taxonomies. Our results further suggest that speciation and morphological evolution of fishes in this region may be more complex than those previously expected.     

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.     

Length–weight relationships of three endemic fish species from the upper Yellow River,China

Length–weight relationships (LWRs) of three Chinese endemic fish species (Gymnocypris eckloni eckloni Herzenstein, 1891, Platypharodon extremus Herzenstein, 1891 and Triplophysa pseudoscleroptera (Zhu & Wu, 1981)) were analyzed, collecting from the upper Yellow River (Xunhua and Guide Counties, Qinghai Province). Totally 347 specimens monthly collected by gill nets with 40 mm mesh size and traps with 5 m length from August to October 2014, were used to estimate the relationship parameters. The allometric factor b values ranged from 2.96 in T. pseudoscleroptera to 3.33 in G. eckloni eckloni. The condition factor a values varies between 0.0063 and 0.0175 in three species. Three WLRs of fishes distributed in the upper Yellow River were firstly reported and will fill in some of the empty spaces on endemic species in the Fishbase.