几种鲟鱼基因组大小、倍体的特性及鲟形目细胞进化的探讨

采用Ｆｅｕｌｇｅｎ－显微分光光度计方法,以鸡红细胞为标准ＤＮＡ（３．２２ｐｇ／２Ｃ）测定了长江白鲟、达氏鲟、中华鲟、史氏鲟和北美匙吻鲟的体细胞基因组大小（ＤＮＡ含量）。结果表明,上述五种鲟鱼ＤＮＡ含量分别为４．１１、８．２６、９．０７、６．０７和３．９６ｐｇ。长江白鲟和北美匙吻鲟均属于四倍体鱼类,分布在长江水系的中华鲟和达氏鲟两种鲟属鱼类为八倍体类型。史氏鲟初步判断为八倍体,据分析可能存在四倍体的     

Functional morphology of prey capture in the sturgeon,Scaphirhynchus albus

Acipenseriformes (sturgeon and paddlefish) are basal actinopterygians with a highly derived cranial morphology that is characterized by an anatomical independence of the jaws from the neurocranium. We examined the morphological and kinematic basis of prey capture in the Acipenseriform fish Scaphirhynchus albus, the pallid sturgeon. Feeding pallid sturgeon were filmed in lateral and ventral views and movement of cranial elements was measured from video sequences. Sturgeon feed by creating an anterior to posterior wave of cranial expansion resulting in prey movement through the mouth. The kinematics of S. albus resemble those of other aquatic vertebrates: maximum hyoid depression follows maximum gape by an average of 15 ms and maximum opercular abduction follows maximum hyoid depression by an average of 57 ms. Neurocranial rotation was not a part of prey capture kinematics in S. albus, but was observed in another sturgeon species, Acipenser medirostris. Acipenseriformes have a novel jaw protrusion mechanism, which converts rostral rotation of the hyomandibula into ventral protrusion of the jaw joint. The relationship between jaw protrusion and jaw opening in sturgeon typically resembles that of elasmobranchs, with peak upper jaw protrusion occurring after peak gape.     

Unusual intraindividual variation of the nuclear 18S rRNA gene is widespread within the Acipenseridae

Significant intraindividual variation in the sequence of the 18S rRNA gene is unusual in animal genomes. In a previous study, multiple 18S rRNA gene sequences were observed within individuals of eight species of sturgeon from North America but not in the North American paddlefish, Polyodon spathula, in two species of Polypterus (Polypterus delhezi and Polypterus senegalus), in other primitive fishes (Erpetoichthys calabaricus, Lepisosteus osseus, Amia calva) or in a lungfish (Protopterus sp.). These observations led to the hypothesis that this unusual genetic characteristic arose within the Acipenseriformes after the presumed divergence of the sturgeon and paddlefish families. In the present study, a survey of nearly all Eurasian acipenseriform species was conducted to examine 18S rDNA variation. Intraindividual variation was not found in the polyodontid species, the Chinese paddlefish, Psephurus gladius, but variation was detected in all Eurasian acipenserid species. The comparison of sequences from two major segments of the 18S rRNA gene and identification of sites where insertion/deletion events have occurred are placed in the context of evolutionary relationships within the Acipenseriformes and the evolution of rDNA variation in this group.     

Evolution of MHC class I genes in two ancient fish, paddlefish (Polyodon spathula) and Chinese sturgeon (Acipenser sinensis)

Here we present the first isolation of major histocompatibility complex (MHC) class I genes from two ancient fish, paddlefish (Polyodon spathula) and Chinese sturgeon (Acipenser sinensis). Seventeen sequences obtained showed high polymorphism and positive natural selection with d_N/d_S > 1. Evolutionary relationships revealed that sequences from paddlefish and Chinese sturgeon distinguished from other vertebrate class I and had an intermingling of alleles, which indicates that Acipenseriformes have a common ancestral gene of class I and a trans-species polymorphism across Acipenseriformes. We also found clear evidence of recombination among class I genes of paddlefish and Chinese sturgeon.     

基于线粒体基因组全序列的鲟形目鱼类(Pisces: Acipenseriformes)的分子系统发育重建

为厘清鲟形目鱼类的系统发育, 研究新测定了中华鲟(Acipenser sinensis)、长江鲟(A. dabryanus)、短吻鲟(A. brevirostrum)、纳氏鲟(A. naccarii)、鳇(Huso dauricus)和匙吻鲟(Polyodon spathula)共6种鲟类的线粒体全基因组序列。联合已测的17种鲟类的线粒体基因组数据, 利用最大似然法和贝叶斯法重建了鲟形目鱼类的分子系统发育关系, 并采用似然值检验对不同的树拓扑结构进行了评价。结果表明, 6种新测鲟类的线粒体基因组大小为16521—16766 bp, 编码13个蛋白质编码基因、22个转运RNA基因和2个核糖体基因, 与大多数已测的鲟类的线粒体基因组结构高度相似。基于23种鲟形目鱼类线粒体基因组数据, 系统发育分析的结果表明: (1)鲟形目的两个科, 匙吻鲟科(Polyodontidae)和鲟科(Acipenseridae)均为单系; (2)鲟科的内部亲缘关系复杂, 鲟属和鳇属的物种均不构成单系群。鲟科鱼类按分子系统发育重建结果可以分为3个类群: 尖吻鲟类(A. sturio - A. oxyrinchus clade)、大西洋鲟类(Atlantic clade)和太平洋鲟类(Pacific clade)。树拓扑结构的检验结果表明, 鲟科的系统发育关系为(尖吻鲟类(太平洋鲟类, 大西洋鲟类))。铲鲟属(Scaphirhynchus)是大西洋鲟类的基部类群。研究也说明线粒体基因组数据在鲟形目鱼类系统与进化研究方面具有重要应用价值。     

Phylogeny of the Acipenseriformes: cytogenetic and molecular approaches

The review of the data on karyology and DNA content in Acipenseriformes shows that both extant families, the Polyodontidae and Acipenseridae, originated from a tetraploid ancestor which probably had a karyotype consisting of 120 macro- and microchromosomes and DNA content of about 3.2–3.8 pg per nucleus. The tetraploidization of the presumed 60-chromosome ancestor seems to have occurred at an early time of evolution of the group. The divergence of the Acipenseridae into Scaphirhyninae and Acipenserinae occurred without polyploidization. Within the genus Acipenser, polyploidization was one of the main genetic mechanisms of speciation by which 8n and 16n-ploid species were formed. Individual gene trees constructed for sequenced partial fragments of the 18S rRNA (230 base pairs, bp), 12S rRNA (185 bp), 16S rRNA (316 bp), and cytochrome b (270 bp) genes of two Eurasian (A. baerii and A. ruthenus) and two American (A. transmontanus and A. medirostris) species of Acipenser, Huso dauricus, Pseudoscaphirhynchus kaufmanni, Scaphirhynchus albus, and Polyodon spathula showed a low level of resolution; the analysis of a combined set of data for the four genes, however, gave better resolution. Our phylogeny based on molecular analysis had two major departures from existing morphological hypotheses: Huso dauricus is a sister-species to Acipenser instead of being basal to all acipenseriforms, and Scaphirhynchus and Pseudoscaphirhynchus do not form a monophyletic group. The phylogenetic tree constructed for the cytochrome b gene fragments (with inclusion of 7 additional species of Acipenser) supported the conclusion that octoploid species appeared at least three times within Acipenser.     

中华鲟(Acipenser sinensis)及相关种类的mtDNA控制区串联重复序列及其进化意义

采用 PCR技术和 DNA测序技术 ,发现了我国一级珍稀保护动物中华鲟 ( Acipensersinensis)线粒体 DNA( mt DNA)的控制区 ( D- loop)存在数目不等的串联重复序列 ,该重复序列造成了中华鲟广泛的异质性现象 .从分子水平进行了不同类型重复序列变化规律的研究 ,同时还初探了重复序列在我国其它几种鲟鱼类的存在情况 ,发现在白鲟 ( Psephurus gladius)、达氏鲟 ( A.dabryanus)和史氏鲟 ( A.schrenckii)均存在类似的重复序列结构 .序列比较分析表明 ,不同鲟鱼类重复序列在鲟鱼类进化过程中扮演着一定的角色 ,很有可能碱基差异大小与它们的亲缘关系的远近呈正相关 .     

Viability and fertilizing capacity of cryopreserved sperm from three North American acipenseriform species: a retrospective study

Populations of sturgeon across the globe are threatened due to unregulated harvest and habitat loss, and the status varies among species across North America. Ready access to viable and functional sperm would contribute to recovery programmes for these species. In this study, we examined the motility, viability (cell membrane integrity) of cryopreserved sperm from three North American acipenseriform species and fertilizing capacity. Milt samples were collected from captive shortnose sturgeon (Acipenser brevirostrum), wild paddlefish (Polyodon spathula) and pallid sturgeon (Scaphirhynchus albus) and cryopreserved using combinations of Modified Tsvetkova’s (MT) extender, Original Tsvetkova’s extender, and modified Hanks’ balanced salt solution, along with the cryoprotectants methanol (MeOH) or dimethyl sulfoxide (DMSO). A dual‐staining technique using the fluorescent stains SYBR‐14 and propidium iodide was employed with flow cytometry to determine the percentages of spermatozoa that were viable by virtue of having intact membranes. The percentage of viable spermatozoa ranged from 5% to 12% in shortnose sturgeon, 30–59% in paddlefish, and 44–58% in pallid sturgeon. In the first experiment with shortnose sturgeon sperm, methanol allowed for higher values for dependent variables than did DMSO, and sperm viability generally correlated with post‐thaw motility. However, fertilization rate, neurulation, or hatching rates were independent from these factors. In the second experiment with shortnose sturgeon, 5% MeOH combined with MT yielded higher values for all parameters tested than the other combinations: viability was correlated with motility, fertilization rate, and hatching rate. Overall, viability and post‐thaw motility was not affected by the use of hyperosmotic extenders (OT) or cryoprotectants (DMSO), but their use decreased fertilization percentages. For paddlefish sperm (experiment 3), MT combined with 10% MeOH was clearly a good choice for cryopreservation; viability and motility results were correlated, but independent of fertilization. For pallid sturgeon sperm (experiment 4), MT with 5–10% MeOH showed significantly higher sperm quality and fertilization parameters. Membrane integrity can be used as a predictor of fertilization by cryopreserved sperm, however additional sperm quality parameters, supplementary to motility and membrane integrity, would be useful in the refining and optimizing cryopreservation protocols with acipenseriform sperm.     

Length Variation, Heteroplasmy and Sequence Divergence in the Mitochondrial DNA of Four Species of Sturgeon (Acipenser)

The extent of mtDNA length variation and heteroplasmy as well as DNA sequences of the control region and two tRNA genes were determined for four North American sturgeon species: Acipenser transmontanus, A. medirostris, A. fulvescens and A. oxyrhnychus. Across the Continental Divide, a division in the occurrence of length variation and heteroplasmy was observed that was concordant with species biogeography as well as with phylogenies inferred from restriction fragment length polymorphisms (RFLP) of whole mtDNA and pairwise comparisons of unique sequences of the control region. In all species, mtDNA length variation was due to repeated arrays of 78-82-bp sequences each containing a D-loop strand synthesis termination associated sequence (TAS). Individual repeats showed greater sequence conservation within individuals and species rather than between species, which is suggestive of concerted evolution. Differences in the frequencies of multiple copy genomes and heteroplasmy among the four species may be ascribed to differences in the rates of recurrent mutation. A mechanism that may offset the high rate of mutation for increased copy number is suggested on the basis that an increase in the number of functional TAS motifs might reduce the frequency of successfully initiated H-strand replications.     

Chromosomal mapping of 18S-28S and 5S rRNA genes by two-colour fluorescent in situ hybridization in six sturgeon species.

The number and distribution of the 18S-28S and 5S rRNA (rDNA) gene sequences were examined on mitotic chromosomes of six sturgeon species by two-colour in situ hybridization. Four of the six species, Huso huso, Acipenser stellatus, Acipenser sturio, and Acipenser ruthenus, with about 120 chromosomes, showed from six to eight 18S-28S rDNA signals, while 5S rDNA signals were on only one chromosome pair. The two species with 250-270 chromosomes, Acipenser baerii and Acipenser transmontanus, showed from 10 to 12 18S-28S sites and two chromosome pairs bearing 5S rDNA signals. In all examined species, the rather intense 5S rDNA signals apparently overlapped those of 18S-28S rDNA. These data support the diploid-tetraploid relationships between the two chromosome groups of sturgeons. The close association between the two rDNA families in species belonging to an ancestral fish order, such as Acipenseriformes, supports the hypothesis that the association represents a primitive condition.     

Pectoral fin and girdle development in the basal actinopterygians Polyodon spathula and Acipenser transmontanus

The pectoral fins of Acipenseriformes possess endoskeletons with elements homologous to both the fin radials of teleosts and the limb bones of tetrapods. Here we present a study of pectoral fin development in the North American paddlefish, Polyodon spathula, and the white sturgeon, Acipenser transmontanus, which reveals that aspects of both teleost and tetrapod endoskeletal patterning mechanisms are present in Acipenseriformes. Those elements considered homologous to teleost radials, the propterygium and the mesopterygial radials, form via subdivision of an initially chondrogenic plate of mesenchymal cells called the endoskeletal disc. In Acipenseriformes, elements homologous to the sarcopterygian metapterygium develop separately from the endoskeletal disc as an outgrowth of the endoskeletal shoulder girdle that extends into the posterior margin of the finbud. As in tetrapods, the elongating metapterygium and the metapterygial radials form in a proximal to distal order as discrete condensations from initially nonchondrogenic mesenchyme. Patterns of variation seen in the Acipenseriform fin also correlate with putative homology: all variants from the "normal" fin bauplan involved the metapterygium and the metapterygial radials alone. The primary factor distinguishing Polyodon and Acipenser fin development from each other is the composition of the endoskeletal extracellular matrix. Proteoglycans (visualized with Alcian Blue) and Type II collagen (visualized by immunohistochemistry) are secreted in different places within the mesenchymal anlage of the fin elements and girdle and at different developmental times. Acipenseriform pectoral fins differ from the fins of teleosts in the relative contribution of the endoskeleton and dermal rays. The fins of Polyodon and Acipenser possess elaborate endoskeletons overlapped along their distal margins by dermal lepidotrichia. In contrast, teleost fins generally possess relatively small endoskeletal radials that articulate with the dermal fin skeleton terminally, with little or no proximodistal overlap.     

Effects of body mass and water temperature on routine metabolism of American paddlefish Polyodon spathula

This study quantified the effects of temperature and fish mass on routine metabolism of the American paddlefish Polyodon spathula. Thermal sensitivity, as measured by Q₁₀ value, was low in P. spathula. Mean Q₁₀ was 1·78 while poikilotherms are generally expected to have Q₁₀ values in the 2·00–2·50 range. Mass‐specific metabolism did not decrease with increased fish size to the extent that this phenomenon is observed in teleosts, as evidenced by a mass exponent (β) value of 0·92 for P. spathula compared with 0·79 in a review of teleost species. Other Acipenseriformes have exhibited relatively high β values for mass‐specific respiration. Overall P. spathula metabolism appears to be more dependent on body mass and less dependent on temperature than for many other fishes. An equation utilizing temperature and fish mass to estimate gross respiration for P. spathula was derived and this equation was applied to respiratory data from other Acipenseriformes to assess inter‐species variation. Polyodon spathula respiration rates across water temperature and fish mass appear most similar to those of Atlantic sturgeon Acipenser naccarii and white sturgeon Acipenser transmontanus.     

Sensitivity of North American sturgeons and paddlefish to fishing mortality

Sturgeons and paddlefish exhibit unusual combinations of morphology, habits, and life history characteristics, which make them highly vulnerable to impacts from human activities, particularly fisheries. Five North American sturgeons (shortnose, Gulf, pallid, Alabama, and green sturgeon) are listed as endangered or threatened by management authorities. Managers have instituted fishery closures for the three other species of North American sturgeons (Atlantic, white, and shovelnose) and paddlefish because of low stock abundance at some point in this century. Reproductive potential in four species I examined (Atlantic, white, and shortnose sturgeon, and paddlefish) is more sensitive to fishing mortality than it is for three other intensively-fished coastal species in North America: striped bass, winter flounder, and bluefish. The sturgeons and paddlefish are generally longer-lived than the three other coastal species, and also have an older age at full maturity, lower maximum fecundity values, and older ages at which 50% of the lifetime egg production is realized with no fishing mortality.     

Immunoglobulin heavy chain constant region of five Acipenseridae: cDNA sequence and evolutionary relationship

The immunoglobulin M (IgM) heavy chain constant region genes of Russian sturgeon (Acipenser gueldenstaedtii), Sterlte sturgeon (Acipenser ruthenus), Amur sturgeon (Acipenser schrenckii), Chinese sturgeon (Acipenser sinensis) and Great sturgeon (Huso huso) were cloned and analyzed with molecular biology and bioinformatics methods. We cloned IGH nucleic acid sequences by RT-PCR using the specific primer, then determined the characteristics and functions of the amino acid sequences. The gene contains four constant region domain-encoding exons (CH1-4), of which CH4 sub-regions were the most conserved in IgM heavy chain constant region domain and had the highest identity within all the experimental species. According to the analysis of the phylogenetic tree, the variation expectation value (K(aa)), and species differentiation time (T) in the CH4 sub-region, we found that Chinese sturgeon and the other five sturgeon form one whole bifurcation of the tree, while, among the five left, Amur sturgeon and Huso sturgeon, Russian sturgeon and Siberian sturgeon (data from GenBank), Sterlte itself forms three other bifurcations. This result can clearly explain the relations of taxonomic status, geographical distribution and evolution among the species studied.     

从线粒体DNA控制区核苷酸序列论达式鲟、亚洲和北美洲中吻鲟的分类地位

尽管古老的鲟形目鱼类的分类及系统演化一直是中外学者感兴趣的研究课题,但迄今仍有诸多谜团未解。其中,关于亚洲远东地区和北美地区的中吻鲟(Acipenser medirostris)的分类地位争论已久。长江水系的达式鲟(A.dabryanus)和中华鲟(A.sinensis)及其它鲟属(Acipenser)鱼类之间的亲缘关系近年来也有异议。为了给上述争议提供更多的科学依据,作者测定了线粒体DNA(mtDNA)控制区 (D-loop) 的核苷酸序列,并进行了分子系统学和遗传差异分析。研究结果表明：⑴UPGMA,NJ和MP分子系统学分析方法以及遗传差异分析都支持了亚洲远东地区中吻鲟和北美中吻鲟为一个有效种的观点;⑵同样研究方法并结合相关群体遗传资料表明,长江达式鲟与中华鲟关系最近,提出了达式鲟为中华鲟的一陆封类群的假说。最后,作者认为本文提出的观点和假说还需要更多的研究来证实。     

从线粒体DNA控制区核苷酸序列论达式鲟、亚洲和北美洲中吻鲟的分类地位

尽管古老的鲟形目鱼类的分类及系统演化一直是中外学者感兴趣的研究课题 ,但迄今仍有诸多谜团未解。其中 ,关于亚洲远东地区和北美地区的中吻鲟 (Acipensermedirostris)的分类地位争论已久。长江水系的达式鲟 (A .dabryanus)和中华鲟 (A .sinensis)及其它鲟属 (Acipenser)鱼类之间的亲缘关系近年来也有异议。为了给上述争议提供更多的科学依据 ,作者测定了线粒体DNA (mtDNA)控制区(D loop) 的核苷酸序列 ,并进行了分子系统学和遗传差异分析。研究结果表明 :⑴UPGMA ,NJ和MP分子系统学分析方法以及遗传差异分析都支持了亚洲远东地区中吻鲟和北美中吻鲟为一个有效种的观点 ;⑵同样研究方法并结合相关群体遗传资料表明 ,长江达式鲟与中华鲟关系最近 ,提出了达式鲟为中华鲟的一陆封类群的假说。最后 ,作者认为本文提出的观点和假说还需要更多的研究来证实。     

Mitochondrial genome of the Siberian sturgeon Acipenser baerii

The complete mitochondrial genome of Siberian sturgeon Acipenser baerii had been determined. The total length of the mitogenome was 16,763?bp and had 13 protein-coding, 22 tRNA, and 2 rRNA genes. Except for the eight tRNA and Nd6 genes, all other mitochondrial genes are encoded on the heavy strand. The overall base composition of the heavy strand is 30.3% A, 24.3% T, 29.1% C, 16.3% G, with an AT content of 54.6%. The DNA sequence of A. baerii shared 97.8, 97.6, 93.5, 91.6, 93.6, and 87.1% sequence homology with that of Acipenser sinensis, Acipenser gueldenstaedtii, Acipenser transmontanus, Acipenser dabryanus, Acipenser stellatus, and Polyodon spathula. Molecular data presented here provide a useful tool for evolutionary as well as population genetics studied.     

The photoreceptors and visual pigments of two species of Acipenseriformes, the shovelnose sturgeon (Scaphirhynchus platorynchus ) and the paddlefish (Polyodon spathula )

Scanning electron microscopy, microspectrophotometry, and spectrophotometry of digitonin extracts were employed to characterize the photoreceptors and visual pigments of two freshwater Acipenseriformes. The retinas of the shovelnose sturgeon, Scaphirhynchus platorynchus (Acipenseridae), and the paddlefish, Polyodon spathula (Polyodontidae) are dominated by large rods with long, broad outer segments. A second rod, rare and much narrower than the dominant rod, is present in Scaphirhynchus but not seen in Polyodon. The absorbance maximum of the visual pigment in the rods of Polyodon is near 540 nm; that of Scaphirhynchus near 534 nm. The retinas of both species contain substantial numbers of large, single cones, about 33% of the photoreceptors in Scaphirhynchus; 37% in Polyodon. Scaphirhynchus cone pigments have absorbance maxima near 610 nm, 521 nm and 470 nm, respectively. Polyodon cone pigments absorb maximally near 607 nm and 535 nm, respectively. All visual pigments are based on vitamin A₂. The data are compared to those from other Acipenseriformes and are discussed in terms of lifestyle and behavior. Accepted: 7 October 1998