High‐throughput SNP‐genotyping analysis of the relationships among Ponto‐Caspian sturgeon species

Legally certified sturgeon fisheries require population protection and conservation methods, including DNA tests to identify the source of valuable sturgeon roe. However, the available genetic data are insufficient to distinguish between different sturgeon populations, and are even unable to distinguish between some species. We performed high‐throughput single‐nucleotide polymorphism (SNP)‐genotyping analysis on different populations of Russian (Acipenser gueldenstaedtii), Persian (A. persicus), and Siberian (A. baerii) sturgeon species from the Caspian Sea region (Volga and Ural Rivers), the Azov Sea, and two Siberian rivers. We found that Russian sturgeons from the Volga and Ural Rivers were essentially indistinguishable, but they differed from Russian sturgeons in the Azov Sea, and from Persian and Siberian sturgeons. We identified eight SNPs that were sufficient to distinguish these sturgeon populations with 80% confidence, and allowed the development of markers to distinguish sturgeon species. Finally, on the basis of our SNP data, we propose that the A. baerii‐like mitochondrial DNA found in some Russian sturgeons from the Caspian Sea arose via an introgression event during the Pleistocene glaciation.     

Organization of diencephalon of the sturgeons. Preoptic area

Cytoarchitectonics of preoptic area of diencephalon of chondroid ganoid fish (the sturgeons) was studied in serial sections using Nissl and Bilschowski techniques, the latter in Viktorov’s modification). The preoptic area of the hausen, Huso huso L., Kura sturgeon, Acipenser guldenstaedtii persicus n. kurensis Belyaeff, Caspian sturgeon, Acipenser stellatus Pall. and the sturgeon Acipenser nudioventris Lov. was shown to be one of the largest parts of diencephalon. It is located along the rostral excess of the third ventricle and the anterior third of the ventricle thalamic part. Eight nuclei are identified in the preoptic area: (1) anterior microcellular preoptic nucleus, (2) suprachiasmatic nucleus, (3) accessory preoptic nucleus, (4) intrachiasmatic nucleus, (5) magnocellular preoptic nucleus, (6) posterior microcellular preoptic nucleus, (7) caudal preoptic nucleus, (8) entopeduncular nucleus. Besides, microcellular, magnocellular, and gigantocellular parts are identified in the magnocellular preoptic nucleus. The obtained results indicate high differentiation of this area of the sturgeons among Actinopterygii.     

Organization of diencephalon of the sturgeons. Posterior tubercular area. Caudal region

Cytoarchitectonics of caudal region of the posterior tubercular area of diencephalon was studied in four species of the true sturgeons: the giant sturgeon, Huso huso L., Kura sturgeon, Acipenser güldenstädtii persicus n. Kurensis Belyaeff, stellate sturgeon, Ac. stellatus Pall. and the barbel sturgeon Ac. nudiventris Lov. Morphologic organization of the brain region was studied by using staining techniques of Nissl and Bielschowski (Viktorov’s modification). Five nuclei were identified in the caudal region of the posterior tubercular area in all studied species: caudomedial, caudal and lateral thalamic nuclei forming the medial group, as well as lateral torus and posterior thalamic nuclei forming the lateral group. The latter two nuclei have their subdivisions. Characteristic peculiarity of the caudal region is the presence of only migrated nuclei. It is concluded that the level of differentiation of the diencephalon posterior tubercular area of the sturgeons allows considering it as one of the most evolutionary advanced structures among those in the actinopterygian fish line.     

Polymorphism of the myogens locus in some representatives of sturgeons (Acipenseridae)

This study investigates muscle proteins in Russian (Acipenser gueldensiaedtii Brandt), Siberian (Acipenser baerii Brandt), and Amur (Acipenser schrenckii Brandt) sturgeons using the method of polyacrylamide gel electrophoresis. A group of fractions, which is presumably the product of a polyallelic duplicated MY-1* locus, is recorded among the general pool of proteins. Data from densitometric analysis makes it possible to put forward a hypothesis about an eight-gene determination of this system of proteins. The highest heterogeneity and polymorphism was revealed in the Russian sturgeon. Analysis of the genotype distribution in samples of the studied species allowed us to make an assumption that the genetic structure of spawning sturgeon stocks is significantly metamorphosed under the influence of anthropogenic and natural factors.     

Organization of the diencephalon ventrothalamic area in the sturgeons

Cytoarchitectonics of ventrothalamic area of the diencephalon of the sturgeons was studied in four species of the sturgeons, the great sturgeon Huso huso L., the Kura sturgeon Acipenser guelden-staedtii persicus n. Kurensis Belyaeff, the Caspian sturgeon, Ac. stellatus Pall., and the barbel sturgeon Ac. nudioventris Lov. using Nissl staining and Bilschowski impregnation techniques in Viktorov’s modification. The ventral thalamus of the sturgeons was found to include seven nuclei: ventromedial, ventrolateral, intermediate and ventral thalamic nuclei, thalamic eminence, and nuclei of ventral and medial optic tracts. The main attention was paid to comparative analysis of this area in the sturgeons and other actinopterygian fish. The structures common to all ray-finned fish or only to the lower teleosts and ganoids as well as characterizing only the sturgeons.     

鲟分枝杆菌病及其病原研究

20092010年间,我国人工养殖的中华鲟（Acipenser sinensis）、史氏鲟（Acipenser schrencki）和杂交鲟（hybrid sturgeon:A. baeri-A. gueldenstaedtii）暴发了细菌性疾病。患病鲟通过组织切片观察,病原菌的分离、鉴定以及组织样品中病原菌的检测,结果显示从19条患病鲟中分离到49株分枝杆菌。病原菌经过多个保守基因的测序分析和部分生理生化特征的鉴定,共发现有7种分枝杆菌,分别为龟分枝杆菌（Mycobacterium chelonae）、海分枝杆菌（Mycobacterium marinum）、戈登氏分枝杆菌（Mycobacterium gordonae）、偶发分枝杆菌（Mycobacterium fortuitum）、苏尔加分枝杆菌（Mycobacterium szulgai）、猪分枝杆菌 （Mycobacterium porcinum）和Mycobacterium arpuense。在诊断过程中发现两种或三种分枝杆菌同时存在于同一样品中,分子生物学的诊断结果表明分枝杆菌复合感染十分常见,而海分枝杆菌是分枝杆菌复合感染中最为常见的分枝杆菌。分离的病原菌对斑马鱼的攻毒试验结果表明在以上7种分枝杆菌中海分枝杆菌的毒力最强。以上结果表明海分枝杆菌是鲟分枝杆菌病的主要致病菌,分枝杆菌复合感染是鲟分枝杆菌病的主要感染形式。研究中史氏鲟和中华鲟的分枝杆菌病,以及在病鱼体内分离的猪分枝杆菌和M. arupense在国内外均尚未见报道。       

Genetic Identification of Western Mediterranean Sturgeons and its Implication for Conservation

To date, the only native sturgeon species in Western Europe was believed to be Acipenser sturio. However, this species is currently restricted to the Gironde River (Southern France), and it poses serious difficulties for rearing in captivity and for using in recovery programme. Furthermore, it has been questioned whether A. sturio is in fact the only species within the rivers of Western Europe, as A. naccarii, a species previously considered endemic of the Adriatic region, has been reported from the Iberian Peninsula in recent years. Here, we have used forensic techniques to obtain DNA from several museum specimens of sturgeons caught in the Spanish Guadalquivir River and in other European rivers. We analysed DNA sequences from these specimens for five genetic markers (three nuclear and two mitochondrial regions), which were subsequently compared with sequences obtained from A. sturioand A. naccarii. Our study demonstrates that A. naccarii coexists withA. sturio, from the Adriatic Sea to the Iberian Peninsula, a finding that could be taken into account in future sturgeon recovery programmes in Western Europe.     

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     

Formation of populations of Acipenseridae sturgeons in the Volga-Caspian basin

Analysis of dynamics of commercial catches and the state of populations of Russian sturgeon Acipenser gueldenstaedtii, starred sturgeon A. stellatus, and great sturgeon A. huso (Huso huso) in the Caspian Sea is presented. In the retrospective, the role of natural and artificial reproduction in formation of populations of Acipenseridae sturgeons is considered. The main principles of rational organization of practical activity for preservation and restoration of populations of Acipenseridae sturgeons are formulated.     

Morphology and ecology of Pacific sturgeons

The six North Pacific members of the genus Acipenser have generally similar environmental requirements and share several specific morphological features. This Pacific group consists of green sturgeon, Acipenser medirostris, Sakhalin sturgeon, A. mikadoi, white sturgeon, A. transmontanus, Chinese sturgeon, A. sinensis, Dabry’s sturgeon, A. dabryanus and Amur sturgeon, A. schrenkii. A qualitative morphological examination of these species revealed synapomorphic characters that suggest close phyolgenetic relationships resulting from common ancestry of eastern and western species within the Pacific Rim. An evolutionary taxonomic approach, considering a reduction of characters from east to west, would suggest an Asian ancestry for all Pacific sturgeons. In contrast however, a phylogenetic approach using derived character states suggests a North American ancestry. Besides a common ancestry for Chinese and American sturgeons, the taxonomic status of A. mikadoi is discussed.     

The low level of differences between mitogenomes of the Sakhalin sturgeon <Emphasis Type="Italic">Acipenser mikadoi</Emphasis> Hilgendorf, 1892 and the green sturgeon <Emphasis Type="Italic">A. medirostris</Emphasis> Ayeres, 1854 (Acipenseridae) indicates their recent divergence

The complete mitochondrial genome of the Sakhalin sturgeon Acipenser mikadoi and two mitogenomes of the Amur sturgeon A. schrenckii were sequenced using Roche 454 technology. The mitogenomes of the green sturgeon A. medirostris (obtained from GenBank) and the Sakhalin sturgeon differ as much as the mitogenomes of two mtDNA haplogroups (SM and BG) found in the same population of the Amur sturgeon: 0.0042 ± 0.0006 and 0.0036 ± 0.0005 substitutions per site (Tamura–Nei distance, TrN), respectively. The differences of these mitogenome pairs from mitogenomes of sister species (kaluga A. dauricus and white sturgeon A. transmontanus) are 3–6 times larger: 0.0260 ± 0.0015 and 0.0102 ± 0.0008, respectively. Thus, the differences between the mitogenomes of the Sakhalin and green sturgeons can be attributed to the variability at the intraspecific level. The time that has passed since the divergence of the Sakhalin and green sturgeons is considered to be much shorter than was previously believed: approximately 0.16 rather than 9.60 million years.     

Organization of diencephalon of the sturgeons. External nuclei of the pretectal area

Cytoarchitectonics of the external nuclei of the pretectal area was studied in four species of the sturgeons: great sturgeon, Huso huso L., Kura sturgeon, Acipenser gueldenstaedtii persicus n. Kurensis Belyaeff, stellate sturgeon, Ac. stellatus Pall. and barbel sturgeon, Ac. nudiventris Lov. Study of morphological organization of the structures was carried out using routine Nissl staining and Bilschowski impregnation technique in Viktorov’s modification. Similar structure of this pretectal area was found in the species. Five nuclear formations were described in this pretectal area: parvocellular and magnocellular external pretectal nuclei, laminiform nucleus of pretectum, dorsal pretectal nucleus, and the accessory optic nucleus. Comparison of the obtained data with the literature revealed a high level of differentiation of the area of pretectum in the sturgeons. Also, a general conclusion is given on the organization of the whole pretectal area in the sturgeons.     

Genetic variation and relationships of seven sturgeon species and ten interspecific hybrids

Background

Sturgeon cultivation is important for both industry and aquaculture in China. To date, more than 17 species or strains have been farmed for fillets and caviar production. Crossbreeding among different sturgeon species is frequent and the F2 hybrids are fertile. However, large-scale farming can have negative impacts on wild populations i.e. escape of exotic sturgeons and must be taken into consideration. Escape of exotic sturgeons can cause severe ecological problems, including threatening native sturgeon species once the exotic varieties become established or hybridize with native individuals. However, little is known about their genetic resources and variation.

Methods

Genetic diversity and introgression of seven sturgeon species were analyzed using mitochondrial DNA cytochrome oxidase subunit I (COI) and nine microsatellite markers. This study included 189 individuals from seven sturgeon species and 277 individuals from ten lineages of F2 hybrid strains.

Results

MtDNA COI sequences (632 bp long) were generated from 91 individuals across the 17 sturgeon strains and produced 23 different haplotypes. Haplotype diversity was high (h = 0.915 ± 0.015) and nucleotide diversity was low (π = 0.03680 ± 0.00153) in the seven sturgeon species and ten interspecific hybrids. Phylogenetic analyses resulted in almost identical tree topologies, and different haplotype structures were mainly related with sturgeons of different female parents. Analysis of molecular variance revealed that 81.73% of the genetic variance was due to matrilineal differences, while 9.40% resulted from strain variation. Pairwise Fst values obtained with POLYSAT software, were high among strains and ranged from 0.031 to 0.164. Admixture analysis assigned seven distinct groups and ten genotypes of admixed clusters composed of hybrid strains using STRUCTURE when assuming K = 7.

Conclusions

The interspecific mtDNA gene tree corresponded to the expected taxonomic divisions. These relationships were also supported by the results from the microsatellite analysis and contributed to unambiguously identify seven sturgeon species and ten F2 hybrid strains from sturgeon farms in China. Moreover, we found that introgressive hybridization is pervasive, exists in both purebred and hybrid sturgeons, and may reflect widespread mismanagement in sturgeon breeding in China.     

Phylogenetic relationships of the North American sturgeons (order Acipenseriformes) based on mitochondrial DNA sequences

The evolutionary relationships of the extant species within the order Acipenseriformes are not well understood. Nucleotide sequences of four mitochondrial genes (12S rRNA, COII, tRNA(Phe), and tRNA(Asp) genes) in North American sturgeon and paddlefish were examined to reconstruct a phylogeny. Analysis of the combined gene sequences suggests a basal placement of the paddlefish with regard to the sturgeons. Nucleotide sequences of all four genes for the three Scaphirhynchus species were identical. The position of Scaphirhynchus based on our data was uncertain. Within the genus Acipenser, the two Acipenser oxyrinchus subspecies were very similar in sequence and found to be basal to the remaining Acipenser species examined. Based on our data, Acipenser transmontanus and Acipenser medirostris were sister taxa, as were Acipenser fulvescens and Acipenser brevirostrum. Comparison of our results with hypotheses of sturgeon relationships proposed by previous authors is presented. The sequence data presented here are phylogenetically useful and provide a solid foundation of genetic information for the North American Acipenseriformes that can be expanded to include Eurasian species to provide a global picture of sturgeon evolution.     

Meiotic Gynogenesis in the Stellate and Russian Sturgeons and Sterlet

Diploid gynogenetic progenies were obtained in the stellate sturgeon Acipenser stellatus, Russian sturgeon, A. gueldenstaedtii, and sterlet A. ruthenus by means of insemination of the eggs with UV-irradiated spermatozoa and suppression of the second meiotic division by heat shock. The gynogenetic nature of experimental fish was confirmed by RAPD-PCR analysis of DNA. Effective photoreactivation of UV-induced lesions of spermatozoa was shown in the case of illumination of the fertilized eggs with visible light. This phenomenon should be taken into account when determining the doses of irradiation that allow inactivation of the male chromosomes and incubating gynogenetic embryos. Gynogenetic stellate and Russian sturgeons are viable and can be reared in order to study the mechanism of sex determination in sturgeons.     

Comparative morphology of the pectoral fin spine of the Persian sturgeon Acipenser persicus,the Russian sturgeon Acipenser gueldenstaedtii,and the Starry sturgeon Acipenser stellatus in Iranian waters of the Caspian Sea

The morphological characteristics of the pectoral fin spine were compared in three species of sturgeon, the Persian sturgeon (Acipenser persicus), the Russian sturgeon (Acipenser gueldenstaedtii), and the Starry sturgeon (Acipenser stellatus), all sampled from the Caspian Sea. On the basis of morphological characters of the pectoral fin spine, 62.2% of the individuals were correctly classified into separate groups. The cluster analysis also divided the three species into two major subgroups. Acipenser persicus and A. gueldenstaedtii were grouped together, suggesting a similar evolutionary basis. Significant morphological heterogeneity in pectoral fin spine characteristics was observed among the three sturgeon species. Principal component analysis identified the largest differences were in the pectoral fin spine size and the angle between distal pectoral fin spine and the horizontal line (A°). The first and second principal components (PC1 and PC2) of all observations accounted for 64.19% and 14.33% of the total variation, respectively. The combination of all analyses showed the relevance of applying pectoral fin spine shape for interspecific distinction of the three species of sturgeons.     

Genetic monitoring of progeny derived from natural spawning of Acipenser ruthenus Linnaeus, 1758 and Acipenser stellatus Pallas, 1771 as well as their hybrids in the Lower Volga river during the period 2017 to 2019

The state of natural spawning of sturgeons in the Lower Volga River was investigated through genetic monitoring of juveniles (larvae, fingerlings, subadults) captured at seven traditionally used monitoring locations. Sampling was performed during the period 2017–2019. In total, 460 individuals were caught and genotyped using a set of six microsatellite loci (Afug41, Afug51, Afug135, An20, AoxD161, AoxD165) and mtDNA control region sequencing. The observed species-specific microsatellite alleles revealed the presence of juvenile sturgeons originating from natural spawning also included some hybrids between Acipenser stellatus and A. ruthenus. Thus, the majority of sterlet breeding takes place in natural spawning grounds but also sterlet hybridizes with a small number of stellate sturgeons. Juveniles of other sturgeon species were not identified, and it is concluded that natural spawning activities of Russian sturgeon, beluga and ship sturgeon were insignificant or even absent in the Lower Volga during the observation period.     

First archaeozoological identification of Atlantic sturgeon (Acipenser oxyrinchus Mitchill 1815) in France

To this day, the only sturgeon to be listed on the French vertebrate inventory is the European sturgeon (Acipenser sturio Linnaeus, 1758). The recent study of sturgeon remains on various French archaeological sites shows the presence of another species: the Atlantic sturgeon (A. oxyrinchus Mitchill 1815). This species already existed in the French Atlantic region at the end of the Neolithic Age 5000 years ago and was still to be found 3000 years later. Thus the A. oxyrinchus determined in several Baltic medieval sites are neither the only nor the first sturgeons to have inhabited European waters. Sturgeon restoration projects in European rivers necessitate a precise determination of the native species. In the case of relict or extinct species, the bone remains found on archaeological sites represent the most reliable source of information. This discovery will also be the starting point of palaeogenetical research (mitochondrial and cellular aDNA) and will give information about the genetic diversity of these threatened or recently extinct populations.     

Organization of hypothalamic area of diencephalon in the sturgeons

Cytoarchitectonics of hypothalamic area of diencephalon of the sturgeons was studied in serial sections by techniques of Nissl staining and Bielschowski impregnation in Viktorov’s modification. The hypothalamus was shown to be the most expanded area of diencephalon and forms its the most ventral part. The hypothalamic area of four studied sturgeons, the hausen, Huso huso L., Kura sturgeon, Acipenser guldenstaedtii persicus n. Kurensis Belyaeff, Caspian sturgeon, Ac. stellatus Pall. and the barbel sturgeon Ac. nudioventris Lov. was found to have similar structure. Eleven nerve structures are identified and described in the hypothalamic area: dorsal, ventral, and caudal periventricular zones, rostral and dorsal hypothalamic nuclei, ventral and ventrolateral hypothalamic nuclei, diffuse and central nuclei of the inferior lobes, nucleus of the vascular sac, and mammillary nucleus. Peculiarities and common features of organization of four major parts of hypothalamus of the sturgeons are considered in comparison with those of other ray-finned fish. The performed analysis indicates a high level of development of hypothalamus of the sturgeons.