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.     

Natural genetic polymorphism and phylogeography of Siberian sturgeon <Emphasis Type="Italic">Acipenser baerii</Emphasis> Brandt, 1869

Five populations of Siberian sturgeon Acipenser baerii collected throughout the species distribution range (Lake Baikal, Lena, Yenisei, Kolyma, and Irtysh rivers) were examined for genetic polymorphism using five tetraploid microsatellite loci and the mitochondrial DNA control region. It was demonstrated that Siberian sturgeon was represented by genetically well-differentiated groups that corresponded to hydrographic basins, Ob–Irtysh, Baikal–Yenisei, Lena, and Kolyma. Population assignment of the Siberian sturgeon broodstock should be considered in the restocking programs.     

Polymorphism of microsatellite loci in cultivars and species of pear (<Emphasis Type="Italic">Pyrus</Emphasis> L.)

Using five SSR markers, polymorphism of microsatellite loci was examined in 46 cultivars and five species of pear (Pyrus ussuriensis, P. bretscgneideri, P. pyraster, and P. elaegnifolia). Most of the accessions examined showed the presence of unique allele sets. The degree of relationship between Russian and Western European pear cultivar was established. It was demonstrated that P. ussuriensis and its first generation progeny were genetically distant from typical cultivars of P. communis, as well as from the P. communis × P. ussuriensis hybrids of later generations. SSR estimates of the cultivar relatedness were shown to correlate with the corresponding pedigree-based estimates. A number of SSR alleles specific to P. ussuriensis were identified. Based on the analysis of microsatellite loci, the allelic composition was determined for each cultivar examined. These data can serve as a molecular certificate of the cultivar.     

Development of new microsatellite primers for green and white sturgeon

Sixty-eight primer sets for microsatellite loci were developed from microsatellite motif enriched genomic libraries of pooled DNA from the polyploid green and white sturgeon (Acipenser medirostris and A. transmontanus). Four individuals from each species were screened for polymorphism at these loci. Forty-eight loci amplified in both species, and some exhibited species-specific amplification for white or green sturgeon (8 and 12 loci, respectively). The number of alleles per locus ranged from one to 12. At least 68% of the green and 65% of the white sturgeon loci we developed are polysomic.     

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.     

Polyphyly of mtDNA lineages in the Russian sturgeon, Acipenser gueldenstaedtii: forensic and evolutionary implications

Molecular species identification methods are an important component of CITES monitoring programs for trade in sturgeon and caviar. To date, obtaining molecular evidence for distinguishing caviar from four closely related Eurasian sturgeon species Acipenser baerii (Siberian sturgeon), A. gueldenstaedtii (osetra), A. persicus (Persian sturgeon), A. naccarii (Italian sturgeon) remains problematic. Using approximately 2.3 kb of mtDNA sequence data (cytochrome b, NADH5, control region), we find this to be attributable to the polyphyletic nature of these mitochondrial DNA markers in the Russian sturgeon, A. gueldenstaedtii. Two mitochondrial lineages are present within this species: one is phylogenetically affiliated with A. persicus and A. naccarii, while the other clusters with A. baerii. These findings have a direct impact on molecular testing of commercial caviar and demonstrate the necessity of using large sample sizes when constructing forensic databases. Furthermore, the results affect current taxonomic designations for these species as well as hypotheses concerning their evolutionary origins.     

Dispermic androgenesis in sturgeons with the use of cryopreserved sperm: Production of androgenetic siberian sturgeon and androgenetic hybrids between Siberian and Russian sturgeons

Dispermic androgenesis was used to produce, for the first time, an androgenetic progeny of the Siberian sturgeon (Acipenser baerii) and the androgenetic nucleo-cytoplasmic hybrids (Siberian sturgeon, A. baerii × Russian sturgeon, A. gueldenstaedtii) using cryopreserved sperm. Microsatellite DNA analysis confirmed exclusively paternal inheritance in the androgenetic progeny of Siberian sturgeon. Heterozygotes for certain microsatellite loci were detected among the androgenetic hybrids, thereby confirming a dispermic nature of androgenesis. According to the data of comparative morphological analysis, the obtained androgenetic hybrid, by the age of 15 months old, was completely identical to the paternal species. Both a female and a male were detected in the androgenetic sturgeon progenies, which is of interest for producing bisexual progenies via androgenesis. The data of this study confirm the feasibility of dispermic androgenesis using cryopreserved sperm to preserve and recover the gene pools of endangered sturgeon species.     

Identification of microsatellite loci in lake sturgeon,Acipenser fulvescens,and their variability in green sturgeon,A. medirostris

From (CATC)_n, (GATA)_n, (AAAC)_n, and (CA)_n–enriched libraries for the lake sturgeon Acipenser fulvescens, 254 primer pairs were developed. These primer pairs resulted in the identification of 128 microsatellite loci in either A. fulvescens or A. medirostris. Polymorphic loci were identified in both sturgeon species for 48 of the primer pairs and 14 of the primer pairs amplified polymorphic loci only in A. medirostris. Most of the identified loci appear to be tetrasomic (79.1% in A. fulvescens and 64.5% in A. medirostris). These results offer estimates of the degree of diploidization in each of these species.     

Improved genetic identification of acipenseriform embryos with application to the endangered pallid sturgeon Scaphirhynchus albus

We produced pallid sturgeon Scaphirhynchus albus embryos at five pre-hatch developmental stages and isolated and quantified genomic DNA from four of the stages using four commercial DNA isolation kits. Genomic DNA prepared using the kit that produced the largest yields and concentrations were used for microsatellite DNA analyses of 10–20 embryos at each of the five developmental stages. We attempted to genotype the hatchery-produced embryos at 19 microsatellite loci and confirmed reliable genotyping by comparing the microsatellite genotypes to those of known parents. Embryos at stages 5 and 8 did not produce reliable genotyping while those at stages 14, 24 and 33 did. We used the same DNA isolation method on 262 wild-caught acipenseriform embryos collected from the lower Yellowstone River. A total of 200 of the wild embryos were successfully identified to stages 8 to 34 and the rest could not be staged. Using a combination of single nucleotide polymorphism and microsatellite markers, 249 of the wild-caught embryos were genetically identified as paddlefish Polyodon spathula, five were identified as shovelnose sturgeon Scaphirhynchus platorynchus and eight failed to amplify. None were identified as pallid sturgeon. This study demonstrates that early-stage wild-spawned acipenseriform embryos can be genetically identified less than 24 h post-spawn. This methodology will be useful for recovery efforts for endangered pallid sturgeon and can be applied to other acipenseriform species.     

Stock structure of shovelnose sturgeon analyzed with microsatellite DNA and morphological characters

Shovelnose sturgeon (Scaphirhynchus platorhynchus) caviar fisheries exist in several states throughout the Mississippi River drainage. Management of these fisheries may benefit from information about genetic stock structure. Sixteen microsatellite loci and morphological analysis were used to examine geographic stock structure of shovelnose sturgeon among seven geographic locations: five within continuous shovelnose sturgeon habitat, and two isolated by artificial barriers. Tissue samples were collected from 1999 to 2006 from the upper Missouri, Platte, lower Missouri, middle Mississippi, Ohio, Wabash, and Atchafalaya rivers. Geographic samples of shovelnose sturgeon samples could be separated into three groups with discriminate function analysis of four morphological characters. The microsatellite loci were highly variable (allelic richness range 5.65–13, observed heterozygosity range 0.64–0.89). Bayesian clustering did not identify multiple groups in the genetic data. However, significant genetic differentiation (θ_ST = 0.017, P < 0.0001) was observed among a priori defined geographic samples and all pairwise estimates of θ_ST were significant. Assignment testing among a priori defined groups indicated that the sturgeon from the upper Missouri, Platte, and Atchafalaya rivers had the highest assignment scores and thus were most distinct, while the lower Missouri and the middle Mississippi were less distinct and a larger fraction of the sturgeon from these rivers was genetically assigned to other rivers. The Ohio and Wabash rivers were genetically most similar. A Mantel test revealed a positive relationship between genetic and geographic distance (r = 0.464, P = 0.055) that was not statistically significant. The level of genetic differentiation observed at both molecular and morphological characters suggests that multiple shovelnose sturgeon populations may exist within the studied area, yet demographic factors and possible gene flow may have minimized the amount of genetic differentiation among locations.     

Behavioral response of mature males of acipenseridae to female sex pheromone

It was established that, as in many other representatives of Teleostei, there is a pheromonal regulation of reproductive behavior in Acipenseridae. The postovulatory releasing sex pheromone in Russian sturgeon Acipenser gueldenstaedtii, Persian sturgeon A. persicus, and starred sturgeon A. stellatus is in the ovarian fluid of females, whose odor has an interspecific efficiency and causes manifestation of typical spawning behavior (increase in locomotor activity, attraction to the site of solution presentation, circular swimming, and slightly convulsive movements) in mature males of its own and closely related species. A relationship was found between the ability of males injected with suspension of acetonized hypotheses of acipenserids to respond to odor of ovarian fluid and by both quality and quantity of sperm obtained from them. Data obtained extend our concepts of the role of chemical reception in biology of acipenserids and the range of their chemocommunications.     

Genetic discrimination of middle Mississippi River <Emphasis Type="Italic">Scaphirhynchus</Emphasis> sturgeon into pallid,shovelnose, and putative hybrids with multiple microsatellite loci

The pallid sturgeon (Scaphirhynchus albus), which is protected under the US endangered species act, and shovelnose sturgeon (S. platorhynchus), which is legally harvested in some locations, are sympatric throughout the range of pallid sturgeon. There is considerable morphological overlap between the species making discrimination problematic. The inability to reliably differentiate between species across all life stages has hampered pallid sturgeon recovery efforts. Furthermore, the two species are believed to hybridize. This study used allele frequency data at multiple microsatellite loci to perform Bayesian and likelihood-based assignment testing and morphological measures and meristics to discriminate pallid, shovelnose, and putative hybrid sturgeons from the middle Mississippi River. Bayesian model-based clustering of the genetic data indicated that two natural genetic units occur in the region. These units correspond to morphologically identified pallid and shovelnose sturgeon. Some individuals were morphologically intermediate and many of these failed to strongly assign genetically as either pallid or shovelnose sturgeon, suggesting they may be hybrids. These data indicate that pallid sturgeon and shovelnose sturgeon are genetically distinct in the middle Mississippi River (F _ST = 0.036, P < 0.0001) and suggest that hybridization between pallid sturgeon and shovelnose sturgeon has occurred in this region with genetic distance estimates indicating the greatest distance is between pallid and shovelnose sturgeon, while hybrid sturgeon are intermediate but closer to shovelnose. This study demonstrates that assignment testing with multiple microsatellite markers can be successful at discriminating pallid sturgeon and shovelnose sturgeon, providing a valuable resource for pallid sturgeon recovery and conservation.

Stock structure of pallid sturgeon analyzed with microsatellite loci

Recovery efforts for the endangered pallid sturgeon (Scaphirhynchus albus) include supplementation of wild stocks with hatchery reared progeny. Identifying the extent of genetic stock structure, which has previously been detected in samples from the range extremes, will help to determine whether stock transfers might be harmful. DNA microsatellite genotypes were screened in pallid sturgeon from the upper Missouri River, lower Missouri River, middle Mississippi River and Atchafalaya River and analyzed using a combination of Bayesian model‐based and more traditional F‐statistic based methods to characterize genetic differentiation. Scaphirhynchus specimens were collected by researchers active in the recovery effort and genotypes were screened at 16 microsatellite loci. Because there is considerable genetic and morphological overlap between pallid sturgeon, shovelnose sturgeon, and their hybrids, a combination of morphological and genetic techniques were used to eliminate shovelnose and possible hybrids from the sample. Genetic differentiation was detected among samples (overall θ = 0.050, P = 0.001). Pairwise θ, genetic distances, and Bayesian assignment testing reveal that pallid sturgeon from the upper Missouri River are the most distinct group with pairwise comparisons of pallid sturgeon among all the remaining samples exhibiting lower θ values, higher genetic distances, and self assignment scores. Our results indicate that using local broodstock, when available, should be used for pallid sturgeon propagation. If local broodstock are not available, geographically proximate individuals would limit genetic differences between native and stocked individuals.     

Development and standardization of disomic microsatellite markers for lake sturgeon genetic studies

Lake sturgeon (Acipenser fulvescens) are of conservation concern in North America. To facilitate the recovery of this fish species, an understanding of their population genetic structure is necessary to develop and implement spatially and temporally appropriate management actions. Until recently, few genetic data using nuclear loci have been collected, primarily due to the paucity of suitable genetic markers because most microsatellite loci in lake sturgeon appeared to be tetrasomic. The authors identified nine microsatellite loci (from 254 examined) that were putative polymorphic disomic loci and tested their conformance to a disomic mode of inheritance using three lake sturgeon families. The objectives of the study were to: (i) confirm the disomic status of the nine loci through inheritance testing, and (ii) standardize the genetic markers among participating laboratories. At all nine loci, disomic inheritance were confirmed, and all nine loci segregated independently in the 26 of 36 loci pairs possible to test. One of the nine loci showed non‐Mendelian segregation, possibly due to meiotic drive and/or selection. Three progeny had peak patterns inconsistent with disomy at one or more loci. The nine loci when combined with four microsatellite loci previously confirmed in other studies as disomic in lake sturgeon now yield a suite of 13 microsatellite markers. These 13 markers have been standardized among four other laboratories to facilitate building an inter‐laboratory genetic database for lake sturgeon.     

Species identification in the trematomid family using nuclear genetic markers

The Trematominae, a subfamily of the Nototheniidae, are typical of the high-Antarctic shelf waters. Within the Trematominae examples of phenotypic plasticity and possible cryptic speciation have been observed. Morphological identification of adult stages can be problematic in cases of high phenotypic plasticity or cryptic speciation. Additionally, postlarval and juvenile stages often have traits still under development and which lack distinction. A microsatellite DNA multiplex of six markers has been developed for Trematomus newnesi (Van Houdt et al. 2006). This multiplex was tested on five additional trematomid taxa: Pagothenia borchgrevinki, Trematomus bernacchii, Trematomus eulepidotus, Trematomus hansoni and Trematomus scotti. We used these six microsatellite loci to assess the genetic differentiation among species and the resolution power of these loci for individual-based assignment methods. The six species could be well discriminated by conventional methods such as principal component analysis and distance-based methods, and individual Bayesian assignment methods. This marker set can be used for a number of purposes, including the identification of eggs and larval and adult stages. It is also useful for the investigation of recent phylogenetic patterns, as well as the detection of cryptic speciation, which has been suggested for T. bernacchii and T. newnesi but never confirmed with high polymorphic genetic markers.     

Interspecific hybridization in natural sturgeon populations of the Eastern Black Sea: the consequence of drastic population decline?

The eastern part of the Black Sea and its tributaries are suitable habitats for several sturgeon species, among which Acipenser gueldenstaedtii, A. stellatus, A. nudiventris, A. persicus, A. sturio, and H. huso are well documented. However, different threats have led these species to a dramatic decline, all of them are currently listed as Critically Endangered, and some Locally Extinct, in that area. We tested 94 wild sturgeon samples from the Black Sea and Rioni River by analyzing the mitochondrial Control Region and nuclear markers for hybrid identification. The data analyses (1) assessed mitochondrial diversity among samples, (2) identified their species, as well as (3) indicated instances of hybridization. The data collected, besides confirming a sharp decrease of catches of Beluga and Stellate sturgeon in recent years, also revealed four juvenile hybrids between Russian and Stellate sturgeon, providing the first evidence of natural interspecific hybridization in the Rioni. The present communication raises concerns about the status of sturgeon species in this area and underlines the urgent need for conservation programs to restore self-sustaining populations.

     

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.     

Taste attractiveness of free amino acids for juveniles of Persian sturgeon <Emphasis Type="Italic">Acipenser persicus</Emphasis>

Taste preferences towards 20 free amino acids (L-isomers, 0.1–0.0001 M) were determined in juveniles of Persian sturgeon Acipenser persicus. It was found that most amino acids (16) had a positive effect on extraoral gustatory reception (increased the frequency of catching artificial pellets by fishes). The most efficient amino acids were the following: threonine, histidine, arginine, asparagine, phenylalanine, cysteine, glutamine, and glycine. Fifteen amino acids were efficient for intraoral gustatory receptors: serine, arginine, cysteine, histidine, alanine, and some others. The presence of these amino acids in pellets increased the consumption. Amino acids decreasing the frequency of catching or consumption of pellets were not found. Highly significant positive correlation was found between the amino acid ranges mediated by the extraoral and intraoral gustatory reception. The fishes demonstrated maximum sensitivity to aspartic acid, 0.01 and 0.001 M, respectively, for intraoral and extraoral gustatory systems. The threshold concentrations of arginine and glycine were higher for the intraoral gustatory system (0.01 M) than for the extraoral one (0.1 M). The comparison of Persian sturgeon with sturgeon fishes (Russian sturgeon A. gueldenstaedtii, Siberian sturgeon A, baerii, and starred sturgeon A. stellatus) studied earlier confirmed high species specificity of intraoral taste preferences in representatives of Acipenser genus. Species specificity of extraoral taste ranges was less pronounced.     

Characterization of microsatellite loci isolated from the blacktip shark and their utility in requiem and hammerhead sharks

We isolated and characterized 16 microsatellite loci from the blacktip shark, Carcharhinus limbatus, and tested cross‐species amplification in 11 Carcharhinus species and five additional shark genera. Thirty‐six (1.6%) and 180 (48%) colonies were positive for dinucleotide repeat motifs from unenriched and enriched libraries, respectively. Heterozygosities of polymorphic loci ranged from 0.04 to 0.96 with two to 22 alleles per locus. Amplification products were observed at nine to 13 loci (five to 11 of which where polymorphic) in 10 Carcharhinus species. Several loci were also polymorphic in each of the additional genera examined.