Genetic variation and phylogenetic relationships among domesticated and wild paddlefish (Polyodon spathula) populations

© 2020 K. M. Kurta et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 602.6:639.212:639.31


Introduction
The purposeful management of the genetic resources is becoming increasingly important in animal husbandry and aquaculture to preserve biodiversity in hatchery-reared stocks. Growing demand for fishery products requires the expansion of the production of highly valuable objects for the produ cing of marketable products in a relatively short time [1,2].
Currently, American paddlefish (Рolyodon spathula), as a representative of Acipen seriformes species, is of high commercial importance, and its cultivation in aquaculture allows an increase in the productivity of fishing enterprises by rearing in polyculture with other valuable fishes [3,4].
Artificial paddlefish reproduction is widespread in the United States for restocking and conservation of natural genetic resources, producing marketable products, and for sport fishing of this species. A similar strategy has evolved since the creation of the Mississippi Interstate Cooperative Resource Association (MICRA) in 1991 providing the management of natural paddlefish populations through artificial reproduction and maintenance of its genetic diversity [5,6].
In turn, the development of paddlefish aquaculture particularly in Europe, Asia and Cuba, is mainly focused on the production of commodity products as black caviar and delicatessen meat to meet the needs of the domestic market and export [7,8]. Meanwhile, in Ukraine the paddlefish aquaculture is becoming increasingly popular as an optimal alternative to sturgeon species. However, the main challenge in the aquaculture sector is the risk of reduction of genetic variability in farmed paddlefish populations due to small quantities of the genetic material imported from the USA and Russian Federation to establish its stocks. Likewise, in neighboring country Poland the broodstocks are relatively small and were formed from the progeny of a few spawners from Hungary [4,12,16]. Because of this, it is especially significant to evaluate genetic variation and identify genetic differences among paddlefish populations.
At present, the study of genetic processes occurring in paddlefish populations due to natural geographical isolation or under the influence of cultivation practice is at high of its development. The first main contribution proposed in the investigation of such genetic processes is the microsatellite DNA markers [9][10][11][12][13][14].
In population-genetic studies, microsatellites are widely used to determine genetic differentiation, carry out DNA identification and certification of species, and ma nage selection and breeding of valuable objects during their artificial reproduction [9,[15][16][17][18].
Based on the DNA analysis of paddlefish individuals, the controlled selection of spawner pairs can be performed in order to purposefully restore local populations and maintain genetic diversity of populations under artificial reproduction [11][12][13][14]. Additionally, the identification and assessment of genetic differences among the populations of natural reservoirs and farmed species will allow the establishment of phylogenetic relationships and determination of the origin of target objects for effective management of the genetic resources [13,[19][20][21][22].
The purpose of this study is to determine the genetic variation and phylogenetic relationships among the domesticated and wild paddlefish (Polyodon spathula) populations by STRloci polymorphism.
The selected polymorphic microsatellites, namely Psp21, Psp26 Psp28, which are presented in GenBank ® (Table 1), were co-amplified in a single multiplex PCR reaction. One primer (forward or reverse) from each pair was end-labeled with a fluorescent dye.

Genotyping
Amplified products were denatured with formamide (Sigma, USA), then separated and detected via capillary electrophoresis (ABI Prism 3130xl Genetic Analyzer, Applied Biosystems, USA). The DNA fragment size  [27][28][29][30]. Based on the allele frequencies, the genetic distances and coefficient of genetic identity were computed in agreement with Nei [31].
The Principal Coordinate Analysis (PCoA) was performed using GENALEX 6.5 program. The Neighbor-joining method (NJ) was used to construct the dendrogram of genetic distances between the studied fish groups using the POPTREEW Software [32].

Results
The comparative study were carried out between three cultured stock from Ukrainian regions (obtained as a result of own studies), two stocks from Poland studied by Kaczmarczyk D. (Kaczmarczyk, 2012): Pogórze, Silesian Voivodeship (n = 24) and Wasosze, Kujawsko-Pomorskie Voivodeship (n = 29) and wild populations from the United States investigated by Heist E. J. (Heist, 2002). For the comparative studies, the populations were chosen from different geographic regions, including Bayou Nezpique, Mermentau River, Louisiana (n = 45); Big Muddy and Mississippi, Illinoice (n = 28); Missouri, North Dakota (n = 43); Red River near Natchitoches, Louisiana (n = 48); Red River below Lake Texoma, Oklahoma (n = 41); Grand Lake Neosho River, Oklahoma (n = 35). Table 2 shows that the mean number of alleles per locus (Na) varied from 5.5 to 6.6 in the Polish and Ukrainian paddlefish stocks, respectively, though it demonstrated a lower genetic polymorphism compared to that found in wild population from the USA (11.1 alleles per locus).
The mean observed heterozygosity (Ho) estimated in the Ukrainian stocks (0.709) was relatively close to those in the Polish stocks (0.809) and in wild populations (0.817). Meanwhile, the average observed heterozygosity was higher than expected for both Ukrainian (Но = 0.709; Не = 0.616) and Polish (Но = 0.809; Не =0.699) paddlefish stocks. Similarly, the Wright's fixation index (F IS ) on average confirmed the predominance of heterozygous genotypes in domesticated populations from Ukraine (-0.124) and Poland (-0.108). The results for the populations from the USA showed that the average observed and expected heterozygosity values were at 0.817 and 0.813, respectively, indicating that the genotype frequencies in wild stocks were in Hardy-Weinberg equilibrium (HWE), as evidenced by the fixation index (F IS = -0.002).
Based on the previously calculated frequencies for the identified allelic variants (Kurtа, 2019), the Nei's genetic distances and genetic identity among Ukrainian paddlefish populations were revealed (Table 3). It has been established that local populations demonstrated a high level of genetic similarities, which signifies the common origin of these fish groups. The maximum level of the genetic similarity was determined between Chernihiv and Vinnytsia populations at 0.981, whereas the minimum level of the genetic similarity was observed between Kherson and Chernihiv populations at 0.908. The genetic similarity among investigated groups of paddlefish from Kherson and Vinnytsia regions was 0.937. According to the analysis of genetic distances for the studied populations, this index ranged from 0.019 (Chernihiv and Vinnytsia regions) to 0.096 (Kherson and Chernihiv regions), whereas among paddlefish populations from Kherson and Vinnytsia it was 0.065. The increased genetic identity of Ukrainian paddlefish populations is characterized by the presence of predominantly similar allelic variants and their frequency values at the studied loci.
The Principal Coordinate Analysis (PCoA) has been used to visualize the genetic relationships measured by Nei's genetic distance (Fig. 1).
The dissimilarity matrix used as input depicts on the plot shows that the population originated from Kherson region is more distant from those originated from the farms in Chernihiv and Vinnytsia regions.
Phylogenetic analysis of Ukrainian populations using the neighbor-joining method (Fig. 2) showed that Chernihiv and Vinnytsia populations constitute a single cluster. In turn, the Kherson population formed a separate cluster.
Overall, the results of the research showed that the detected genetic distances are a consequence of the features of introduction of domesticated paddlefish populations within different geographic location in Ukraine. A high genetic identity among the three paddlefish populations confirms their common origin. The phylogenetic analysis showed that the investigated individuals of paddlefish populations from the Chernihiv and Vinnytsia regions were grouped into a single cluster. The data obtained indicate that these populations originate from one stock and have common parental individuals. At the same time, the population from the Kherson region appeared more genetically distant, perhaps, due to the genetic material additionally imported to this region, which is confirmed by historical aspects of paddlefish introduction in Ukrainian fish farms [8,32].
Meanwhile, the additional comparison allowed the establishing of the genetic relationships between paddlefish population from Ukraine and Poland and natural populations from the United States based on three microsatellite DNA-loci (Psp21, Psp26, Psp28).
The distribution of genetic heterogeneity among paddlefish populations from Ukraine, Poland and natural populations from the USA  showed that domesticated and natural populations fall into different clusters according to their population membership. At the same time, the Polish population was more genetically distant from the studied Ukrainian paddlefish populations. Such results are similar to those obtained in the D. Kaczmarczyk studies [14] and could be a consequence of the formation of Ukrainian and Polish brood populations from different parental individuals.
Analysis of the genetic relationships among paddlefish populations indicated the genetic delimitation of Ukrainian individuals from natural groups. Similarly, the genetic distances were found between Polish and natural populations. Such results are observed as a consequence of differences in the allele frequency spectrum identified in the gene pool of analyzed populations.

Discussion
The evaluated genetic variation in Ukrainian domesticated populations was compared with the respective values observed in Polish paddlefish populations and natural populations of this species from the USA. The mean number of alleles per locus was significantly lower in Ukrainian (6.6) and Polish (5.5) populations in comparison to the natural populations (11.1). These results indicate that almost a two-fold lower genetic variation was observed within domesticated populations from Ukraine and Poland than for the populations from natural reservoirs. The analysis of heterozygosity across the examined populations showed that the mean observed heterozygosity (Ho) estimated in Ukrainian populations (0.709) was relatively close to those in Polish (0.809) and wild populations (0.817). Moreover, the pro-portion of Ho to He rates within domesticated stocks indicated the excess of heterozygous genotypes over the homozygous for both Ukrainian (Но = 0.709; Не = 0.616) and Polish (Но = 0.809; Не =0.699) paddlefish stocks. These findings were confirmed by values of Wright's fixation index (F IS ) in Ukrainian (-0.124) and Polish (-0.108) populations. Despite the fact that the data obtained show high heterozygosity levels for the examined populations, keen attention should be paid to the genetic control of paddlefish breeding in order to prevent the loss of its genetic variability in the farmed populations.
The phylogenetic relationships of cultured paddlefish populations from Ukraine and Poland with natural populations from the United States were determined using three microsatellite DNA markers: Psp21, Psp26 and Psp28. The results demonstrate the close genetic relationships among Ukrainian paddlefish populations, confirming their common origin. Meanwhile, the genetic differences were found among Ukrainian and Polish populations, which allows considering the fish groups as a potential source for the exchange and enrichment of the paddlefish genetic resources.
The genetic distinctiveness between the paddlefish populations from Ukraine and Poland and natural populations from the United States indicate differences in the allele pool, in particular, low allelic diversity and allele frequency spectrum, due to cultivation of this species of fish in aquaculture.