Unidirectional hybridization between American paddlefish Polyodon spathula (Walbaum, 1792) and sterlet Acipenser ruthenus (Linnaeus, 1758)

Introduction

Acipenserid fishes have a high rate of interspecific hybridization (Birstein, Hanner & De Salle, 1997). Family Acipenseridae, comprising the sturgeons, is the only vertebrate family in which all species can hybridize with each other in their natural overlapping habitats (Birstein & De Salle, 1998). However, hybrids between sturgeon from the family Acipenseridae with other Acipenseriformes family (Polyodontidae) are hardly known (Káldy et al., 2020). The diploid chromosome number of the ancient common ancestor of acipenseriform was approximately 60 (Birstein, Hanner & De Salle, 1997; Ludwig et al., 2001). During the evolution of acipenseriform, several polyploidization events occurred and resulted in three ploidy levels in sturgeons and paddlefishes: (1) 120 chromosomes; (2) 250–270 chromosomes and (3) 370 chromosomes (Vasil’v et al., 2014). Depending on the maximum ploidy levels, during the evolution of each species, can be distinguished the evolutionary and the current functional scale of the chromosome number for all acipenseriform species (Rajkov, Shao & Berreb, 2014). The evolutionarily tetraploids with approximately 120 chromosomes function as diploids and the evolutionarily octoploids with approximately 240 chromosomes function as tetraploids and the evolutionarily dodecaploids with approximately 360 chromosomes function as hexaploids (Havelka et al., 2011). In this article, the functional ploidy levels were applied as described above).

Successful hybridization between taxonomically distant species is rare due to genetic incompatibility of different chromosome numbers (Arnold, 1997; Parker, Philipp & Whitt, 1985a; Parker, Philipp & Whitt, 1985b). The unique character of natural evolutional polyploids in acipenseriform taxa makes hybridization between sturgeon species feasible (Birstein, Hanner & De Salle, 1997). Also, the success of interspecific hybridization between sturgeon species is dependent on the ploidy levels of the parent species (Rachek et al., 2022), i.e., hybridization occurs between diploids and diploids, tetraploids and tetraploids, diploids and tetraploids (resulting in triploid hybrids) (Shivaramu, 2019). These hybridizations were mostly reported bidirectional in natural habitats and in domestic farms by artificial fertilization. However, unidirectional hybridization of female Kaluga (Huso dauricus) and Amur sturgeon (Acipenser schrenckii) was reported in natural habitat (Shedko & Shedko, 2016; Shedko, Miroshnichenko & Nemkova, 2020), and viable hybrids were observed only in the hybridization of female Kaluga and male Amur sturgeon. However, in aquaculture conditions viable hybrid offspring were obtained from bidirectional hybridization. Moreover, viable hybrids have been produced from three interspecific crosses through polyspermic fertilization (Iegorova, 2018).

Hybridizations between species from the Family Polyodontidae and species from the Family Acipenseridae were also reported. For example, through artificial fertilization male American paddlefish (Family Polyodontidae) could hybridize with female Russian sturgeon (Acipenser gueldenstaedtii) (Káldy et al., 2020) and with female pallid sturgeon (Scaphirhynchus albus) (Flamio Jr et al., 2021). From the families’ point of view Acipenseridae and Polyodontidae shared the same ancestor, the extinct Family Peipiaosteidae (Birstein & De Salle, 1998), and diverged in the Jurassic period (184.4 Mya) (Peng et al., 2007). In order Acipenseriformes all species have natural polyploids, which contribute to the feasibility of interspecific and intergeneric hybridization such as the hybridization of Family Acipenseridae (Birstein & De Salle, 1998). The polyploid state of acipenserids originates from up to three allopolyploid/autopolyploid whole-genome duplications (WGDs), while the polyodontids originate from one autopolyploid WGD event (Lebeda et al., 2020). The allopolyploid origin contributes to the formation of viable dispermic androgenetic hybrids between phylogenetically close sturgeon species (Grunina & Recoubratsky, 2005). Although, the maternal-effect genes can affect the early embryogenesis before zygotic gene activation (Kishimoto et al., 2004), normal early embryogenesis occurs in closely related sturgeon species with a paternal genome containing homologous silent or not compatible genes (Grunina & Recoubratsky, 2005).

Next to the evolutionary differences, the two species are separated geographically and biologically as well. The sterlet is a Ponto-Caspian distributed species, the smallest sturgeon in the family Acipenseridae, which is a typical bottom feeder (benthic) fish (Fieszl et al., 2011). The American paddlefish is a zooplankton feeding species, native to North America, which is reaching up to 2 m in length and 70 kg in body weight (Mims & Shelton, 2015). Both species are important for aquaculture production and are farmed in ponds and intensive systems in many countries around the world. The eggs of these species are also used to produce black caviar, but their meat is the important market product. Both species are listed on the International Union for Conservation of Nature (IUCN) Red List; the sterlet as “endangered” and the American paddlefish as “vulnerable” conservation status (http://iucnredlist.org/).

The goal of this study was to explore the feasibility of hybridization between two functionally diploid acipenseriform species from different families: the American paddlefish and the sterlet. The objectives were to: (1) examine the fertilization; (2) test the hybrid offspring viability, and (3) analyse of the parentage by use of microsatellite markers in bidirectional hybridization crosses.

Materials & Methods

Results

Overall comparison of hybrid and purebred fishes

In comparison, the purebred crosses produced significantly higher fertilization and larval survival than that of all hybrid crosses (Fig. 1). Hybridization crosses yielded variable offspring survivals and the crosses with female paddlefish and male sterlet did not show embryo development to the early neurula stage and thus no percentage of fertilization was collected.

On the boxplots, the middle line represents the median value; and the box borders show the lower and upper quartiles. Different letters indicate significant differences between groups regarding fertility (blue) or survival (green). After fitting a logistic regression model, multiple comparisons of means (Tukey’s contrasts) were performed to analyze the differences between groups. P < 0.05 was considered significant (* P < 0.05, **P < 0.01, ***P < 0.001).

Discussion

Hybridization among paddlefish and sturgeons has been attempted before. For example, Mims et al. (1997) reported that American paddlefish eggs could be fertilized with shovelnose sturgeon (Scaphirhynchus platorynchus) sperm and that the fertilized eggs developed to the gastrula stage. While, Zou, Wei & Pan (2011) showed that embryogenesis reached the gastrula and early neurula stages for American paddlefish eggs fertilized by Amur sturgeon (Acipenser schrenckii) sperm. Overall hybrid crosses with female paddlefish and male Acipenseridae sturgeon did not result in viable offspring, but in contrast, more reciprocal hybrid crosses between these taxa, resulted in viable hybrid offspring. There are several successful American paddlefish gynogenesis studies used by inactive Amur sturgeon (Zou, Wei & Pan, 2011) and shovelnose sturgeon (Mims et al., 1997) sperm. However, gynogenesis could be successful not only with inactivated sperm, but also with active foreign sperm as the natural gynogenetically reproductive form of silver crucian carp (Carassius auratus) (Fan & Liu, 1990). After (normal or gynogenetic) fertilization, the male and female pronuclei fuse to form the zygotic nucleus; the second meiosis resumes first and then the zygote enters the first mitotic division stage (Lindemanand & Pelegri, 2010). If the invasive sperm caused gynogenesis, the paternal genome in the fertilized egg is lost before the first mitotic division (Beukeboom & Vrijenhoek, 1998). The first mitotic division in the paddlefish egg occurs about 180 min after fertilization at 16 °C (Mims & Shelton, 2015), and gastrulation begins approximately after day two, and neurulation lasts until day six (Bemis & Grande, 1992). Our results show that in American paddlefish eggs fertilized by sterlet sperm, the paternal chromatin was present in early cell cleavage and also in the early embryonic stage (not lost before first mitotic division), because the paternal genome was detected 120 h post fertilization. It suggests that unidirectional hybridization is not caused by the death of the unfertilized haploid egg (for example in gynogenesis by hybridization), but by a genetic process or processes that occur during embryogenesis.

In embryogenesis, gastrulation is the first major block of development (Merriner, 1971), where the eggs without paternal nuclei die during gastrulation. The genetic incompatibilities between gametes is less investigated in fishes than in other animal species, but it has been established that this incompatibility enables hybridization in one direction while hindering it in the other direction, thereby resulting in unidirectional hybridization (Álvarez & Garcia-Vazquez, 2011).

Within this, the genetic explanation may be an abnormal gene expression (regulation). If the mutational differences between species reach a saturation point, gene regulation/expression divergence between species will be too large for the embryos to survive the gastrula stage (Parker, Philipp & Whitt, 1985a). For example, hybridization between species belonging to the genus Lepomis (i.e., green sunfish Lepomis cyanellus) and Micropterus (i.e., largemouth bass Micropterus salmoides) (Centrarchidae, Perciformes, Teleostei) led to unidirectional hybridization, which was described as being caused by normal gene expression in one direction and an abnormal pattern of gene regulation in the other direction, leading to embryo lethality (Whitt, Philipp & Childers, 1977). This result highlights that the gene expression of related species does not necessarily have the same molecular and genetic basis, so when genes from different species are placed in a common nucleus, their mutational differences can lead to incompatibility in the hybrid offspring (Whitt, Philipp & Childers, 1977), or affect the transcription of multiple genes, which may induce stress signalling (Landry, Hartl & Ranz, 2007). Altered gene expression may also be caused by repetitive DNA and inappropriate activity of silenced genes, which may be caused by defects in several epigenetic mechanisms in hybrids (Maheshwari & Barbash, 2011). The gene expression in the early development of embryos is regulated by the maternal cytoplasm; in the case of hybridization of triangular bream (Megalobrama terminalis) and grass carp (Ctenopharyngodon idellus), the hybrid is viable if the maternal species is grass carp, otherwise, the hybrid offspring will not develop normally (Liu et al., 2020). This is not due to the incompatibility of the nucleic DNA of the parent species, but to the abnormal coordination of the nuclear DNA of the triangular bream and the sperm of the grass carp (Liu et al., 2020). In addition, the divergence of gene expression between increasingly distant species increases further (Philipp, Childers & Whitt, 1979; Parker, Philipp & Whitt, 1985b).

Further genetic explanation could be the cytogenetic maternal influence, namely by the transcription of maternal mRNA into functional proteins in the embryo, which can affect the development of the embryo in the early stages of embryogenesis. Embryonic development begins under maternal regulation of mRNA and protein synthesis, and then this regulation shifts to zygotic transcript levels (referred to as the maternal-zygotic transition). However, incompatibilities can then arise between paternal alleles and the maternal factors that halt embryonic development (Turelli & Moyle, 2007). The paternal genome does not have genes that are appropriately silenced to allow full expression of maternally derived mRNA, and poor interaction of maternal and paternal genes can impede development (Grunina & Recoubratsky, 2005). But genetic imbalances may be caused by uniparental mitochondria, mRNAs, proteins and noncoding RNAs inherited from maternal cytoplasm, which may affect epigenetic processes that critically depend on maternally inherited factors (Maheshwari & Barbash, 2011), such as when maternally inherited material fails to silence embryonic lethality induced by paternally inherited blockade of satellite DNA (Maheshwari & Barbash, 2011).

On the other hand, crossing a male American paddlefish with a female sterlet resulted in viable hybrid offspring, as did crossing a male American paddlefish with a female Russian (Káldy et al., 2020) or pallid (Flamio Jr et al., 2021) sturgeon. The chromosome number of the hybrids was identical to the chromosome number of the parent species, as in the case of hybrid offspring with pallid sturgeon, where the chromosome number of the parent species was also identical (Flamio Jr et al., 2021). In contrast, the hatching rate of viable hybrids of sterlet and paddlefish is much lower than that of purebred sterlet and paddlefish. This is not surprising, as there is already a big difference in successful crossing between inter- and intrageneric hybridization. The hatching rates between salmonid species were equivalent to the purebred control in 18 out of 43 intrageneric hybridizations, while in intergeneric hybridization, it was equivalent to the purebred control in three out of 46 hybridizations (Chevassus, 1979).

Conclusions

The hybridization between female sterlet and male American paddlefish was proved with viable verified offspring harvested, but embryos from reciprocal hybridization do not survive the stage of gastrula or early neurula. To date, successful hybridization with viable offspring between the Acipenseridae and Polyodontidae families has been reported only when the American paddlefish were applied as paternal parents. Possibly, the unidirectional hybridization observed in this study was attributed to genetic incompatibility during early embryonic development (and not to the fertile incompatibility), as indicated by microsatellite marker analysis. Further investigation of the unidirectional hybridization is warranted.

Supplemental Information