Event Abstract Back to Event Interspecies hybridization is not the end: the lesson from asexual fishes from European spined loaches complex (Cobitis) Dmitry Dedukh1*, Zuzana Majtanova1, Anatolie Marta1, Martin Psenicka2, Jan Kotusz3, Jiri Klima4, Dorota Juchno5, Alicja Boron5 and Karel Janko1 1 Laboratory of Fish Genetics, Institute of Animal Physiology and Genetics (ASCR), Czechia 2 Faculty of Fishery and Protection of Waters, University of South Bohemia, Czechia 3 University of Wrocław, Poland 4 Institute of Animal Physiology and Genetics (ASCR), Czechia 5 University of Warmia and Mazury in Olsztyn, Poland Biological species are fundamental evolutionary units separated from each other via different prezygotic and postzygotic barriers (Coyne, Orr, 1998). Such barriers are frequently disrupted leading to interspecies hybrids, although such hybrids are generally sterile (Mallet, 2007; Abbott et al., 2013). One of the outcomes of hybrid sterility is the failure of chromosomes from different species to pair and recombine in meiotic prophase causing abruption of meiosis and/or aneuploid gametes (Arnold, 1997; Chapman, Burke, 2007). Nevertheless, interspecies hybridization allows instant creation of novel gene combinations which ultimately have a great impact on evolution and speciation process (Mallet, 2007; Abbott et al., 2013; Seehausen et al., 2014). In plant hybrids, spontaneous tetraploidization can rescue the meiosis of hybrids via the pairing of identical chromosomal sets and formation of diploid gametes (Abbott et al., 2013). However, in animal hybrids, spontaneous polyploidization is a quite rare phenomenon whereas fertility can be restored by particular gametogenetic alterations ultimately connected with so-called asexual reproductive modes (Dawley, Bogart 1989; Schön et al., 2009). Such alterations allowing hybrid organisms produce gametes vary from complete loss of meiotic attributes such as chromosomal pairing and recombination (automixis) to those with modified but retained meiosis (apomixis) (Schön et al., 2009; Neaves, Baumann, 2011; Stenberg, Saura, 2013). These evolutionary novelties give rise to a great variety of clonal and hemiclonal reproductive strategies such as parthenogenesis (occurring in some fishes, reptiles), gynogenesis (spread in various fishes), kleptogenesis (amphibians) and hybridogenesis (fishes, amphibians) (Dawley, Bogart 1989; Schön et al., 2009). However, the connection of interspecies hybridization with asexuality still remains a big mystery. Here, we describe the consequence of interspecies hybridization for males and females on the model of European spined loaches fishes from Cobitis taenia hybrid complex. European spined loaches complex includes several sexually reproducing species among which C. taenia (TT, 2n = 48 chromosomes) and C. elongatoides (EE 2n = 50 chromosomes) can easily hybridize producing diploid (ET, 2n = 49 chromosomes) and triploid (ETT, EET, 3n = 73 and 74 chromosomes correspondingly) hybrid progeny (Bohlen, Rab, 2001). Notably, hybrid females and males drastically differ in their ability to reproduce: diploid and triploid hybrid males are sterile while females are fertile and are able to produce progeny asexually via gynogenesis (Choleva et al., 2012). To find out while males are sterile we analyzed meiosis of diploid and triploid hybrids and sexual species. Using flow cytometry and histology of gonads from hybrid males we found aberrant nonreduced sperm and enrichment of primary spermatocytes probably accumulated due to problems during chromosomal pairing comparatively to sexual species. To answer why hybrids cannot produce sperm and accumulated cells during meiotic prophase we focused on the analysis of meiosis in details. After analysis of more than 250 metaphase plates from each of 4 diploids and 5 triploid hybrid male, we observe the high number of cells during prophase and first metaphase of meiosis not only with bivalents but also with improperly paired chromosomes. Moreover, we didn’t found any evidence of an increased number of chromosomes during meiosis, indicating the absence of premeiotic genome doubling. Using fluorescent in situ hybridization with PNA probes to telomeric sequences we proved the presence of bivalents and also detected univalents and multivalent during metaphase one of diploid and triploid hybrid males. Comparative genome hybridization made on the same slides demonstrated that some bivalents consist of chromosomes from separate species while univalents are formed by chromosomes from both parental genomes. To confirm abnormal bivalent formation we performed analysis of chromosomes during pachytene using immunofluorescent detection of lateral and central components of synaptonemal complexes (SYCP3 and SYCP1 proteins) as well as recombination (MLH1 protein) in diploid and triploid hybrid males and sexual species. Diploid hybrids demonstrated numerous abnormalities of chromosomal pairing with just a few normally formed bivalents and significantly decreased the number of recombination. In triploid hybrids, we observed partially restored bivalent formation and increased recombination, probably because of homospecific chromosome pairing. However, in triploid hybrids, chromosomal pairing is still insufficient to accomplish meiosis and form reduced gametes. We suggest that abnormal pairing of some chromosomes prevents further propagation of meiosis during metaphase 1 checkpoint and leads to the sterility of hybrid males. In order to investigate why hybrid females are able to reproduce we analysed diploid (ET) and triploid (ETT) hybrid females. After analysis of synaptonemal complexes during pachytene and chromosomes during diplotene (also known as “lampbrush chromosomes”), we found that oocytes of sexual species include the number of bivalents which is two times less than their chromosomal number: we observed 24 bivalents in C. taenia and 25 bivalents in C. elongatoides. However after analysis of oocytes from diploid ET and triploid ETT hybrids we found 49 and 73 bivalents correspondingly. Analysis of synaptonemal complexes formation confirmed our results from diplotene chromosome analysis for sexual species as well as diploid and triploid hybrid females. We conclude that all hybrid females are able to form oocytes with the number of bivalents, which coincide with the number of chromosomes in somatic cells. Our results suggest doubling of chromosomes prior to the meiosis during diploid and triploid hybrid female gametogenesis. According to unique bivalent morphology during diplotene, we conclude that in hybrids recombination occurred between identical copies of chromosomes but not between orthologous chromosomes. Analysis of recombination points during pachytene and chiasmata during diplotenes bivalents showed two and three times increased recombination in diploid and triploid hybrids correspondingly comparatively to sexual species. In summary, hybrid females are able to reproduce because of premeiotic endoreplication during their gametogenesis rescuing them from sterility. Both premeiotic endoreplication in hybrid females and sterility of hybrid males successfully prevent gene flow between two parental species.