Figures and data
(a) Phylogenetic tree
based on exome-wide SNP data of the species used in the current crossing experiments. Red color indicates Bicanestrini group; green color indicates Cobitis sensu stricto group; yellow color indicates Adriatic group. (b) Distribution map of the spined loaches (Cobitis) included in this study. While Cobitis elongatoides, C. taenia, C. tanaitica, are known to act as parental species in hybridization events and emergence of clonal lineages, the other species (C. strumicae, C. ohridana, C. taurica, C. pontica) are known to be involved in secondary hybridization events. Abbreviations for all species in brackets. (c) Reproduction scheme of the clonal hybrids in the C. taenia complex, where E represents a haploid genome of C. elongatoides (orange), and T – of C. taenia (blue). Conventionally, hybridization between EE female and TT male lead to ET sterile males and clonal ET females that reproduce via gynogenesis. Gynogenetic females are pseudogamous, i.e. females produce diploid eggs via premeiotic genome endoreplication. Such eggs require sperm from a parental species (e.g. T sperm) to activate the embryonic development. The sperm genome is not incorporated in the hybrid’s genome but is instead eliminated after the egg’s activation, skipping karyogamy. All hybrid males are sterile because they do not produce spermatozoa or their extremely rare spermatozoa are aberrant and unable to fertilize eggs.
Matrix with successfully established and analyzed crosses between eight species of Cobitis.
Each cell represents a particular cross between parental species including the information about maternal and paternal species (i.e. the direction of the cross). Phylogenomic tree plotted along margins indicates the relationships among crossed species based on exome-wide SNP data. Each color represents a particular reproductive output in F1 hybrids; green labeling indicates production of haploid gametes via normal meiosis; yellow color indicates the F1 progeny which produce unreduced gametes and present fully developed gonads; Red color denotes sterile progeny, predominantly referring to hybrid males. Grey labels potentially sterile hybrids for which we did not obtain diplotenic cells to fully confirm their reproductive output. The striped cell represents the F1 hybrid combination which was previously described 17 and involves the species used in this study.
Comparison of pachytene spermatocytes between male of sexual diploid species (a), hybrids’ genotypes (b,c), and corresponding gametogenic pathways (d-f).
The spread of pachytene spermatocytes of C. elongatoides with 25 completely paired bivalents (a), and diploid NP hybrid with almost paired chromosomes (b) and OE hybrid with aberrant pairing, including bivalents and univalents (c). Thick arrows indicate bivalents; thin arrows indicate univalents. Scale bar = 10 µm. Schematic representation of the gametogenic pathway including presumptive karyotype composition in gonocytes and pachytene cells in males of sexual species (d) and NP hybrids (e) that are able to complete pairing and form gametes meiotically, and OE hybrids (f) which exhibit abnormal pairing leading to sterility.
Comparison of pachytene oocytes between female of sexual diploid species (a) and hybrids genotypes (b,c,d) and corresponding gametogenic pathways (e-h).
The SC spreads from pachytene oocyte of C. ohridana with fully paired 25 bivalents (a), diploid OB hybrid with almost paired chromosomes (b) and OE hybrid female which have pachytene cells with aberrant pairing (c) and cells with fully paired chromosomes emerged after premeiotic genome endoreplication (d). Thick arrows indicate bivalents; thin arrows indicate univalents. Scale bar = 10 µm. Schematic representation of gametogenic pathway including presumptive karyotype composition in gonocytes and pachytene cells. Females of sexual species (e) and OB hybrid (f) which are able to fully or partially complete pairing of chromosomes and form gametes meiotically; OE hybrids (g) exhibit two populations of pachytene oocytes: oocytes with unduplicated genomes and oocytes with duplicated genome. Oocytes with unduplicated genome (g, left) have abnormal pairing leading to the inability of proceed beyond pachytene and thus sterility. Oocytes with duplicated genomes (g, right) have normal pairing and thus leading to the formation of unreduced gametes.
Effect of karyotype dissimilarity on numbers of bivalents in hybrids.
Boxplots showing the number of synaptonemal complexes per cell (y-axis) of the F1 hybrids ranked along x-axis according to the morphological divergence of karyotypes between parental species expressed by AKD index 28. Numbers indicate the AKD index values, subscripts F and M indicate females and males, respectively and experimental crosses are indicated by respective letter combinations.
Reproductive outcomes of hybrids from crosses between species from the subfamily Cobitinae (a).
Plot demonstrating the relationship between reproductive outcome and viability of F1 hybrid loaches and the genetic and karyotype divergences among parental species. Data on F1 hybrids have been obtained in this study as well as from the literature. Karyotype divergence is marked as AKD index and genetic distance is estimated from published RAG1 sequences (K2P distance). Green color shows the ranges of hybrids with sexual reproduction; yellow color shows the ranges of F1 hybrids, which produce unreduced gametes; purple color indicates the ranges of sterility in both sexes; red color shows the ranges of inviable hybrids. To rank reproductive outcomes for every type of cross we labeled them with additional symbols: 
