Impairment of reproductive capabilities in three subsequent generations of asymmetric hybrids between Eisenia andrei and E. fetida from French, Hungarian and Polish laboratory colonies

Abstract

Eisenia andrei (Ea) and E. fetida (Ef) lumbricid earthworms are simultaneous hermaphrodites potentially capable of self-fertilization and hybridization. We have shown previously that reproductive isolation in these species is incomplete in Ea and Ef earthworms of French provenance, as viable offspring appeared in inter-specific pairs. Fertile asymmetric hybrids developed from Ea-derived ova fertilized by Ef-derived spermatozoa, as well as Ea or Ef specimens derived after self-fertilization (resulting from admixture of endogenously produced spermatozoa with sperm from a partner), but never Ef-hybrids from Ef-ova fertilized by Ea-spermatozoa. The latter appeared only in backcrosses of Ea-hybrids with the Ef. Here we show that these phenomena are not unique for French Ea/Ef earthworms, but are shared by earthworms from French, Hungarian, and Polish laboratory cultures. Semi-quantitative studies on fertility of Ea-derived hybrids revealed gradually decreasing numbers of offspring in three successive generations, more rapid in backcrosses with Ef than with Ea, and the absence of progeny in pairs of hybrids, despite the presence of cocoons in almost all pairs. Based on species specific mitochondrial and nuclear DNA sequences, we provide the first examples of two unique sterile hybrids with mitonuclear mismatch and potential mitonuclear incompatibility among offspring of one of the hybrid+Ef pairs. Earthworms from the investigated populations did not reproduce when kept from hatching in isolation or with representatives of Dendrobaena veneta but started reproducing upon recognition of a related partner, such as Ea, Ef or their hybrids. The existence of Ea or Ef specimens among offspring of hybrid+Ea/Ef pairs might be explained either by partner-induced self-fertilization

Polymorphic microsatellite markers demonstrate hybridization and interspecific gene flow between lumbricid earthworm species, Eisenia andrei and E. fetida

“…Lumbricid earthworms Eisenia andrei (Ea) and E. fetida (Ef) originally derived from laboratory stocks at the University in Lille (France) were cultured for a decade in the laboratories of the Institute of Zoology and Biomedical Research of the Jagiellonian University (Krakow, Poland), and in parallel for the last five years in the College of Natural Sciences, University of Rzeszo ´w [7,9,10,28,29]. For several years they were used as convenient models for studies of hybridization of these simultaneous hermaphrodites by genotyping them by species-specific sequences of mitochondrial COI gene ('a' for Ea and 'f' for Ef) and diploid nuclear sequences of 28s rRNA gene of maternal/paternal origin ('A' for Ea and 'F' for Ef) as aAA, fFF and aAF or fFA interspecific hybrids [7][8][9][10]. Every genotyped specimen from these experiments was marked by the genetic symbols followed by a unique numerical code and all sequences have been deposited in GeneBank [7,9,10,28] while ethanol-fixed posterior segments were preserved for further use.…”

“…For several years they were used as convenient models for studies of hybridization of these simultaneous hermaphrodites by genotyping them by species-specific sequences of mitochondrial COI gene ('a' for Ea and 'f' for Ef) and diploid nuclear sequences of 28s rRNA gene of maternal/paternal origin ('A' for Ea and 'F' for Ef) as aAA, fFF and aAF or fFA interspecific hybrids [7][8][9][10]. Every genotyped specimen from these experiments was marked by the genetic symbols followed by a unique numerical code and all sequences have been deposited in GeneBank [7,9,10,28] while ethanol-fixed posterior segments were preserved for further use.…”

“…Then another 15 specimens per species were used to establish numbers of alleles of potential microsatellite markers. Finally DNA samples from ethanol-fixed posterior segments of 24 earthworms from previous laboratory studies on hybridization [7,9] were used for a pilot application of microsatellite markers in tracking interspecific gene flow between Ea and Ef.…”

“…Asymmetrical hybridization has been experimentally proven in the progeny of laboratory-paired Ea and Ef virgin specimens delimited by species-specific sequences of the mitochondrial COI gene of maternal origin and the 28s rRNA gene of maternal/paternal origin [7,8]. The first generation offspring of these hermaphroditic species included self-fertilized Ea and Ef specimens and fertile hybrids derived from Ea ova fertilized by Ef spermatozoa, while sterile hybrids from Ef ova fertilized by Ea spermatozoa appeared only among progeny of back-crossed Ea-derived hybrids with Ef parental specimens [7][8][9][10]. Although the reproductive capabilities of hybrids was gradually impaired [9], interspecies gene flow was documented by intermediate pigmentation patterns and by the presence of Ea-specific fluorophore not only in hybrids but also in a few Ef specimens [4,8].…”

“…The first generation offspring of these hermaphroditic species included self-fertilized Ea and Ef specimens and fertile hybrids derived from Ea ova fertilized by Ef spermatozoa, while sterile hybrids from Ef ova fertilized by Ea spermatozoa appeared only among progeny of back-crossed Ea-derived hybrids with Ef parental specimens [7][8][9][10]. Although the reproductive capabilities of hybrids was gradually impaired [9], interspecies gene flow was documented by intermediate pigmentation patterns and by the presence of Ea-specific fluorophore not only in hybrids but also in a few Ef specimens [4,8]. Novel species-specific genetic markers are needed for detailed studies on mechanisms of hybridization and gene introgression in these convenient model species.…”