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Uniformly pigmented  Eisenia andrei  (Ea) and striped  E .  fetida  (Ef) lumbricid earthworms are hermaphrodites capable of self-fertilization, cross-fertilization, and asymmetrical hybridization. The latter was detected by genotyping of F1 and F2 progeny of the controlled Ea+Ef pairs by species-specific sequences of maternal mitochondrial COI genes and maternal/paternal nuclear S28 rRNA genes. Among F1offspring there were self-fertilized Ea (aAA), Ef (fFF), and cross-fertilized fertile Ea-derived hybrids (aAF); the latter mated with Ea and gave new generation of Ea and hybrids, while mated with Ef gave Ea, Ef, Ea-derived hybrids and sterile Ef-derived hybrids (fFA). Coelomic fluid of Ea exhibits unique fluorescence spectra called here the M-fluorescence considered as a molecular biomarker of this species. Since similar fluorescence was detected also in some Ef (hypothetical hybrids?), the aim of present investigations was to identify the M-positive earthworms among families genotyped previously. It was assumed that factor/s responsible for metabolic pathways leading to production of undefined yet M-fluorophore might be encoded/controlled by alleles of hypothetical nuclear gene of  Eisenia  sp. segregating independently from species-specific S28 rRNA nuclear genes, where ‘MM’ or ‘Mm’ alleles determine M-positivity while ‘mm’ alleles determine M-negative phenotypes. Spectra of M-fluorescence were detected in all 10 Ea (aAAMM) and 19 Ea-derived hybrids (aAFMm), three of four Ef-derived hybrids (fFAMm) and one ‘atypical’ Ef (fFFMm) among 13 Ef earthworms. Among progeny of ‘atypical’ M-positive Ef (fFFMm) reappeared ‘typical’ M-negative Ef (fFFmm), confirming such hypothesis. Alternatively, the M-fluorescence might be dependent on unknown gene products of vertically-transmitted  Ea -specific symbiotic bacteria sexually transferred to the Ef partner. Hypotheses of intrinsic and external origin of M-fluorescence might complement each other. The presence/absence of M-fluorophore does not correspond with body pigmentation patterns; Ef-characteristic banding appeared in posterior parts of hybrids body. In conclusion, Ea/Ef hybridization may serve for further studies on bi-directional gene flow.

Asymmetrical hybridization and gene flow between Eisenia andrei and E. fetida lumbricid earthworms