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

Barbara Plytycz,Tomasz Panz,Paweł Grzmil,Janusz Bigaj,P Pardha-Saradhi

doi:10.1371/journal.pone.0204469

Abstract

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.

Highlights

Lumbricid earthworms from Eisenia sp. are valuable models in various scientific disciplines like biochemistry, ecotoxicology, and biomedicine [1,2,3,4,5] where proper species delimitation is crucial
Body pigmentation is often not conclusive, during our earlier studies of Eisenia andrei (Ea)/E. fetida (Ef) delivered from France we have used various methods for proper distinction of specimens of these two species [10]; among others, coelomic fluid was analyzed in respect of presence of fluorescence spectra considered to be a fingerprint of E. andrei, hypothetically derived from 4-methylumbelliferyl β-D-glucoronide [11], called the MUG fluorophore [10; 12; 13], and here shortly the M-fluorophore
As visible on phylogenetic tree of 46 descendants of Ea+Ef earthworms arranged on the basis of mitochondrial COI gene of maternal origin, all 29 specimens derived from E. andrei ova, i.e. 10 aAA and19 aAF hybrids, were M-positive

Summary

Introduction

Lumbricid earthworms from Eisenia sp. are valuable models in various scientific disciplines like biochemistry, ecotoxicology, and biomedicine [1,2,3,4,5] where proper species delimitation is crucial. We have detected such fluorescent biomarker not exclusively in Ea and in some Ef specimens– we considered them as hypothetical hybrids [10; 12] Just this observation, together with a wide spectrum of pigmentation patterns of earthworms from our Ea/Ef cultures prompted us to test a hypothesis about the existence of inter-specific hybrids between Ea and Ef, both of them being simultaneous hermaphrodites [14] capable to self-fertilization [15]. Using the methods of the combined mitochondrial and nuclear markers we detected on the Ea branch of the COIbased phylogram both the ‘pure’ Ea specimens (aAA) and relatively common inter-specific hybrids (aAF), while on the Ef branch there were both ‘pure’ fFF specimens and a few sterile fFA hybrids [18]

Methods

Results

Discussion

Conclusion