Historical events of population fragmentation, expansion and admixture over geological time may result in complex patterns of reproductive isolation and may explain why, for some taxa, the study of mitochondrial (mt) and nuclear (nu) genetic data results in discordant evolutionary patterns. Complex patterns of taxonomic diversity were recently revealed in earthworms for which distribution is largely the result of paleogeographical events. Here, we investigated reproductive isolation patterns in a complex of cryptic species of earthworms in which discordant patterns between mt and nu genetic lineages were previously revealed, the Allolobophora chlorotica aggregate. Using four nu microsatellite markers and a fragment of the cytochrome c oxidase subunit I mt gene we carried out a parentage analysis to investigate the mating patterns (i) between individuals belonging to two divergent mt lineages that cannot be distinguished with nu markers and (ii) between individuals belonging to lineages that are differentiated both at the mt and nu levels. Among the 157 field collected individuals, 66 adults were used in cross-breeding experiments to form 22 trios based on their assignment to a mt lineage, and 453 obtained juveniles were genotyped. We showed that adults that mated with both their potential mates in the trio produced significantly more juveniles. In crosses between lineages that diverged exclusively at the mt level, a sex-specific pattern of reproduction characteristic to each lineage was observed, suggesting a possible conflict of interest concerning the use of male/female function between mating partners. In crosses between lineages that diverged both at the mt and nu level, a high production of cocoons was counterbalanced by a low hatching rate, suggesting a post-zygotic reproductive isolation. Different degrees of reproductive isolation, from differential sex allocation to post-zygotic isolation, were thus revealed. Lineages appear to be at different stages in the speciation process, which likely explain the observed opposite patterns of mito-nuclear congruence.

The sampling was carried out at a farm site (Walton Hall Farm, Preston, UK) in a field grazed by cattle. The soil is an alluvial, sandy clay with a pH of 8.3. At this site, A. chlorotica were found in high density clusters (i.e. patches). Regardless of their colour morph, we collected a total of 157 A. chlorotica individuals: 46 juveniles in 2012, and 71 juveniles and 40 adults (i.e. clitellate) in 2015. From each individual, we removed the last segments of the caudal section and preserved it in 96% ethanol before DNA extraction using the NucleoSpin® 96 Tissue kit (Macherey-Nagel).

To explore the nuclear genetic variation in the field population, we genotyped individuals at four highly polymorphic microsatellite loci, Ac127, Ac170, Ac418 and Ac476, as described in Dupont et al. (2011). We amplified the loci by polymerase chain reaction (PCR) in one multiplex set and in 12 μl reactions using 10 ng of DNA and the Qiagen ® Multiplex Kit, according to the manufacturer’s protocol. The migration of the PCR products was carried out on an ABI 3130 xl Genetic Analyzer using the LIZ500 size standard (Applied Biosystems); alleles were scored using GeneMapper 5 software (Applied Biosystems). 

We kept the 71 juveniles collected in 2015 in individual pots and placed them in an incubator until they reached adulthood, in order to have virgin adults. We performed cross-breeding experiments in trios to test for multiple paternity and reproductive success between individuals from divergent lineages. On the basis of the available individuals from the different lineages, we used a subset of 66 adults to form 22 trios that we labelled from letter A to V . We formed six types of trios with up to two adults per lineage. In details , these trios were composed of: (A-E) two adults of L2 and one adult of L3, (F-J) two adults of L3 and one adult of L2, (K- L) three adults of L2, (M-N) three adults of L3, (O-R) two adults of L2 and one adult of L1, (S-V) two adults of L3 and one adult of L1. We collected the cocoons produced by each trio monthly for four months and kept them in an incubator until hatching. Upon hatching, the juveniles were fixed in ethanol before we performed DNA extraction, as described above.