DOI: 10.5061/dryad.xksn02vj4
title: Microsatellite genotypes of A. chlorotica parentage analysis

description: 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.
description: 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, <em>A. chlorotica</em> were found in high density
 clusters (i.e. patches). Regardless of their colour morph, we collected a
 total of 157 <em>A. chlorotica</em> 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.