In organisms where individuals mate multiply, knowledge on the proportion of offspring sired by the last male to mate (P₂) under field conditions is important for a thorough understanding of how sexual selection works in nature. In many insect groups, pronounced intraspecific variation in P₂ is commonplace. Interestingly, however, in stark contrast to these observations, compilation of P₂ data in dragonflies and damselflies (Odonata) indicate that a high P₂, seldom below 0.95, is a feature of this taxon. Here we used double digest restriction-site associated DNA sequencing (ddRADseq) to generate a panel of single nucleotide polymorphisms (SNPs) with which we could determine paternity and estimate values of P₂ in the offspring of 19 field-collected pairs of the emerald damselfly Lestes sponsa. We also estimated the relationship between P₂ and male genital shape of 16 males using geometric morphometric analysis. P₂ was variable (range=0.0-1.0; mean=0.5), and there was a marginally non-significant (P=0.069) relationship between genital shape and P2, suggesting that male with a high P₂ had an aedeagus with a broader tip. We suggest that the high P₂-values reported in past studies in Odonata is partly due to the methods used to infer paternity. Use of SNPs to determine patterns of paternity and P₂ in odonates is needed for a better appraisal of fitness in odonates, and would open many future avenues for use of odonates as models of sexual selection.

Genotyping by double digest restriction associated DNA sequencing (ddRADseq). Libraries were created by digesting DNA with EcoRI-HF and MseI, after which adaptors with individual tags were ligated with T4 DNA ligase, and PCR with Q5 DNA polymerase (New England Biolabs, Massachusetts, USA). Samples were then pooled and size selected on agarose gels. Libraries were sequenced on a single lane of an Illumina HiSeq2500 flow-cell (125 bp paired end) to generate 180 M read pairs. Single nucleotide polymorphisms (SNPs) were called using STACKS v.2.2 denovo-map pipeline. We set the number of mismatches allowed between stacks within individuals (-M) and between individuals (-n) to 2. Final data were filtered in the populations program of STACKS to retain only the first SNP of each RAD-tag that could be found in 80% of all the individuals at a minor allele frequency ≥0.05. 

Data in DRYAD are the raw data (SNPs called from ddRAD genotyping) in VCF format, which were used to determine parentage in the emerald damselfly. Note that these data were filtered as described in the manuscript before conducting paternity assignment.

Gra / Gni indicate the population

m / f indicate the parent’s sex (male / female)

o indicates that these individuals were offspring  

For example, Gra03m and Gra03f would be a pair, who produced (may have sired in the case of the male) the clutch of offspring oGra_03_1, oGra_03_2, oGra_03_3 … etc.