In eusocial Hymenoptera, haplodiploidy and polyandry may facilitate selection for hybridization. Interspecific hybridization is widespread in ants and can lead to hybrid inviability as well as the formation of new species through hybrid speciation. However, in ants, polyandry is uncommon. By analyzing microsatellite markers on 15 ant workers per colony, we show that the mating system of 28 pure colonies of Tetramorium immigrans, 15 pure colonies of Tetramorium caespitum and 27 hybrid colonies is a monogyne/polyandrous mating system, with a higher mating rate in T. caespitum (mean = 2.4 males vs. 1.7 in T. immigrans). Hybrid queens, but no hybrid fathers, were deduced from workers’ genotypes, in accordance to Haldane’s rule extended to haplodiploid organisms, which states that the haploid sex should more often be sterile or inviable. In five colonies, hybridization and multiple mating allowed the simultaneous production of both hybrid and nonhybrid offspring. Although rare, these situations hinted at asymmetrical, larger contributions of T. immigrans vs. T. caespitum males to offspring production. Together, these findings point towards a complex and dynamic mating system in T. immigrans and T. caespitum and contribute to better understand interspecific hybridization mechanisms and their consequences on genetic and taxonomic diversity. The study of polyandry within a hybrid zone is unprecedented and opens new opportunities to better understand interspecific hybridization mechanisms and their short- to long-term consequences.

DNA was extracted from whole individuals, which were crushed and then mixed with 150 μL of Chelex® 100 and 10 μL of proteinase K (15 mg.mL-1) at room temperature; the solution was incubated at 55°C overnight. The 17 microsatellites developed for T. immigrans or T. tsushimae by Steiner et al. (2008) were organized in three multiplex PCR mixes. All three mixes had a total volume of 10 μL with 1X MasterMix (kit type-it microsatellite PCR Qiagen n°206246) and 2 μL DNA. Mix1 contained 0.08 μM of tspE53a primers, and 0.2 μM of tspE52b, tspE52d, tspE52k, ttsU55a and ttsU56d primers. Mix2 contained 0.15 μM of tspE51oR3 primers, 0.2 μM of tspE51a, tspE51b, tspE51d, ttsU58i and ttsU59j primers, and 0.3 μM of tspE51i primers. Mix 3 contained 0.08 μM of ttsU54e, 0.15 μM of tspE52a, and 0.2 μM of tspE53b and ttsU57l primers. Amplifications consisted in 5 min at 95°C, then 32 cycles (30 s at 95°C, 90 s at 60°C, 30 s at 72°C), and 30 min at 60°C. All PCR products were analyzed with an ABI 3730xl sequencer. Electropherograms were read and interpreted with Genemarker v.1.95 (SoftGenetics). One marker was discarded because it presented risks of misinterpretation at the reading stage. In addition, workers where alleles were not clearly legible for at least 12 markers were removed from the analysis, and colonies with less than 12 (pure T. immigrans or T. caespitum) or 14 (hybrids colonies) legible worker genotypes were discarded. The dataset included genotypes for 16 microsatellite markers (Tspe52d, Tspe52b, Tspe52k, Ttsu56d, Tspe53a, Ttsu55a, Ttsu59j, Tspe51i, Ttsu58i, Tspe51a, Tspe51o, Tspe51b, Tspe51d, Tspe52a, Tspe53b, Ttsu57l) for 28 putative T. immigrans colonies, 15 putative T. caespitum colonies, and 27 putative hybrid colonies (14 F1 and 13 backcrosses) with an average of 14.8 worker genotypes per colony, and a total 1038 genotypes for 16 markers. Based on the worker genotypes, the genotypes of queens and their mates have been inferred in each colony. At each locus two alleles shared by all the workers were assigned to the queen, while haploid fathers’ genotypes were determined by the alleles unassigned to the mother. This pattern was then iterated over the 16 markers until reaching a minimal number of sires per queen.

Localities : X,Y coordinates of the sampled families

Microsatellites data : Table of the microsatellites; Missing data coded as -9; Family : name of the family sampled; Cast: F = female, M = male, W = worker; Tspe52d, Tspe52b, Tspe52k, Ttsu56d, Tspe53a, Ttsu55a, Ttsu59j, Tspe51i, Ttsu58i, Tspe51a, Tspe51o, Tspe51b, Tspe51d, Tspe52a, Tspe53b, Ttsu57l: names of the microsatellites developed for Tetramorium immigrans or T. tsushimae by Steiner et al. (2008).