Habitat fragmentation threatens wildlife populations and may cause reduced genetic variation, fitness, and even local extinction, but effects differ across species.

Here, we addressed effects of habitat fragmentation using the harvester ant Messor structor, a habitat specialist in natural xerothermic grassland, in 54 habitat islands of a fragmented 200 km² area in Austria analysing microsatellites, mitochondrial DNA, infection with the endocellular bacterium Wolbachia, and morphology.

We found (a) pronounced genetic diversity and two mitochondrial lineages that we infer to originate from separate glacial refugia. (b) Wolbachia infection rates were high, but the two strains detected, correlating with the mitochondrial lineages, do not seem to have induced a reproductive barrier. (c) Habitat islands with fewer ant nests had less allelic richness, and the ones with less allelic richness had reduced heterozygosity, as measured by mean locus heterozygosity; this indicated inbreeding. (d) Fluctuating asymmetry of workers, known to be low in fit individuals, positively correlated with heterozygosity, as measured by the interallelic distance of heterozygous loci; high levels of this distance probably are due to the mixing of lineages, and facing such a potential outbreeding depression, vulnerability for inbreeding may increase. (e) Gyne mesosoma size in less connected habitat was smaller than that of gynes in better connected habitat, indicating reduced flight ability.

Our work highlights the use of multiple approaches to evaluate species responses to habitat fragmentation, showcasing the importance of historical colonization and current habitat connectivity to the maintenance of genetic and phenotypic diversity.