Alpine ecosystems are frequently characterized by an abundance of wing‐reduced insect species, but the drivers of this biodiversity remain poorly understood. Insect wing reduction in these environments has variously been attributed to altitude, temperature, isolation, habitat stability or decreased habitat size. We used fine‐scale ecotypic and genomic analyses, along with broad‐scale distributional analyses of ecotypes, to unravel the ecological drivers of wing reduction in the wing‐dimorphic stonefly Zelandoperla fenestrata complex. Altitudinal transects within populations revealed dramatic wing reduction over very fine spatial scales, tightly linked to the alpine treeline. Broad biogeographical analyses confirm that the treeline has a much stronger effect on these ecotype distributions than altitude per se. Molecular analyses revealed parallel genomic divergence between vestigial‐winged (high altitude) and full‐winged (low altitude) ecotypes across distinct streams. These data thus highlight the role of the alpine treeline as a key driver of rapid speciation, providing a new model for ecological diversification along exposure gradients.

Recent genomic analyses have highlighted parallel divergence in response to ecological gradients, but the extent to which altitude can underpin such repeated speciation remains unclear. Wing reduction and flight loss have apparently evolved repeatedly in montane insect assemblages, and have been suggested as important drivers of hexapod diversification. We test this hypothesis using genomic analyses of a widespread wing-polymorphic stonefly species complex in New Zealand. We identified over 50,000 polymorphic genetic markers generated across almost 200 Zelandoperla fenestrata stonefly specimens using a newly generated plecopteran reference genome, to reveal widespread parallel speciation between sympatric full-winged and wing-reduced ecotypes. Rather than the existence of a single, widespread, flightless taxon (Zelandoperla pennulata), evolutionary genomic data reveal that wing-reduced upland lineages have speciated repeatedly and independently from full-winged Z. fenestrata. This repeated evolution of reproductive isolation between local ecotype pairs that lack mitochondrial DNA differentiation suggests that ecological speciation has evolved recently. A cluster of outlier SNPs detected in independently wing-reduced lineages, tightly linked in an approximately 85 kb genomic region that includes the developmental ‘supergene’ doublesex, suggests that this ‘island of divergence’ may play a key role in rapid ecological speciation.

The secondary loss of flight in previously winged insect lineages has long fascinated biologists. Habitat stability and isolation are thought to play important roles in driving wing reduction (Roff 1990, 1994), with exposure to high winds suggested to accelerate this process (Darwin 1859), although the role exposure plays in insect wing loss has never been empirically demonstrated. Here we assess fine‐scale distributional records from a diverse regional stonefly assemblage, to demonstrate a widespread association between wing loss and the treeline in New Zealand. The observed pattern suggests that exposure plays a crucial role driving wing loss in alpine insects.

Wing polymorphism is a prominent feature of numerous insect groups, but the genomic basis for this diversity remains poorly understood. Wing reduction is a commonly observed trait in many species of stoneflies, particularly in cold or alpine environments. The widespread New Zealand stonefly Zelandoperla fenestrata species group (Z. fenestrata, Z. tillyardi, Z. pennulata) contains populations ranging from fully winged (macropterous) to vestigial-winged (micropterous), with the latter phenotype typically associated with high altitudes. The presence of flightless forms on numerous mountain ranges, separated by lowland fully winged populations, suggests wing reduction has occurred multiple times. We use Genotyping by Sequencing (GBS) to test for genetic differentiation between fully winged (n = 62) and vestigial-winged (n = 34) individuals, sampled from a sympatric population of distinct wing morphotypes, to test for a genetic basis for wing morphology. While we found no population genetic differentiation between these two morphotypes across 6,843 SNP loci, we did detect several outlier loci that strongly differentiated morphotypes across independent tests. These findings indicate that small regions of the genome are likely to be highly differentiated between morphotypes, suggesting a genetic basis for wing reduction. Our results provide a clear basis for ongoing genomic analysis to elucidate critical regulatory pathways for wing development in Pterygota.

Events of inbreeding are inevitable in critically endangered species. Reduced population sizes and unique life history traits can increase the severity of inbreeding, leading to declines in fitness and increased risk of extinction. Here, we investigate levels of inbreeding in a critically endangered flightless parrot, the kākāpō (Strigops habroptilus), wherein a highly inbred island population and one individual from the mainland of New Zealand founded the entire extant population. Genotyping-by-sequencing (GBS), and a genotype calling approach using a chromosome-level genome assembly, identified a filtered set of 12,241 single nucleotide polymorphisms (SNPs) among 161 kākāpō, which together encompass the total genetic potential of the extant population. Multiple molecular-based estimates of inbreeding were compared, including genome-wide estimates of heterozygosity (FH), the diagonal elements of a genomic-relatedness matrix (FGRM), and runs of homozygosity (RoH, FRoH). Additionally, we compared levels of inbreeding in chicks from a recent breeding season to examine if inbreeding is associated with offspring survival. The density of SNPs generated with GBS was sufficient to identify chromosomes that were largely homozygous with RoH distributed in similar patterns to other inbred species. Measures of inbreeding were largely correlated and differed significantly between descendants of the two founding populations. However, neither inbreeding nor ancestry were found to be associated with reduced survivorship in chicks, owing to unexpected mortality in chicks exhibiting low levels of inbreeding. Our study highlights important considerations for estimating inbreeding in critically endangered species, such as the impacts of small population sizes and admixture between diverse lineages.