The chromosome-level genome assembly of the giant dobsonfly Acanthacorydalis orientalis (McLachlan, 1899).

Acanthacorydalis orientalis (McLachlan, 1899) (Megaloptera: Corydalidae) is an important freshwater-benthic invertebrate species that serves as an indicator for water-quality biomonitoring and is valuable for conservation from East Asia. Here, a high-quality reference genome for A. orientalis was constructed using Oxford Nanopore sequencing and High throughput Chromosome Conformation Capture (Hi-C) technology. The final genome size is 547.98 Mb, with the N50 values of contig and scaffold being 7.77 Mb and 50.53 Mb, respectively. The longest contig and scaffold are 20.57 Mb and 62.26 Mb in length, respectively. There are 99.75% contigs anchored onto 13 pseudo-chromosomes. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis showed that the completeness of the genome assembly is 99.01%. There are 10,977 protein-coding genes identified, of which 84.00% are functionally annotated. The genome contains 44.86% repeat sequences. This high-quality genome provides substantial data for future studies on population genetics, aquatic adaptation, and evolution of Megaloptera and other related insect groups.

Anatomical, phenological and genetic aspects of the host-parasite relationship between Andrena vaga (Hymenoptera) and Stylops ater (Strepsiptera).

Stylops ater is an endoparasite of the mining bee Andrena vaga with extreme sexual dimorphism and hypermetamorphosis. Its population structure, parasitization mode, genetic diversity and impact on host morphology were examined in nesting sites in Germany to better understand this highly specialized host­parasite interaction. The shift in host emergence due to stylopization was proven to be especially strong in A. vaga. Around 10% of bees hosted more than 1 Stylops, with at maximum 4. A trend in Stylops' preference for hosts of their own sex and a sex-specific position of extrusion from the host abdomen was found. Invasion of Andrena eggs by Stylops primary larvae was depicted for the first time. Cephalothoraces of female Stylops were smaller in male and pluristylopized hosts, likely due to lower nutrient supply. The genes H3, 18S and cytochrome c oxidase subunit 1 were highly conserved, revealing near-absence of local variation within Stylops. Ovaries of hosts with male Stylops contained poorly developed eggs while those of hosts with female Stylops were devoid of visible eggs, which might be due to a higher protein demand of female Stylops. Male Stylops, which might have a more energy-consuming development, led to a reduction in head width of their hosts. Host masculinization was present in the leaner shape of the metabasitarsus of stylopized females and is interpreted as a by-product of manipulation of the host's endocrine system to shift its emergence. Stylopization intensified tergal hairiness, most strongly in hosts with female Stylops, near the point of parasite extrusion, hinting towards substance-induced host manipulation.

Morphological description of a new species of Capnia (Plecoptera: Capniidae) with DNA barcoding of genus members from the Russian Far East.

Capnia yavorskayae, a new species of the stonefly family Capniidae, is described from the Low Amur River Basin, Khabarovskiy Kray of the Russian Far East, on the basis of female morphological features. Confirmation of the uniqueness of the new species was also molecularly compared to other Capnia, including a few Far Eastern species, C. aligera Zapekina-Dulkeit, C. bargusinica Zapekina-Dulkeit, C. khingana Teslenko, C. kurnakovi Zhiltzova, C. nearctica Banks, C. nigra (Pictet), and C. rara Zapekina-Dulkeit for which DNA barcodes were obtained. We support the distinctiveness of the new species with mitochondrial DNA sequences, comparing it to Capnia from the eastern Palaearctic and Nearctic realms and one Zwicknia species. The new species forms a common clade with C. khingana, C. kurnakovi from the Russian Far East, and an undetermined Capnia species from Honshu, Japan. Each species from the Russian Far East has high interspecific distances from other Capnia species except C. nearctica which was close to C. atra Morton.

X chromosomes show relaxed selection and complete somatic dosage compensation across Timema stick insect species.

Sex chromosomes have evolved repeatedly across the tree of life. As they are present in different copy numbers in males and females, they are expected to experience different selection pressures than the autosomes, with consequences including a faster rate of evolution, increased accumulation of sexually antagonistic alleles and the evolution of dosage compensation. Whether these consequences are general or linked to idiosyncrasies of specific taxa is not clear as relatively few taxa have been studied thus far. Here, we use whole-genome sequencing to identify and characterize the evolution of the X chromosome in five species of Timema stick insects with XX:X0 sex determination. The X chromosome had a similar size (approximately 12% of the genome) and gene content across all five species, suggesting that the X chromosome originated prior to the diversification of the genus. Genes on the X showed evidence of relaxed selection (elevated dN/dS) and a slower evolutionary rate (dN + dS) than genes on the autosomes, likely due to sex-biased mutation rates. Genes on the X also showed almost complete dosage compensation in somatic tissues (heads and legs), but dosage compensation was absent in the reproductive tracts. Contrary to prediction, sex-biased genes showed little enrichment on the X, suggesting that the advantage X-linkage provides to the accumulation of sexually antagonistic alleles is weak. Overall, we found the consequences of X-linkage on gene sequences and expression to be similar across Timema species, showing the characteristics of the X chromosome are surprisingly consistent over 30 million years of evolution.

Convergent consequences of parthenogenesis on stick insect genomes.

The shift from sexual reproduction to parthenogenesis has occurred repeatedly in animals, but how the loss of sex affects genome evolution remains poorly understood. We generated reference genomes for five independently evolved parthenogenetic species in the stick insect genus Timema and their closest sexual relatives. Using these references and population genomic data, we show that parthenogenesis results in an extreme reduction of heterozygosity and often leads to genetically uniform populations. We also find evidence for less effective positive selection in parthenogenetic species, suggesting that sex is ubiquitous in natural populations because it facilitates fast rates of adaptation. Parthenogenetic species did not show increased transposable element (TE) accumulation, likely because there is little TE activity in the genus. By using replicated sexual-parthenogenetic comparisons, our study reveals how the absence of sex affects genome evolution in natural populations, providing empirical support for the negative consequences of parthenogenesis as predicted by theory.

The phylogenic position of aschiphasmatidae in euphasmatodea based on mitochondrial genomic evidence.

The mitochondrial genome (mitogenome) has been regarded as significant source of data to better understand the phylogenetic relationships within the Euphasmatodea, but no mitogenome in Aschiphasmatoidea has been sequenced to date. In this study, two mitogenomes of Orthomeria smaragdinum and Nanhuaphasma hamicercum of Aschiphasmatidae were sequenced and annotated for the first time. The same mitochondrial gene rearrangement structure was present in the two mitogenomes sequenced, showing as the translocation of tRNA-Arg and tRNA-Asn, which conformed to the tandem duplication-random loss and could be used as a possible synapomorphy for Aschiphasmatidae. The phylogenetic results based on the maximum likelihood (ML) and bayesian inference (BI) methods both showed that Aschiphasmatidae and Neophasmatodea in Euphasmatodea are sister taxa. Although the monophyly of Oriophasmata, Occidophasmata, Diapheromeridae, Phasmatidae, Lonchodidae and Bacilloidea has not been solved, the monophyly of Neophasmatodea and Phyllioidea was well supported.

Calculating Orthologous Protein-Coding Sequence Set Probability Using the Poisson Process.

We extend the popular Jukes-Cantor evolution model and calculate the probability of an orthologous nucleotide sequence set [a reference sequence (B1) stays with the other sequences (B-1)], where the sequence evolution [from a last common ancestral sequence (ɑ)] follows the (prospective) Poisson process with the overall event rate λ prorated among mutation types (nucleotide/codon substitution, insertion, and deletion) and sites along each sequence. The corresponding retrospective process (reversing the prospective process) facilitates developing algorithms to calculate the marginal probability [Pr(B1)] (Monte Carlo integration) and sample ɑ (given B1). We calculate probability Pr(B-1|ɑ) based on the identified events (during "ɑ→B-1") from pairwise sequence alignment to implement Pr(B-1|B1) calculation (Monte Carlo integration). Event queue sampling and probability magnifiers are used to improve the computational efficiency when the number of events is large. We finally test our procedure on both simulated and recently studied hexapod transcriptome data (Brandt et al.), where each asexual lineage pairs with its closest related sexual lineage. Rate estimates (for Phasmatodea and Zygentoma) and model comparison indicate that the asexual lineages likely mutate several times faster than their sexual relatives.

Mapping the interaction surface of scorpion ß-toxins with an insect sodium channel.

The interaction of insect-selective scorpion depressant ß-toxins (LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus) with the Blattella germanica sodium channel, BgNav1-1a, was investigated using site-directed mutagenesis, electrophysiological analyses, and structural modeling. Focusing on the pharmacologically defined binding site-4 of scorpion ß-toxins at the voltage-sensing domain II (VSD-II), we found that charge neutralization of D802 in VSD-II greatly enhanced the channel sensitivity to Lqh-dprIT3. This was consistent with the high sensitivity of the splice variant BgNav2-1, bearing G802, to Lqh-dprIT3, and low sensitivity of BgNav2-1 mutant, G802D, to the toxin. Further mutational and electrophysiological analyses revealed that the sensitivity of the WT = D802E < D802G < D802A < D802K channel mutants to Lqh-dprIT3 correlated with the depolarizing shifts of activation in toxin-free channels. However, the sensitivity of single mutants involving IIS4 basic residues (K4E = WT << R1E < R2E < R3E) or double mutants (D802K = K4E/D802K = R3E/D802K > R2E/D802K > R1E/D802K > WT) did not correlate with the activation shifts. Using the cryo-EM structure of the Periplaneta americana channel, NavPaS, as a template and the crystal structure of LqhIT2, we constructed structural models of LqhIT2 and Lqh-dprIT3-c in complex with BgNav1-1a. These models along with the mutational analysis suggest that depressant toxins approach the salt-bridge between R1 and D802 at VSD-II to form contacts with linkers IIS1-S2, IIS3-S4, IIIP5-P1 and IIIP2-S6. Elimination of this salt-bridge enables deeper penetration of the toxin into a VSD-II gorge to form new contacts with the channel, leading to increased channel sensitivity to Lqh-dprIT3.

Perlidae (Plecoptera) from the Paranapiacaba Mountains, Atlantic Forest, Brazil: Diversity and implications of the integrative approach and teneral specimens on taxonomy.

The study of complementary sources of biological variation (e.g. morphological, molecular) has allowed a better understanding of biodiversity through the construction of an integrative taxonomy. Using this approach, specimens from the Paranapiacaba Mountains, southeastern Brazil, were studied to update the knowledge on the stonefly family Perlidae from the region, characterize the species, and make associations between nymphs and adults using a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. The study also discusses the implications of integrative taxonomy and teneral specimens for the study of South American Perlidae. The molecular data were analyzed using Bayesian inference, Neighbor-joining, and delimiting species methods. Our results revealed that, in general, there was a morphological and molecular congruence between species. In the Paranapiacaba Mountains, three genera and 15 species were recorded: Anacroneuria boraceiensis Froehlich 2004, A. debilis (Pictet 1841) (new record), A. fiorentini De Ribeiro and Froehlich 2007 (new record), A. flintorum Froehlich 2002, A. iporanga Bispo and Froehlich 2004, A. itajaimirim Bispo and Froehlich 2004, A. polita (Burmeister 1913), A. subcostalis Klapálek 1921, A. tupi Bispo and Froehlich 2004 (with a description of the nymph), Kempnyia auberti Froehlich 1996, K. colossica (Navás 1934), K. flava Klapálek 1916, K. neotropica (Jacobson and Bianchi 1905) (including its new junior synonym K. petersorum Froehlich 1996), Kempnyia sp., and Macrogynoplax veneranda Froehlich 1984. COI sequences were obtained for 11 species, five of which had nymphs associated with adults. Among the five associated nymphs, the nymph of A. tupi is described here. The results of this study indicate that the color of adult teneral specimens differs from that of mature specimens. Given this, the synonym of K. neotropica and K. petersorum was proposed since these species have high morphological and molecular similarities and differ only in color patterns. In addition, the previous record of A. petersi Froehlich 2002 from the Paranapiacaba Mountains was invalidated since it was considered a teneral specimen of A. flintorum. These results suggest that the development of an integrative taxonomy is essential to continue advancing the study of Perlidae diversity in South America.

Hemocyanin and hexamerins expression in response to hypoxia in stoneflies (Plecoptera, Insecta).

Many freshwater ecosystems worldwide undergo hypoxia events that can trigger physiological, behavioral, and molecular responses in many organisms. Among such molecular responses, the regulation of the hemocyanin (Hc) protein expression which plays a major role in oxygen transportation within aquatic insects remains poorly understood. The stoneflies (Plecoptera) are aquatic insects that possess a functional Hc in the hemolymph similar to crustacean that co-occurs with a nonfunctional Hc protein, hexamerins (Hx). However, the role of both proteins during hypoxia remains undetermined. Here, we evaluated the effect of hypoxia on the expression of Hc and Hx proteins via a comparison between hypoxia and normoxia amino acid sequence variation and protein expression pattern within 23 stonefly species. We induced short-term hypoxia in wild-caught stoneflies species, sequenced the target region of Hc and Hx by complementary DNA synthesis, characterized the protein biochemistry using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ultrafiltration, and polarographic fluorometric method, and amplified the genome region of the hypoxia-inducible factor (HIF) transcriptional response element that regulated Hc using genome walking library approach. We found a lack of Hc expression in all examined species during hypoxia conditions, despite recognition of the HIF gene region as a possible regulatory factor of Hc, suggesting that compensatory responses as metabolic changes or behavioral tracheal movements to enhance respiratory efficiency could be possible mechanics to compensate for hypoxia. A short Hc-like novel isoform was detected instead in these 23 species, possibly due to either protein degradation or alternative splicing mechanisms, suggesting that the protein could be performing a different function other than oxygen transportation. Hx during hypoxia was expressed and exhibited species-level amino acid changes, highlighting a possible role during hypoxia. Our results demonstrate that hypoxia could enable a similar potential adaptive response of multiple species regarding specific physiological requirements, thereby shedding light on community behavior in stress environments that may help us to improve conservation practices and biomonitoring.

Comparative analysis of five mitogenomes of Osmylinae (Neuroptera: Osmylidae) and their phylogenetic implications.

In this study, five mitogenomes of Osmylinae species were sequenced: Osmylus fulvicephalus, Osmylus lucalatus, Plethosmylus atomatus, Plethosmylus zheanus, and Plethosmylus sp. These mitogenomes vary from 15,401 bp to 17,136 bp in size. The nucleotide compositions of Osmylinae mitogenomes are biased towards A/T. The gene arrangement of five mitogenomes is congruent with the putative ancestral pattern of the insects. Most of PCGs initiate with typical start codon ATN and terminate with TAA as the stop codon. All tRNA genes are folded into the typical cloverleaf secondary structure with an exception of tRNASer(AGN). The phylogenetic relationship was reconstructed by both maximum likelihood and Bayesian methods based on 13 PCGs and two rRNA genes. The sister group relationships between Osmylinae and the clade of Spilosmylinae and Protosmylinae were recovered as expected. The monophyly of Osmylinae was corroborated, but within the subfamily, three species (P. atomatus, P. zheanus, and Plethosmylus sp.) originally belonging to Plethosmylus (transferred to Osmylus by Winterton) were restored as a clade and sister to the clade of Osmylus. The current results implied that it is necessary to reassess the systematic status of Plethosmylus in the future.

Evolution of Neuropeptide Precursors in Polyneoptera (Insecta).

Neuropeptides are among the structurally most diverse signaling molecules and participate in intercellular information transfer from neurotransmission to intrinsic or extrinsic neuromodulation. Many of the peptidergic systems have a very ancient origin that can be traced back to the early evolution of the Metazoa. In recent years, new insights into the evolution of these peptidergic systems resulted from the increasing availability of genome and transcriptome data which facilitated the investigation of the complete neuropeptide precursor sequences. Here we used a comprehensive transcriptome dataset of about 200 species from the 1KITE initiative to study the evolution of single-copy neuropeptide precursors in Polyneoptera. This group comprises well-known orders such as cockroaches, termites, locusts, and stick insects. Due to their phylogenetic position within the insects and the large number of old lineages, these insects are ideal candidates for studying the evolution of insect neuropeptides and their precursors. Our analyses include the orthologs of 21 single-copy neuropeptide precursors, namely ACP, allatotropin, AST-CC, AST-CCC, CCAP, CCHamide-1 and 2, CNMamide, corazonin, CRF-DH, CT-DH, elevenin, HanSolin, NPF-1 and 2, MS, proctolin, RFLamide, SIFamide, sNPF, and trissin. Based on the sequences obtained, the degree of sequence conservation between and within the different polyneopteran lineages is discussed. Furthermore, the data are used to postulate the individual neuropeptide sequences that were present at the time of the insect emergence more than 400 million years ago. The data confirm that the extent of sequence conservation across Polyneoptera is remarkably different between the different neuropeptides. Furthermore, the average evolutionary distance for the single-copy neuropeptides differs significantly between the polyneopteran orders. Nonetheless, the single-copy neuropeptide precursors of the Polyneoptera show a relatively high degree of sequence conservation. Basic features of these precursors in this very heterogeneous insect group are explained here in detail for the first time.

The complete mitochondrial genome of Amphinemura claviloba (Wu, 1973) (Plecoptera: Nemouridae).

We sequenced the complete mitochondrial genome (mitogenome) of a stonefly, Amphinemura claviloba (Wu, 1973), of the family Nemouridae (Insecta: Plecoptera). The mitogenome was 15,707 bp long and contained typical 37 genes with an A+T content of 68.5%. All protein-coding genes (PCGs) use standard initiation codons (methionine and isoleucine), except ND1 and ND5 which starts with TTG and GTG, respectively. Two of the 13 PCGs harbor the incomplete termination codon. All tRNA genes have typical clover secondary structures, except the dihydrouridine (DHU) arm of tRNASer(AGN) forms a simple loop. Secondary structure models of the ribosomal RNA genes of A. claviloba are similar to those proposed for other insects. We also found some structural elements in the control region, such as tandem repeats, poly-C stretch and microsatellite-like elements, etc. Phylogenetic analyses showed the clades for the Nemoura, Amphinemura, and (Mesonemoura + Sphaeronemoura + Indonemoura + Protonemura) are well supported in a polytomy.

The genetic mechanism of selfishness and altruism in parent-offspring coadaptation.

The social bond between parents and offspring is characterized by coadaptation and balance between altruistic and selfish tendencies. However, its underlying genetic mechanism remains poorly understood. Using transcriptomic screens in the subsocial European earwig, Forficula auricularia, we found the expression of more than 1600 genes associated with experimentally manipulated parenting. We identified two genes, Th and PebIII, each showing evidence of differential coexpression between treatments in mothers and their offspring. In vivo RNAi experiments confirmed direct and indirect genetic effects of Th and PebIII on behavior and fitness, including maternal food provisioning and reproduction, and offspring development and survival. The direction of the effects consistently indicated a reciprocally altruistic function for Th and a reciprocally selfish function for PebIII. Further metabolic pathway analyses suggested roles for Th-restricted endogenous dopaminergic reward, PebIII-mediated chemical communication and a link to insulin signaling, juvenile hormone, and vitellogenin in parent-offspring coadaptation and social evolution.

Evolutionary origin and functioning of pregenital abdominal outgrowths in a viviparous insect, Arixenia esau.

Although pregenital abdominal outgrowths occur only rarely in pterygote insects, they are interesting from the evolutionary viewpoint because of their potential homology to wings. Our previous studies of early development of an epizoic dermapteran, Arixenia esau revealed that abdominal segments of the advanced embryos and larvae, growing inside a mother's uterus, are equipped with paired serial outgrowths. Here, we focus on the origin and functioning of these outgrowths. We demonstrate that they bud from the lateral parts of the abdominal nota, persist till the end of intrauterine development, and remain in contact with the uterus wall. We also show that the bundles of muscle fibers associated with the abdominal outgrowths may facilitate flow of the haemolymph from the outgrowths' lumen to the larval body cavity. Following completion of the intrauterine development, abdominal outgrowths are shed together with the larval cuticle during the first molt after the larva birth. Using immunohistochemical and biochemical approaches, we demonstrate that the Arixenia abdominal outgrowths represent an evolutionary novelty, presumably related to intrauterine development, and suggest that they are not related to serial wing homologs.

Molecular identity of Nemoura lacustris (Plecoptera: Nemouridae) throughout its distributional range.

The recent report of Nemoura lacustris Pictet, 1865 in Great Britain has raised doubts on its identity, given the isolation with respect to the Mediterranean and continental populations of this species. Using molecular analyses, we tested if populations from  the United Kingdom and the Iberian Peninsula were conspecific and tested the hypotheses of a recent colonization event versus a more ancient origin for the British populations. Phylogenetic analysis of the mitochondrial marker COI allowed us to conclude that the United Kingdom specimens morphologically ascribed to N. lacustris were conspecific with populations from France and the Iberian Peninsula. Based on the genetic divergence of the two reciprocally monophyletic clades from the Iberian Peninsula and Great Britain/France, respectively, the present distribution of N. lacustris can be postulated as a relatively recent dispersal or introduction into Great Britain from France.                Finally, we note the isolated position displayed by N. lacustris in the phylogenetic tree of Nemoura species based on COI sequences, as the sister to all included species of the genus. This isolated position corresponds with the specific morphology of N. lacustris genitalia and requires additional studies to ascertain clearer generic boundaries within the Nemouridae.

The mitochondrial genomes of palaeopteran insects and insights into the early insect relationships.

Phylogenetic relationships of basal insects remain a matter of discussion. In particular, the relationships among Ephemeroptera, Odonata and Neoptera are the focus of debate. In this study, we used a next-generation sequencing approach to reconstruct new mitochondrial genomes (mitogenomes) from 18 species of basal insects, including six representatives of Ephemeroptera and 11 of Odonata, plus one species belonging to Zygentoma. We then compared the structures of the newly sequenced mitogenomes. A tRNA gene cluster of IMQM was found in three ephemeropteran species, which may serve as a potential synapomorphy for the family Heptageniidae. Combined with published insect mitogenome sequences, we constructed a data matrix with all 37 mitochondrial genes of 85 taxa, which had a sampling concentrating on the palaeopteran lineages. Phylogenetic analyses were performed based on various data coding schemes, using maximum likelihood and Bayesian inferences under different models of sequence evolution. Our results generally recovered Zygentoma as a monophyletic group, which formed a sister group to Pterygota. This confirmed the relatively primitive position of Zygentoma to Ephemeroptera, Odonata and Neoptera. Analyses using site-heterogeneous CAT-GTR model strongly supported the Palaeoptera clade, with the monophyletic Ephemeroptera being sister to the monophyletic Odonata. In addition, a sister group relationship between Palaeoptera and Neoptera was supported by the current mitogenomic data.

Sex-biased gene expression is repeatedly masculinized in asexual females.

Males and females feature strikingly different phenotypes, despite sharing most of their genome. A resolution of this apparent paradox is through differential gene expression, whereby genes are expressed at different levels in each sex. This resolution, however, is likely to be incomplete, leading to conflict between males and females over the optimal expression of genes. Here we test the hypothesis that gene expression in females is constrained from evolving to its optimum level due to sexually antagonistic selection on males, by examining changes in sex-biased gene expression in five obligate asexual species of stick insect, which do not produce males. We predicted that the transcriptome of asexual females would be feminized as asexual females do not experience any sexual conflict. Contrary to our prediction we find that asexual females feature masculinized gene expression, and hypothesise that this is due to shifts in female optimal gene expression levels following the suppression of sex.

Active mode of excretion across digestive tissues predates the origin of excretory organs.

Most bilaterian animals excrete toxic metabolites through specialized organs, such as nephridia and kidneys, which share morphological and functional correspondences. In contrast, excretion in non-nephrozoans is largely unknown, and therefore the reconstruction of ancestral excretory mechanisms is problematic. Here, we investigated the excretory mode of members of the Xenacoelomorpha, the sister group to Nephrozoa, and Cnidaria, the sister group to Bilateria. By combining gene expression, inhibitor experiments, and exposure to varying environmental ammonia conditions, we show that both Xenacoelomorpha and Cnidaria are able to excrete across digestive-associated tissues. However, although the cnidarian Nematostella vectensis seems to use diffusion as its main excretory mode, the two xenacoelomorphs use both active transport and diffusion mechanisms. Based on these results, we propose that digestive-associated tissues functioned as excretory sites before the evolution of specialized organs in nephrozoans. We conclude that the emergence of a compact, multiple-layered bilaterian body plan necessitated the evolution of active transport mechanisms, which were later recruited into the specialized excretory organs.

Ecological gradients drive insect wing loss and speciation: The role of the alpine treeline.

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.