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The availability of public genomic resources can greatly assist biodiversity assessment, conservation, and restoration efforts by providing evidence for scientifically informed management decisions. Here we survey the main approaches and applications in biodiversity and conservation genomics, considering practical factors, such as cost, time, prerequisite skills, and current shortcomings of applications. Most approaches perform best in combination with reference genomes from the target species or closely related species. We review case studies to illustrate how reference genomes can facilitate biodiversity research and conservation across the tree of life. We conclude that the time is ripe to view reference genomes as fundamental resources and to integrate their use as a best practice in conservation genomics., Trends in Genetics, 39 (7), ISSN:0168-9525

Background Teleost fishes comprise more than half of the vertebrate species. Within teleosts, most phylogenies consider the split between Osteoglossomorpha and Euteleosteomorpha/Otomorpha as basal, preceded only by the derivation of the most primitive group of teleosts, the Elopomorpha. While Osteoglossomorpha are generally species poor, the taxon contains the African weakly electric fish (Mormyroidei), which have radiated into numerous species. Within the mormyrids, the genus Campylomormyrus is mostly endemic to the Congo Basin. Campylomormyrus serves as a model to understand mechanisms of adaptive radiation and ecological speciation, especially with regard to its highly diverse species-specific electric organ discharges (EOD). Currently, there are few well-annotated genomes available for electric fish in general and mormyrids in particular. Our study aims at producing a high quality genome and to use this to examine genome evolution in relation to other teleosts. This will facilitate further understanding of the evolution of the osteoglossomorpha fish in general and of electric fish in particular. Results A high quality weakly electric fish (C. compressirostris) genome was produced from a single individual with a genome size of 862Mb, consisting of 1,497 contigs with an N50 of 1,399 kb and a GC-content of 43.69%. Gene predictions identified 34,492 protein-coding genes, which is a higher number than in the two other available Osteoglossomorpha genomes of Paramormyrops kingsleyae and Scleropages formosus. A CAFE5 analysis of gene family evolution comparing 33 teleost fish genomes suggests an overall faster gene family turnover rate in Osteoglossomorpha than in Otomorpha and Euteleosteomorpha. Moreover, the ratios of expanded/contracted gene family numbers in Osteoglossomorpha are significantly higher than in the other two taxa, except for species that had undergone an additional genome duplication (Cyprinus carpio and Oncorhynchus mykiss). As potassium channel proteins are hypothesized to play a key role in EOD diversity among species, we put a special focus on them, and manually curated 16 Kv1 genes. We identified a tandem duplication in the KCNA7a gene in the genome of C. compressirostris. Conclusions We present the fourth genome of an electric fish and the third well-annotated genome for Osteoglossomorpha, enabling us to compare gene family evolution among major teleost lineages. Osteoglossomorpha appear to exhibit rapid gene family evolutiona, with more gene family expansions than contractions. The curated Kv1 gene family showed seven gene clusters, which is more than in other analyzed fish genomes outside Osteoglossomorpha. The KCNA7a, encoding for a potassium channel central for EOD production and modulation, is tandemly duplicated which may related to the diverse EOD observed among Campylomormyrus species.

Progress in genome sequencing now enables the large-scale generation of reference genomes. Various international initiatives aim to generate reference genomes representing global biodiversity. These genomes provide unique insights into genomic diversity and architecture, thereby enabling comprehensive analyses of population and functional genomics, and are expected to revolutionize conservation genomics., Trends in Ecology & Evolution, 37 (3), ISSN:0169-5347, ISSN:1872-8383

Intergenerational effects, also known as parental effects in which the offspring phenotype is influenced by the parental phenotype, can occur in response to factors that occur not only in early but also in late parental life. However, little is known about how these parental life stage–specific environments interact with each other and with the offspring environment to influence offspring phenotypes, particularly in organisms that realize distinct niches across ontogeny. We examined the effects of parental larval starvation and adult reproductive environment on offspring traits under matching or mismatching offspring larval starvation conditions using the holometabolous, haplodiploid insect Athalia rosae (turnip sawfly). We show that parental larval starvation had trait-dependent intergenerational effects on both life history and consumption traits of offspring larvae, partly in interaction with offspring conditions, while there was no significant effect of parental adult reproductive environment. In addition, while offspring larval starvation led to numerous gene-and pathway-level expression differences, parental larval starvation impacted far fewer genes and only the ribosomal pathway. Our findings reveal that parental starvation evokes complex intergenerational effects on offspring life history traits, consumption patterns, and gene expression, although the effects are less pronounced than those of offspring starvation.

Intergenerational effects, also known as parental effects in which the offspring phenotype is influenced by the parental phenotype, can occur in response to factors that occur not only in early but also in late parental life. However, little is known about how these parental life stage-specific environments interact with each other and with the offspring environment to influence offspring phenotype, particularly in organisms that realize distinct niches across ontogeny. We examined the effects of parental larval starvation and adult reproductive environment on offspring traits under matching or mismatching offspring larval starvation conditions using the holometabolous, haplo-diploid insect Athalia rosae (turnip sawfly). We show that the parental larval starvation treatment had trait-dependent intergenerational effects on both life-history and consumption traits of offspring larvae, partly in interaction with offspring conditions and sex, while there was no significant effect of parental adult reproductive environment. In addition, while offspring larval starvation led to numerous gene- and pathway-level expression differences, parental larval starvation impacted fewer genes and only the ribosomal pathway. Our findings reveal that parental starvation evokes complex intergenerational effects on offspring life-history traits, consumption patterns as well as gene expression, although the effects are less pronounced than those of offspring starvation.

Pharmacophagy involves the sequestration of specialised plant metabolites for non-nutritive purposes and commonly occurs in insects. Here we investigate pharmacophagy in the turnip sawfly, Athalia rosae, where adults not only collect specialised metabolites (clerodanoids) from a plant (Ajuga reptans), but also from the exterior of conspecifics via fighting. Using behavioural assays, chemical analytics, and RNAseq we show that when individuals nibble on conspecifics that have already acquired clerodanoids from A. reptans leaves, this nibbling results in the transfer of compounds between individuals. Furthermore, unlike other pharmacophagous insects, the acquisition of clerodanoids by A. rosae from the leaves of A. reptans does not induce the upregulation of known detoxification or sequestration genes and pathways. In contrast, pharmacophagous nibbling on conspecifics results in the upregulation of metabolic pathways associated with elevated metabolic rates and increased energy consumption. It therefore seems that individuals attack conspecifics to acquire clerodanoids despite the apparent metabolic costs of this form of pharmacophagy compared to clerodanoid uptake from a plant. Changes in the metabolic phenotype of A.rosae individuals consequently has profound consequences for social interactions with possible ramifications for their social niche.Summary statementThe turnip sawfly (Athalia rosae) gains potentially beneficial compounds from the leaf surface of non-food plants (e.g. Ajuga reptans), but can also steal these compounds from conspecifics via aggressive nibbling.

The harbour porpoise (Phocoena phocoena) is a highly mobile cetacean found across the Northern hemisphere. It occurs in coastal waters and inhabits basins that vary broadly in salinity, temperature and food availability. These diverse habitats could drive subtle differentiation among populations, but examination of this would be best conducted with a robust reference genome. Here, we report the first harbour porpoise genome, assembled de novo from an individual originating in the Kattegat Sea (Sweden). The genome is one of the most complete cetacean genomes currently available, with a total size of 2.39 Gb and 50% of the total length found in just 34 scaffolds. Using 122 of the longest scaffolds, we were able to show high levels of synteny with the genome of the domestic cattle (Bos taurus). Our draft annotation comprises 22,154 predicted genes, which we further annotated through matches to the NCBI nucleotide database, GO categorization and motif prediction. Within the predicted genes, we have confirmed the presence of >20 genes or gene families that have been associated with adaptive evolution in other cetaceans. Overall, this genome assembly and draft annotation represent a crucial addition to the genomic resources currently available for the study of porpoises and Phocoenidae evolution, phylogeny and conservation.

Reciprocal selection between aphids, their protective endosymbionts, and the parasitoid wasps that prey upon them offers an opportunity to study the basis of their coevolution. We investigated adaptation to symbiont-conferred defense by rearing the parasitoid wasp Lysiphlebus fabarum on aphids (Aphis fabae) possessing different defensive symbiont strains (Hamiltonella defensa). After ten generations of experimental evolution, wasps showed increased abilities to parasitize aphids possessing the H. defensa strain they evolved with, but not aphids possessing the other strain. We show that the two symbiont strains encode different toxins, potentially creating different targets for counter-adaptation. Phenotypic and behavioral comparisons suggest that neither life-history traits nor oviposition behavior differed among evolved parasitoid lineages. In contrast, comparative transcriptomics of adult female wasps identified a suite of differentially expressed genes among lineages, even when reared in a common, symbiont-free, aphid host. In concurrence with the specificity of each parasitoid lineages' infectivity, most differentially expressed parasitoid transcripts were also lineage-specific. These transcripts are enriched with putative venom toxins and contain highly expressed, potentially defensive viral particles. Together, these results suggest that wild populations of L. fabarum employ a complicated offensive arsenal with sufficient genetic variation for wasps to adapt rapidly and specifically to their hosts' microbial defenses.

Coevolving taxa offer the opportunity to study the genetic basis of rapid reciprocal adaptation. We have used experimental evolution to examine adaptation in the parasitoid waspLysiphlebus fabarumto resistance conferred by the protective endosymbiontHamiltonella defensain its aphid hostAphis fabae. To examine a key stage in parasitoid infection, we have used RNA-seq to study gene expression in 4-5 day old parasitoid larvae contained in still living aphids. With this dual RNA-seq we can simultaneously view expression in individual experimentally evolved parasitoids and the aphids that house them. This gives a view of the sweeping changes in both taxa accompanying successful or unsuccessful infections. Among successful larvae, we find that experimentally evolved populations adapted toH. defensa-protected hosts differ in the expression of genes that include putative toxins and genes to cope with stress. These differences remain even when the larvae are developing in aphids possessing no defensive endosymbionts, suggesting that they are genetically based. In contrast, plastic responses between parasitoids reared in hosts with and withoutH. defensaare relatively small. Although aphids rely largely on their secondary endosymbionts for defense against parasitoids, we identify expression differences in aphids housing different parasitoid phenotypes. Together, these results demonstrate that wild parasitoid populations possess the genetic variation for rapid adaptation to host resistance, resulting in genetically based differences in gene expression that increase their success in parasitizing symbiont-protected host aphids.

We report on a novel RNA virus infecting the wasp Lysiphlebus fabarum, a parasitoid of aphids. This virus, tentatively named “Lysiphlebus fabarum virus” (LysV), was discovered in transcriptome sequences of wasps from an experimental evolution study in which the parasitoids were allowed to adapt to aphid hosts (Aphis fabae) with or without resistance-conferring endosymbionts. Based on phylogenetic analyses of the viral RNA-dependent RNA polymerase (RdRp), LysV belongs to the Iflaviridae family in the order of the Picornavirales, with the closest known relatives all being parasitoid wasp-infecting viruses. We developed an endpoint PCR and a more sensitive qPCR assay to screen for LysV in field samples and laboratory lines. These screens verified the occurrence of LysV in wild parasitoids and identified the likely wild-source population for lab infections in Western Switzerland. Three viral haplotypes could be distinguished in wild populations, of which two were found in the laboratory. Both vertical and horizontal transmission of LysV were demonstrated experimentally, and repeated sampling of laboratory populations suggests that the virus can form persistent infections without obvious symptoms in infected wasps.

BackgroundParasitoid wasps have fascinating life cycles and play an important role in trophic networks, yet little is known about their genome content and function. Parasitoids that infect aphids are an important group with the potential for biocontrol, and infecting aphids requires overcoming both aphid defenses and their defensive endosymbionts.ResultsWe present thede novogenome assemblies, detailed annotation, and comparative analysis of two closely related parasitoid wasps that target pest aphids:Aphidius erviandLysiphlebus fabarum(Hymenoptera: Braconidae: Aphidiinae). The genomes are small (139 and 141 Mbp), highly syntenic, and the most AT-rich reported thus far for any arthropod (GC content: 25.8% and 23.8%). This nucleotide bias is accompanied by skewed codon usage, and is stronger in genes with adult-biased expression. AT-richness may be the consequence of reduced genome size, a near absence of DNA methylation, and age-specific energy demands. We identify expansions of F-box/Leucine-rich-repeat proteins, suggesting that diversification in this gene family may be associated with their broad host range or with countering defenses from aphids’ endosymbionts. The absence of some immune genes (Toll and Imd pathways) resembles similar losses in their aphid hosts, highlighting the potential impact of symbiosis on both aphids and their parasitoids.ConclusionsThese findings are of fundamental interest for insect evolution and beyond. This will provide a strong foundation for further functional studies including coevolution with respect to their hosts, the basis of successful infection, and biocontrol. Both genomes are available athttps://bipaa.genouest.org.

The harbour porpoise (Phocoena phocoena) is a highly mobile cetacean found in waters across the Northern hemisphere. It occurs in coastal water and inhabits water basins that vary broadly in salinity, temperature, and food availability. These diverse habitats could drive differentiation among populations. Here we report the first harbour porpoise genome, assembled de novo from a Swedish Kattegat individual. The genome is one of the most complete cetacean genomes currently available, with a total size of 2.7 Gb and 50% of the total length found in just 34 scaffolds. Using the largest 122 scaffolds, we were able to validate a high level of homology to the chromosome-level genome assembly of the closest related species for which such resource was available, the domestic cattle (Bos taurus). The draft annotation comprises 22,154 predicted gene models, which we further annotated through matches to the NCBI nucleotide database, GO categorization, and motif prediction. To infer the adaptive abilities of this species, as well as their population history, we performed a Bayesian skyline analysis, and produced results that are concordant with the demographic history of this species, including expansion and fragmentation events. Overall, this genome assembly, together with the draft annotation, represents a crucial addition to the limited genetic markers currently available for the study of porpoises and Phocoenidae conservation, phylogeny, and evolution.

There is growing interest in biological control as a sustainable and environmentally friendly way to control pest insects. Aphids are among the most detrimental agricultural pests worldwide, and parasitoid wasps are frequently employed for their control. The use of asexual parasitoids may improve the effectiveness of biological control because only females kill hosts and because asexual populations have a higher growth rate than sexuals. However, asexuals may have a reduced capacity to track evolutionary change in their host populations. We used a factorial experiment to compare the ability of sexual and asexual populations of the parasitoid Lysiphlebus fabarum to control caged populations of black bean aphids (Aphis fabae) of high and low clonal diversity. The aphids came from a natural population, and one‐third of the aphid clones harbored Hamiltonella defensa, a heritable bacterial endosymbiont that increases resistance to parasitoids. We followed aphid and parasitoid population dynamics for 3 months but found no evidence that the reproductive mode of parasitoids affected their effectiveness as biocontrol agents, independent of host clonal diversity. Parasitoids failed to control aphids in most cases, because their introduction resulted in strong selection for clones protected by H. defensa. The increasingly resistant aphid populations escaped control by parasitoids, and we even observed parasitoid extinctions in many cages. The rapid evolution of symbiont‐conferred resistance in turn imposed selection on parasitoids. In cages where asexual parasitoids persisted until the end of the experiment, they became dominated by a single genotype able to overcome the protection provided by H. defensa. Thus, there was evidence for parasitoid counteradaptation, but it was generally too slow for parasitoids to regain control over aphid populations. It appears that when pest aphids possess defensive symbionts, the presence of parasitoid genotypes able to overcome symbiont‐conferred resistance is more important for biocontrol success than their reproductive mode.

Reciprocal selection between aphids, their protective endosymbionts, and the parasitoid wasps that prey upon them offers an opportunity to study the basis of their coevolution. We investigated adaptation to symbiont-conferred defense by rearing the parasitoid wasp Lysiphlebus fabarum on aphids (Aphis fabae) possessing different defensive symbiont strains (Hamiltonella defensa). After ten generations of experimental evolution, wasps showed increased abilities to parasitize aphids possessing the H. defensa strain they evolved with, but not aphids possessing the other strain. We show that the two symbiont strains encode different toxins, potentially creating different targets for counter-adaptation. Phenotypic and behavioral comparisons suggest that neither life-history traits nor oviposition behavior differed among evolved parasitoid lineages. In contrast, comparative transcriptomics of adult female wasps identified a suite of differentially expressed genes among lineages, even when reared in a common, symbiont-free, aphid host. In concurrence with the specificity of each parasitoid lineages' infectivity, most differentially expressed parasitoid transcripts were also lineage-specific. These transcripts are enriched with putative venom toxins and contain highly expressed, potentially defensive viral particles. Together, these results suggest that wild populations of L. fabarum employ a complicated offensive arsenal with sufficient genetic variation for wasps to adapt rapidly and specifically to their hosts' microbial defenses.

Cold tolerance is a key determinant of insect distribution and abundance, and thermal acclimation can strongly influence organismal stress tolerance phenotypes, particularly in small ectotherms like Drosophila. However, there is limited understanding of the molecular and biochemical mechanisms that confer such impressive plasticity. Here, we use high-throughput mRNA sequencing (RNA-seq) and liquid chromatography – mass spectrometry (LC-MS) to compare the transcriptomes and metabolomes of D. melanogaster acclimated as adults to warm (rearing) (21.5 °C) or cold conditions (6 °C). Cold acclimation improved cold tolerance and led to extensive biological reorganization: almost one third of the transcriptome and nearly half of the metabolome were differentially regulated. There was overlap in the metabolic pathways identified via transcriptomics and metabolomics, with proline and glutathione metabolism being the most strongly-supported metabolic pathways associated with increased cold tolerance. We discuss several new targets in the study of insect cold tolerance (e.g. dopamine signaling and Na+-driven transport), but many previously identified candidate genes and pathways (e.g. heat shock proteins, Ca2+ signaling, and ROS detoxification) were also identified in the present study, and our results are thus consistent with and extend the current understanding of the mechanisms of insect chilling tolerance.

Phasmatodea, more commonly known as stick insects, have been poorly studied at the molecular level for several key traits, such as components of the sensory system and regulators of reproduction and development, impeding a deeper understanding of their functional biology. Here, we employ de novo transcriptome analysis to identify genes with primary functions related to female odour reception, digestion, and male sexual traits in the New Zealand common stick insect Clitarchus hookeri (White). The female olfactory gene repertoire revealed ten odorant binding proteins with three recently duplicated, 12 chemosensory proteins, 16 odorant receptors, and 17 ionotropic receptors. The majority of these olfactory genes were over-expressed in female antennae and have the inferred function of odorant reception. Others that were predominantly expressed in male terminalia (n = 3) and female midgut (n = 1) suggest they have a role in sexual reproduction and digestion, respectively. Over-represented transcripts in the midgut were enriched with digestive enzyme gene families. Clitarchus hookeri is likely to harbour nine members of an endogenous cellulase family (glycoside hydrolase family 9), two of which appear to be specific to the C. hookeri lineage. All of these cellulase sequences fall into four main phasmid clades and show gene duplication events occurred early in the diversification of Phasmatodea. In addition, C. hookeri genome is likely to express γ-proteobacteria pectinase transcripts that have recently been shown to be the result of horizontal transfer. We also predicted 711 male terminalia-enriched transcripts that are candidate accessory gland proteins, 28 of which were annotated to have molecular functions of peptidase activity and peptidase inhibitor activity, two groups being widely reported to regulate female reproduction through proteolytic cascades. Our study has yielded new insights into the genetic basis of odour detection, nutrient digestion, and male sexual traits in stick insects. The C. hookeri reference transcriptome, together with identified gene families, provides a comprehensive resource for studying the evolution of sensory perception, digestive systems, and reproductive success in phasmids., PLoS ONE, 11 (6), ISSN:1932-6203

Geographic range differences among species may result from differences in their physiological tolerances. In the intertidal zone, marine and terrestrial environments intersect to create a unique habitat, across which physiological tolerance strongly influences range. Traits to cope with environmental extremes are particularly important here because many species live near their physiological limits and environmental gradients can be steep. The snail Melampus bidentatus occurs in coastal salt marshes in the western Atlantic and the Gulf of Mexico. We used sequence data from one mitochondrial (COI) and two nuclear markers (histone H3 and a mitochondrial carrier protein, MCP) to identify three cryptic species within this broad-ranging nominal species, two of which have partially overlapping geographic ranges. High genetic diversity, low population structure, and high levels of migration within these two overlapping species suggest that historical range limitations do not entirely explain their different ranges. To identify microhabitat differences between these two species, we modelled their distributions using data from both marine and terrestrial environments. Although temperature was the largest factor setting range limits, other environmental components explained features of the ranges that temperature alone could not. In particular, the interaction of precipitation and salinity likely sets physiological limits that lead to range differences between these two cryptic species. This suggests that the response to climatic change in these snails will be mediated by changes to multiple environmental factors, and not just to temperature alone.

The acquisition of physiological strategies to tolerate novel thermal conditions allows organisms to exploit new environments. As a result, thermal tolerance is a key determinant of the global distribution of biodiversity, yet the constraints on its evolution are not well understood. Here we investigate parallel evolution of cold tolerance in New Zealand stick insects, an endemic radiation containing three montane-occurring species. Using a phylogeny constructed from 274 orthologous genes, we show that stick insects have independently colonized montane environments at least twice. We compare supercooling point and survival of internal ice formation among ten species from eight genera, and identify both freeze tolerance and freeze avoidance in separate montane lineages. Freeze tolerance is also verified in both lowland and montane populations of a single, geographically widespread, species. Transcriptome sequencing following cold shock identifies a set of structural cuticular genes that are both differentially regulated and under positive sequence selection in each species. However, while cuticular proteins in general are associated with cold shock across the phylogeny, the specific genes at play differ among species. Thus, while processes related to cuticular structure are consistently associated with adaptation for cold, this may not be the consequence of shared ancestral genetic constraints., Scientific Reports, 5, ISSN:2045-2322

Lingcod, Ophiodon elongatus Girard, have a 3-month pelagic larval stage and are an important recreational and commercial species on the west coast of North America. Cytochrome-c oxidase I sequences from tissue samples were used to characterize population structure and infer patterns of gene flow from California to Alaska. No significant genetic structure was found when estimates of Wright’s FST (i.e., ΦST) were generated among all populations sampled. Nesting populations within regions, however, indicated that the inner coast of Washington State is distinct, a result corroborating previous allozyme work. Coalescent-based estimates of gene flow indicate that although migration can be high from an evolutionary perspective, nearly half of all comparisons among populations showed no gene flow in at least one direction. From an ecological perspective, moderate migration rates (Nm < 10) among most populations provide surprisingly limited connectivity at large (∼ 1,000 km) and small (∼100 km) spatial scales. Coalescent-based estimates also show that gene flow between the inner and the outer coasts is asymmetric, a result consistent with prevailing surface currents. Because the expected inter-locus variances for coalescent-based estimates of gene flow are likely large, future work will benefit from analyses of nuclear DNA markers. However, limited demographic connectivity on large spatial scales may help explain why stock recovery has been uneven, with greater recovery in the northern (87% rebuilt) than in the southern (24% rebuilt) fishery region, supporting a regional management strategy. These results suggest that despite a 3-month pelagic larval stage, some areas may be effectively closed with respect to both population dynamics and fishery management issues.

Low temperatures limit the poleward distribution of many species such that the expansion of geographic range can only be accomplished via evolutionary innovation. We have tested for physiological differences among closely related species to determine whether their poleward latitudinal ranges are limited by tolerance to cold. We measured lower temperature tolerance (LT50) among a group of intertidal pulmonate snails from six congeneric species and nine locales. Differences in tolerance are placed in the context of a molecular phylogeny based on one mitochondrial (cytochrome oxidase subunit I) and two nuclear (histone 3 and a mitochondrial phosphate carrier protein) markers. Temperate species from two separate lineages had significantly lower measures of LT50 than related tropical species. Range differences within the temperate zone, however, were not explained by LT50. These results show that multiple adaptations to cold and freezing may have enabled range expansions out of the tropics in Melampus. However, northern range limits within temperate species are not governed by cold tolerance alone.

In widespread and genetically structured populations, temperature variation may lead to among-population differentiation of thermal biology. The New Zealand stick insect genus Micrarchus contains four species that inhabit different thermal environments, two of which are geographically widespread. RNA-Seq and quantitative PCR were used to investigate the transcriptional responses to cold shock among lowland and alpine species to identify cold-responsive transcripts that differ between the species and to determine whether there is intraspecific geographical variation in gene expression. We also used mitochondrial DNA, nuclear 28S ribosomal DNA and transcriptome-wide SNPs to determine phylogeographic structure and the potential for differences in genetic backgrounds to contribute to variation in gene expression. RNA-Seq identified 2160 unigenes that were differentially expressed as a result of low-temperature exposure across three populations from two species (M. hystriculeus and M. nov. sp. 2), with a majority (68% ± 20%) being population specific. This extensive geographical variation is consistent across years and is likely a result of background genetic differences among populations caused by genetic drift and possibly local adaptation. Responses to cold shock shared among alpine M. nov. sp. 2 populations included the enrichment of cuticular structure-associated transcripts, suggesting that cuticle modification may have accompanied colonization of low-temperature alpine environments and the development of a more cold-hardy phenotype.

Stick insects are found in a variety of habitats throughout New Zealand, including at least four species that occur at high altitudes. Here they face physiological challenges that differ from their typically warmer lowland habitats, but their strategies to deal with harsh winter conditions are not known. Autumn and winter field surveys, coupled with caging experiments, were conducted to determine which life stages are overwintering in montane and lowland habitats. Data loggers were placed for approximately one year at each site to measure the leaf litter and canopy microhabitat temperatures. From this, we have found that alpine and lowland stick insects persist in a variety of life stages throughout the year despite multiple exposure to freezing temperatures.

The endemic New Zealand alpine stick insect Micrarchus nov. sp. 2 regularly experiences sub-zero temperatures in the wild. 454-based RNA-Seq was used to generate a de novo transcriptome and differentiate between treatments to investigate the genetic basis of cold tolerance. Non cold-treated individuals were compared to those exposed to 0°C for 1 h followed by a 1 h recovery period at 20°C. We aligned 607,410 Roche 454 reads, generating a transcriptome of 5235 contigs. Differential expression analysis ranked candidate cold responsive genes for qPCR validation by P-value. The top nine up-regulated candidates, together with eight a priori targets identified from previous studies, had their relative expression quantified using qPCR. Three candidate cold responsive genes from the RNA-Seq data were verified as significantly up-regulated, annotated as: prolyl 4-hydroxylase subunit alpha-1 (P4HA1), staphylococcal nuclease domain-containing protein 1 (snd1) and cuticular protein analogous to peritrophins 3-D2 (Cpap3-d2). All three are novel candidate genes, illustrating the varied response to low temperature across insects.

Reproductive proteins commonly show signs of rapid divergence driven by positive selection. The mechanisms driving these changes have remained ambiguous in part because interacting male and female proteins have rarely been examined. We isolate an egg protein the vitelline envelope receptor for lysin (VERL) from Tegula, a genus of free-spawning marine snails. Like VERL from abalone, Tegula VERL is a major component of the VE surrounding the egg, includes a conserved zona pellucida (ZP) domain at its C-terminus, and possesses a unique, negatively charged domain of about 150 amino acids implicated in interactions with the positively charged lysin. Unlike for abalone VERL, where this unique VERL domain occurs in a tandem array of 22 repeats, Tegula VERL has just one such domain. Interspecific comparisons show that both lysin and the VERL domain diverge via positive selection, whereas the ZP domain evolves neutrally. Rates of nonsynonymous substitution are correlated between lysin and the VERL domain, consistent with sexual antagonism, although lineage-specific effects, perhaps owing to different ecologies, may alter the relative evolutionary rates of sperm- and egg-borne proteins.

Background The glycolytic pathway is central to cellular energy production. Selection on individual enzymes within glycolysis, particularly phosphoglucose isomerase (Pgi), has been associated with metabolic performance in numerous organisms. Nonetheless, how whole energy-producing pathways evolve to allow organisms to thrive in different environments and adopt new lifestyles remains little explored. The Lanceocercata radiation of Australasian stick insects includes transitions from tropical to temperate climates, lowland to alpine habitats, and winged to wingless forms. This permits a broad investigation to determine which steps within glycolysis and what sites within enzymes are the targets of positive selection. To address these questions we obtained transcript sequences from seven core glycolysis enzymes, including two Pgi paralogues, from 29 Lanceocercata species. Results Using maximum likelihood methods a signature of positive selection was inferred in two core glycolysis enzymes. Pgi and Glyceraldehyde 3-phosphate dehydrogenase (Gaphd) genes both encode enzymes linking glycolysis to the pentose phosphate pathway. Positive selection among Pgi paralogues and orthologues predominately targets amino acids with residues exposed to the protein’s surface, where changes in physical properties may alter enzyme performance. Conclusion Our results suggest that, for Lancerocercata stick insects, adaptation to new stressful lifestyles requires a balance between maintaining cellular energy production, efficiently exploiting different energy storage pools and compensating for stress-induced oxidative damage.