Historical museum samples reveal signals of selection and drift in response to changing insecticide use in an agricultural pest moth.

In response to environmental and human-imposed selective pressures, agroecosystem pests frequently undergo rapid evolution, with some species having a remarkable capacity to rapidly develop pesticide resistance. Temporal sampling of genomic data can comprehensively capture such adaptive changes over time, for example, by elucidating allele frequency shifts in pesticide resistance loci in response to different pesticides. Here, we leveraged museum specimens spanning over a century of collections to generate temporal contrasts between pre- and post-insecticide populations of an agricultural pest moth, Helicoverpa armigera. We used targeted exon sequencing of 254 samples collected across Australia from the pre-1950s (prior to insecticide introduction) to the 1990s, encompassing decades of changing insecticide use. Our sequencing approach focused on genes that are known to be involved in insecticide resistance, environmental sensation, and stress tolerance. We found an overall lack of spatial and temporal population structure change across Australia. In some decades (e.g., 1960s and 1970s), we found a moderate reduction of genetic diversity, implying stochasticity in evolutionary trajectories due to genetic drift. Temporal genome scans showed extensive evidence of selection following insecticide use, although the majority of selected variants were low impact, and alternating trajectories of allele frequency change were suggestive of potential antagonistic pleiotropy. Our results provide new insights into recent evolutionary responses in an agricultural pest and show how temporal contrasts using museum specimens can improve mechanistic understanding of rapid evolution.

The effect of missing data on evolutionary analysis of sequence capture bycatch, with application to an agricultural pest.

Sequence capture is a genomic technique that selectively enriches target sequences before high throughput next-generation sequencing, to generate specific sequences of interest. Off-target or 'bycatch' data are often discarded from capture experiments, but can be leveraged to address evolutionary questions under some circumstances. Here, we investigated the effects of missing data on a variety of evolutionary analyses using bycatch from an exon capture experiment on the global pest moth, Helicoverpa armigera. We added > 200 new samples from across Australia in the form of mitogenomes obtained as bycatch from targeted sequence capture, and combined these into an additional larger dataset to total > 1000 mitochondrial cytochrome c oxidase subunit I (COI) sequences across the species' global distribution. Using discriminant analysis of principal components and Bayesian coalescent analyses, we showed that mitogenomes assembled from bycatch with up to 75% missing data were able to return evolutionary inferences consistent with higher coverage datasets and the broader literature surrounding H. armigera. For example, low-coverage sequences broadly supported the delineation of two H. armigera subspecies and also provided new insights into the potential for geographic turnover among these subspecies. However, we also identified key effects of dataset coverage and composition on our results. Thus, low-coverage bycatch data can offer valuable information for population genetic and phylodynamic analyses, but caution is required to ensure the reduced information does not introduce confounding factors, such as sampling biases, that drive inference. We encourage more researchers to consider maximizing the potential of the targeted sequence approach by examining evolutionary questions with their off-target bycatch where possible-especially in cases where no previous mitochondrial data exists-but recommend stratifying data at different genome coverage thresholds to separate sampling effects from genuine genomic signals, and to understand their implications for evolutionary research.

Population structure and interspecific hybridisation of two invasive blowflies (Diptera: Calliphoridae) following replicated incursions into New Zealand.

Rates of biological invasion are increasing globally, with associated negative effects on native biodiversity and ecosystem services. Among other genetic processes, hybridisation can facilitate invasion by producing new combinations of genetic variation that increase adaptive potential and associated population fitness. Yet the role of hybridisation (and resulting gene flow) in biological invasion in invertebrate species is under-studied. Calliphora hilli and Calliphora stygia are blowflies proposed to have invaded New Zealand separately from Australia between 1779 and 1841, and are now widespread throughout the country. Here, we analysed genome-wide single nucleotide polymorphisms (SNPs), generating genotyping-by-sequencing data for 154 individuals collected from 24 populations across New Zealand and Australia to assess the extent of gene flow and hybridisation occurring within and between these blowflies and to better understand their overall population structure. We found that New Zealand populations of both species had weak genetic structure, suggesting high gene flow and an absence of dispersal limitations across the country. We also found evidence that interspecific hybridisation is occurring in the wild between C. hilli and C. stygia in both the native and invasive ranges, and that intraspecific admixture is occurring among populations at appreciable rates. Collectively, these findings provide new insights into the population structure of these two invasive invertebrates and highlight the potential importance of hybridisation and gene flow in biological invasion.

Genomic signals of local adaptation across climatically heterogenous habitats in an invasive tropical fruit fly (Bactrocera tryoni).

Local adaptation plays a key role in the successful establishment of pest populations in new environments by enabling them to tolerate novel biotic and abiotic conditions experienced outside their native range. However, the genomic underpinnings of such adaptive responses remain unclear, especially for agriculturally important pests. We investigated population genomic signatures in the tropical/subtropical Queensland fruit fly, Bactrocera tryoni, which has an expanded range encompassing temperate and arid zones in Australia, and tropical zones in the Pacific Islands. Using reduced representation sequencing data from 28 populations, we detected allele frequency shifts associated with the native/invasive status of populations and identified environmental factors that have likely driven population differentiation. We also determined that precipitation, temperature, and geographic variables explain allelic shifts across the distribution range of B. tryoni. We found spatial heterogeneity in signatures of local adaptation across various climatic conditions in invaded areas. Specifically, disjunct invasive populations in the tropical Pacific Islands and arid zones of Australia were characterised by multiple significantly differentiated single nucleotide polymorphisms (SNPs), some of which were associated with genes with well-understood function in environmental stress (e.g., heat and desiccation) response. However, invasive populations in southeast Australian temperate zones showed higher gene flow with the native range and lacked a strong local adaptive signal. These results suggest that population connectivity with the native range has differentially affected local adaptive patterns in different invasive populations. Overall, our findings provide insights into the evolutionary underpinnings of invasion success of an important horticultural pest in climatically distinct environments.

Genomic Tools in Biological Invasions: Current State and Future Frontiers.

Human activities are accelerating rates of biological invasions and climate-driven range expansions globally, yet we understand little of how genomic processes facilitate the invasion process. Although most of the literature has focused on underlying phenotypic correlates of invasiveness, advances in genomic technologies are showing a strong link between genomic variation and invasion success. Here, we consider the ability of genomic tools and technologies to (i) inform mechanistic understanding of biological invasions and (ii) solve real-world issues in predicting and managing biological invasions. For both, we examine the current state of the field and discuss how genomics can be leveraged in the future. In addition, we make recommendations pertinent to broader research issues, such as data sovereignty, metadata standards, collaboration, and science communication best practices that will require concerted efforts from the global invasion genomics community.

Current stewardship practices in invasion biology limit the value and secondary use of genomic data.

Invasive species threaten native biota, putting fragile ecosystems at risk and having a large-scale impact on primary industries. Growing trade networks and the popularity of personal travel make incursions a more frequent risk, one only compounded by global climate change. With increasing publication of whole-genome sequences lies an opportunity for cross-species assessment of invasive potential. However, the degree to which published sequences are accompanied by satisfactory spatiotemporal data is unclear. We assessed the metadata associated with 199 whole-genome assemblies of 89 invasive terrestrial invertebrate species and found that only 38% of these were derived from field-collected samples. Seventy-six assemblies (38%) reported an 'undescribed' sample origin and, while further examination of associated literature closed this gap to 23.6%, an absence of spatial data remained for 47 of the total assemblies. Of the 76 assemblies that were ultimately determined to be field-collected, associated metadata relevant for invasion studies was predominantly lacking: only 35% (27 assemblies) provided granular location data, and 33% (n = 25) lacked sufficient collection date information. Our results support recent calls for standardized metadata in genome sequencing data submissions, highlighting the impact of missing metadata on current research in invasion biology (and likely other fields). Notably, large-scale consortia tended to provide the most complete metadata submissions in our analysis-such cross-institutional collaborations can foster a culture of increased adherence to improved metadata submission standards and a standard of metadata stewardship that enables reuse of genomes in invasion science.

Genome-wide analysis reveals distinct global populations of pink bollworm (Pectinophora gossypiella).

The pink bollworm (Pectinophora gossypiella) is one of the world's most destructive pests of cotton. This invasive lepidopteran occurs in nearly all cotton-growing countries. Its presence in the Ord Valley of North West Australia poses a potential threat to the expanding cotton industry there. To assess this threat and better understand population structure of pink bollworm, we analysed genomic data from individuals collected in the field from North West Australia, India, and Pakistan, as well as from four laboratory colonies that originated in the United States. We identified single nucleotide polymorphisms (SNPs) using a reduced-representation, genotyping-by-sequencing technique (DArTseq). The final filtered dataset included 6355 SNPs and 88 individual genomes that clustered into five groups: Australia, India-Pakistan, and three groups from the United States. We also analysed sequences from Genbank for mitochondrial DNA (mtDNA) locus cytochrome c oxidase I (COI) for pink bollworm from six countries. We found low genetic diversity within populations and high differentiation between populations from different continents. The high genetic differentiation between Australia and the other populations and colonies sampled in this study reduces concerns about gene flow to North West Australia, particularly from populations in India and Pakistan that have evolved resistance to transgenic insecticidal cotton. We attribute the observed population structure to pink bollworm's narrow host plant range and limited dispersal between continents.

The blowfly Chrysomya latifrons inhabits fragmented rainforests, but shows no population structure.

Climate change and deforestation are causing rainforests to become increasingly fragmented, placing them at heightened risk of biodiversity loss. Invertebrates constitute the greatest proportion of this biodiversity, yet we lack basic knowledge of their population structure and ecology. There is a compelling need to develop our understanding of the population dynamics of a wide range of rainforest invertebrates so that we can begin to understand how rainforest fragments are connected, and how they will cope with future habitat fragmentation and climate change. Blowflies are an ideal candidate for such research because they are widespread, abundant, and can be easily collected within rainforests. We genotyped 188 blowflies (Chrysomya latifrons) from 15 isolated rainforests and found high levels of gene flow, a lack of genetic structure between rainforests, and low genetic diversity - suggesting the presence of a single large genetically depauperate population. This highlights that: (1) the blowfly Ch. latifrons inhabits a ~ 1000 km stretch of Australian rainforests, where it plays an important role as a nutrient recycler; (2) strongly dispersing flies can migrate between and connect isolated rainforests, likely carrying pollen, parasites, phoronts, and pathogens along with them; and (3) widely dispersing and abundant insects can nevertheless be genetically depauperate. There is an urgent need to better understand the relationships between habitat fragmentation, genetic diversity, and adaptive potential-especially for poorly dispersing rainforest-restricted insects, as many of these may be particularly fragmented and at highest risk of local extinction.

Population genomic insights into invasion success in a polyphagous agricultural pest, Halyomorpha halys.

Invasive species are increasingly threatening ecosystems and agriculture by rapidly expanding their range and adapting to environmental and human-imposed selective pressures. The genomic mechanisms that underlie such rapid changes remain unclear, especially for agriculturally important pests. Here, we used genome-wide polymorphisms derived from native, invasive, and intercepted samples and populations of the brown marmorated stink bug (BMSB), Halyomorpha halys, to gain insights into population genomics processes that have promoted the successful global invasion of this polyphagous pest. Our analysis demonstrated that BMSB exhibits spatial structure but admixture rates are high among introduced populations, resulting in similar levels of genomic diversity across native and introduced populations. These spatial genomic patterns suggest a complex invasion scenario, potentially with multiple bridgehead events, posing a challenge for accurately assigning BMSB incursions to their source using reduced-representation genomic data. By associating allele frequencies with the invasion status of BMSB populations, we found significantly differentiated single nucleotide polymorphisms (SNPs) located in close proximity to genes for insecticide resistance and olfaction. Comparing variations in allele frequencies among populations for outlier SNPs suggests that BMSB invasion success has probably evolved from standing genetic variation. In addition to being a major nuisance of households, BMSB has caused significant economic losses to agriculture in recent years and continues to expand its range. Despite no record of BMSB insecticide resistance to date, our results show high capacity for potential evolution of such traits, highlighting the need for future sustainable and targeted management strategies.

Genomic data is missing for many highly invasive species, restricting our preparedness for escalating incursion rates.

Biological invasions drive environmental change, potentially threatening native biodiversity, human health, and global economies. Population genomics is an increasingly popular tool in invasion biology, improving accuracy and providing new insights into the genetic factors that underpin invasion success compared to research based on a small number of genetic loci. We examine the extent to which population genomic resources, including reference genomes, have been used or are available for invasive species research. We find that 82% of species on the International Union for Conservation of Nature "100 Worst Invasive Alien Species" list have been studied using some form of population genetic data, but just 32% of these species have been studied using population genomic data. Further, 55% of the list's species lack a reference genome. With incursion rates escalating globally, understanding how genome-driven processes facilitate invasion is critical, but despite a promising trend of increasing uptake, "invasion genomics" is still in its infancy. We discuss how population genomic data can enhance our understanding of biological invasion and inform proactive detection and management of invasive species, and we call for more research that specifically targets this area.

Assessing population genetic structure of three New Zealand stream insects using mitochondrial and nuclear DNA markers.

Assessing genetic differentiation among natural populations can aid understanding of dispersal patterns and connectivity among habitats. Several molecular markers have become increasingly popular in determining population genetic structure for this purpose. Here, we compared the resolution of mitochondrial cytochrome c oxidase subunit I (COI) and nuclear single nucleotide polymorphism (SNP) markers for detecting population structure among stream insects at small spatial scales. Individuals of three endemic taxa-Coloburiscus humeralis (Ephemeroptera), Zelandobius confusus (Plecoptera), and Hydropsyche fimbriata (Trichoptera)-were collected from forested streams that flow across open pasture in the North Island of New Zealand. Both COI and SNP data indicated limited population structure across the study area, and small differences observed among these species were likely related to their putative dispersal abilities. For example, fine-scale genetic differentiation between and among neighbouring stream populations for H. fimbriata suggests that gene flow, and hence dispersal, may be more limited for this species relative to the others. Based on the generally similar results provided by both types of markers, we suggest that either COI or SNP markers can provide suitable initial estimates of fine-scale population genetic differentiation in stream insects.

Adaptation to environmental temperature in divergent clades of the nematode Pristionchus pacificus.

Because of ongoing climate change, populations of organisms are being subjected to stressful temperatures more often. This is especially problematic for ectothermic organisms, which are likely to be more sensitive to changes in temperature. Therefore, we need to know if ectotherms have adapted to environmental temperature and, if so, what are the evolutionary mechanisms behind such adaptation. Here, we use the nematode Pristionchus pacificus as a case study to investigate thermal adaptation on the Indian Ocean island of La Réunion, which experiences a range of temperatures from coast to summit. We study the evolution of high-temperature tolerance by constructing a phylogenetic tree of strains collected from many different thermal niches. We show that populations of P. pacificus at low altitudes have higher fertility at warmer temperatures. Most likely, this phenotype has arisen recently and at least twice independently, consistent with parallel evolution. We also studied low-temperature tolerance and showed that populations from high altitudes have increased their fertility at cooler temperatures. Together, these data indicate that P. pacificus strains on La Réunion are subject to divergent selection, adapting to hot and cold niches at the coast and summit of the volcano. Precisely defining these thermal niches provides essential information for models that predict the impact of future climate change on these populations.

Insights into invasive species from whole-genome resequencing.

Studies of invasive species can simultaneously inform management strategies and quantify rapid evolution in the wild. The role of genomics in invasion science is increasingly recognised, and the growing availability of reference genomes for invasive species is paving the way for whole-genome resequencing studies in a wide range of systems. Here, we survey the literature to assess the application of whole-genome resequencing data in invasion biology. For some applications, such as the reconstruction of invasion routes in time and space, sequencing the whole genome of many individuals can increase the accuracy of existing methods. In other cases, population genomic approaches such as haplotype analysis can permit entirely new questions to be addressed and new technologies applied. To date whole-genome resequencing has only been used in a handful of invasive systems, but these studies have confirmed the importance of processes such as balancing selection and hybridization in allowing invasive species to reuse existing adaptations and rapidly overcome the challenges of a foreign ecosystem. The use of genomic data does not constitute a paradigm shift per se, but by leveraging new theory, tools, and technologies, population genomics can provide unprecedented insight into basic and applied aspects of invasion science.

Evolutionary Responses to Warming.

Climate change is predicted to dramatically alter biological diversity and distributions, driving extirpations, extinctions, and extensive range shifts across the globe. Warming can also, however, lead to phenotypic or behavioural plasticity, as species adapt to new conditions. Recent genomic research indicates that some species are capable of rapid evolution as selection favours adaptive responses to environmental change and altered or novel niche spaces. New advances are providing mechanistic insights into how temperature might accelerate evolution in the Anthropocene. These discoveries highlight intriguing new research directions - such as using geothermal and polar systems combined with powerful genomic tools - that will help us to understand the processes underpinning adaptive evolution and better project how ecosystems will change in a warming world.

Oxidative stress delays development and alters gene expression in the agricultural pest moth, Helicoverpa armigera.

Stress is a widespread phenomenon that all organisms must endure. Common in nature is oxidative stress, which can interrupt cell homeostasis to cause cell damage and may be derived from respiration or from environmental exposure through diet. As a result of the routine exposure from respiration, many organisms can mitigate the effects of oxidative stress, but less is known about responses to oxidative stress from other sources. Helicoverpa armigera is a major agricultural pest moth that causes significant damage to crops worldwide. Here, we examined the effects of oxidative stress on H. armigera by chronically exposing individuals to paraquat-a free radical producer-and measuring changes in development (weight, developmental rate, lifespan), and gene expression. We found that oxidative stress strongly affected development in H. armigera, with stressed samples spending more time as caterpillars than control samples (>24 vs. ~15 days, respectively) and therefore living longer overall. We found 1,618 up- and 761 down-regulated genes, respectively, in stressed versus control samples. In the up-regulated gene set, was an over-representation of biological processes related to cuticle and chitin development, glycine metabolism, and oxidation-reduction. Oxidative stress clearly impacts physiology and biochemistry in H. armigera and the interesting finding of an extended lifespan in stressed individuals could demonstrate hormesis, the phenomenon whereby toxic compounds can actually be beneficial at low doses. Collectively, our findings provide new insights into physiological and gene expression responses to oxidative stress in invertebrates.

Effects of sample age on data quality from targeted sequencing of museum specimens: what are we capturing in time?

BACKGROUND: Next generation sequencing (NGS) can recover DNA data from valuable extant and extinct museum specimens. However, archived or preserved DNA is difficult to sequence because of its fragmented, damaged nature, such that the most successful NGS methods for preserved specimens remain sub-optimal. Improving wet-lab protocols and comprehensively determining the effects of sample age on NGS library quality are therefore of vital importance. Here, I examine the relationship between sample age and several indicators of library quality following targeted NGS sequencing of ~ 1300 loci using 271 samples of pinned moth specimens (Helicoverpa armigera) ranging in age from 5 to 117 years. RESULTS: I find that older samples have lower DNA concentrations following extraction and thus require a higher number of indexing PCR cycles during library preparation. When sequenced reads are aligned to a reference genome or to only the targeted region, older samples have a lower number of sequenced and mapped reads, lower mean coverage, and lower estimated library sizes, while the percentage of adapters in sequenced reads increases significantly as samples become older. Older samples also show the poorest capture success, with lower enrichment and a higher improved coverage anticipated from further sequencing. CONCLUSIONS: Sample age has significant, measurable impacts on the quality of NGS data following targeted enrichment. However, incorporating a uracil-removing enzyme into the blunt end-repair step during library preparation could help to repair DNA damage, and using a method that prevents adapter-dimer formation may result in improved data yields.

Genome-wide SNP data reveal improved evidence for Antarctic glacial refugia and dispersal of terrestrial invertebrates.

Antarctica is isolated, surrounded by the Southern Ocean and has experienced extreme environmental conditions for millions of years, including during recent Pleistocene glacial maxima. How Antarctic terrestrial species might have survived these glaciations has been a topic of intense interest, yet many questions remain unanswered, particularly for Antarctica's invertebrate fauna. We examine whether genetic data from a widespread group of terrestrial invertebrates, springtails (Collembola, Isotomidae) of the genus Cryptopygus, show evidence for long-term survival in glacial refugia along the Antarctic Peninsula. We use genome-wide SNP analyses (via genotyping-by-sequencing, GBS) and mitochondrial data to examine population diversity and differentiation across more than 20 sites spanning >950 km on the Peninsula, and from islands both close to the Peninsula and up to ~1,900 km away. Population structure analysis indicates the presence of strong local clusters of diversity, and we infer that patterns represent a complex interplay of isolation in local refugia coupled with occasional successful long-distance dispersal events. We identified wind and degree days as significant environmental drivers of genetic diversity, with windier and warmer sites hosting higher diversity. Thus, we infer that refugial areas along the Antarctic Peninsula have allowed populations of indigenous springtails to survive in situ throughout glacial periods. Despite the difficulties of dispersal in cold, desiccating conditions, Cryptopygus springtails on the Peninsula appear to have achieved multiple long-distance colonization events, most likely through wind-related dispersal events.

Mitochondrial DNA genomes of five major Helicoverpa pest species from the Old and New Worlds (Lepidoptera: Noctuidae).

Five species of noctuid moths, Helicoverpa armigera, H. punctigera, H. assulta, H. zea, and H. gelotopoeon, are major agricultural pests inhabiting various and often overlapping global distributions. Visual identification of these species requires a great deal of expertise and misidentification can have repercussions for pest management and agricultural biosecurity. Here, we report on the complete mitochondrial genomes of H. assulta assulta and H. assulta afra, H. gelotopoeon, H. punctigera, H. zea, and H. armigera armigera and H. armigera conferta' assembled from high-throughput sequencing data. This study significantly increases the mitogenome resources for these five agricultural pests with sequences assembled from across different continents, including an H. armigera individual collected from an invasive population in Brazil. We infer the phylogenetic relationships of these five Helicoverpa species based on the 13 mitochondrial DNA protein-coding genes (PCG's) and show that two publicly available mitogenomes of H. assulta (KP015198 and KR149448) have been misidentified or incorrectly assembled. We further consolidate existing PCR-RFLP methods to cover all five Helicoverpa pest species, providing an updated method that will contribute to species differentiation and to future monitoring efforts of Helicoverpa pest species across different continents. We discuss the value of Helicoverpa mitogenomes to assist with species identification in view of the context of the rapid spread of H. armigera in the New World. With this work, we provide the molecular resources necessary for future studies of the evolutionary history and ecology of these species.

Multiple recombination events between two cytochrome P450 loci contribute to global pyrethroid resistance in Helicoverpa armigera.

The cotton bollworm, Helicoverpa armigera (Hübner) is one of the most serious insect pest species to evolve resistance against many insecticides from different chemical classes. This species has evolved resistance to the pyrethroid insecticides across its native range and is becoming a truly global pest after establishing in South America and having been recently recorded in North America. A chimeric cytochrome P450 gene, CYP337B3, has been identified as a resistance mechanism for resistance to fenvalerate and cypermethrin. Here we show that this resistance mechanism is common around the world with at least eight different alleles. It is present in South America and has probably introgressed into its closely related native sibling species, Helicoverpa zea. The different alleles of CYP337B3 are likely to have arisen independently in different geographic locations from selection on existing diversity. The alleles found in Brazil are those most commonly found in Asia, suggesting a potential origin for the incursion of H. armigera into the Americas.

Variation in rates of spontaneous male production within the nematode species Pristionchus pacificus supports an adaptive role for males and outcrossing.

BACKGROUND: The nematode species Pristionchus pacificus has an androdioecious mating system in which populations consist of self-fertilizing hermaphrodites and relatively few males. The prevalence of males in such a system is likely to depend on the relative pros and cons of outcrossing. While outcrossing generates novel allelic combinations and can therefore increase adaptive potential, it may also disrupt the potentially beneficial consequences of repeated generations of selfing. These include purging of deleterious alleles, inheritance of co-adapted allele complexes, improved hermaphrodite fitness and increased population growth. Here we use experimental and population genetic approaches to test hypotheses relating to male production and outcrossing in laboratory and natural populations of P. pacificus sampled from the volcanic island of La Réunion. RESULTS: We find a significant interaction between sampling locality and temperature treatment influencing rates of spontaneous male production in the laboratory. While strains isolated at higher altitude, cooler localities produce a higher proportion of male offspring at 25 °C relative to 20 or 15 °C, the reverse pattern is seen in strains isolated from warmer, low altitude localities. Linkage disequilibrium extends across long physical distances, but fails to approach levels reported for the partially selfing nematode species Caenorhabditis elegans. Finally, we find evidence for admixture between divergent genetic lineages. CONCLUSIONS: Elevated rates of laboratory male generation appear to occur under environmental conditions which differ from those experienced by populations in nature. Such elevated male generation may result in higher outcrossing rates, hence driving increased effective recombination and the creation of potentially adaptive novel allelic combinations. Patterns of linkage disequilibrium decay support selfing as the predominant reproductive strategy in P. pacificus. Finally, despite the potential for outcrossing depression, our results suggest admixture has occurred between distinct genetic lineages since their independent colonization of the island, suggesting outcrossing depression may not be uniform in this species.