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The frequency and severity of outbreaks by pestiferous insects is increasing globally, likely as a result of human-mediated introductions of non-native organisms. However, it is not always apparent whether an outbreak is the result of a recent introduction of an evolutionarily naïve population, or of recent disturbance acting on an existing population that arrived previously during natural range expansion.  Here we use approximate Bayesian computation to infer the colonization history of a pestiferous insect, the winter moth, Operophtera brumata L. (Lepidoptera: Geometridae), which has caused widespread defoliation in northern Fennoscandia. We generated genotypes using a suite of 24 microsatellite loci and find that populations of winter moth in northern Europe can be assigned to five genetically distinct clusters that correspond with 1) Iceland, 2) the British Isles, 3) Central Europe and southern Fennoscandia, 4) Eastern Europe, and 5) northern Fennoscandia. We find that the northern Fennoscandia winter moth cluster is most closely related to a population presently found in the British Isles, and that these populations likely diverged around 2,900 years ago. This result suggests that current outbreaks are not the result of a recent introduction, but rather that recent climate or habitat disturbance is acting on existing populations that may have arrived to northern Fennoscandia via pre-Roman traders from the British Isles, and/or by natural dispersal across the North Sea likely using the Orkney Islands of northern Scotland as a stepping-stone before dispersing up the Norwegian coast.

In light of the current biodiversity crisis, molecular barcoding has developed into an irreplaceable tool. Barcoding has been considerably simplified by developments in high throughput sequencing technology, but still can be prohibitively expensive and laborious when community samples of thousands of specimens need to be processed. Here, we outline an Illumina amplicon sequencing approach to generate multilocus data from large collections of arthropods. We reduce cost and effort up to 50-fold, by combining multiplex PCRs and DNA extractions from pools of presorted and morphotyped specimens and using two levels of sample indexing. We test our protocol by generating a comprehensive, community wide dataset of barcode sequences for several thousand Hawaiian arthropods from 14 orders, which were collected across the archipelago using various trapping methods. We explore patterns of diversity across the Archipelago and compare the utility of different arthropod trapping methods for biodiversity explorations on Hawaii, highlighting undergrowth beating as highly efficient method. Moreover, we show the effects of barcode marker, taxonomy and relative biomass of the targeted specimens and sequencing coverage on taxon recovery. Our protocol enables rapid and inexpensive explorations of diversity patterns and the generation of multilocus barcode reference libraries across whole ecosystems.

New genetic diagnostic approaches have greatly aided efforts to document global biodiversity and improve biosecurity. This is especially true for organismal groups in which species diversity has been underestimated historically due to difficulties associated with sampling, the lack of clear morphological characteristics, and/or limited availability of taxonomic expertise. Among these methods, DNA sequence barcoding (also known as "DNA barcoding") and by extension, meta-barcoding for biological communities, has emerged as one of the most frequently utilized methods for DNA-based species identifications. Unfortunately, the use of DNA barcoding is limited by the availability of complete reference libraries (i.e., a collection of DNA sequences from morphologically identified species), and by the fact that the vast majority of species do not have sequences present in reference databases. Such conditions are critical especially in tropical locations that are simultaneously biodiversity rich and suffer from a lack of exploration and DNA characterization by trained taxonomic specialists. To facilitate efforts to document biodiversity in regions lacking complete reference libraries, we developed a novel statistical approach that categorizes unidentified species as being either likely native or likely nonnative based solely on measures of nucleotide diversity. We demonstrate the utility of this approach by categorizing a large sample of specimens of terrestrial insects and spiders (collected as part of the Moorea BioCode project) using a generalized linear mixed model (GLMM). Using a training data set of known endemic (n = 45) and known introduced species (n = 102), we then estimated the likely native/nonnative status for 4,663 specimens representing an estimated 1,288 species (412 identified species), including both those specimens that were either unidentified or whose endemic/introduced status was uncertain. Using this approach, we were able to increase the number of categorized specimens by a factor of 4.4 (from 794 to 3,497), and the number of categorized species by a factor of 4.8 from (147 to 707) at a rate much greater than chance (77.6% accuracy). The study identifies phylogenetic signatures of both native and nonnative species and suggests several practical applications for this approach including monitoring biodiversity and facilitating biosecurity.

Coevolution may be an important component of the sustainability of importation biological control, but how frequently introduced natural enemies coevolve with their target pests is unclear. Here we explore whether comparative population genetics of the invasive walnut aphid, Chromaphis juglandicola, and its introduced parasitoid, Trioxys pallidus, provide insights into the localized breakdown of biological control services in walnut orchards in California. We found that sampled populations of C. juglandicola exhibited higher estimates of genetic differentiation (F ST) than co-occurring populations of T. pallidus. In contrast, estimates of both the inbreeding coefficient (G IS) and contemporary gene flow were higher for T. pallidus than for C. juglandicola. We also found evidence of reciprocal outlier loci in some locations, but none showed significant signatures of selection. Synthesis and applications. Understanding the importance of coevolutionary interactions for the sustainability of biological control remains an important and understudied component of biological control research. Given the observed differences in gene flow and genetic differentiation among populations of T. pallidus and C. juglandicola, we suspect that temporary local disruption of biological control services may occur more frequently than expected while remaining stable at broader regional scales. Further research that combines genomewide single nucleotide polymorphism genotyping with measurements of phenotypic traits is needed to provide more conclusive evidence of whether the occurrence of outlier loci that display significant signatures of selection can be interpreted as evidence of the presence of a geographic mosaic of coevolution in this system.

Background: Illumina sequencing with its high number of reads and low per base pair cost is an attractive technology for development of molecular resources for non-model organisms. While many software packages have been developed to identify short tandem repeats (STRs) from next-generation sequencing data, these methods do not inform the investigator as to whether or not candidate loci are polymorphic in their target populations.Results: We provide a python program iMSAT that uses the polymorphism data obtained from mapping individual Illumina sequence reads onto a reference genome to identify polymorphic STRs. Using this approach, we identified 9,119 candidate polymorphic STRs for use with the parasitoid wasp Trioxys pallidus and 2,378 candidate polymorphic STRs for use with the aphid Chromaphis juglandicola. For both organisms we selected 20 candidate tri-nucleotide STRs for validation. Using fluorescent-labeled oligonucleotide primers, we genotyped 91 female T. pallidus collected in nine localities and 46 female C. juglandicola collected in 4 localities and found 15 of the examined markers to be polymorphic for T. pallidus and 12 of the examined markers to be polymorphic for C. juglandicola.Conclusions: We present a novel approach that uses standard Illumina barcoding primers and a single Illumina HiSeq run to target polymorphic STR fragments to develop and test STR markers. We validate this approach using the parasitoid wasp T. pallidus and its aphid host C. juglandicola. This approach, which would also be compatible with 454 Sequencing, allowed us to quickly identify markers with known variability. Accordingly, our method constitutes a significant improvement over existing STR identification software packages.

At the same time that molecular researchers are improving techniques to extract DNA from museum specimens, this increased demand for access to museum specimens has created tension between the need to preserve specimens for maintaining collections and morphological research and the desire to conduct molecular analyses. To address these concerns, we examined the suitability of non-invasive DNA extraction techniques on three species of parasitic Hymenoptera (Braconidae), and test the effects of body size (parasitoid species), age (time since collection), and DNA concentration from each extract on the probability of amplifying meaningful fragments of two commonly used genetic loci. We found that age was a significant factor for determining the probability of success for sequencing both 28S and COI fragments. While the size of the braconid parasitoids significantly affected the total amount of extracted DNA, neither size nor DNA concentration were significant factors for the amplification of either gene region. We also tested several primer combinations of various lengths, but were unable to amplify fragments longer than ~150 base pairs. These short fragments of 28S and COI were however sufficient for species identification, and for the discovery of within species genetic variation.

The dynamic structure of ecological communities results from interactions among taxa that change with shifts in species composition in space and time. However, our ability to study the interplay of ecological and evolutionary processes on community assembly remains relatively unexplored due to the difficulty of measuring community structure over long temporal scales. Here, we made use of a geological chronosequence across the Hawaiian Islands, representing 50 years to 4.15 million years of ecosystem development, to sample 11 communities of arthropods and their associated plant taxa using semiquantitative DNA metabarcoding. We then examined how ecological communities changed with community age by calculating quantitative network statistics for bipartite networks of arthropod–plant associations. The average number of interactions per species (linkage density), ratio of plant to arthropod species (vulnerability) and uniformity of energy flow (interaction evenness) increased significantly in concert with community age. The index of specialization H2′ has a curvilinear relationship with community age. Our analyses suggest that younger communities are characterized by fewer but stronger interactions, while biotic associations become more even and diverse as communities mature. These shifts in structure became especially prominent on East Maui (~0.5 million years old) and older volcanos, after enough time had elapsed for adaptation and specialization to act on populations in situ. Such natural progression of specialization during community assembly is probably impeded by the rapid infiltration of non‐native species, with special risk to younger or more recently disturbed communities that are composed of fewer specialized relationships. [ABSTRACT FROM AUTHOR]

Islands make up a large proportion of Earth's biodiversity, yet are also some of the most sensitive systems to environmental perturbation. Biogeographic theory predicts that geologic age, area, and isolation typically drive islands' diversity patterns, and thus potentially impact non‐native spread and community homogenization across island systems. One limitation in testing such predictions has been the difficulty of performing comprehensive inventories of island biotas and distinguishing native from introduced taxa. Here, we use DNA metabarcoding and statistical modelling as a high throughput method to survey community‐wide arthropod richness, the proportion of native and non‐native species, and the incursion of non‐natives into primary habitats on three archipelagos in the Pacific – the Ryukyus, the Marianas and Hawaii – which vary in age, isolation and area. Diversity patterns largely match expectations based on island biogeography theory, with the oldest and most geographically connected archipelago, the Ryukyus, showing the highest taxonomic richness and lowest proportion of introduced species. Moreover, we find evidence that forest habitats are more resilient to incursions of non‐natives in the Ryukyus than in the less taxonomically rich archipelagos. Surprisingly, we do not find evidence for biotic homogenization across these three archipelagos: the assemblage of non‐native species on each island is highly distinct. Our study demonstrates the potential of DNA metabarcoding to facilitate rapid estimation of biogeographic patterns, the spread of non‐native species, and the resilience of ecosystems. [ABSTRACT FROM AUTHOR]

The European winter moth, Operophtera brumata L. (Lepidoptera: Geometridae), was accidentally introduced to North America on at least 4 separate occasions, where it has been hybridizing with the native Bruce spanworm, O. bruceata Hulst, at rates up to 10% per year. Both species are known to respond to the same sex pheromones and to produce viable offspring, but whether they differ in the seasonal timing of their mating flights is unknown. Therefore, we collected adult male moths weekly along 2 transects in the northeastern United States and genotyped individuals using polymorphic microsatellite markers as males of these 2 species cannot be differentiated morphologically. Along each transect, we then estimated the cumulative proportions (i.e. the number of individuals out of the total collected) of each species on each calendar day. Our results indicate that there are significant differences between the species regarding their seasonal timing of flight, and these allochronic differences likely are acting to promote reproductive isolation between these 2 species. Lastly, our results suggest that the later flight observed by winter moth compared to Bruce spanworm may be limiting its inland spread in the northeastern United States because of increased exposure to extreme winter events. [ABSTRACT FROM AUTHOR]

Species of knotweeds, Reynoutria spp. Houtt. (Caryophyllales: Polygonaceae), including Japanese knotweed (R. japonica Houtt.), are among the most invasive and ecologically destructive plant species introduced to North America and Europe. The Kyushu strain of the psyllid Aphalara itadori Shinji (Hemiptera: Aphalaridae) has been approved as a biological control agent for release against Japanese knotweed in the United Kingdom, Canada, and the United States. However, recent reports from Canada suggest that both biotic and abiotic factors may limit its establishment. Therefore, we examined the potential role of predation by comparing open- versus closed-sleeve treatments, and climate mis-matches by collecting temperature data from release sites and performing climate suitability analyses using MaxEnt. Our results indicated that populations of the Kyushu strain could only be maintained in the field in closed-sleeve treatments, suggesting that predation is likely limiting the establishment success of this strain. In addition, we noted that daily maximum temperatures at our field sites might exceed documented developmental thresholds for this strain, and that MaxEnt species distribution modeling indicates no climate similarities between locations in eastern North America and Kyushu. Combined with previous results, our study suggests that the establishment of the Kyushu strain of A. itadori as a biological control agent for Japanese knotweed may be limited in eastern North America. We suggest that one strategy to increase the probability of establishment of the Kyushu strain could be to increase the number of release sites in an effort to find a more optimal niche with predator-free space. [ABSTRACT FROM AUTHOR]

Research pertaining to the two closely‐related microsporidian genera Nosema and Vairimorpha is hindered by inconsistencies in species differentiation within and between the two clades. One proposal to better delimit these genera is to restructure the Nosema around a "True Nosema" clade, consisting of species that share a characteristic reversed ribosomal DNA operon arrangement and small subunit (SSU) ribosomal DNA sequences similar to that of the Nosema type species, N. bombycis. Using this framework, we assess two distinct microsporidia recovered from the forest insect Bruce spanworm (Operophtera bruceata) by sequencing their SSU and internal transcribed spacer regions. Phylogenetic analyses place one of our isolates within the proposed True Nosema clade close to N. furnacalis and place the other in the broader Nosema/Vairimorpha clade close to N. thomsoni. We found that 25% of Bruce spanworm cadavers collected over the four‐year study period were infected with microsporidia, but no infections were detected in cadavers of the Bruce spanworm's invasive congener, the winter moth (O. brumata), collected over the same period. We comment on these findings as they relate to the population dynamics of the Bruce spanworm‐winter moth system in this region, and more broadly, on the value of ribosomal DNA operon arrangement in Nosema systematics. [ABSTRACT FROM AUTHOR]

Gall wasps (Hymenoptera: Cynipidae) are phytophagous insects that often go unnoticed; however, when they are introduced to a new area or released from their natural enemies, they have the capacity to outbreak and cause extensive foliar damage. One such outbreaking pest, Zapatella davisae (Cynipidae: Cynipini), causes significant damage and mortality to black oak, Quercus velutina, in the northeastern United States. In this study, we aimed to identify the parasitoid community associated with Z. davisae, compare differences in percent parasitism of Z. davisae in Cape Cod and Long Island, and determine which parasitoid species contribute most to parasitism in each region. From both locations, we reared parasitoids, identified morphological groups, analyzed percent parasitism rates for each group, and used DNA barcoding to provide species‐level identifications. On Long Island, there was nearly 100% parasitism in 2015 followed by a near total collapse of the population in 2016. In contrast, parasitism rates were lower and remained consistent on Cape Cod between 2015 and 2016, which may explain the greater canopy damage observed in that region. Species of Sycophila were the dominant parasitoids, with one species Sycophila nr. novascotiae representing ~65% of reared parasitoids from Long Island, and two species of Sycophila (S. nr. novascotiae and S. foliatae) with near equal representations on Cape Cod. In order to manage an insect pest, it is important to understand factors that influence its mortality and survival. An understanding of how these infestations progress overtime can help predict the impact that newer infestations in Nantucket, MA, and coastal Rhode Island will have on black oak populations and will aid in the management of this rapidly spreading gall wasp pest. Gall wasps (Hymenoptera: Cynipidae) are phytophagous insects that often go unnoticed; however, when they are introduced to a new area or released from their natural enemies, they have the capacity to outbreak and cause extensive foliar damage. In order to manage an insect pest, it is important to understand factors that influence its mortality and survival. Our results suggest that a particular parasitoid community assemblage for the gall wasp Zapatella davisae is linked to lower gall wasp numbers and healthier black oak communities, thereby increasing local ecosystem productivity and resilience. [ABSTRACT FROM AUTHOR]