Interspecific gene flow obscures phylogenetic relationships in an important insect pest species complex.

As genomic data proliferates, the prevalence of post-speciation gene flow is making species boundaries and relationships increasingly ambiguous. Although current approaches inferring fully bifurcating phylogenies based on concatenated datasets provide simple and robust answers to many species relationships, they may be inaccurate because the models ignore inter-specific gene flow and incomplete lineage sorting. To examine the potential error resulting from ignoring gene flow, we generated both a RAD-seq and a 500 protein-coding loci highly multiplexed amplicon (HiMAP) dataset for a monophyletic group of 12 species defined as the Bactrocera dorsalis sensu lato clade. With some of the world's worst agricultural pests, the taxonomy of the B. dorsalis s.l. clade is important for trade and quarantines. However, taxonomic confusion confounds resolution due to intra- and interspecific phenotypic variation and convergence, mitochondrial introgression across half of the species, and viable hybrids. We compared the topological convergence of our datasets using concatenated phylogenetic and various multispecies coalescent approaches, some of which account for gene flow. All analyses agreed on species delimitation, but there was incongruence between species relationships. Under concatenation, both datasets suggest identical species relationships with mostly high statistical support. However, multispecies coalescent and multispecies network approaches suggest markedly different hypotheses and detected significant gene flow. We suggest that the network approaches are likely more accurate because gene flow violates the assumptions of the concatenated phylogenetic analyses, but the data-reductive requirements of network approaches resulted in reduced statistical support and could not unambiguously resolve gene flow directions. Our study highlights the importance of testing for gene flow, particularly with phylogenomic datasets, even when concatenated approaches receive high statistical support.

Cryptic diversity in a vagile Hawaiian moth group suggests complex factors drive diversification.

Allopatric speciation should be the dominant model of diversification across archipelagos because islands naturally promote isolation. It also follows that ecologically similar, vagile species should be more resistant to this kind of isolation due to dispersal and unifying selection. In a closely-related group of endemic Hawaiian hawkmoths, we found confounding patterns of inter-island connectivity and speciation that did not correlate with vagility, ecological specialization, or island age. Speciation occurred both in allopatric and sympatric taxa, with only the oldest and youngest islands fostering single-island endemic species. The intermediately-sized, central islands supported a combination of endemic and more widely-occurring lineages, suggesting no clear pattern leading to the current diversity in Hawaii. While some species are relatively common, others are apparently extinct or very rare, even on the same island. Further research into the specific mechanisms for these patterns in Hyles may prove broadly informative for understanding both cladogenesis and improving conservation planning. Our study identifies one new species endemic to Kauai and unique mitochondrial lineages in H. perkinsi, which may prove to be new species.

Transpacific coalescent pathways of coconut rhinoceros beetle biotypes: Resistance to biological control catalyses resurgence of an old pest.

Biological control agents have several advantages over chemical control for pest management, including the capability to restore ecosystem balance with minimal non-target effects and a lower propensity for targets to develop resistance. These factors are particularly important for invasive species control. The coconut rhinoceros beetle (Oryctes rhinoceros Linnaeus) is a major palm pest that invaded many Pacific islands in the early 20th century through human-mediated dispersal. Application of the Oryctes nudivirus in the 1960s successfully halted the beetle's first invasion wave and made it a textbook example of successful biological control. However, a recently discovered O. rhinoceros biotype that is resistant to the nudivirus appears to be correlated with a new invasion wave. We performed a population genomics analysis of 172 O. rhinoceros from seven regions, including native and invasive populations, to reconstruct invasion pathways and explore correlation between recent invasions and biotypes. With ddRAD sequencing, we generated data sets ranging from 4,000 to 209,000 loci using stacks and ipyrad software pipelines and compared genetic signal in downstream clustering and phylogenetic analyses. Analysis suggests that the O. rhinoceros resurgence is mediated by the nudivirus-resistant biotype. Genomic data have been proven essential to understanding the new O. rhinoceros biotype's invasion patterns and interactions with the original biotype. Such information is crucial to optimization of strategies for quarantine and control of resurgent pests. Our results demonstrate that while invasions are relatively rare events, new introductions can have significant ecological consequences, and quarantine vigilance is required even in previously invaded areas.

Incongruence between molecules and morphology: A seven-gene phylogeny of Dacini fruit flies paves the way for reclassification (Diptera: Tephritidae).

Molecular and morphological research often suggest conflicting results. Selective pressure on certain morphologies can confound understanding of evolutionary relationships. Dacini is one of the most diverse tribes of tephritid flies and contains many economically important pest species. Their black and yellow patterned body markings are presumed to act as wasp mimicry, and the characters separating species and groups are limited and in some cases phenotypically plastic. The traditional taxonomy of the tribe is controversial because groupings are based on unique combinations of morphological characters without the use of cladistic methods, though recent phylogenetic and taxonomic analyses have resulted in significant changes to their taxonomy. The monophyly of the three largest genera in the tribe has been tested with only small numbers of representatives per genus and a limited number of genes. To further understand the taxonomy and evolution of Dacini we sequenced seven genes from 167 Dacini species and five dipteran outgroups to construct a robust phylogeny and test phylogenetic relationships between genera, subgenera, and species complexes. Our phylogeny confirms the monophyly of Dacus, Bactrocera, and Zeugodacus. However, most groups below the genus level are not monophyletic, and only through further revision will we be able to understand their evolution and clarify the taxonomy within this tribe.

Sex-biased secondary contact obscures ancient speciation onto relictual host trees in central California moths (Syndemis: Tortricidae).

The tortricid moth genus Syndemis has ten described species, with two polyphagous species in Europe and North America respectively. We sequenced five nuclear and four mitochondrial genes for Syndemis samples across both continents and discovered unexpected, extensive diversification restricted to California. DNA evidence supports five new, undescribed, species endemic to California, while the rest of North America and Europe have only one species each. Further, the nuclear genes are less variable and yield contrasting phylogenetic signal compared to mitochondrial DNA for basal relationships between taxa across the genus. Such conflict strongly suggests that male and female moths exhibit radically different levels of philopatry. Our results highlight the importance of sex-specific behavior, and the need for inclusion of multiple genes to fully understand species boundaries, their causes, and the process of speciation. While mtDNA introgression often is invoked to explain incongruous haplotype distributions, our study shows that nuclear DNA selective sweeps, or swamping, can occur while mtDNA and ecology preserve an ancient divergence that is not discernable in nuclear DNA. This study further demonstrates that diversification of herbivores may occur on relictual, declining hostplants, which contrasts with the dominant co-speciation model.

Genetic Diversity of Bactrocera dorsalis (Diptera: Tephritidae) on the Hawaiian Islands: Implications for an Introduction Pathway Into California.

Population genetic diversity of the oriental fruit fly, Bactrocera dorsalis (Hendel), on the Hawaiian islands of Oahu, Maui, Kauai, and Hawaii (the Big Island) was estimated using DNA sequences of the mitochondrial cytochrome c oxidase subunit I gene. In total, 932 flies representing 36 sampled sites across the four islands were sequenced for a 1,500-bp fragment of the gene named the C1500 marker. Genetic variation was low on the Hawaiian Islands with >96% of flies having just two haplotypes: C1500-Haplotype 1 (63.2%) or C1500-Haplotype 2 (33.3%). The other 33 flies (3.5%) had haplotypes similar to the two dominant haplotypes. No population structure was detected among the islands or within islands. The two haplotypes were present at similar frequencies at each sample site, suggesting that flies on the various islands can be considered one population. Comparison of the Hawaiian data set to DNA sequences of 165 flies from outbreaks in California between 2006 and 2012 indicates that a single-source introduction pathway of Hawaiian origin cannot explain many of the flies in California. Hawaii, however, could not be excluded as a maternal source for 69 flies. There was no clear geographic association for Hawaiian or non-Hawaiian haplotypes in the Bay Area or Los Angeles Basin over time. This suggests that California experienced multiple, independent introductions from different sources.

Towards a better future for DNA barcoding: Evaluating monophyly- and distance-based species identification using COI gene fragments of Dacini fruit flies.

The utility of a universal DNA 'barcode' fragment (658 base pairs of the Cytochrome C Oxidase I [COI] gene) has been established as a useful tool for species identification, and widely criticized as one for understanding the evolutionary history of a group. Large amounts of COI sequence data have been produced that hold promise for rapid species identification, for example, for biosecurity. The fruit fly tribe Dacini holds about a thousand species, of which 80 are pests of economic concern. We generated a COI reference library for 265 species of Dacini containing 5601 sequences that span most of the COI gene using circular consensus sequencing. We compared distance metrics versus monophyly assessments for species identification and although we found a 'soft' barcode gap around 2% pairwise distance, the exceptions to this rule dictate that a monophyly assessment is the only reliable method for species identification. We found that all fragments regularly used for Dacini fruit fly identification >450 base pairs long provide similar resolution. 11.3% of the species in our dataset were non-monophyletic in a COI tree, which is mostly due to species complexes. We conclude with recommendations for the future generation and use of COI libraries. We revise the generic assignment of Dacus transversus stat. rev. Hardy 1982, and Dacus perpusillus stat. rev. Drew 1971 and we establish Dacus maculipterus White 1998 syn. nov. as a junior synonym of Dacus satanas Liang et al. 1993.

Genomics reveals widespread hybridization across insects with ramifications for species boundaries and invasive species.

As the amount of genomic data for nonmodel taxa grows, it is increasingly clear that gene flow across species barriers in insects is much more common than previously thought. In recent years, the decreased cost and increased accuracy of long-read sequencing has enabled the assembly of high-quality reference genomes and chromosome maps for nonmodel insects. With this long-read data, we can now not only compare variation across the genome among homologous genes between species, which has been the basis of phylogenetics for more than 30 years, but also tease apart evidence of ancient and recent hybridization and gene flow. The implications of hybridization for species adaptation may be more positive than previously considered, explaining its prevalence across many groups of insects. Unfortunately, due to anthropogenic actions, some pest species appear to be benefitting from hybridization and gene flow, facilitating future invasions.

Highly variable COI haplotype diversity between three species of invasive pest fruit fly reflects remarkably incongruent demographic histories.

Distance decay principles predict that species with larger geographic ranges would have greater intraspecific genetic diversity than more restricted species. However, invasive pest species may not follow this prediction, with confounding implications for tracking phenomena including original ranges, invasion pathways and source populations. We sequenced an 815 base-pair section of the COI gene for 441 specimens of Bactrocera correcta, 214 B. zonata and 372 Zeugodacus cucurbitae; three invasive pest fruit fly species with overlapping hostplants. For each species, we explored how many individuals would need to be included in a study to sample the majority of their haplotype diversity. We also tested for phylogeographic signal and used demographic estimators as a proxy for invasion potency. We find contrasting patterns of haplotype diversity amongst the species, where B. zonata has the highest diversity but most haplotypes were represented by singletons; B. correcta has ~7 dominant haplotypes more evenly distributed; Z. cucurbitae has a single dominant haplotype with closely related singletons in a 'star-shape' surrounding it. We discuss how these differing patterns relate to their invasion histories. None of the species showed meaningful phylogeographic patterns, possibly due to gene-flow between areas across their distributions, obscuring or eliminating substructure.

Six years of fruit fly surveys in Bangladesh: a new species, 33 new country records and discovery of the highly invasive Bactrocera carambolae (Diptera, Tephritidae).

We engaged in six years of snap-shot surveys for fruit flies in rural environments and ten protected forest areas of Bangladesh, using traps baited with male lures (cue-lure, methyl eugenol, zingerone). Our work has increased the recorded number of species of Tephritidae in the country from seven to 37. We summarize these surveys and report eight new country occurrence records, and a new species (Zeugodacus madhupuri Leblanc & Doorenweerd, sp. nov.) is described. The highlight among the new records is the discovery, and significant westward range extension, of Bactrocera carambolae Drew & Hancock, a major fruit pest detected in the Chattogram and Sylhet Divisions. We rectify the previously published erroneous record of Bactrocera bogorensis (Hardy), which was based on a misidentification of Zeugodacus diaphorus (Hendel). We also report the occurrence in Bangladesh of nine other Tephritidae, the rearing of three primary fruit fly parasitoids from Zeugodacus, and records of non-target attraction to fruit fly lures.

Tracking the Origins of Fly Invasions; Using Mitochondrial Haplotype Diversity to Identify Potential Source Populations in Two Genetically Intertwined Fruit Fly Species (Bactrocera carambolae and Bactrocera dorsalis [Diptera: Tephritidae]).

Bactrocera carambolae Drew and Hancock and Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) are important pests of many fruits. These flies have been spread across the world through global travel and trade, and new areas are at risk of invasion. Whenever new invasive populations are discovered, quick and accurate identification is needed to mitigate the damage they can cause. Determining invasive pathways can prevent further spread of pests as well as subsequent reinvasions through the same pathway. Molecular markers can be used for both species identification and pathway analysis. We analyzed 1,601 individuals from 19 populations using 765 base pairs of the mitochondrial cytochrome oxidase I (COI) gene to infer the haplotype diversity and population structure within these flies from across their native and invasive ranges. We analyzed these samples by either grouping by species or geographic populations due to the genetic similarity in the mitochondrial genome. We found no genetic structure between B. dorsalis and B. carambolae and our findings suggest recent and most likely ongoing, genetic exchange between these two species in the wild. Hyper-diverse mitochondrial genetic diversity in the native range suggests large population sizes and relatively high mutation rates. Only 52% of the haplotypes found in the trap captures from California are shared with haplotypes from flies found in our global survey, indicating significant genetic diversity in the native range that is missing from our samples. However, these results provide a foundation for the accurate determination of the provenance of invasive populations around the world.

HiMAP: Robust phylogenomics from highly multiplexed amplicon sequencing.

High-throughput sequencing has fundamentally changed how molecular phylogenetic data sets are assembled, and phylogenomic data sets commonly contain 50- to 100-fold more loci than those generated using traditional Sanger sequencing-based approaches. Here, we demonstrate a new approach for building phylogenomic data sets using single-tube, highly multiplexed amplicon sequencing, which we name HiMAP (highly multiplexed amplicon-based phylogenomics) and present bioinformatic pipelines for locus selection based on genomic and transcriptomic data resources and postsequencing consensus calling and alignment. This method is inexpensive and amenable to sequencing a large number (hundreds) of taxa simultaneously and requires minimal hands-on time at the bench (<1/2 day), and data analysis can be accomplished without the need for read mapping or assembly. We demonstrate this approach by sequencing 878 amplicons in single reactions for 82 species of tephritid fruit flies across seven genera (384 individuals), including some of the most economically important agricultural insect pests. The resulting filtered data set (>150,000-bp concatenated alignment, ~20% missing character sites across all individuals and amplicons) contained >40,000 phylogenetically informative characters, and although some discordance was observed between analyses, it provided unparalleled resolution of many phylogenetic relationships in this group. Most notably, we found high support for the generic status of Zeugodacus and the sister relationship between Dacus and Zeugodacus. We discuss HiMAP, with regard to its molecular and bioinformatic strengths, and the insight the resulting data set provides into relationships of this diverse insect group.

Description of a new species and new country distribution records of Bactrocera (Diptera: Tephritidae: Dacinae) from Cambodia.

Bactrocera (Bactrocera) kohkongiae Leblanc (Diptera: Tephritidae: Dacinae), from the Koh Kong Province of Cambodia, is described as new. This species belongs to the Oriental fruit fly (B. dorsalis) complex. Genetic sequences (mitochondrial COI and nuclear EF1α and Period) are deposited in GenBank. A haplotype network, based on the COI sequences for 21 specimens, shows high genetic diversity. New country records from Cambodia are included for 22 species.

A phylogenetic assessment of the polyphyletic nature and intraspecific color polymorphism in the Bactrocera dorsalis complex (Diptera, Tephritidae).

The Bactrocera dorsalis complex (Tephritidae) comprises 85 species of fruit flies, including five highly destructive polyphagous fruit pests. Despite significant work on a few key pest species within the complex, little has been published on the majority of non-economic species in the complex, other than basic descriptions and illustrations of single specimens regarded as typical representatives. To elucidate the species relationships within the Bactrocera dorsalis complex, we used 159 sequences from one mitochondrial (COI) and two nuclear (elongation factor-1α and period) genes to construct a phylogeny containing 20 described species from within the complex, four additional species that may be new to science, and 26 other species from Bactrocera and its sister genus Dacus. The resulting concatenated phylogeny revealed that most of the species placed in the complex appear to be unrelated, emerging across numerous clades. This suggests that they were placed in the Bactrocera dorsalis complex based on the similarity of convergent characters, which does not appear to be diagnostic. Variations in scutum and abdomen color patterns within each of the non-economic species are presented and demonstrate that distantly-related, cryptic species overlap greatly in traditional morphological color patterns used to separate them in keys. Some of these species may not be distinguishable with confidence by means other than DNA data.

Geographic proximity not a prerequisite for invasion: Hawaii not the source of California invasion by light brown apple moth (Epiphyas postvittana).

BACKGROUND: The light brown apple moth (LBAM), Epiphyas postvittana (Walker), is native to Australia but invaded England, New Zealand, and Hawaii more than 100 years ago. In temperate climates, LBAM can be a major agricultural pest. In 2006 LBAM was discovered in California, instigating eradication efforts and quarantine against Hawaiian agriculture, the assumption being that Hawaii was the source of the California infestation. Genetic relationships among populations in Hawaii, California, and New Zealand are crucial to understanding LBAM invasion dynamics across the Pacific. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced mitochondrial DNA (mtDNA) from 1293 LBAM individuals from California (695), Hawaii (448), New Zealand (147), and Australia (3) to examine haplotype diversity and structure among introduced populations, and evaluate the null hypothesis that invasive populations are from a single panmictic source. However, invasive populations in California and New Zealand harbor deep genetic diversity, whereas Hawaii shows low level, shallow diversity. CONCLUSIONS/SIGNIFICANCE: LBAM recently has established itself in California, but was in Hawaii and New Zealand for hundreds of generations, yet California and New Zealand show similar levels of genetic diversity relative to Hawaii. Thus, there is no clear relationship between duration of invasion and genetic structure. Demographic statistics suggest rapid expansion occurring in California and past expansions in New Zealand; multiple introductions of diverse, genetically fragmented lineages could contribute to these patterns. Hawaii and California share no haplotypes, therefore, Hawaii is not the source of the California introduction. Paradoxically, Hawaii and California share multiple haplotypes with New Zealand. New Zealand may be the source for the California and Hawaii infestations, but the introductions were independent, and Hawaii was invaded only once. This has significant implications for quarantine, and suggests that probability of invasion is not directly related to geographic distance. Surprisingly, Hawaiian LBAM populations have much lower genetic diversity than California, despite being older.