Michael San Jose

An Evaluation of the Species Status of Bactrocera invadens and the Systematics of the Bactrocera dorsalis (Diptera: Tephritidae) Complex

ABSTRACT The genus Bactrocera (Tephritidae) contains >500 species, including many severe pests of fruits and vegetables. Although native to tropical and subtropical areas of Africa, India, Southeast Asia, and Australasia, a number of the pest species, largely members of the Bactrocera dorsalis (Hendel) complex, have become widespread through accidental introduction associated with agricultural trade. The B. dorsalis complex includes several morphologically and ecologically similar pests, making species designations uncertain. One of these, Bactrocera invadens Drew, Tsuruta, and White, endemic to Sri Lanka, has spread across Africa in the last decade and become a major agricultural pest. We sequenced one mitochondrial and two nuclear genes from 73 specimens, belonging to 19 species to construct phylogenies and examine species relationships and limits within the genus Bactrocera and several species of the B. dorsalis complex-specifically addressing the placement of B. invadens. Results indicate the B. dorsalis complex is polyphyletic. B. invadens and several other species within the B. dorsalis complex (B. dorsalis, Bactrocera papayae Drew &Hancock, and Bactrocera philippinensis Drew & Hancock) are also paraphyletic with respect to each other and probably represent a single genetically indistinguishable, phenotypically plastic, pest species that has spread throughout the world.

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

Abstract 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.

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

ABSTRACT 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.

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.

Phylogenetics and Species Status of Hawai'i's Endangered Blackburn's Sphinx Moth, Manduca blackburni (Lepidoptera: Sphingidae)

Abstract: Manduca blackburni, commonly known as Blackburns Sphinx Moth, is a federally listed endangered species restricted to localized habitats on three islands in the Hawaiian archipelago. Manduca blackburni was thought to be closely related to the widely distributed New World species M. quinquemaculatus, but this has never been formally tested, and shortly after its description, many authors dismissed it as a subspecies or form of M. quinquemaculatus. We used one mitochondrial gene, COI, and two nuclear genes, CAD and EF-1&agr; (2,975 bp total), to examine the phylogenetic relationships between M. blackburni and putative sister species in the genus. The phylogeny resulting from two single-gene analyses (CAD, COI) and the concatenation of all three genes suggest that M. blackburni + M. quinquemaculatus are sister taxa, and the monophyly of each species is supported with relatively high branch support under parsimony, maximum likelihood, and Bayesian inference. Manduca blackburni and M. quinquemaculatus also differ in genetic distance for CAD and COI, and we therefore consider them separate species. Thus, our molecular results corroborate previous studies on the morphology of M. blackburni and retain the species rank of this taxon. Our results also indicate that one or more South American subspecies of M. sexta may merit elevation to species.

Multi-gene phylogeny of the Hemileuca maia complex (Saturniidae) across North America suggests complex phylogeography and rapid ecological diversification

The Hemileuca maia species complex occurs across the North American continent and consists of six named taxa, and several others that were recently synonymized. Taxa exhibit a wide span of adult flight periods, dramatic shifts in host‐plant use and occur in a range of habitats, all of which would suggest unrecognized diversity. We used one mitochondrial and three nuclear genes to generate 3900 bp per individual, including samples from every ecotype in the species group across the United States from New England to central Florida to California. We assessed phylogenetic relationships using both maximum likelihood and Bayesian phylogenetic methods. Results suggest very low levels of divergence across most of the continent and low levels of genetic structure – even between some recognized species that maintain clear ecological difference in sympatry. Our results suggest that meaningful and localized ecological divergence may occur in the absence of easily recognizable genetic divergence, due to either ongoing gene flow or the recent diversification in the group.

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.

A global checklist of the 932 fruit fly species in the tribe Dacini (Diptera, Tephritidae)

Abstract The correct application of the scientific names of species is neither easy nor trivial. Mistakes can lead to the wrong interpretation of research results or, when pest species are involved, inappropriate regulations and limits on trade, and possibly quarantine failures that permit the invasion of new pest species. Names are particularly challenging to manage when groups of organisms encompass a large number of species, when different workers employ different philosophical views, or when species are in a state of taxonomic flux. The fruit fly tribe Dacini is a species-rich taxon within Tephritidae and contains around a fifth of all known species in the family. About 10% of the 932 currently recognized species are pests of commercial fruits and vegetables, precipitating quarantines and trade embargos. Authoritative species lists consist largely of scattered regional treatments and outdated online resources. The checklist presented here is the first global overview of valid species names for the Dacini in almost two decades, and includes new lure records. By publishing this list both in paper and digitally, we aim to provide a resource for those studying fruit flies as well as researchers studying components of their impact on agriculture. The list is largely a consolidation of previous works, but following the results from recent phylogenetic work, we transfer one subgenus and eight species to different genera: members of the Bactrocera subgenus Javadacus Hardy, considered to belong to the Zeugodacus group of subgenera, are transferred to genus Zeugodacus; Bactrocera pseudocucurbitae White, 1999, stat. rev., is transferred back to Bactrocera from Zeugodacus; Zeugodacus arisanicus Shiraki, 1933, stat. rev., is transferred back to Zeugodacus from Bactrocera; and Z. brevipunctatus (David & Hancock, 2017), comb. n.; Z. javanensis (Perkins, 1938), comb. n.; Z. montanus (Hardy, 1983), comb. n.; Z. papuaensis (Malloch, 1939), comb. n.; Z. scutellarius (Bezzi, 1916), comb. n.; Z. semisurstyli (Drew & Romig, 2013), comb. n.; and Z. trilineatus (Hardy, 1955), comb. n. are transferred from Bactrocera to Zeugodacus.

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.

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.