Norman B. Barr

Episodic radiations in the fly tree of life

Flies are one of four superradiations of insects (along with beetles, wasps, and moths) that account for the majority of animal life on Earth. Diptera includes species known for their ubiquity (Musca domestica house fly), their role as pests (Anopheles gambiae malaria mosquito), and their value as model organisms across the biological sciences (Drosophila melanogaster). A resolved phylogeny for flies provides a framework for genomic, developmental, and evolutionary studies by facilitating comparisons across model organisms, yet recent research has suggested that fly relationships have been obscured by multiple episodes of rapid diversification. We provide a phylogenomic estimate of fly relationships based on molecules and morphology from 149 of 157 families, including 30 kb from 14 nuclear loci and complete mitochondrial genomes combined with 371 morphological characters. Multiple analyses show support for traditional groups (Brachycera, Cyclorrhapha, and Schizophora) and corroborate contentious findings, such as the anomalous Deuterophlebiidae as the sister group to all remaining Diptera. Our findings reveal that the closest relatives of the Drosophilidae are highly modified parasites (including the wingless Braulidae) of bees and other insects. Furthermore, we use micro-RNAs to resolve a node with implications for the evolution of embryonic development in Diptera. We demonstrate that flies experienced three episodes of rapid radiation—lower Diptera (220 Ma), lower Brachycera (180 Ma), and Schizophora (65 Ma)—and a number of life history transitions to hematophagy, phytophagy, and parasitism in the history of fly evolution over 260 million y.

Molecular systematics of nuclear gene period in genus Anastrepha (Tephritidae)

Abstract The nuclear gene period is used to reconstruct the phylogeny of Anastrepha, a genus of tephritid fruit flies. Specifically, a region of period termed C3C5 that is located in the dCLK:CYC inhibition domain (CCID) is analyzed. This is the first study using this C-terminal region of the period gene for phylogenetics. Variation in the gene is informative for this genus, and period trees are compared with phylogenies based on mitochondrial and morphological data sets. In general, branches that are supported in the other data sets are recovered in the period tree; moreover, trees built using the period gene support branching patterns previously unresolved with 16S rRNA gene data. Important relationships recovered in the period gene tree include a paraphyletic placement of Anastrepha with respect to Toxotrypana, support for the separation of Anastrepha species groups into two evolutionary sections (with the exception of the pseudoparallela species group), and evidence that several species groups are not monophyletic.

Molecular evaluation of nominal species in the Ceratitis fasciventris, C. anonae, C. rosa complex (Diptera: Tephritidae)

Ceratitis fasciventris, C. anonae and C. rosa form a complex of economically important fruit fly pests infesting a variety of crops in African countries. Hitherto only adult males of these species can be distinguished easily by morphological characters. Other stages cannot, and for some taxa the taxonomic interpretation and species boundaries remain unclear. In order to clarify phylogenetic relationships and taxonomic status of these species, sequences of mitochondrial (16S, COI, ND6) and nuclear markers (period, ITS1) were analysed in specimens of the three morphospecies throughout the distribution of the complex. Maximum likelihood trees did not recover monophyletic groups corresponding to the morphospecies. Conversely, ND6 and COI divided West African C. fasciventris specimens in two consistent and bootstrap supported clades, involving specimens from Benin and from Mali/Ivory Coast, while the nuclear gene fragments per and ITS1 recovered a well-supported clade corresponding to C. fasciventris from Kenya/Uganda. Hence, the phylogenetic relationships and taxonomic interpretation of the complex appear more intricate than previously hypothesised. The current molecular data do not allow to identify C. fasciventris, C. anonae and C. rosa as distinct phylogenetic species but rather suggest that the morphospecies C. fasciventris is itself a complex of cryptic taxa.

Pathway Analysis of Ceratitis capitata (Diptera: Tephritidae) Using Mitochondrial DNA

ABSTRACT The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method described in a previous publication to document worldwide population structure of Mediterranean fruit flies and analyze probable sources of fly introductions is re-evaluated. Analysis of representatives of the RFLP-based markers using the ND5-tRNA-ND4 mitochondrial locus through DNA sequencing shows that the original markers produced haplotypes that are homoplasic and exclude valuable information. DNA sequences also identified errors in data generated with the protocol because of failure of the XbaI enzyme to efficiently cut PCR product. In all, the PCR-RFLP method is able to document haplotypes present in medfly populations outside of Sub-Saharan Africa and is a cost-effective approach to pathway analysis. New primer pairs for analysis of this locus are reported that reduce the number of PCR steps required to analyze the RFLP marker and the time of each PCR run. For comparison of the PCR-RFLP method, the utility of a DNA sequence-based approach to Mediterranean fruit fly pathway analysis is reported using the ND5-ND4 locus and the proposed “barcode region” of the COI gene. Both gene sequences are more informative than the PCR-RFLP method and document high genetic variation within Sub-Saharan Africa. Application of these methods as tools and their interpretation is discussed.

Molecular Identification of Ceratitis capitata (Diptera: Tephritidae) using DNA Sequences of the COI Barcode Region

ABSTRACT The utility of the cytochrome oxidase I gene barcode region for diagnosis of the Mediterranean fruit fly, Ceratitis capitata (Weidemann), is evaluated using African fruit fly collections. The method fails to discern C. capitata from its close relative Ceratitis caetrata Munro, based on genetic distances, parsimony networks, or nucleotide diagnostic characters observed in the DNA barcode sequences. When treated as a single taxon, it is possible to discern the C. capitata + C. caetrata lineage from other Ceratitis species. Levels of intraspecific diversity vary within the genus Ceratitis and multiple copies of the mitochondrial gene are reported for Ceratitis cosyra (Walker). The DNA barcoding method based on genetic distance is compared with a molecular identification method using restriction fragment length polymorphism. The DNA barcode and restriction fragment-length polymorphism methods provide similar identification results, but the DNA sequence information is more suitable for quantitative analysis of the information.

Phylogeography of Anastrepha obliqua inferred with mtDNA sequencing.

ABSTRACT Anastrepha obliqua (Macquart) (Diptera: Tephritidae), the West Indian fruit fly, is a frugivorous pest that occasionally finds its way to commercial growing areas outside its native distribution. It inhabits areas in Mexico, Central and South America, and the Caribbean with occasional infestations having occurred in the southern tier states (California, Florida, and Texas) of the United States. This fly is associated with many plant species and is a major pest of mango and plum. We examine the genetic diversity of the West Indian fruit fly based on mitochondrial COI and ND6 DNA sequences. Our analysis of 349 individuals from 54 geographic collections from Mexico, Central America, the Caribbean, and South America detected 61 haplotypes that are structured into three phylogenetic clades. The distribution of these clades among populations is associated with geography. Six populations are identified in this analysis: Mesoamerica, Central America, Caribbean, western Mexico, Andean South America, and eastern Brazil. In addition, substantial differences exist among these genetic types that warrants further taxonomic review.

Patterns of mitochondrial haplotype diversity in the invasive pest Epiphyas postvittana (Lepidoptera: Tortricidae).

ABSTRACT The light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), is a horticultural pest of Australia and New Zealand that has more recently invaded Hawaii, Europe, and California. A 2,216-bp region of the mitochondrial genome containing the cytochrome oxidase I and II genes was sequenced from 752 individuals. Haplotype network analyses revealed a major split between a predominantly Western Australian clade and all other samples, suggestive of either a deep genetic divergence or a cryptic species. Nucleotide and haplotype diversity were highest in the country of origin, Australia, and in New Zealand populations, with evidence of haplotype sharing between New Zealand and Tasmania. Nucleotide and haplotype diversity were higher in California than within the British Isles or Hawaii. From the total of 96 haplotypes, seven were found in California, of which four were private. Within California, there have been at least two introductions; based on genetic diversity we were unable to assign a likely source for a single moth found and eradicated in Los Angeles in 2007; however, our data suggest it is unlikely that Hawaii and the British Isles are sources of the major E. postvittana population found throughout the rest of the state since 2006.

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.

Phylogenetic relationships of Ceratitis fruit flies inferred from nuclear CAD and tango/ARNT gene fragments: testing monophyly of the subgenera Ceratitis (Ceratitis) and C. (Pterandrus).

Systematic studies of Ceratitis (Tephritidae) fruit flies using molecular (i.e., COI, ND6, and period genes) and morphological (plus host-use characters) data have recently challenged the monophyly of the subgenera Ceratitis (Ceratitis) and Ceratitis (Pterandrus). In this paper, we report on the phylogenetic utility of three single-copy nuclear gene regions (two non-overlapping fragments of the carbamoylphosphate synthetase, CPS, locus of CAD, and a fragment of tango) within these taxa and investigate evolutionary relationships based on a concatenated ca. 3.4kb data set that includes the six protein encoding gene regions. Results indicate that the CAD and tango genes provide useful phylogenetic signal within the taxa and are compatible with the previously studied genes. The two subgenera, as currently classified, are not monophyletic. Our molecular phylogenetic analyses support a revised classification in which (1) the subgenus C. (Pterandrus) comprises two lineages called A and B, (2) the C. (Pterandrus) B species should be included in C. (Ceratitis), and (3) the newly defined subgenera C. (Pterandrus) (=Pterandrus section A) and C. (Ceratitis) [=C. (Ceratitis)+C. (Pterandrus) section B] are reciprocally monophyletic.

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.