The evolution of marine dwelling in Diptera.

Marine dwelling in Diptera has been relatively unexplored and the frequency of transitions to the marine environment and the evolutionary history remain poorly understood. By reviewing records from the World Register of Marine Species and using ancestral state reconstruction methods, we build on the fly tree of life phylogeny and ecological descriptions of marine life history. Our ancestral state reconstruction analyses suggest marine dwelling is lacking as an ancestral trait for the most recent common ancestor to Diptera. While many transitions in Empidoidea, Sciomyzoidea, Tipulomorpha, and Culicomorpha seem to have been gradual, other transitions in Tephritoidea and Tabanomorpha were found likely to have been stochastic occurrences. From the collection of 532 marine species, we reveal several independent transitions to the marine environment throughout the fly tree of life. Considering the results from our analysis, we outline potential adaptations for marine flies and discuss the barriers of colonizing the marine environment and the implications to the mechanisms for salt tolerance.

Diptera of Canada.

The Canadian Diptera fauna is updated. Numbers of species currently known from Canada, total Barcode Index Numbers (BINs), and estimated numbers of undescribed or unrecorded species are provided for each family. An overview of recent changes in the systematics and Canadian faunistics of major groups is provided as well as some general information on biology and life history. A total of 116 families and 9620 described species of Canadian Diptera are reported, representing more than a 36% increase in species numbers since the last comparable assessment by JF McAlpine et al. (1979). Almost 30,000 BINs have so far been obtained from flies in Canada. Estimates of additional number of species remaining to be documented in the country range from 5200 to 20,400.

Large-Scale Biomonitoring of Remote and Threatened Ecosystems via High-Throughput Sequencing.

Biodiversity metrics are critical for assessment and monitoring of ecosystems threatened by anthropogenic stressors. Existing sorting and identification methods are too expensive and labour-intensive to be scaled up to meet management needs. Alternately, a high-throughput DNA sequencing approach could be used to determine biodiversity metrics from bulk environmental samples collected as part of a large-scale biomonitoring program. Here we show that both morphological and DNA sequence-based analyses are suitable for recovery of individual taxonomic richness, estimation of proportional abundance, and calculation of biodiversity metrics using a set of 24 benthic samples collected in the Peace-Athabasca Delta region of Canada. The high-throughput sequencing approach was able to recover all metrics with a higher degree of taxonomic resolution than morphological analysis. The reduced cost and increased capacity of DNA sequence-based approaches will finally allow environmental monitoring programs to operate at the geographical and temporal scale required by industrial and regulatory end-users.

Discrimination of grasshopper (Orthoptera: Acrididae) diet and niche overlap using next-generation sequencing of gut contents.

Species of grasshopper have been divided into three diet classifications based on mandible morphology: forbivorous (specialist on forbs), graminivorous (specialist on grasses), and mixed feeding (broad-scale generalists). For example, Melanoplus bivittatus and Dissosteira carolina are presumed to be broad-scale generalists, Chortophaga viridifasciata is a specialist on grasses, and Melanoplus femurrubrum is a specialist on forbs. These classifications, however, have not been verified in the wild. Multiple specimens of these four species were collected, and diet analysis was performed using DNA metabarcoding of the gut contents. The rbcLa gene region was amplified and sequenced using Illumina MiSeq sequencing. Levins' measure and the Shannon-Wiener measure of niche breadth were calculated using family-level identifications and Morisita's measure of niche overlap was calculated using operational taxonomic units (OTUs). Gut contents confirm both D. carolina and M. bivittatus as generalists and C. viridifasciata as a specialist on grasses. For M. femurrubrum, a high niche breadth was observed and species of grasses were identified in the gut as well as forbs. Niche overlap values did not follow predicted patterns, however, the low values suggest low competition between these species.

Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform.

Genetic information is a valuable component of biosystematics, especially specimen identification through the use of species-specific DNA barcodes. Although many genomics applications have shifted to High-Throughput Sequencing (HTS) or Next-Generation Sequencing (NGS) technologies, sample identification (e.g., via DNA barcoding) is still most often done with Sanger sequencing. Here, we present a scalable double dual-indexing approach using an Illumina Miseq platform to sequence DNA barcode markers. We achieved 97.3% success by using half of an Illumina Miseq flowcell to obtain 658 base pairs of the cytochrome c oxidase I DNA barcode in 1,010 specimens from eleven orders of arthropods. Our approach recovers a greater proportion of DNA barcode sequences from individuals than does conventional Sanger sequencing, while at the same time reducing both per specimen costs and labor time by nearly 80%. In addition, the use of HTS allows the recovery of multiple sequences per specimen, for deeper analysis of genetic variation in target gene regions.

Mitochondrial and nuclear phylogenetic analysis with Sanger and next-generation sequencing shows that, in Área de Conservación Guanacaste, northwestern Costa Rica, the skipper butterfly named Urbanus belli (family Hesperiidae) comprises three morphologically cryptic species.

BACKGROUND: Skipper butterflies (Hesperiidae) are a relatively well-studied family of Lepidoptera. However, a combination of DNA barcodes, morphology, and natural history data has revealed several cryptic species complexes within them. Here, we investigate three DNA barcode lineages of what has been identified as Urbanus belli (Hesperiidae, Eudaminae) in Área de Conservación Guanacaste (ACG), northwestern Costa Rica. RESULTS: Although no morphological traits appear to distinguish among the three, congruent nuclear and mitochondrial lineage patterns show that "Urbanus belli" in ACG is a complex of three sympatric species. A single strain of Wolbachia present in two of the three cryptic species indicates that Urbanus segnestami Burns (formerly Urbanus belliDHJ01), Urbanus bernikerni Burns (formerly Urbanus belliDHJ02), and Urbanus ehakernae Burns (formerly Urbanus belliDHJ03) may be biologically separated by Wolbachia, as well as by their genetics. Use of parallel sequencing through 454-pyrosequencing improved the utility of ITS2 as a phylogenetic marker and permitted examination of the intra- and interlineage relationships of ITS2 variants within the species complex. Interlineage, intralineage and intragenomic compensatory base pair changes were discovered in the secondary structure of ITS2. CONCLUSION: These findings corroborate the existence of three cryptic species. Our confirmation of a novel cryptic species complex, initially suggested by DNA barcode lineages, argues for using a multi-marker approach coupled with next-generation sequencing for exploration of other suspected species complexes.

Next-generation DNA barcoding: using next-generation sequencing to enhance and accelerate DNA barcode capture from single specimens.

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large-scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next-generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high-target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next-generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10-mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full-length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full-length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next-generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.

A phylogenetic analysis of relationships among genera of Conopidae (Diptera) based on molecular and morphological data.

Members of the family Conopidae (Diptera) have been the focus of little targeted phylogenetic research. The most comprehensive test of phylogenetic support for the present subfamily classification of Conopidae is presented here using 66 specimens, including 59 species of Conopidae and seven outgroup taxa. Relationships among subfamily clades are also explored. A total of 6824 bp of DNA sequence data from five gene regions (12S ribosomal DNA, cytochrome c oxidase subunit I, cytochrome b, 28S ribosomal DNA and alanyl-tRNA synthetase) are combined with 111 morphological characters in a combined analysis using both parsimony and Bayesian methods. Parsimony analysis recovers three shortest trees. Bayesian analysis recovers a nearly identical tree. Five monophyletic subfamilies of Conopidae are recovered. The rarely acknowledged Zodioninae is restored, including the genera Zodion and Parazodion. The genus Sicus is removed from Myopinae. Morphological synapomorphies are discussed for each subfamily and inter-subfamily clade, including a comprehensive review of the character interpretaions of previous authors. Included are detailed comparative illustrations of male and female genitalia of representatives of all five subfamilies with new morphological interpretation.

Next-generation sequencing technologies for environmental DNA research.

Since 2005, advances in next-generation sequencing technologies have revolutionized biological science. The analysis of environmental DNA through the use of specific gene markers such as species-specific DNA barcodes has been a key application of next-generation sequencing technologies in ecological and environmental research. Access to parallel, massive amounts of sequencing data, as well as subsequent improvements in read length and throughput of different sequencing platforms, is leading to a better representation of sample diversity at a reasonable cost. New technologies are being developed rapidly and have the potential to dramatically accelerate ecological and environmental research. The fast pace of development and improvements in next-generation sequencing technologies can reflect on broader and more robust applications in environmental DNA research. Here, we review the advantages and limitations of current next-generation sequencing technologies in regard to their application for environmental DNA analysis.

Observations on hilltopping in thick-headed flies (Diptera: Conopidae).

Direct observations of hilltopping behaviour in the thick-headed flies (Diptera: Conopidae) have only been mentioned once in the literature. Hilltop collecting, however, may be an effective way to survey these endparasitoids. The first evidence of hilltopping in species belonging to the subfamilies Myopinae and Dalmanniinae is presented and discussed. Field observations were conducted on Colle Vescovo, Italy and Mount Rigaud, Canada, and museum specimens were examined. Observations and records indicate that four species in the genera Dalmannia, Myopa, and Zodion are hilltoppers on Colle Vescovo, while three species in the genera Myopa and Physocephala are hilltoppers on three hilltops near Ottawa, Canada. Fifteen additional species of conopids have been collected on hilltops and could possibly utilize hilltops in some years as a part of their mating strategy. Detailed phenologies and observations of mating and perching behaviours are given for species in the genera Dalmannia, Myopa, Physocephala, and Zodion. The importance of hilltop habitat preservation is stressed.

Band-cutting no more: A method for the isolation and purification of target PCR bands from multiplex PCR products using new technology.

A procedure for the isolation of target PCR bands from multiplex PCR products using the E-Gel iBase Power System, an E-Gel Safe Imager Real-Time Transilluminator, and E-Gel CloneWell 0.8% SYBR Safe agarose cassettes is presented. The collected isolates are suitable for direct use in sequencing reactions without need of further purification. Reductions in time spent per sample and cost per sequence produced compared to traditional "band-cutting" methods are demonstrated. An added benefit is that the new procedure does not require exposure to ethidium bromide or ultraviolet radiation.

Placement of Conopidae (Diptera) within Schizophora based on mtDNA and nrDNA gene regions.

The first attempt to phylogenetically place Conopidae using molecular characters, as well as the largest molecular analysis of relationships within Schizophora (Diptera) to date, is presented. Twenty-eight taxa from 11 acalyptrate families and seven acalyptrate superfamilies are represented. Nearly 12,800 bp of sequence data from 10 genes representing both mitochondrial (cytochrome oxidase I (COI), cytochrome b (cytB), and 12S) and nuclear genes (28S, the carbamoyl phosphate synthetase region of CAD (CAD), elongation factor-1alpha (EF-1alpha), white, alanyl-tRNA synthetase (AATS), triose phosphate isomerase (TPI), and phosphogluconate dehydrogenase (PGD)) are analysed. Parsimony and Bayesian analyses strongly support the monophyly of both Conopidae and Schizophora. While in the parsimony analysis, Conopidae are placed as sister to the remaining Schizophora, the Bayesian analysis recovers a Conopidae+Lauxaniidae clade. The value of nuclear, mitochondrial, ribosomal, and protein-coding gene sequence data for answering phylogenetic questions at different levels of divergence is evaluated.