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.
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.
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.
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.
We reviewed the availability of cytochrome c oxidase subunit I (COI) sequences for 2534 North American freshwater invertebrate genera in public databases (GenBank and Barcode of Life Data Systems) ...and assessed representation of genera commonly encountered in the Canadian Aquatic Biomonitoring Network (CABIN) database. COI sequence records were available for 61.2% of North American genera and 72.4% of Insecta genera in public databases. Mollusca (73.9%) and Nematoda (15.4%) were the best and worst represented groups, respectively. In CABIN, 85.4% of genera had COI sequence records, and 95.2% of genera occurring in >1% of samples were represented. Genera absent from CABIN tended to be uncommon or members of groups not routinely used for biomonitoring purposes. On average, 94.1% of genera in well-identified samples had associated sequence data. To leverage the full potential of genomics approaches, we must expand DNA-barcode reference libraries for poorly described components of freshwater food webs. Some genera appear to be well represented (e.g., Eukiefferiella), but deposited sequences represent few sampling localities or few species and lead to underestimation of sequence diversity at the genus level and reduced confidence in identifications. Public COI libraries are sufficiently populated to permit routine application of genomics tools in biomonitoring, and ongoing quality assurance/quality control should include re-evaluation as new COI reference sequences are added or taxonomic hierarchies change. Next, we must understand whether and how established biomonitoring approaches can capitalize on high-throughput sequencing tools. Biomonitoring approaches that use genomics data to facilitate structural and functional assessments are fertile ground for future investigation and will benefit from continued improvement of publicly available sequence libraries.
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.
Highlights of the marine fly tree of life show several independent transitions to the marine environment. The distribution of marine‐dwelling Diptera has challenged the notion that marine flies are a rare phenomenon. Multiple mechanisms for adapting to the marine conditions are likely at play.
DNA sequence data from a variety of mitochondrial and nuclear gene regions are significant components of phylogenetic research in entomology. Polymerase chain reaction (PCR) amplification primers for ...many gene regions have been developed that are specific to a range of dipteran groups. Here, we review the existing Diptera-specific PCR amplification primers that have been published for 11 mitochondrial and nuclear gene regions: 12S small ribosomal subunit, cytochrome b, cytochrome oxidase c subunit I, 28S ribosomal RNA, alanyl-tRNA synthetase, the carbamoyl phosphate synthase region of CAD, elongation factor-1α, 6-phosphogluconate dehydrogenase, triose phosphate isomerase, white, and wingless. We also have designed in total 94 new PCR amplification primers for use in these same gene regions. Our new primers have been developed and tested using our DNA sequence database of > 1,600 specimens representing 40 families of Diptera. All of the past and newly developed primer sequences are presented in tables, and their locations are shown on gene maps. This combined data will facilitate future molecular phylogenetic research within Diptera.
Insects are usually considered to be excluded from the marine environment. A small number of species, however, are considered to be marine, due to spending some portion of their life cycle in salt ...water. We use natural history collection specimens, in-field observations, and molecular analysis to generate new locale records and natural history data for seven insect species. All seven species are associated with barnacles (Balanomorpha: Balanidae, Chthamalidae) along the Pacific coast of Canada, the United States of America, or Japan. Use of DNA barcode analysis confirms the monophyly of three species of Oedoparena (Diptera: Dryomyzidae). Natural history collection specimens expand the geographical range and illuminate the phenology of Oedoparena spp. In-field observations record direct associations between three species of Thalassosmittia (Diptera: Chironomidae), Diaulota densissima (Casey) (Coleoptera: Staphylinidae), and intertidal barnacles from various locations in British Columbia, Canada. Barnacle host associations and microhabitat preferences are proposed for all species. A new definition of what constitutes a marine insect is offered.
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.
Multiple specimens of four species of grasshopper 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. Niche overlap values did not follow predicted patterns, however, the low values suggest low competition between these species.
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-1α (EF-1α),
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
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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.
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