Interrogating 1000 insect genomes for NUMTs: A risk assessment for estimates of species richness.

The nuclear genomes of most animal species include NUMTs, segments of the mitogenome incorporated into their chromosomes. Although NUMT counts are known to vary greatly among species, there has been no comprehensive study of their frequency/attributes in the most diverse group of terrestrial organisms, insects. This study examines NUMTs derived from a 658 bp 5' segment of the cytochrome c oxidase I (COI) gene, the barcode region for the animal kingdom. This assessment is important because unrecognized NUMTs can elevate estimates of species richness obtained through DNA barcoding and derived approaches (eDNA, metabarcoding). This investigation detected nearly 10,000 COI NUMTs ≥ 100 bp in the genomes of 1,002 insect species (range = 0-443). Variation in nuclear genome size explained 56% of the mitogenome-wide variation in NUMT counts. Although insect orders with the largest genome sizes possessed the highest NUMT counts, there was considerable variation among their component lineages. Two thirds of COI NUMTs possessed an IPSC (indel and/or premature stop codon) allowing their recognition and exclusion from downstream analyses. The remainder can elevate species richness as they showed 10.1% mean divergence from their mitochondrial homologue. The extent of exposure to "ghost species" is strongly impacted by the target amplicon's length. NUMTs can raise apparent species richness by up to 22% when a 658 bp COI amplicon is examined versus a doubling of apparent richness when 150 bp amplicons are targeted. Given these impacts, metabarcoding and eDNA studies should target the longest possible amplicons while also avoiding use of 12S/16S rDNA as they triple NUMT exposure because IPSC screens cannot be employed.

Enhancing DNA barcode reference libraries by harvesting terrestrial arthropods at the Smithsonian's National Museum of Natural History.

The use of DNA barcoding has revolutionised biodiversity science, but its application depends on the existence of comprehensive and reliable reference libraries. For many poorly known taxa, such reference sequences are missing even at higher-level taxonomic scales. We harvested the collections of the Smithsonian's National Museum of Natural History (USNM) to generate DNA barcoding sequences for genera of terrestrial arthropods previously not recorded in one or more major public sequence databases. Our workflow used a mix of Sanger and Next-Generation Sequencing (NGS) approaches to maximise sequence recovery while ensuring affordable cost. In total, COI sequences were obtained for 5,686 specimens belonging to 3,737 determined species in 3,886 genera and 205 families distributed in 137 countries. Success rates varied widely according to collection data and focal taxon. NGS helped recover sequences of specimens that failed a previous run of Sanger sequencing. Success rates and the optimal balance between Sanger and NGS are the most important drivers to maximise output and minimise cost in future projects. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, the Global Genome Biodiversity Network Data Portal and the NMNH data portal.

A workflow for expanding DNA barcode reference libraries through 'museum harvesting' of natural history collections.

Natural history collections are the physical repositories of our knowledge on species, the entities of biodiversity. Making this knowledge accessible to society - through, for example, digitisation or the construction of a validated, global DNA barcode library - is of crucial importance. To this end, we developed and streamlined a workflow for 'museum harvesting' of authoritatively identified Diptera specimens from the Smithsonian Institution's National Museum of Natural History. Our detailed workflow includes both on-site and off-site processing through specimen selection, labelling, imaging, tissue sampling, databasing and DNA barcoding. This approach was tested by harvesting and DNA barcoding 941 voucher specimens, representing 32 families, 819 genera and 695 identified species collected from 100 countries. We recovered 867 sequences (> 0 base pairs) with a sequencing success of 88.8% (727 of 819 sequenced genera gained a barcode > 300 base pairs). While Sanger-based methods were more effective for recently-collected specimens, the methods employing next-generation sequencing recovered barcodes for specimens over a century old. The utility of the newly-generated reference barcodes is demonstrated by the subsequent taxonomic assignment of nearly 5000 specimen records in the Barcode of Life Data Systems.

Culicoides species community composition and feeding preferences in two aquatic ecosystems in northern Spain.

BACKGROUND: Aquatic ecosystems provide breeding sites for blood-sucking insects such as Culicoides biting midges (Diptera: Ceratopogonidae), but factors affecting their distribution and host choice are poorly understood. A study was undertaken at two nature reserves in northern Spain to examine the abundance, species composition, population dynamics and feeding patterns of biting midges between 2018 and 2019. METHODS: Culicoides were captured by light suction traps baited with CO2 and by sweep netting vegetation. Blood meals and species identification of blood-fed specimens were determined using cytochrome c oxidase I subunit (COI) DNA barcoding. Multivariate generalized linear models were used to evaluate the associations between the abundance of Culicoides, the species richness and other parameters. RESULTS: The 4973 identified specimens comprised 28 species of Culicoides. These included two species reported for the first time in northern Spain, thus raising to 54 the number of Culicoides species described in the region. Specimens of all 28 species and 99.6% of the total specimens collected were caught in suction traps, while sweep netting vegetation revealed just 11 species and 0.4% of the total specimens. Midge abundance peaked in June/early July, with five species comprising > 80% of the captures: Culicoides alazanicus (24.9%), Culicoides griseidorsum (20.3%), Culicoides poperinghensis (16.2%), Culicoides kibunensis (10.7%) and Culicoides clastrieri (9.6%). DNA barcode analysis of blood meals from eight Culicoides species revealed that they fed on 17 vertebrate species (3 mammals and 14 birds). Species in the subgenus Avaritia were primarily ornithophilic, except for C. griseidorsum and C. poperinghensis. Host DNA from blood meals was successfully amplified from 75% of blood-fed females. A pictorial blood meal digestion scale is provided to accurately assess the blood-fed status of female Culicoides. CONCLUSIONS: The large number of different blood meal sources identified in the midges captured in this study signals the likely importance of wild birds and mammals (e.g. red deer and wild boar) as reservoir/amplifying hosts for pathogens. Available hosts are more exposed to being bitten by biting midge populations in aquatic ecosystems in late spring and early summer.

Drought at a coastal wetland affects refuelling and migration strategies of shorebirds.

Droughts can affect invertebrate communities in wetlands, which can have bottom-up effects on the condition and survival of top predators. Shorebirds, key predators at coastal wetlands, have experienced widespread population declines and could be negatively affected by droughts. We explored, in detail, the effects of drought on multiple aspects of shorebird stopover and migration ecology by contrasting a year with average wet/dry conditions (2016) with a year with moderate drought (2017) at a major subarctic stopover site on southbound migration. We also examined the effects of drought on shorebird body mass during stopover across 14 years (historical: 1974-1982 and present-day: 2014-2018). For the detailed comparison of two years, in the year with moderate drought we documented lower invertebrate abundance at some sites, higher prey family richness in shorebird faecal samples, lower shorebird refuelling rates, shorter stopover durations for juveniles, and, for most species, a higher probability of making a subsequent stopover in North America after departing the subarctic, compared to the year with average wet/dry conditions. In the 14-year dataset, shorebird body mass tended to be lower in drier years. We show that even short-term, moderate drought conditions can negatively affect shorebird refuelling performance at coastal wetlands, which may carry-over to affect subsequent stopover decisions. Given shorebird population declines and predicted changes in the severity and duration of droughts with climate change, researchers should prioritize a better understanding of how droughts affect shorebird refuelling performance and survival.

A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)-patterns of nucleotide misincorporation.

Natural history collections are a valuable resource for molecular taxonomic studies and for examining patterns of evolutionary diversification, particularly in the case of rare or extinct species. However, the recovery of sequence information is often complicated by DNA degradation. This article describes use of the Sequel platform (Pacific Biosciences) to recover the 658 bp barcode region of the mitochondrial cytochrome c oxidase I (COI) gene from 380 butterflies with an average age of 50 years. Nested multiplex PCR was employed for library preparation to facilitate sequence recovery from extracts with low concentrations of highly degraded DNA. By employing circular consensus sequencing (CCS) of short amplicons (circa 150 bp), full-length barcodes could be assembled without a reference sequence, an important advance from earlier protocols which required reference sequences to guide contig assembly. The Sequel protocol recovered COI sequences (499 bp on average) from 318 of 380 specimens (84%), much higher than for Sanger sequencing (26%). Because each read derives from a single molecule, it was also possible to quantify the incidence of substitutions arising from DNA damage. In agreement with past work on sequence changes induced by DNA degradation, the transition C/G → T/A was the most prevalent category of change, but its rate of occurrence (4.58E-4) was so low that it did not impede the recovery of reliable sequences. Because the current protocol recovers COI sequence from most museum specimens, and because sequence fidelity is unaffected by nucleotide misincorporations, large-scale sequence characterization of museum specimens is feasible.

Vertebrate-Aedes aegypti and Culex quinquefasciatus (Diptera)-arbovirus transmission networks: Non-human feeding revealed by meta-barcoding and next-generation sequencing.

BACKGROUND: Aedes aegypti mosquito-borne viruses including Zika (ZIKV), dengue (DENV), yellow fever (YFV), and chikungunya (CHIKV) have emerged and re-emerged globally, resulting in an elevated burden of human disease. Aedes aegypti is found worldwide in tropical, sub-tropical, and temperate areas. The characterization of mosquito blood meals is essential to understand the transmission dynamics of mosquito-vectored pathogens. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report Ae. aegypti and Culex quinquefasciatus host feeding patterns and arbovirus transmission in Northern Mexico using a metabarcoding-like approach with next-generation deep sequencing technology. A total of 145 Ae. aegypti yielded a blood meal analysis result with 107 (73.8%) for a single vertebrate species and 38 (26.2%) for two or more. Among the single host blood meals for Ae. aegypti, 28.0% were from humans, 54.2% from dogs, 16.8% from cats, and 1.0% from tortoises. Among those with more than one species present, 65.9% were from humans and dogs. For Cx. quinquefasciatus, 388 individuals yielded information with 326 (84%) being from a single host and 63 (16.2%) being from two or more hosts. Of the single species blood meals, 77.9% were from dogs, 6.1% from chickens, 3.1% from house sparrows, 2.4% from humans, while the remaining 10.5% derived from other 12 host species. Among those which had fed on more than one species, 11% were from dogs and humans, and 89% of other host species combinations. Forage ratio analysis revealed dog as the most over-utilized host by Ae. aegypti (= 4.3) and Cx. quinquefasciatus (= 5.6) and the human blood index at 39% and 4%, respectively. A total of 2,941 host-seeking female Ae. aegypti and 3,536 Cx. quinquefasciatus mosquitoes were collected in the surveyed area. Of these, 118 Ae. aegypti pools and 37 Cx. quinquefasciatus pools were screened for seven arboviruses (ZIKV, DENV 1-4, CHIKV, and West Nile virus (WNV)) using qRT-PCR and none were positive (point prevalence = 0%). The 95%-exact upper limit confidence interval was 0.07% and 0.17% for Ae. aegypti and Cx. quinquefasciatus, respectively. CONCLUSIONS/SIGNIFICANCE: The low human blood feeding rate in Ae. aegypti, high rate of feeding on mammals by Cx. quinquefasciatus, and the potential risk to transmission dynamics of arboviruses in highly urbanized areas of Northern Mexico is discussed.

Next generation sequencing-aided comprehensive geographic coverage sheds light on the status of rare and extinct populations of Aporia butterflies (Lepidoptera: Pieridae).

The Black-veined White Aporia crataegi (Linnaeus, 1758), a common and widespread butterfly ranging from northwestern Africa to Europe and Asia, has been extinct in Britain since the 1920s and is on a steady decline in several other parts of its range. In order to investigate genetic diversity within A. crataegi and its correspondence with current subspecies-level taxonomy, we barcoded 173 specimens from across its range including, for the first time, extinct populations from Britain and Korea. Using next generation sequencing we also obtained a sequence for Aporia joubini, a peculiar taxon from China known only by its type specimen collected in the early twentieth century. Our phylogenetic analysis placed A. joubini sister to A. oberthuri, although further taxon sampling may reveal a different scheme. Within A. crataegi, we observed a shallow and weak mitogenomic structure with only a few distinct lineages in North Africa, Sicily, Iran, and Japan. Eurasian populations, including those extinct in Britain and Korea, clustered into a large set of closely allied lineages, consistent with a recent expansion during the Late Pleistocene glacial period. This study highlights the importance of museum collections and the unique opportunities they provide in documenting species diversity and helping conservation efforts.

Dynamics of a host-parasitoid interaction clarified by modelling and DNA sequencing.

It has been hypothesised that the 2-year oscillations in abundance of Xestia moths are mediated by interactions with 1-year Ophion parasitoid wasps. We tested this hypothesis by modelling a 35-year time series of Xestia and Ophion from Northern Finland. Additionally, we used DNA barcoding to ascertain the species diversity of Ophion and targeted amplicon sequencing of their gut contents to confirm their larval hosts. Modelling of the time-series data strongly supported the hypothesised host-parasitoid dynamics and that periodic occurrence of Xestia moths is mediated by Ophion. DNA barcodes revealed that Ophion included five species rather than just one while targeted amplicon sequencing verified that Ophion does parasitise Xestia. At least one Ophion species employs 1-year Syngrapha interrogationis as an alternate host, but it did not detectably affect Xestia-Ophion dynamics. We also demonstrate the previously unrecognised complexity of this system due to cryptic parasitoid diversity.

An Integrated Molecular Approach to Untangling Host-Vector-Pathogen Interactions in Mosquitoes (Diptera: Culicidae) From Sylvan Communities in Mexico.

There are ~240 species of Culicidae in Mexico, of which some are vectors of arthropod-borne viruses such as Zika virus, dengue virus, chikungunya virus, and West Nile virus. Thus, the identification of mosquito feeding preferences is paramount to understanding of vector-host-pathogen interactions that, in turn, can aid the control of disease outbreaks. Typically, DNA and RNA are extracted separately for animal (insects and blood meal hosts) and viral identification, but this study demonstrates that multiple organisms can be analyzed from a single RNA extract. For the first time, residual DNA present in standard RNA extracts was analyzed by DNA barcoding in concert with Sanger and next-generation sequencing (NGS) to identify both the mosquito species and the source of their meals in blood-fed females caught in seven sylvan communities in Chiapas State, Mexico. While mosquito molecular identification involved standard barcoding methods, the sensitivity of blood meal identification was maximized by employing short primers with NGS. In total, we collected 1,634 specimens belonging to 14 genera, 25 subgenera, and 61 morphospecies of mosquitoes. Of these, four species were new records for Mexico (Aedes guatemala, Ae. insolitus, Limatus asulleptus, Trichoprosopon pallidiventer), and nine were new records for Chiapas State. DNA barcode sequences for >300 bp of the COI gene were obtained from 291 specimens, whereas 130 bp sequences were recovered from another 179 specimens. High intraspecific divergence values (>2%) suggesting cryptic species complexes were observed in nine taxa: Anopheles eiseni (5.39%), An. pseudopunctipennis (2.79%), Ae. podographicus (4.05%), Culex eastor (4.88%), Cx. erraticus (2.28%), Toxorhynchites haemorrhoidalis (4.30%), Tr. pallidiventer (4.95%), Wyeomyia adelpha/Wy. guatemala (7.30%), and Wy. pseudopecten (4.04%). The study increased the number of mosquito species known from 128 species to 138 species for Chiapas State, and 239 for Mexico as a whole. Blood meal analysis showed that Aedes angustivittatus fed on ducks and chicken, whereas Psorophora albipes fed on humans. Culex quinquefasciatus fed on diverse hosts including chicken, human, turkey, and Mexican grackle. No arbovirus RNA was detected by reverse transcriptase-polymerase chain reaction in the surveyed specimens. This study demonstrated, for the first time, that residual DNA present in RNA blood meal extracts can be used to identify host vectors, highlighting the important role of molecular approaches in both vector identification and revealing host-vector-pathogen interactions.

A reference library for Canadian invertebrates with 1.5 million barcodes, voucher specimens, and DNA samples.

The reliable taxonomic identification of organisms through DNA sequence data requires a well parameterized library of curated reference sequences. However, it is estimated that just 15% of described animal species are represented in public sequence repositories. To begin to address this deficiency, we provide DNA barcodes for 1,500,003 animal specimens collected from 23 terrestrial and aquatic ecozones at sites across Canada, a nation that comprises 7% of the planet's land surface. In total, 14 phyla, 43 classes, 163 orders, 1123 families, 6186 genera, and 64,264 Barcode Index Numbers (BINs; a proxy for species) are represented. Species-level taxonomy was available for 38% of the specimens, but higher proportions were assigned to a genus (69.5%) and a family (99.9%). Voucher specimens and DNA extracts are archived at the Centre for Biodiversity Genomics where they are available for further research. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, and the Global Genome Biodiversity Network Data Portal.

Validation of COI metabarcoding primers for terrestrial arthropods.

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40-60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents).

DNA metabarcoding allows non-invasive identification of arthropod prey provisioned to nestling Rufous hummingbirds (Selasphorus rufus).

Hummingbirds consume sugars from nectar, sap and honeydew, and obtain protein, fat and minerals from arthropods. To date, the identity of arthropod taxa in hummingbird diets has been investigated by observation of foraging or examination of alimentary tract contents. Direct examination of nestling provisioning adds the extra complication of disturbance to the young and mother. Here, we show that arthropod food items provisioned to Rufous hummingbird (Selasphorus rufus) nestlings can be identified by a safe and non-invasive protocol using next-generation sequencing (NGS) of DNA from nestling fecal pellets collected post-fledging. We found that females on southern Vancouver Island (British Columbia, Canada) provisioned nestlings with a wide range of arthropod taxa. The samples examined contained three Classes, eight Orders, 48 Families, and 87 Genera, with from one to 15 Families being identified in a single pellet. Soft-bodied Dipterans were found most frequently and had the highest relative abundance; hard-bodied prey items were absent from almost all samples. Substantial differences in taxa were found within season and between years, indicating the importance of multi-year sampling when defining a prey spectrum.

Metabarcoding a diverse arthropod mock community.

Although DNA metabarcoding is an attractive approach for monitoring biodiversity, it is often difficult to detect all the species present in a bulk sample. In particular, sequence recovery for a given species depends on its biomass and mitome copy number as well as the primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 species. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body segments (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicons generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, although the Miseq delivered the highest sequence quality. As expected, species recovery was most efficient from the Single Leg treatment because amplicon abundance varied little among taxa. Among the three treatments where PCR occurred after pooling, the Bulk Abdomen treatment produced a more uniform read abundance than the Bulk Leg or Composite Leg treatment. Primer choice also influenced species recovery and evenness. Our results reveal how variation in protocols can have substantial impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote.

A Sequel to Sanger: amplicon sequencing that scales.

BACKGROUND: Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system. RESULTS: By examining templates from more than 5000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL can reduce greatly reduce sequencing costs in comparison to first (Sanger) and second generation platforms (Illumina, Ion). CONCLUSIONS: SMRT analysis generates high-fidelity sequences from amplicons with varying GC content and is resilient to homopolymer tracts. Analytical costs are low, substantially less than those for first or second generation sequencers. When implemented on the SEQUEL platform, SMRT analysis enables massive amplicon characterization because each instrument can recover sequences from more than 5 million DNA extracts a year.

DNA barcoding as an aid for species identification in austral black flies (Insecta: Diptera: Simuliidae).

In this paper, the utility of a partial sequence of the COI gene, the DNA barcoding region, for the identification of species of black flies in the austral region was assessed. Twenty-eight morphospecies were analyzed: eight of the genus Austrosimulium (four species in the subgenus Austrosimulium s. str., three species in the subgenus Novaustrosimulium, and one species unassigned to subgenus), two of the genus Cnesia, eight of Gigantodax, three of Paracnephia, one of Paraustrosimulium, and six of Simulium (subgenera Morops, Nevermannia, and Pternaspatha). The neighbour-joining tree derived from the DNA barcode sequences grouped most specimens according to species or species groups recognized by morphotaxonomic studies. Intraspecific sequence divergences within morphologically distinct species ranged from 0% to 1.8%, while higher divergences (2%-4.2%) in certain species suggested the presence of cryptic diversity. The existence of well-defined groups within S. simile revealed the likely inclusion of cryptic diversity. DNA barcodes also showed that specimens identified as C. dissimilis, C. nr. pussilla, and C. ornata might be conspecific, suggesting possible synonymy. DNA barcoding combined with a sound morphotaxonomic framework would provide an effective approach for the identification of black flies in the region.

Rapid identification of the botanical and entomological sources of honey using DNA metabarcoding.

Honey is generated by various bee species from diverse plants, and because the value of different types of honey varies more than 100-fold, it is a target for fraud. This paper describes a protocol that employs DNA metabarcoding of three gene regions (ITS2, rbcLa, and COI) to provide an inexpensive tool to simultaneously deliver information on the botanical and entomological origins of honey. This method was used to examine seven varieties of honey: light, medium, dark, blended, pasteurized, creamed, and meliponine. Plant and insect sources were identified in five samples, but only the botanical or insect source could be identified in the other two. Two samples were found to be misrepresented. Although this method was generally successful in determining both plant and insect sources, honeys rich in polyphenolic compounds or subject to crystallization were recalcitrant to analysis, so further research is required to combat honey adulteration and mislabeling.

Calibrating the taxonomy of a megadiverse insect family: 3000 DNA barcodes from geometrid type specimens (Lepidoptera, Geometridae).

It is essential that any DNA barcode reference library be based upon correctly identified specimens. The Barcode of Life Data Systems (BOLD) requires information such as images, geo-referencing, and details on the museum holding the voucher specimen for each barcode record to aid recognition of potential misidentifications. Nevertheless, there are misidentifications and incomplete identifications (e.g., to a genus or family) on BOLD, mainly for species from tropical regions. Unfortunately, experts are often unavailable to correct taxonomic assignments due to time constraints and the lack of specialists for many groups and regions. However, considerable progress could be made if barcode records were available for all type specimens. As a result of recent improvements in analytical protocols, it is now possible to recover barcode sequences from museum specimens that date to the start of taxonomic work in the 18th century. The present study discusses success in the recovery of DNA barcode sequences from 2805 type specimens of geometrid moths which represent 1965 species, corresponding to about 9% of the 23 000 described species in this family worldwide and including 1875 taxa represented by name-bearing types. Sequencing success was high (73% of specimens), even for specimens that were more than a century old. Several case studies are discussed to show the efficiency, reliability, and sustainability of this approach.

DNA Barcoding of Marine Metazoans.

The accumulation of DNA barcode sequences will provide an increasingly useful and comprehensive library for species identification and discovery of marine metazoans. Here we present a summary of protocols designed to obtain DNA barcodes of marine metazoans from diverse phyla.

DNA barcodes from century-old type specimens using next-generation sequencing.

Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain. This study addresses this challenge by employing next-generation sequencing (NGS) to recover sequences for the barcode region of the cytochrome c oxidase 1 gene from small amounts of template DNA. DNA quality was first screened in more than 1800 century-old type specimens of Lepidoptera by attempting to recover 164-bp and 94-bp reads via Sanger sequencing. This analysis permitted the assignment of each specimen to one of three DNA quality categories--high (164-bp sequence), medium (94-bp sequence) or low (no sequence). Ten specimens from each category were subsequently analysed via a PCR-based NGS protocol requiring very little template DNA. It recovered sequence information from all specimens with average read lengths ranging from 458 bp to 610 bp for the three DNA categories. By sequencing ten specimens in each NGS run, costs were similar to Sanger analysis. Future increases in the number of specimens processed in each run promise substantial reductions in cost, making it possible to anticipate a future where barcode sequences are available from most type specimens.