Here we demonstrate a cost effective and scalable microbial ecology sequencing platform using the Ion Torrent Personal Genome Machine (PGM). We assessed both PCR amplified 16S rRNA and shotgun ...metagenomic approaches and generated 100,000+ to 1,000,000+ reads using ‘post-light’ based sequencing technology within different sized semi-conductor chips. Further development of Golay barcoded Ion Tags allowed multiplex analyses of microbial communities with substantially reduced costs compared with platforms such as 454/GS-FLX. Using these protocols we assessed the bacterial and archaeal dynamics within covered anaerobic digesters used to treat piggery wastes. Analysis of these sequence data showed that these novel methanogenic waste treatment systems are dominated by bacterial taxa, in particular Clostridium, Synergistia and Bacteroides that were maintained as a stable community over extended time periods. Archaeal community dynamics were more stochastic with the key methanogenic taxa more difficult to resolve, principally due to the poor congruence seen between community structures generated either by nested PCR or metagenomic approaches for archaeal analyses. Our results show that for microbial community structure and function analyses, the PGM platform provides a low cost, scalable and high throughput solution for both Tag sequencing and metagenomic analyses.
► ‘Post light’ DNA sequencer producing analyses in 8 hours with low reagent costs ► Scalable chip based sequencing ranging from 100,000 to 1,000,000+ reads ► Multiplex 16S rRNA analyses developed and demonstrated in anaerobic digesters. ► Novel covered anaerobic ponds dominated by Bacteroidia, Synergistia and Clostridia.
In light of the current biodiversity crisis, molecular barcoding has developed into an irreplaceable tool. Barcoding has been considerably simplified by developments in high throughput sequencing ...technology, but still can be prohibitively expensive and laborious when community samples of thousands of specimens need to be processed. Here, we outline an Illumina amplicon sequencing approach to generate multilocus data from large collections of arthropods. We reduce cost and effort up to 50-fold, by combining multiplex PCRs and DNA extractions from pools of presorted and morphotyped specimens and using two levels of sample indexing. We test our protocol by generating a comprehensive, community wide dataset of barcode sequences for several thousand Hawaiian arthropods from 14 orders, which were collected across the archipelago using various trapping methods. We explore patterns of diversity across the Archipelago and compare the utility of different arthropod trapping methods for biodiversity explorations on Hawaii, highlighting undergrowth beating as highly efficient method. Moreover, we show the effects of barcode marker, taxonomy and relative biomass of the targeted specimens and sequencing coverage on taxon recovery. Our protocol enables rapid and inexpensive explorations of diversity patterns and the generation of multilocus barcode reference libraries across whole ecosystems.
DNA barcoding is a well‐established tool for rapid species identification and biodiversity monitoring. A reliable and traceable DNA barcode reference library with extensive coverage is necessary but ...unavailable for many geographical regions. The arid region in northwestern China, a vast area of about 2.5 million km2, is ecologically fragile and often overlooked in biodiversity studies. In particular, DNA barcode data from the arid region in China are lacking. We develop and evaluate the efficacy of an extensive DNA barcode library for native flowering plants in the arid region of northwestern China. Plant specimens were collected, identified and vouchered for this purpose. The database utilized four DNA barcode markers, namely rbcL, matK, ITS and ITS2, for 1816 accessions (representing 890 species from 385 genera and 72 families), and consisted of 5196 barcode sequences. Individual barcodes varied in resolution rates: species‐ and genus‐level rates for rbcL, matK, ITS and ITS2 were 79.9%–51.1%/76.1%, 79.9%–67.2%/88.9%, 85.0%–72.0%/88.2% and 81.0%–67.4%/84.9%, respectively. The three‐barcode combination of rbcL + matK + ITS (RMI) revealed a higher species‐ and genus‐level resolution (75.5%/92.1%, respectively). A total of 110 plastomes were newly generated as super‐barcodes to increase species resolution for seven species‐rich genera, namely Astragalus, Caragana, Lactuca, Lappula, Lepidium, Silene and Zygophyllum. Plastomes revealed higher species resolution compared to standard DNA barcodes and their combination. We suggest future databases include super‐barcodes, especially for species‐rich and complex genera. The plant DNA barcode library in the current study provides a valuable resource for future biological investigations in the arid regions of China.
Hummingbird flower mites are assumed to monopolize single host plant species owing to sexual selection for unique mating rendezvous sites. We tested the main assumption of the mating rendezvous ...hypothesis—extreme host specialization—by reconstructing interactions among tropical hummingbird flower mites and their host plants using DNA barcoding and taxonomic identifications. We collected 10,654 mites from 489 flowers. We extracted DNA from 1,928 mite specimens and amplified the cytochrome c oxidase I (CO1) DNA barcode. We analyzed the network structure to assess the degree of generalization or specialization of mites to their host plants. We recorded 18 species of hummingbird flower mites from three genera (Proctolaelaps, Rhinoseius, and Tropicoseius) interacting with 14 species of plants. We found that generalist mites are common, and congeneric mite species often share host plants. Our results challenge the assumption of strict specialization that supports this system as an example of mating rendezvous evolution.
Morphological identification of gastropods can be difficult considering the larva of species and high similarity among the same genera. DNA barcoding has been widely used in species identification ...and biodiversity research. The purpose of this study was to explore the feasibility of using the cytochrome c oxidase subunit I (COI) gene for the identification of gastropod species and to construct a reliable DNA barcoding reference database of gastropods in Hainan island, China. A total of 306 mitochondrial COI barcode sequences were obtained from 120 species, 35 families and 7 orders of gastropods. The average length of the sequence was 640 base pairs. The average genetic distances based on Kimura two parameter (K2P) within species, genera, families, orders and classes were 0.9 %, 14.7 %, 18.9 %, 24.5 % and 28.6 %, respectively. Most of the gastropod species could be identified using COI sequences. Our results confirmed that the identification method combining morphology and DNA barcode greatly improved the efficiency of species identification. In this study, we found three new record species in China, namely Semiricinula tissoti (Petit de la Saussaye, 1852), Engina alveolata (Kiener, 1836) and Wallaconchis ater (Lesson, 1831). Overall, this study revealed that the identification of gastropods by DNA barcoding is efficient, and COI sequencing technology can be used for the identification of gastropod species and thereby can be used to manage fisheries and assess biodiversity.
The black species of weak-veined Lasioglossum (or Hemihalictus series) in eastern North America are revised to clarify their taxonomy and nomenclature and to facilitate identification. A subgeneric ...classification based upon available phylogenetic data is applied. Lasioglossum (Sphecodogastra) is applied more broadly than any previous usage to include many species typically classified as L. (Evylaeus). The subgenus L. (Evylaeus) is retained but applied narrowly in agreement with phylogenetic results. Lasioglossum (Hemihalictus) has historically been considered monotypic but is here applied to many species of L. (Dialictus) sensu lato (equivalent to the carinaless L. (Evylaeus) of some authors). Usage of L. (Dialictus) is restricted primarily to species with metallic integument. Additional subgeneric synonymies for extralimital taxa are formalized and discussed. Descriptions are provided for each species with a synonymic list, diagnosis, and notes on taxonomy and biology. The recently revised Onagraceae-specialist species of L. (Sphecodogastra) are given abbreviated treatments. Notes on available DNA barcode data are given, with diagnostic characters supplied for closely related spe-cies. One new species is described: L. (Sphecodogastra) seillean Gibbs and Packer and the males of L. fedorense (Crawford) and L. pectinatum (Robertson) are described for the first time. The following three new synonymies are proposed: Lasioglossum (Hemihalictus) sopinci (Crawford), senior subjective synonym of Evylaeus bradleyi Mitchell; Lasioglosum (Hemihalictus) macoupinense (Robertson), senior subjective synonym of Halictus divergens Lovell; and Lasioglossum (Hemihalictus) inconditum (Cockerell), senior subjective synonym of Halictus tracyi Cockerell. Lasioglossum inconditum is here considered to be distinct from the Palaearctic species L. rufitarse (Zetterstedt). A lectotype is designat-ed for Halictus quebecensis Crawford. We present the first record of L. lustrans (Cockerell) and L. swenki (Crawford) in Canada and the first record of L. lusorium (Cresson) east of the Mississippi River. Updated keys to species are provided for the fauna of eastern North America.
Flake and shark samples were purchased from outlets in several coastal Australian regions and genetically barcoded using the cytochrome oxidase subunit 1 (CO1) gene to investigate labelling ...reliability and species‐specific sources of ambiguously labelled fillets. Of the 41 shark fillet samples obtained, 23 yielded high‐quality CO1 sequences, out of which 57% (n = 13) were labelled ambiguously (misleading) and 35% (n = 8) incorrectly. In contrast, barramundi fillets, which are widely available and sought after in Australian markets, were shown to be accurately labelled. Species identified from shark samples, including the shortfin mako (n = 3) and the scalloped hammerhead (n = 1), are assessed by the IUCN as endangered and critically endangered, respectively, with several others classified as vulnerable and near threatened.
The syngnathiform genus Corythoichthys comprises a group of taxonomically complex, tail‐brooding (Syngnathinae) pipefishes widely distributed in the Indo‐Pacific region. Due to the presence of ...overlapping interspecific morphological characters, reliable taxonomic information on Corythoichthys is still lacking. Using 52 CO1 sequences, including seven newly generated, a phylogenetic analysis was carried out to understand the genetic diversity, distribution and ‘species groups’ within the genus Corythoichthys. Species delimitation using Automatic Barcode Gap Discovery (ABGD) analysis confirmed the presence of 13 species which include ‘species‐complexes’ previously considered as a single taxon. Our results revealed the presence of three species groups, ‘C. amplexus’, ‘C. conspicillatus’ and ‘C. haematopterus’ and four unidentified/undescribed species in the wider Indo‐Pacific realm. Interestingly, 60 sequences and a mitogenome identified as Corythoichthys in GenBank are misidentified at the genus level. Based on our findings, we suggest that the taxonomy and systematics of Corythoichthys need to be re‐examined and validated using integrative methods, and care should be taken while selecting specimens for genetic studies.
Metabarcoding of feces has revolutionized the knowledge of animal diets by providing unprecedented resolution of consumed resources. However, it is still unclear how different methodological ...approaches influence the ecological conclusions that can be drawn from such data. Here, we propose a critical evaluation of several data treatments on the inferred diet of the bat Plecotus auritus using guano regularly collected from various colonies throughout the entire active season. First and unlike previous claims, our data indicates that DNA extracted from large amounts of fecal material issued from guano accumulates yield broader taxonomic diversity of prey than smaller numbers of pellets would do, provided that extraction buffer volumes are adapted to such increased amounts of material. Second, trophic niche analyses based on prey occurrence data uncover strong seasonality in the bat's diet and major differences among neighboring maternity colonies. Third, while the removal of rare prey items is not always warranted as it introduces biases affecting particularly samples with greater prey species richness. Fourth, examination of distinct taxonomic depths in diet analyses highlights different aspects of food consumption providing a better understanding of the consumer's diet. Finally, the biologically meaningful patterns recovered with presence-absence approaches are virtually lost when attempting to quantify prey consumed using relative read abundances. Even in an ideal situation where reference barcodes are available for most potential prey species, inferring realistic patterns of prey consumption remains relatively challenging. Although best practice in metabarcoding analyses will depend on the aims of the study, several previous methodological recommendations seem unwarranted for studying such diverse diets as that of brown long-eared bats.
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