Monday reads

New reads, hot of the press. Very diverse spread of application, news and research.

Ten years of barcoding at the African Centre for DNA barcoding

The African Centre for DNA Barcoding (ACDB) was established in 2005 as part of a global initiative to accurately and rapidly survey biodiversity using short DNA sequences. The mitochondrial cytochrome c oxidase 1 gene (CO1) was rapidly adopted as the de facto barcode for animals. Following the evaluation of several candidate loci for plants, the Plant Working Group of the Consortium for the Barcoding of Life in 2009 recommended that the two plastid genes, rbcLa and matK, be adopted as core DNA barcodes for terrestrial plants. To date, numerous studies continue to test the discriminatory power of these markers across various plant lineages. Over the past decade, we at the African Centre for DNA Barcoding, have used these core DNA barcodes to generate a barcode library for southern Africa. To date, the ACDB has contributed more than 21 000 plant barcodes and over 3 000 CO1 barcodes for animals to the Barcode of Life Database (BOLD). Building upon this effort, we at the ACDB have addressed questions related to community assembly, biogeography, phylogenetic diversification, and invasion biology. Collectively, our work demonstrates the diverse applications of DNA barcoding in ecology, systematics, evolutionary biology, and conservation.

Mapping global biodiversity connections with DNA barcodes: Lepidoptera of Pakistan

Sequences from the DNA barcode region of the mitochondrial COI gene are an effective tool for specimen identification and for the discovery of new species. The Barcode of Life Data Systems (BOLD) (www.boldsystems.org) currently hosts 4.5 million records from animals which have been assigned to more than 490,000 different Barcode Index Numbers (BINs), which serve as a proxy for species. Because a fourth of these BINs derive from Lepidoptera, BOLD has a strong capability to both identify specimens in this order and to support studies of faunal overlap. DNA barcode sequences were obtained from 4503 moths from 329 sites across Pakistan, specimens that represented 981 BINs from 52 families. Among 379 species with a Linnaean name assignment, all were represented by a single BIN excepting five species that showed a BIN split. Less than half (44%) of the 981 BINs had counterparts in other countries; the remaining BINs were unique to Pakistan. Another 218 BINs of Lepidoptera from Pakistan were coupled with the 981 from this study before being compared with all 116,768 BINs for this order. As expected, faunal overlap was highest with India (21%), Sri Lanka (21%), United Arab Emirates (20%) and with other Asian nations (2.1%), but it was very low with other continents including Africa (0.6%), Europe (1.3%), Australia (0.6%), Oceania (1.0%), North America (0.1%), and South America (0.1%). This study indicates the way in which DNA barcoding facilitates measures of faunal overlap even when taxa have not been assigned to a Linnean species.

Demonstrating the value and importance of combining DNA barcodes and discriminant morphological characters for polystome taxonomy (Platyhelminthes, Monogenea).

Polystomes are monogenean parasites that infest mainly semi aquatic vertebrates, such as amphibians and chelonians. Owing to the lack of discriminative morphological characters and because polystomes are considered to be strictly host- and site-specific, host identity is often used as an additional character for parasite identification. Recent genetic studies, however, show that polystomes infecting freshwater turtles in outdoor turtle enclosures and natural environments, are not strictly host-specific. Therefore, we proposed a new procedure for turtle polystome taxonomy based on the combination of Cytochrome c Oxydase I sequences and two discriminant morphological characters, namely the number of genital spines and the testis shape. We tested the validity of this procedure with Polystomoides oris, which was collected from the pharyngeal cavity of the American painted turtle Chrysemys picta and two undescribed species, both collected from the pharyngeal cavity of the American slider Trachemys scripta and two other European turtles, namely the European pond turtle Emys orbicularis and the Mediterranean turtle Mauremys leprosa. A Principal Component Analysis based on both morphological characters allowed the separation of all specimens in three morphological groups, which matched well with the molecular data. As a result, we describe two new polystome species, i.e., Polystomoides soredensis n. sp. and Polystomoides scriptanus n. sp.

New and poorly known Palaearctic fungus gnats (Diptera, Sciaroidea).

BACKGROUND:

Fungus gnats (Sciaroidea) are a globally species rich group of lower Diptera. In Europe, Fennoscandian peninsula in particular holds a notable diversity, ca. 1000 species, of which 10 % are still unnamed. Fungus gnats are predominantly terrestrial insects, but some species dwell in wetland habitats.

NEW INFORMATION:

Eight new fungus gnat species, belonging to the families Keroplatidae (Orfelia boreoalpina Salmela sp.n.) and Mycetophilidae (Sciophila holopaineni Salmela sp.n., S. curvata Salmela sp.n., Boletina sasakawai Salmela & Kolcsár sp.n., B. norokorpii Salmela & Kolcsár sp.n., Phronia sompio Salmela sp.n., P. reducta Salmela sp.n., P. prolongata Salmela sp.n.), are described. Four of the species are known from Fennoscandia only whilst two are supposed to have boreo-alpine disjunct ranges, i.e. having populations in Fennoscandia and the Central European Alps. One of the species probably has a boreal range (Finnish Lapland and Central Siberia). Type material of Boletina curta Sasakawa & Kimura from Japan was found to consist of two species, and a further species close to these taxa is described from Finland. Phronia elegantula Hackman is redescribed and reported for the first time from Norway. DNA barcodes are provided for the first time for five species.

DNA extraction and barcode identification of development stages of forensically important flies in the Czech Republic

Several methods of DNA extraction, coupled with 'DNA barcoding' species identification, were compared using specimens from early developmental stages of forensically important flies from the Calliphoridae and Sarcophagidae families. DNA was extracted at three immature stages - eggs, the first instar larvae, and empty pupal cases (puparia) - using four different extraction methods, namely, one simple 'homemade' extraction buffer protocol and three commercial kits. The extraction conditions, including the amount of proteinase K and incubation times, were optimized. The simple extraction buffer method was successful for half of the eggs and for the first instar larval samples. The DNA Lego Kit and DEP-25 DNA Extraction Kit were useful for DNA extractions from the first instar larvae samples, and the DNA Lego Kit was also successful regarding the extraction from eggs. The QIAamp DNA mini kit was the most effective; the extraction was successful with regard to all sample types - eggs, larvae, and pupari.

Evolution of group I introns in Porifera: new evidence for intron mobility and implications for DNA barcoding

BACKGROUND:

Mitochondrial introns intermit coding regions of genes and feature characteristic secondary structures and splicing mechanisms. In metazoans, mitochondrial introns have only been detected in sponges, cnidarians, placozoans and one annelid species. Within demosponges, group I and group II introns are present in six families. Based on different insertion sites within the cox1 gene and secondary structures, four types of group I and two types of group II introns are known, which can harbor up to three encoding homing endonuclease genes (HEG) of the LAGLIDADG family (group I) and/or reverse transcriptase (group II). However, only little is known about sponge intron mobility, transmission, and origin due to the lack of a comprehensive dataset. We analyzed the largest dataset on sponge mitochondrial group I introns to date: 95 specimens, from 11 different sponge genera which provided novel insights into the evolution of group I introns.

RESULTS:

For the first time group I introns were detected in four genera of the sponge family Scleritodermidae (Scleritoderma, Microscleroderma, Aciculites, Setidium). We demonstrated that group I introns in sponges aggregate in the most conserved regions of cox1. We showed that co-occurrence of two introns in cox1 is unique among metazoans, but not uncommon in sponges. However, this combination always associates an active intron with a degenerating one. Earlier hypotheses of HGT were confirmed and for the first time VGT and secondary losses of introns conclusively demonstrated.

CONCLUSION:

This study validates the subclass Spirophorina (Tetractinellida) as an intron hotspot in sponges. Our analyses confirm that most sponge group I introns probably originated from fungi. DNA barcoding is discussed and the application of alternative primers suggested.

Reminder: Abstract deadline ends in 10 days

The 7th International Barcode of  Life Conference will be held from November 20 - 24, 2017 at the Nombolo Mdhluli Conference Centre, Skukuza, located within the heart of African wildlife at Kruger National Park, South Africa. 

On March 31st the abstract submission will end and because I know that we researchers like to cut it close I like to remind you to send in your's quickly. How about setting aside a couple of minutes this week and write it down? Here you go.

Monday reads

Now back to Monday reads on a Monday. Some new studies for you to catch up.

An rbcL Reference Library to Aid in the Identification of Plant Species Mixtures by DNA Metabarcoding

Premise of the study: DNA metabarcoding has broad-ranging applications in ecology, aerobiology, biosecurity, and forensics. A bioinformatics pipeline has recently been published for identification using a comprehensive database of ITS2, one of the common plant DNA barcoding markers. There is, however, no corresponding database for rbcL, the other primary marker used in plants.

Methods: Using publicly available data, we compiled a reference library of rbcL sequences and trained databases for use with UTAX and RDP classifier algorithms. We used this reference library, along with the existing bioinformatics pipeline and ITS2 reference library, to identify species in an artificial mixture of nine species of pollen. We have made this database publicly available in multiple formats, to allow use with multiple bioinformatics pipelines, now and in the future.

Results: Using the rbcL database, in addition to the ITS2 database, we succeeded in making species-level identifications for eight species and a family-level identification of the ninth species. This is an improvement on ITS2 sequence alone.

Discussion: The reference library described here will assist with identification of plant species using rbcL. By making another gene region available for standard barcoding, this will increase the resolution and accuracy of identifications.

Feeding by Leucopis argenticollis and Leucopis piniperda (Diptera: Chamaemyiidae) from the western USA on Adelges tsugae (Hemiptera: Adelgidae) in the eastern USA

Leucopis argenticollis (Zetterstedt) and Leucopis piniperda (Malloch) are known to feed on the lineage of Adelges tsugae Annand that is native to western North America, but it is not known if they will survive on the lineage that was introduced from Japan to the eastern USA. In 2014, western Leucopis spp. larvae were brought to the laboratory and placed on A. tsugae collected in either Washington (North American A. tsugae lineage) or Connecticut (Japanese lineage). There were no significant differences in survival or developmental times between flies reared on the two different adelgid lineages. In 2015 and 2016, western Leucopis spp. adults were released at two different densities onto enclosed branches of A. tsugae infested eastern hemlock (Tsuga canadensis (L.) Carr.) in Tennessee and New York. Cages were recovered and their contents examined 4 weeks after release at each location. Leucopis spp. larvae and puparia of the F1 generation were recovered at both release locations and adults of the F1 generation were collected at the Tennessee location. The number of Leucopis spp. offspring collected increased with increasing adelgid density, but did not differ by the number of adult flies released. Flies recovered from cages and flies collected from the source colony were identified as L.argenticollis and L. piniperda using DNA barcoding. These results demonstrate that Leucopis spp. from the Pacific Northwest are capable of feeding and developing to the adult stage on A. tsugae in the eastern USA and they are able to tolerate environmental conditions during late spring and early summer at the southern and northern extent of the area invaded by A. tsugae in the eastern USA.

Deceit with decapods? Evaluating labelling accuracy of crustacean products in South Africa

Despite the high value of decapod crustaceans, relatively little research has focused on assessing the transparency in the marketing of these species. This study represents the first comprehensive evaluation of the quality of labelling, and the extent of mislabelling, of decapod crustacean products on the South African market. Data collected through surveys of supermarkets and seafood shops in three provinces (KwaZulu-Natal [KZN], Western Cape [WC] and Gauteng [GP]), indicated that the large majority of domestically available crustacean products were imported, but that 18% of these failed to comply with locally applicable country of origin labelling regulations. Voluntary information relating to the scientific name, production method (wild caught or farmed), and capture method of the species was supplied more frequently in supermarkets than in seafood shops, more frequently in the WC and GP than in KZN, and more frequently on shrimp products than on crab and lobster products. DNA sequencing of 77 products collected from the surveyed outlets revealed that 24 (31%) were misrepresented in some way. Species misrepresentations were most pronounced for shrimps, with Litopenaeus vannamei and Pleoticus muelleri being confirmed as the most common substitute species. One shrimp product was found to contain at least three different species, none of which matched the declared species, whereas a product labelled as crab turned out to be a member of the phylum Mollusca rather than the subphylum Crustacea. Overall, these findings demonstrate that the misrepresentation of crustaceans is commonplace on the South African market, signalling the need for a revision of the current seafood labelling and traceability legislation, as well as monitoring and enforcement efforts.

Barcode DNA length polymorphisms vs fatty acid profiling for adulteration detection in olive oil

The aim of this study was to compare the performance of a DNA-barcode assay with fatty acid profile analysis to authenticate the botanical origin of olive oil. To achieve this aim, we performed a PCR-capillary electrophoresis (PCR-CE) approach on olive oil: seed oil blends using the plastid trnL (UAA) intron barcode. In parallel to genomic analysis, we subjected the samples to gas chromatography analysis of fatty acid composition. While the PCR-CE assay proved equally efficient as gas chromatography analysis in detecting adulteration with soybean, palm, rapeseed, sunflower, sesame, cottonseed and peanut oils, it was superior to the widely utilized analytical chemistry approach in revealing the adulterant species and detecting small quantities of corn and safflower oils in olive oil. Moreover, the DNA-based test correctly identified all tested olive oil: hazelnut oil blends whereas it was not feasible to detect hazelnut oil adulteration through fatty acid profile analysis. Thus, the present research has shown the feasibility of a PCR-CE barcode assay to detect adulteration in olive oil.

Molecular scatology and high-throughput sequencing reveal predominately herbivorous insects in the diets of adult and nestling Western Bluebirds (Sialia mexicana) in California vineyards

Determining the ecosystem function of high-order predators is critical for evaluation of food web interactions. Insectivorous birds are abundant predators in many ecosystems yet because they forage upon small taxa, it remains largely unknown whether birds are providing ecosystem services in the form of pest control or disservices by preying upon predaceous arthropod species. We extracted DNA from noninvasive fecal samples of adult and nestling Western Bluebirds (Sialia mexicana) in California vineyards. Using universal arthropod-specific primers, we sequenced prey items via massively parallel sequencing on the Illumina MiSeq platform. Bluebirds consumed a broad diet comprising 66 unique arthropod species from 6 orders and 28 families. Aedes sp. (mosquitoes: Culicidae), a previously unknown prey, was the most common item recovered, occurring in 49.5% of the fecal samples. Ectoparasitic bird blowfly (Protocalliphora) DNA was found in 7% of adult and 11% of nestling samples, presenting clear evidence of active feeding by the avian hosts on adult or larval ectoparasites. Herbivorous insects, primarily from the orders Hemiptera and Lepidoptera, represented over half (56%) of the prey items in bluebird diets. Intraguild predation (consumption of predator or parasitoid arthropods) represented only 3% of adult and nestling dietary items. Diets of adults were significantly different from nestlings as were diets from birds sampled in different vineyard blocks. Sex, date, number of young, and individual bird (based on resampled individuals) were all insignificant factors that did not explain diet variability. Nestling age was a significant factor in explaining a small amount of the variability in dietary components. In addition, our analysis of subsampling larger fecal samples and processing them independently revealed highly dissimilar results in all 10 trials and we recommend avoiding this common methodology. Molecular scatology offers powerfully informative techniques that can reveal the ecosystem function and services provided by abundant yet cryptic avian foragers.

Evaluation of the Agilent Technologies Bioanalyzer-based DNA Fish Identification Solution

A wide variety of DNA based methods have been developed to identify fish species, including those that employ a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. One such method developed by Dooley, Sage, Clarke, Brown, and Garrett (2005) used amplification of a portion of the mitochondrial cytochrome B (cytB) gene, with a three enzyme digestion, visualized and identified using the Agilent 2100 Bioanalyzer System. More recently this method was modified by Agilent Technologies to target a section of the cytochrome c oxidase 1 (CO1) gene, within the 655 base pair (bp) "barcoding" fragment, using a two enzyme digestion to increase sample throughput and to exploit publically available CO1 data generated through the Barcode of Life initiative (Mueller et al. 2015). Here we evaluate this method on fifteen different commercial fish species with five replicate specimens of each. DNA barcoding of the CO1 gene was used as an orthogonal confirmatory method and also to further understand the results found using the modified Agilent PCR-RFLP method.

Monday reads

Monday reads on a Wednesday - just the result of some conference travel. For the same reason there are a few more articles than usual. Interesting reads as always.

Environmental DNA metabarcoding reveals primary chemical contaminants in freshwater sediments from different land-use types.

Land-use intensification threatens freshwater biodiversity. Freshwater eukaryotic communities are affected by multiple chemical contaminants with a land-use specific manner. However, biodiversities of eukaryotes and their associations with multiple chemical contaminants are largely unknown. This study characterized in situ eukaryotic communities in sediments exposed to mixtures of chemical contaminants and assessed relationships between various environmental variables and eukaryotic communities in sediments from the Nanfei River. Eukaryotic communities in the sediment samples were dominated by Annelida, Arthropoda, Rotifera, Ochrophyta, Chlorophyta and Ciliophora. Alpha-diversities (Shannon entropy) and structures of eukaryotic communities were significantly different between land-use types. According to the results of multiple statistical tests (PCoA, distLM, Mantel and network analysis), dissimilarity of eukaryotic community structures revealed the key effects of pyrethroid insecticides, manganese, zinc, lead, chromium and polycyclic aromatic hydrocarbons (PAHs) on eukaryotic communities in the sediment samples from the Nanfei River. Furthermore, taxa associated with land-use types were identified and several sensitive eukaryotic taxa to some of the primary contaminants were identified as potential indicators to monitor effects of the primary chemical contaminants. Overall, environmental DNA metabarcoding on in situ eukaryotic communities provided a powerful tool for biomonitoring and identifying primary contaminants and their complex effects on benthic eukaryotic communities in freshwater sediments.

How to know the fungi: combining field inventories and DNA-barcoding to document fungal diversity.

no abstract

Bayesian Species Identification under the Multispecies Coalescent Provides Significant Improvements to DNA Barcoding Analyses

DNA barcoding methods use a single locus (usually the mitochondrial COI gene) to assign unidentified specimens to known species in a library based on a genetic distance threshold that distinguishes between-species divergence from within-species diversity. Recently developed species delimitation methods based on the multispecies coalescent (MSC) model offer an alternative approach to individual assignment using either single-locus or multi-loci sequence data. Here we use simulations to demonstrate three features of an MSC method implemented in the program bpp. First, we show that with one locus, MSC can accurately assign individuals to species without the need for arbitrarily determined distance thresholds (as required for barcoding methods). We provide an example in which no single threshold or barcoding gap exists that can be used to assign all specimens without incurring high error rates. Second, we show that bpp can identify cryptic species that may be mis-identified as a single species within the library, potentially improving the accuracy of barcoding libraries. Third, we show that taxon rarity does not present any particular problems for species assignments using bpp, and that accurate assignments can be achieved even when only one or a few loci are available. Thus, concerns that have been raised that MSC methods may have problems analyzing rare taxa (singletons) are unfounded. Currently barcoding methods enjoy a huge computational advantage over MSC methods and may be the only approach feasible for massively large datasets, but MSC methods may offer a more stringent test for species that are tentatively assigned by barcoding.

Relationships between metal compartmentalization and biomarkers in earthworms exposed to field-contaminated soils.

Partitioning tissue metal concentration into subcellular compartments reflecting toxicologically available pools may provide good descriptors of the toxicological effects of metals on organisms. Here we investigated the relationships between internal compartmentalization of Cd, Pb and Zn and biomarker responses in a model soil organism: the earthworm. The aim of this study was to identify metal fractions reflecting the toxic pressure in an endogeic, naturally occurring earthworm species (Aporrectodea caliginosa) exposed to realistic field-contaminated soils. After a 21 days exposure experiment to 31 field-contaminated soils, Cd, Pb and Zn concentrations in earthworms and in three subcellular fractions (cytosol, debris and granules) were quantified. Different biomarkers were measured: the expression of a metallothionein gene (mt), the activity of catalase (CAT) and of glutathione-s-transferase (GST), and the protein, lipid and glycogen reserves. Biomarkers were further combined into an integrated biomarker index (IBR). The subcellular fractionation provided better predictors of biomarkers than the total internal contents hence supporting its use when assessing toxicological bioavailability of metals to earthworms. The most soluble internal pools of metals were not always the best predictors of biomarker responses. metallothionein expression responded to increasing concentrations of Cd in the insoluble fraction (debris + granules). Protein and glycogen contents were also mainly related to Cd and Pb in the insoluble fraction. On the other hand, GST activity was better explained by Pb in the cytosolic fraction. CAT activity and lipid contents variations were not related to metal subcellular distribution. The IBR was best explained by both soluble and insoluble fractions of Pb and Cd. This study further extends the scope of mt expression as a robust and specific biomarker in an ecologically representative earthworm species exposed to field-contaminated soils. The genetic lineage of the individuals, assessed by DNA barcoding with cytochrome c oxidase subunit I, did not influence mt expression.

Advances of Community-Level Plant DNA Barcoding in China

DNA barcoding is a commonly used bio-technology in multiple disciplines including biology, environmental science, forensics and inspection, etc. Forest dynamic plots provide a unique opportunity to carry out large-scale, comparative, and multidisciplinary research for plant DNA barcoding. The paper concisely reviewed four previous progresses in China; specifically, species discrimination, community phylogenetic reconstruction, phylogenetic community structure exploration, and biodiversity index evaluation. Further, we demonstrated three major challenges; specifically, building the impetus to generate DNA barcodes using multiple plant DNA markers for all woody species at forest community levels, analyzing massive DNA barcoding sequence data, and promoting theoretical innovation. Lastly, we raised five possible directions; specifically, proposing a "purpose-driven barcode" fit for multi-level applications, developing new integrative sequencing strategies, pushing DNA barcoding beyond terrestrial ecosystem, constructing national-level DNA barcode sequence libraries for special plant groups, and establishing intelligent identification systems or online server platforms. These efforts will be potentially valuable to explore large-scale biodiversity patterns, the origin and evolution of life, and will also facilitate preservation and utilization of biodiversity resources.