Monday reads

As per usual some new reads at the beginning of the week. Enjoy reading.

New Evidences of Mitochondrial DNA Heteroplasmy by Putative Paternal Leakage between the Rock Partridge (Alectoris graeca) and the Chukar Partridge (Alectoris chukar)

The rock partridge, Alectoris graeca, is a polytypic species declining in Italy mostly due to anthropogenic causes, including the massive releases of the closely related allochthonous chukar partridge Alectoris chukar which produced the formation of hybrids. Molecular approaches are fundamental for the identification of evolutionary units in the perspective of conservation and management, and to correctly select individuals to be used in restocking campaigns. We analyzed a Cytochrome oxidase I (COI) fragment of contemporary and historical A. graeca and A. chukar samples, using duplicated analyses to confirm results and nuclear DNA microsatellites to exclude possible sample cross-contamination. In two contemporary specimens of A. graeca, collected from an anthropogenic hybrid zone, we found evidence of the presence of mtDNA heteroplasmy possibly associated to paternal leakage and suggesting hybridization with captive-bred exotic A. chukar. These results underline significant limitations in the reliability of mtDNA barcoding-based species identification and could have relevant evolutionary and ecological implications that should be accounted for when interpreting data aimed to support conservation actions.

DNA Barcoding of an Assembly of Montane Andean Butterflies (Satyrinae): Geographical Scale and Identification Performance

DNA barcoding is a technique used primarily for the documentation and identification of biological diversity based on mitochondrial DNA sequences. Butterflies have received particular attention in DNA barcoding studies, although varied performance may be obtained due to different scales of geographic sampling and speciation processes in various groups. The montane Andean Satyrinae constitutes a challenging study group for taxonomy. The group displays high richness, with more of 550 species, and remarkable morphological similarity among taxa, which renders their identification difficult. In the present study, we evaluated the effectiveness of DNA barcodes in the identification of montane Andean satyrines and the effect of increased geographical scale of sampling on identification performance. Mitochondrial sequences were obtained from 104 specimens of 39 species and 16 genera, collected in a forest remnant in the northwest Andes. DNA barcoding has proved to be a useful tool for the identification of the specimens, with a well-defined gap and producing clusters with unambiguous identifications for all the morphospecies in the study area. The expansion of the geographical scale with published data increased genetic distances within species and reduced those among species, but did not generally reduce the success of specimen identification. Only in Forsterinaria rustica (Butler, 1868), a taxon with high intraspecific variation, the barcode gap was lost and low support for monophyly was obtained. Likewise, expanded sampling resulted in a substantial increase in the intraspecific distance in Morpho sulkowskyi (Kollar, 1850); Panyapedaliodes drymaea (Hewitson, 1858); Lymanopoda obsoleta (Westwood, 1851); and Lymanopoda labda Hewitson, 1861; but for these species, the barcode gap was maintained. These divergent lineages are nonetheless worth a detailed study of external and genitalic morphology variation, as well as ecological features, in order to determine the potential existence of cryptic species. Even including these cases, DNA barcoding performance in specimen identification was 100% successful based on monophyly, an unexpected result in such a taxonomically complicated group.

Mitochondrial chromosome as a marker of animal migratory routes: DNA barcoding revealed Asian (non-African) origin of a tropical migrant butterfly Junonia orithya in south Israel.

The blue pansy Junonia orithya Linnaeus, 1758 (Lepidoptera, Nymphalidae) is widely distributed along the tropical areas of Africa, Asia and Australia. It is also known as a migrant species in the Levant. Here we record Junonia orithya in south Israel and provide a DNA-barcode-based evidence for its Asian (non-African) origin.

What can we do with 1000 plastid genomes?

The plastid genome of plants is the smallest and most gene-rich of the three genomes in each cell and the one generally present in the highest copy number. As a result, obtaining plastid DNA sequence is a particularly cost-effective way of discovering genetic information about a plant. Until recently, the sequence information gathered in this way was generally limited to small portions of the genome amplified by PCR, but recent advances in sequencing technology have stimulated a substantial rate of increase in the sequencing of complete plastid genomes. Within the last year, the number of complete plastid genomes accessible in public sequence repositories has exceeded 1000. This sudden flood of data raises numerous challenges in data analysis and interpretation but also offers the keys to potential insights across large swathes of plant biology. We examine what has been learnt so far, what more could be learnt if we look at the data in the right way, and what we might gain from the tens of thousands more genome sequences that will surely arrive in the next few years. The most exciting new discoveries are likely to be made at the interdisciplinary interfaces between molecular biology and ecology.

Mark your calendar: Conference Abstract submission

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. 

Next week, Tuesday 31 January the abstract submission will open. Deadline will be 31 March. I just had the pleasure to be test user for the submission system - very nice and user friendly. Check it out next week when you submit your contribution.

Tree identification by laser scanning

LIDAR, which stands for Light Detection and Ranging, is a remote sensing method that uses light in the form of a pulsed laser to measure variable distances. 

LiDAR provides measurements of the horizontal and vertical vegetation structure of ecosystems. The light pulses generate precise, three-dimensional information that, alone, or in combination with satellite multispectral images, allows to automatically and accurately predict forest characteristics, such as tree height, single tree detection, stem diameter, basal area, stem volume, biomass etc.

A joint research project by the Tampere University of Technology's mathematics laboratory and the Natural Resources Institute Finland has developed a new method of identifying tree species based on laser scanning measurements. 

With their method, individual trees can be extracted from forest plot level point cloud data, and the structure of their crowns can be reconstructed as comprehensive 3D models. The created tree models consist of consecutive cylinders, which determine the structure of the tree stem and branches as well as the branching structure.

Previously, it was possible to make a rough distinction between the stem and the crown, based on the point cloud. Now, we are able to make out individual branches and analyse the characteristics of their diameters, volumes and branch angles.

For species identification, the researchers defined 15 classification features, the values of which were then calculated for each tree. Some of these features are completely new and some have been used in previous studies. The new aspect is that now their value can be calculated more accurately, as the colleagues were able to utilize information about the tree's entire crown. They tested three of the most common tree species in Finland, birch, pine and spruce, but they already plan to extend their test set to more species.

According to our results, automatic species recognition is possible with more than 95% accuracy. The purpose was not to find the best possible combination of features, but only to prove that classification based on detailed tree models is possible. However, several combinations produced good results and all the classification methods had a maximum accuracy over 95%. The results also showed that just 30 trees per species is enough learning material for the classification.

Future tests will also include measurements taken from more diverse forests. The tree models calculated based on the laser scanning data will be stored in a database, which can be accessed for even more accurate species recognition when the number of included samples grows.

Monday reads

Welcome to another week. This weekend saw the birth of 'alternative facts', an expression most people better know as 'falsehood', but never mind. We also witnessed a very powerful expression of democracy in the Women's march on Washington and all the Sister marches around the world.

Its Monday, time for science again, so here are some good reads for the week.

Alert! Shameless self-promoting to follow. The first two reads include me as co-author. Number one is the result of collaborative work of colleagues around the Mediterranean Sea.

Improving the Conservation of Mediterranean Chondrichthyans: The ELASMOMED DNA Barcode Reference Library

Cartilaginous fish are particularly vulnerable to anthropogenic stressors and environmental change because of their K-selected reproductive strategy. Accurate data from scientific surveys and landings are essential to assess conservation status and to develop robust protection and management plans. Currently available data are often incomplete or incorrect as a result of inaccurate species identifications, due to a high level of morphological stasis, especially among closely related taxa. Moreover, several diagnostic characters clearly visible in adult specimens are less evident in juveniles. Here we present results generated by the ELASMOMED Consortium, a regional network aiming to sample and DNA-barcode the Mediterranean Chondrichthyans with the ultimate goal to provide a comprehensive DNA barcode reference library. This library will support and improve the molecular taxonomy of this group and the effectiveness of management and conservation measures. We successfully barcoded 882 individuals belonging to 42 species (17 sharks, 24 batoids and one chimaera), including four endemic and several threatened ones. Morphological misidentifications were found across most orders, further confirming the need for a comprehensive DNA barcoding library as a valuable tool for the reliable identification of specimens in support of taxonomist who are reviewing current identification keys. Despite low intraspecific variation among their barcode sequences and reduced samples size, five species showed preliminary evidence of phylogeographic structure. Overall, the ELASMOMED initiative further emphasizes the key role accurate DNA barcoding libraries play in establishing reliable diagnostic species specific features in otherwise taxonomically problematic groups for biodiversity management and conservation actions.

Number two is the result of some follow-up emails to a blog post I wrote a while ago.

No homology means there can be no analyses; a comment on Jose & Harikrishnan

no abstract as it is a commentary paper

And now to all the good ones.

Building a DNA barcode library of Alaska's non-marine arthropods

Climate change may result in ecological futures with novel species assemblages, trophic mismatch, and mass extinction. Alaska has a limited taxonomic workforce to address these changes. We are building a DNA barcode library to facilitate a metabarcoding approach to monitoring non-marine arthropods. Working with the Canadian Centre for DNA Barcoding, we obtained DNA barcodes from recently collected and authoritatively identified specimens in the University of Alaska Museum (UAM) Insect Collection and the Kenai National Wildlife Refuge collection. We submitted tissues from 4776 specimens, of which 81% yielded DNA barcodes representing 1662 species and 1788 Barcode Index Numbers (BINs), of primarily terrestrial, large-bodied arthropods. This represents 84% of the species available for DNA barcoding in the UAM Insect Collection. There are now 4020 Alaskan arthropod species represented by DNA barcodes, after including all records in Barcode of Life Data Systems (BOLD) of species that occur in Alaska - i.e., 48.5% of the 8277 Alaskan, non-marine-arthropod, named species have associated DNA barcodes. An assessment of the identification power of the library in its current state yielded fewer species-level identifications than expected, but the results were not discouraging. We believe we are the first to deliberately begin development of a DNA barcode library of the entire arthropod fauna for a North American state or province. Although far from complete, this library will become increasingly valuable as more species are added and costs to obtain DNA sequences fall.

Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

DNA metabarcoding is a promising approach for rapidly surveying biodiversity and is likely to become an important tool for measuring ecosystem responses to environmental change. Metabarcoding markers need sufficient taxonomic coverage to detect groups of interest, sufficient sequence divergence to resolve species, and will ideally indicate relative abundance of taxa present. We characterized zooplankton assemblages with three different metabarcoding markers (nuclear 18S rDNA, mitochondrial COI, and mitochondrial 16S rDNA) to compare their performance in terms of taxonomic coverage, taxonomic resolution, and correspondence between morphology- and DNA-based identification. COI amplicons sequenced on separate runs showed that operational taxonomic units representing >0.1% of reads per sample were highly reproducible, although slightly more taxa were detected using a lower annealing temperature. Mitochondrial COI and nuclear 18S showed similar taxonomic coverage across zooplankton phyla. However, mitochondrial COI resolved up to threefold more taxa to species compared to 18S. All markers revealed similar patterns of beta-diversity, although different taxa were identified as the greatest contributors to these patterns for 18S. For calanoid copepod families, all markers displayed a positive relationship between biomass and sequence reads, although the relationship was typically strongest for 18S. The use of COI for metabarcoding has been questioned due to lack of conserved primer-binding sites. However, our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well-developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment.

High phylogenetic diversity is preserved in species-poor high-elevation temperate moth assemblages

Understanding the diversity and composition of species assemblages and identifying underlying biotic and abiotic determinants represent great ecological challenges. Addressing some of these issues, we investigated the α-diversity and phylogenetic composition of species-rich geometrid moth (Lepidoptera: Geometridae) assemblages in the mature temperate forest on Changbai Mountain. A total of 9285 geometrid moths representing 131 species were collected, with many species displaying wide elevational distribution ranges. Moth α-diversity decreased monotonously, while the standardized effect size of mean pairwise phylogenetic distances (MPD) and phylogenetic diversity (PD) increased significantly with increasing elevation. At high elevations, the insect assemblages consisted largely of habitat generalists that were individually more phylogenetically distinct from co-occurring species than species in assemblages at lower altitudes. This could hint at higher speciation rates in more favourable low-elevation environments generating a species-rich geometrid assemblage, while exclusion of phylogenetically closely related species becomes increasingly important in shaping moth assemblages at higher elevations. Overall, it appears likely that high-elevation temperate moth assemblages are strongly resilient to environmental change, and that they contain a much larger proportion of the genetic diversity encountered at low-elevation assemblages in comparison to tropical geometrid communities.

...and some bioinformatics

DAMe: a toolkit for the initial processing of datasets with PCR replicates of double-tagged amplicons for DNA metabarcoding analyses.

BACKGROUND:

DNA metabarcoding is an approach for identifying multiple taxa in an environmental sample using specific genetic loci and taxa-specific primers. When combined with high-throughput sequencing it enables the taxonomic characterization of large numbers of samples in a relatively time- and cost-efficient manner. One recent laboratory development is the addition of 5'-nucleotide tags to both primers producing double-tagged amplicons and the use of multiple PCR replicates to filter erroneous sequences. However, there is currently no available toolkit for the straightforward analysis of datasets produced in this way.

RESULTS:

We present DAMe, a toolkit for the processing of datasets generated by double-tagged amplicons from multiple PCR replicates derived from an unlimited number of samples. Specifically, DAMe can be used to (i) sort amplicons by tag combination, (ii) evaluate PCR replicates dissimilarity, and (iii) filter sequences derived from sequencing/PCR errors, chimeras, and contamination. This is attained by calculating the following parameters: (i) sequence content similarity between the PCR replicates from each sample, (ii) reproducibility of each unique sequence across the PCR replicates, and (iii) copy number of the unique sequences in each PCR replicate. We showcase the insights that can be obtained using DAMe prior to taxonomic assignment, by applying it to two real datasets that vary in their complexity regarding number of samples, sequencing libraries, PCR replicates, and used tag combinations. Finally, we use a third mock dataset to demonstrate the impact and importance of filtering the sequences with DAMe.

CONCLUSIONS:

DAMe allows the user-friendly manipulation of amplicons derived from multiple samples with PCR replicates built in a single or multiple sequencing libraries. It allows the user to: (i) collapse amplicons into unique sequences and sort them by tag combination while retaining the sample identifier and copy number information, (ii) identify sequences carrying unused tag combinations, (iii) evaluate the comparability of PCR replicates of the same sample, and (iv) filter tagged amplicons from a number of PCR replicates using parameters of minimum length, copy number, and reproducibility across the PCR replicates. This enables an efficient analysis of complex datasets, and ultimately increases the ease of handling datasets from large-scale studies.

Machine Learned Replacement of N-Labels for Basecalled Sequences in DNA Barcoding

This study presents a machine learning method that increases the number of identified bases in Sanger Sequencing. The system post-processes a KB basecalled chromatogram. It selects a recoverable subset of N-labels in the KB-called chromatogram to replace with basecalls (A,C,G,T). An N-label correction is defined given an additional read of the same sequence, and a human finished sequence. Corrections are added to the dataset when an alignment determines the additional read and human agree on the identity of the N-label. KB must also rate the replacement with quality value of > 60 in the additional read. Corrections are only available during system training. Developing the system, nearly 850 000 N-labels are obtained from Barcode of Life Datasystems, the premier database of genetic markers called DNA Barcodes. Increasing the number of correct bases improves reference sequence reliability, increases sequence identification accuracy, and assures analysis correctness. Keeping with barcoding standards, our system maintains an error rate of < 1%. Our system only applies corrections when it estimates low rate of error. Tested on this data, our automation selects and recovers: 79% of N-labels from COI (animal barcode); 80% from matK and rbcL (plant barcodes); and 58% from non-protein-coding sequences (across eukaryotes).

Metabarcoding and Metagenomics - a new journal, have your say

Some of you might have heard about the EU-COST Action DNAqua-Net. It is based on a fundamental problem in morpho-taxonomic approaches of aquatic biodiversity assessment and monitoring. These have many drawbacks such as being time consuming, limited in temporal and spatial resolution, and error-prone due to variation of individual taxonomic expertise of analysts. Novel genomic tools could circumvent many of the aforementioned problems and thereby complement traditional strategies. Yet, a plethora of protocols are independently developed in different institutions, thereby hampering any concerted routine application.

DNAqua-Net brings together researchers across disciplines in order to identify gold-standard genomic tools (aka barcodes) and novel eco-genomic indices and metrics for routine application for biodiversity assessments and biomonitoring of European water bodies. Furthermore, DNAqua-Net will provide a platform for training of the coming generation of researchers preparing them for the new technologies. Jointly with water managers, politicians and other stakeholders, the group will develop a conceptual framework for the standard application of eco-genomic tools as part of legally binding assessments, and here we are talking about things like the EU-Water Framework Directive and the Marine Strategy Framework Directive.

I am a co-proposer of the project and representative for Canada. The project's core focus is of course the EU but the project leads wanted to ensure that their efforts are embedded in the international community. After all we are talking about a shift in the way we do biomonitoring in general. 

The project is only a few months old, the kick-off conference will happen in March but the group is already very active. Together with Pensoft Publishers the team is planning to launch a new journal, named Metabarcoding and Metagenomics. The journal would feature a set of tailored publication types to address the needs of the community. The hope is that it will serve as a community-organizing focal point far beyond the scope and duration of the DNAqua-Net project.

In order to turn this idea into a perfect fit for the needs of the metabarcoding and metagenomic community, the team is currently soliciting opinions on the features that they envision. 

They would greatly appreciate if you take a few minutes for a short Questionnaire to provide them with your feedback and ideas.

More sticklebacks through climate change?

It has been speculated that conditions brought on by a warming climate may allow animals to breed more often in a single year. However, this has only been empirically shown in insects. The problem is that such predictions and vulnerability assessments require comprehensive and high-quality long-term datasets which are not widely available. But if such long term studies have been done they can deliver some exciting and perhaps concerning results.

A good example is a new study that for the first time documented multiple breeding cycles for fish in a single season due to climate change. Research conducted by the University of Washington showed that one of Alaska's most abundant freshwater fish species, the three-spined stickleback (Gasterosteus aculeatus) is altering its breeding patterns in response to earlier spring ice breakup and longer ice-free summers.

The data were collected from 1963 to 2015 in Alaska's Lake Aleknagik, home to one of the University of Washington's Alaska Salmon Program research stations. For 52 years, the abundance of juvenile sockeye salmon and other fish that live in the region's freshwater lakes was recorded by capturing fish along the lakeshore at 10 different sites every seven days between June and September. All fish were identified and measured.

While the program's monitoring was designed to track the commercially important sockeye salmon population, scientists also meticulously recorded every other fish present, including three-spine stickleback. Stickleback represent almost half of the fish found in Lake Aleknagik, with juvenile sockeye salmon nearly matching that percentage. Three-spine stickleback make up a large percentage of the fish communities in many northern lakes, so these findings could be relevant throughout the region. 

Stickleback are born near the shore, then move to the middle of the lake to feed on zooplankton. Adults return to the shore in the summer to spawn; males will build the nest and attract a female, who then lays the eggs. Males guard the nest until the fish hatch, usually after about two weeks. This behaviour made them great study objects for fish nerds like me. I remember keeping some of them in a tank at home fascinated by textbook knowledge becoming reality in front of my eyes.

By analyzing decades of data showing fish sizes throughout each summer, the colleagues could determine roughly when certain fish were born -  a larger fish captured in August was indicative of an early season brood, while a smaller fish captured on the same day likely came from a brood that hatched later in the summer. Using these data and additional environmental data, they found that the fish spawned earlier in years when ice breakup occurred earlier, and in some years, the fish produced more than one brood. Given the short summers in Alaska, most stickleback have time and stamina for only one brood, but increasingly they are rearing two broods a summer as climate change ushers in earlier springs.

If stickleback are increasing in abundance because of their modified reproduction strategy, this can have ecosystem implications for the productivity of species we commercially care about, like sockeye salmon. We don't know exactly what this means for demographics of this species. It could also mean that fish are living shorter lives because there's a higher physiological cost to breeding more than once. In the lower-latitude extent of their range, fish mature earlier and die earlier.

From the Inbox: BioSyst.EU conference Barcoding Symposium

Dear colleagues, I would like to draw your attention to the upcoming BioSyst.EU conference in August 2017 in Sweden, Gothenburg, where we will organize a half-day symposium on “DNA-barcoding and the future of biodiversity monitoring”, with a special focus on metabarcoding approaches for biodiversity monitoring. We are aiming at bringing together the experts on this topic and would like to cover as many aspects as possible in a balanced fashion - given the time limitation for approx. 1 plenary talk and 8 regular talks. There is of course room for poster contributions as well. Registration for presentations closes at February 1st. It would be great if you let us know if you are interested in bringing the audience up to date with the latest findings from your field of research. 

Application for presenters is done by sending an e-mail . The e-mail should contain the following information: -Which symposium you would prefer to be assigned to -The title of your presentation -If it is an Oral presentation or Poster -An abstract in either doc/pdf/txt -format. Please find further details on the conference here.

Monday reads

Another week, another suite of interesting papers to read.

I wrote about this one in a separate blog post:

Testing the Efficacy of DNA Barcodes for Identifying the Vascular Plants of Canada

Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers (rbcL, matK, ITS2). Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada. To test identification success, we employed a DNA barcode reference library with sequence records for 96% of the 5108 vascular plant species known from Canada, but coverage varied from 94% for rbcL to 60% for ITS2 and 39% for matK. Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance (MPD) and mean nearest taxon distance (MNTD) were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity. All three genes performed strongly in assigning the taxa present in local floras to the correct genus with values ranging from 91% for rbcL to 97% for ITS2 and 98% for matK. However, matK delivered the highest species discrimination (~81%) followed by ITS2 (~72%) and rbcL (~44%). Despite the low number of plant taxa in the Canadian Arctic, DNA barcodes had the least success in discriminating species from this biogeographic region with resolution ranging from 36% with rbcL to 69% with matK. Species resolution was higher in the other settings, peaking in the Woodland region at 52% for rbcL and 87% for matK. Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is highest.

Level of environmental threat posed by horticultural trade in Cactaceae

Ornamental horticulture has been identified as an important threat to plant biodiversity and the major pathway for plant invasions worldwide. In this context, the family Cactaceae is particularly interesting and challenging for three main reasons-it is considered the fifth most threatened major taxonomic group in the world; several cactus species are amongst the most widespread and damaging invasive species; and Cactaceae is one of the most popular horticultural plant groups. Based on CITES trade data and the eleven main auction sites selling cacti on the internet we document a substantial global trade from and to almost all continents. While less than 20 % of this trade involves threatened species, and less than 3% involves known invasive species, many species are sold without a valid scientific name. Importantly, however, hardly any of the globally traded cacti are collected from wild populations. In order to provide an in-depth look at the dynamics of the industry, we surveyed the businesses involved in the cactus trade in South Africa (one of the main hotspots of cactus trade and invasions). Despite a large commercial network, all South African imports (of which only 15 % and 1.5 % were of species listed as threatened and invasive, respectively) came from the same source. We purchased seeds of every available species and, based on DNA-barcoding techniques, could only identify 24 % of the species to genus level. If trade restrictions are placed on the small proportion of cacti that are invasive and there is no major increase in harvesting of native populations, the commercial cactus horticultural trade will pose a negligible environmental threat. However, there are currently no effective methods for easily identifying which cacti are traded, and both the illicit harvesting of cacti from the wild and the informal trade in invasive taxa pose on-going conservation challenges.

This one made some news headlines:

Using DNA barcoding to track seafood mislabeling in Los Angeles restaurants

Seafood mislabeling is common in both domestic and international markets. Previous studies on seafood fraud often report high rates of mislabeling (e.g. >70%), but these studies have been limited to a single sampling year, making it difficult to assess the impact of stricter governmental truth-in-labeling regulations. This study uses DNA barcoding to assess seafood mislabeling in Los Angeles over a four-year period. Sushi restaurants had a consistently high percentage of mislabeling (47%) from 2012 to 2015, yet mislabeling was not homogenous across species. Menu-listed halibut, red snapper, yellowfin tuna, and yellowtail had consistently high occurrences of mislabeling, whereas mislabeling of salmon and mackerel were typically low. All sampled sushi restaurants had at least one case of mislabeling. Mislabeling of sushi-grade fish from high-end grocers was also identified in red snapper, yellowfin tuna, and yellowtail, but at a slightly lower frequency (42%) than sushi restaurants. Results show that despite increased regulatory measures and media attention, seafood mislabeling continues to be prevalent. 

Global food and fibre security threatened by current inefficiencies in fungal identification

Fungal pathogens severely impact global food and fibre crop security. Fungal species that cause plant diseases have mostly been recognized based on their morphology. In general, morphological descriptions remain disconnected from crucially important knowledge such as mating types, host specificity, life cycle stages and population structures. The majority of current fungal species descriptions lack even the most basic genetic data that could address at least some of these issues. Such information is essential for accurate fungal identifications, to link critical metadata and to understand the real and potential impact of fungal pathogens on production and natural ecosystems. Because international trade in plant products and introduction of pathogens to new areas is likely to continue, the manner in which fungal pathogens are identified should urgently be reconsidered. The technologies that would provide appropriate information for biosecurity and quarantine already exist, yet the scientific community and the regulatory authorities are slow to embrace them. International agreements are urgently needed to enforce new guidelines for describing plant pathogenic fungi (including key DNA information), to ensure availability of relevant data and to modernize the phytosanitary systems that must deal with the risks relating to trade-associated plant pathogens.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'.

Reference Library for Canadian vascular plants

Just finished reading a paper by some of my colleagues here at the institute. They summarize the results of a large study which employed a DNA barcode library for the vascular plants of Canada to determine the method with the best species resolution and the barcode marker (rbcL, matK, ITS2) with the highest performance. 

The colleagues build a barcode reference library for 4923 of the 5108 species of non-hybrid origin (~96%) with coverage for all 1153 genera and 171 families in the Database of Vascular Plants of Canada. Of course coverage for the three markers differs. The rbcL dataset is most complete with almost 94% coverage. The ITS2 library includes almost 60% of the species and the matK dataset 39%. Overall, 78% of the species possess records for some combination of two markers, but only 1074 species (22%) have data for all three. Despite such gaps the results are more than promising and certainly very impressive. For almost all vascular plants in Canada the library contains barcode sequences for at least one marker and given their individual effectiveness it is possible to make species and genus assignments at a considerable level:

Analyses based on this library indicate that any one of the three barcode regions is very effective (>90%) in delivering a generic assignment while species resolution is often possible with ITS2 (72%) and matK (80%). BLAST demonstrated higher performance than mothur in assigning specimens to a species in all datasets, including those at a community level and for 1074 species with data for all three barcode regions. The higher performance of BLAST reflects its consideration of indel variation and absolute length of the marker, leading matK to deliver the highest resolution. Although ITS2 showed slightly lower performance, it has two important advantages; its short length makes it suitable for HTS-based applications, and it is readily recovered from diverse taxa, including vascular plants and fungi.

Saving two birds with one stone

Across the planet, poachers have reached into the last remote habitats to harvest wildlife populations used for clothing, eaten, or kept as pets in faraway cities. In some cases, the traded organisms have escaped and are now thriving in their introduced habitats.

The exhaustive international trade of wildlife has pushed many species to the brink of extinction. Coincidentally, many of the same species have been introduced to urban centres or wilderness areas outside their natural ranges. Ironically, a recent study shows that these introduced populations may provide hope for these threatened species. 

A research team from Hong Kong and Australia identified 49 globally threatened species (listed by the International Union for the Conservation of Nature as Vulnerable, Endangered, or Critically Endangered) which have established introduced populations outside their native distribution. These include amphibians, reptiles, mammals, and birds, as well as insects and plants, with introduced populations found on all continents except Antarctica.

One example is the Yellow-crested Cockatoo (Cacatua sulphurea), a species in peril due to extensive pet trade. Many of these pet birds were accidentally or deliberately released in their new environments and as a result about 200 of them, an estimated 10% of the bird's global population, are now found on Hong Kong Island although the native range of them lies in Indonesia and East Timor.

Reintroduction of this species to its native range in Indonesia and East Timor could help to buffer populations there, which are rapidly declining due to poaching.

In addition to the reintroduction of birds to their native range harvesting of introduced cockatoos in Hong Kong could offset pressure on their native counterparts. Both approaches could also eliminate threats the introduced population might pose to native species in its introduced environment, such as monopolising nesting sites and triggering population declines of local birds.

Combined, augmenting declining populations in their native ranges and eliminating the threats to native ecosystems could save two birds with one stone. This creative tactic could be essential to save species imperiled by wildlife trade as well as eliminate threats the same species pose in their adopted territories.

Monday reads

A very happy New Year to all readers out there. I am back to posting after a brief holiday hibernation. Lots of interesting papers have been published in the last few weeks:

DNA barcoding analysis of more than 9 000 yeast isolates contributes to quantitative thresholds for yeast species and genera delimitation.

DNA barcoding is a global initiative for species identification through sequencing of short DNA sequence markers. Sequences of two loci, ITS and LSU, were generated as barcode data for all (ca. 9k) yeast strains included in the CBS collection, originally assigned to ca. 2 000 species. Taxonomic sequence validation turned out to be the most severe bottleneck due to the large volume of generated trace files and lack of reference sequences. We have analysed and validated CBS strains and barcode sequences automatically. Our analysis shows that there were 6 and 9.5 % of CBS yeast species that could not be distinguished by ITS and LSU, respectively. Among them, ∼3 % were indistinguishable by both loci. Except for those species, both loci were successfully resolving yeast species as the grouping of yeast DNA barcodes with the predicted taxonomic thresholds was more than 90 % similar to the grouping with respect to the expected taxon names. The taxonomic thresholds predicted to discriminate yeast species were 98.41 % for ITS and 99.51 % for LSU. To discriminate current yeast genera, thresholds were 96.31 % for ITS and 97.11 % for LSU. Using ITS and LSU barcodes, we were also able to show that the recent reclassifications of basidiomycetous yeasts in 2015 have made a significant improvement for the generic taxonomy of those organisms. The barcodes of 4 730 (51 %) CBS yeast strains of 1 351 (80 %) accepted yeast species that were manually validated have been released to GenBank and the CBS-KNAW website as reference sequences for yeast identification.

Spatial subsidies in spider diets vary with shoreline structure: Complementary evidence from molecular diet analysis and stable isotopes.

Inflow of matter and organisms may strongly affect the local density and diversity of organisms. This effect is particularly evident on shores where organisms with aquatic larval stages enter the terrestrial food web. The identities of such trophic links are not easily estimated as spiders, a dominant group of shoreline predator, have external digestion. We compared trophic links and the prey diversity of spiders on different shore types along the Baltic Sea: on open shores and on shores with a reed belt bordering the water. A priori, we hypothesized that the physical structure of the shoreline reduces the flow between ecosystem and the subsidies across the sea-land interface. To circumvent the lack of morphologically detectable remains of spider prey, we used a combination of stable isotope and molecular gut content analyses. The two tools used for diet analysis revealed complementary information on spider diets. The stable isotope analysis indicated that spiders on open shores had a marine signal of carbon isotopes, while spiders on reedy shores had a terrestrial signal. The molecular analysis revealed a diverse array of dipteran and lepidopteran prey, where spiders on open and reedy shores shared a similar diet with a comparable proportion of chironomids, the larvae of which live in the marine system. Comparing the methods suggests that differences in isotope composition of the two spider groups occurred because of differences in the chironomid diets: as larvae, chironomids of reedy shores likely fed on terrestrial detritus and acquired a terrestrial isotope signature, while chironomids of open shores utilized an algal diet and acquired a marine isotope signature. Our results illustrate how different methods of diet reconstruction may shed light on complementary aspects of nutrient transfer. Overall, they reveal that reed belts can reduce connectivity between habitats, but also function as a source of food for predators.

Meeting the challenge of DNA barcoding Neotropical amphibians: Polymerase chain reaction optimization and new COI primers.

Amphibians are one of the most threatened vertebrate classes, yet at the same time new species are being described every year, demonstrating that the number of existing species is grossly underestimated. In groups such as amphibians, with high extinction rates and poorly known species boundaries, DNA barcoding is a tool that can rapidly assess genetic diversity and estimate species richness for prioritizing conservation decisions. However, reliable recovery of the 5' region of the cytochrome c oxidase subunit 1 (COI) gene is critical for the ongoing effort to gather DNA barcodes for all amphibian species. Here we provide new PCR conditions and tested new primers that increase the efficiency of barcode recovery in amphibians. We found that a low extension temperature for PCR cycles significantly improves the efficiency of amplification for all combinations of primers. Combining low PCR extension temperature and primers AnF1 + AnR1 we were able to recover COI sequences for 100% of the species analyzed (N=161), encompassing ~15% of the species known from Brazil (representing 77 genera and 23 families), which is an important improvement over previous studies. The preliminary assessment of species diversity suggested that number of species might be underestimated by about 25%. We conclude that DNA barcoding is an efficient, simple, and standardized protocol for identifying cryptic diversity in amphibians and advocate for its use in biodiversity inventories and across widespread populations within known species.

DNA barcoding uncovers cryptic diversity in 50% of deep-sea Antarctic polychaetes

The Antarctic marine environment is a diverse ecosystem currently experiencing some of the fastest rates of climatic change. The documentation and management of these changes requires accurate estimates of species diversity. Recently, there has been an increased recognition of the abundance and importance of cryptic species, i.e. those that are morphologically identical but genetically distinct. This article presents the largest genetic investigation into the prevalence of cryptic polychaete species within the deep Antarctic benthos to date. We uncover cryptic diversity in 50% of the 15 morphospecies targeted through the comparison of mitochondrial DNA sequences, as well as 10 previously overlooked morphospecies, increasing the total species richness in the sample by 233%. Our ability to describe universal rules for the detection of cryptic species within polychaetes, or normalization to expected number of species based on genetic data is prevented by taxon-specific differences in phylogenetic outputs and genetic variation between and within potential cryptic species. These data provide the foundation for biogeographic and functional analysis that will provide insight into the drivers of species diversity and its role in ecosystem function.

Highlighting Astyanax Species Diversity through DNA Barcoding

DNA barcoding has been used extensively to solve taxonomic questions and identify new species. Neotropical fishes are found in a wide variety of shapes and sizes, with a large number of species yet to be described, many of which are very difficult to identify. Characidae is the most species-rich family of the Characiformes, and many of its genera are affected by taxonomic uncertainties, including the widely-distributed, species-rich genus Astyanax. In this study, we present an extensive analysis of Astyanax covering almost its entire area of occurrence, based on DNA barcoding. The use of different approaches (ABGD, GMYC and BIN) to the clustering of the sequences revealed ample consistency in the results obtained by the initial cutoff value of 2% divergence for putative species in the Neighbor-Joining analysis using the Kimura-2-parameter model. The results indicate the existence of five Astyanax lineages. Some groups, such as that composed by the trans-Andean forms, are mostly composed of well-defined species, and in others a number of nominal species are clustered together, hampering the delimitation of species, which in many cases proved impossible. The results confirm the extreme complexity of the systematics of the genus Astyanax and show that DNA barcoding can be an useful tool to address these complexes questions.

Molecular determination of kleptoplast origins from the sea slug Plakobranchus ocellatus (Sacoglossa, Gastropoda) reveals cryptic bryopsidalean (Chlorophyta) diversity in the Hawaiian Islands.

The sacoglossan sea slug species complex Plakobranchus ocellatus is a common algivore throughout the tropical Pacific, including the Hawaiian Islands. Plakobranchus ocellatus is kleptoplastic - it sequesters and retains algal chloroplasts - a characteristic that can be exploited to molecularly characterize diminutive bryopsidalean algae that are typically difficult to locate, collect, and identify. Previous DNA barcode analyses of both P. ocellatus and its kleptoplasts have been conducted primarily in the western Pacific and have only minimally sampled the most eastern populations in the Hawaiian Islands. Using two chloroplast markers rbcL and tufA, kleptoplast samples from an O'ahu population of P. ocellatus were amplified and cloned to identify their algal sources. Plakobranchus ocellatus sequester chloroplasts from up to 11 bryopsidalean algal species, all but one being diminutive in thallus size. Notably, eight of the detected algal species were new records to the Hawaiian Islands. A sequestration preference study demonstrated that the O'ahu population of P. ocellatus preferentially sequesters chloroplasts from diminutive, epilithic taxa. Using coxI barcoding of P. ocellatus we showed the O'ahu population to be part of a clade that includes sequences from the neighboring island Maui, Australia, and the Philippines. The use of P. ocellatus as a novel sampling tool allows the exploration of the green algal community diversity and composition at a fine scale.