Barcode Bulletin - December issue

The brand new Barcode Bulletin hot off the press:

Enjoy reading.

New discoveries at Dragon's breath

I am always excited to see reports about new discoveries from the deep sea. Today I saw a news release on a study published in Scientific Reports:

The Southwest Indian Ridge is the longest section of very slow to ultraslow-spreading seafloor in the global mid-ocean ridge system, but the biogeography and ecology of its hydrothermal vent fauna are previously unknown. We collected 21 macro- and megafaunal taxa during the first Remotely Operated Vehicle dives to the Longqi vent field at 37° 47′S 49° 39′E, depth 2800 m

Longqi also known as 'Dragon's Breath' lies 2000 km southeast of Madagascar and the research team explored an area the size of a football stadium on the ocean floor, pinpointing the locations of more than a dozen mineral spires known as 'vent chimneys'. These spires, many of which rise more than two storeys above the seabed, are rich in copper and gold that is now attracting interest for future seafloor mining. However, the spires are also populated with deep-sea animals, nourished by hot fluids gushing out of the vent chimneys.

The expedition, which took place in November 2011, provides a record of what lives on the ocean floor in the area, which is licensed for mineral exploration by the International Seabed Authority of the United Nations, before any mining surveys are carried out. 

The deep-sea animals that are so far only known from Longqi include: a species of hairy-chested 'Hoff' crab, closely related to 'Hoff' crabs at Antarctic vents; two species of snail and a species of limpet; a species of scaleworm; and another species of deep-sea worm. Apart from one species of snail, which has been given the scientific name Gigantopelta aegis, most have not yet been formally described. By the way, the colleagues used DNA barcoding (without using the term) to help with these discoveries.

We can be certain that the new species we've found also live elsewhere in the southwest Indian Ocean, as they will have migrated here from other sites, but at the moment no-one really knows where, or how well-connected their populations are with those at Longqi. Our results highlight the need to explore other hydrothermal vents in the southwest Indian Ocean and investigate the connectivity of their populations, before any impacts from mineral exploration activities and future deep-sea mining can be assessed.

The researchers also found species at Longqi that are known at other vents far away in other oceans. Another new species of scaleworm lives at vents on the East Scotia Ridge in the Antarctic, 6,000 km away, while a species of ragworm lives at vents in the eastern Pacific, more than 10,000 km away.

Finding these two species at Longqi shows that some vent animals may be more widely distributed across the oceans than we realised.

Just a Chinese Hoax?

Current climate change may be a major threat to global biodiversity, but the extent of species loss will depend on the details of how species respond to changing climates. For example, if most species can undergo rapid change in their climatic niches, then extinctions may be limited. Numerous studies have now documented shifts in the geographic ranges of species that were inferred to be related to climate change, especially shifts towards higher mean elevations and latitudes.

Climate change is predicted to threaten many species with extinction, but determining how species will respond in the future is difficult. A way to understand this better is to study the extinctions caused by the climate change which have already occurred. This also includes local extinctions as they document the loss of populations at the climate edge of species’ ranges e.g at lower elevations and latitudes. Many studies already demonstrated that species are shifting their geographic ranges over time as the climate warms, towards cooler habitats at higher elevations and latitudes. A new study used these range-shift studies to show that local extinctions have already happened in the warmest parts of the ranges of more than 450 plant and animal species. This result is particularly concerning because so far global warming has increased mean temperatures by less than 1 degree Celsius. These extinctions will almost certainly become much more widespread over time, because temperatures are predicted to increase by an additional 1 to 5 degrees in the next several decades no matter if ignorant politicians believe it or not. These local extinctions could also soon extend to species that humans depend on for food and resources.

The study also tested the frequency of local extinction across different regions, habitats, and groups of organisms. It found that local extinctions occurred in about half of the species surveyed across different habitats and taxonomic groups. However, the results showed that local extinctions varied by region and were almost twice as common among tropical species as among temperate species. This is important as the majority of plant and animal species live in the tropics. 

A major conclusion of this study is that populations of many species are already unable to undergo niche shifts that are fast enough to prevent local extinction from climate change. The rate is emphasized here because even if the absolute amount of niche change needed to avoid extinction might be attainable, it might require more time to achieve than is allowed by the rapid pace of anthropogenic climate change.

Monday reads

Although it is not long ago that I posted some new publications, there are a few more:

A dataset of fishes in and around Inle Lake, an ancient lake of Myanmar, with DNA barcoding, photo images and CT/3D models.

BACKGROUND:

Inle (Inlay) Lake, an ancient lake of Southeast Asia, is located at the eastern part of Myanmar, surrounded by the Shan Mountains. Detailed information on fish fauna in and around the lake has long been unknown, although its outstanding endemism was reported a century ago.

NEW INFORMATION:

Based on the fish specimens collected from markets, rivers, swamps, ponds and ditches around Inle Lake as well as from the lake itself from 2014 to 2016, we recorded a total of 948 occurrence data (2120 individuals), belonging to 10 orders, 19 families, 39 genera and 49 species. Amongst them, 13 species of 12 genera are endemic or nearly endemic to the lake system and 17 species of 16 genera are suggested as non-native. The data are all accessible from the document "A dataset of Inle Lake fish fauna and its distribution", as well as DNA barcoding data (mitochondrial COI) for all species being available from the DDBJ/EMBL/GenBank (Accession numbers: LC189568-LC190411). Live photographs of almost all the individuals and CT/3D model data of several specimens are also available at the graphical fish biodiversity database. The information can benefit the clarification, public concern and conservation of the fish biodiversity in the region.

Mosquito Surveillance and the First Record of the Invasive Mosquito Species Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae) in Southern Iran.

BACKGROUND:

Epidemics of mosquito-borne viral infections such as dengue, chikungunya, West Nile and Rift Valley fevers in neighbouring countries and risk of introduction of exotic vectors into Iran have placed this country at a significant risk for these mosquito-borne diseases.

METHODS:

After the first dengue case reported in Iran in 2008, active entomological surveillance of Aedes albopictus (Skuse) and Ae. aegypti (Linnaeus) were conducted in May/Jun, Sep, and Oct/Nov, 2008-2014. Based on occurrence of dengue cases and the presence of potential entry sides including ports and boarder gates, 121 sites in eight provinces were monitored for mosquito vectors. Larval collections were carried out using droppers or dippers and adult collections with CDC light traps, human landing catches, aspirator and Pyrethrum spray space catches.

RESULTS:

A total of 8,186 larvae and 3,734 adult mosquitoes were collected belonging to 23 Culicinae species, including 13 of the genus Culex, 1 Culiseta, 1 Uranotaenia, and 8 of the genus Aedes. Five Aedes albopictus larvae were identified from the Sistan & Baluchestan province bordering Pakistan in 2009. In 2013, seven Ae. albopictus adult mosquitoes were also collected in a coastal locality near the city of Chabahar in the same province.

CONCLUSION:

The detection of larvae and adults of this species in different parts of this province reveal its probable establishment in southeast Iran, which has implications for public health and requires active entomological surveillance as well as the implementation of vector control to prevent the further spread of this critical vector.

DNA barcodes identify medically important tick species in Canada.

Medically important ticks (Acari: Ixodidae) are often difficult to identify morphologically. A standardized, molecular approach using a 658 base pair DNA barcode sequence (from the 5' region of the mitochondrial cytochrome c oxidase subunit I gene) was evaluated for its effectiveness in discriminating ticks in North America, with an emphasis on Canadian ticks. DNA barcodes were generated for 96 of 154 specimens representing 26 ixodid species. A genetic cluster analysis was performed on the barcode sequences, which separated specimens into haplogroups closely corresponding with morphologically identified species. The tree topology was further supported by a BIN analysis. COI sequences generated were found to have a mean maximum intraspecific divergence of 1.59% and a mean nearest neighbour divergence of 12.8%, indicating a significant "barcode gap". This study also revealed possible cryptic diversity among specimens morphologically identified as Ixodes soricis and Ixodes texanus. A PCR-based test for Borrelia burgdorferi determined that 18.1% of Lyme-competent ticks in this study were positive. This study is also the first to record a B. burgdorferi-positive exoskeleton. In conclusion, DNA barcoding is a powerful tool that clinicians can use to determine the identification of tick specimens which can help them to suggest whether an attached tick is a potential health risk.

DNA Barcoding Coupled with High Resolution Melting Analysis Enables Rapid and Accurate Distinction of Aspergillus species.

We describe a high-resolution melting (HRM) analysis method that is rapid, reproducible, and able to identify reference strains and further 40 clinical isolates of Aspergillus fumigatus (14), A. lentulus (3), A. terreus (7), A. flavus (8), A. niger (2), A. welwitschiae (4), and A. tubingensis (2). Asp1 and Asp2 primer sets were designed to amplify partial sequences of the Aspergillus benA (beta-tubulin) genes in a closed-, single-tube system. Human placenta DNA, further Aspergillus (3), Candida (9), Fusarium (6), and Scedosporium (2) nucleic acids from type strains and clinical isolates were also included in this study to evaluate cross reactivity with other relevant pathogens causing invasive fungal infections. The barcoding capacity of this method proved to be 100% providing distinctive binomial scores; 14, 34, 36, 35, 25, 15, 26 when tested among species, while the within-species distinction capacity of the assay proved to be 0% based on the aligned thermodynamic profiles of the Asp1, Asp2 melting clusters allowing accurate species delimitation of all tested clinical isolates. The identification limit of this HRM assay was also estimated on Aspergillus reference gDNA panels where it proved to be 10-102 genomic equivalents (GE) except the A. fumigatus panel where it was 103 only. Furthermore, misidentification was not detected with human genomic DNA or with Candida, Fusarium, and Scedosporium strains. Our DNA barcoding assay introduced here provides results within a few hours, and it may possess further diagnostic utility when analyzing standard cultures supporting adequate therapeutic decisions.

School Malaise Trap program - one last time

We were scrambling a little in the last weeks but here they are (and all the participating schools have them already) - the results of our School program fall run which will for the time being our last one.

In 67 traps students collected 780 specimens on average for the two week period in September. Our collections group sorted 52,447 specimens and selected 12,029 to be barcoded. Our final dataset was made up of 10,478 DNA Barcodes (not all  worked and short barcodes were discarded). Using BOLDs BIN analysis we could  determine that 3,301 species were collected over the two week period of the program, 209 of which were brand new to BOLD. The map below shows all of these collection sites, which include elementary schools, secondary schools, and comparison sites (blue markers). You might notice that we also had some participants from the US. A shout out to our friends from the Western Center Academy and Mount San Jacinto College in California.

Our overall pie chart shows the typical species composition (largely grouped in orders):

Once more a great accomplishment by a large number of young citizen scientists. After three years still many surprises and new finds. Impressive.

As mentioned in a recent post, this will be the last run for now. We don't have any funds to continue the program in the future. Our hope for a comeback lies in the advancement of technology. New HTS technology and metabarcoding might come to our rescue. At some point they will allow us to reduce the costs for the analysis of a single trap catch to a point that it becomes affordable for schools even on a shoestring budget. Unfortunately, we are not there yet but hopefully soon.

"Monday" reads

Another post Monday issue on new DNA barcoding articles. Once again my Monday was too busy to find a few minutes to assemble the post. Nevertheless, here we go - some interesting reads for the week:

Forecasting pollination declines through DNA barcoding: the potential contributions of macroecological and macroevolutionary scales of inquiry.

While pollinators are widely acknowledged as important contributors to seed production in plant communities, we do not yet have a good understanding of the importance of pollinator specialists for this ecosystem service. Determination of the prevalence of pollinator specialists is often hindered by the occurrence of cryptic species and the limitations of observational data on pollinator visitation rates, two areas where DNA barcoding of pollinators and pollen can be useful. Further, the demonstrated adequacy of pollen DNA barcoding from historical records offers opportunities to observe the effects of pollinator loss over longer timescales, and phylogenetic approaches can elucidate the historical rates of extinction of specialist lineages. In this Viewpoint article, we review how advances in DNA barcoding and metabarcoding of plants and pollinators have brought important developments to our understanding of specialization in plant-pollinator interactions. We then put forth several lines of inquiry that we feel are especially promising for providing insight on changes in plant-pollinator interactions over space and time. Obtaining estimates of the effects of reductions in specialists will contribute to forecasting the loss of ecosystem services that will accompany the erosion of plant and pollinator diversity.

Metabarcoding of Fecal Samples to Determine Herbivore Diets: A Case Study of the Endangered Pacific Pocket Mouse

Understanding the diet of an endangered species illuminates the animal’s ecology, habitat requirements, and conservation needs. However, direct observation of diet can be difficult, particularly for small, nocturnal animals such as the Pacific pocket mouse (Heteromyidae: Perognathus longimembris pacificus). Very little is known of the dietary habits of this federally endangered rodent, hindering management and restoration efforts. We used a metabarcoding approach to identify source plants in fecal samples (N = 52) from the three remaining populations known. The internal transcribed spacers (ITS) of the nuclear ribosomal loci were sequenced following the Illumina MiSeq amplicon strategy and processed reads were mapped to reference databases. We evaluated a range of threshold mapping criteria and found the best-performing setting generally recovered two distinct mock communities in proportions similar to expectation. We tested our method on captive animals fed a known diet and recovered almost all plant sources, but found substantial heterogeneity among fecal pellets collected from the same individual at the same time. Observed richness did not increase with pooling of pellets from the same individual. In field-collected samples, we identified 4–14 plant genera in individual samples and 74 genera overall, but over 50 percent of reads mapped to just six species in five genera. We simulated the effects of sequencing error, variable read length, and chimera formation to infer taxon-specific rates of misassignment for the local flora, which were generally low with some exceptions. Richness at the species and genus levels did not reach a clear asymptote, suggesting that diet breadth remained underestimated in the current pool of samples. Large numbers of scat samples are therefore needed to make inferences about diet and resource selection in future studies of the Pacific pocket mouse. We conclude that our minimally invasive method is promising for determining herbivore diets given a library of sequences from local plants.

Assessing Bacterial and Fungal Diversity in the Plant Endosphere.

Plants are colonized various microorganisms including endophytes. These microbes can play an important role in agricultural production as they promote plant growth and/or enhance the resistance of their host plant against diseases and environmental stress conditions. Although culture-independent molecular approaches such as DNA barcoding have greatly enhanced our understanding of bacterial and fungal endophyte communities, there are some methodical problems when investigating endophyte diversity. One main issue are sequence contaminations such as plastid-derived rRNA gene sequences which are co-amplified due to their high homology to bacterial 16S rRNA genes. The same is true for plant and fungal ITS sequences. The application of highly specific-primers suppressing co-amplification of these sequence contaminations is a good solution for this issue. Here, we describe a detailed protocol for assessing bacterial and fungal endophyte diversity in plants using these primers in combination with next-generation sequencing.

Discrete phenotypes are not underpinned by genome-wide genetic differentiation in the squat lobster Munida gregaria (Crustacea: Decapoda: Munididae): a multi-marker study covering the Patagonian shelf.

BACKGROUND:

DNA barcoding has demonstrated that many discrete phenotypes are in fact genetically distinct (pseudo)cryptic species. Genetically identical, isogenic individuals, however, can also express similarly different phenotypes in response to a trigger condition, e.g. in the environment. This alternative explanation to cryptic speciation often remains untested because it requires considerable effort to reject the hypothesis that the observed underlying genetic homogeneity of the different phenotypes may be trivially caused by too slowly evolving molecular markers. The widespread squat lobster Munida gregaria comprises two discrete ecotypes, gregaria s. str. and subrugosa, which were long regarded as different species due to marked differences in morphological, ecological and behavioral traits. We studied the morphometry and genetics of M. gregaria s. l. and tested (1) whether the phenotypic differences remain stable after continental-scale sampling and inclusion of different life stages, (2) and whether each phenotype is underpinned by a specific genotype.

RESULTS:

A total number of 219 gregaria s. str. and subrugosa individuals from 25 stations encompassing almost entire range in South America were included in morphological and genetic analyses using nine unlinked hypervariable microsatellites and new COI sequences. Results from the PCA and using discriminant functions demonstrated that the morphology of the two forms remains discrete. The mitochondrial data showed a shallow, star-like haplotype network and complete overlap of genetic distances within and among ecotypes. Coalescent-based species delimitation methods, PTP and GMYC, coherently suggested that haplotypes of both ecotypes forms a single species. Although all microsatellite markers possess sufficient genetic variation, AMOVA, PCoA and Bayesian clustering approaches revealed no genetic clusters corresponding to ecotypes or geographic units across the entire South-American distribution. No evidence of isolation-by-distance could be detected for this species in South America.

CONCLUSIONS:

Despite their pronounced bimodal morphologies and different lifestyles, the gregaria s. str. and subrugosa ecotypes form a single, dimorphic species M. gregaria s. l.. Based on adequate geographic coverage and multiple independent polymorphic loci, there is no indication that each phenotype may have a unique genetic basis, leaving phenotypic plasticity or localized genomic islands of speciation as possible explanations.

A glimpse into the Global Malaise Trap Program

from original publication

These results illustrate how a comprehensive DNA barcode reference library can identify unknown specimens, but also reveal how this potential is constrained by gaps in the quantity and quality of records in BOLD, especially for Hymenoptera and Diptera. As voucher specimens are available for morphological study, we invite taxonomic experts to assist in the identification of unnamed BINs.

This is taken from an abstract of a new paper that came out yesterday in the Biodiversity Data Journal. It involves data collected as part of the so called Global Malaise Trap program which is an international collaboration between the Centre for Biodiversity Genomics and a number of international partners. The program started in 2012 and represents the a first attempt at the acquisition of detailed temporal and spatial information on terrestrial arthropod communities across the globe. Malaise traps are deployed over an entire season in order to obtain tissue material and subsequently determine species diversity using DNA barcoding.

The number of arthropod specimens such surveys produce are quite impressive, e.g. the study done at two sites in Germany in 2012 and 2013 resulted in 37,274 specimens that were DNA barcoded. In total they found 5301 different BINs which represent about 63% of the total diversity collected in a single experiment. The Global Malaise Trap Program has partnered with 30 different sites across the planet. There is a lot of data to look forward to.