At the museum

Lot's of kids at the Royal Ontario Museum and all love DNA Barcoding. Well not entirely true - they love our buttons and bookmarks:

Curators' Corner

In case you are in Toronto today. I am at the Royal Ontario Museum for a few hours to join the:

Curators' Corner: Nature in the City

Over 6 million people live within the Greater Toronto Area, making it the most populated region of the country.  However, we are not alone in the city!  Many different species of plants and animals live in and around the city, ranging from the familiar raccoons and squirrels, to the less well-known, such as coyotes and possums.  Toronto has a large diversity of nature to experience, right outside your door!

On August 31st, come join us at the museum for this Curators’ Corner and learn more about nature in the city!  You’ll have an opportunity to talk to experts from nature and wildlife organizations including the Toronto Zoo, Ontario Nature, Rouge Park, and BIObus.  You’ll also have an opportunity to hear some info about the upcoming BioBlitz on September 14th and 15th.  The BioBlitz is 24 hour campout in Rouge Park focused on identifying and cataloguing as many of the different species of plants and animals within the park as possible.  This is a great way to meet up with real scientists, get some experience about what biologists do “in the field”, and to have a great time outdoors experiencing the wonderful nature of Toronto!

If you’d like to find out more about the BioBlitz, or learn about the biodiversity of the city in general, then be sure to join us on Saturday, August 31st, in the Earth Rangers Studio at the ROM (11:00 am - 4:00 pm) !

A new diamondback moth

The tiny diamondback moth (Plutella xylostella) gets its common name from the array of diamond-shapes along the margin of its forewing. Despite their small size, the caterpillars of the diamondback moth exert tremendous damage on many crucifers including cabbage and broccoli. More than $1 billion is spent globally each year in efforts to control damage by this moth, reflecting its amazing capacity to evolve resistance to both insecticides and biological control agents.

A global study of DNA Barcodes revealed unexpected complexity: the occurrence of two distinct species among Australian diamondback moths. One of them is the well-known diamondback pest which is found nearly everywhere. The other is a new species, named Plutella australiana by Jean-François Landry of the Canadian National Collection in Ottawa and Paul Hebert from BIO, the authors of the study. The new species has so far been found only in Australia, where it occurs together with typical Plutella xylostella.

The new species was initially detected by Paul Hebert in a general survey of Australian moths aimed at developing a library of DNA Barcodes representing all the species of the fauna. One part of this effort (sequencing the lepidoptera of the Australian National Insect Collection) I described a few days ago but the specimens of this study were caught in the wild. Subsequent study of the anatomy revealed significant, previously unsuspected, differences in internal reproductive organs between typical diamondbacks and the new species.

Although the new species of diamondback moth has now gained recognition and a name, key aspects of its biology remain uncertain. For example, what is its role as a crop pest in Australia and does it pose a threat to agriculture? The presence of these two species also has implications for past evaluations of biological control strategies, particularly since both species appear to be abundant and widely distributed in eastern Australia. The next step could be to find the host plant(s) of Plutella australiana to ascertain if it is also a crop pest. If so, it likely represents a new risk to international trade which should be subsequently evaluated.

Italian fungi

As promised earlier here another example for ongoing and past efforts to barcode existing collections. 

Over the last couple of years the University of California, Berkeley and the Venice Museum of Natural History in Italy were collaborating to extensively catalog a museum collection using DNA Barcoding. The Venice Museum hosts the largest and best preserved fungal collection in Italy with more than 25,000 samples, representing approximately 6,000 species of fungi including many rare specimens. A diverse representation of the Venice collection was sampled and sent to Berkeley, where the fungal DNA Barcode region (ITS) was sequenced.

The Venice collection is one of the first ones in the world to be used for building an extensive DNA Barcode library. The collection is unique as it is entirely generated and curated by volunteers, and in fact it is the outlet of the largest amateur mycological association in the world, the Associazione Micologica Bresadola, which currently has about 13,000 active members.

The results of this work have been published this spring and they are indeed promising:

Our goals were to investigate issues related to large sequencing projects, develop heuristic methods for assessing the overall performance of such a project, and evaluate the prospects of such efforts to reduce the current gap in fungal biodiversity knowledge. The effort generated 1107 sequences submitted to GenBank, including 416 previously unrepresented taxa and 398 sequences exhibiting a best BLAST match to an unidentified environmental sequence. Specimen age and taxon affected sequencing success, and subsequent work on failed specimens showed that an ITS1 mini-barcode greatly increased sequencing success without greatly reducing the discriminating power of the barcode. 

An interactive map showing the geographic location of all sequenced accessions, linked to collection data and searchable by taxon, is available for viewing (see image above). The researchers made the DNA Barcodes avaiable in an organized fashion (e.g. by genera) on their project website and they explicitly invite researchers to use the sequences in their analyses and provide feedback that could be useful for refining the taxonomic identifications attached to the data.

Natural history collections

In yesterdays post I had mentioned that there are more examples of initiatives that aim to build reference DNA Barcode libraries by sequencing type specimens and historical material wherever possible. 

Natural history collections would seem an ideal resource for barcode library construction, but they have never seen large-scale analysis because of concerns linked to DNA degradation. The present study examines the strength of this barrier, carrying out a comprehensive analysis of moth and butterfly (Lepidoptera) species in the Australian National Insect Collection (ANIC). 

This is from the abstract of a publication that came out earlier this summer describing protocols that were developed to enable tissue samples, specimen data, and images to be assembled in a short time. The results are quite impressive:

A total of 41,650 specimens were processed over 375 person-days of work, an average of 111 specimens per person-day. These specimens provided coverage for 8245 named species and for 4454 presumptive species for a total of 12,699 taxa. The specimens had an average age of 30.4 years (median = 28.9 years), but ranged from 1 to 112 years.

Some 20,000 species of the order lepidoptera are thought to live in Australia. Only about have of those (10,500) have been described in the last 235 years. Without formal documentation, all the undescribed species contribute nothing towards a deeper understanding of distributional patterns and biodiversity despite the fact that this information is of high importance to both research and conservation. The answer to the problem could be large scale efforts to barcode large collections but before one would do such a thing he would need to find out if it is feasible. It seems it actually is (at least for a collection with largely pinned insects):

The results from analysis of the ANIC specimens indicate that existing collections of museum specimens, especially insects, can enable the construction of a comprehensive DNA barcode reference library. They also establish that progress can be rapid – a team of five individuals can sample tissues, photograph and digitize data from 10,000 species in three months and a matching team with sequencing expertise can barcode these samples in nine months. Three teams of this size could complete barcode libraries for the Lepidoptera of Australia, Europe, and North America in a year. Coverage for Asia, Africa and South America would require more time, but could still be accomplished in less than a decade. If similar teams were established to lead work on each of the other insect orders, progress would be rapid. We conclude that a comprehensive DNA barcode library for insects is not only feasible, but is almost irresistible.

My first birthday

I almost didn't notice myself but this blog is now one year old. The vast majority of bloggers give up within first year of blogging but 230 posts later I can proudly say: "I survived". I could not have done it without the support of all the people who read my posts, share my blog with others, and provide me with new topics. So, a big THANK YOU to my readers for making this all possible. 

Barcoding P.A.T.H.S.

A group of Italian researchers made their sequences of type and historical material publicly available through a new database, called “Barcoding P.A.T.H.S.” (Plant & Algal Type & Historical Specimens). It represents a library of reference sequences, easy to update, and against which putative new species or other plant material can be compared (they use a BLAST tool on the site). As the acronym P.A.T.H.S suggests, specimens comprise water, and land plants and algae, interpreted sensu lato to include not only microalgae and seaweeds, but also cyanobacteria. 

Most importantly, the project focuses solely on reference specimens and includes type specimens and historical material. The intention is to tackle the problem of frequent misidentifications, e.g. for sequences available in public databases obtained from incorrectly identified specimens or with a number of superfluous and synonymised species entities recorded for some taxa. It has been stated very often that the ideal approach to DNA Barcoding would be to sequence type specimens and historical material wherever possible. This new initiative is a great example. I'll try to find more for this week's blog posts as we are long past the stage of having just great ideas.

The Italian group is now reaching out to the community through a small paper published in Taxon. Here a small excerpt:

Our project has just begun and our group is willing to establish  collaborations with other research groups, museums, and botanical  gardens involved in similar DNA barcoding studies. Such collaborations would increase the molecular data regarding type and  historical specimens and make them accessible to everyone. In the  P.A.T.H.S. database each sample that represents the type strain of a  newly described species (supported by publication on ISI journals)  can be included. In addition, it is possible for collaborators to add  unpublished sequences so they are available for comparison with  BLAST, but not visible or downloadable by other users, this would  facilitate the rapid sharing of data. However, the sequences have to be submitted to one of the public databases, like GenBank.

Diversification in hotspots

A common view is that species in biodiversity hotspots diversify more quickly than species in less biodiverse areas. However, that seems not to be the case for a group of Australian wildflowers, the spikey-flowered Banksia. This genus is found in many parts of Australia, including the biodiverse Southwest Botanical Province, which is home to more than 7300 plant species in the midst of shrubland, semi-arid heath, and Mediterranean-climate forests. But although Banksia species richness in this hotspot is ten times higher than in the rest of the continent, it's not diversifying any more quickly than congeneric plants in other parts of Australia.

How did the biodiversity of the Southwest Botanical Province arose? If new species aren't arising more rapidly, perhaps they go extinct less frequently compared with similar plants in less diverse regions. A team from the Australian National University, Canberra constructed the a near-complete phylogeny of Banksia to test whether diversification rates have differed between lineages confined to the southwest Australian hotspot and those found throughout southern, eastern and northern Australia. 

The geographic pattern of diversification in Banksia appears more complex than can be characterized by a simple hotspot vs. non-hotspot comparison, but in general, these findings contrast with the view that the high diversity of Mediterranean hotspots is underpinned by rapid radiations. Steady accumulation of species at unexceptional rates, but over long periods of time, may also have contributed substantially to the great botanical richness of these regions.

The team also looked at biodiversity within the confines of the Southwest Province where they found that Banksia plants in semi-arid heath and shrublands were diversifying more quickly than plants in the high-rainfall forests. Diversity is likely generated in these semi-arid regions, then migrates out to boost diversity in the adjacent forest. 

Biodiversity hotspots are frequently found in Mediterranean-climate regions, where they rival tropical rainforests for flowering plant biodiversity. But these environments typically lack features such as high rainfall or productivity that are usually linked with high plant diversity. Indeed, some of the most species-rich Mediterranean communities are found in dry regions, on nutrient-poor soils. Understanding these apparent outliers on global biodiversity gradients may yield insights into the factors driving the diversification of flowering plants.

Addendum to Lemming post

Just found this New Yorker cartoon on Jerry Coyne's blog. Funny coincidence.

Lemming diet

Lemmings became the subject of a popular misconception that they commit mass suicide when they migrate. However, it is not a mass suicide, but the result of their migratory behavior. Some species of the small rodents (e.g. the Norwegian lemming, Lemmus lemmus) may migrate in very large groups when population density becomes too great. Lemmings can swim and may choose to cross a body of water in search of a new habitat. In such cases, many lemmings may drown if the body of water is so wide as to stretch their physical capability to the limit. This fact, combined with unexplained fluctuations in the population of Norwegian lemmings, gave rise to the misconception. Lemmus lemmus can reproduce so quickly that population fluctuations become chaotic, rather than following linear growth to a carrying capacity or regular oscillations. It is unknown why lemming populations fluctuate with such great variance on average every four years. Only recently new evidence suggested that populations of lemming predators, particularly the stoat (Mustela erminea), may be more closely involved in changing the lemming population. 

As lemmings function as the main trophic link between vegetation and predators in most tundra ecosystems, their high amplitude population density cycles often have a major impact on tundra food webs. A new study now aimed to correctly evaluate the effect of lemmings on vegetation by identifying what they feed on in the wild:

Lemmings are key herbivores in many arctic food webs, and their population dynamics have major impacts on the functioning of tundra systems. However, current knowledge of lemming diet is limited, hampering evaluation of lemming–vegetation interactions. This lack of knowledge is mainly due to methodological challenges, as previously used microhistological methods result in large proportions of poorly resolved plant taxa. We analyzed diets of Norwegian lemmings (Lemmus lemmus) in three different habitats using a new method, DNA metabarcoding of stomach contents. To achieve detailed information on ingested vascular plants, bryophytes, and fungi, we amplified short fragments of chloroplast DNA (for plants; P6 loop of the trnL intron) and nuclear ribosomal DNA (for fungi; ITS1-region). Our results revealed that lemming diets were dominated by grasses, mainly Avenella flexuosa, and mosses, mainly Dicranum spp., but that a variety of other food items were also eaten. Vascular plant composition of the diets differed between heath, meadow, and wetland habitats, whereas bryophyte composition did not. Also, a variety of fungal taxa were retrieved, but as most of the identified taxa belong to micromycetes, they were unlikely to be consumed as food. The role of fungi in the diet of lemmings remains to be investigated. We suggest that there may be substantial variation between habitats and regions in lemming diet.

Barcode of Life Conference - deadline for abstract submissions extended to August 23rd

Only 10 more weeks until the 5th International Barcode of Life Conference starts in Kunming, China. 

Registrations are rolling in (already some 300 from 40 countries) and a lot of abstracts have been submitted already. As a consequence of the great interest in the conference the organizing committee has decided to extend the deadline for abstract submission once more. 

So, if you were thinking of submitting an abstract and haven't done it already, you have one more week to do it. You'll find the submission page on the conference website.

MALDI-Tof-MS

Molecular data show less variability than protein spectra, but are more time-consuming and cost-intensive. On the contrary, mass spectra are able to discriminate between some developmental stages. As a result, we suggest the application of species typing based on proteomic fingerprinting as an alternative approach for a rapid and cost-effective method. Once an accessible reference library for metazoan proteome fingerprints is established and by working with adequate software, species can be identified straightforwardly within minutes. We suggest that this method is especially useful for analysing a high number of samples or when specimens are difficult to determine/discriminate with respect to congeners or life history stages.

So far the authors of a new study which compares DNA-based identification methods (e.g. DNA Barcoding) with proteome fingerprints generated through so called matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-Tof-MS). This technology has had a major impact in many fields of the life sciences over the last two decades. Samples (cells, molecules, peptides or proteins) co-crystallize with a matrix solution on a target plate. A pulsed laser causes desorption of the sample mixture, followed by the ionization of the sample. In a strong electric field, the ions are accelerated along a vacuum tube. Based on the time of flight, the different masses of the single molecules are represented as spectra. 

MALDI-Tof-MS studies for rapid species identification were e.g conducted using species-specific proteome profiles of fish, flies, and copepods. It has been argued that for a rapid identification of individuals, techniques based on nucleotide sequences still have the disadvantage of requiring a number of steps, i.e. DNA extraction, polymerase chain reaction, gel electrophoresis, and are therefore considered more costly and time-consuming.

As other methods that are proposed for rapid species identification MALDI-Tof-MS could be complementary to what we already have in place. While methods based on molecular biology generally demand some a priori knowledge of the sample under investigation (e.g. for primer selection) and, more importantly, can be slow and laborious with results that may depend on experimental variables, a mass spectral approach can be much more straightforward, requiring minimal time and relatively low costs. However, a prerequisite is the creation of a taxonomically comprehensive molecular profile database for known species. That reminds me of something...

Ray Wings

DNA Barcoding has revealed which species are sold under the commercial term 'ray wings' in Ireland and the UK. The research was conducted at the University of Salford and University College Dublin, as part of an international project called "LabelFish" that is currently investigating seafood labelling and traceability within the European Union (EU). 

The findings showed that none of the ray wings analysed originated from endangered species that were prohibited from being landed in the EU. Good news for the conservation of groups of skate that are of most critical conservation concern. However, the results were not all positive; the most commonly identified species in the study was the blonde ray (Raja brachyura), a large-growing species that has a decreasing population trend and which the marine conservation society (MSC) awards its lowest sustainability rating, suggesting consumers should avoid purchasing it. In fact, size has previously been suggested as a good proxy for risk to overfishing, and the blonde ray was just one of three large growing species identified during investigations. All three are associated with declining abundance and concerns over sustainability.

The study concludes that the use of ambiguous or amalgamated sales terms makes it impossible for consumers to exercise the right to choose species of lesser conservation concern helping to protect these declining species. According to the authors the EU generally requires skates to be identified to species level when landed, but this information is not being passed onto consumers. This remains a real obstacle in allowing those to make informed choices. Interestingly, some supermarket chains have already made commitments to supply smaller growing species that are viewed as more sustainable. One retailer even went as far as labelling its ray wings as originating from these smaller growing groups. Analysis of a limited number of wings purchased from this supermarket showed a third were actually mislabelled; they really belonged to a more vulnerable larger growing species. This highlights the importance of on-going efforts to improve labelling of foods, not only to protect consumers, but also to allow informed choice and promote the conservation of vulnerable species.

Metabarcoding

Metabarcoding is a rapid method of biodiversity assessment that combines two technologies: DNA based identification and high-throughput DNA sequencing. It uses universal PCR primers to mass-amplify DNA Barcodes from mass collections of organisms or from environmental DNA. The PCR product is sent to a next generation sequencer and the result is a wealth of DNA sequences. Such sequence collections are auditable, because sites can be sampled by independent parties, or samples can be split, and analysed by certified entities following a standardized protocol. They can also be verified by fieldwork to confirm the presence or absence of particular species. These metabarcode data sets are taxonomically more comprehensive, many times quicker to produce, and less reliant on taxonomic expertise.

However, the reliability of such datasets has not been tested fully: In general, studies have found that not every species is recovered from samples and that the ecological patterns do not perfectly match those found using standard data sets. Can these discrepancies be ignored? Are the metabarcode data sets in fact revealing higher resolution ecological patterns? Most importantly, can the information that is recovered by metabarcoding be used to answer policy and management questions reliably?

A new study published in Ecology Letters two days ago aims to answer those questions:

Here, we validate metabarcoding by testing it against three high-quality standard data sets that were collected in Malaysia (tropical), China (subtropical) and the United Kingdom (temperate) and that comprised 55,813 arthropod and bird specimens identified to species level with the expenditure of 2,505 person-hours of taxonomic expertise. The metabarcode and standard data sets exhibit statistically correlated alpha- and beta-diversities, and the two data sets produce similar policy conclusions for two conservation applications: restoration ecology and systematic conservation planning. Compared with standard biodiversity data sets, metabarcoded samples are taxonomically more comprehensive, many times quicker to produce, less reliant on taxonomic expertise and auditable by third parties, which is essential for dispute resolution.

Needless to say that this study was very successful and provided some interesting numbers, e.g. on the amount of work the went into both the standard approach and into the next generation sequencing method. Even at such an experimental stage only about a fourth of the person-hours is necessary and under standard conditions not a full sample was analysed. The authors close with some recommendations for further metabarcoding work which I like to share:

Naturally, metabarcode data sets are subject to error and loss of information, so most research effort to date has been to validate metabarcoding against standard biodiversity censuses (see Introduction), and to develop more efficient and reliable pipelines that take advantage of advances in sequencing technology. Another focus has been devising clever ways to collect the DNA of difficult-to-trap taxa: water, soil, pollen traps, faeces and parasites. We expect both of these areas to continue to consume research effort.

In addition, we see the following three directions as especially important if metabarcoding is to bridge the science-practitioner divide:

1. Developing statistical and laboratory methods to allow robust inference of species abundances in samples and across landscapes. Related to this is the development of PCR-free methods that reduce read-number biases and allow the detection of taxa that do not amplify well, such as the Hymenoptera (Yu et al. 2012).

2. Robust methods of taxonomic assignment and phylogenetic placement, with confidence estimates at each taxonomic level, while minimising false-positive assignments (Matsen et al. 2010; Zhang et al. 2012).

3. Deeper connection with the end-users of biodiversity data (Cook et al. 2013), including the development of chain-of-evidence and bioinformatic-reporting protocols to increase the credibility of the data.

Blastocystis in Colombia

Blastocystis is a genus of single-celled protozoan parasites belonging to a group of organisms known as the Stramenopiles (also called Heterokonts) that includes algae, diatoms, and water molds. Blastocystis comprises several species, living in the gastrointestinal tracts of mostly vertebrate species (including humans) although some were also discovered in insects, such as Cockroaches. 

Human infections are found at a rate of 5-10% in most developed countries with elevated rates for individuals that work with animals. In developing countries the rate can be as high as 50%. However, only 50% and 80% of individuals infected with Blastocystis will show symptoms. Symptoms associated with the infection are diarrhea, nausea, abdominal cramps, bloating, excessive gas, and anal itching. Most cases of the infection appear to become diagnosed as irritable bowel syndrome, which is a symptom-based diagnosis with no known organic cause but characterized by the very same symptoms. 

For many years, scientists believed one species of Blastocystis infected humans and called it Blastocystis hominis. Consequently they thought that different species of Blastocystis infected other animals. However, genetic analyses in recent years it have shown shown that Blastocystis hominis as a unique entity does not exist. In fact, a number of distinct genetic types of Blastocystis can infect humans and other animals. In 2007 it was proposed to drop the name Blastocystis hominis entirely and refer to Blastocystis from humans and animals as Blastocystis sp. subtype nn where nn is a number assigned to each group according to the degree of genetic identity of the Blastocystis organisms, based on small subunit ribosomal RNA sequences, essentially a DNA Barcoding based taxonomy. Today there are at least 17 genetically distinct small subunit ribosomal RNA lineages known. So far, no strict associations between those subtypes and their hosts have been reported, although moderate host specificity is seen.

A team of researchers from Columbia and Denmark have now published a study that shows the distribution of Blastocystis subtypes in different Colombian hosts. Six subtypes were identified by DNA Barcoding. Humans, domesticated mammals and rats shared the same subtypes, while birds and opossums had subtypes, which are usually rare in humans. Noticeably, some subtypes were absent in cattle and in humans, indicating significant phylogeographic differences.

New Whipray

A new species of Whipray from the Himantura genus has been described with the help of DNA Barcoding.

Leopard Whipray (Himantura leoparda)

The researchers took tissue samples from 115 spotted whiprays which were collected throughout the Indio-Pacific region. The group was initially thought to contain 3 species (Himantura leoparda, Himatura uarnak, Himantura undulata). However, after analysis it became clear that there was a previously unidentified species among the DNA samples. These new leopard-skin whiprays — which have been reproductively isolated from the other species for quite some time — have been named Himantura tutul. All four species occupy the same costal habitats but apparently subsist in separate ecological niches.

These studies should help to assess the state of these whipray populations and improve their conservation. Knowing the biological characteristics of each species will for instance help to redefine a minimum size for fishing purposes to avoid the catching of juveniles that belong to the larger species. Determining their geographical distribution and habitats will also make it possible to protect the breeding and nursery habitats of each species. 

Ocellated whiprays can grow over 1.50 meters wide. These large animals start breeding fairly late, at the age of 5 or 10 years, and only in small numbers. Their populations are therefore very vulnerable. Fished for food and especially for their skin that is sold to tanneries in South-East Asia, they are threatened almost throughout the tropical Indo-West Pacific. Their overfishing will in time jeopardise a whole segment of the economy in Indonesia, which is the largest shark and whipray exploiter with 30% of all catches worldwide. In less than twenty years, the amount fished in the Java Sea has been divided by ten! As high-level predators, whiprays also play an important role in regulating ecosystems. Their extinction will threaten the functioning of coastal marine environments. Ocellated whiprays have one or two venom glands at the base of their tail to protect them against their natural predators, namely sharks and killer whales. Their sting is painful and potentially infectious, with serious consequences if not treated correctly.

According to the International Union for Conservation of Nature's Red List over 36% of the 650 known ray species known are currently at least endangered. This also includes the leopard whipray (Himantura leoparda) , which is currently classified as ‘vulnerable’. There is quite some confusion within the genus Himantura as they are really difficult to tell apart and a couple of new species have been described in the last years. This could mean that the current status might not reflect the true situation in this group.