Nematodes and genetically-modified crops

Once in a while one comes across applications of DNA Barcoding you would never thought of in the first place although they perfectly make sense. It is nice to see that so many creative researchers are picking up the method and using it for questions they are interested in.

This post looks at the potential of DNA Barcoding for ecological risk assessment of genetically-modified crops. Dutch researchers from the National Institute for Public Health and the Environment were interested in crop induced changes in nematode assemblages in agricultural soils. Nematodes constitute a very diverse group within the soil fauna, feeding on microbes, invertebrates (including other nematodes) and plant roots. Such an intimate feeding relationship with all major players in agro-ecosystems implies that any effect genetically modified crop might have will likely be reflected in the nematode community. Better knowledge of nematode assemblage compositions could be used to extrapolate impacts on soil systems.

Nematodes in a sample

The classical methods to characterize nematode communities is based on morphological identification using light microscopy. However, this method is both very time consuming (on average several hours for 150 individuals per sample) and the resolution depends on the level of taxonomic expertise. For me they are all white to translucent small inconspicuous worms.

The overall goal of the study was to implement DNA Barcoding into a long-term monitoring network for soil impact of pest management products and the use of genetically-modified crops. Three parts are supposed to serve as baseline for future application. A first part represents the control and looks at seasonal fluctuations of nematode communities as a measure for natural variability over time. The second part focuses on the effects of current pest management (bio-fumigation using Brassica juncea) on nematodes, while the last part investigates the effects of genetically-modified crops (pathogen-resistant potatoes and amylase-potatoes) on soil nematode community structure.

The DNA-based approach to identification allowed for the analysis of nematode communities without microscopic pre-selection because it was based on a considerably broad (2,400 taxa) genetic database that covers the majority of terrestrial and freshwater nematode taxa. For the Dutch researchers one of the main advantages of using molecular methods is the time saving aspect as it allows for more intense and frequent sampling schedules.

A whitefly puzzle solved

Bemisia tabaci or maybe not

The silverleaf whitefly (Bemisia tabaci, which is also referred to as Silverleaf whitefly strain B) is one of several whiteflies that are currently important agricultural pests.Their nymphs use parts of their mouth to stab into the plant and consume the plant’s juices. The honeydew they leave behind can induce the growth of sooty molds, which can then reduce the plants ability to absorb light. This results in less growth, lower yield, and poor quality plants.It is thought that the United States alone has suffered crop and ornamental plant damages in excess of $1 billion through this pest.

For 100 years, outbreaks were sporadic and relatively small, but this changed in the mid-1980s with widespread outbreaks occurring across the south western USA. This was odd because the whitefly was well known across the region as a minor pest yet here it was destroying large amounts of crops.  Although there were no morphological differences, molecular and biological data indicated that the outbreak pest was a different species. Using this information, it was proposed that rather than one species, Bemisia tabaci was composed of at least 28 different morphologically indistinguishable species, all separated by at least 3.5% divergence in their DNA Barcode. To date 34 species have been delimited using the same metrics.

These findings are supported by mating compatibility studies. These have shown that crosses between individuals identified as different cryptic species are reproductively isolated to the point that in most cases copulation does not occur and where it does, the resulting progeny are either sterile or reproductively inferior to their parents.

There was only one question left: Who is the the real Bemisia tabaci originally described in 1889?

A group of researchers has now solved this mystery. They were able to sequence a 496 bp DNA Barcode fragment belonging to a single whitefly taken from the original 1889 collection by Panayiotis Gennadius who first described the species. He had travelled to Greece to identify a small fly-like pest that was devastating tobacco crops there. It is clear now that it wasn't this species that invaded the US but rather a species that was provisionally called "Mediterranean". This species has now begun its own global journey of invasion spreading from its Mediterranean home range to at least 10 different countries in Europe.

Bigfoot does not exist!

It is a myth as much as the yeti, the unicorn, mermaids, giant octopus that attack ships and drag them to the bottom of the ocean, the Bermuda triangle, aliens at area 51, little green aliens on Mars, Santa Claus (sorry kids), the Easter bunny and so on. I could probably fill the entire blog post with mystical creatures and suspicious encounters of similar credibility. 

Why would I do that? Because some really bad science is making the headlines again.

A dubious scientist claims to have sequenced Bigfoot DNA. Dr. Melba Ketchum from Nacogdoches (her company) claims:

“Our study has sequenced 20 whole mitochondrial genomes and utilized next generation sequencing to obtain 3 whole nuclear genomes from purported Sasquatch samples. The genome sequencing shows that Sasquatch mtDNA is identical to modern Homo sapiens, but Sasquatch nuDNA is a novel, unknown hominin related to Homo sapiens and other primate species. Our data indicate that the North American Sasquatch is a hybrid species, the result of males of an unknown hominin species crossing with female Homo sapiens."

First and foremost, these results are nowhere published and Ketchum is not sharing them not even more details on the methods used. No reputable scientist makes a press release before a paper has been published. If true any big journal would take the story and insist on doing the PR using their own machinery. Obviously this didn't happen which makes these 'results' not more reliable than any of the shaky videos, blurry images or dubious witness accounts that are called evidence for the existence of Bigfoot.

Good science is shared with the scientific community to allow colleagues a critical view at results and the conclusions. Currently we are left with a pretty much meaningless press release. 

Dr. Ketchum, if you want anyone to believe your findings release them! Until that happens I and hopefully anybody else with some common sense sees Bigfoot as just another hoax. Unfortunately, these 'news' are currently all over the place and many outlets take the outrageous claim for granted. Bye bye critical thinking. Dear press - you shouldn't buy everything just because it says DNA on the label. The fact that these researchers claim they used DNA analyses doesn't mean a thing. The new hominid nuclear DNA might as well be chimeric and the human mtDNA from a researcher that sneezed on the samples. Most importantly, where did the samples come from? I hardly dare ask, but is there a voucher specimen?

That leaves us with two unproven claims. (1) There is such a being like Bigfoot, and (2) the hybrid between an unknown hominid and a human was actually fertile and evolved into a species that survived the last 15,000 years in North America.

I don't buy it!

PeerJ

When I recently did my little number crunching exercise I was also looking at the percentage of DNA Barcoding papers that have been published in open access journals. Among the major ones was of course PLoSONE but there were also a few others that have papers available without subscription fees. However, it only amounts to a total of 12% of the publications that are available to everyone. In turn that means that about 88% of all DNA Barcoding papers are not available to every researcher on the planet. I find that very frustrating as I thought that especially in this new field there is more inclusiveness!

In 2006 Marguerite Holloway published an article in Conservation in Practice in which she tried to capture the innovative thinking behind the at that time very new method called DNA Barcoding. The title was "Democratizing Taxonomy" referring to Dan Janzen and Winnie Hallwachs who believe passionately that greater access to knowledge and technology will transform the future of conservation. I've heard this term from Dan in various of his talks and I am always reminded of it when it comes to publishing as researchers do not live in a free publishing world. This is especially true for all of us in early phases of our careers where an insufficient measure (impact factor) is dictating where to publish. The result is that many researchers, maybe even the majority, have no access to a lot of what we've published over the last 10 years just because they or their institution can't afford the often ridiculously high subscription fees. That's a shame!

The more happy I am to announce the birth of a new journal with a new business model of open access publishing. PeerJ is a new multidisciplinary open access journal that announced today the first formal call for papers. Unique among academic publishers, PeerJ provides authors with low cost lifetime memberships giving them the rights to publish their papers freely thereafter. A basic membership plan is only $99 that would give an author the right to publish once a year for the rest of their life. That seems reasonable and compared to some of the usual fees of other open access journals it is very cheap. Access to the articles is of course free!

Agarwood barcoding

Aquilaria crassna

Aquilaria crassna is a tree species that has been of great ethnobotanical importance to people throughout the Greater Mekong region and beyond. Its heartwood and resin are highly valued commodities that have been transported along long-established trade routes for thousands of years. However, this species has now become Critically Endangered, owing to over-exploitation.

The wood from Aquilaria crassna (Agarwood) contains aromatic resin, known as ‘gaharu’. This is produced by the tree in response to injury if the production of callus tissue is inhibited. It acts as a chemical barrier to attack by insects and fungi. However, under natural conditions gaharu is not produced by trees at all. This resin is often used in temples as perfume wood. The wood can also be distilled to yield a valuable essential oil, which is widely used in Chinese and Southern Asian medicine and also in the Middle East for making perfumes and cosmetics. 

Currently the majority of the wood comes from wild populations and there is now a very real danger this species may become extinct if wild harvesting continues at the current rate. To conserve this species, it is vital that this plant becomes more widely grown in cultivation, to reduce the pressure on the few wild populations that remain. Cultivation requires high quality seed and especially in Vietnam there is concern that a range of hybrid species or other members of the genus could lead to production loss and disappearance of the original species. Proper species identification starts with the seeds used and Vietnamese researchers have now tested if DNA Barcoding could help. Not surprisingly it worked quite well and one of the official plant markers, rbcL along with ITS was tested most effective.

Probably it needs to reemphasized that DNA Barcoding cannot identify hybrids (oh well, I probably should say mostly, but that's a different story...) but it can help to distinguish between seeds of different Aquilaria species obtained in the wild and destined to be used in cultivation.

Helpful weevils

Eurasian milfoil (Myriophyllum spicatum)

Eurasian milfoil (Myriophyllum spicatum), a submerged aquatic plant that grows in still or slow-moving water, is one of the worst aquatic plant pests in North America. Like native aquatic milfoils, it has feather-like underwater leaves and emergent flower spikes. Usually leaf shape and size can be used to distinguish it from other milfoil species. However, Eurasian milfoil is a variable species, often making it difficult to identify without chemical or DNA analysis. It has been known to crowd out native plants and create dense mats that interfere with recreational activity. Eurasian milfoil can grow from broken off stems which increases the rate in which the plant can spread and grow. 

One of the many lakes that are heavily invested with this invasive plant is Christina Lake in British Columbia, Canada. In the lake milfoil is currently controlled by having dive teams who manually extract the plant from June until October. Those teams work seven days a week with one overlapping day just to keep the nasty aquatic weed at bay. 

Milfoil weevil (Euhrychiopsis lecontei)

Obviously it would be much easier and cost-effective to develop biological pest control strategies and there are a few species that have been discussed and used. The water veneer moth (Acentria ephemerella), feeds upon and damages this water milfoil. It has been used as biocontrol but carefully, as it lacks host specificity and attacks other plant species, including natives. In addition it is also an introduced species. Another method for biocontrol is grass carp, (Ctenopharyngodon idella), is sometimes released into affected areas, since these fish primarily feed on aquatic plants and have proven effective at controlling the spread. However, the carp prefers native species before turning against Eurasian milfoil and more imporantly it has become a serious pest in North American lakes as well. That leaves us with the milfoil weevil (Euhrychiopsis lecontei), a native species that specifically loves to eat the new growth on the pest thereby stunting the plant’s growth and propagation.  that a weevil that lives in the region is actually this native species, making it possible to rear and release more of the bug. In the case of Christina Lake it needed DNA tests to confirm that the right kind of weevil naturally occurs in the lake. This makes it possible to rear and release more of the bug as it will be easier to get permission to introduce more of them to help control the ever growing milfoil. Regional authorities now hope that a pilot project can be started as early as coming summer. Eventually they would like to see a business created that will raise weevils to release not only into Christina Lake, but other lakes around the province to naturally control the alien species of Eurasian Milfoil.

Barcoding types

When describing a new species, taxonomists provide us with a species name and designate a type specimen to which this name will be permanently attached. In other words, a type is an example that serves to anchor the defining features of a particular taxon. 

DNA Barcodes can be used both to attribute species names to a given specimen and to flag genetic clusters for which no name is available. In an ideal situation this can be achieved by sequencing type specimens. However, the majority of the type specimens that are in our Natural History Museums is fairly old and not necessarily stored under DNA-friendly conditions. Many are conserved in formalin which destroys DNA over time. Others, like many insects, are pinned and boxed. From time to time researchers were able to extract DNA from such specimens but mostly they focused on considerably younger specimens (collected and described more recently) or addressed a very small number of samples as it is a more  laborious process.

Eois isographata (credit discoverlife.org)

In a new study researchers from Austria and Germany report successful sequencing of DNA Barcodes from 96 historical specimens (92 type specimens +4 non-types) of the moth genus Eois (in the 79 to 157 year age range). They used six primer combinations (recovering between 109 and 130 bp each) to assemble a full-length barcode sequence for each specimen. And indeed they were able to generate sequences with an average length of ~500 bp. Furthermore, they developed a non-destructive DNA extraction which means that the often very valuable type is not destroyed in the sense that no additional damage compared to conventional preparation of genitalia slides is inflicted on the specimen. Genitalia slides are usually necessary for morphology-based identification and description. This is still possible after the extraction procedure.

What strikes me is the fact that they were still able to retrieve enough DNA to apply the primer sets they had designed to come up with DNA Barcodes of reasonable length albeit they had to admit that the work is considerably more expensive than 'regular' DNA Barcoding. Nevertheless, a big step forward to anchor another defining feature to some types. Only this time it is one far more objective than many morphological characters out there.

Oak killer

The goldspotted oak borer, Agrilus auroguttatus, is a woodboring beetle best known for destroying stands of oak trees in San Diego County, California, in the United States. This pest is native to southeastern Arizona, although a related species occurs in southern Mexico and northern Guatemala. Little is known about the life history of the insect, whether it is introduced or moving into the area as part of a natural range expansion, nor do we know anything about its current distribution. What is even more puzzling is the fact that it has never been known to cause the kind of oak destruction as it was observed in California. As of 2010, Agrilus auroguttatus has killed an estimated 21,500 trees covering 1,893 square miles in San Diego County but some estimates put the numbers at 80,000 trees.

So far the problem was restricted to San Diegio County but now the species was found 65 km north in Riverside County which is very alarming for local authorities. Larvae extracted from under the bark of a recently-killed California black oak were subjected to DNA analysis at the University of California Riverside and confirmed to be Agrilus auroguttatus. 

For a while DNA Barcoding has been suggested as preventive tool by researchers from e.g. New Zealand and Canada. Earlier this year researchers from California have tested the potential of DNA Barcoding for biological control of this particular pest.

As for the one discovered in Riverside County it is believed to have made the jump from San Diego through the movement of infested firewood. It is my hope that this was an isolated find and the early discovery helps to stop it from spreading further North. Its close relatives, the emerald ash (Agrilus planipennis) and bronze birch borer (Agrilus anxius) have caused more than enough havoc among North American trees. This species now poses a major threat to susceptible oak species throughout California, southern Oregon, and beyond. Currently there are no effective tools for protecting trees once infestation occurs.

Midden barcoding

For most of us midden is an expression for an old dump for domestic waste. Archaeologists use it to describe any kind of feature containing waste products relating to everyday human life. 

In the animal kingdom, ground burrows are also known as middens although they are primarily used for food storage. A midden may also be a regularly used toilet area or dunghill, created by many mammals, such as the hyrax, and also serving as a territorial marker..

In the case of some 30 000 year old American and Australian middens, the animals urinated and defecated on their nests during the course of habitation, and organic material such as plant and animal tissue, bone, hair and eggshell gathered from the local surroundings, became cemented together into a hard, impermeable mixture.

With the advent of affordable and accessible high-throughput sequencing and methods to extract ancient DNA it is now possible to genetically screen a wide range of complex modern and ancient substrates, with an unprecedented depth of sequence coverage. Recently studies have attempted the isolation of DNA from samples – including fossil rodent middens – collected in cool and dry environments but so far not from hot and dry regions. Cool and frozen environments were considered more suitable to long-term DNA preservation. However, an international team of researchers has now managed to isolate DNA from 30 000 year old faeces matter to ascertain which plants and animals existed at that time in a couple of hot and dry regions in Australia and South Africa. 

Using several genetic markers and next generation sequencing they discovered a range of plant and animals, some of which are now locally extinct, such as the common brushtail possum found in one midden in the Cavenagh Range in Western Australia. The group found flora and fauna that had previously gone undetected in different types of studies, such as a range of previously indistinguishable dry zone grasses.

Eco-Immersion

Imagine a course where students learn about a country’s delicate ecosystem by exploring the various geographical zones, their inhabitants and the challenges they face. The key to creating an authentic experience rather than providing typical student “destination” travel is meeting with local guides, many of whom are naturalists possessing an intimate knowledge of the ecology and culture of a country or region. Students get acquainted with these so called visionary collaborators, providing them with an opportunity to be inspired by their work. In Costa Rica student groups have the chance to work with the renowned ecologist Dan Janzen and his team, whose conservation field the Area de Conservacion de Guanacaste (ACG) includes more than 325,000 species. Ultimate goal is to generate a DNA Barcode for each one.

The two former teachers Crawford Hill and Eddie Rodriguez are running Chill Expeditions a company that offers teenage students an adrenal rush on their trips, such as whitewater rafting, climbing active volcano peaks and zip-lining hundreds of feet above a rain forest to take in a “monkey’s-eye” view of the jungle canopy. But they are quick to point out the emphasis is just as much an in-your-face cerebral experience as described above. They call this unique experience eco-immersion. To further deepen their interaction with the local communities, the students take part in service projects when available. Hill said, for instance, that about two dozen student groups from all over America recently participated in building a school in Costa Rica over a period of a year. Students have also helped local indigenous people to build and install clean-burning stoves, which use less wood and produce less smoke, replacing those that have caused respiratory illness in their villages. Other projects have included planting organic gardens, laying sidewalks and using recycled materials to build walls.

A great hands-on experience on how ecology impacts a country and its people and a wonderful way to introduce teenagers to an entirely different world.

Lonesome George’s kin

Lonesome George (Source: BBC)

In June 2012 the presumably last individual of the Pinta Island Giant Tortoise species (Chelonoidis abingdonii) died at about 100 years of age at the Charles Darwin Research Station on the Galapagos. Its name was Lonesome George, and over the years it had become famous as the rarest creature in the world. George served as a potent symbol for conservation efforts in the Galápagos and internationally.

Over the years all attempts at mating Lonesome George with other closely related species had been unsuccessful. Therefore, everybody mourned the extinction of this tortoise subspecies in this summer.

And today the potentially good news: On the remote northern tip of Isabella Island, a group of researchers of Yale University collected DNA from more than 1,600 tortoises and discovered by comparison with DNA from museum specimens that 17 of them were ancestors of Chelonoidis abingdoni, native to Pinta Island of which Lonesome George was the last known survivor. The 17 tortoises are hybrids, but evidence suggested a few might be the offspring of a purebred Chelonoidis abingdoni parent. Five of these tortoises are juveniles, which suggested to researchers that purebred individuals may still live on the rocky cliffs of Isabella in an area called Volcano Wolf.

The researchers and the Galapagos Conservancy now hope to collect the hybrids and any surviving purebred members and begin a captive breeding program that would restore this species.

The publication is unfortunately not accessible yet but I will update the link when Elsevier decides to release it. Did I mention that I think that all press releases should always be accompanied by full open access to the publication for everyone? Bloggers like me love these kind of news but we also want to read more than the digested version that goes to the press. Especially after the ENCODE disaster.

Numbers Part II

Here it is - episode two of me playing with numbers of publications on DNA Barcoding. If you recall I was looking at over 2000 publications of the last 10 years to provide some insight into the progress the discipline was making within such a short time. I was very happy to see that Mark Stoeckle has added to this look at the big picture by sharing some DNA Barcode stats from his treasure box. Next time you have to prepare a talk on DNA Barcoding and you need some convincing figures on progress and development over the last years feel free to 'steal' my graphs and I am sure Mark would be happy to see his material used as well. Just don't forget to tell everybody where you got it from :-)

When looking at the publication data I was also very interested on how DNA Barcoding has been incorporated into the more applied areas of science. Therefore, I grabbed 208 that were clearly representing studies of applied DNA Barcoding, and categorized them into four different groups I considered most interesting. The largest number of publications I was able to find in the field of pest research and management. The majority of those were focusing on crop damaging animals and ways to identify them. A lot of papers were also published on health related topics mostly the identification of disease vectors. Both conservation and forensics are dominated by studies that assess the value of DNA Barcoding for regulatory purposes but you can even find research on potential applications in criminal forensics. These numbers show that DNA Barcoding is not just an effort of a few 'stamp collectors' travelling around the globe in order to complete their little collection books but rather a still young discipline that has already been translated into applications that are directly beneficial to society.

My only other graphic today demonstrates how diverse the field has become as I was looking at the number of journals that actually had published DNA Barcoding papers and if there were actually some preferences among authors or even publishers. In total 157 different journals have published at least one DNA Barcoding paper. Most did more than that. Therefore, the first take home message is that DNA Barcoding is widely accepted and appeals to a variety of topic journals. Two journals are leading the pack and that's not a surprise as both have been trying to become home for DNA Barcoding research publications. Molecular Ecology Resources has a strong focus and PLoS ONE with its inclusive concept is catching up quickly.  The third place is another very good indicator how DNA Barcoding has impacted taxonomic research. Zootaxa is the world leading journal on taxonomy and main home for species descriptions and revisions. If they would just starting to use DOI's but that's another story for another post...

The good news is that researchers can actually choose where to publish depending on their needs and perhaps even strategically. DNA Barcoding is certainly not an exotic field anymore. This is also good news for anyone like me who prefers options that are open access even if it comes with the price of a lower impact factor.

Tea protection

The tea mosquito bug (Helopeltis theivora) is one of the most serious pests in tea cultivations in India. The loss of crop can be substantial (10-50%). Before the use of chemical pesticides the loss on tea plantations sometimes reached 100% and in commercially grown cashew, losses sometimes reached 30-40%. This translates in a huge loss of revenue in many parts of the country. India is after China the second largest tea producer of the planet. Furthermore, India is the world's largest tea-drinking nation, although the per capita consumption of tea remains a modest 750 grams per person every year. 

Species of the genus Helopeltis are very similar in coloration and morphology, and not all of them are actually equally dangerous to plantations. The only reliable identification based on morphology is the inspection of male and female genital structures but for integrated pest management on a large scale this is not a suitable option. 

That's were DNA Barcoding came in handy as a group of Indian researchers recently barcoded the most common pest species of the genus and furthermore developed some COI primers that amplify species specific regions. If the sequencing of the barcode region is too costly such methods are very good alternatives as a simple PCR run with a subsequent gel check will do it. Species specific markers such as the ones in this study are usually of different length and will migrate differently. This is depicted in the figure on the right for two Helopeltis species.

For someone who has his cup of tea every morning this is very good news.

Protist barcoding

Protists are a diverse and loose grouping of disparate eukaryotic microorganisms. Thy do not have much in common besides a relatively simple organization - either they are unicellular, or they are multicellular without specialized tissues. This simple cellular organization distinguishes the protists from other eukaryotes. They diverged after Archaea and Bacteria evolved but before plants, animals, or fungi appeared on Earth.

Given their mostly single-celled nature, discovering and describing new species has been difficult, and many lineages contain a relatively small number of formally described species, despite their critical importance as pathogens, environmental quality indicators, and markers of past environmental changes. 

It would seem natural to use DNA barcoding in order to shed more light on the taxonomy of protists especially because of  the lack of diagnostic morphological features, but this has been hampered by the extreme diversity within the group. So far nobody was able to find a single set of molecular markers that works for all lineages as the genetic divergence observed between and within major protistan groups greatly exceeds that found in plants, fungi, and animals.

Proposed workflow by ProWG

But there is hope. The Protist Working Group (ProWG), initiated by the Consortium for the Barcode of Life (CBOL ) has assessed the efforts to identify the barcode regions across all protist lineages and now introduced a two-step barcoding approach to assess protistan biodiversity. A suggested workflow (see figure on the right) follows already well established best practice and adds a standardized barcoding step. They propose the V4 region of 18S rDNA as the universal eukaryotic pre-barcode. In a second step group-specific barcodes will then have to be defined separately for each major group of protists. Much of this work is still to be done, however, having a standard to start with is a very big step forward (politically probably the biggest). 

Field book - there is an app for that

For many years there was only one way to keep track of field data during expeditions and collecting trips. Explorer and scientists alike used field books and I remember well my second year at university when a professor tried to explain the importance of proper field data written in special field books to us students. Not that we really listened but when it came to the first own collecting trips some of us remembered that we had been told what to keep track of and how to do it. Everything was of course not digital. A notebook and a pencil would do it. Not that there weren't any computers and even laptops available but even today I find a laptop in the field rather bulky. As a result I still take notes in a little notebook and transfer those into an electronic spreadsheet at night. 

But we have the age of mobile phones, tablets, and other gadgets. So what would be more obvious than using those tools to collect all data.

The DNA Barcoding Assistant is a mobile technician application that provides an intuitive interface to acquire specimen metadata that contain provisional user-assigned taxonomic identifications, digital images, geospatial data, and collection event details for specimens obtained in the field. In other words the basic data required for proper DNA Barcode library assembly.  The specimen records created with the DNA Barcoding Assistant can be stored and converted into an Excel spreadsheet which can be send out via the standard email app. Imported text and linked image data can then be authenticated, edited, and uploaded to the Barcode of Life Data Systems (BOLD) using its standard processes.

This tool is already available for a while but only for the iPhone and I have to admit that I am not a member of the fellowship of the Apple. However, I currently have the privilege to test the beta version of the Android version on my tablet, and it is a really neat little tool. I love to be able to include an image into every record. It used to be a problem to keep track of image file name and associated specimen as it added another layer of complexity to the field data collection. Now I use the phone or the tablet to make images and link them to my data on the spot. Similarly geospatial information is retrieved through the build-in GPS of most gadgets. This is a huge time saver and it minimizes the chance of error creeping in while copying data from field book to spreadsheet to database.

It is my hope that this tool and similar ones are used more often and tested in the field by researchers and students. I know that for the hardcore collector there can't be enough data fields but only with the help of a user community these tools can get better adapted to the needs of every single one. I can only encourage iPhone owner to get it from from the app store. Android users - stay tuned - shouldn't take much longer.

 

Numbers Part I

For the last few years I have been collecting citations of all papers related to DNA Barcoding. Main reason was initially to make my life easier when it came to manuscript writing. Modern citation management software is really a big help and when I started my work on this blog it became even more helpful. The last I want to claim is that I have them all but I think I have most of them and I thought I start sharing some numbers that I was able to extract from my dataset as they show an impressive picture of a still rather young field in biology.

The most impressive number first: Since the publication of the famous Hebert et al. paper in 2003, 2014 publications somehow involving DNA barcoding have been produced. The first figure shows the steady increase over the last nine years. The small decline in 2012 is likely due to the fact that the year isn't over yet.

 

Within two years the barcoding movement had started global campaigns to start building libraries of groups that were considered feasible or of relevance for society. Those were either chosen based on taxonomy (e.g. Lepidoptera, Fish, Birds) or habitat (e.g. marine or polar). Here are some numbers of publications with taxonomic focus which are perhaps more a measure of impact in the particular community. The high number of plant related publications is surprising as the majority of papers (87%) has been published after 2008. Around the same time the community of marine researchers formed a global campaign and as its former leader I am proud to report that 410 barcoding publications on marine life have seen the light of the day. Studies focusing on the Arctic and Antarctic regions amassed 107 papers over the years.

I'd like to show one more example in today's post and a few more in future ones. Over the years DNA Barcoding has also been heavily criticized (actually only in 28 papers). One of the criticism was that it would not help to improve current taxonomic knowledge. I picked the lepidopera dataset and had a closer look. The result is that 31% of all papers on butterflies and moths are either discoveries of cryptic species, new descriptions or taxonomic revisions. If that doesn't qualify for some impact of DNA Barcoding on taxonomy I don't know what does.

I know of course that simple counting of numbers of publications in a particular field of science is not 100% accurate but I believe it is enough to show some trends and for sure it demonstrates how rapidly the discipline grew within the last 9 years. Who would have predicted that?

Feed them better!

School canteens around the world don't have the best reputation although there are certainly exceptions to this. They are often considered places that provide our kids with unhealthy junk food. I still remember Jaime Oliver's almost heroic fight for more healthy food in Britain's schools with his project "Feed Me Better".F act is that many are indeed serving low quality food because it is cheaper and easier to prepare in large quantities. Or as in many North American Universities you find a lot of the standard fast food franchises instead of a real kitchen on site.

Take a pick! Halibut or flounder?*

I have been posting about market substitution in Natural Health Products and food before and, especially for fish DNA Barcoding has been used widely to unravel some very bad habits in the business. But now a group of Italian researchers report a case of substitution at a school canteen in Tuscany. The children had been served fish that was supposedly halibut (Reinhardtius hippoglossoides) but turned out to be a much cheaper flounder of the genus Paralichthys. Fillets were reported by the cooks to have an unusual reaction to cooking and researchers of the University of Pisa used mitochondrial DNA markers to show that the suspicion was indeed correct.

It is already bad enough that fish mislabeling was found to be rampant in the marketplace but it gets worse if our children are dragged into this right at their schools. It is reassuring to see that some cooks like the ones in that Tuscan school are doing a proper and caring job.

However, I have to admit that I am glad that we prepare the lunch for our kids our self at home.

 

* The filet on top is halibut and the bottom one is from a flounder 

h/t Andy Bentley 

Mite capital of the world

Maybe you have heard of Churchill, Manitoba. If so, I assume you know it as the world's capital of the mighty polar bear. Far from it! I believe we have to change this. A new study shows that this small town at the tundra-taiga transition zone is rather the capital of mites.

Mites are among the most diverse and successful of all invertebrate groups. They have exploited a broad array of habitats, and because of their small size (most are minute) go largely unnoticed. Many live freely in the soil or water, but there are also a large number of species that live as parasites on plants, animals, and some that feed on mold. There are plenty of them living on the human body. A single hair follicle for example can support a family of 25 mites. Some 45000 species have been described but estimates go as high as perhaps 1 Million species.

Despite their diversity and abundance, mites are rarely included in biodiversity assessments because of serious taxonomic barriers. The status of many species is uncertain and immature life stages were mostly excluded from surveys as they lack diagnostic morphological characters. Aside from these challenges, there is a scarcity of taxonomic experts. 

Back to Churchill. DNA Barcodes for over 6000 specimens revealed nearly 900 presumptive species of mites with high species turnover between substrates and between forested and non-forested sites. However, species accumulation curves estimated to get a sense for the coverage of the sampling indicate that there might be as many as 1200 species living at this remote place thereby rivalling the most diverse of temperate habitats.

I've been up there twice and have watched the polar bears from a safe distance, collected marine invertebrates, fishes, and even insects. Little did I know about this incredible diversity underneath my feet. Next time I look closer!

It's Movember

Have you ever heard of Movember?

No? Then it is about time that you do!

During November each year, Movember is responsible for the growth of moustaches on thousands of men’s faces around the world - no matter how strange their appearance might become. With their “Mo’s”, these men intend to raise funds and awareness for men's health, specifically prostate cancer and male mental health initiatives both often ignored issues.

This year I joined our departmental team called Biostashes because I think it is a great (and very easy) way to spread the word and do fundraising for something very important. Well, it's early in the month, the moustache is growing slowly but steadily with lots of family support at home. I'll report on progress here (maybe with documenting images). Stay tuned.

Raising awareness - one hair at a time!

Barcode Bulletin Reader's Digest

For a few years I am now collecting all citations for DNA Barcoding related publications. With the work on this blog the need to have a database with this information grew and I started to categorize and to keep track of progress. 

DNA Barcoding has long matured. This is supported by the fact that every single month about 40-60 new peer-reviewed scientific studies utilizing DNA-based identification, mostly DNA Barcoding, are published. For me that was reason enough to create The Barcode Bulletin Reader’s Digest which is a monthly electronic newsletter intended to provide everyone interested with up to date information on new publications in the field sorted by the main topics of interest. The first issue went out today. Have a click if you are interested. All citations in this pdf are hyperlinked. A simple mouse click will bring you to the article page on the publishers website.

The world’s rarest whale

Mesoplodon traversii (New Zeland Dept. of Conservation)

The spade-toothed whale (Mesoplodon traversii) represented a big mystery for 140 years. This species has never been spotted in the wild and the only previously known specimens of this whale were a single mandible with teeth from an adult male (the holotype), collected from the Chatham Islands, New Zealand in 1872, and two skulls without mandibles, one from White Island, collected in the 1950s and one from Robinson Crusoe Island, Chile, collected in 1986. 

This changed in December 2010 when two beaked whales, a mother and calf, stranded and died on Opape Beach on the North Island of New Zealand. However, it took 2 years to eventually find out the truth. These two stranded whales were in fact Mesoplodon traversii.

From their initial description the whales were identified as Gray’s beaked whales (Mesoplodon grayi), the most commonly stranding beaked whale around New Zealand. However, to confirm their identity, researchers from New Zealand amplified and sequenced two mitochondrial DNA regions. Sequences were compared to other species of the genus, among those Gray's beaked whale and DNA fragments retrieved from the reference specimens from Mesoplodon traversii. The latter matched the sequences of the two stranded animals and given that these are the only two complete specimens of the species ever spotted we can truly assume that this is the world's rarest whale.

Kleptomaniac slugs

Sacoglossan sea slugs have the unique ability to incorporate functional chloroplasts over weeks or months during starvation and thus are able to survive by maintaining photosynthetic activity. One of those so-called long-term retention forms is Plakobranchus ocellatus a common inhabitant of shallow coral reef pools and lagoons. It is seldom seen as it is well-camouflaged, half buried in the coral sand. It shows an exceptional ability to starve over months with a relatively slow loss of photosynthetic activity. For this reason, it is of high interest for our understanding of the mechanism of functional long-term photosynthesis in Sacoglossa. It is known that plastids from algae are sequestered by the sea slugs. The alga is eaten normally and partially digested, leaving the plastid intact. The plastids are maintained within the host, temporarily retaining functional photosynthesis for use by the predator. This phenomenon is called kleptoplasty.

Little is known regarding the taxonomy and food sources of Plakobranchus ocellatus, but it was suggested that it is a species complex and feeds on a broad variety of green algae of the family Ulvophyceae.

A group of researchers from Germany now identified the species of algal food sources depending on starvation time and light treatment by means of DNA-barcoding. They used for the first time the combination of two algal chloroplast markers, rbcL and tufA. Furthermore, they used COI and 16S sequences of specimens from various localities to show that Plakobranchus is indeed a species complex with likely four distinct clades. Food analyses do not indicate an ecological separation of the clades into differing foraging strategies. The combined results from both algal markers suggest that, in general, Plakobranchus ocellatus has a broad food spectrum. 

Who ate all the twigs?

Moose browsing

Browser we call herbivores which eat primarily leaves, shoots, twigs of trees, bushes, forbs, and other vegetation which is up off the ground. The impact of ungulate herbivores on tree regeneration and its possible consequences for long-term forest dynamics has raised concerns worldwide. Browsing can have a strong effect on ecological processes by affecting plant community structure and composition, with cascading effects on animal communities.

However, without direct observations of foraging, species-specific foraging behaviour is difficult to quantify. As a result we know relatively little especially about species-specific patterns in systems with several browsers that are perhaps competing for resources.

A group of Swedish researchers have now developed a browser specific species diagnostic kit that utilizes environmental DNA (eDNA). They took advantage of the fact that during browsing, a small amount of saliva containing some cells is deposited at the bite site, providing a source for DNA that can be used for species identification. They were able to distinguish between four sympatric ungulates (moose, Alces alces; roe deer, Capreolus capreolus; fallow deer, Cervus dama; and red deer, Cervus elaphus) that occur in deciduous forests in central Sweden. I would call that DNA Barcoding if they hadn't decided to use Cytochrome b primers for their study but that's more a problem of standardization. The group was able to show that false positives were extremely uncommon and that about half of the samples could be amplified even if the browsing happened up to 12 weeks before. Some samples as old as 24 weeks could also be amplified.