Citizen science

Citizen Scientists have helped to answer serious scientific questions, provide vital data to the scientific community, and have discovered thousands of species. I already wrote how high school students got engaged in citizen science projects that used DNA Barcoding, and there have been several bioblitzes that had a barcoding component.

The U.S. National Science Foundation has awarded $250,000 to the Mount Desert Island Biological Laboratory, the National Park Service, and the Schoodic Education and Research Center Institute for a new project that will involve visitors to the Acadia National Park in hands-on scientific research.

The project, called "Pathway to BioTrails," will involve members of the public in monitoring animal and plant species in Acadia National Park and Frenchman Bay using DNA Barcoding. Currently many citizen science projects spend more time on species identification and less on the actual scientific and educational goals of the project. The idea is to validate tentative identifications made by citizen scientists using DNA Barcodes and thereby increasing both the scientific and educational value of a project.

BioTrails will offer a range of citizen science projects organized around hiking, cycling and sea-kayaking trails to people who visit Acadia National Park. The trails will serve as consistent observation points where specimens and other data can be collected. Research scientists will use this information to address important ecological research questions, such as the relationship between climate change and changes in biodiversity.

 The two-year initial project will feature four, five-day citizen science events – two in 2013 and two in 2014 – that will help build and use DNA Barcode 'libraries' for a selection of Acadia's invertebrate animal species. Volunteers for each of the citizen science events will be recruited through education and outreach channels already established by the participating institutions. Volunteers will also be recruited online through a website that connects educators and students to researchers through citizen science projects.

The principal investigator on the project, Karen James, hopes that the BioTrails concept, once tested in Acadia National Park, can be expanded to other national parks and long-distance trails. "A network of local, regional and national BioTrails programs, helping citizen scientists contribute to ecological questions of national and international importance," said James, "could aid in monitoring and managing wildlife in a rapidly changing world."

Congratulations to Karen and her team!

Larva Thursday (3)

Quite a few families and species of beetles have larval forms that prefer to eat wood which often puts them at odds with us humans. Unfortunately wood boring beetles are commonly detected only a few years after new construction. The lumber supply may have contained wood infected with beetle eggs or larvae, and since beetle life cycles can last one or more years, several years may pass before the presence of beetles becomes noticeable. This also puts them on top of the list of  potential pest species. Lumber trade is a global business and as a result the beetle larvae become globetrotters. One of the worst examples of these days is the Emerald ash borer native to Asia but accidentally introduced to North America in the 1990s. It was detected as late as 2002 and has killed some 100 million ash trees so far and threatens to kill most of the 7.5 billion ash trees throughout North America.

Emerald Ash Borer
(Agrilus planipennis)

Therefore it is essential that biosecurity authorities have reliable species diagnostic tools available in order to detect incursions of these species early enough. However, taxonomic literature on relevant species is scattered and sparse, and the lack of molecular diagnostic methods means that identification of eggs and larvae has been impossible to date because the immature life stages are morphologically homogeneous. 

The great potential DNA Barcoding has been recognized by a few researchers and confirmed by studies that e.g. recorded other invaders to North America, emphasized the importance for the nut production, or simply investigated its applicability in a regulatory context. In one of the studies the prospects are nicely summarized:

Furthermore, while it is crucial that appropriate taxonomic specialists confirm the identity of specimens used to develop DNA barcodes, once developed such data provide a generic method of identification that easily could be adopted by a molecular diagnostician without the need for specialised taxonomic training. This could free taxonomists from routine identification duties allowing more time to concentrate on much needed taxonomic research.

Ubiquist - really?

Parisotoma notabilis (Credit: J. van Duinen)

Meet Parisotoma notabilis - a tiny fellow that belongs to a group that largely goes unnoticed by most of us. Collembola or Springtails live on our planet already for some 400 Million years and are reputed to be one of the most abundant of all macroscopic animals, with estimates of 100,000 individuals per cubic meter of topsoil, essentially everywhere on Earth where soil and related habitats occur.

Most soil springtails are considered beneficial to agriculture. They have the capacity to carry spores of mycorrhizal fungi and mycorrhiza-helper bacteria on their body and thereby help to establish plant-fungal symbioses. They also contribute to controlling plant fungal diseases through their active consumption of mycelia and spores of pathogenic fungi. It even has been suggested that they could be reared to be used for the control of pathogenic fungi in greenhouses and other indoor cultures. Such plans make accurate species delineation indispensable.

Our little friend, P. notabilis has paradoxically been reported to both tolerate and being negatively impacted by industrial pollution, pesticides, and heavy metal. It was also considered a poor indicator for pH, reacting positively to either low or high pH values. This broad range of ecological and life history parameters raised some doubts about its status as a unique species. This species has been extensively used in numerous studies and was considered well characterized based on its morphology.

Now a new study has a very good explanation for these ambiguous findings. By using DNA Barcoding the researchers were able to postulate that this species likely consists of four species. They could confirm all four lineages by using the nuclear gene 28S and geographical distribution patterns.Their study also included specimens from the type locality which enables them to assign the original species name to one of the lineages. So what has been reported as discordance in response to pollution could just be attributed to the fact that scientists have been looking at several cryptic species.

Big scam

Something funny for a change. I found this on Jonathan Eisen's Tree of Life blog.
Caution - this is meant as satire.

Barcoding at Warpspeed

Imagine an interception of potentially endangered species at a border or pest detection in shipments scheduled for transboundary movement. In both cases a rapid identification of organisms is paramount in order to facilitate border control decisions. DNA Barcoding seems the best option for identification in such situations, but its utility will further increase if results are delivered in hours rather than days. 

The quest for fast procedures to obtain useful sequences for identification is already underway fro a few years. In 2009 researchers from the University of Guelph proposed simple modified protocols that would enable to shorten a process that often takes 2 days to just 2 hours. The only drawback was that they were targeting smaller fragments with a maximum length around 400 bp and they were still using the classical steps of sequencing: DNA extraction followed by PCR and Sanger-Sequencing (with cleanup prior to sequencing). The trick was to reduce the time for each step as much as possible.

Now imagine you are able to skip a step in the process such as the isolation of the DNA which is often the most time-consuming one. Direct amplification is the buzz word for this and the community of forensic scientists are leading the way here. Companies offer rapid DNA services for law enforcement and biometric applications. They utilize commercial off-the-shelf equipment and reagents that already commonly used in forensic laboratories and capitalize on advancements in both rapid thermal cycling and direct amplification. A big step forward as with the DNA isolation out of the way you not only save time but also money on instrumental investments and chemical supplies. The question is if this can be used in a normal laboratory setting. After all most researchers are not working in a well funded crime lab. 

Do you remember the DNA Barcoding story of the Mezcal Worm? Certain mezcals, usually from the state of Oaxaca, are sold con gusano (with worm), a practice that began as a marketing gimmick in the 1940s. The worm is actually the larval form of the moth Hypopta agavis that lives on the agave plant. A group of researchers were able to retrieve DNA Barcodes from the booze (not the larva) and indeed they used direct amplification as most DNA isolation methods would have failed.

And in the not so distant future?

Direct Sequencing will be the future. No DNA isolation, no PCR reaction to provide enough target sequence copies, just sequencing. Science Fiction? Not really - certainly not at a point where we can buy kits for the every day use but for my part I am watching developments closely.

h/t Mark Stoeckle

A mysterious crazy ant

Nylanderia fulva (Credit: Alex Wild)

The Rasberry Crazy Ant is an invasive ant in North America that was first noticed infesting areas around Houston, Texas about ten years ago, but its species identity has remained undetermined until now.

Ants of the 'crazy ant' species complex got their name because of the ants' random, nonlinear movements. This particular species was called "Rasberry" after the exterminator Tom Rasberry who first noticed the ants were a problem in 2002. This species is able to out-compete fire ants because they reproduce faster. They are not attracted to ordinary ant baits, cannot be controlled by over-the-counter pesticides, and are harder to fully exterminate because their colonies have multiple queens. But what's the problem? Well, they are infesting electrical equipment which can cause short circuits when they chew through insulation. Overheating and mechanical failures are other problems caused by high numbers of dead worker ants in electrical devices. They were attracted by a pheromone released by a dying ant which signals potential danger by attackers.

Previous attempts at identifying this species have resulted in widely different conclusions with respect to its native range, source, and biology, let alone its species name. A new study identified the invader as Nylanderia fulva by using morphometric data, molecular sequence data  from six independent loci, one being COI, and more traditional morphological comparisons with type specimens. Previous attempts at identification using DNA Barcoding and morphometric analyses could not exclude closely related species but placed it with a closely related sister species. A good example for the fact that a DNA Barcode is not the all-encompassing miracle tool - nobody ever claimed that - but rather one of many in an integrated framework available to modern biology. Even more so because now with the knowledge of the species identity the search for some single diagnostic characters in the COI sequence can begin.

The researchers were also able to document the utility of the new knowledge. They could show that the species  is distributed more widely than previously thought and has likely invaded all Gulf Coast states. "This study demonstrates the invaluable role that taxonomy, an often underappreciated discipline, plays in our understanding of emerging pests. Now that we know just what species the Rasberry Crazy Ant really is, we can better understand its biology to improve control of this invasive species", says John LaPolla, one of the authors of the study.

Natural Health Products

Not long ago I was posting on how DNA Barcoding can help to ensure the quality of ingredients of traditional Chinese medicine. Two new studies corroborate this once more  and cast some doubt on Natural Health Products in general.

Natural Health Products are often considered safe due to their natural origin, however, adulterated, counterfeit and low quality products pose serious safety threats to consumers. In some countries such as the U.S., unlike drugs, they are not required to be tested for safety or efficacy before they hit the market. Furthermore, testing to make sure the contents match the label are much more lax than it is for pharmaceuticals, opening the opportunity for mislabeling, whether it is accidental or intentional.

One study conducted here at the Biodiversity Institute of Ontario consisted of tests on 95 plant and animal products bought in Toronto and New York City. The sampling included a variety of products such as capsules, tablets, roots, extracts, teas and shredded products. Of the 26 animal DNA Barcodes obtained, 21 correctly matched their commercial label. The remaining five were either cheaper alternatives or fragments of protected species such as some sharks.

Panax ginseng

Half of the plant products labelled as Korean ginseng (Panax ginseng) were in fact American ginseng (Panax quinquefolius) which is cheaper and is sold for different medicinal benefits. More concerning is one case were a product labelled as Echinacea (Echinacea purpurea), closely matched species from the walnut family (Juglandaceae). Bad news for people with nut allergies.

Panax quinquefolius

Study number two comes from New York City and provides a more focused look at a herbal menopause supplement. Black cohosh  (Actaea racemosa) is a consumer favorite especially since hormone replacement therapy was found to put some women at increased risk of cancer and cardiovascular disease. However, controlled trials with the popular remedy showed mixed results. Sometimes it had been effective in the treatment of menopause symptoms whereas other times it was revealed to be ineffective, and a few cases even suggested that it can be toxic. The researchers found that 25% of the 36 dietary supplements tested were in fact members of three Asian Actaea species. By the way the study described above also contained such a case.

Actaea racemosa

This might explain the contradictory results in the clinical trials. Reasons could be variation in the concentration of black cohosh, adulteration that affects the action of the active ingredients, or even harmful compounds.

Two more studies that unfortunately tell us that no matter what and where you buy you cannot always be sure you get what the label on a product says. The good news is that with DNA Barcoding we have an instrument that could put an end to this.

Larva Thursday (2)

Chironomus plumosus male

Welcome to episode 2 of the series Larva Thursday on this blog. Today we will be looking at the non-biting midges - the Chironomidae, a family of nematoceran flies comprising of perhaps some 10000 species. 

Chironomidae are important as indicator organisms. Their presence, absence, or quantities of various species in a body of water can indicate whether pollutants are present. Larval stages of them can be found in almost any aquatic habitat in which they usually form an important fraction of the macro zoobenthos.

polytene chromosomes

However they are notoriously difficult to identify and ecologists often record them only by species groups. Each morphologically distinct group comprises a number of morphologically similar species that can only be further identified by rearing adult males or by cytogenetic analysis of the polytene chromosomes. Morphology-based identification of females is often impossible. Hence, environmental assessments and bio-monitoring of freshwater habitats have much to gain if the larvae and other life history stages could be more readily identified to species. The use of DNA Barcoding seems to be a logical consequence and indeed it has been used in a number of studies. Researchers were able to conduct biodiversity assessments that included females and to describe hitherto undescribed larval stages of some species. New species were discovered and described for Brazil, Norway, and Tibet.

All studies show that a DNA Barcode library for chironomids is very useful for species identification especially the larval stages that serve so well as environmental indicators. As effective and rapid identification is essential for the success of modern freshwater bio-monitoring projects, DNA Barcoding represents a powerful tool to do exactly this.

A message from the past

C. carolinensis by John James Audubon

Once it was the only parrot species native to the eastern United States. It was found from southern New York and Wisconsin to the Gulf of Mexico, and lived in old forests along rivers. But then its habitat was taken away in favor of more agricultural land. The bird's colorful feathers were also in demand as decorations in ladies' hats. Many were killed because farmers considered them a pest. The end was inevitable - the last bird of the species died at the Cincinnati Zoo in 1918.

The Carolina parakeet (Conuropsis carolinensis) went extinct before we knew much about its ecology and behavior, let alone anything about the evolutionary relationships between this and other neotropical parrot species. 

Aratinga solstitialis

94 years later a group of researchers from the New State Museum and the New Mexico State University opened a door into the past. They were able to extract DNA from slivers of skin and connective tissue from the toes of some museum specimens. Two mitochondrial DNA fragments were sequenced, ND2 and COI mainly to gain insight into the evolutionary relationships of this group of parrots. The authors could for example show that the Carolina parakeet is a sister species of the Sun Parakeet (Aratinga solstitialis) and not as previously hypothesized of the monk parakeet, Myiopsitta monachus which seems more like a distant cousin.

The COI sequences retrieved for this study may be useful as references against which controversial remains of putative Carolina Parakeets can be evaluated. Of particular interest to historians are two egg sets in the collection of the Florida Museum of Natural History that were collected several years after the last uncontroversial sightings.

New Malaria-Transmitting Mosquito

Anopheles sp.

Potentially bad news for the fight against malaria in Kenya. A group of Scientists from the London School of Hygiene and Tropical Medicine, have discovered  what looks like a new species of Anopheles. They used DNA Barcodes (COI) and the nuclear marker ITS2. Their analysis indicated that the DNA from mosquitoes that did not resemble ay other specimens differed from sequences available for known malaria-transmitting mosquitoes in Africa.

About half of the mosquitoes collected did not match the morphologic descriptions of any of the more recently identified species nor did their DNA Barcode match any sequences in the public databases. Some of those specimens were also tested positive for Plasmodium falciparum sporozoites. 

The most alarming find is that this putative new species was found to be active outdoors and bite people earlier in the evening soon after sunset while the commonly caught Anopheles mosquitoes that transmit malaria in Africa, generally, prefer to rest indoors and feed on humans at night. This led to the development of programs to stop the spread of malaria such as spraying insecticide in homes and issuing bed nets for people. The outdoor activity of these mosquitoes could lead to the failure of current indoor-based interventions to control this species, and it could therefore contribute to malaria parasite transmission in the area.

These findings highlight the value of an integrated approach, here including behavior and DNA Barcoding. Morphology alone would not have given the scientists this essential piece of information which will hopefully allow the implementation of appropriate, and therefore successful, malaria control interventions.

Lady Gaga and a small wasp

Aleiodes gaga

A little parasitoid wasp goes through the news today. Not because it looks particularly different or has any features that make it somehow special. No - it bears a famous name: Aleiodes gaga. The small braconid wasp from Thailand has been named "in honour of popular singer and performer Stefani Joanne Angelina Germanotta, known professionally as Lady Gaga". Not that there is any resemblance between the two (see images) although I consider it possible that one day Gaga returns the favor and appears in a corresponding costume.

The eponym

As usual the really important scientific message is well hidden behind the headlines. The wasp is only one of 179 newly described species of a single genus in a monograph published in September in Zootaxa. What makes this paper one that stands out is the way the authors approached the description of those species. It is a well known fact that the description of a new species is a time consuming process due to its thoroughness. Some taxonomists are trying to find ways to speed up the course of action without compromising quality. DNA Barcoding was one tool brought into the discussion early on and this new paper showcases how barcoding information for an extremely diverse genus of cosmopolitan wasps can be used to provide a framework, supplemented by minimalist morphology, to describe a large number of new species in a relatively short time. Interestingly, this study perhaps covers just a small proportion of the Thai fauna of Aleiodes. According to the authors there might be as many as 438 species in this genus in Thailand alone. With conventional methods species description would take decades. The turbo-taxonomy presented here might be a very good alternative.

What I find amusing is the fact that this was publicly announced by someone who is rather critical of DNA Barcoding and parts of the article in the Guardian do reflect some of the suspicion and hesitance this newly proposed way of doing taxonomy is met with. Unfortunately, Quentin Wheeler, the author of the article couldn't let the matter rest and closes his contribution with some pointed remarks. I am not going to comment on those. I only hope that one day everybody will be able to find back to objective scientific dialogue.

Barcoding in Schools

Mader, Biology, 11th Edition, 2013

It started in 2008 with a freelance science project of two high school students. Kate Stoeckle and Louisa Strauss checked samples of seafood using DNA Barcoding to see whether the fish New Yorkers buy is what they think they are getting. They found that one-fourth of the fish samples with identifiable DNA were mislabeled and created a big hype in the press - the term "Sushigate" went around the world and found its way in a High School textbook .

This was the start to a couple of projects that involve high school students in DNA Barcoding projects. I think it is a great idea to bring the method into the classrooms and provide students with a hands-on experience in biodiversity science. 

Most high school students are accustomed to learning science in a very different way than it is actually practiced in real-world scientific research settings. As a consequence, students develop an unrealistic perception of science that overlooks many of its most valuable and exciting elements, leaving them disengaged and uninspired.

This excerpt from the website of the Coastal Marine Biolabs in Ventura, California illustrates the reasons for a shift in High School Science education. Their mission is research based science education and DNA Barcoding fits in this concept extremely well as it is hands-on from the field collection all the way to the work in the laboratory. Students can participate in all stages of the process and at the end stands a tangible result -  a barcode as valuable as all the other ones in the databases of the 'real' scientists.The very successful program "Barcoding the Kelp forest" runs already for a few years.

The winners of the Urban Barcode Project

High School students in New York City recently participated in the Urban Barcode Project, a science competition in which student research teams used DNA Barcoding to explore biodiversity in their city. The projects ranged from studies on the ant diversity in the South Bronx to surveys of the fungal biodiversity in Central Park. The winning team used DNA Barcodes to identify the content of Gingko products. 

No matter what example one looks at, the question - can High School students do DNA Barcoding -  is easy to answer: Of course they can! Actually they should as it is a great way to learn the basics of modern genetic methods and develop an appreciation for the biodiversity around us.

Larva Thursday (1)

One of the many promising applications of DNA Barcoding was always the ability to assign larval stages of animals to their adult form. There are many groups where the larval forms are distressingly uniform in appearance and the young don't look at all like the adults. It is no surprise that a significant number of researchers who are interested in larval identification have tested DNA Barcoding and its value for their work. Therefore, I will use every Thursday in the weeks to come to present their research and how DNA Barcoding has changed their workflow.

Zu cristatus  (Credit: Allan D. Connell)

Our first episode of the series "Larva Thursday" will be on fish - maybe not a surprise to readers that know me a bit better as this is a group of animals close to my heart :-)

Unfortunately not many fish larvae do resemble juveniles and adults in basic form, and, even more helpful for the identifier, in the number of fin spines and rays and myomeres, which represent commonly used traits. More often they are transparent, very small, and, while they are alive, look just like tiny slivers of glass with eyes. Therefore, identification to species or genus is very often only possible for real taxonomic experts who perhaps spend a good time of the their life to look at larvae and describing their appearance. There are indeed quite a few monumental books out there with drawings and keys to the regional ichthyofauna but frankly the average non-taxonomist won't use them very often. More accessible are online keys and listings with images of alive animals and when researchers also went through the lengths to confirm their identifications by matching larval DNA Barcodes to adult ones I personally find that very appealing. 

Of the various websites documenting fish larvae I picked two that utilized DNA Barcoding results to confirm assignments and even discover new species.

Malacoctenus gilli (Credit: Ben Victor)

One is a guide to the larval reef fishes of the Caribbean. As most sites this is not a complete list yet but as it's owner Ben Victor states himself, "there is no reason in the age of the internet not to have a work in progress made generally accessible". Quite a few families, genera and species are already represented by short descriptions, lots of photos, and explanations of key characters. Many have been barcoded and are available as references on BOLD and GenBank. While Ben travels the Caribbean (and other places) to dive and collect larvae, one of his colleagues, Allan Connell, collects fish eggs almost every day in his "backyard" (Indian Ocean) close to Durban, South Africa. He hatches the eggs, rears the larvae and documents every stage with images and measurements. Species assignments are made morphological were possible but more and more by using DNA Barcoding because he actively helps building a reference library of DNA Barcodes of adult fishes in South African waters. All his data are publicly available through his website. 

Both researchers do something exceptional. They are sharing their findings openly prior to any formal publication. It would be great if many of us could follow their lead.

Cave Barcoding

It has been conventional wisdom for many years to see caves as depauperate ecosystems in terms of biodiversity. This notion derives from a confluence of factors: the lack of primary producers for the most part, the limitations of space, and the fact that most studies have been done in temperate latitudes, in which caves are rather poor in terms of biodiversity when compared with their tropical counterparts. However, caves are teeming with live which is often well adapted to a permanent life underground.

Compared to surface species, cave-adapted faunas generally have small geographic ranges and high levels of endemism at all scales of measurement, making their biogeography distinct. There are quite a few records of single cave endemics in cave-adapted species and it is not rare that adjacent cave systems with similar conditions host entirely different faunal communities.

When it comes to caves our knowledge about their biodiversity is very limited and the discovery of new species is quite common. Just recently a new family of spiders was discovered in caves in Oregon and California. 

One common problem though is the fact that many organisms have shown morphological stasis as they acquired specific adaptations to a highly selective environment. The result is a lot of undiscovered cryptic species as scientists only start to look at cave life more systematically. DNA Barcoding could be of great help in this undertaking.

Therefore, members of the Biodiversity Institute of Ontario, the Biospeleological Register of the the Hesse Federation  for Cave and Karst Research, the German Federation for Cave and Karst Research, and the Goethe University, Frankfurt am Main have joined forces to build up a DNA Barcode library for all described species that are known to occur in caves in Germany. This project is part of the German Barcode Initiative GBOL. The study will first utilize material collected over the past years in German caves and eventually include more freshly collected material. It is the hope of the participating researchers that this project will serve as a model for other countries and regions to survey the biodiversity of their caves

The sixth extinction

Not a DNA Barcoding sensu stricto post today but I really like to introduce a series I discovered at The Guardian which is very well done and worth some recurrent visits.

The Cretaceous-Tertiary (or K-T) extinction event 65 Million years ago is probably the most well-known because it wiped out the dinosaurs. However, a series of other mass extinction events has occurred throughout the history of the Earth, some even more devastating than K-T. Mass extinctions are periods in Earth's history when abnormally large numbers of species die out simultaneously or within a limited time frame. The most severe occurred at the end of the Permian period (248 Million years ago) when 96% of all species perished. This along with K-T are two of the Big Five mass extinctions, each of which wiped out at least half of all species. While the reasons for these five major events are still under discussion and many models have been proposed, there is a sixth major extinction event happening at present and the reasons for this one are quite well understood. Earth is currently losing something on the order of 30,000 species per year. Statistically more dramatically expressed: three species per hour. The so-called Sixth Extinction is the first one that is caused by a species living on the planet: humans are the direct cause of ecosystem stress and species destruction in the modern world and therefore the main driver.

The series at the Guardian is called "The sixth extinction" and presents the current state of knowledge and provides examples for species at the brink of extinction but also conservation efforts that let one hope. Very good articles, well researched, and diverse. I believe the series is already running since beginning of September but all articles and audio files are still available. Today a rather sad record list has been published - the World's 100 most endangered species which was released today by the Zoological Society of London and the International Union for Conservation of Nature.

Reading these contributions makes you pause for a moment and wonder if there will be an end to it. Back in 2005 the renowned Paleontologist Niles Eldrege wrote the following on the question if this extinction can be stopped:

Though it is true that life, so incredibly resilient, has always recovered (though after long lags) after major extinction spasms, it is only after whatever has caused the extinction event has dissipated. That cause, in the case of the Sixth Extinction, is ourselves — Homo sapiens. This means we can continue on the path to our own extinction, or, preferably, we modify our behavior toward the global ecosystem of which we are still very much a part. The latter must happen before the Sixth Extinction can be declared over, and life can once again rebound.

Dangerous herbs

DNA Barcoding and traditional Chinese medicine

Chinese herbs have been used as medicine for over 2000 years. There are some 13,000 natural medicinals used in China with over 100,000 recipes recorded in the ancient literature. More and more these traditional drugs are used outside of China and some producers of Chinese herbal medicines are pursuing FDA clinical trials to market their products as drugs in U.S. and European markets. Therefore, the authentication of the ingredients is becoming a critical, international issue because mistakes can cause illness and even death. Plant elements and extracts are by far the most common elements used and there is confusion and substitution happening. Not a surprise given the large number of potential ingredients for these products.

A couple of years ago a 60 year-old man from Hong-Kong was diagnosed with kidney failure and cancer of the urinary tract. He had been taking an herbal prescription and the case was investigated by the local Department of Health. The investigations revealed that the patient was mistakenly given a product called Herba Aristolochiae Mollissimae (made from Aristolochia mollissima) instead of Herba Solani Lyrati (made from Solanum lyratum). Herba Aristolochiae Mollissimae is known to contain the poisonous aristolochic acid which indeed can cause kidney failure and is known to be cancerogen when taken over a longer period of time.

Aristolochia mollissima

Solanum lyratum

In the wild both plants can be distinguished rather easily (see pictures) but some characteristics may vary with growing stages, environmental properties, physical forms, and post-harvest processing of the herbs. Confusion is further facilitated by the fact that both herbs share the same Chinese common name Bai Mao Teng (meaning  ‘white hair-bearing vine’).

Now a group of researchers from Hong Kong tested if DNA barcoding and chemical fingerprinting are useful alternatives to the use of microscopy and elaborate chemical analysis. Indeed they are. The chemical profiling was able to detect the harmful component aristolochic acid. However, the chemical composition of a herb can vary between different life stages and can be altered during processing. In contrast, DNA barcoding  is not affected by these factors and usually a small amount of sample is sufficient for DNA extraction. The study also showed that the smallest DNA fragment amplified was about 50 bp long which demonstrates that even partially degraded DNA could be retrieved. The researchers tested a variety of markers (the standards matK and rbcL, as well as ITS, trnH-psbA and trnL-trnF) which provides us with an arsenal of possible tests that will help to avoid such fatal confusions.

An unconventional field site

Scientists from the Florida museum published the first study on butterflies and moths of Guantanamo Bay Naval Station and discovered large biodiversity in an area previously unknown to researchers and one you wouldn't normally think of when it comes to plan field work. Appearing hopefully soon in the Bulletin of the Allyn Museum, the study creates a baseline for understanding how different plant and animal species have spread throughout the Caribbean.

Guantanamo Bay Naval Station

 

One of the authors, Jacqueline Miller says, "Biodiversity studies are extremely important because they give us clues about where things were and how they evolved over time so we can better understand what may happen in the future.We're also looking at climate change over time, and butterflies are biological indicator species since they are associated with particular plants as caterpillars and often found in particular habitats."

In January, researchers collected 1,100 specimens representing 192 moth and 41 butterfly species, including the invasive lime swallowtail whose proximity to the U.S. poses a threat to citrus plants. The researchers also froze tissue samples from many of the collected specimens for future DNA Barcoding analysis.

Leased to the United States in 1903 (although this is disputed by Cuba), the land has unintentionally become a wildlife refuge, offering researchers the opportunity to better understand the island's natural habitats. "Because it is a military base -- and this is true for many military bases, which typically have large areas of land -- people are not trampling, bulldozing or developing the land," says Roger Portell, another author of the study. "So there is a large area of land in the southeast corner of the island that has basically been untouched for 100 years."

Indeed such pristine environments can often be found on many military bases or similarly protected areas. A good example for the latter is the former border separating West and East Germany. It was estimated that around 600 threatened species of animals and plants were given a free rein in a no man’s land overshadowed by minefields, metal fences and watchtowers. Today the former border is turned into a unique chain of nature reserves running for nearly 1,400km in a gentle zigzag from the Vogtland region, near the German-Czech border in the south, to the Baltic Sea in the north, to form what is called the green belt.

This could serve as a good example what to do with such areas once the military has given them up. I haven't given up the hope that this happens to Guantanamo Bay one day.

Standards

A DNA Barcode is a short standardized sequence enabling species discrimination

This short definition contains all important elements of DNA Barcoding and this post is about one very important word in it: standardized

Standards are paramount in our work and it is concerning that an increasing number of studies use different gene regions and their authors call it DNA Barcoding. I find it this even more puzzling when it comes to vertebrates where the reasons for not using COI are either traditional or even worse - the result of convenience. 

It is always exciting when new species are discovered by using DNA methods and I fully support the idea that any phylogenetic approach needs additional genetic markers, the same is true if a sufficient placement can't be made using DNA Barcoding alone. However, I will never be able to compare my barcode data on fishes with data of colleagues who decided to use cyt b instead. This is already frustrating when you try to build phylogenies. 

Phylogenetics is a good example what can go wrong when you don't agree on standards. In essence everybody can use whatever marker they think works best for them. Fortunately, that started to change over the last few years. Well, we are living in a free world, and I am the last who is going to tell somebody else how to do their job but the result is that most data that is publicly available isn't necessarily comparable. With years of experience in trying to assemble datasets for particular taxonomic groups in order to build robust phylogenies I consider it a waste of money and ignorant not to use data other researchers have generated before. However, often I simply can't do that as the allowable amount of unknown data in an analysis is limited. There isn't sufficient overlap between datasets as communities of researchers haven't agreed on common gene regions for this kind of analysis. On the contrary there were long lasting disputes about which region(s) to use. Some battles still continue.

For me DNA Barcoding represented a big leap forward by proposing standard regions (and features) and in some cases it took longer find a consensus among all scientists (e.g. the plant barcode) but they did. Nevertheless, currently we have standard regions for 3 major groups: 

• Cytochrome Oxidase I (COI or COXI) for animals
• Ribulose-bisphosphate carboxylase gene (rbcL) and the chloroplast maturase K gene (matK) for plants
• The complete ITS1 spacer, the 5.8S gene, and the ITS2 spacer as a single contiguous sequence (ITS) for fungi

There might be compelling reasons (especially technical ones) not to use one of those in the particular groups and people are of course always entitled to chose whatever works best for them but I would like to make two suggestions:

(1) Use the term DNA Barcoding only if you use one of the markers listed above to indicate that you follow agreed upon community standards.

(2) Give the markers at least a try to ensure that you've done what you can to contribute to a global effort and the community of your colleagues.

More on DNA quality

A few posts ago I was talking about a new publication that put DNA Barcoding sequences to some quality tests. Today the journal MycoKeys published a paper that represents a compilation of best practices which can help with the quality management of sequence data. 

The lead author of the paper, Henrik Nilsson, on the motivation to write the publication: "Many researchers find sequence quality control difficult, though. There just isn't any straightforward document to put in their hands to give them a flying start. As a result, scientists differ in the degree to which they are aware of the need to exercise sequence quality control and in what measures they take."

The authors focus on the DNA Barcode for fungi, ITS but all suggestions and recommendations are broadly applicable to other markers and organisms. The paper is a collection of really good guidelines and especially useful for starters. This is one of the papers I would give any freshman student to read if I had any :-)

Here a summary of their 5 guidelines (Table 1 of their publication).

Target of guideline	Way of getting there
1. Establish that the sequences come from the intended gene or marker	Do a multiple alignment of the sequences and verify that they all feature some suitable, conserved sub-region (here the 5.8S gene)
2. Establish that all sequences are given in the correct (5’ to 3’) orientation	Examine the alignment for any sequences that do not align at all to the others; re-orient these; re-run the alignment step; and examine them again
3. Establish that there are no (bad cases of) chimeras in the dataset	Run the sequences through BLAST in INSD/UNITE and verify that the best match comprises more or less the full length of the query sequences
4. Establish that there are no other major technical errors in the sequences	Examine the BLAST results carefully, particularly the graphical overview and the pairwise alignment, for anomalies
5. Establish that any taxonomic annotations given to the sequences make sense	Examine the BLAST hit list to see that the species names produced make sense

Thanks!

I would like to take the opportunity to thank everyone for the support in the first week of my blog. I was very surprised about the extremely positive response and I am very proud to have reached 500 unique page views on some days, and that I already have 11 followers, not counting all RSS feeds :-)

This is very encouraging and motivating and I will continue with my goal of a post per day (maybe not on weekends though - family time comes first). There is certainly enough out there that deserves a post. I might miss things but I strive to be fast (my "CSI: Rainforest" was the second entry on that news in the web - not bad for a journalism rookie). If you have something interesting that you think might deserve a post e.g. something you are currently working on and are about to publish please let me know. I am happy to beat the press once more :-)

Please feel free to post comments. Feedback is always welcome. Blogs are usually very short (for good reasons) and commenting is a way to add information to a post or discuss the topic as such. I am all up for that. For now I haven't put many restrictions on that function as my site is currently far from being popular enough to attract spam or comments of strange people. Of course an ambitious blogger strives for many more readers but I am realistic enough not to expect to be able to compete with the big science blogs. Nevertheless, tell your family, friends, colleagues, students, sponsors... there is a blog on DNA Barcoding not only for scientists.

Thanks a lot.