Scandinavian Barcodes

More than a thousand new species –nearly one-quarter of which are new to science – have been discovered in Norway since the Norwegian Taxonomy Initiative began in 2009. This unique effort to find and name all of the country's species  is one of just two government efforts worldwide where research has been funded solely to find and catalog the country's species diversity.

The Norwegian initiative is focused on describing poorly known species groups across the country's varied habitats, which range from alpine plateaus to the arctic. Scientists believe that there are roughly 55 000 species in Norway, but until now only 41 000 of them have been discovered. The 1165 new species discovered by the initiative over the last four years are thus an important addition to this number. Among them are new insect species, lichens, molluscs and cold-water sponges. 

A sub-set of the material collected by the Taxonomy Initiative has been made available for DNA barcoding in collaboration with the Norwegian Barcode of Life network (NorBOL). To date, NorBOL has registered barcodes from approximately 3 800 species in Norway, over half of which have come via the Taxonomy Initiative. This progress likely helped to convince the Research Council of Norway to provide more funding towards national DNA Barcoding. They just announced that NorBOL has been granted about $4.2 to develop the network into a national research infrastructure. 

The project starts in January 2014 and will focus on knowledge transfer and capacity building in all NorBOL member institutions in addition to continue to build a DNA Barcode library for the Norwegian fauna and flora (including fungi). 

Congratulations to the colleagues in Norway - it would be great if other nations would follow this example.

Potato eaters

Flea beetles are small, jumping beetles of the leaf beetle family Chrysomelidae. Many major agricultural crops are attacked by flea beetles. Species of the genus Epitrix, for example, feed mostly on plants from the family Solanaceae (Nightshades) and some of them are major pests of potato crops. What makes pest management so difficult is the fact that Epitrix species are morphologically very similar. Species delineation usually requires dissection and study of genitalia and that in turn requires a high level of expertise (and time, and patience). 

It comes to no surprise that DNA Barcoding is proposed to help with the identification of at least all the economically important Epitrix species especially when considering all their developmental stages at which it is usually impossible to tell what species is which. There are five species of the genus that feed on potato in Europe and North America (Epitrix cucumeris, Epitrix similaris, Epitrix tuberis, Epitrix subcrinita and Epitrix hirtipennis) and they are considered major pests. 

A group of French researchers has tested not only the standard COI barcode region but also ITS2. The latter was used to test for potential ambigous results with the mitochondrial marker. They couldn't detect any mitochondrial introgression. Once the reliability of the DNA Barcode was confirmed the researchers developed an RFLP-based diagnostic method and showed that unambiguous species discrimination can be achieved by using the sole restriction enzyme TaqI on COI polymerase chain reaction products. Now that makes the identification rather simple and considerable cheaper. 

The concluding paragraph of the paper shows how timely this study was:

During the final stages of review of this manuscript, the European commission published a decision (212/270/EU) to prevent the introduction of E. tuberis and E. subcrinita in Europe and limit the spread of E. cucumeris and E. similaris (Official Journal of the European Union, 2012). By enabling the identification of Epitrix specimens at different periods of the life cycle, this diagnostics tool should help member states conducting survey for the presence of potato flea beetles on potato crops as well as other host plant species, as recommended by the European commission. Fast and accurate detection of Epitrix potato flea beetles would help study their potential spread and contribute to their management with minimal disruption to Solanaceae trade.

By they way, BOLD contains quite a few sequences of those species in question some of which actually collected by the School Malaise traps, e.g. this one.

December Bulletin

Another accomplishment of the day:

The new Barcode Bulletin is out! 20 pages that showcase what is currently going on in this field of research, and an Editorial written by yours truly.

A lot of articles represent research and projects that were presented in Kunming. So, this is an excellent opportunity to read over the Holidays what you might have missed because you couldn't attend.

School Malaise Trap Program Fall results

It's time again to share some results of a School Malaise Trap program.Yes, we did it again this fall. From September 23rd to October 4th we had 21 schools catching flying insects with Malaise Traps. All participating teachers will receive an email this afternoon with a big results package.

You might remember that the first run of program involved 60 schools in 42 cities. We had to reduce this for the fall for financial and logistical reasons and we also skipped our usual visit by the BIOBus. Nevertheless, the results were impressive especially given that it was quite late in the season. We were quite curious because by that time of the year we usually remove our traps in the National and Provincial Parks. This would give us an indication what we potentially miss by doing so.

Indeed the trap catches were quite high and despite having only about a third of the spring numbers of traps out, we caught more species. The 21 traps on average collected 1,338 specimens for the collecting period. Our staff sorted 28,110 specimens and selected 5,985 to be barcoded. Our final dataset was made up of 4,736 DNA Barcodes (not all worked and short barcodes were discarded). An impressive number of 1,493 putative species were collected over the two week period of the program. Here a breakdown:

113 of these species were new to BOLD which could either mean that they are known but have not been sampled yet or that they are indeed new to the region or even to science. We also had a look at the amount of overlap between our spring and the fall runs and found out that 404 species occur at both events which leaves us with 2500 species found in two weeks in early spring and two weeks in late fall in 70 schools in south-western Ontario. 

This project is unique in many respects. Firstly, it is a great discovery based science project for classrooms in both elementary and secondary schools. The teacher and student feedback is very positive and the fact that with the help of BOLD we can give credit to each participating group for their contribution to scientific knowledge, is invaluable. Secondly, we have an unprecedented surveillance network in our immediate vicinity. Both runs unveiled a good number of firsts for Ontario, Canada, North America. Just one example encountered last week when looking at the data: The hemipteran Dicyphus errans has only been known from Europe so far where it is used as a biocontrol agent. The new School Malaise Trap dataset contains a record that matches some European counterparts on BOLD. A first quick morphological inspection confirms this. Now a colleague at the CNC (Canadian National Collection of Insects) will look closer and subsequently we will officially report this find. This might be an escaped biocontrol agent used by a local farmer to eradicate aphids. Problem with this species is that once they eat all the aphids or flies they suppose to control they can switch their diet and become a pest themselves.

For the researchers here at BIO the fun starts now. We have so much data to look at and it even if those two events are just taxonomically restricted snap shots they allow us insights into diversity patterns on a scale never possible before. Furthermore, the program has not only excited almost 3000 kids in the region but also considerably helped building our DNA Barcode reference library.

100 pigeons and their diet

Due to a high degree of faunal and floral endemism oceanic island ecosystems are of high priority when it comes to biodiversity conservation. Evolution under isolated conditions formed unique ecosystems, but those are highly vulnerable to human disturbances, such as deforestation or the introduction of invasive species.

For example knowing the diet of an endangered animal might become very important for adequate restoration efforts on oceanic islands because introduced species may already be a major component of the diets of some endangered species.

The red-headed wood pigeon Columba janthina nitens is a critically endangered subspecies endemic to the Ogasawara Islands, a chain of oceanic islands located 1000 km south of the main islands of Japan. The current population of this species might only comprise of 100 individuals. Furthermore, the native forest of the Ogasawara Islands has been destroyed as a result of human settlements in the 19th century and World War II. As a result the islands are now home to several introduced plants which have expanded their natural range replacing the eradicated local flora. Researchers supected that the red-headed wood pigeon might have shifted its diet in favor of the introduced species in order to cope with the lack of native food resources. 

A group of Japanese colleagues went ahead and tested this assumption by using next-generation sequencing of fecal samples from the birds. They called it DNA Barcoding although they used a different marker (the chloroplast trnL P6 loop) for their analysis. It is not clear why this marker was chosen although it had been used for similar studies before.The disadvantage of using a non-standard becomes clear when reading their publication. In order to get some useful results they first had to construct their own library of reference sequences (over 200 species) before moving on to the fecal samples. Not only does that sound like a lot of extra work but for all other scientists following agreed upon standards this library is not very useful unless they shift their attention to trnL P6. I find this very frustrating as the study as such is great and the results very important for the planning of any restoration efforts:

In this study, a diet analysis using DNA barcoding provided a high-resolution identification of food plants and clearly overcame the bias of traditional microhistological analysis. The results of the DNA barcoding indicated frequent consumption of introduced species, rather than only native species, by the pigeons. The rapid eradication of some introduced species without restoration of the native seed plants may reduce available food resources for this pigeon. Thus, a strategy that balances the eradication of introduced plants and the restoration of native food resources is important. Differences between the composition of pigeon diets on Chichijima and those on Hahajima should also be considered during the restoration of each island. Although some existing technical problems must still be solved (e.g., the discrimination rate of the P6 loop database, sampling strategy), the NGS DNA barcoding approach will provide a better understanding of the food web, including the interactions between native and introduced species and appropriate nature restoration planning for oceanic island ecosystems.

Overall a great example showing the advantages of a deeper understanding of food webs provided through next generation sequencing. If it helps those birds even better. They just shouldn't call it DNA Barcoding because it isn't. 

Frosty cockroaches

With winter’s arrival comes the kind of news that may give New Yorkers the creeps. A species of cockroach never found in the United States before has been positively identified in Manhattan and even worse, this variety can survive not just indoors where it’s warm, but also outdoors in freezing temperatures. This species (Periplaneta japonica) is known in Asia, but was never confirmed in the United States.

The Asian species was first spotted in New York in 2012 by an exterminator working on the High Line, an elevated walkway and park on Manhattan’s West Side (see photo). These cockroaches looked different to him, so he sent the carcasses to the University of Florida for analysis. Researchers there used DNA Barcoding to confirm the presence of this new cockroach species. It is not clear how the cockroach arrived in New York City but it is suspected that one or more of the ornamental plants that are planted on the High Line arrived in soil that contained the new pest.

The original paper has been published in the Journal of Economical Entomology but I couldn't find it on their website yet. As soon as I discover a valid link I will share it through the comments.

Termites

Like their hymenopteran cousins (ants, bees and wasps) termites (isopterans) live in colonies and divide labor among castes, produce overlapping generations, and take care of their young collectively. A typical colony contains nymphs (half-mature young), workers, soldiers, and reproductive individuals of both sexes, sometimes even several egg-laying queens.There are termite species that do not have a soldier caste at all, and they are fairly well represented in tropical regions of the world, especially South America and Africa. 

Workers take on the defense role by using their gut contents to either seal up nest damage, or trap invading ants which are the predominant invaders. At first glance, it may appear that this defensive method is inadequate, as casualties are often high on the termite’s side, especially in the event that a large breach has occurred and ants manage to invade in large numbers. However, most of these soldierless termite species feed on decaying wood or humus, and thereby largely avoid contact with ants while foraging. 

Species boundaries in termites are traditionally inferred by using morphological characters, although morphology sometimes fails to correctly delineate species. Another commonly used non-morphological taxonomic character is a profile of the cuticular hydrocarbons. They have been proved useful to distinguish a couple of insect species, especially among social insects. Using cuticular hydrocarbon profiles for species recognition is thought to be a direct way to compare the chemical characters through which social insects recognize each other. However, more recently researchers have taken advantage of alternative methods such as DNA Barcoding but nobody really tried using all of those methods simultaneously in an integrative taxonomic approach. 

An international group of researchers has now done this more extensive testing and their paper was published this week:

We sampled soldierless termites in various forest types of the Nouragues Nature Reserve, French Guiana. Our results show that morphological species determination generally matches DNA barcoding, which only suggests the existence of three cryptic species in the 31 morphological species. Among them, Longustitermes manni is the only species whose splitting is corroborated by ecological data, other widely distributed species being supported by DNA Barcoding. On the contrary, although cuticular hydrocarbon profiles provide a certain taxonomic signal, they often suggest inconsistent groupings which are not supported by other methods. Overall, our data support DNA Barcoding and morphology as two efficient methods to distinguish soldierless termite species.

The experienced barcoder is likely not surprised about how well COI sequences work to delineate soldierless termite species. It is remarkable though that cuticular hydrocarbon profiles are not very reliable although they have been portrait as representative for chemical cues the animals actually use to identify members of their own species. Probably there aren't as good as thought.

Very old DNA

Sima de los Huesos, the "bone pit," is a cave site in Northern Spain that has yielded the world's largest assembly of Middle Pleistocene hominin fossils, consisting of at least 28 skeletons, which have been excavated and pieced together over the course of more than two decades. The fossils are classified as Homo heidelbergensis but also carry traits typical of Neandertals. Until now it had not been possible to study the DNA of these unique hominins.

A team from the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, has developed new techniques for retrieving and sequencing highly degraded ancient DNA. Together with a Spanish team of paleontologists they sampled two grams of bone powder from a hominin thigh bone from the cave. They extracted its DNA and sequenced the mitochondrial genome. By comparing those DNA sequences those of Neandertals, Denisovans, present-day humans, and apes, the researchers calculated that the Sima hominin lived about 400,000 years ago. They also found that Sima hominin shared a common ancestor with the Denisovans, an extinct group from Asia related to the Neandertals, that lived about 700,000 years ago. This find was rather unexpected since skeletal remains carry some Neandertal-derived features.

What I find most intriguing is the simple fact that the colleagues were able to determine an almost complete mitochondrial genome sequence of a 400,000-year-old representative of the genus Homo not buried (and preserved) in Permafrost. I am thinking of all the type specimens we try to obtain DNA Barcodes from. They are much younger but most likely less well preserved. Nevertheless, worth to have a close look at all the methods and tricks used to come up with a mitochondrial genome that old. One thing for sure - they were able to retrieve the full DNA Barcode - just checked it.

Tree barcodes

Podocarpaceae is the second largest family of conifers and mainly found in the Southern Hemisphere. The group comprises about 198 species of evergreen trees and shrubs that are sometimes cultivated as ornamentals in suitably warm climates.

It isn't well known and as a result their use is very limited but Podocarpaceae contain a variety of bio–active compounds such as antioxidants, nordi–terpenes, podocarpic acid, and tatarol. Some of these compounds are known to be antimicrobial, fungistatic, or bacteriostatic, others are cytotoxic and could be used in cancer treatment. However, any potential medical use needs further more detailed research. Furthermore, species of Podocarpaceae are of conservation concern primarily as a result of small population sizes and limited available habitat. Twenty–seven Podocarpaceae species are included in the International Union for the Conservation of Nature (IUCN) red list under the categories of vulnerable (10 species), endangered (14 species), and critically endangered (three species). Two species are included in the appendices of the Convention on International Trade in Endangered Species (CITES): Podocarpus parlatorei is listed in Appendix I (trade is not allowed) and Podocarpus neriifolius is listed in Appendix III (trade with, some limitations, is allowed). That and a slow rate of growth for most species makes harvest of Podocarpaceae generally unsustainable and would require extensive conservation management efforts.

In addition accurate identification of tropical forest trees, such as Podocarpaceae, is often very difficult. The most easily accessed material is usually sterile. If fertile material is present, it is frequently either inaccessible or detached from the tree making it difficult to convincingly associate the fertile and sterile portions. Although sterile material of Podocarpaceae can usually be identified to genus using phyllotaxis and leaf form, accurate species identification often requires careful microscopic examination of internal and external characteristics. Proper use of the existing identification tools requires training in botanical terminology, skill in microtechnique, and familiarity with Podocarpaceae.

Now a team of US and German researchers build a DNA Barcode library for 145 of the species (73% of the family). They generated matK, rbcL, and nrITS2 sequences. In addition, they created a living Podocarpaceae collection in the Botanic Garden of the Ruhr-Universität in Bochum, Germany which promotes the protection of rare and endangered species. There the species will be propagated and passed on to other botanic gardens worldwide.

The discrimination power of matK, rbcL, and nrITS2, individually and in combination was relatively low (56.7%) in this study. The authors also state that this is not unusal and that plant DNA Barcoding studies that heavily sample within taxonomic groups usually report low rates of species discrimination. They suggest that sequences of some unlinked genetic markers may however increase the rate of species discrimination. 

An overlooked giant

Giant clams are among the most conspicuous marine invertebrates on coral reefs due to their large size. Some species can grow up to 230 kg. Species of the genus Tridacna also show a brilliantly colored mantle that contains photosynthesizing symbionts (dinoflagellates of the genus Symbiodinium).

Many of those clams have been harvested for their meat and their shells They are also in demand on the ornamental aquarium market. All that led to widely depleted stocks. Consequently, most Tridacna species are listed by CITES and the IUCN Redlist. A crucial part of conservation management of those giant clams is the proper identification of different species and an estimation of the size of their breeding populations. Many of them are so called broadcast spawners which means eggs and sperm meet freely in the water column. This in turn means that a critical mass of individuals in an area is needed to ensure that eggs and sperm are likely to meet.

A new species of giant clam, thought to have been another well-known species (Tridacna maxima), has now been discovered on reefs at Ningaloo in Western Australia and the Solomon Islands. Individuals looked almost identical to the common species Tridacna maxima, but were actually genetically quite different. The Australian researchers used DNA Barcodes and other DNA regions to study gene flow of different Tridacna species and came across this cryptic species by chance.

This could turn out to be a very important discovery as chances are high that scientists have been overestimating the population of related species of clams. If this new species was mistaken and counted for Tridacna maxima the population size of that species would likely be estimated to be larger than it actually is. Given the conservation status of these majestic animals this is potentially bad news as population estimates need to be adjusted downwards.