Project Metabarpark in IRBio Seminars

Project Metabarpark will be present in the talk session hosted by IRBio (Institute for Research in Biodiversity) at the School of Biology of the University of Barcelona, this Thursday 26th of November.

Xavier Turon will talk about "Biodiversity crisis and the taxonomic impediment: what the metabarcoding of communities has to offer" and Owen Wangensteen will follow with a talk about "Metabarcoding of marine hard-bottom communities, the unseen biodiversity".

The entrance is free and you are all invited!

Metabarpark at the Course “New Technologies applied to the control and monitoring of invasive species”

In October, 19 to 23, the Spanish National Parks managing organisation (“Organismo Autónomo Parques Nacionales”) organised a course on new technologies applied to the study of invasive species in Vigo (Galicia). This course was attended by staff, technicians, and rangers of the National Parks in Spain.

Xavier Turon was invited to give a lecture about “Genetic tools and invasive species”. It was an enjoyable experience, with interested audience that posed clever questions. It was also a pleasure to meet again the friends of the Parque Nacional de las Islas Atlánticas (Pepín, Vicente, Mercedes…), thanks for your hospitality, as always!

Second collecting trip in Cabrera done!

In October, 2 to 8, we made the second sampling foreseen in the workplan in the Cabrera Archipelago National Park. As in the previous year, we organized a joint trip with members of the CORCLIM project. In this way we optimized costs and logistics, and of course it was an excellent opportunity for interaction and exchange of experiences.

From left to right: Xavier Turon, Pol Capdevila, Kike Ballesteros... in action (foto: Eneko Aspillaga)

The always blue waters of Cabrera (foto: Eneko Aspillaga)

In this trip we worked again in the Imperial Islet, where we sampled the same communities as in the previous year. In addition, we got samples from a community dominated by the invasive seaweed Caulerpa cylindracea at 30 m depth. Weather was far from perfect, and we had to cope with some unexpected problems such as the breakdown of the compressor, but we finally managed to get back with all samples foreseen.

An image of the community dominated by Caulerpa cylindracea (foto: Eneko Aspillaga)

By now, all samples collected in Islas Cíes in May and in Cabrera in October have been processed, sieved into size classes, their DNA extracted, and we are working in the amplification of the two genes chosen, 18S and COI. In one-two weeks we expect to send the samples to Fasteris (Switzerland) for parallel sequencing.

The importance of being barcoded

by Owen S. Wangensteen

When, back in 2003, Canadian biologist Paul Hebert proposed the creation of a public database of short sequences aimed at identifying biological specimens by means of just these small sequences (DNA-barcodes), he did not find the unanimous response anyone would expect nowadays. Detractors were many and they actually made a lot of scientific noise. In fact, as Laurence Packer once pointed, scientific papers published from 2004 to 2007 criticizing and opposing DNA-barcoding were more numerous (and had more citations) than papers written by the first “barcoders” who embraced the idea. Then, a miracle occurred: as the public databases grew, DNA-barcoding started to prove its utility in a myriad of forms. From long-disputed basic taxonomy issues to medico-legal forensics, passing through river ecology, agricultural sciences, control of disease outbreaks, food security or biodiversity conservation, all took advantage of DNA-barcoding in ways only a few would have dreamt of some years before. 

The reticences were mainly based in some false concerns and a few ancestral fears. Morphological taxonomists argued that no small-sequence could replace their meticulous, morphological observations and thorough interpretations, while internally fearing to lose their jobs. Geneticists argued that genomes were way too complex to be reduced to just a three-hundred base-pairs sequence. Ecologists thought that molecular methods were too expensive and beyond their field of expertise. All of them were basically wrong. 

DNA-barcoding does not plan (and is never going) to replace taxonomists. At its best, DNA-barcoding is meant to replace traditional identification keys (which were probably bound to disappear anyway!). Identification keys are usually a nightmare even for expert taxonomists and are completely useless for specious groups including thousands or tens of thousands of species. DNA-barcoding will only facilitate the work of taxonomists, which are still needed (in fact, more needed than ever) for identifying problematic specimens. Given the estimations for the global number of species and the current description rate of new species, we will need taxonomists, at least, for the next 500 years, or probably much longer. This is more than most scientists working in other areas of knowledge could bet. 

It is true that genomes of non-model organisms are large, complex, gigantic monsters waiting to be tamed. But the fact is that all you need for unambiguously identify the species where a specimen belongs is a fragment of just a few hundreds base-pairs of a highly variable genomic region (in most cases, just 200 base-pairs will do!). Mitochondrial DNA (and, specifically, the cytochrome oxidase I gene, COI) has proven its power to achieve this task in many groups of organisms (including animals; other genes are most used for green plants or fungi). If you need your sequences just for identification purposes, all you need is to sequence but 200 base-pairs of COI. Fast and easy. 

And unexpensive. Sequencing costs have done nothing but plummeting over the last three decades and they are predicted to keep decreasing in a month-to-month basis. Astounding sequencing technologies that grew up under the big umbrella of Human Genome Project have already arrived to average-sized laboratories. Nowadays, DNA-barcoding of an unknown specimen is something that a freshman biology student could perform during his/her first lab course. Costs and complexity are no longer valid excuses. 

I recently understood the pivotal importance of barcoding (i.e. the urgent need for getting the sequences from as many different species as you can and depositing them in a public database as soon as possible) when I started analyzing the data we got from bulk-sequencing environmental samples collected in shallow marine hard-bottom communities of selected marine reserves in Spain. This kind of research is crucial to understand how marine communities are changing (and they are changing indeed, probably at a faster pace than we use to think). We need to sample these communities and characterize them as soon as possible, if we want to be able to detect any changes in the near future, before it's too late. The sensible way to do this is by metabarcoding them. That is: get the sample inside a bag, blend it with a kitchen blender, extract the DNA of every organism present in the mixture, use this DNA to amplify a useful genetic marker (COI is the usual choice), and then try to match the millions of sequences you get to the current contents of public barcoding databases, in order to identify the organisms that are present in your samples (and probably their abundances, but that's another story I will be writing some other day). The first steps of this procedure proved to be surprisingly easy. Notably, it is the last identification step which is currently limiting the utility of this technique. We have literally millions of different COI sequences, representing all the biodiversity hidden in our samples, but we are able to identify (give a species name) just a small percentage of these sequences. Marine barcoding databases are not as developed as terrestrial ones. Small and microscopic organisms are much more underrepresented in the databases than big, macroscopic ones. A surprisingly high percentage of marine diversity is yet unknown, even for shallow ecosystems in the Mediterranean or temperate Atlantic European shores, which have been extensively studied by marine biologists, at least since Aristotle. 

If we want to begin to understand the biodiversity changes which are happening now in our beloved marine ecosystems, we really need to undertake the titanic task of sequencing the DNA-barcodes of as many marine species as possible, and release the data to our public databases. I am confident that metabarcoding will become the main tool of marine ecologists in the next few years. We need to extract its full power as soon as possible by expanding our databases and filling the main gaps. This is no task for an individual or a small group of people. Given the overwhelmingly high number of different marine species, this is a task for several hundreds of marine taxonomists, working across the World, identifying species in their different groups of expertise, sequencing data, curating taxonomies, releasing data, and waiting for no bigger reward on these data release but contributing to help other marine biologists in building the greatest and most useful marine taxonomy enterprise of all time. 

As Harry Truman once said, it is amazing what you can accomplish if you do not care who gets the credit. 

Comparing DNA-barcoding vs environmental-DNA metabarcoding approaches to biodiversity monitoring. In DNA-barcoding, a single individual is analyzed, whereas metabarcoding can analyze whole communities by using the power of next-generation sequencing technologies.

References:
Hebert et al. 2003. Biological identifications through DNA barcodes. Proc. R. Soc. Lond. B 270, 313–321. doi: 10.1098/rspb.2002.2218

Metabarpark at the 6th International Barcode of Life Conference at Guelph (Canada)

Metabarpark Project will be present in the 6th International Barcode of Life Conference that will be held at Guelph (Canada), next August 18th - 21st 2015. Our postdoc researcher Owen S. Wangensteen will be presenting our most recent developments of metabarcoding pipelines to study the biodiversity of marine hard-bottom communities with a level of detail never seen before.

Working with the second gene

In March we received the results of the Illumina run performed on our samples amplified for the COI gene. Unfortunately the quality of the reads was no good enough and we had to discard many of them. This led us to ask for a repetition of the run with a new protocol for preparation of the genetic libraries. Fasteris was up to the challenge and repeated the run for free. The results of the new run arrived in May, and this time most of the reads could be used.

With another 14 milion sequences to work with, we are still adjusting our pipelines to meet the specificities of COI. We had to construct a new reference database using the Barcode Of Life Data (BOLD) systems. We hope to have results of this second gene in short!

Recent dissemination activities of Metabarpark

We are committed to disseminate our results to management bodies, researchers, and to the grand public. For this reason, we have recently engaged in a series of divulgative actions that we would like to share here:

# In the recent edition of the International Festival Pint of Science, intended to share scientific knowledge in the fun and relaxed atmosphere of pubs, Xavier Turon presented a talk (with a beer at hand, for sure) at the bar Cara B in the district of Gràcia (Barcelona), the 18th May. The talk was about "deciphering the enigma of biodiversity with genetics", and a good deal of it was centered on the progress of Metabarpark. Cara B was fully packed with interested people!

 http://pintofscience.es/event/barcelona-cara-b-falta-2/

Billboard anouncing the Festival 

# Likewise, Xavier Turon delivered a talk about the project to the managers and staff of the Organismo Autónomo Parques Nacionales (OAPN), funder of Metabarpark, in Madrid, the 22nd May

# Owen Wangensteen participated in the XV International Echinoderm Conference, held at Playa del Carmen, Mexico (25th-29th May), and presented a talk about the applicability of metabarcoding to echinoderms, stemming largely from results of our Project: "Metabarcoding of Echinoderms, promises and pitfalls"

# Xavier Turon spent a couple of weeks at the National Oceanography Centre (NOC) in Southampton for sampling and manuscript writing with colleagues. He was the speaker at the Friday Seminar of the 5th June presenting the talk "Biodiversity crisis and taxonomic impediment: what the genetic metabarcoding of marine communities has to offer", where he presented results of Metabarpark

We made it to the front page of Faro de Vigo

The second sampling trip to the Cíes Islands was showcased by the newspaper Faro de Vigo in his front page of the 15th May edition, followed by an interview to Xavier Turon in the inner pages

http://www.farodevigo.es/gran-vigo/2015/05/15/csi-remata-criba-cies/1240243.html

We are happy to collaborate with Alberto Otero from Faro de Vigo and we acknowledge his help in disseminating what we are doing to the wide readership of this newspaper

Second sampling in Cíes

In May 13th-16th, members of the Metabarpark team (Creu Palacín, Owen Wangensteen, Xavier Turon) went to Islas Cíes again for a second sampling. Our goal was to assess seasonal differences in the communities, by comparing this sampling with the one done in September.
We are thankful to Alex Macía and Buceo Islas Cíes, who provided logistic support and took us with his boat every day from Vigo to the islands.
We re-sampled the photophilous and sciaphilous communities, as well as the Maërl bed and the Lago dos Nenos sediment, at exactly the same places as in September. In addition, we obtained samples from a shallow community of Asparagopsis armata, an introduced seaweed.

From left to right, Xavier, Owen, and Creu ready to leave from Vigo

The samples will be processed in exactly the same way as those from September, and we expect to find significant changes in species composition and relative abundances linked to the seasonal component... let's see!

In the news again!!

METABARPARK was again in the news (El Faro de Vigo) this last week-end.  Thanks to Alberto Otero for his editorial work

Here are the links:

http://www.farodevigo.es/gran-vigo/2015/04/05/xavier-turon-inventario-biodiversidad-base/1214419.html

http://www.farodevigo.es/gran-vigo/2015/04/05/csic-identifica-huella-genetica-3000/1214431.html

Got a second gene!

We could finally amplify a second gene from the DNA of the samples collected last year. This was a fragment of the mitochondrial gene Cytochrome Oxidase I (COI). This gene is the standard for the Barcode of Life project (at least for metazoans), but problems with the universality of the primers and the obtained sequence length made it less used in metabarcoding applications.

The agarose gel of the COI amplification

Starting from published primers and modifying them through database mining, we obtained highly degenerate primers that amplified successfully in all samples. We will now send this DNA for high throughput sequencing

We cannot wait to have the results and compare them with what we get with the 18S gene. It is reasonable to expect that a multi-locus approach will give us the best picture of the biodiversity present.

The comparison of molecules with morphology

One of the objectives of the project was to validate the obtained results from molecular data by using a traditional morphological approach. We welcome in the project Manuel Orobitg, a student in the Master in Oceanography and Marine Environment Management of the University of Barcelona. In his MSc Thesis Manuel will analyse morphologically the samples from the Maërl community in Islas Cíes and Cabrera that were collected at the same time than the samples for genetics and were preserved in formalin.

Manuel will identify morphospecies of the major groups, and will identify to species level the polychaetes with the help of Dr. Daniel Martin. He will also obtain sequences from the main species so that we will be able to identify them unequivocally in our genetic metabarcodes.

Aspect of a Maërl sample

Manuel in action sieving samples

We expect that this morphological dataset will confirm the usefulness and completeness of molecular techniques, but it might also uncover any bias, if present.

We filtered the samples with the same protocol used for the genetic samples, and Manuel is now doing the long and time-consuming process of sample sorting and group separation...best of lucks, Manuel!

Drowned in sequences!

Well, we did receive in February the sequences of 18S of the first round of samples from Islas Cíes and Cabrera... not bad to start with: over 14 million sequences!!

Now we are working in the analyses of this huge amount of data. We had to filter out bad quality reads, sequencing errors, and contaminants. Then we combined all sequences that were equal.

From this we went on and grouped similar sequences in Molecular Operational Taxonomic Units, our proxy for species, and our raw data for ecological analyses. These MOTUs were assigned a taxonomy using reference databases in GenBank

We had to fight a little bit with the different programs and pipelines, we wrote some code, and kept the processors of the CEAB computer cluster busy for some time. But we got finally over 4200 MOTUs!!! our preliminary analyses showed a clear distinction in biodiversity structure in both parks, and also between communities.

We are excited about the amount of MOTUs we obtained, and are looking forward to start the ecological/biodiversity analyses... will keep you updated!