Category Archives: BOLD

Guest researcher: Marla Spencer

Ready for fieldwork!

Marla, a PhD student supervised by Dr Tammy Horton (NOC), Dr Andrew Gates (NOC), Dr Lawrence Hawkins (UoS), Dr Miranda Lowe (NHM) and Dr Gordon Paterson (NHM) has spent 6 weeks in the invertebrate collections at UiB.

Marla was studying the amphipods from the family Phoxocephalidae from the Western African Waters, focussing particularly on the amphipods from the sub-family Harpiniinae [crustacea; Amphipoda; Phoxocephalidae; Harpiniinae].

: Basuto stimpsoni, barcoded

: Harpiniopsis sp, barcoded

: Metaphoxus sp., barcoded

Phoxocephalid amphipods are highly speciose and abundant in deep sea sediments globally. Species identity is critical to understanding mechanisms driving observed biodiversity patterns and to asses community change. The aim of the project while in Bergen, was to use both DNA barcoding and traditional morphological taxonomic approaches in order to create a robust library of Phoxocephalidae species from the poorly known West African waters. Large scale projects such as Marine Invertebrates of West Africa (MIWA) provide the perfect opportunity for collaborative work.

The MIWA project submitted over 2700 tissue samples from over 600 morphospecies for DNA barcode sequencing, including Crustaceans, Echinoderms, Molluscs and Polychaetes. Out of these, 45 samples were from the family Phoxocephalidae, the target taxa. Working with Dr Anne-Helene Tandberg and Prof Endre Willassen, the sequenced MIWA Phoxcephalid voucher specimens were dissected and mounted as permanent microscope slides to morphologically score them. Later, the phylogenetic analysis based on molecular and morphological characters will be compared. Each appendage was photographed on the modular (Leica CTR6000) microscope and the images were stacked, resulting in incredible photos!

Harpinia abyssi P7. Photo: M. Spencer

Harpinia abyssi Photo: M. Spencer

Out of the 2700 tissue samples, a total of 1450 sequences were obtained (54% sequencing success rate). This is not uncommon as the ‘Universal’ barcode protocol often needs to be adapted for different taxon groups.

At work in the DNA lab

Working with Anne Helene within the molecular biology labs at the University of Bergen, currently developing taxon specific primers and PCR conditions for the Harpiniinae MIWA specimens which were not successfully sequenced with the Universal primers. As a starting point, an additional 13 MIWA specimens had tissue extracted for DNA, then dissected and permanent slides were made in order to morphologically score them. Each appendage was photographed and the images stacked. At this time the primers and PCR conditions are a work in progress, but we will keep you posted. However, this was a very successful trip resulting in a lot of data to analyse!

-Marla

DNA-barcoding: update

Yesterday, Endre and I attended an event where the different units of the University of Bergen were invited to “..present ongoing digitalisation projects, tools and methods, and future digital solutions.”

We brought with us a poster titled “Data con carne – sources of new knowledge on biodiversity” (in Norwegian), where we presented how our barcoding efforts on both African and Norwegian material are parts of a global undertaking of building a “library of life”, and how using huge databases such as BOLD can help us gain better understanding of biodiversity – and on where to focus our efforts in unraveling the taxonomy.

There is a very real challenge connected with estimating biodiversity when many of the species are still undescribed, as is the case with many invertebrates, especially the more obscure and diminutive groups. In such cases, barcoding can serve as a tool in screening for biodiversity, and aid the taxonomists in identifying areas where the taxonomic resolution is poor.

There is a global effort underway to establish a library of short, species specific genetic sequences. These standardized genetic sequences (“barcodes”) consists of a segment of approximately 650 base pairs of the mitochondrial gene cytochrome oxidase c subunit 1 (COI). You can read more about DNA barcoding on WIKIPEDIA.

For MIWA, we have submitted over 2700 tissue samples from over 600 morphospecies for DNA barcode sequencing through the BOLD-pipeline, where the lab work is done in Guelph, Canada, and the data is uploaded to the Barcode of Life Data Systems (BOLD Systems) .

Out of these, 1450 sequences have been obtained (54% sequencing success rate), and these cluster into close to 550 BINs (OTUs) – suggesting that the diversity may be (much!) higher than what our preliminary investigations reveal. This is not so surprising, considering how hugely diverse and little studied the invertebrate fauna of the GCLME and the CCLME is.

Below is an interactive map of the submitted samples, you can click on the stations to see the specimens that have been sent from that location, and whether or not they have gotten a barcode. You can also follow this link to find the map

What we find is that it is crucial to be able to go back and reexamine the material post-barcoding, and that is where the “con carne” part of our poster came from;

Our barcoding revealed several examples of mismatching taxonomic identifications amongst researchers in different labs and institutions in different countries. This illustrates a major challenge, as it has been shown that when benthic fauna is used in standardized methods for quality assessment and monitoring, mismatching identifications produced by different identifiers can have strong effects on indexing and valuation of ecological conditions in the same habitat.

For DNA barcoding to be useful, it is absolutely neccessary that the correlation between species name, specimen, and barcode is correct (or at least as close to it as we can get, if the species is undescribed).

We must first build this reference library before barcoding can be reliably used for identification of unknown organisms. Hence it is imperative that the voucher specimens that correspond to the genetic barcodes are deposited in a museum where it will be preserved and made available for research.

-Endre & Katrine

Indeed we did!

(get DNA from our faded stars, that is)

As explained in a previous post, we submitted tissues samples from the sea stars (class Asteroidea) that were suitable for DNA-analysis to the CCDB lab and the BOLD database. We just recieved the preliminary results, which are very promising!

The next step now will be to collaborate further with our asteroid taxonomist, and evaluate the genetic data combined with traditional morphology based taxonomy. Combining barcoding and morphology in this way helps us explore and understand the biodiversity better.

Stay tuned for updates!

Will we get DNA from our faded stars?

We have done substantial amounts of COI barcoding on various animal groups through the MIWA-project. You can find all the specimens that we have submitted for barcoding here. Of the Echinodermata we have previously submitted Echinoids (sea urchins) and Ophiuroids (brittle stars). Currently we are focusing on the class Asteroidea, the sea stars.

There were not terribly many sea stars in our material, and all of the Asteroids were identified when we had asteroid specialist Anna Dilman visiting last spring.

Part of this material is fixated in ethanol and therefore available for genetic work, and we’ve been waiting for some more material to come along so that we would have enough samples to fill the 95 wells in a plate for barcoding and uploading to the BOLD-database. Now we’ve gotten some supplemental material, and are preparing a plate of mainly Asteroidea. We are also including a few brittle stars, as we had six Gorgonocephalidae (basket stars) waiting to be barcoded, and they are plain too cool to pass up. We did a blog post about basket stars in our InvertebrateCalendar, click here to read more about the head of the Medusa.

Why “faded”? Well, in real life they are amazingly beautiful critters, looking something like this:

By Philippe Guillaume – Flickr, CC BY 2.0, https://commons.wikimedia.org/w/index.php?curid=29948486

But once a specimen of the same species – this is a Astropecten aranciacus – has been dragged up by a trawl and marinated in ethanol for a while, it looks more like this:

Astropecten aranciacus from Sao Tome & Principe, collected at 54 m depth

Thankfully, the colour loss does not mean that the animal is “ruined” – it still retains its key identifying characters and DNA – they just looks a bit less exiting for us non-sea-star-experts!

The cast of characters so far – there’s a few waiting to be photographed still

We’ll finish in the “photo booth” and get the tissue sampling done over the next couple of days, and hopefully our faded stars will shine (as barcode vouchers) after all!

Stay tuned for updates.

#InvertebrateCalendar

We are running a calendar count down from the 1st to the 24th of December on our collections blog, and MIWA is frequently featured!

Some of the topics covered in the first half – ranging from sea spiders to sciart!

Click to have a look

Guest researcher: Mario

Mario

Mario (who also visited us in January) came back to continue his work on the Terebellids and Pista (polychaeta) in October. In his own words: This time, I take to my home two papers close to completion; one about species of the genus Pista (Terebellidae) with additional information to what I found during my last visit in January. The second paper is about species in the subfamily Polycirrinae (Terebellide) from the West coast of Africa.

Pista cristata

The idea is combine drawings, digital photos of specimens with methyl-green staining pattern and SEM pictures, as well as molecular information that will hopefully help us separate species and make better estimates of the region’s biodiversity.

You can read more about Mario’s visit in our Invertebrate Advent Calendar, which is running from December 1-24th on our collections blog. Click here to find all the calendar posts!

Mapping our barcoding efforts

Here is a interactive map of all the samples (2175 as we speak!) that we have submitted to the BOLD-database for genetic barcoding.

You can also follow this link to find it.

All the stations from which we have submitted specimens for barcoding

By clicking around on the map you can see how many specimens we have submitted from each station, as well as photos of the animals and wether or not the sequencing was successful and resulted in a genetic barcode.

By clicking on a station, you get the information about which animals have been sequenced – here two brittle stars that were both successfully barcoded

The samples we have submitted (so far – there are still plans to do more!) include several animal main taxa; Crustaceans, Echinoderms, Molluscs and Polychaetes. These animals have been sorted out and identified by employees at the invertebrate collections, and by visiting guest researchers who have come here to work in the material – so it is very much a combined effort behind this.

# of submitted specimens (animals) from each phylum

Not all our material is suited for genetic analyses; fixation in formaldehyde gives well preserved specimens that are well suited for morphological examinations – which is the backbone of taxonomy – but it distorts the DNA so that the samples are not eligible for molecular work.
Provided that the material has been fixated in a DNA-friendly way (i.e. in ethanol), there is a lot of work to be done before we are left with identified specimens. We wrote a bit about the sorting of samples her: “biodiversity in a dish”.

We are still working actively with this material and with the results we are getting – some of it has already been published – se our list of publications here – and more is on the way.

New species of West African snails

Our studies of mollusks have revealed new species of philinid snails. They are described in a paper that was published today in the Zoological Journal of the Linnean Society with open access: http://onlinelibrary.wiley.com/doi/10.1111/zoj.12478/full

We used both morphological techniques and DNA-based species discrimination methods in this study and DNA-barcodes have been uploaded to the BOLD-database. Laona nanseni was named as a tribute to the Nansen programme and Philine schrammi in honour of J. R. Schramm, who founded JRS Biodiversity Foundation, an important sponsor of our work.

Laona nanseni new species

Philine schrammi new species

New knowledge on the Glyceriformia through an integrated approach

Integrating morphological studies with DNA barcoding is indicating significantly higher species diversity than previously known in the polychaete families Goniadidae and Glyceridae (Glyceriformia) from the Western coast of Africa.

Our multi-toothed acquaintance from a previous blog post makes an appearance here, together with a multitude of other species. The animals were initially identified to species or genus level using available literature with keys or morphological descriptions. Several specimens did not match any species description and may be new species.

For each recorded species, a number of specimens were selected for DNA barcoding and uploaded into the BOLD database. You can read more about how this work was carried out in this blog post from when guest researchers Lloyd and Williams visited in November 2015.

Our preliminary findings were presented as a poster at the 12^th International Polychaete Conference in Cardiff, Wales during the first week of August. You can read about the University Museums attendance here. Next we are hoping to invite one of the taxonomic experts on the group to come visit and work on the material – there is certainly a lot to be done!

Glyceriformia©University_Museum_of_Bergen

The poster presented at the IPC2016. Click to enlarge!

Mysterious limpet

Management of species requires information about their habitat, ecology, population size, geographical range, exploitation and environmental threats. It usually should go without saying that confident species identification is a key factor in the acquisition of such knowledge. DNA barcoding may help to establish a relative objective identity tag on taxa and to place local and regional populations in a global context with their closest relatives.

We recently published a paper about a species of Cellana that is closely related to C. toreuma in the Indo-Pacific. More research is needed to assess if the Gulph of Guinea population is a separate species or a population with relatively recent connections to the Indian Ocean.

The paper and Additional file are Open Access here:

http://mbr.biomedcentral.com/articles/10.1186/s41200-016-0059-9

Cellana species near torreuma from Nigeria

Cellana species near toreuma from Nigeria

Marine Invertebrates of Western Africa

– an ongoing survey