Galindo LA, Puillandre N, Strong EE, Bouchet P (2014) Using microwaves to prepare gastropods for DNA barcoding. Molecular Ecology Resources, 14(4): 700-705.

This paper is so simple, yet so epic in so many ways:

We have experimented with a method traditionally used to clean shells that involves placing the living gastropods in a microwave (MW) oven; the electromagnetic radiation very quickly heats both the animal and the water trapped inside the shell, resulting in separation of the muscles that anchor the animal to the shell. Done properly, the body can be removed intact from the shell and the shell voucher is preserved undamaged.

To reiterate: these researchers put snails in the microwave and got a paper out of it. Now of course, this is actually a brilliant method – the scientists stumbled across this quick fix because they need to preserve BOTH the shell and DNA from their specimens. With such thick shells, preservatives can’t get into the tissue very easily, and other methods (boiling the snails alive! or using chemical relaxants to pull out the muscle) are time consuming, sloooowwwwww, and downright dangerous:

To some extent, [these methods] can also represent a hazard (electrical drill and boiling water) on an unstable research vessel at sea.

Microwaves can zap lots of animals quickly and keep all their DNA intact!

This paper also wins for the most unnecessary use of acronyms, shortening the terms for microwaves (MW) and microwave ovens (MWO). So in everyday life I guess we can now further reduce MWs to a hand signal, and just say that we’re going to heat up our coffee in the “Muah”.

The post Putting snails in the microwave…for science! first appeared on Deep Sea News.

Everyone knows I despise charismatic megafauna (especially dolphins). I will now secretly admit that I also don’t care much for charismatic invertebrates. I mean, Yeti crabs are pretty much the Lindsay Lohan of marine creatures – they’re just too damn attention-seeking with their bristly claws and all. And they’re everywhere…

What I do love are cryptic species – nondescript, gooey specimens that all look the same, but have surprisingly distinct genomes. Candlelight and soul forever, I dream of myself and them together!

Looking at the DNA of microscopic animals is full of mystery and intrigue – it feels a bit like solving a crime (albeit a PG-13, Nancy Drew type of crime). Traditional taxonomy just doesn’t have that thill for me – it is slow and steady, requires hours under the microscope, and has an unsettling degree of subjectivity. DNA, on the other hand, is extremely objective – you can’t argue* with good data sequenced from a trustworthy spot in the genome. Those ATCGs just don’t lie.

But that doesn’t mean DNA is a magic bullet. DNA provides a new type of evidence for making decisions about species, but that evidence still has to be robustly analyzed and interpreted in the context of historical knowledge (taxonomic classifications and anatomical features of specimens).

A recent paper by Jörger et al. (2012) displays the power of DNA to differentiate amongst gelatinous blobs. The authors analyzed molecular evidence alongside body features to investigate sea slug species in the genus Pontohedyle, a group which taxonomists thought they had described LIKE A BOSS. Anatomy didn’t tell much of an exiting story – specimens could be lumped into two different groups based on appearance, and both these species were presumed to live pretty much anywhere in the ocean. [Sidenote: this is a pretty common assumption for microscopic animals and is known as the “meiofaunal paradox”. Victorian scientists would think “Oh, they all look the same, so lets describe these as the same species.” because they didn’t have any badass knowledge about DNA, and they didn’t have a time machine. But nowadays, this thinking is in direct conflict with our knowledge about these species–microscopic animals have been shown to have limited ability to move around, even during larval stages. They pretty much get stuck in the marine neighborhood where they grew up, so how can they spread their sweet DNA all around the world?]

The researchers started to look a little bit closer, baby, and study how sea slugs get it on, get it on. Because…

Uncovering putative cryptic lineages is fundamental not only for our advances in understanding speciation processes in meiofaunal taxa [microscopic multicellular animals <1mm long, such as nematodes, copepods, and tardigrades], but also to understanding historical biogeography.

Jörger et al. analyzed the sea slug DNA in every way they could think of. First off, they looked at the story from three different genes: Cytocrhome c Oxidase subunit 1 (the “barcoding” locus in the mitochondrial genome), 16S rRNA (a gene encoding a ribosome subunit in the mitochondria), and 28S rRNA (a ribosome subunit gene in the nuclear genome).

All three genes told the same story. The results of different data analysis methods were consistent. And wow, they sure threw all the data analysis acronyms they could think of at that DNA:

…we apply four independent methods of molecular based species delineation: General Mixed Yule Coalescent model (GMYC), statistical parsimony, Bayesian Species Delineation (BPP) and Automatic Barcode Gap Discovery (ABGD). The secondary species hypothesis (SSH) is gained by relying only on uncontradicted results of the different approaches (‘minimum consensus approach’).

In the end, 2 morphological species became 12 cryptic species (each color in the this tree below representing a different cryptic species):

The power of antatomical features was, er, not so powerful after all.

Previously used external morphological characters such as the shape of oral tentacles, body length and width, or color of the digestive gland (e.g., [68]) depend highly on the stage of contraction and nutrition, and are variable through time for each individual [40,41] and therefore inappropriate for species delineation.

The color of the digestive gland?! Really, guys?

So think about what this means for our understanding of biodiversity on earth. This is just one study, from one genus out of thousands in the taxonomic classification of animals (and there’s still a lot of microscopic critters we haven’t even described). Evidence like this comes out all the time. For example, a study focused on the nematode Pellioditis marina (a well known estuarine species living in Northern Europe) found that this “species” is actually a complex of at least 4 genetically distinct cryptic species. Just when we think we’re making good progress on taxonomy, DNA is routinely telling us that we probably have to split every known microbial eukaryote species into 3-4 cryptic entities.

And on top of that, Jörger et al. remind us of how little of the earth’s surface we’ve actually sampled:

Although we present the first study on meiofaunal slugs with representative worldwide taxon sampling, we know our dataset is likely to represent only a fragment of the hidden diversity in the genus because a) tropical sands still are largely unsampled, b) suitable habitats are patchy and disjunct, and c) the indication of accumulated diversity in geographically small areas (e.g., three distinct genetic Pontohedyle lineages on the island of Moorea).

Scientists–and humans–are on an eternal quest to talk about species. It helps us organize our knowledge of the world, and helps us to understand historical processes which have influenced how things evolve and how they are distributed geographically. There’s always a lot of press surrounding the latest paper claiming the official and absolute estimate of the number of species on earth (here’s one hyped up example). Personally, I don’t take much stock in these, because I don’t think we have nearly enough information to even make ballpark guesses. We have NO genetic information about most microscopic animals on earth. Hell, the Guinea Worm can’t even get its genome sequenced. For small non-parasitic wormy things, we’re just lumping similar-looking blobs into categories after a quick peek down the microscope. Its like saying all blonde haired people belong to one species, and all redheads belong to another – a.k.a, surface appearances take precedence, end of story.

Next time you hear a “global species estimate”, don’t say you believe it, please don’t say you believe it!

*Ok, well you can argue, and people do. But that is another blog post for another day. Just work with me here, people.

Reference:
Jörger KM, Norenburg JL, Wilson NG, Schrödl M. (2012) Barcoding against a paradox? Combined molecular species delineations reveal multiple cryptic lineages in elusive meiofaunal sea slugs. BMC Evol Biol,12(1):245.

The post When 2 becomes 12: Cryptic species need some love like they’ve never needed love before first appeared on Deep Sea News.

When all the shiz went down in the Gulf of Mexico, yours truly and collaborators had their nose to the grind, madly running around collecting samples and spending late nights in the lab listening to the hum of PCR machines. We were awarded an NSF RAPID grant in August 2010 to use parallel taxonomic and high-throughput sequencing approaches to characterize the impacts of the Deepwater Horizon oil spill on microscopic eukaryote communities inhabiting marine sediments. In English: we used both DNA and old skool microscopy to compare species living on beaches before and after the oil spill. This grant funded some hardcore sampling trips (September 2010, where I went on a boat and drove 1700+ miles along the Gulf Coast in one week, and March 2011 when we returned to sample sites a year after the oil spill) and a kickass undergraduate workshop about the “Bioinformatics of Biodiversity” which may or may not have involved YouTube Karaoke sessions and a randomly acquired cowboy hat.

So today, I’m pleased to announce that the first (of hopefully many) papers from this project has officially been published in PLoS One. For our first manuscript, we focused on a set of pre- and post-spill samples collected around Dapuhin Island, Alabama in May and September 2010, respectively. Our pre-spill samples represented our baseline, collected by our collaborator Ken Halanych at Auburn University days after the oil started gushing (his team quickly drove down to the coast before any of it had come close to the shore). Post-spill samples were collected 4 months later, after sheets of sticky crude were pitched ashore during a summertime of heavy beach oiling and BP’s cleanup efforts doggedly wiped away the blackness. We also included another post-spill sample site from Grand Isle, Louisiana, where I suspected that the the state of the beach would produce some intriguing data.

Before I describe our results, I’ll show you what the beaches looked like at the time of post-spill sampling. In September 2010, Dauphin Island was pretty serene: quiet, yes, because tourists were eschewing the region, but the shoreline itself showed little evidence of the BP fiasco. If you looked closely (which I certainly did), you could find little splotches of oil, an isolated tarball, or a buried “dirty” layer of sand. But if you had spent the past year on a media blackout, never heard of Deepwater Horizon, you would think that the Alabama coast looked pretty ordinary.

Grand Isle was a scene from another planet. A far cry from the parallel, reestablished tranquility on other Gulf shores. Grand Isle was a beach that was clearly impacted at the time of sampling. In fact, I specifically made a 6-hour detour to this site after hearing local news reports lamenting those tarnished Louisiana shores. Upon my arrival, I found the beach to be so impacted that I was hardly allowed near the sea. The ranger on duty turned me away at the local park. Fluorescent orange mesh blocked my attempts to cross the dunes. When I finally found a gap that led me to water, I made haste for fear of being chased away. There was heavy machinery humming up and down the shore (the park ranger noted that beach access was restricted for safety concerns), and piles of oiled sand awaiting a mechanical cleanse.

At each site we collected replicate sediment cores for DNA work (frozen immediately on dry ice) and taxonomic analysis (archived in 4% formalin, which is better for preserving morphology). The sequencing itself was a piece of cake (although the bioinformatics, not so much). I carried out standard environmental DNA protocols at the Hubbard Center for Genome Studies at the University of New Hampshire, where I was previously working as a postdoc under Kelley Thomas (senior author on our paper). First we separated the microbial eukaryote species from the sediment by suspending them in water and concentrating these organisms on a 45um sieve. Next we broke open all the cells using a beadbeather: think “Will it Blend” with ball bearings and soft tissue. Nothing stays intact. Finally, we used the Polymerase Chain Reaction (PCR) to broadly amplify two different regions of the 18S rRNA gene from the entire biological community present at each sample site. PCR amplicons were sent off for 454 sequencing, and we waited. In the meantime, Jo Sharma at UTSA was spending long hours at the microscope carrying out taxonomic identifications for nematodes at each site being sequenced.

I’ll pause for a moment here to offer more context. When I say we are studying “microbial eukaryote” species, I’m talking about puny things with a body size <1mm. You know, the ones no one cares about. And the ones I happen to be obsessed with (they’re so much more interesting than dolphins). We’re talking about taxonomic groups like meiofaunal metazoans (e.g. Nematoda, Platyhelminthes, Gastrotricha and Kinorhyncha, etc.), microbial representatives of fungi and deep protist lineages (Alveolata, Rhizaria, Amoebozoa, algal taxa in the Chlorophyta and Rhodophyta, etc.), and eggs and juvenile stages of some larger metazoan species. The reason why we chose to focus on these groups is precisely because they tend to be ignored. Most of the awesome genomic investigations only look at Bacteria and Archaea. But small eukaryotes are equally ubiquitous as their non-nucleated counterparts, and in marine ecosystems they play key roles as decomposers, predators, producers and parasites–yet we know little about their biology, ecology and diversity. By describing species changes in the Gulf of Mexico, we wanted to infer something about the potential for large-scale or long-term repercussions for Gulf ecosystems.

Sorry to keep you waiting–lets get down to the juicy stuff. Our results were pretty dramatic. After analyzing 1.2 million DNA sequences alongside nematode taxonomy, we found shockingly significant shifts in microbial communities between pre- and post-spill sites.

The first thing we saw was a stark shift in the diversity and abundance of taxa between pre- and post-spill sites. Pre-spill sites showed a typical marine community: dominated by nematodes, but containing a mishmash of other taxa such as arthropods, polychaetes, protists, algae, and fungi. Post-spill sites, in contrast, were almost exclusively dominated by a few species of fungi, with a spattering of some other metazoan species.

After looking at the charts summarizing the overall taxonomic assemblages, we moved on to ecological analyses such as Unifrac. We built a phylogenetic tree with our DNA sequences, computed some metrics about the branching topology, and got an overarching indication of how similar our samples sites were at the community level (e.g. for all species sequenced at each site).

We also did something similar with taxonomic data, using the Bray-Curtis similarity metric to analyze the list of visually-identified nematode species which were present (or not) across our sample sites.

Both our DNA and taxonomic analyses were relaying the same story: our samples clustered together according to pre- and post-spill time points: the before/after communities at the SAME site weren’t closely related to each other. Principal Coordinates Analysis (PCoA, Unifrac figure B, above) also underlined biodiversity distinctions across pre-spill sites. Even though pre-spill sites were characterized by nematode dominance, it wasn’t the same group of nematode species present at every site. In contrast, post-spill sites converged towards a similar community structure–these trends were likely driven by oil-associated fungal taxa that were common across post-spill sites.

You’ll also note that we observed a few outlier sites; Ryan Court (a sandy beach in front of residential property, on the Gulf coast of Dauphin Island) and Dauphin Bay (an inlet on the opposite site of the island, facing the Alabama mainland). Although we did observe community shifts in these post-spill samples, the shifts weren’t characterized by the typical fungal dominance seen at other sites. We think this has something to do with the geography and human-mediated cleanup efforts. To protect residents, Ryan Court had waterborne barriers going up and down during the heaviest oiling–this might have mitigated the worst effects in the sediment perhaps, preventing a shift to fungal dominance. Oil also might not have penetrated the inland Dauphin Bay site very well, since it was inherently sheltered by its location and some nearby marshland on Dauphin Island.

For me, the most convincing evidence of oil impacts was the data from Grand Isle. That 5-hour drive (and accompanying True Blood soundtrack) was the best sampling decision I made. Although I was pretty scared of Vampires when I was driving back through Louisiana that night. The beach was unarguably facing heavy oil impacts when I took samples. DNA analysis showed that the fungal-dominated post-spill assemblage in LA contained the same taxa as the Shellfish Lab, Dauphin Island community. Same putative species (Operational Taxonomic Units, a.k.a. OTUs), found 250 miles apart. To the casual observer, the scene at Dauphin Island didn’t look anything like Grand Isle. But thanks to the deep insight afforded by high-throughput sequencing, we were able to capture a snapshot of post-spill microbial assemblages that was highly indicative of environmental disturbance.

So we started looking closer at the data. Looking within phylogenetic tree topologies, I manually examined what taxa were most closely related to our fungal OTUs. Evolutionary relationships seemed to hint that post-spill fungi could survive using environmental hydrocarbons as an energy source:

Two distinct fungal community structures were recovered at post-spill sites: one assemblage dominated by Cladosporium OTUs (recovered at Shellfish Lab and Grand Isle), showing a close relationship to C. cladosporioides sequences in phylogenetic topologies, and a second assemblage dominated by OTUs in the fungal genus Alternaria (Belleair Blvd and Bayfront Park).  Fungal taxon dominance may be dictated by the physical marine environment; Alternaria OTUs dominated in brackish Mobile Bay, while Cladosporium was recovered in higher-salinity sediments on the outer shores of Dauphin Island.  These highly dominant post-spill OTUs appear as rare taxa in diverse pre-spill fungal assemblages, suggesting that oil-induced environmental stress may have favoured the rise of resilient, opportunistic species (able to capitalize on the large input of new resources).  Although the diversity and ecological role of marine fungi is not well understood, previous evidence suggests that observed fungal assemblages denote a signature of crude oil in Gulf sediments. Cladosporium contains ubiquitous, opportunistic species that can extensively utilize hydrocarbon compounds and thrive in hostile, polluted conditions that appear to be intolerable for other marine fungi [9,10].  Compared to many other fungi, marine Altenaria demonstrate increased activity of lignocellulose-degrading enzymes [11] that have been implicated in breakdown of industrial toxins [12,13].  In addition to these dominant OTUs, we recovered a variety of fungi at post-spill sites (including OTUs phylogenetically related to Apergillus, Acremonium, Acarospora, Rhodocollybia, and Rhizopus) that rarely comprised a significant component of pre-spill fungal communities. A number of these marine groups have also been shown to metabolize hydrocarbon compounds [14,15]. (Bik et al. 2012)

This paper has been a long time coming, and I’ve been dying to blog about it for the better part of a year. We went through the rounds and rejections at several top-tier journals before a lengthy review process at PLoS ONE, so I’m now pleased that these results are finally seeing the light of day. Our work in the Gulf of Mexico is still ongoing — unfortunately this was one study that raised a hell of a lot more questions than answers. We’ve continued to collect post-spill samples at regular intervals (including the samples I collected one-year after the original pre-spill samples). We want to figure out if the community shifts we saw in this study were really due to oil (as suggested by the dominance of oil-associated fungal taxa), mechanical beach cleanup efforts (which may have physically damaged and killed fragile microbial species), or whether they might be influenced by seasonal and temporal variation in the Gulf region (a topic where there isn’t much existing data). Another motivation is to study the longevity of these patterns–additional sampling time points will allow us to track the post-spill recovery, or lack thereof, of microbial eukaryote communities. Will assemblages begin to resemble pre-spill communities again, or will these beaches remain depauperate and/or be replenished with a different set of fauna? The post-spill fungi are cool and intriguing too. Were they thriving in oiled beach sands, or just weakly persisting after other species were killed off? Transcriptomics (studying gene expression by sequencing mRNA) will help us to answer this question and determine the species that were alive and kicking at the time of sampling. We’re also going to use random, shotgun sequencing to look deeply into the genomes of sparsely-populated, fungal-dominated beaches at sites such as Grand Isle: if post-spill species are eating hydrocarbon compounds, perhaps their genetic machinery will give an indication of the metabolic pathways that enable them to use oil as an energy source.

So really, this first paper is just a prelude. The main act will be as grand as Beethoven’s 5th.

Reference: Bik, H.M., Halanych, K.M., Sharma, J. & Thomas, W.K. (2012) Dramatic shifts in benthic microbial eukaryote communities following the Deepwater Horizon oil spill, PLoS ONE http://dx.plos.org/10.1371/journal.pone.0038550

The post Dramatic impacts on beach microbial communities following the Deepwater Horizon oil spill first appeared on Deep Sea News.

The western Pacific is broken land, plates are crashing every which way creating earthquakes and volcanoes from Russian Kamchatka to New Zealand. At these volcanic arcs exist a unique and abundant flatfish at hydrothermal areas. Symphurus thermophilus was described as one species along the entire western Pacific volcanic arcs, which begged the study that Tunnicliffe and colleagues did – study how divergent population of this species are! The authors used 2 genes, COI and 16S, both commonly used in population genetics studies, on Symphurus species from near Japan and near New Zealand. They found that between those individuals inhabiting the Mariana Arc and those inhabiting the Tonga-Kermedec arc, population were diverged by 9% and 14% for COI and 16S, respectively.  Previous Barcode of Life projects found 3% (butterflies) to 9-10% (fishes) divergence to be sufficient for delimiting new species. Within each arc system, populations appeared to be well-mixed, but there are indeed migrants between arcs.

Why is this study cool? Besides their barcoding of the vent flatfish from Japan and New Zealand vent, they used a real integrative approach with morphology, gut contents, ecology and behavior to conclude that this is likely to be 2 or 3 species total.

Tunnicliffe V, Koop BF, Tyler J, So S (2010) Flatfish at seamount hydrothermal vents show strong genetic divergence between volcanic arcs. Marine Ecology doi:10.1111/j.1439-0485.2010.00370.x

This paper by Milner and colleagues studied whether male fiddler crabs could sense there were receptive females around when they could no see them by observing the courtship displays of neighboring crabs (the fiddler crab claw wave). In a simple, but elegant, experiment they placed a barrier in front of the males so they could not see if receptive females were present or not. They found that the number of claw waves by males increased (see graph on left) in the presence of females with or without barriers. This means that they were eavesdropping on the courtship behavior of their neighbors in order to prepare themselves for waving their arms about madly to attract the opposite. Sound familiar?

Why is this study cool? OK, let’s be honest here, I picked this article because of the title: “Eavesdropping in crabs: an agency for lady detection”. WIN!

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Is it a coincidence that there were mass die-offs of urchins off Nova Scotia after 2 eastern US hurricane landfalls? Scheibling and colleagues find it curious at least and show interesting data correlating the timing of two hurricanes and the urchinicide, which occurred 2-3 weeks after the hurricanes made landfall. They found that disease-causing amoebas were present in dying urchins, confirmed it by taking urchins form colder waters, a more harsh environment for the Paramoeba to develop in, and exposing them to water from infected urchins. All urchins died off between 17 and 21 days from all treatments.

Why is this study cool? They show that “… hurricane-induced mixing can deliver a nonresident pathogenic agent to the Atlantic coast of Nova Scotia.” If a pteropod flaps its mantl “wings” off of Florida is there a mass urchinicide in Canada?

The post The Tide Pool: Divergent Flatfish, Eavesdropping Fiddler Crabs, Hurricanes Kill Urchins first appeared on Deep Sea News.

“… join scientists from South America, UK and USA to identify key opportunities for using sailing vessels for modern research with a focus on marine biology, the Barcode of Life and Earth monitoring. As part of the British Council-supported workshop, participants will embark on two short scientific voyages aboard the Brazilian tall ship Tocorimé into coastal waters to coincide with passes of the International Space Station, and local schoolchildren will experience a real-time Q&A session with astronauts aboard the station.”

The workshop participants came from Brazil, Venezuela, Chilé and Argentina. Representatives from other countries were invited too, but were unable to come and unable to find replacements on short notice. It was a great group and each person had a lot to offer.

The morning started with introductions Markus Lehmann and Michal Nesvara of the Tocorimé. Unfortunately, I had to miss Markus’ presentation because I needed to set up and test out equipment for linking up to astronaut Mike Barrett in the International Space Station (lame excuse I know ;p). I managed to catch most of Michal’s talk though and it was very fascinating and inspiring. He and some colleagues rebuilt Magellan’s Victoria and retraced his route around the world. If you can understand Czech you can read about it here, there are also some pictures of the boat. The video was great and there is a wonderful book about the expedition. Unfortunately it is in Czech and only a small number of copies came off the press. I hope he translates it though as I think it could sell more copies in English speaking countries as a fun travel narrative.

Afterward we went around the room and heard small presentations about everyone and what their research interests were. Lots of specialties and taxa were represented. Some were ecologists, some were purely taxonomists, some were both. Some did not do any molecular biology or DNA barcoding, some did mostly population genetics, some did a little of everything. Organizations, besides the universities where people worked, represented were NASA, Census of Marine Life and Consortium for the Barcoding of Life. It was a nice amalgamation where everyone had something unique to bring to table. Much of the discussion was focused on how sailing ships could be used in the research or educational programs, focused on the Tocorimé.

The outcomes of the meeting will hopefully be published in a peer-reviewed journal as an editorial or perspective. We agreed that a network of South American marine scientists was needed and they would seek funding and more collaborators to use the Tocorimé as a science and educational flagship. A theme along the lines of “biodiversity without borders” was chosen as a unifying research framework. The participants were very enthusiastic and are doing great research that really compliments each other. It will be exciting to see how it all will unfold in the coming months as we work out the details!

The post Darwin and the Adventure – The Workshop first appeared on Deep Sea News.