Work has continued on the DNA extraction and barcoding front, and once things were working this all scaled up rapidly. I have now done 770 DNA extractions (650 of those in the last 4 months), with the main focus being on the fish larvae. In total, I had nearly 4000 fish larvae that were looked at ‘fresh’ (within 2 days of the sampling day, before preservation), and all of this DNA extraction and the subsequent PCRs have been to get an idea of which fish species are present in the different sampling months. Using the data, I can also see which species are more abundant, and how the abundance and diversity of the ichthyoplankton community changes. I have sent off 4 PCR plates for DNA barcoding (a plate is when you send off 96 samples at once, rather than individually sending off samples), so have the DNA results for around 400 fish larvae. With so many barcode results coming back, I am now able to compare the genetic results to the photos I took of the fresh larvae and can combine this data to confirm what the different fish larvae species look like. All of this identification data has been showing some really interesting results, which has been great fun to look at.
Fish larvae identified using DNA barcoding
Example of where species are branching unexpectedly on the phylogenetic trees
Red cod egg - you can make out the larvae on the yolk sac!
So far, there have been 19 confirmed species according to the matches of my samples to those on GenBank (a genetic database where people submit their barcode results). As well as comparing my results to these samples, I also put my results on phylogenetic trees to make sure I can trust the result. This is because when I’m comparing my results to the database, I am limited by what samples are in the database – so the top species match will be the closest genetic match to my sample, but if my sample is a species that hasn’t been uploaded to GenBank, the top result won’t be the same species. This has been less of a problem with the fish larvae (fish are generally better studied than invertebrates) but is still something worth considering and checking! Making the phylogenetic trees for the fish has been especially interesting for the Patagonotothen (rock cod) species, as there are a couple of samples that have the same top species match, but branch separately, suggesting that they may be different species! If this is true, then I have at least 11 Patagonotothen species (rather than 8) in these samples, and at least 21 different fish species are using the coastal environment of the Falkland Islands during their early-life stages!
The Patagonotothens weren’t the only interesting fishy finds. As well as fish larvae, I also had a few fish eggs, which were more difficult to successfully get DNA out of but, when they finally did work, were confirmed to be the eggs of red cod. My favourite find was to do with lantern fish larvae! In previous blog posts, I’ve mentioned that we found a couple of different species of lantern fish which were both in autumn / winter samples (the quiet period for fish larvae). I was very excited when the DNA results started coming back from some of the spring samples that a third species of lantern fish was also present!
These are particularly exciting as lantern fish are a deep-sea species and got their name due to the presence of bioluminescent photophores along their body. As they live in deeper waters, less is known about them, so it was really interesting to be finding them in coastal waters.
As well as fish, I have been working on extracting DNA from other species within the zooplankton. There is a reason why this has mainly been about fish though – unfortunately, the invertebrates have not been behaving nearly as well as the fish, so I’ll have to pick up the PCR trial and error with these again in the New Year!