“The Darwin Tree of Life (DToL) project aims to sequence the genomes of all 70,000+ species of eukaryotic organisms in Britain and Ireland. It is a collaboration between biodiversity, genomics and analysis partners that hopes to transform the way we do biology, conservation and biotechnology.”Darwin Tree of Life (DToL) project
In 2020, Liam Crowley from the University of Oxford contacted me about sequencing earthworms in the British Isles. Liam’s work on the DToL project focuses on invertebrates at Wytham woods, a 1,000-acre semi-natural woodland owned and maintained by the University of Oxford. Currently, he is working with others on the first phase of the project – to sequence the full genome of 2,000 species from as many different taxonomic families as possible. They are also focussing in greater depth on certain groups of particular ecological or evolutionary interest. Later phases will aim to ramp up this sequencing to eventually sequence every species!
My area of expertise is very much in earthworm identification, and not at all in genome sequencing. The truth is that my understanding of genomics is somewhere between basic and non-existent, so below is a brief overview of some of the terminology (for my benefit as much as the reader!):
DNA (Deoxyribonucleic acid) A self-replicating molecule that is present in the cells of nearly all living organisms. It is the carrier of genetic information and exists in the form of a double-stranded helix held together by weak hydrogen bonds between base pairs of nucleotides.
Nucleotides DNA contains four different nucleotides – adenine (A), guanine (G), cytosine (C) and thymine (T). The order of these four nucleotides in the DNA sequence forms a code which determines the function of the DNA.
Gene A unit of heredity which is transferred from a parent to offspring and is held to determine some characteristic of the offspring. Each gene is a distinct sequence of nucleotides forming part of a chromosome. All genes are made up of DNA, but not all DNA forms genes.
Chromosome A long molecule containing genes and other sequences of DNA. The number of chromosomes a species has differs greatly. For example, humans have 46 whereas ferns can have over 1,000!
Genome The full complement of genetic material held by an organism. This includes all of the DNA within the chromosomes (including the genes), as well as any mitochondrial DNA in animals and chloroplast DNA in plants.
The DToL project is interested in two forms of recording this genetic information regarding British and Irish eukaryotic organisms:
- DNA barcoding A method of species identification using a short section of DNA from a specific gene or genes. This ‘barcode region’ is a tiny part of the genome specifically chosen as it is consistent enough across different species to easily find it, yet varies just enough to differentiate different species.
- Genome sequencing The process of determining the entirety (or nearly the entirety) of the DNA sequence of an organism’s genome at a single time, including the chromosomal DNA as well as the mitochondrial DNA (in animals) or chloroplast DNA (in plants).
Recording earthworm genetic material
In the British Isles we have just 31 species of earthworm that occur in natural environments. This makes earthworms an easy target group for getting a good head start with during phase 1 of the project. Twenty-nine of those species belong to a single family, Lumbricidae, with the remaining two species being the only species within their respective families within the UK (Acanthodrilidae and Sparganophilidae) and are both very rare and difficult to find (I’ve never personally come across either).
As live specimens are required to obtain the genetic material , my task was to find as many different species as I could over a two-day period while working on the project with Liam at Wytham woods (University of Oxford). I collected live specimens from my garden early in the morning on 27th May 2021 before heading to the Woods to undertake further sampling throughout the afternoon of 27th and morning of 28th May. On 28th May we were joined by Michael Tansley, an Oxford PhD student studying earthworm.
In addition to my garden, we explored ancient woodland, calcareous grassland and fen wetland habitats, looking within soil, in and under deadwood and in the leaf litter layer. Identifying earthworms in the field live is extremely difficult and rarely even possible, so it was difficult to know how many species we had collected. Any juveniles (earthworms with no saddle/clitellum) were released as it is not possible to identify these to species – though this may be possible in the future using DNA barcoding.
Extracting genetic material
The DToL project is using an exciting and relatively new sequencing technology known as ‘long-read’ sequencing, which reads and works out the order of nucleotides in much longer fragments of DNA than other methods. In much the same way as a jigsaw with fewer, larger pieces is easier to assemble than one with many small pieces, longer DNA fragments allow for a more accurate genome assembly.
The catch, however, is that DNA is a very unstable molecule, and starts breaking down into smaller fragments very quickly in dead tissue. To allow successful long-read sequencing, therefore, living tissue needs to be flash frozen to preserve long chunks of DNA. We achieved this for our earthworm samples by removing and immediately flash freezing a small section of the tail from each specimen at -80oC, before the specimens were euthanised and preserved in 80% ethanol.
A further small piece of the tail was preserved in 70% ethanol, to be submitted to the Natural History Museum for DNA barcoding. The purpose of also barcoding specimens is three-fold – firstly it allows us to populate the species barcode reference databases, allows matching of barcode ID against the identification made by collectors (preventing unnecessary expensive sequencing of the same species multiple times) and finally, allows a sense check that samples have not got mixed up during the sequencing process and each genome is matched to the correct specimen.Liam Crowley, Oxford University
Once the relevant material was preserved for each molecular method, the remainder of the specimens (everything but a small piece of the tail) were identified under a stereomicroscope. I identified the specimen using the Key to the Earthworms of the UK & Ireland (2nd Edition) by Emma Sherlock. This key uses external morphological features such as the type of head, location of the male pore, location/shape of the TP and the spatial distance between the setae.
Ideally, we’d like to sequence every specimen to build up a more complete library of earthworm genomes. However, there is a cost to sequencing each sample, so we prioritised up to 3 specimens per species for sequencing. Those specimens that will not be sequenced were still identified and contribute important records to the National Earthworm Recording Scheme.
Earthworm species records All specimens were identified and records submitted to the National Earthworm Recording Scheme. In total this contributed 43 species records across 14 species of earthworm, including 13 new species records for a previously unrecorded site (Wytham Woods). These records will be publicly available through the National Earthworm Recording Scheme (UK) dataset on the NBN Atlas.
Genome sequencing 34 specimens were sent for genome sequencing across 14 different earthworm species (see list below). This will enable different DNA extraction methodology to be tried and tested. This is necessary because earthworm biochemistry is quite different to other taxa such as insects, for which we have the most knowledge of suitable methods, therefore different variations on extraction methods need to be tested. Any successfully sequenced genomes will be published with open access, making all the data publicly available for everyone: https://www.darwintreeoflife.org/interim-data/
- Allolobophora chlorotica
- Aporrectodea caliginosa
- Aporrectodea rosea
- Bimastos eiseni
- Bimastos rubidus
- Eisenia fetida
- Eiseniella tetraedra
- Lumbricus castaneus
- Lumbricus rubellus
- Lumbricus terrestris
- Murchieona muldali
- Octolasion cyaneum
- Octolasion lacteum
- Satchellius mammalis
Preserved specimens 34 preserved specimens submitted to the Natural History Museum (London) earthworm collection, where they will be curated to a high standard and available for further inspection and research.
I’d like to say a huge thank you to Liam Crowley for hosting me at Oxford University and for reviewing and contributing to this blog.