Susanna successfully defended her Master thesis last week. In her project, she wanted to address the evolution of the mitochondrial genome order of hairybellies (Gastrotricha). Gastrotricha are very tiny and flat animals, which are usually not longer than 1 mm. Nonetheless, they can have very delicate structures on their skin in the form of spines, thrones and scales. They occur both in marine and freshwater habitats. More about these interesting animals you read in one of our previous blog posts.
Susanna’s journey to her final thesis was quite a challenging one as the small critters did not make it easy to get their mitochondrial genomes. Susanna concentrated her efforts on one of the two orders of Gastrotricha, Macrodasyida. First, we had samples of different species of Macrodasyida. Second, the mitochondrial genomes were already available for two species the other order, Chaetonotida. One was published for Liognotus ghinii recently and the other one of Lepidodermella squamata had been available for some time. The gene order of both mitochondrial genomes was identical suggesting maybe the presence of a conserved gene order in Gastrotricha in general. This has also been seen in Annelida (earthworms and alike) before.
Given the success with obtaining the entire genome of Lepidodermella recently, Susanna set out to also use a genome approach as we had used for Lepidodermella. This worked quite well and she was able to assemble genomes of relative good quality for most of the species attempted. Hence, the start turned out to be very nice. The next step was then that Susanna pulled out the mitochondrial genome from the genome assemblies; or better said, the genomes. As she got back much more than one contig (=genome) for her genome. It turned out that the whole genome amplification had caused palindromic stretches of just the mitochondrial genome.
What does this mean? The mitochondrial genome was amplified in one direction, then the amplification switched back (seemingly random) and continued into the opposite direction. Hence, single reads had parts of the genome duplicated and in reverse order. This became apparent when one looked at the gene order of the different contigs of a gastrotrich species showing high degrees of duplication and the reversed gene order for these parts. However, the pattern was random across the different contigs of the same species. This indicated that this is an artifact and not a natural variation in the gene order.
The next step was there for to detect the switching points in the contigs, cut them in to smaller sub-units and then reverse-complement the ones with the reversed gene order. These sub-units were then used to assemble the mitochondrial genome anew. This was quite work intensive. Depending on the number of the contigs, the process could be done using available bioinformatic tools such as Pegasus and MitoHifi, but in the end almost all species needed careful manual curation. However, in all cases except one, Susanna was successful to obtain the mitochondrial genome of the species with good coverage.
When she then compared the mitochondrial genomes with each other it turned out that the gene order in each of them was completely different. This means that in Macrodasyida the gene order is substantially more variable than in Chaetonotida. Unfortunately, this also meant that the gene order could not be used to inform on the phylogeny of Gastrotricha or their phylogenetic position within the larger group Lophotrochozoa (also known as Spiralia). As we lack a robust phylogeny of Gastrotricha, it was also not possible to reconstruct the ancestral order of the gastrotrich mitochondrial gene order.
