A new, high-quality tardigrade genome refutes extensive horizontal gene transfer, and identifies new radioresistance genes.

Takuma Hashimoto et al (2016). Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nature Communications DOI:10.1038/ncomms12808

Tardigrades are famous for their ability to enter cryptobiosis, a state of suspended animation during which they can resist extremes of environmental insult. For example, many species can be dehydrated, stored dry for years, and brought back to life merely by adding water. While the story that tardigrades were revived from a herbarium specimen of moss a century old appears to be an urban (or scientific) myth, credible reports of survival for over 20 years have been published. We have been exploring the genomes of tardigrades to approach answers to how these tiny animals can do this (and other questions), but got caught up in a claim in late 2015, from a team at the University of North Carolina, that the genome of one tardigrade, Hypsibius dujardini, was full of genes it had acquired from other species by horizontal gene transfer (HGT) and that these transfers might underpin cryptobiotic abilities. We (Koutsovoulos et al 2016) and others (Delmont and Eren 2016, Arakawa 2016, Bemm et al 2016) roundly (and quickly) refuted these claims, showing that they were due to unrecognised bacterial contamination, and the controversy got called “tardigate” during the vigorous social media discussion that followed.

When asked about tardigate (or #tardigate…), I have frequently commented that, in the end, it was not a very attractive show. True, it was a “victory for open science”, but – imagine you are a new postdoc, wondering what field to make your mark in… However cool tardigrades are (and they are cool), the field looks, from the outside, like a difficult mess, with conflicting claims and data that is hard to get your head round. Is there a lot of horizontal gene transfer, or not? Is the genome contaminated or not? Have genes for cool cryptobiotic biology been identified, or are they analysis errors? Maybe I should work on something safer…

That all changes now. Today, Takuma Hashimoto and colleagues from the University of Tokyo and a number of other Japanese institutes published the genome of the tardigrade Ramazzottius varieornatus. The assembly is spectacular. The 55.8 Mb genome is in only 199 scaffolds, with a scaffold N50 of 4.74 Mb. Just under 20,000 protein coding genes are predicted, and nearly all of the genes expected from a good eukaryote are present. This leaps ahead of our best efforts on our favourite tardigrade H. dujardini, where we achieved an N50 of only 50 kb. By all measures the genome of R. varieornatus is excellent, and becomes, de facto, the reference tardigrade genome.

Importantly, R. varieornatus is a “good” cryptobiont, readily going into anhydrobiosis, and being resistant to doses of radiation that would kill most animals. Again, in comparison H. dujardini is lacking – it enters anhydrobiosis only with difficulty (after extended preconditioning) and does not survive rehydration very well. If anhydrobiosis is a driver of HGT as suggested by the U. North Carolina team (and others), R. varieornatus ought to be stuffed with bits of DNA it picked up from its environment.

It is hard, when working with minute organisms (tardigrades are mostly less than 1 mm long) that eat algae, fungi and bacteria, to prepare a DNA sample that is pure. Even if we wash the animals extensively, and then starve them, the bacteria in their guts may persist and biofilms of bacteria may remain stuck to their surfaces. Separating target from contaminant or cobiont is essential once the sequence data are in. The R. varieornatus team carefully assembled the genome without explicitly screening the sequence for HGT or contaminants. Rather, they used coverage to identify assembly components that belonged with the tardigrade. Coverage is a sensitive measure in these kinds of assays because all the genes in one genome will be at the same concentration, and this concentration will differ between the target (the tardigrade in this case) and the contaminants or cobionts. Using coverage alone, and several independently generated sequence datasets, the Tokyo team were able to eliminate a large number of sequences that could not, biologically, belong with the tardigrade genome.

With the “true”, high-quality genome to hand, Hashimoto and colleagues could ask what the true level of HGT in this tardigrade was. Was it similar to that proposed by the U. North Carolina team – over 6000 genes, 17% of the total – or the values we estimated from our H. dujardini analysis – 149 genes, 0.8%? Or, as one reviewer of our manuscript desired, somewhere in between? By careful analysis, and in particular based on the high contiguity of their assembly, the Tokyo team identified just 234 genes that passed a numerical test of “foreignness”, and even fewer of these – 138 – were expressed (based on their extensive EST and transcriptome data). So there is no evidence for massive HGT in this tardigrade, either. The “true” value for HGT appears to be around the 150 mark, or 0.8% of all genes. Importantly, well over half of these genes had closest similarity to genes in fungi, making assimilation from another eukaryotic genome more likely. (The U. North Carolina 6000-gene HGT set included mostly bacteria-like genes.)

Rather obviously (given #tardigate) this finding is to me a vindication of our analyses of H. dujardini: it now behoves those still wedded to “extensive” functional or neutral HGT to provide positive evidence for their claims. HGT is an important but not an overriding component of tardigrade (and other animal) evolution.

There is a lovely, biological twist to this tale, however. HGT allows organisms to acquire new functions rapidly, avoiding the slow burn of “Nature’s great tinkerers”, random mutation and natural selection. Thus in plant-parasitic nematodes, plant cell-wall digesting enzymes were acquired from other rhizosphere organisms, and now underpin the ability of these clever parasites to live in and eat plants. What functional roles do the HGT candidates in the R. varieornatus genome bring to the tardigrade? One set of three genes (and a pseudogene) were identified as belonging to a particular bacterial class of catalase (class II). Metazoan catalases are class III. R. varieornatus has thus acquired (and duplicated) a gene that plays a role in resistance to oxidant stress – and thus could be involved in cryptobiotic physiology.

Hashimoto and colleagues went on to identify a series of genes associated with cryptobiosis, and especially radiation resistance, and showed that one R. varieornatus-unique gene (not identified as an HGT candidate) conferred radiation resistance when transgenically expressed in human cells in culture. There are 8,023 R. varieornatus-unique genes in the genome.

A future career for the postdoc I invented in the opening paragraphs could be built on finding out where and when the HGT transfers happened in tardigrade evolution, pinning down the potential functions of the remaining HGT candidates, and identifying functions for the remaining 8,022 new genes. Rich pickings, and new biology!

———-

Image credit: Image of a tardigrade that undergoes cryptobiosis, from the blog of the joint first author of the paper: http://horikawad.hatenadiary.com/entry/2016/01/12/162306