No evidence for extensive horizontal gene transfer in the genome of the tardigrade Hypsibius dujardini

Tardigrades – An Evolutionary Success Story Tardigrades (also known as a moss piglet or water bear) are resilient, microscopic animals in the phylum Tardigrada (Superphylum Ecdysozoa, which also includes arthropods and nematodes) Have been found in almost every single environment: deep sea, mountaintops, rainforests, Antarctica, etc. Can withstand pressure six times greater than the deepest part of the ocean, radiation hundreds of times than the lethal dose for a human, the vacuum of outer space, temperatures as low as 1 kelvin and as high as 420 kelvin, and can go without food or water for up to 30 years. Earliest fossils dating to the Cambrian period

Boothby et al. Published a genome analysis on the tardigrade species Hybsibius dujardini and concluded that horizontal acquisition of genes from bacteria and other sources is extensive and may be key to cryptobiosis (metabolic activity is reduced to an almost undetectable level). This paper, Koutsovoulos et al., independently sequenced the genome of H. dujardini and detected a low level of horizontal gene transfer, concluding that Boothby et al. failed to eliminate contaminants from sequence data.

Justification for Sequencing Genome Environmental and biogeographic markers Insight from crytobiotic mechanisms Exploration of relationships among ecdysozoans Why Hypsibius dujardini? Easily cultured in the laboratory Transparent Rapid life cycle Genomic model system

A Word on Horizontal Gene Transfer Animal genomes can take on horizontally transferred DNA, especially from germ line-transmitted symbionts Majority of transfers are nonfunctional and thus evolve neutrally – Dead on Arrival Horizontal Gene Transfer (doaHGT) Functional Horizontal Gene Transfer (fHGT) can bring new biochemical capacities – bdelloid rotifers of the genus Adineta attribute their survival as asexual organisms and their ability to undergo cryptobiosis Adaptation to the new transcriptional environment – active transcription, acquisition of introns, and acclimatization to host base composition and codon use bias.

Genome Assembly Propidium Iodide flow cytometry – estimated ~110 Mb Sequenced and assembled using Illumina short-read technology. Having only about 1000 cells, highly susceptible to effects of contamination Read pairs contributing to contigs with bacterial identification and no evidence of being tardigrade were removed; contigs and scaffolds below 500 bp were removed. 23,021 protein-coding genes Authors admit there may still be some contamination.

Boothby et al. Estimated genome at 252 Mb, with 39,532 protein coding genes, with more than 17% derived from fHGTs. One-third of this assembly does not appear to be derived from the tardigrade. Scaffolds had low coverages, with different relative coverages in different libraries, were not represented in the second genome assembly, and were noneukaryote. Used an HGT index – only valid when there is independent evidence of host genome incorporation.

Analysis Out of 107 HGT candidates, 38 were bacterial-bacterial and 8 archaeal-bacterial – with no expression. 571 bacterial-metazoan HGT candidates, 355 of which were on 166 scaffolds that contained only other bacterial genes – likely remaining contaminants. 216 HGT candidates were linked to eukaryotic on 162 scaffolds with GC content and coverage similar to the tardigrade genome. Of 23,021 protein coding genes predicted, only 13,500 had sequence similarity matching to other organisms – 10,161 metazoan HGT: 0.2-0.9% (as opposed to 17%)

Conclusion Boothby et al. did a poor job assembling the genome and removing contaminants – with a superior assembly, the biological conclusions reached by Boothby et al. were found to be unsupported. Questions: Was there a response by Boothby et al.?