Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade

Abstract

Horizontal gene transfer (HGT), or the transfer of genes between species, has been recognized recently as more pervasive than previously suspected. Here, we report evidence for an unprecedented degree of HGT into an animal genome, based on a draft genome of a tardigrade, Hypsibius dujardini. Tardigrades are microscopic eight-legged animals that are famous for their ability to survive extreme conditions. Genome sequencing, direct confirmation of physical linkage, and phylogenetic analysis revealed that a large fraction of the H. dujardini genome is derived from diverse bacteria as well as plants, fungi, and Archaea. We estimate that approximately one-sixth of tardigrade genes entered by HGT, nearly double the fraction found in the most extreme cases of HGT into animals known to date. Foreign genes have supplemented, expanded, and even replaced some metazoan gene families within the tardigrade genome. Our results demonstrate that an unexpectedly large fraction of an animal genome can be derived from foreign sources. We speculate that animals that can survive extremes may be particularly prone to acquiring foreign genes.

Keywords: genome; horizontal gene transfer; lateral gene transfer; stress tolerance; tardigrade.

Fig. 1.

Genome of the tardigrade H. dujardini. (A) Light micrograph of a tardigrade specimen. Image courtesy of S. Stammers, used with permission. (B) Percentage of coverage (complete + partial) for core eukaryotic genes in our H. dujardini genome, as well as genome assemblies from recently sequenced and model organisms. A. gambiae, Anopheles gambiae; CEGMA, core eukaryotic genes mapping approach; D. pulex, Daphnia pulex; I. scapularis, Ixodes scapularis; P. pacificus, Pristionchus pacificus; S. maritima, Strigamia maritima; T. urticae, Tetranychus urticae. (C) Source of genes in the H. dujardini genome as determined by HGT index calculations following Galaxy tools taxonomy extraction. (D) Proportion of horizontally transferred genes vs. total number of genes by scaffold size in the H. dujardini genome. The red line indicates the proportion of HGT genes in the total assembly (17.5%).

Fig. S1.

Comparison of H. dujardini genome statistics. (A) Genomic sequencing resources generated by this study. A comparison of the genome size (B), number of genes (C), average coding sequence length (D), guanine-cytosine content (E), average exon number per gene (F), and average exon size (G) for our H. dujardini assembly with the genome assemblies of several other model organisms was performed. Complete (H) and partial (I) core eukaryotic gene (CEG) coverage within our H. dujardini genome assembly is shown. A. gambiae, Anopheles gambiae; A. vaga, Adineta vaga; D. pulex, Daphnia pulex; I. scapularis, Ixodes scapularis; P. pacificus, Pristionchus pacificus; S. maritima, Strigamia maritima; T. urticae, Tetranychus urticae.

Fig. S2.

Most foreign tardigrade genes score well above the HGT index threshold. (A) Graph showing the number of genes with a given HGT index score (to the nearest integer) for the tardigrade H. dujardini, rotifer A. ricciae, and nematode C. elegans. (B) Graph showing the cumulative percentage of foreign genes accounted for as the HGT index threshold is lowered to 30. For example, with an HGT index threshold of 30, 100% of H. dujardini foreign genes are accounted for, whereas increasing the threshold to 250 still accounts for 50% of all H. dujardini foreign genes. (C) Average coverage for genes of metazoan (Met) or foreign origin. (D) Average coverage for scaffolds containing genes of metazoan or foreign origin. (E) Plot showing the percentage of horizontally acquired genes vs. scaffold size. (F) Graph showing the percentage of scaffolds with no (black bars), all (dark gray bars), or some (light gray bars) horizontally acquired genes, as the minimal gene count per scaffold is increased. As the minimal number of genes per scaffold increases, the number of scaffolds with only horizontally acquired genes decreases.

Fig. 2.

HGT in the genome of H. dujardini. (A) Schematic representation of a portion of scaffold 962. Boxes represent exons, red arches represent U2 spliceosomal introns, and black arrows represent primer sites. (B) Gene 0.37 (orange, bases 9,344–10,094) encodes a putative bacterially derived DOPA dioxygenase with no identifiable animal homologs. (C) Gene 0.3 (dark blue, bases 12,404–17,273) encodes a tardigrade (metazoan) FUT8 gene. Supports on trees in B and C are given as Bayesian/bootstrap (500) supports. Trees for the remaining 102 genes and associated information are available in Dataset S2.

Fig. S3.

Genes of metazoan and foreign origin are physically linked in the H. dujardini genome. Results and a schematic of PCRs performed with primers bridging genes predicted to be present on the same genomic scaffold (associated information is available in Dataset S2) are shown. Green and orange boxes highlight foreign-metazoan gene pairs and foreign-foreign gene pairs, respectively, that produced PCR products of the correct size. Large (red) boxes highlight PCR reactions failing to recover products of the correct size.

Fig. S4.

Comparison with PacBio long reads supports the accuracy of the H. dujardini genome assembly. A heat map shows the concordance between the PacBio and Illumina assemblies. The independently sequenced PacBio sequences are highly similar to the Illumina assembly, and there is no significant off-diagonal homology that would indicate misassembly or residual heterozygosity. Both sets of contigs are highly congruent. Synteny within contigs is clearly preserved, and assemblies are 98.53% concordant per base.

Fig. 3.

Horizontally transferred genes have acquired characteristics of the metazoan genes. (A) For a particular horizontally transferred gene from bacteria, its closest metazoan homolog (Met) in the H. dujardini genome and its closest bacterial homolog (Bac) were found, and codon use statistics for each codon were calculated and compared. (Lower Right) Additionally, for each of the 64 codons, the difference in codon use between genes of foreign origin in the H. dujardini genome and metazoan genes from the H. dujardini genome, the bacterium Niastella koreensis, and the bacterium Fluviicola taffensis (bacterial species with the highest representation in the H. dujardini genome) was calculated. Horizontal lines represent the average difference between the codon use in Hypsibius genes of foreign origin and each other corresponding dataset. Unpaired t test: **P < 0.01; ***P < 0.001; ****P < 0.0001. (B) Sequence logos generated for U2 intron 5′ and 3′ splice sites for genes of metazoan (Top) and bacterial (Bottom) origin.

Fig. 4.

Protein families in the H. dujardini genome have been expanded, supplemented, and replaced by genes acquired through horizontal transfer. (A) Raw counts for the 20 most abundant InterPro domains and families represented by H. dujardini genes of metazoan origin (blue), along with comparative data from two closely related species, D. melanogaster (dark gray) and C. elegans (light gray). The count for each InterPro domain contributed by genes of foreign origin is also shown (orange). ABC, ATP-binding cassette transporter-like; ANF lig-bd rcpt, ligand binding receptor region; Ankyrin rpt-contain dom, Ankyrin repeat-containing domain; Chitin-bd dom, chitin binding domain; Cyt P450, Cytochrome P450; DEAD_N, DEAD-box N-terminal domain; DH_sc/Rdtase SDR, short-chain dehydrogenases/reductases family; GPCR rhodpsn, rhodopsin-like G protein-coupled receptor domain; HATPase ATP-bd, histidine kinase-like ATPase C-terminal domain; MFS, major facilitator superfamily; Prot kinase dom, Protein kinase domain; RRM dom, RNA recognition motif; Sig transdc resp-reg receiver, signal transduction response regulator receiver domain. (B) Cladogram showing evolutionary relationships between foreign H. dujardini (gray), bacterial (orange), and metazoan (blue) genes implicated in stress tolerance. Numbers on branches indicate Bayesian followed by bootstrap (500) supports.

Fig. S5.

Foreign genes implicated in stress tolerance have been transferred into the H. dujardini genome. Cladograms show the evolutionary relationships between foreign H. dujardini (gray), bacterial (orange), and metazoan (blue) genes implicated in stress tolerance. Numbers on branches indicate Bayesian followed by bootstrap (500) supports. recA, DNA recombination and repair A; spds, spermidine synthase.

Fig. S6.

Model of HGT in desiccation-tolerant organisms. Speculative model of HGT acquisition in desiccation-tolerant organisms. Prolonged desiccation induces dsDNA breaks. During rehydration, membranes become transiently leaky, allowing the transfer of large macromolecules into and out of cells, including fragmented foreign DNA. Anhydrobiotic organisms possess robust DNA repair mechanisms for fixing desiccation-induced DNA damage. If foreign DNA is present in the nucleus of an anhydrobiotic cell, it may be accidentally incorporated during postrehydration genomic repair.