The genome of a subterrestrial nematode reveals adaptations to heat

Abstract

The nematode Halicephalobus mephisto was originally discovered inhabiting a deep terrestrial aquifer 1.3 km underground. H. mephisto can thrive under conditions of abiotic stress including heat and minimal oxygen, where it feeds on a community of both chemolithotrophic and heterotrophic prokaryotes in an unusual ecosystem isolated from the surface biosphere. Here we report the comprehensive genome and transcriptome of this organism, identifying a signature of adaptation: an expanded repertoire of 70 kilodalton heat-shock proteins (Hsp70) and avrRpt2 induced gene 1 (AIG1) proteins. The expanded Hsp70 genes are transcriptionally induced upon growth under heat stress, and we find that positive selection is detectable in several members of this family. We further show that AIG1 may have been acquired by horizontal gene transfer (HGT) from a rhizobial fungus. Over one-third of the genes of H. mephisto are novel, highlighting the divergence of this nematode from other sequenced organisms. This work sheds light on the genomic basis of heat tolerance in a complete subterrestrial eukaryotic genome.

Fig. 1

Genomic comparison of H. mephisto protein-coding genes. a Multilocus phylogeny of H. mephisto using 99 single-copy orthologous genes (SCOGS). b Pfam domain comparison of nonredunant protein domain content in C. elegans versus H. mephisto at an e-value cutoff of 1e-10. Expanded AIG1 and Hsp70 domain families are marked, along with the MATH, FTH, and FBA_2 domains known C. elegans-specific expansions. c Examination of Hsp70 family expansion across species, quantifying proteins, not domains, identified using e-values as in (b). d Venn diagram comparing orthologous gene clusters between D. melanogaster, C. elegans, P. redivivus, and H. mephisto. Abbreviations: MATH meprin and TRAF-homology domain, FTH Fog-2 homology domain, FBA_2 F-box associated domain

Fig. 2

Analysis of Hsp70. a Bayesian phylogenetic tree of Hsp70. H. mephisto sequences marked with an asterisk (*) and D. pachys with arrows. Branch numbers indicate posterior probabilities; scale bar represents substitutions per site. b Hsp70 protein Bayesian tree used for dN/dS analysis of coding sequence. Branch letters A–M correspond to Table 3 for ω (dN/dS) value. Bold branches indicate statistical significance of dN/dS p value after correcting for multiple hypothesis testing, and the long branch (F) is semi-bold because it is statistically significant prior to multiple testing correction with 89% of sites under positive selection (Table 3). Branch numbers indicate posterior probabilities; scale bar represents substitutions per site. The sequences used in both trees are indicated with Wormbase (nematode) or Genbank Accessions (other species)

Fig. 3

Phylgenetic analysis of AIG1. a Nematode-only RAxML tree of 17 species showing two clusters: MTG1-related and AIG1-like groups. b Nematode, human, Arabidopsis, and fungal RAxML tree illustrating potential HGT event. IAN/GIMAP are synonyms of AIG1, the original names given to plant and vertebrate sequences. Two fungal (Rhizophagus irregularis) AIG1-like sequences highlighted in light red boxes. For both trees, branch numbers indicate bootstrap support from 200 replicates, and scale bar represents substitutions per site. The sequences used in both trees are indicated with Wormbase (nematode), UniProtKB (Rhizophagus irregularis) or Genbank Accessions (other species)

Fig. 4

Transcriptome analysis of gene expression in H. mephisto. a Boxplot showing that Hsp70 transcripts are induced on heat while AIG1 transcripts are unchanged. Box shows median (center line) and first and third quartiles, while whiskers indicate the 15th to 85th percentiles, and notches represent confidence intervals. p Values obtained by two-tailed Mann–Whitney test. Source data are provided as a Source Data file. b Volcano plot of gene expression fold change comparing heat (combined replicates: 38°, n = 3, and 40°, n = 6) versus 25 °C control (replicates, n = 3). Statistically altered transcripts, defined as q value less than 0.05 and upregulated or downregulated at least twofold under heat-stress conditions (38–40 °C) relative to 25 °C controls, are indicated with luminosity of 1 while nonsignificant are luminosity 0.1. The q value was obtained from the stattest() function within the Ballgown R package. Genes labeled “Unknown” are novel, as discussed in the text. Proteins encoding no recognizable domain but matching other nematodes by blastp are indicated with the number of nematode species matched (out of the 28 used in constructing the blast database). For genes matching only a single other nematode, the species is given in parenthesis. Color key: red: Hsp70, blue: AIG1, green: ARMET, and magenta: peptidases. Abbreviations: Sax-2 sensory axon guidance 2, LSM like SM. BI-1, Bax Inhibitor—1, ARMET arginine-rich, mutated in early-stage tumors. Source data are provided as a Source Data file