Fidelity varies in the symbiosis between a gutless marine worm and its microbial consortium

Abstract

Background: Many animals live in intimate associations with a species-rich microbiome. A key factor in maintaining these beneficial associations is fidelity, defined as the stability of associations between hosts and their microbiota over multiple host generations. Fidelity has been well studied in terrestrial hosts, particularly insects, over longer macroevolutionary time. In contrast, little is known about fidelity in marine animals with species-rich microbiomes at short microevolutionary time scales, that is at the level of a single host population. Given that natural selection acts most directly on local populations, studies of microevolutionary partner fidelity are important for revealing the ecological and evolutionary processes that drive intimate beneficial associations within animal species.

Results: In this study on the obligate symbiosis between the gutless marine annelid Olavius algarvensis and its consortium of seven co-occurring bacterial symbionts, we show that partner fidelity varies across symbiont species from strict to absent over short microevolutionary time. Using a low-coverage sequencing approach that has not yet been applied to microbial community analyses, we analysed the metagenomes of 80 O. algarvensis individuals from the Mediterranean and compared host mitochondrial and symbiont phylogenies based on single-nucleotide polymorphisms across genomes. Fidelity was highest for the two chemoautotrophic, sulphur-oxidizing symbionts that dominated the microbial consortium of all O. algarvensis individuals. In contrast, fidelity was only intermediate to absent in the sulphate-reducing and spirochaetal symbionts with lower abundance. These differences in fidelity are likely driven by both selective and stochastic forces acting on the consistency with which symbionts are vertically transmitted.

Conclusions: We hypothesize that variable degrees of fidelity are advantageous for O. algarvensis by allowing the faithful transmission of their nutritionally most important symbionts and flexibility in the acquisition of other symbionts that promote ecological plasticity in the acquisition of environmental resources. Video Abstract.

Keywords: Animal-bacterial symbiosis; Intraspecific genetic variation; Microbiome; Phylosymbiosis; Symbiont transmission.

Fig. 1

The O. algarvensis population in two bays off the island of Elba was dominated by two mitochondrial haplotypes. a Light microscopy image of Olavius algarvensis. b Fluorescence in situ hybridization image of an O. algarvensis cross section, highlighting the symbionts just below the cuticle of the host (gammaproteobacterial symbionts in green and deltaproteobacterial symbionts in red, using general probes for these two phyla). Reproduced with permission from Kleiner et al. [67]. c and d Location of the two collection sites, Sant’ Andrea and Cavoli, two bays off the island of Elba in the Mediterranean. e Haplotype network of mitochondrial cytochrome c oxidase subunit 1 (COI) gene sequences of O. algarvensis individuals from the two collection sites. The two dominant COI haplotypes A and B co-occurred in both bays. The size of the pie charts corresponds to COI haplotype frequencies. Hatch marks correspond to the number of point mutations between COI haplotypes. Nodes depicted by small red points indicate unobserved intermediates predicted by the algorithm in the haplotype network software. The number of individuals identified as COI haplotype A or B in each bay is in parentheses in the box below the network.

Fig. 2

The composition of the symbiont community in 80 O. algarvensis individuals. a The number of O. algarvensis host individuals from two mitochondrial lineages (COI haplotypes A and B) and two locations (Sant’ Andrea and Cavoli) in which the respective symbiont species was detected (n = 80 in total; 20 replicates per location and COI haplotype; Supplementary text 1.1). b Relative read abundances of symbionts in the 80 O. algarvensis individuals. Each column shows the reads from a single host individual. The sulphur-oxidizing symbionts Ca. Thiosymbion (Ca. Thiosym.) and Gamma3 were the most abundant across host individuals, while the abundances of the sulphate-reducing symbionts (Delta1a, Delta1b, Delta3, Delta4) and the spirochete symbiont (Spiro) were consistently lower. Relative abundances of each symbiont were estimated based on metagenomic sequencing reads that mapped to the single-copy genes of each symbiont (Supplementary text 1.1). Relative symbiont abundances based on 16S rRNA gene sequences in the metagenomes were similar (Supplementary text 1.2; Supplementary Fig. S5)

Fig. 3

Comparative phylogenetic analyses of O. algarvensis and its microbial consortium members revealed variable patterns of congruence across the six symbionts. Phylogenetic trees based on SNPs across genomes of a host mitochondria (166 SNPs, n = 80), b Ca. Thiosymbion (2872 SNPs, n = 80), c Gamma3 symbiont (618 SNPs, n = 80), d Delta1a symbiont (375 SNPs, n =37), e Delta1b symbiont (624 SNPs, n = 46), f Delta4 symbiont (675 SNPs, n = 67) and g spirochete symbiont (88 SNPs, n = 41). Phylogenies were inferred from genetic distances calculated from posterior genotype probabilities. Scale bars indicate 0.1 substitution per SNP site. Bootstrap support values > 95% are shown in black circles. Support for branches internal to each coloured leaf was omitted for visibility

Fig. 4

Host mitochondrial lineage and geographic location had a significant effect on the genetic divergence of some but not all symbionts. Host mitochondrial lineages explained genetic divergence in Ca. Thiosymbion and Gamma3, while geographic location explained divergence in the Gamma3, Delta1b and Delta4 symbionts. Pairwise genetic distances in O. algarvensis mitochondrial genomes and symbionts were calculated from pairs of O. algarvensis individuals within the same combination of host lineage (A- or B-host) and location (“Within”), between individuals of A- and B-hosts from the same location (“Between A- and B-hosts”) and between individuals from the two locations, Sant’ Andrea and Cavoli, but from the same host lineage (“Between locations”). Pairwise genetic distances were compared among these three categories for a mitochondria, b Ca. Thiosymbion, c Gamma3 symbiont, d Delta1a symbiont, e Delta1b symbiont, f Delta4 symbiont and g spirochete symbiont. Genetic distances were normalized per SNP site and log scaled. Thick horizontal lines and grey boxes respectively indicate the median and interquartile range (IQR) of observations. Vertical lines show the IQR ± 1.5 IQR range, and outliers out of this range are shown as circles. Numbers in brackets indicate numbers of pairwise comparisons per category tested. Asterisks respectively denote statistical significance (*p < 0.05, **p < 0.01, see Supplementary Table S5). Orange and blue brackets highlight a significant effect on genetic divergence by the mitochondrial lineage and location, respectively. “N.S.” indicates no significant differences among categories (p > 0.05)