Evolutionary history influences the microbiomes of a female symbiotic reproductive organ in cephalopods

Abstract

Many female squids and cuttlefishes have a symbiotic reproductive organ called the accessory nidamental gland (ANG) that hosts a bacterial consortium involved with egg defense against pathogens and fouling organisms. While the ANG is found in multiple cephalopod families, little is known about the global microbial diversity of these ANG bacterial symbionts. We used 16S rRNA gene community analysis to characterize the ANG microbiome from different cephalopod species and assess the relationship between host and symbiont phylogenies. The ANG microbiome of 11 species of cephalopods from four families (superorder: Decapodiformes) that span seven geographic locations was characterized. Bacteria of class Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia were found in all species, yet analysis of amplicon sequence variants by multiple distance metrics revealed a significant difference between ANG microbiomes of cephalopod families (weighted/unweighted UniFrac, Bray-Curtis, P = 0.001). Despite being collected from widely disparate geographic locations, members of the family Sepiolidae (bobtail squid) shared many bacterial taxa including (~50%) Opitutae (Verrucomicrobia) and Ruegeria (Alphaproteobacteria) species. Furthermore, we tested for phylosymbiosis and found a positive correlation between host phylogenetic distance and bacterial community dissimilarity (Mantel test r = 0.7). These data suggest that closely related sepiolids select for distinct symbionts from similar bacterial taxa. Overall, the ANGs of different cephalopod species harbor distinct microbiomes and thus offer a diverse symbiont community to explore antimicrobial activity and other functional roles in host fitness.IMPORTANCEMany aquatic organisms recruit microbial symbionts from the environment that provide a variety of functions, including defense from pathogens. Some female cephalopods (squids, bobtail squids, and cuttlefish) have a reproductive organ called the accessory nidamental gland (ANG) that contains a bacterial consortium that protects eggs from pathogens. Despite the wide distribution of these cephalopods, whether they share similar microbiomes is unknown. Here, we studied the microbial diversity of the ANG in 11 species of cephalopods distributed over a broad geographic range and representing 15-120 million years of host divergence. The ANG microbiomes shared some bacterial taxa, but each cephalopod species had unique symbiotic members. Additionally, analysis of host-symbiont phylogenies suggests that the evolutionary histories of the partners have been important in shaping the ANG microbiome. This study advances our knowledge of cephalopod-bacteria relationships and provides a foundation to explore defensive symbionts in other systems.

Keywords: Alphaproteobacteria; Verrucomicrobia; accessory nidamental gland; cephalopod; microbial communities; microbiomes; phylosymbiosis; symbiosis.

Fig 1

Representative photos of the ANGs from different families of cephalopods (left) and sampling locations (right). The ANG is situated inside the white inset box. Scale bars: Sepiidae 1 cm; Idiosepiidae 0.1 cm; Sepiolidae 0.5 cm; Loliginidae 0.5 cm. Right: sampling locations and species from which ANGs were collected. Colors correspond to different cephalopod families.

Fig 2

Alpha and beta diversity of ASVs between cephalopod families. (A) Alpha diversity indices of ASVs by cephalopod family. Asterisks indicate significant differences between groups (Wilcoxon test, Bonferroni-corrected ^****P < 0.0001, ^***P = 0.001, ^**P = 0.01, and ^*P = 0.05). Principal coordinate analysis of (B) weighted UniFrac distances and (C) Bray–Curtis distances across cephalopod families and (D) weighted UniFrac and (E) Bray–Curtis distances across cephalopod families and species, where ellipses represent 95% confidence intervals between families.

Fig 3

Percent relative abundance of taxa with ASVs greater than 0.1% of normalized data set. Representative figures of cephalopods are above the names of the cephalopod families (where Idio- indicates Idiosepiidae). “n” represents the number of biological replicates from each species.

Fig 4

Bubble plot of the top 44 most abundant ASVs that made up approximately 40%–50% of each cephalopod species. The ASVs were identified to the closest taxonomic level assigned. Similar colors of the bubbles represent ASVs from the same class/phylum. The size of the bubble reflects the relative abundance of the ASV in each sample. The bar plot at the bottom corresponds to the percent relative abundance of the ASVs in the bubble plot for each sample.

Fig 5

Phylogenetic tree of abundant Opitutae spp. and Ruegeria spp. in ANG microbiomes. Maximum likelihood phylogenetic tree from FastTree (Newick format) of 240 bp of 16S rRNA gene sequences identified as (A) bacterial class Opitutae spp. of phylum Verrucomicrobia in sepiolids, Sepia esculenta, and Idiosepius pygmaeus and (B) genus Ruegeria spp. in sepiolids and I. pygmaeus. The symbols represent the sources of the sequences.

Fig 6

Comparison of the dendrograms of hosts and associated ANG microbiomes. The microbiome trees were generated with (A) weighted UniFrac distances and (C) Bray–Curtis distances. The topological congruence of the microbiome and host trees were calculated with nMC. (B, D) The rate of microbiome divergence across phylogenetic distance (B: weighted UniFrac, D: Bray–Curtis) with divergence times in Mya. The pink line represents distance decay. The P-values and Mantel r statistics are shown in the bottom right corners of the plot.