High partner specificity in an algal-salamander mutualism at continental scale

Shannon Evelyn Wallace¹John A Burns²Rebecca Hale³Ryan Robert Kerney⁴Cy Mott⁵Cory Douglas Bishop^1*

• ¹Biology, St. Francis Xavier University, Antigonish, Nova Scotia, Canada
• ²Bigelow Laboratory For Ocean Sciences, Boothbay, Maine, United States
• ³University of North Carolina at Asheville, Asheville, North Carolina, United States
• ⁴Gettysburg College, Gettysburg, Pennsylvania, United States
• ⁵Eastern Kentucky University, Richmond, Virginia, United States

The mutualism between the green alga Oophila amblystomatis ("Oophila") (Lambert, 1905 ex Wille, 1909) and egg masses of the spotted salamander Ambystoma maculatum (Shaw) involves acquisition of algal cells from water, followed by their population growth within individual egg capsules of the egg mass. The host range spans over 20 degrees latitude and longitude. High site fidelity and fine scale genetic structure of host populations has been resolved, but genetic diversity of green algal symbionts across this range has not been investigated. We sampled egg capsule fluid harvested from five locales in North America, each separated by ~2 latitude and used both PacBio and Illumina sequencing of the 18S rRNA gene to investigate (i) whether algal symbionts are restricted to the Oophila clade and (ii) examine geographic variation in Oophila diversity. We also re-analyzed 18S (V4) reads from previous work using contemporary denoising protocols, which, along with current data, produced 16.37 million reads. We detected no chlorophytes outside Oophila clade B and detected cercozoan protists and rhizophydialean fungi in some capsules. There was a mean of 10.14  3.40 amplicon sequence variants (ASVs) per sample corresponding to O. amblystomatis, ranging in prevalence from single ponds within a locale to all samples. We observed among-locale variation in unique ASVs, but not in a latitudinal pattern. However, we detected two highly prevalent ASVs (ASVs A and B), which varied in abundance, relative to each other, as a function of latitude. Pairwise alignments of ASVs from the V4 region revealed that, on average, more divergent ASVs were rarer. Haplotype network analysis revealed ASV A as the ancestral variant and amplicon sequence analysis from newly isolated cultures of O. amblystomatis demonstrate that ASVs A and B are intragenomic variants that can segregate. We conclude that, at the scale of the host range, partner specificity in this algal-salamander mutualism is very high and reveal an intriguing effect of latitude on the relative abundance of intragenomic 18S rRNA gene variants.