Museum Genomics Illuminate the High Specificity of a Bioluminescent Symbiosis for a Genus of Reef Fish

Abstract

Symbiotic relationships between bioluminescent bacteria and fishes have evolved multiple times across hundreds of fish taxa, but relatively little is known about the specificity of these associations and how stable they are over host generations. This study describes the degree of specificity of a bioluminescent symbiosis between cardinalfishes in the genus Siphamia and luminous bacteria in the Vibrio family. Primarily using museum specimens, we investigated the codivergence of host and symbiont and test for patterns of divergence that correlate with both biogeography and time. Contrary to expectations, we determined that the light organ symbionts of all 14 Siphamia species examined belong to one genetic clade of Photobacterium mandapamensis (Clade II), indicating that the association is highly specific and conserved throughout the host genus. Thus, we did not find evidence of codivergence among hosts and symbionts. We did observe that symbionts hosted by individuals sampled from colder water regions were more divergent, containing more than three times as many single nucleotide polymorphisms than the rest of the symbionts examined. Overall, our findings indicate that the symbiosis between Siphamia fishes and P. mandapamensis Clade II has been highly conserved across host taxa and over a broad geographic range despite the facultative nature of the bacterial symbiont. We also present a new approach to simultaneously recover genetic information from a bacterial symbiont and its vertebrate host from formalin-fixed specimens, enhancing the utility of museum collections.

Highly contiguous genome assemblies ofPhotobacteriumstrains isolated from fish light organs using nanopore sequencing technology

“…Leiognathid fishes appear to associate with a wide range of both P. leiognathi and P. mandapamensis strains, as do Acrompomatids, which also associate with 'Candidatus' Photobacterium acropomis described here. In contrast, Apogonid hosts in the genus Siphamia associate with a much narrower range of only P. mandapamensis strains (Kaeding et al 2007, Gould et al 2021. It remains unclear why this degree of specificity exists for the bioluminescent symbiosis with Siphamia hosts and not for the other hosts examined, but it could be due the host's distinct behavioral ecology as a cryptic reef fish (Gould et al 2014).…”

“…Despite the divergence between P. leiognathi and P. mandapamensis in certain genes, the two groups remain indistinguishable at the 16S rRNA gene , Wada et al 2006, and the average nucleotide identity between the two are slightly above the 95% cut-off of the bacterial species definition, indicating the two should be considered the same species (Urbanczyk et al 2013). Furthermore, cardinalfish in the genus Siphamia appear to only associate with P. mandapamensis (Kaeding et al 2007, Gould et al 2021, indicating there may be important ecological and/or physiological differences between the two groups that are recognizable by Siphamia hosts. For example, they differ in their growth and luminescence responses to salinity as well as the color of light produced (Ast and Dunlap 2004).…”

Unlocking inaccessible historical genomes preserved in formalin

“…Originally developed for DNA extraction from FFPE sections, the HA method relies on high heat (120°C) under alkaline conditions (pH = 13) to break strong inter-and intramolecular cross links and utilizes organic extraction to maximize capture of fragmented gDNA from formalin-preserved tissues (50)(51)(52). This method has been applied to museum specimens to successfully recover sections of the mitochondrial genome in trout ( 53) and full mitochondrial genomes from lizards (40) and bacterial symbionts (41). Here we show the HA yields gDNA in adequate quantities for WGS from higher-quality formalin-preserved museum specimens.…”

“…Hot alkaline extraction followed by WGS of a single 30-year-old formalin-preserved Anolis lizard yielded sufficient coverage to reconstruct the entire mitochondrial genome (40). Using the same method, whole genomes were recovered for the bioluminescent bacterial symbionts contained within light organs of formalin-preserved cardinalfish (41). Using a proteinase K digestion method, sufficient gDNA was recovered for capture and sequencing of ultra-conserved elements from formalin-preserved snakes (42).…”

The mitochondrial genome of the red icefish (Channichthys rugosus) casts doubt on its species status

“…Moreover, most specimens of these species were caught decades ago and fixed in formalin, which leads to degradation and chemical modification of DNA, making the recovery of genetic information challenging. To overcome this limitation, protocols for DNA extraction and sequencing library preparation tailored for formalin-fixed specimens have recently become available and proved to be remarkably successful (Gansauge, Aximu-Petri, Nagel, & Meyer, 2020; Gansauge et al, 2017; Gould, Fritts-Penniman, & Gaisner, 2021).…”