Sepiola COI and Vibrio gapA gene sequences

Symbiotic marine bacteria that are transmitted through the environment are susceptible to possible abiotic factors (salinity, temperature, physical barriers) that separate them from their hosts. Given that many symbioses are driven by host specificity, environmentally transmitted symbionts can alter symbiont preference depending on conditions over space and time. In order to determine whether the population structure of environmentally transmitted beneficial associations reflects host specificity or biogeography, we analyzed the genetic structure of Sepiola atlantica (Cephalopoda: Sepiolidae) and their Vibrio symbionts (V. fischeri and V. logei) in four Galician Rías (Spain). This geographical location is characterized by a jagged coastline with a deep-sea entrance into the land, ideal for testing whether such population barriers exist due to genetic isolation. We used haplotype estimates combined with nested clade analysis to determine the genetic relatedness for both S. atlantica and Vibrio bacteria. Analyses of molecular variance (AMOVA) were used to estimate variation within and between populations for both host and symbiont genetic data. Our analyses reveal a low percentage of variation among and between host populations, suggesting that these populations are panmictic. In contrast, Vibrio symbiont populations show a certain degree of genetic structure, demonstrating that the hydrology of the rias is driving bacterial distribution (and not host specificity). Thus, for environmentally transmitted symbioses such as the sepiolid squid-Vibrio association, abiotic factors can be a major selective force for determining the population structure of one of the partners.

Methods
Samples were obtained via SCUBA at approximately 6 to 10 meters deep at each site. Animals were transported and maintained at the Centro de Investigación Mariña-Estación de Ciencias Mariñas de Toralla (CIM-ECIMAT) facilities and subsequently euthanized one day later following protocols previously approved by the Animal Experimentation Ethics Committee from the University of Vigo (Spain) and the IACUC at New Mexico State University (NMSU; Andrews et al., 2013; Moltschaniwskyj et al., 2007). Single colonies of Vibrio bacteria were isolated from host light organs that were removed via ventral dissection and homogenized and diluted onto seawater tryptone agar (SWT; 0.5% tryptone, 0.3% yeast extract, 0.3% glycerol, 1.5% agar, and 70% seawater at 32 ppt.