Phylogenetic diversity and temporal variation in the Spirochaeta populations from two Mediterranean microbial mats

Spirochetes are among the bacterial groups often observed in hydrogen-sulfide-rich layers of coastal microbial mats. However, relatively few spirochetes from these microbial mats have been described and characterized. We used 16S rDNA phylogenetic analysis to investigate the spirochetal diversity of microbial mats from two locations in the western Mediterranean (Ebro Delta, Spain, and Camargue, France). Samples from each location were monitored in the spring and winter over a period of 1 to 2 years. In the sequence analysis of 332 clones derived from samples of both locations, 42 novel phylotypes of not-yet-cultivated spirochetes belonging to the genus Spirochaeta were detected. None of the phylotypes were identified as known culturable species of Spirochaeta or previously identified phylotypes cloned from other hypersaline microbial mat such as Guerrero Negro, Mexico. Eight of the phylotypes were common to Ebro and Camargue mats, and two (IF058 and LL066) were present both in spring and winter. Some phylotypes appeared to show seasonal variation, i.e., they were found only in the spring, but not in the winter. Ebro and Camargue phylotypes, like phylotypes from Guerrero Negro, grouped according to the vertical gradient of oxygen and sulfide in the mat. Some phylotypes, such as LH073, IE028, LH042, or LG013 were harbored in low H2S or H2S-O2 interface zone. In contrast, major phylotypes were detected in deeper layers and they were likely strict anaerobes and high tolerant to H2S. The presence of spirochetes in differently located microbial mats suggests that they constitute very diverse and stable populations involved in a well-integrated metabolic symbiosis (i.e., permanent physiological cooperation) with other guild populations in the mats, where they maintain a coordinated functional and stable community.