Niphargus–Thiothrix associations may be widespread in sulphidic groundwater ecosystems: evidence from southeastern Romania

Abstract

Niphargus is a speciose amphipod genus found in groundwater habitats across Europe. Three Niphargus species living in the sulphidic Frasassi caves in Italy harbour sulphur-oxidizing Thiothrix bacterial ectosymbionts. These three species are distantly related, implying that the ability to form ectosymbioses with Thiothrix may be common among Niphargus. Therefore, Niphargus–Thiothrix associations may also be found in sulphidic aquifers other than Frasassi. In this study, we examined this possibility by analysing niphargids of the genera Niphargus and Pontoniphargus collected from the partly sulphidic aquifers of the Southern Dobrogea region of Romania, which are accessible through springs, wells and Movile Cave. Molecular and morphological analyses revealed seven niphargid species in this region. Five of these species occurred occasionally or exclusively in sulphidic locations, whereas the remaining two were restricted to nonsulphidic areas. Thiothrix were detected by PCR on all seven Dobrogean niphargid species and observed using microscopy to be predominantly attached to their hosts' appendages. 16S rRNA gene sequences of the Thiothrix epibionts fell into two main clades, one of which (herein named T4) occurred solely on niphargids collected in sulphidic locations. The other Thiothrix clade was present on niphargids from both sulphidic and nonsulphidic areas and indistinguishable from the T3 ectosymbiont clade previously identified on Frasassi-dwelling Niphargus. Although niphargids from Frasassi and Southern Dobrogea are not closely related, the patterns of their association with Thiothrix are remarkably alike. The finding of similar Niphargus–Thiothrix associations in aquifers located 1200 km apart suggests that they may be widespread in European groundwater ecosystems.

Zoogeography of epigean freshwater Amphipoda (Crustacea) in Romania: <br />fragmented distributions and wide altitudinal variability

“…The presence of 11 epigean freshwater amphipod species or species complexes previously recognized in Romania, and the two presently synonymized subspecies of G. balcanicus, was confirmed by our data and recent literature. The only species not encountered during our field survey was N. hrabei; however, a recent study reported it in south-eastern Romania (Flot et al 2014), confirming its presence after more than 50 years.…”

Niphargus dancaui sp. nov. (Amphipoda, Niphargidae) – a new species thriving in sulfidic groundwaters in southeastern Romania

“…Niphargus thrives in all types of subterranean waters, from shallow hypotelminorheic ones (i.e., shallow seeps filled with groundwater; Fišer et al 2010) to epikarst (Sket 1981), sinking streams and phreatic lakes (Trontelj et al 2012). Niphargid amphipods have also been noted for their dominant presence in hypogenic caves, i.e., caves that result from limestone dissolution by hydrogen sulfide arising from deep reservoirs (Latella et al 1999;Sarbu et al 2000;Forti et al 2002;Flot et al 2010Flot et al , 2014. These sulfidic caves are of particular interest from an ecological viewpoint: instead of sunlight (photoautotrophy) or allochthonous organic material (organoheterotrophy), they derive their energy from inorganic chemical compounds (chemoautotrophy; Sarbu et al 1996Sarbu et al , 2000.…”

“…This genus is taxonomically demanding and its species richness is far from being completely described. The morphology of niphargids is not a very good proxy for the phylogenetic relationships among these species (Fišer et al 2008;Trontelj et al 2012;Flot et al 2014), and cryptic species (i.e., species that are nearly indistinguishable from a morphological point of view but possess distinctive DNA sequences) appear common in this group ).…”

“…The sulfidic waters of Movile Cave are host to two of these species. One of them, P. racovitzai, was originally described as belonging to the distinct genus Pontoniphargus Dancău, 1970, although molecular analyses show it clearly nested within the genus Niphargus (Flot et al 2014). The second species was called Niphargus cf.…”

“…A similar molecular study conducted in Romania revealed the presence of seven niphargid species in and around Movile Cave: Niphargus cf. stygius (Schiödte, 1847), N. decui Karaman & Sarbu, 1995, N. dobrogicus Dancău, 1964, N. gallicus Schellenberg, 1935, N. hrabei Karaman, 1932, Pontoniphargus racovitzai Dancău, 1970, and P. ruffoi Karaman & Sarbu, 1993(Flot et al 2014Fišer et al 2015). The sulfidic waters of Movile Cave are host to two of these species.…”