Table 1. Sampling details of Niphargus spp. (Niphargus ictus, Niphargus frasassianus and Niphargus montanarius), Beggiatoa and ɛ-Proteobacteria biofilms and sediment in the Frasassi caves.

Sample	Sampling time	Experiments
Niphargus spp.
N. frasassianus (ST)	June 2009	RNA extraction from the whole animals, reverse transcriptase (RT)-PCR amplification of nifH homologs
N. ictus (PC)	July 2010
N. frasassianus (ST)a	May 2009	nifH amplification from DNA extracted from the symbiotic microbial community associated with separated legs
N. frasassianus (RS)a	May 2009
N. montanarius A (BG)a	January 2008
N. montanarius B (BG)a	January 2008
Beggiatoa-dominated biofilms
PC-BeggA	July 2010	Total RNA extraction, and RT-PCR amplification of 16S rRNA and nifH
PC-BeggB	February 2011	Acetylene reduction assay
PC-BeggC	May 2011	Acetylene reduction assay, Isotope Ratio Mass Spectrometry (IRMS) and RT-PCR amplification of nifH homologs
ɛ-Proteobacteria biofilm
PC-EpsilA	July 2010	IRMS
Sulfidic sediment
PC-SedA	February 2011	Acetylene reduction assay
PC-SedB	March 2011	15N2 isotope labeling and IRMS
PC-SedC	May 2011	Acetylene reduction assay and IRMS
PC-SedD	July 2011	Total RNA extraction and RT-PCR amplification of nifH homologs

Abbreviations: BG, Il Bugianardo; PC, Pozzo dei Cristalli; RS, Ramo Sulfureo; ST, Sorgente del Tunnel.

The experiments performed with the samples are mentioned in brief (for details, refer to Materials and methods). Collection locations within the Frasassi cave complex are provided in parentheses (see Bauermeister et al., 2012, for a cave map). In March and July 2011, no Beggiatoa biofilms were present at PC; moreover, water levels were elevated in the cave by ∼1 m as compared with the levels observed during other collections.

^aThese samples were collected as a part of other studies and the same DNA preparations were used here (Flot et al., 2010; Bauermeister et al., 2012).