Table 1.
Summary of cruise information and sample preparation.
Fig 1.
Maximum likelihood tree of γ-proteobacterial nifH OTUs.
To improve visibility only OTUs ≥3 sequences are shown (400 of 1492 OTUs) and sequences names are not displayed (see S4 Fig for the sequence names). Clades of uncultured sequences from marine environments are shaded blue and from a variety of environments are shaded green. Clades containing several qPCR target sequences are labeled. Clades encompassing sequences isolated from PCR reagents are colored red. Probes used in qPCR studies are labeled and include Gamma 1, 2, 4, P, ETSP1, and ETSP3, and Zhang (S2 Fig). Tree was constructed using neighbor joining and RAxML methods. Significant branching (agreement between both algorithms and bootstrap >60) is colored black.
Fig 2.
Maximum-likelihood tree of the nearest cultured neighbors to the γ-proteobacterial nifH OTUs targeted in marine qPCR studies.
Sequences that have been used as targets for qPCR studies are colored blue and labeled as in the original publications: Gamma 1–4 from Halm et al. (2011) [13], Gamma A and P from Langlois et al. (2008) [18], ETSP1-3 from Turk-Kubo et al. 2014 [25], and Zhang et al. (2011) [26]. Tree was constructed using neighbor joining and RAxML methods. Significant branching (agreement between both algorithms and bootstrap >70) is colored black and bootstrap values are shown.
Fig 3.
Maximum-likelihood tree of Marine 1 nifH OTUs.
For visualization, only OTUs of ≥3 sequences are shown. Maximum likelihood and neighbor joining algorithms were compared. Bootstrap values above 70 are shown and significant branches (bootstrap >70 and agreement between both algorithms) are shown in black. Sequences are identified by accession number, first word in title, the number of sequences in the OTU, and then the number of studies where the sequence was found. For comparison purposes, sequences used as probes and the closest related organisms are included. The qPCR phylotype targets in the Marine 1 clade are labeled (black), as are potential phylotype targets (grey).
Table 2.
Comparison of Gamma A primer and probe sequences to the other γ–proteobacterial nifH sequences used as targets in qPCR studies.
Fig 4.
Surface abundances of Gamma A nifH DNA (a) and cDNA (b) estimated by TaqMan qPCR.
Abundances are presented as log (nifH copies l-1) where red colors indicate high abundances and purple near detection limit abundances.
Fig 5.
Principal components analysis of environmental parameters for samples where Gamma A nifH abundances were estimated.
The cluster analysis distance of 3.1 is shown in panels ‘a-c’ and 2 in ‘b’. Samples are identified by collection depth (surface- <20 m, mid- 20–100 m, deep- >100 m) in ‘a’ and distance to the continental margin (coastal- <1000 km or open- >1000 km) in ‘b’. The Gamma A abundances (nifH copies l-1) are displayed in ‘c’.
Fig 6.
Activity ratio (cDNA:DNA) where Gamma A nifH cDNA and DNA were detected by TaqMan qPCR as a function of measured environmental parameters.
The cDNA:DNA ratio is graphed versus temperature (°C, a), depth (m, b), O2 concentration (μM, c) and N* (μM, d). The cDNA:DNA ratio was calculated only for samples in which both Gamma A DNA and cDNA abundances were >80 nifH copies l-1. Please note that not all parameters were available for all samples.