Diversity of ammonia-oxidizing archaea and bacteria in the sediments of a hypernutrified subtropical estuary: Bahía del Tóbari, Mexico

Abstract

Nitrification within estuarine sediments plays an important role in the nitrogen cycle, both at the global scale and in individual estuaries. Although bacteria were once thought to be solely responsible for catalyzing the first and rate-limiting step of this process, several recent studies have suggested that mesophilic Crenarchaeota are capable of performing ammonia oxidation. Here we examine the diversity (richness and community composition) of ammonia-oxidizing archaea (AOA) and bacteria (AOB) within sediments of Bahía del Tóbari, a hypernutrified estuary receiving substantial amounts of ammonium in agricultural runoff. Using PCR primers designed to specifically target the archaeal ammonia monooxygenase alpha-subunit (amoA) gene, we found AOA to be present at five sampling sites within this estuary and at two sampling time points (January and October 2004). In contrast, the bacterial amoA gene was PCR amplifiable from only 40% of samples. Bacterial amoA libraries were dominated by a few widely distributed Nitrosomonas-like sequence types, whereas AOA diversity showed significant variation in both richness and community composition. AOA communities nevertheless exhibited consistent spatial structuring, with two distinct end member assemblages recovered from the interior and the mouths of the estuary and a mixed assemblage from an intermediate site. These findings represent the first detailed examination of archaeal amoA diversity in estuarine sediments and demonstrate that diverse communities of Crenarchaeota capable of ammonia oxidation are present within estuaries, where they may be actively involved in nitrification.

FIG. 1.

Locations of Bahía del Tóbari and sampling transect (left) and Yaqui Valley-Gulf of California region of northwest Mexico (right). Locations of sampling points within Bahía del Tóbari plotted on 10-m resolution panchromatic data from the Advanced Land Imager (data provided by National Aeronautics and Space Administration and the Carnegie Institution, courtesy of G. P. Asner). The filled white circles show the locations of agricultural-drain inputs into Tóbari. The scale bar is at the lower left.

FIG. 2.

Rarefaction curves showing relative richnesses of archaeal and bacterial amoA genes at site 4 within Tóbari. January libraries are shown in black and October libraries in gray, with archaeal amoA data shown as solid lines and bacterial data as dashed lines. OTUs were defined based on a 5% cutoff; 95% confidence intervals for the curves are not shown, as these values are identical to the observed richnesses at the end points of the curves.

FIG. 3.

Phylogenetic relationships among bacterial amoA sequences from Bahía del Tóbari, closely related database sequences, and cultivated AOB (accession numbers are in parentheses). Clusters containing Tóbari sequences referred to in the text are labeled on the right. Bootstrap values (>60%) are indicated at branch points, with distance bootstrap values above the line and parsimony values below. The scale bar is at the lower left. The tree is neighbor joining, based on Jukes-Cantor-corrected DNA distances, and rooted with Nitrosospira and Nitrosospira-like sequences and Nitrosomonas cryotolerans.

FIG. 4.

Phylogenetic relationships among archaeal amoA sequences from Bahía del Tóbari and previously reported environmental sequences. The sequences are color coded according to the sampling site within Tóbari. The sequence names denote the overall location (Mexico), the sampling site (2 to 6), the sampling time point (January or October), and the individual sequence number. For example, “MX-2-JAN-1” indicates Mexico site 2, January sampling, sequence 1. Previously reported site 4 and 6 October sequences do not contain “OCT” within the sequence names. Other environmental sequences are shown in black, and critical database sequences in boldface (see the text). The clusters are color coded by the most abundant sampling site represented in the cluster, and the sampling sites and times of the represented sequences are indicated, followed by the number of corresponding sequences in parentheses. These are ordered by abundance within the cluster. Next to large clusters, January sequences appear on the first line and October sequences on the lower line. Bootstrap values (>60%) are indicated at branch points, with distance bootstrap values above the line and parsimony values below. The scale bar is at the lower left. This tree is a neighbor joining tree based on Jukes-Cantor-corrected DNA distances and is midpoint rooted. Accession numbers corresponding to the 694 sequences represented in this tree are listed in Materials and Methods. The sequence group from Elkhorn Slough (Hummingbird Island) referred to in the text is indicated by an asterisk.