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Metagenomic characterization of soil microbial communities in the Luquillo experimental forest (Puerto Rico) and implications for nitrogen cycling

June 2020

DOI:10.1101/2020.06.15.153866

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Authors:

Smruthi Karthikeyan

University of California, San Diego

Luis H Orellana

Max Planck Institute for Marine Microbiology

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The phylogenetic and functional diversity of microbial communities in tropical rainforests, and how these differ from temperate communities remain poorly described but are directly related to the increased fluxes of greenhouse gases such as nitrous oxide (N 2 O) from the tropics. Towards closing these knowledge gaps, we analyzed replicated shotgun metagenomes representing distinct life zones from four locations in the Luquillo Experimental Forest (LEF), Puerto Rico. These soils had a distinct microbial community composition and lower species diversity when compared to temperate grasslands or agricultural soils. Unlike temperate soils, LEF soils showed little stratification with depth in the first 0-30cm, with ~45% of community composition differences explained solely by location. The relative abundances and nucleotide sequences of N 2 O reductases ( nosZ ) were highly similar between tropical forest and temperate soils. However, respiratory NO reductase ( norB ) was 2-fold more abundant in the tropical soils, which might be relatable to their greater N 2 O emissions. Nitrogen fixation ( nifH ) also showed higher relative abundance in rainforest compared to temperate soils (20% vs. 0.1-0.3% of bacterial genomes in each soil type harbored the gene, respectively). Collectively, these results advance our understanding of spatial diversity and metabolic repertoire of tropical rainforest soil communities, and should facilitate future ecological modeling efforts. Importance Tropical rainforests are the largest terrestrial sinks of atmospheric CO 2 and the largest natural source of N 2 O emissions, two critical greenhouse gases for the climate. The microbial communities of rainforest soils that directly or indirectly, through affecting plant growth, contribute to these fluxes remain poorly described by cultured-independent methods. To close this knowledge gap, the present study applied shotgun metagenomics to samples selected from 3 distinct life zones within the Puerto Rico rainforest. The results advance our understanding of microbial community diversity in rainforest soils and should facilitate future studies of natural or manipulated perturbations of these critical ecosystems.

Proportion of total microbial community diversity explained by measured soil 568

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Metagenomic characterization of soil microbial communities in the Luquillo experimental

forest (Puerto Rico) and implications for nitrogen cycling

Smruthi Karthikeyan1, Luis H. Orellana1, Eric R. Johnston1, Janet K. Hatt1, Frank E. Löffler3,4,

Héctor L. Ayala-del-Río5, Grizelle González6, Konstantinos T Konstantinidis1,2*

1 School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta,

Georgia, USA

2 School of Biological Sciences, Georgia Institute of Technology, Atlanta, Georgia, USA

3 Center for Environmental Biotechnology, Department of Microbiology, Department of Civil

and Environmental Engineering, Department of Biosystems Engineering and Soil Science,

University of Tennessee, Knoxville, Tennessee, USA

4 Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

5 Department of Biology, University of Puerto Rico at Humacao, Puerto Rico, USA

6 USDA Forest Service, International Institute of Tropical Forestry, Río Piedras, Puerto Rico,

USA

*Address correspondence to Konstantinos Konstantinidis, kostas@ce.gatech.edu

School of Civil & Environmental Engineering, Georgia Institute of Technology. 311 Ferst Drive,

ES&T Building, Room 3321, Atlanta, GA, 30332. Telephone: 404-639-4292

The authors declare no conflict of interest.

.CC-BY-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprintthis version posted June 16, 2020. . https://doi.org/10.1101/2020.06.15.153866doi: bioRxiv preprint

ABSTRACT

The phylogenetic and functional diversity of microbial communities in tropical rainforests, and

how these differ from temperate communities remain poorly described but are directly related to

the increased fluxes of greenhouse gases such as nitrous oxide (N2O) from the tropics. Towards

closing these knowledge gaps, we analyzed replicated shotgun metagenomes representing

distinct life zones from four locations in the Luquillo Experimental Forest (LEF), Puerto Rico.

These soils had a distinct microbial community composition and lower species diversity when

compared to temperate grasslands or agricultural soils. Unlike temperate soils, LEF soils showed

little stratification with depth in the first 0-30cm, with ~45% of community composition

differences explained solely by location. The relative abundances and nucleotide sequences of

N2O reductases (nosZ) were highly similar between tropical forest and temperate soils. However,

respiratory NO reductase (norB) was 2-fold more abundant in the tropical soils, which might be

relatable to their greater N2O emissions. Nitrogen fixation (nifH) also showed higher relative

abundance in rainforest compared to temperate soils (20% vs. 0.1-0.3% of bacterial genomes in

each soil type harbored the gene, respectively). Collectively, these results advance our

understanding of spatial diversity and metabolic repertoire of tropical rainforest soil

communities, and should facilitate future ecological modeling efforts.

Importance:

Tropical rainforests are the largest terrestrial sinks of atmospheric CO2 and the largest natural

source of N2O emissions, two critical greenhouse gases for the climate. The microbial

communities of rainforest soils that directly or indirectly, through affecting plant growth,

contribute to these fluxes remain poorly described by cultured-independent methods. To close

this knowledge gap, the present study applied shotgun metagenomics to samples selected from 3

distinct life zones within the Puerto Rico rainforest. The results advance our understanding of

microbial community diversity in rainforest soils and should facilitate future studies of natural or

manipulated perturbations of these critical ecosystems.

INTRODUCTION

Soil microbiomes are one of the most complex ecosystems owing to microenvironments

and steep physicochemical gradients, which can change on a micrometer or millimeter scale (1-

3). Temporal and spatial heterogeneity, demographic stochasticity, ecotype mixing, dispersion

and biotic interactions are the major drivers of soil microbial diversity in these ecosystems (4, 5).

The formation of such “metacommunities” coupled with biogeography and other edaphic factors

greatly influence the functional and taxonomic profile of a soil ecosystem at any given location

(6).

Tropical rainforests (“forests” hereafter) are characterized by humid and wet climate

patterns and account for a large portion of the world’s total forest cover (7). These forests have

high levels of primary productivity (~30% of the total global production) due to large amounts of

precipitation coupled with year-long warm temperatures and high levels of light (8).

Consequently, high levels of biodiversity are observed in these forest soils with unique microbial

genotypic signatures being exclusive to this habitat/location, along with only a few cosmopolitan

taxa that are shared with other (non-tropical forest) habitats (9, 10). Although tropical forest soils

are critical ecosystems that host a plethora of distinct ecological niches, little is known about the

metabolic potential of tropical soils, especially, across elevation and depth gradients. Describing

this metabolic diversity is important for studying and monitoring the microbial activities related

to greenhouse gas fluxes, namely, nitrous oxide (N2O) and carbon dioxide (CO2) from the

tropical soils (11).

Notably, tropical forests represent the largest terrestrial sinks of atmospheric CO2 and the

largest natural source of N2O emissions (12-15). Natural soils have been reported to contribute

over 43% of the total global N2O emissions, with tropical ecosystems being the highest

contributors, having 2 to 4 times higher contributions compared to natural temperate ecosystems

(16-19). These soils are also responsible for about 70% of terrestrial nitrogen fixation, which

underlies, at least in part, their high rates of net primary productivity (11, 20).

Microbially-mediated nitrification and denitrification are the biotic processes contributing

the most to global N2O soil emissions (60-70%) (19, 21, 22), although chemodenitrification, i.e.,

ferrous iron generated by ferric iron-reducing bacteria reacting with nitrite to produce N2O

abiotically, is also likely high in iron-rich tropical soils (23). In soils, N2O is biologically

produced as a result of incomplete nitrification, DNRA (dissimilatory nitrite reduction to

ammonium) or denitrification respiratory pathways (22, 24, 25). Respiratory nitric oxide

reductase (nor) is a key contributor to the microbial production of N2O and is commonly

encoded in the genome of denitrifying bacteria as well as some ammonia-oxidizing organisms

(22, 26-30).

While both biotic and abiotic processes contribute to N2O production, consumption of

N2O is exclusively mediated by microbial N2O reductase (NosZ) activity (31-34). Yet, whether

the denitrifying microorganisms in these soils differ from their counterparts in temperate soils

and, if their functional genes present in the community reflect the high nitrogen fluxes, remain

unanswered questions despite their apparent importance for better management and modeling of

tropical soil ecosystems. It has also been demonstrated that tropical forests have significantly

higher rates of nitrogen fixation (~70% of total terrestrial nitrogen fixation) compared to other

ecosystems, significantly affecting the nitrogen budgets in these ecosystems (3, 35-37). For

instance, higher rates of nitrogen fixation in soils have been linked to nitrous oxide emissions (N

loss) due to reduced N retention capacities (11, 38, 39). How these ecosystem rates translate to

the nitrogen-fixing microbial (sub)community diversity and gene potential remains unclear.

The Luquillo Experimental Forest (LEF), also known as the El Yunque National Forest in

100

Puerto Rico (PR), has been a long term ecological research (LTER) site since 1988. The site is

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dedicated to the assessment of the effects of climate drivers on the biota and biogeochemistry.

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The forest has been subjected to several disturbance regimes over the last few decades, mostly

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natural and -to a smaller extent- anthropogenic such as tourism and experimental manipulations

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(40, 41). This site encompasses distinct “life zones” characterized by sharp environmental

105

gradients even across small spatial scales (40, 42, 43). The broad life zones based on the

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Holdridge classification system include the rain forest, wet forest, lower montane wet forest, and

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lower montane rain forest. These life zones are distinguished by elevation, temperature and

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rainfall patterns in addition to other edaphic factors (44-47). The elevation and rainfall patterns

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also tend to influence oxygen availability, redox potential, nutrient uptake and organic

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decomposition rates (44, 47, 48). The dynamic interplay of existing physicochemical gradients

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and climatic factors gives rise to a complex mosaic of biodiversity patterns observed in this

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forest (45). Hence, LEF represents an ideal environment to study tropical microbial community

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diversity patterns and their impacts on carbon and nitrogen cycling. The four sampling sites of

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this study were chosen to represent the distinct vegetation and life zones within the LEF.

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Previous studies in the LEF, and similar forest regions, have mostly focused on the

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effects of redox dynamics, litter decomposition, nitrogen (N) and other nutrient fertilization on

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microbial community activity through enzyme assays. Few studies have examined microbial

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diversity patterns across an elevation gradient and were only based on low-resolution techniques

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such as terminal restriction fragment length polymorphism analysis (14, 49-53). Furthermore,

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studies linking marker-gene abundances (related to nitrogen cycling) with in-situ flux

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measurements showed very high N2O fluxes in the forest soils (54). However, the nosZ primers

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targeted only the typical (Clade I) clades, thereby introducing a primer bias, which can be

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circumvented by employing metagenomic analyses.

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With recent developments in next generation DNA sequencing and associated

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bioinformatics binning algorithms, near-complete metagenome-assembled genomes (MAGs) can

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been recovered without cultivation (55, 56), opening new windows into studying soil microbial

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communities. Here, shotgun metagenomes originating from soils from the four different

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locations/life zones and three different depths in the LEF were analyzed to describe the microbial

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community diversity, biogeographical patterns, and metabolic potential differences across

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samples. Furthermore, the metagenomic data obtained from these soils were also compared to

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similar data from temperate grasslands in Oklahoma (OK) (57) and agricultural soils from

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Illinois (IL), USA (56) obtained previously by our team. By analyzing near-complete MAGs, we

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show that the most abundant microbial population (based on number of reads recruited) at each

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of the sampling locations represent sequence-discrete populations, similar to those observed in

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other habitats (58). Using such sequence-discrete populations as the fundamental unit of

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microbial communities, we subsequently assess the population distribution at high resolution

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across the sampling sites (biogeography) and the gene content they encoded, with a focus on

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nitrogen metabolism.

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RESULTS

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Diversity of forest microbial communities

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The LEF soil communities were compared to those of intensively studied ecosystems,

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namely the Oklahoma temperate grassland (OK) (57, 59) and Illinois agricultural soils (IL) (56),

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which were previously characterized with similar shotgun metagenomics approaches. Shotgun

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metagenomic sequencing recovered a total of 370 million reads across the 4 sites (Suppl. Table

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S2). Nonpareil 2.0 (60) was used to estimate sequence coverage, i.e., what fraction of the total

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extracted community DNA was sequenced. Nonpareil analysis of community diversity (Suppl.

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Fig. S1) showed that the agricultural Urbana (IL) site had the highest diversity of all the soils

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compared (NP diversity 24.02; note that NP values are given in log scale), and consequently, the

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lowest sequence coverage at (only) 37.23%. El Verde and Pico del Este (20-30cm) were the least

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diverse or most completely sequenced with 87.1% and 73.4% coverage respectively (NP

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diversity of 19.6 and 20.6 respectively or about 2-3 orders of magnitude less diverse). Overall,

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OK and IL soils appear to be more diverse than the PR soils by about two orders of magnitude,

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on average, with an average Nonpareil value of 22.75 0.37. Nearly complete coverage for El

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Verde and Pico del Este (20-30cm samples) would require 2.402e+09bp and 8.735e+09bp,

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respectively, and, for the same level of coverage, the more complex communities in Urbana (IL)

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would require a substantially higher sequencing effort of 1.282e+12bp. The OK soils had an

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estimated sequencing depth of 2.063e+11 1.436e+11 bp.

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Community composition variation across the forest sites based on 16S rRNA gene

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sequences.

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The number of total 16S-rRNA gene-based OTUs (Operational Taxonomic Unit)

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observed in each metagenome as well as the Chao1 estimate of total OTUs present reflected the

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degree of undersampling at each site (Suppl. Fig. S1 and S2), and were also consistent with the

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Nonpareil coverage estimates (Fig. 1). When Puerto Rico tropical soils (PR) were compared with

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the agricultural and grassland soils from the United States at the phylum level, Proteobacteria,

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Acidobacteria and Actinobacteria were the most abundant taxa across all ecosystems. However,

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in the forest soils, a few highly abundant OTUs dominated the entire soil community whereas in

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the OK and IL soils, OTUs were more evenly distributed (Suppl. Fig. S2), consistent with the

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Nonpareil diversity results. Only 1.28% of the total detected OTUs (out of a total 8019, non-

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singleton OTUs) were shared among all PR samples, while 49.95% of OTUs were exclusive to a

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particular sampling site in PR, reflecting partly the under-sampling of the extant diversity by

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sequencing. Only 0.37% of the OTUs (out of a total 13760, non-singleton OTUs) were shared

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among all the sites across all 3 ecosystems, all of which were assignable to Alphaproteobacteria,

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Acidobacteria, Verrucomicrobia and Actinobacteria.

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Further, applying four additional DNA extraction methods on a selected subset of our

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samples, including two manual phenol chloroform-based methods that are often advantageous

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for iron rich soils like those in tropical forest, revealed similar levels of diversity, more or less

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(Suppl.Fig. S3). Hence, the diversity patterns reported here are robust and independent of the

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DNA method used.

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Factors driving community diversity in the forest soils: Multidimensional scaling analysis

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of beta diversity

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The PCoA (Principal Coordinate Analysis) plots, constructed based on the MASH

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distances among whole metagenomes, showed a clustering pattern that was primarily governed

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by site/location. Accordingly, site explained 45.22% of the total diversity (Fig. 2B). The non-

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metric multidimensional scaling (NMDS) analysis of the data revealed only site, pH and soil

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moisture to be statistically significant physicochemical parameters in explaining the observed

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community diversity (Fig. 2C, Suppl. Table S3). ANOSIM values also indicated site to be a

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more important factor than depth, with a P value of 0.001 and 0.94, respectively. Based on the

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distance-based redundancy analysis (dbRDA), site was the most significant factor, even when the

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interplay between site and sampling depth was accounted for (Suppl. Table S4). Table 1 shows

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the partitioning of the variance between the proportion that is explained by constrained axes (i.e.,

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environmental variables measured) and the porportion explained by unconstrained axes (i.e.,

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variance not explained by environmental variables measured). The total variance explained by all

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(measured) environmental variables was 80.2% (Table. 1), which is remarkably high for a soil

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ecosystem (61).

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Major N cycling pathways

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Genes encoding proteins involved in denitrification and nitrogen fixation were the most

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abundant nitrogen (N) cycling pathway genes detected at different sites. Overall, the forest soils

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harbored about a 2-3-fold higher abundance of denitrification genes, i.e., narG, nirK, and norB

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catalyzing the reduction of nitrate, nitrite, and nitric oxide, respectively, compared to the

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grassland and agricultural soils (Fig. 2A). For instance, the norB gene abundance was found to

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be at the highest abundance among the denitrification genes, with ~37% (SD 9.5%) of the

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genomes in the PR soils predicted to contain a norB gene, compared to ~17% (SD 4%) and

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~14% (SD 1.3%) at IL and OK, respectively. Similarly, narG showed a 3-fold higher abundance

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in the PR soils compared to IL and OK soils (Fig.2B). While denitrification gene abundances

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appeared higher in the tropical soils, the relative abundance of nosZ gene, i.e., 11.6% (SD 3%) of

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the total genomes across the four locations in the LEF were predicted to encode nosZ, similar to

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nosZ relative abundance in IL and OK soils, i.e., 11.75% (SD 5%) and 11.08% (SD 3%),

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respectively (not statistically significant at p=0.05). Similar to nosZ, DNRA gene abundances

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(namely, nrfA) was similar across all sites studied herein (9%, SD 1.9%).

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Predominant NosZ clades are shared among soil ecosystems

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Placing nosZ-encoding reads to a reference nosZ phylogenetic tree revealed that atypical

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clades (clade II nosZ), affiliated predominantly with Opitutus, Anaeromyxobacter and other

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closely related genera, dominated the nosZ gene pool in the tropical forests (Figs. 3, Suppl.

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Figs.S4-S7). In contrast, a very small fraction of reads (<10% of total nosZ reads) were recruited

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to typical nosZ clades (or clade I). Members belonging to the clade II nosZ dominated the nosZ

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gene pool in OK and IL soils as well, with IL agricultural soils showing the greatest nosZ

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sequence diversity among the three regions. Notably, O. terrae-affiliated sequences represented

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the most abundant sub-clade (nosZ OTUs/sub-clades were defined at the 95% nucleotide

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sequence identity level) in all regions. Furthermore, most of the O. terrae–affiliated reads in the

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forest soil dataset appeared to be assigned to a single sub-clade, while their counterparts in the

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OK and IL soils appeared to be more evenly distributed among several closely related nosZ sub-

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clades, i.e., showing higher sequence diversity (Fig. 3, Suppl. Figs. S4-S7). O. terrae (strain

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DSM 11246/PB90-1) nosZ reads at >95% identity made up between 20% and 60% of the total

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nosZ reads recovered from the El Verde site and, together with the second most abundant sub-

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clade from Anaeromyxobacter sp., contributed over 30% of the total nosZ reads across all four

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PR locations (Fig. 5). Despite the significant taxonomic diversity observed in these soils (Suppl

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Fig. S2), the soils from PR shared several abundant nosZ gene sequences/sub-clades at >95

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nucleotide identity with soils in OK and IL (Fig. 3). Furthermore, in order to compare the

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predominant nosZ clades across the samples shown here, a new phylogenetic reference tree was

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constructed based on almost full length sequences obtained from the assemblies/MAGs obtained

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from the metagenomes studied here (namely PR,OK,IL). The short-reads identified as nosZ from

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the PR soils were placed on this tree and show that the majority of these reads are recruited by

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the nosZ sequences obtained from these assemblies/MAGs, indicating that the nosZ sequences

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across these ecosystems studies here are similar (Suppl. Fig. S8)

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Nitrogen fixation potential

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The nitrogen fixation genes (mainly nifH) were present at a much lower abundance in the

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lower altitude forest samples. For instance, only ~1-3% of all genomes in the lower altitude

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samples were predicted to encode nifH compared to a ~20% of the genomes in the higher

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elevation samples (Pico del Este) (Fig. 2A), and almost none of the reads from IL and OK

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metagenomes appeared to encode nifH (<0.1%). Therefore, nitrogen fixation gene abundance

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patterns indicated a much stronger selection for nitrogen fixation in the tropical forest relative to

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temperate agricultural or natural prairie soils, especially at higher elevations. Furthermore, no

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ammonia oxidizing genes (amoA) were detected in any of the soils except for Urbana soils (IL),

251

which had a history of fertilizer (N) input.

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Recovery of metagenome-assembled genomes (MAGs) representative of each site

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In order to test the effect of biogeography (i.e., limits to dispersion) of taxa across the

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elevation gradient sampled, the distribution of abundant MAGs recovered from each PR

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sampling site \ (assembly and MAG statistics provided in Suppl. Table S6) were assessed across

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the sites using read-recruitment plots (62). Taxonomic assignment using the Microbial Genomes

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Atlas (63) revealed that the most abundant MAG at site El Verde (lowest elevation), representing

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4.39% of the total metagenome, and was affiliated with an unclassified Verrucomicrobia. The

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second most abundant (1.8% of total) was likely a member of the genus Ca. Koribacter

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(Acidobateria) followed by an unclassified member of Acidobacteria (1.45% of total). The

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Verrucomicrobium MAG was found at an abundance of 1.03% of the total population at Sabana,

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and at 0.07% and 0.03% in Palm Nido and Pico del Este (highest elevation), respectively.

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Uneven coverage across the length of the reference sequence and nucleotide sequence identities

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were observed in the recruitment of short-reads from Palm Nido and Pico del Este as well as

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with all OK datasets, indicating that the related populations in the latter samples were divergent

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from the reference MAG (Suppl. Fig. S10). Therefore, at least this abundant low-elevation

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Verrucomicrobial population did not appear to be widespread in the other samples analyzed here

269

(Suppl. Fig. S10). Similarly, the other abundant MAGs from other sites in the forest soils were

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unique to the corresponding sites (elevation) from which they were recovered. Almost all MAGs

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used in the analyses were assignable to a novel family, if not higher taxonomic rank, according

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to MiGA analysis (when compared to 11,566 classified isolate genomes available in the NCBI

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prokaryotic genome database), underscoring the large unexplored diversity harbored by the PR

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tropical rainforest soils. The sequence diversity/complexity as well as sequencing depth limited

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large-scale recovery of high-quality MAGS.

276

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Functional gene content of the MAGs

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The genome sequences of the most abundant MAGs from each location (n=6) were

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analyzed in more detail to assess the functions they encoded, especially with respect to N cycling

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pathways (Fig. 4). MAGs from Pico del Este (highest elevation) showed a high abundance of N

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metabolism related genes compared to MAGs from other sites (Fig. 4). Most notably, genes

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related to nitrogen fixation were found only in the Pico del Este MAG, which was consistent

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with the short read analysis datasets showing greater relative abundance of nifH at this site.

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Nitrification (ammonia oxidation related genes) gene clusters were not detected in any of the

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recovered MAGs. norB and nosZ genes were found in three out of the six abundant MAGs

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analyzed. The most abundant El Verde MAG, most closely related to O. terrae (AAI = 40 %),

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possessed a nosZ gene, which was congruent with the nosZ phylogeny described above (i.e.,

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~60% of the nosZ-encoded reads from El Verde had a closest match to O. terrae nosZ

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sequences).

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291

DISCUSSION

292

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The present study reported the taxonomic and gene content diversity of the poorly

294

characterized tropical rainforest soils by using whole-community, shotgun metagenomic

295

sequencing of samples from the El Yunque forest, Puerto Rico. The recovered near-complete

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MAGs represented several abundant and widespread organisms within this ecosystem that could

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serve as model organisms for future studies. Furthermore, since the Luquillo Experimental Forest

298

(LEF) within El Yunque is subjected to varying natural as well as experimental (e.g., warming,

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phosphorus fertilization) perturbations, our study could also provide a baseline for these

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perturbations and future soil microbial studies at LEF. Our results revealed that the LEF soils

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harbor distinct microbial communities at sites with distinct elevation from sea-level. In contrast,

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and unlike several other soil ecosystems, sampling depth did not have a substantial impact on

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structuring community diversity, revealing no depth stratification in the LEF soils, at least for the

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depths sampled here (5-30cm). This could be due to the lack of distinct soil horizons within the

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first 30cm of the sampling sites, and indicates that the soil formation and/or physicochemical

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properties in these ecosystems could differ markedly from those in their temperate counterparts

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(44).

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A recent study examining the dominant bacterial phylotypes across the globe found that

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the predominant phylotypes were widespread across ecosystems. The only exception to this

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pattern was the forest tropical soils which harbor distinct phylotypes (10). Consistent with these

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conclusions, the MAGs recovered from each LEF site represented at least novel species and

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genera, further underlining the under-tapped microbial diversity harbored by tropical forest soils.

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Currently, the environmental factors driving these diversity patterns remain poorly understood

314

for tropical forest soils (10), but our study provided several new insights into this issue.

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In particular, sites El Verde and Sabana (lowest elevation sites) had similar community

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structure and diversity compared to the two higher-elevation sampling sites with certain MAGs

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being present at both sites but not in any of the other (higher-elevation) sites examined. This is

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presumably attributable to both sites having similar climate and vegetation patterns (i.e.,

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Tabonuco forest). On the other hand, Pico del Este was the highest elevation site and experiences

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almost continuous cloud cover as well as horizontal precipitation. The unique topology of Pico

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del Este was reflected in distinct and deeply novel MAGs and gene content, which differed

322

markedly from the other three sampling sites within the LEF (PCoA plots, Fig. 2B). The high

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water content of the Pico del Este soils gives rise to a unique ecosystem dominated by epiphytes

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(e.g., moss) (64). The epiphytic community has presumably significant impacts on nutrient (e.g.,

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nitrogen) cycling (65), and influences the water input to the soil, thereby shaping a unique

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habitat/niche for the soil microbes. Free-living microbes have been shown to be one of the

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highest contributors to biological N fixation in these forests with high rates of nitrogenase

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activity associated with the presence of moss/epiphytes (53, 66). Consistent with these previous

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results and interpretations, the Pico del Este showed an extremely high potential for nitrogen

330

fixation, i.e., it was estimated that 1/5 of the total bacterial genomes sampled possessed genes for

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N fixation, which is at least 10 times greater than any other site evaluated herein. Accordingly,

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we found that site (location) alone explained about half (45%) of the beta diversity differences

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observed among the four sampling sites, which reached ~80% when a few physicochemical

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parameters namely pH and moisture were also included in the analyses (Fig. 2B, Table 1). This

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is a remarkably high fraction of beta diversity explained by measured parameters for a soil

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ecosystem (61) and likely reflected that location and the physical properties that characterized

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different locations within LEF structured diversity much stronger than in other soil ecosystems.

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Tropical forests have also been shown to have significantly higher rates of nitrogen fixation

339

compared to other ecosystems, which can exceed the N retention capacity of the soil resulting in

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large N loss as N2O (67). The findings reported here on denitrification gene abundances were

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generally consistent with these previous observations as well.

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Links between soil community structure and nitrogen cycling can help close the

343

knowledge gaps on how the forest ecosystems impact the release and mitigation of certain highly

344

potent greenhouse gases such as N2O. The gene abundances observed here, e.g., more than two-

345

fold higher abundance of norB (associated with NO reduction to N2O) and similar nosZ (N2O

346

consumption) abundances in tropical soils relative to temperate soils were consistent with higher

347

N2O emissions observed previously from the tropics. Further, in acidic soils such as the tropical

348

forest soils evaluated in this study, lack of N limitation can suppress complete denitrification,

349

thereby leading to higher N2O release compared to other soil ecosystems (35). These

350

interpretations were consistent with our observation that the PR soils harbored a relatively high

351

abundance of respiratory (related to denitrification) norB genes as well. Previous studies have

352

also suggested that most denitrifying bacterial genomes possess the genes required to reduce

353

nitrate to nitrous oxide but do not possess the gene responsible for the last step i.e., N2O

354

reduction to N2, leading to the release of N2O gas (Braker and Tiedje, 2003; Richardson et al.,

355

2009; Giles et al., 2012; (22, 26-29), consistent with the findings of our study.

356

It has been established that tropical forest soils are the single highest contributor of

357

natural N2O emissions. While several abiotic and microbial processes can contribute to soil N2O,

358

N2O consumption is an exclusively microbial process, catalyzed by the enzyme product of the

359

nosZ genes (34). Based on the assessment of the nosZ gene phylogeny, it appears that almost all

360

of the nosZ genes from the tropical forest soils studied here belong to a previously overlooked

361

Clade II or atypical nosZ genes (32, 34, 68). This clade consists mainly of non-denitrifying, and

362

secondary denitrifying N2O reducers. Despite the unique phylogenetic diversity harbored by

363

tropical soils in general, the nosZ gene sequence diversity appears to be shared between

364

temperate and agricultural soils (Fig 4). These findings imply strong selection pressure for

365

conservation of nitrous oxide reductase sequences across tropical and temperate soil ecosystems

366

that are not apparently applicable to other N-cycling genes and pathways, which warrants further

367

attention in the future.

368

Integration of functional (e.g., gene expression) data with in-situ rate measurements will

369

provide a more complete picture of the composition and functioning in tropical forest soils. The

370

identification of certain biomarker genes such as nosZ sequences in our study could facilitate

371

future investigations on biogeochemical N-cycling and greenhouse gas emissions. For instance,

372

the assembled MAGs and gene sequences provided here could be useful for the design of

373

specific PCR assays for assessing transcript levels (activity), allowing potential linking of carbon

374

dioxide, methane, nitrogen, SOM, etc. turnover to the activity of individual populations. It would

375

also be interesting to assess how the findings reported here for the LEF apply (or not) to other

376

tropical forests especially because our study is based on a relative small sample size. While the

377

diversity in the Puerto Rico soils appears to be lower than that in temperate grassland and

378

agricultural soils, and different DNA extraction methods, including phenol-chloroform- and kit-

379

based, provided for similar results (Fig. S3), it is important to note that DNA of the temperate

380

soil samples was extracted using different methods (OK soils were extracted using the PowerSoil

381

kit). Therefore, it would be important to confirm these preliminary findings by using the exact

382

same DNA extraction and sequencing procedures in all soils. Despite the sample size, however,

383

our results showed differences along the elevation gradient sampled at the LEF that are

384

independent of DNA extraction (Suppl.Fig. S3) or sequencing methods, and consistent with our

385

metadata (Fig. 2), and previous process rate measurements. As the gradients at the LEF also

386

provide a natural setting to interpret the potential ramifications of climate change scenarios such

387

as altered participation patterns, the DNA sequences provided here could facilitate future

388

manipulation experiments with an emphasis on understanding and predicting the effects of

389

climate change on microbial community dynamics along the elevation gradient.

390

391

MATERIALS AND METHODS

392

393

Sampling sites

394

Soil samples were collected on February 2016 from four locations/sites across the LEF (18.3′ N,

395

65.80′ W). The four sites namely, Sabana, El Verde field station, Palm Nido and Pico del Este,

396

each located at different elevations from the mean sea level, i.e., 265, 434, 634 and 953 m,

397

respectively, were chosen due to their unique landscape and rainfall patterns, thereby creating

398

distinct ecological niches (Fig. 2A).

399

The El Yunque forest is categorized into four distinct vegetation zones namely, the

400

Tabonuco, Palo Colorado, Sierra Palm and Dwarf/Elfin forests. Site Sabana and El Verde, which

401

are located at the lowest elevation among the four sites within the LEF, fall under the Tabonuco

402

forest category in terms of vegetation, dominated by the tree species Dacryodes excelsa (native

403

to Puerto Rico). They are characterized by canopy cover and low light intensities at the ground

404

level which account for the sparsely vegetated forest floor. However, these sites still harbor the

405

richest flora of all sites (69). Palm Nido is characterized by unstable, wetter soils, steeper slopes

406

and the vegetation is dominated by the Sierra Palm (Prestoea montana). The site at the highest

407

elevation, Pico del Este (dwarf forest ecosystem or “elfin woodlands”) is characterized by higher

408

winds, lower temperatures and the vegetation is enveloped by clouds (41, 70) and its main

409

vegetation is comprised of moss and epiphytes. Furthermore, highly acidic soil and continuously

410

water-saturated soils deficient in oxygen are some major characteristics of this ecosystem with

411

most mineral inputs for plants become dissolved in the rain and fog.

412

Three adjacent soil profiles were taken from each of the four LEF sites (4 sites

413

encompassing 3 lifezones, Palo Colorado was not sampled). For each profile, individual soil

414

cores were taken at each depth (0-5cm, 5-20cm, 20-30cm) using a 3-cm diameter x 15-cm length

415

soil corer (AMS Inc, Idaho) that was decontaminated between samplings by washing with 70%

416

ethanol. Soil samples were stored in sterile Whirl-pak bags and kept on ice during transport and

417

until storage at -80º C. The three cores at each sampling depth were pooled together for

418

community DNA extraction, producing a total of twelve samples across the four sites.

419

Soil pH was determined using an automated LabFit AS-3000 pH Analyzer, and soil

420

extractable P, K, Ca, Mg, Mn, and Zn were extracted using the Mehlich-1 method and measured

421

using an inductively coupled plasma spectrograph at the University of Georgia Agricultural and

422

Environmental Services Laboratories (Athens, GA, USA). Soil extractable P using this method is

423

interpreted as the bioavailable fraction of P. NH4-N and NO3-N were measured by first

424

extracting them from soil samples with 0.1 N KCl, followed by the colorimetric phenate method

425

for NH4 + and the cadmium reduction method NO3. The physicochemical conditions at the sites

426

during the time of sampling are provided in Supplementary Table (S1).

427

428

Community DNA extraction and sequencing

429

Total DNA from soil was extracted using the FastDNA SPIN KIT (MP Biomedicals, Solon, OH)

430

following manufacturer’s procedure with the following modifications (71). Soils were air dried

431

under aseptic conditions followed by grinding employing a mortar and pestle. Cells were lysed

432

by bead beating and DNA was eluted in 50 µl of sterile H2O. DNA sequencing libraries were

433

prepared using the Illumina Nextera XT DNA library prep kit according to manufacturer’s

434

instructions except the protocol was terminated after isolation of cleaned double stranded

435

libraries. Library concentrations were determined by fluorescent quantification using a Qubit HS

436

DNA kit and Qubit 2.0 fluorometer (ThermoFisher Scientific), and samples were run on a High

437

Sensitivity DNA chip using the Bioanalyzer 2100 instrument (Agilent) to determine library insert

438

sizes. An equimolar pool of the sequencing libraries was sequenced on an Illumina HiSeq 2500

439

instrument (located in the School of Biological Sciences, Georgia Institute of Technology) using

440

the HiSeq Rapid PE Cluster Kit v2 and HiSeq Rapid SBS Kit v2 (Illumina) for 300 cycles (2 x

441

150 bp paired end). Adapter trimming and demultiplexing of sequenced samples was carried out

442

by the HiSeq instrument. In total, 12 metagenomic datasets were generated (3 per site for the

443

three depths), and statistic details on each dataset are provided in Supplementary Table S2.

444

In order to test for any DNA extraction biases of the kit used above, especially for the

445

high iron/clay content that characterizes tropical forest soils and is known to affect the extraction

446

step, four additional DNA extraction methods were performed in parallel on a small subset of

447

samples collected in 2018 from the same sites (6 samples per extraction method for 5 ecxtraction

448

methods covering the 4 sites). The methods included two manual (as opposed to kit-based)

449

phenol-chloroform based methods (72, 73) as well as two other kit-based methods namely;

450

DNeasy PowerSoil and DNeasy PowerSoil Pro (Qiagen Inc.). For this evaluation, the soils were

451

first homogenized and subsequently in five subsamples to use with each method (including the

452

FastDNA SPIN KIT-based method mentioned above). The libraries were constructed and

453

sequenced the same way as described above for the FastDNA SPIN KIT method.

454

All metagenomic datasets were deposited in the European Nucleotide Archive (ENA) under

455

project PRJEB26500. Additional data is available at http://enve-omics.ce.gatech.edu/data/prsoils.

456

457

Bioinformatics analysis of metagenomic reads and MAGs

458

The paired end reads were trimmed and quality checked using the SolexaQA (74) package with a

459

cutoff of Q>20 (>99% accuracy per base-position) and a minimum trimmed length of 50 bp.

460

i) Assembly and population genome binning: Co-assembly of the short reads from the same

461

location was performed using IDBA-UD (75) and only resulting contigs longer than 500 bp in

462

length were used for downstream analysis (e.g. functional annotation and MyTaxa

463

classification). Genes were predicted on the co-assembled contigs using MetaGeneMark (76) and

464

the predicted protein-coding regions were searched against the NCBI All Genome database using

465

Blastp (77). Since the assembly of individual datasets resulted mostly in short contigs (data not

466

shown), the contigs from the co-assembly (combining metagenomes from the three sampling

467

depths, for each site) were used for population genome binning. Contigs longer than 1Kbp were

468

binned using MaxBin (78) to recover individual MAGs (default settings). The resulting bins

469

were quality checked for contamination and completeness using CheckM (79), and were further

470

evaluated for their intra-population diversity and sequence discreteness using fragment

471

recruitment analysis scripts as part of the Enveomics collection (62) essentially as previously

472

described (80).

473

ii) Functional annotation of MAGs: Genes were predicted for each MAG using MetaGeneMark

474

and the predicted protein-coding regions were searched against the curated Swiss-Prot (81)

475

protein database using Blastp (77). Matches with a bitscore higher than 60 or amino acid identity

476

higher than 40% were used in subsequent analysis. The Swiss-Prot database identifiers were

477

mapped to their corresponding metabolic function based on the hierarchical classification

478

subsystems of the SEED subsystem category (Level 1) (82). The relative abundance of genes

479

mapping to each function was calculated based on the number of predicted genes from each

480

MAG assigned to the function (for read-based assessment, see below). Relative abundance data

481

were plotted in R using the “superheat” package (https://arxiv.org/abs/1512.01524). Individual

482

biomarker genes for each step of the nitrogen cycling pathway were manually verified by

483

visually checking the alignment of the identified sequences by the pipeline outlined above

484

against verified reference sequences.

485

iii) Functional annotation of short reads: Protein-coding sequences present in short reads were

486

predicted using FragGeneScan (83) using the 1% Illumina error model. The predicted genes were

487

then searched against the Swiss-Prot database using Blastp (best match). Low quality matches

488

(bitscore < 60) were excluded, and relative abundance of genes mapping to each function was

489

determined as described in the previous section.

490

491

Community diversity estimation

492

i) Nonpareil: Nonpareil (60) was used to estimate sequence coverage, i.e., what fraction of the

493

total extracted community DNA was sequenced and predict the sequencing effort required to

494

achieve "nearly complete coverage"(≥95%). The default parameters in Nonpareil were used for

495

all datasets. Only one of the two paired reads (forward) for each dataset was used to avoid

496

dependency of the paired reads, which can bias Nonpareil estimates (60).

497

ii) MASH and multidimensional scaling: MASH, a tool employing the MinHash dimensionality

498

reduction technique to compare sample-to-sample sequence composition based on k-mers (84),

499

was used to compute pairwise distances between whole metagenomic datasets and construct the

500

distance matrix to be used in multidimensional scaling. Pairwise MASH distances between the

501

metagenomic datasets were computed from the size-reduced sketches (default parameters).

502

PCoA (Principal coordinate analysis) and NMDS (Non-metric multidimensional scaling) were

503

employed to visualize the distance matrix and evaluate the physicochemical parameters driving

504

community diversity, respectively. Furthermore, dbRDA (distance based redundancy analysis),

505

was used to obtain a finer resolution on the observed compositional variation. All of the above

506

startistical analysis were performed using the vegan package in R (85), with default settings.

507

iii) 16S rRNA gene fragments recovered from shotgun metagenomes: 16S ribosomal rRNA (16S)

508

gene fragments were extracted from the metagenomic datasets using Parallel-META (86). 16S-

509

carrying reads were classified taxonomically using the GreenGenes database.

510

Recovered 16S fragments were clustered (‘closed-reference OTU picking’ strategy using

511

UCLUST (87)) and taxonomically classified based on their best match in the GreenGenes

512

database (88) at an ID ≥ 97% in QIIME (89, 90). The relative abundance of the OTUs were

513

calculated based on the number of reads assigned to each OTU. Community composition was

514

assessed based on OTU taxonomic assignments at the genus and the phylum ranks and was

515

compared between the sites based on the relative abundance of OTUs at each site.

516

517

Identification of N cycling genes using ROCker

518

ROCker (91) was employed for a precise identification and quantification of nosZ (encoding

519

nitrous oxide reductase), norB (encoding respiratory nitric oxide reductase, cytochrome bc

520

complex associated), nirK (encoding nitrite reductase), narG (encoding nitrate reductase), nrfA

521

(encoding nitrite reductase, DNRA related) amoA (encoding ammonia monooxygenase) and nifH

522

(encoding nitrogenase) encoding metagenomic reads (http://enve-

523

omics.ce.gatech.edu/rocker/models). Briefly, the short-read nucleotide sequences were searched

524

(using Blastx) against a training set for each abovementioned protein; training sets were

525

manually curated to encompass experimentally verified reference sequences as suggested

526

previously (91). The resulting matching sequences were then filtered using the ROCker compiled

527

model (model for 150bp-long reads for PR and OK soils and 100 bp model for IL soils). Protein

528

abundances (based on the number of reads assigned to the protein) were normalized by

529

calculating genome equivalents. For the latter, the ROCker-filtered read counts were normalized

530

by the median length of the sequences of each protein reference, and the corresponding genome

531

equivalents were calculated as the ratio of NosZ (or another protein of interest) read counts to the

532

RNA polymerase subunit B (rpoB), a universal single copy marker, read counts.

533

534

NosZ phylogenetic analysis

535

The NosZ reference protein sequences were aligned were aligned using CLUSTAL Omega (92)

536

and a maximum likelihood reference tree was created using RAxML v 8.0.19 (93) with a general

537

time reversible model option, gamma parameter optimization and ‘-f a’ algorithm. The ROCker

538

identified NosZ-encoding reads were extracted from all datasets, translated into protein

539

sequences using FragGeneScan, and then added to the reference alignment using Mafft (94). The

540

reads were placed in the phylogenetic tree using RAxML EPA algorithm and visualized using

541

iTOL (95).

542

543

Intra-population diversity assessment based on recovered MAGs

544

The taxonomic affiliation of individual contig sequences of a MAG was evaluated based on

545

MyTaxa, a homology based classification tool (96). The MiGA (Microbial Genomes Atlas,

546

www.microbial-genomes.org) webserver was used for the taxonomic classification of the whole

547

MAG using the ANI/AAI concept. To assess intra-population diversity and sequence

548

discreteness, each target population MAG was searched against all the reads from each location

549

by Blastn (only contigs longer than 2Kbp were used). Fragment recruitment plots were

550

constructed based on the Blastn matches (threshold values: nucleotide identity 75% and

551

alignment length 80bp) using the Enveomics collection of scripts (62). The evenness of

552

coverage and sequence diversity of the reads across the length of the reference genome sequence

553

were used to evaluate the presence and discreteness of the population in the chosen dataset.

554

555

556

Acknowledgments. This work was supported by the U.S. Department of Energy, Office of

557

Biological and Environmental Research, Genomic Science Program (award DE-SC0006662) and

558

US National Science Foundation (award 1831582). GG was supported by the Luquillo Critical

559

Zone Observatory (National Science Foundation grant EAR-1331841) and the Luquillo Long-

560

Term Ecological Research Site (National Science Foundation grant DEB-1239764). All research

561

at the USDA Forest Service International Institute of Tropical Forestry is done in collaboration

562

with the University of Puerto Rico. We thank María Rivera and Humberto Robles from IITF for

563

their help in soil sampling.

564

565

566

TABLES:

567

Table 1: Proportion of total microbial community diversity explained by measured soil

568

environmental factors.

569

Inertia

Proportion

Rank

Total

0.1092

Constrained

0.0876

0.8021

Unconstrained

0.02161

0.1978

Site, sampling depth, pH, total nitrogen, total carbon, moisture data were considered in the

analysis

570

FIGURE LEGENDS

571

572

Fig. 1: Sampling location map and microbial community diveristy among the study sites. A.

573

Map of the four sampling sites within the Luquillo Experimental Forest (LEF). B. Principal co-

574

ordinate analysis (PCoA) plots based on MASH distances, colored by sampling site, C.

575

Nonmetric multidimensional scaling (NMDS) plot with the soil physicochemical parameters

576

incorporated. The arrow lengths are proportional to the strength of the correlations obtained

577

between measured soil physicochemical parameters and each ordination axis.

578

579

Fig. 2: Abundance of N cycling genes and their distribution across soil ecosystems. A.

580

Abundance of hallmark genes for denitrification, DNRA and nitrogen fixation pathways,

581

represented as genome equivalents (% of total bacterial genomes sampled that carry the gene) in

582

the metagenomes studied (see Figure key). B. Frequency of genomes carrying the respective

583

denitrifying gene across the three ecosystems studied. Genes denoted by the same letter are not

584

statistically significantly different between ecosystems (ANOVA Tukey test). Statistical

585

significance reported at p < 0.05. Note that nitrification genes were not detected in any of the

586

Puerto Rico sites.

587

588

Fig. 3: Phylogenetic diversity of nosZ-encoding sequences recovered in each soil ecosystem.

589

nosZ sequences were identified by the ROCker pipeline and placed in a reference nosZ

590

phylogeny as described in the Materials and Methods section. The radii of the pie charts are

591

proportional to the number of reads assigned to each sub-clade and the colors represent the

592

sampling sites from each ecosystem (see Figure key). Sub-clades highlighted in grey indicate the

593

most abundant sub-clades across all three ecosystems whereas the ones highlighted in blue were

594

abundant only in agricultural soils (IL). A. nosZ reads from every sampling site recruiting to

595

atypical (Clade II) clades. B. nosZ reads recruiting to typical (Clade I) clades. Inset shows the

596

most abundant sub-clade (Opitutus terrae) from panel A and its distribution across all sites. Note

597

that in all three ecosystems most of the reads recruit to atypical sub-clades. Suppl. Fig. S7 shows

598

the distribution of the reads among the most abundant sub-clades in detail.

599

600

Fig. 4: Functions encoded by the recovered population MAGs. Heatmap showing the relative

601

abundance of genes encoding the major metabolic functions (Level 1 of the SEED subsystem

602

category) for each MAG recovered from the four sites in Puerto Rico. The taxonomic

603

classification of each MAG based on MiGA is shown on the bottom left. The symbols at the

604

bottom of the heatmap denote the presence (or absence) of specific N-cycling genes, namely

605

denitrification and nitrogen fixation. No genes involved in nitrification were detected in any of

606

the bins.

607

FIGURES

608

609

610

611

612

Figure 1: Sampling location map and microbial community diveristy among the study sites.

613

614

615

616

617

618

619

620

Figure 2: Abundance of N cycling genes and their distribution across soil ecosystems.

621

622

623

624

625

626

627

Figure 3: Phylogenetic diversity of nosZ-encoding sequences recovered in each soil

628

ecosystem.

629

630

631

632

Figure 4: Functions encoded by the recovered population MAGs.

633

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Phylogenomics Reveal the Dynamic Evolution of Fungal Nitric Oxide Reductases and Their Relationship to Secondary Metabolism

Article

Full-text available

Aug 2018

Fungi expressing P450nor, an unconventional nitric oxide (NO) reducing cytochrome P450, are considered significant contributors to environmental nitrous oxide (N2O) emissions. Despite extensive efforts, fungal contributions to N2O emissions remain uncertain. For example, the majority of N2O emitted from antibiotic-amended soil microcosms is attributed to fungal activity, yet axenic fungal cultures do not couple N-oxyanion respiration to growth and these fungi produce only minor quantities of N2O. To assist in reconciling these conflicting observations and produce a benchmark genomic analysis of fungal denitrifiers, genes underlying denitrification were examined in > 700 fungal genomes. Of 167 p450nor-containing genomes identified, 0, 30, and 48 also harbored the denitrification genes narG, napA or nirK, respectively. Compared to napA and nirK, p450nor was twice as abundant and exhibited two to five-fold more gene duplications, losses, and transfers, indicating a disconnect between p450nor presence and denitrification potential. Furthermore, co-occurrence of p450nor with genes encoding NO-detoxifying flavohemoglobins (Spearman r = 0.87, p = 1.6e-10) confounds hypotheses regarding P450nor's primary role in NO detoxification. Instead, ancestral state reconstruction united P450nor with actinobacterial cytochrome P450s (CYP105) involved in secondary metabolism (SM) and 19 (11%) p450nor-containing genomic regions were predicted to be SM clusters. Another 40 (24%) genomes harbored genes nearby p450nor predicted to encode hallmark SM functions, providing additional contextual evidence linking p450nor to SM. These findings underscore the potential physiological implications of widespread p450nor gene transfer, support the undiscovered affiliation of p450nor with fungal SM, and challenge the hypothesis of p450nor's primary role in denitrification.

The Microbial Genomes Atlas (MiGA) webserver: Taxonomic and gene diversity analysis of Archaea and Bacteria at the whole genome level

Article

Full-text available

Jun 2018
NUCLEIC ACIDS RES

The small subunit ribosomal RNA gene (16S rRNA) has been successfully used to catalogue and study the diversity of prokaryotic species and communities but it offers limited resolution at the species and finer levels, and cannot represent the whole-genome diversity and fluidity. To overcome these limitations, we introduced the Microbial Genomes Atlas (MiGA), a webserver that allows the classification of an unknown query genomic sequence, complete or partial, against all taxonomically classified taxa with available genome sequences, as well as comparisons to other related genomes including uncultivated ones, based on the genome-aggregate Average Nucleotide and Amino Acid Identity (ANI/AAI) concepts. MiGA integrates best practices in sequence quality trimming and assembly and allows input to be raw reads or assemblies from isolate genomes, single-cell sequences, and metagenome-assembled genomes (MAGs). Further, MiGA can take as input hundreds of closely related genomes of the same or closely related species (a so-called 'Clade Project') to assess their gene content diversity and evolutionary relationships, and calculate important clade properties such as the pangenome and core gene sets. Therefore, MiGA is expected to facilitate a range of genome-based taxonomic and diversity studies, and quality assessment across environmental and clinical settings. MiGA is available at http://microbial-genomes.org/.

A global atlas of the dominant bacteria found in soil

Article

Full-text available

Jan 2018

A global map of soil bacteria Soil bacteria play key roles in regulating terrestrial carbon dynamics, nutrient cycles, and plant productivity. However, the natural histories and distributions of these organisms remain largely undocumented. Delgado-Baquerizo et al. provide a survey of the dominant bacterial taxa found around the world. In soil collections from six continents, they found that only 2% of bacterial taxa account for nearly half of the soil bacterial communities across the globe. These dominant taxa could be clustered into ecological groups of co-occurring bacteria that share habitat preferences. The findings will allow for a more predictive understanding of soil bacterial diversity and distribution. Science , this issue p. 320

Denitrification by Anaeromyxobacter dehalogenans , a Common Soil Bacterium Lacking Nitrite Reductase Genes ( nirS/nirK )

Article

Full-text available

Dec 2017

The versatile soil bacterium Anaeromyxobacter dehalogenans lacks the hallmark denitrification genes nirS and nirK (encoding NO2⁻→NO reductases) and couples growth to NO3⁻ reduction to NH4⁺ (respiratory ammonification) and to N2O reduction to N2. A. dehalogenans also grows by reducing Fe(III) to Fe(II), which chemically reacts with NO2⁻ to form N2O (i.e., chemodenitrification). Following the addition of 100 μmol of NO3⁻ or NO2⁻ to Fe(III)-grown axenic cultures of A. dehalogenans, 54 (±7) μmol and 113 (±2) μmol N2O-N, respectively, were produced and subsequently consumed. The conversion of NO3⁻ to N2 in the presence of Fe(II) through linked biotic-abiotic reactions represents an unrecognized ecophysiology of A. dehalogenans. The new findings demonstrate that the assessment of gene content alone is insufficient to predict microbial denitrification potential and N loss (i.e., the formation of gaseous N products). A survey of complete bacterial genomes in the NCBI Reference Sequence database coupled with available physiological information revealed that organisms lacking nirS or nirK but with Fe(III) reduction potential and genes for NO3⁻ and N2O reduction are not rare, indicating that NO3⁻ reduction to N2 through linked biotic-abiotic reactions is not limited to A. dehalogenans. Considering the ubiquity of iron in soils and sediments and the broad distribution of dissimilatory Fe(III) and NO3⁻ reducers, denitrification independent of NO-forming NO2⁻ reductases (through combined biotic-abiotic reactions) may have substantial contributions to N loss and N2O flux. IMPORTANCE Current attempts to gauge N loss from soils rely on the quantitative measurement of nirK and nirS genes and/or transcripts. In the presence of iron, the common soil bacterium Anaeromyxobacter dehalogenans is capable of denitrification and the production of N2 without the key denitrification genes nirK and nirS. Such chemodenitrifiers denitrify through combined biotic and abiotic reactions and have potentially large contributions to N loss to the atmosphere and fill a heretofore unrecognized ecological niche in soil ecosystems. The findings emphasize that the comprehensive understanding of N flux and the accurate assessment of denitrification potential can be achieved only when integrated studies of interlinked biogeochemical cycles are performed.

Year-Round Shotgun Metagenomes Reveal Stable Microbial Communities in Agricultural Soils and Novel Ammonia Oxidizers Responding to Fertilization

Article

Full-text available

Nov 2017

The dynamics of individual microbial populations and their gene functions in agricultural soils, especially after major activities such as nitrogen (N) fertilization, remain elusive but are important for a better understanding of nutrient cycling. Here, we analyzed 20 short-read metagenomes collected at four time points during 1 year from two depths (0 to 5 and 20 to 30 cm) in two Midwestern agricultural sites representing contrasting soil textures (sandy versus silty loam) with similar cropping histories. Although the microbial community taxonomic and functional compositions differed between the two locations and depths, they were more stable within a depth/site throughout the year than communities in natural aquatic ecosystems. For example, among the 69 population genomes assembled from the metagenomes, 75% showed a less than 2-fold change in abundance between any two sampling points. Interestingly, six deep-branching Thaumarchaeota and three complete ammonia oxidizer (comammox) Nitrospira populations increased up to 5-fold in abundance upon the addition of N fertilizer. These results indicated that indigenous archaeal ammonia oxidizers may respond faster (are more copiotrophic) to N fertilization than previously thought. None of 29 recovered putative denitrifier genomes encoded the complete denitrification pathway, suggesting that denitrification is carried out by a collection of different populations. Altogether, our study identified novel microbial populations and genes responding to seasonal and human-induced perturbations in agricultural soils that should facilitate future monitoring efforts and N-related studies. IMPORTANCE Even though the impact of agricultural management on the microbial community structure has been recognized, an understanding of the dynamics of individual microbial populations and what functions each population carries are limited. Yet, this information is important for a better understanding of nutrient cycling, with potentially important implications for preserving nitrogen in soils and sustainability. Here, we show that reconstructed metagenome-assembled genomes (MAGs) are relatively stable in their abundance and functional gene content year round, and seasonal nitrogen fertilization has selected for novel Thaumarchaeota and comammox Nitrospira nitrifiers that are potentially less oligotrophic than their marine counterparts previously studied.

Soil Biology Research across Latitude, Elevation and Disturbance Gradients: A Review of Forest Studies from Puerto Rico during the Past 25 Years

Article

Full-text available

May 2017

Progress in understanding changes in soil biology in response to latitude, elevation and disturbance gradients has generally lagged behind studies of above-ground plants and animals owing to methodological constraints and high diversity and complexity of interactions in below-ground food webs. New methods have opened research opportunities in below-ground systems, leading to a rapid increase in studies of below-ground organisms and processes. Here, we summarize results of forest soil biology research over the past 25 years in Puerto Rico as part of a 75th Anniversary Symposium on research of the USDA Forest Service International Institute of Tropical Forestry. These results are presented in the context of changes in soil and forest floor biota across latitudinal, elevation and disturbance gradients. Invertebrate detritivores in these tropical forests exerted a stronger influence on leaf decomposition than in cold temperate forests using a common substrate. Small changes in arthropods brought about using different litterbag mesh sizes induced larger changes in leaf litter mass loss and nutrient mineralization. Fungi and bacteria in litter and soil of wet forests were surprisingly sensitive to drying, leading to changes in nutrient cycling. Tropical fungi also showed sensitivity to environmental fluctuations and gradients as fungal phylotype composition in soil had a high turnover along an elevation gradient in Puerto Rico. Globally, tropical soil fungi had smaller geographic ranges than temperate fungi. Invertebrate activity accelerates decomposition of woody debris, especially in lowland dry forest, but invertebrates are also important in early stages of log decomposition in middle elevation wet forests. Large deposits of scoltine bark beetle frass from freshly fallen logs coincide with nutrient immobilization by soil microbial biomass and a relatively low density of tree roots in soil under newly fallen logs. Tree roots shifted their foraging locations seasonally in relation to decaying logs. Native earthworms were sensitive to disturbance and were absent from tree plantations, whereas introduced earthworms were found across elevation and disturbance gradients.

Where less may be more: How the rare biosphere pulls ecosystems strings

Article

Full-text available

Jan 2017
ISME J

Rare species are increasingly recognized as crucial, yet vulnerable components of Earth's ecosystems. This is also true for microbial communities, which are typically composed of a high number of relatively rare species. Recent studies have demonstrated that rare species can have an over-proportional role in biogeochemical cycles and may be a hidden driver of microbiome function. In this review, we provide an ecological overview of the rare microbial biosphere, including causes of rarity and the impacts of rare species on ecosystem functioning. We discuss how rare species can have a preponderant role for local biodiversity and species turnover with rarity potentially bound to phylogenetically conserved features. Rare microbes may therefore be overlooked keystone species regulating the functioning of host-associated, terrestrial and aquatic environments. We conclude this review with recommendations to guide scientists interested in investigating this rapidly emerging research area.The ISME Journal advance online publication, 10 January 2017; doi:10.1038/ismej.2016.174.

Effects of Climate Change on CH4 and N2O Fluxes from Temperate and Boreal Forest Soils

Chapter

Mar 2018

Temperate and boreal forest ecosystems cover approximately 13% of the world terrestrial surface and provide a wide range of ecological services to society, including a significant contribution to the regulation of atmospheric greenhouse gas concentrations. Forests do not only function as major sinks (and sources) for atmospheric carbon dioxide (CO2) but also as significant sources and sinks of other atmospheric greenhouse gases, namely, nitrous oxide (N2O) and methane (CH4). The importance of forests as regulators of atmospheric concentrations of these trace gases is undebated, but how this function might change in view of the ongoing climate and associated environmental changes remains a matter of debate. On the one hand, increases in temperature and atmospheric CO2 could lead to permafrost thaw, dramatically increasing N transformation rates in the soil and associated N2O emissions. On the other hand, declining precipitation or changes toward more episodic rainfall events might result in the opposite, through reduced N2O efflux and stimulated uptake of atmospheric CH4 by forest soils. By providing a set of examples from field and laboratory studies, we present the current knowledge and the research perspectives aiming at a better understanding of the current and future role of boreal and temperate forest soils as regulators of the atmospheric concentrations of N2O and CH4 in the frame of global change.

Embracing the unknown: disentangling the complexities of the soil microbiome

Article

Aug 2017

Noah Fierer

Soil microorganisms are clearly a key component of both natural and managed ecosystems. Despite the challenges of surviving in soil, a gram of soil can contain thousands of individual microbial taxa, including viruses and members of all three domains of life. Recent advances in marker gene, genomic and metagenomic analyses have greatly expanded our ability to characterize the soil microbiome and identify the factors that shape soil microbial communities across space and time. However, although most soil microorganisms remain undescribed, we can begin to categorize soil microorganisms on the basis of their ecological strategies. This is an approach that should prove fruitful for leveraging genomic information to predict the functional attributes of individual taxa. The field is now poised to identify how we can manipulate and manage the soil microbiome to increase soil fertility, improve crop production and improve our understanding of how terrestrial ecosystems will respond to environmental change.

Genomics and Ecology of Novel N2O-Reducing Microorganisms

Article

Jan 2018
TRENDS MICROBIOL

Microorganisms withthecapacitytoreducethegreenhousegasnitrousoxide (N2O) toharmlessdinitrogengasarereceivingincreasedattentiondueto increasing N2O emissions(andourneedtomitigateclimatechange)andto recent discoveriesofnovelN2O-reducing bacteriaandarchaea.Thediversityof denitrifying andnondenitrifyingmicroorganismswithcapacityforN2O reduc- tion wasrecentlyshowntobegreaterthanpreviouslyexpected.Aformerly overlooked group(cladeII)intheenvironmentincludealargefractionofnon- denitrifying N2O reducers,whichcouldbeN2O sinkswithoutmajorcontribution to N2O formation.Wereviewtherecentadvancesaboutfundamentalunder- standing ofthegenomics,physiology,andecologyofN2O reducersandthe importance ofthese findings forcurbingN2O emissions.

Metagenomic characterization of soil microbial communities in the Luquillo experimental forest (Puerto Rico) and implications for nitrogen cycling

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