APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2008, p. 2554–2557 0099-2240/08/$08.00⫹0 doi:10.1128/AEM.01482-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 8
Temporal and Spatial Patterns of Eukaryotic and Bacterial Communities Found in Vernal Pools䌤† Sarah R. Carrino-Kyker* and Andrew K. Swanson Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106-7080 Received 2 July 2007/Accepted 15 February 2008
In this study, we examined the effects of physicochemical variability on the microbial communities of vernal pools. Denaturing gradient gel electrophoresis revealed temporal changes to be more pronounced than spatial changes in eukaryotic and bacterial communities. Sequencing revealed high degrees of richness in decomposers, which supports the notion that vernal pools are heterotrophic habitats. bonate filters through which 50 ml of water was filtered. At the time of sampling, dissolved oxygen concentrations, pHs, average depths, and terrestrial light levels were measured in situ. Relative conductivities of water samples were determined in the lab. Community profile analysis was carried out by PCR-denaturing gradient gel electrophoresis (DGGE). DGGE was performed first to compare a subset of pool samples (three pool samples from three different sampling dates) of each individual sample type (water, soil, and organic detritus) separately and then to compare spatial and temporal trends among the five pools (for which the DNA from the three sample types was combined to assess whole-pool changes). DNA from each sample type was extracted such that the total masses (averages for all extracted samples were as follows: water, 103.5 ⫾ 55.9 mg; soil, 666.3 ⫾ 127.9 mg; and detritus, 529.9 ⫾ 96.6 mg) were kept consistent. The fast DNA spin kit for soil was used for DNA extractions according to the protocol of the manufacturer (MP Biomedicals LLC, Solon, OH), except that cell lysis was performed by grinding the samples under liquid nitrogen and shaking them on a vortex genie. For PCR, we targeted the
Vernal pools are seasonally flooded ecosystems that are subject to variability in abiotic conditions (8). Although previous studies have documented the importance of abiotic conditions for vernal-pool macroorganisms (3, 9, 10, 12, 18, 20), little is known about abiotic influences on microorganisms. Given the abundance of vernal pools in the landscape (e.g., reference 5) and their highly variable physicochemical natures (2, 4, 6, 17, 19), vernal pools can serve as a model system to explore abiotic influences on microbial communities. Further, while the diversity of vernal-pool macroorganisms has been welldocumented, comparable studies of microbial biodiversity are rare (see reference 8 for a review). In one survey of a single snowmelt pool, 76% of the identified biota were protists or bacteria (15), which suggests that these organisms dominate temporary water bodies in terms of both species richness and abundance (8). The main objective of this study was to characterize the microbial (eukaryotic and bacterial) communities of vernal pools in northeastern Ohio and to illustrate any spatial and temporal variations in these communities in relation to abiotic factors. This study was conducted at Case Western Reserve University’s Squire Valleevue and Valley Ridge Farms (41°29⬘53⬙N, 81°25⬘27⬙W; elevation, 320 m), where five vernal pools (one man-made pool, called the salamander pond, and four natural pools), which ranged in size from 16 to 20 m2, were studied. The pools were located within an 80-year-old secondarygrowth forest, which is characterized by well-drained, silt loam soils and a canopy dominated by Acer saccharum and Fagus grandifolia. From each pool, overlying water, organic detritus (i.e., leaf matter), and soil were collected on 1 April, 4 May, and 2 June 2004. These three collection times were shortly after snow melting and prior to the drying of the pools. Three replicate samples (of each sample type) were collected from the shore of each pool and combined. Specifically, soil samples were cores collected with sterile 1-ml pipette tips, and water samples were collected with 0.2-m-pore-size polycar-
* Corresponding author. Mailing address: Department of Biology, Case Western Reserve University, 125 Millis Hall, 2080 Adelbert Rd., Cleveland, OH 44106-7080. Phone: (216) 368-8581. Fax: (216) 3684672. E-mail:
[email protected]. † Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 29 February 2008.
FIG. 1. NMDS ordination diagram showing the changes in vernalpool eukaryotic communities from three sample types over 3 months. The letters denote the three sample types (W, water; S, soil; and O, organic detritus). The stress value of the final configuration was 16.13%. 2554
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TABLE 1. Pearson correlation coefficients showing the relationships between vernal-pool physicochemical characteristics and dimensions 1 (EUK dim 1) and 2 (EUK dim 2) from the NMDS analysis of the eukaryotic DGGE data and dimensions 1 (BAC dim 1) and 2 (BAC dim 2) from the NMDS analysis of the bacterial DGGE dataa Coefficient for relationship with: Characteristic
Light pH DO concn Conductivity Depth a
EUK dim 1
EUK dim 2
BAC dim 1
BAC dim 2
Light
pH
DO concn
Conductivity
Depth
0.261 0.563 0.535 ⫺0.107 0.393
0.470 ⫺0.393 ⫺0.223 ⫺0.645 0.265
0.183 ⫺0.234 0.146 ⫺0.781 0.556
⫺0.157 0.621 0.327 0.100 ⫺0.066
1 ⫺0.184 ⫺0.107 ⫺0.192 0.153
1 0.709 0.180 ⫺0.212
1 0.013 ⫺0.008
1 ⫺0.456
1
Light, instantaneous canopy light levels; DO concn, dissolved oxygen concentration; depth, average depth for three arbitrary locations.
18S ribosomal DNA of eukaryotes and the 16S rRNA genes of bacteria with primer sets F37GC/R518 and F1427GC/R1616, respectively (21). PCR amplifications were performed on a MyCycler thermocycler (Bio-Rad Inc., Richmond, CA) using Platinum Taq high-fidelity DNA polymerase (Invitrogen Inc., Chicago, IL). PCRs and cycling were essentially as described by van Hannen et al. (21), except that the total reaction volume was 25 l, the final extension time was 10 min, and the bacterial PCR included 30 cycles, in which the annealing step began at a temperature of 65°C, which decreased by 0.4°C every repeat for the first 25 cycles and remained at 55°C for the final 5 cycles. Because the PCR template concentration can influence the composition and distribution of mixed-species amplification products (7), four dilutions of the template were used: 1:5, 1:10, 1:20, and 1:40. Positive reaction mixtures were combined prior to DGGE, and approximately 800 ng of PCR products per lane, on average, was loaded. DGGE was performed with the DCode universal mutation detection system (Bio-Rad Inc., Richmond, CA) by following the basic protocol of van Hannen et al. (21). Nucleic acids were visualized by staining with a 1:10,000 dilution of Sybr gold (Molecular Probes, Eugene, OR). All visualized bands were excised with separate 1-ml pipette tips and stored in water at ⫺20°C prior to preparations for sequencing. Distinct bands were identified as operational taxonomic units (OTUs) with TotalLab gel analysis software (Nonlinear Dynamics Ltd., Durham, NC). OTU profiles were analyzed by
nonmetric multidimensional scaling (NMDS) to assess changes in community composition (21). NMDS was performed with SAS (version 9.1, 2003; SAS Institute) using PROC MDS and PROC IML to calculate the Bray-Curtis distance metric (11). To assist in interpreting the changes in community profiles, Pearson correlations between dimension scores and the vernalpool characteristics were calculated with SPSS (version 12.0.1, 2003; SPSS Inc.). To assess differences in degrees of richness (numbers of OTU) in the three sample types over time (n ⫽ 9; three pool samples from each of three different sampling dates), a two-way analysis of variance with repeated measures and a Tukey posthoc test were conducted with SigmaStat (version 3.5, 2006; Systat Software, Inc.). Prior to the analysis of variance, tests determined that the data had equality of variances. NMDS revealed differences in the eukaryotic communities among the three sample types (Fig. 1). The eukaryotic communities on detritus formed a fairly distinct cluster, which separated especially from the soil communities. Degrees of eukaryotic richness varied significantly among sample types (P ⫽ 0.036) and were significantly greater in detritus samples than in water and soil (P ⫽ 0.050 for both Tukey posthoc tests), while no other significant differences were observed (comparison by date, P ⫽ 0.672; comparison by a combination of sample type and date, P ⫽ 0.150). In a study of the bacterial communities associated with the water, sediment, and rhizosphere of a wetland, Ibekwe et al. (14) found differences
FIG. 2. NMDS ordination diagrams showing the changes in profiles of eukaryotic communities (stress value, 13.78%) (A) and bacterial communities (stress value, 17.23%) (B) in vernal pools over 3 months. The letters denote the sample months (A, April; M, May; and J, June), and the subscripts denote the pools (S, salamander pond; 1, vernal pool 1; 2, vernal pool 2; 3, vernal pool 3; and 4, vernal pool 4).
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TABLE 2. Sequence similarities of excised OTUsa OTU no.
% Similarity to closest relative sequenceb
Species corresponding to closest relative sequence
Accession no. of closest relative sequence
Taxonomic classification corresponding to closest relative sequence
Eukaryotic sequences 1 2 3 4 6 7 8 9 11 12 13 14 15 16 17 18 19 21 22 23 25 27 28
99 100 99 96 98 97 98 97 100 98 98 100 99 97 100 100 98 99 97 100 99 100 100
Navicula ramosissima Achlya bisexualis Bodomorpha sp. Gymnophrys cometa Uroglena sp. Chrysocapsa paludosa Chaetonotus sp. Hyphodiscus hymeniophilus Sporidiobolales sp. Hyphodiscus hymeniophilus Rhizophydium elyensis Leptodontidium orchidicola Chalara cylindrosperma Troposporella fumosa Leptodontidium orchidicola Dothidea sambuci Xylariales sp. Cyprididae sp. Physoderma dulichi Acidomyces richmondensis Leotia lubrica Bryocamptus pygmaeus Propappus volki
AY485512 DQ403201 DQ211595 AJ14866 EF165132 EF165145 AJ001735 DQ227258 EF060827 DQ227258 DQ536479 DQ521603 AF222507 AY856953 DQ521603 AY544722 AB255193 AY622196 DQ536472 AY374300 AY544687 AY627015 AY365457
Bacillariophyceae Oomycota Cercozoa Cercozoa Synurophyceae Chrysophyceae Gastrotricha Ascomycota Basidomycota Ascomycota Chytridiomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ostracoda Chytridiomycota Ascomycota Ascomycota Copepoda Annelida
Bacterial sequences 6 7 10 11 12 13 14 16 17 18 19 20 21 22 23 28 29 31
99 97 94 97 86 99 98 97 100 100 96 99 97 100 96 92 94 96
Variovorax paradoxus Flavobacteria bacterium Variovorax dokdonensis Rhodoferax sp. Variovorax sp. Acidobacterium capsulatum Terriglobus roseus Amphora delicatissima Erythrobacter sp. Erythrobacter sp. Rhodoferax sp. Pseudomonas sp. Methylobacterium extorquens Asticcacaulis sp. Aquabacterium sp. Pseudonocardia sp. Flavobacterium columnare Sphingomonas sp.
DQ257419 AB269814 DQ178978 EF451708 AM502921 AM086241 DQ660895 U96445 AY371411 EF581001 AB206450 EF541138 DQ870723 AB093140 AF089859 EF588230 DQ005510 AF191021
Betaproteobacteria Flavobacteria Betaproteobacteria Betaproteobacteria Betaproteobacteria Acidobacteria Acidobacteria Diatom chloroplast Alphaproteobacteria Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Alphaproteobacteria Alphaproteobacteria Betaproteobacteria Actinobacteria Flavobacteria Alphaproteobacteria
a b
Sequences were aligned to the closest relatives from the GenBank database. Gaps and ambiguities were not included in the similarity calculations.
among these three substrates, similar to the results of our study. However, the difference between the water and the sediment in the wetland was most notable, while in our vernalpool study, the eukaryotic community varied mostly between the detritus and soil. Our results are somewhat contrary to those of Verb et al. (22), who found little influence of the vernal-pool vegetative substrate on algal communities. For whole-pool comparisons, strong correlations between NMDS dimension scores and physicochemical characteristics were observed (Table 1). This finding suggests that changes in the microbial community composition coincided with shifts in abiotic factors, which also changed over the study period (see Table S1 in the supplemental material). Changes in both eukaryotic and bacterial communities showed strong positive correlations with the pH, dissolved oxygen concentration, and
depth and negative correlations with conductivity. The sampling time also affected microbial communities. NMDS revealed that the eukaryotic communities of all pools changed in June (Fig. 2A). Though the bacterial communities did not show as much clustering as the eukaryotic NMDS pattern, there were essentially three groups of bacterial communities roughly associated with the sampling month (Fig. 2B). From the whole-pool DGGE gels, one excised band representing each OTU was reamplified and used for DNA sequencing (see Fig. S1 in the supplemental material). These band sequences were screened against GenBank sequences last updated in June 2007 (http://www.ncbi.nlm.nih.gov) with BLAST. Biologically relevant sequences with the highest similarities were then aligned with the DGGE band sequences in BioEdit, version 7.0.5.2 (13), and used in a similarity index calculation,
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which omitted gaps and ambiguities. The results were confirmed with phylogenetic tree analyses (data not shown). Of the 28 eukaryotic OTUs (see Fig. S1 in the supplemental material) and 32 bacterial OTUs, 23 eukaryotic OTUs and 18 bacterial OTUs were successfully sequenced (Table 2). The eukaryotic richness was well-represented by fungal groups, which are important to the decomposition of allochthonous carbon input into vernal pools (1). The pools were also rich in Alphaproteobacteria and Betaproteobacteria, which is consistent with the results of Olapade and Leff (16), who found that these groups dominated bacterial biofilm assemblages in a stream. Ibekwe et al. (14) also observed an abundance of Betaproteobacteria in the soil and rhizosphere of a wetland. These authors suggest that Alphaproteobacteria and Betaproteobacteria may be important in such systems for the degradation of organic matter (14, 16). The prevalence of fungal groups and Alphaproteobacteria and Betaproteobacteria in vernal pools suggests that microbial communities are rich in heterotrophic organisms. Overall, our study revealed that vernal-pool microorganism communities are temporally variable and influenced by physicochemical conditions within the pools. The importance of the substrate type for the structure of eukaryotic communities and the prevalence of fungi and Alphaproteobacteria and Betaproteobacteria indicate that microbial communities in these pools are rich in heterotrophic organisms and decomposers. Nucleotide sequence accession numbers. The sequences determined in this study were deposited in GenBank under accession numbers EU000325 through EU000365.
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