Rapid and Stable Microbial Community Assembly in theHeadwaters of a Third-Order Stream

Morgan E. Teachey,a Jacob M. McDonald,b Elizabeth A. Ottesena

aUniversity of Georgia, Department of Microbiology, Athens, Georgia, USAbUniversity of Georgia, Warnell School of Forestry and Natural Resources, Athens, Georgia, USA

ABSTRACT Small streams and their headwaters are key sources of microbial diver-sity in fluvial systems and serve as an entry point for bacteria from surrounding en-vironments. Community assembly processes occurring in these streams shape down-stream population structure and nutrient cycles. To elucidate the development andstability of microbial communities along the length of a first- through third-orderstream, fine-scale temporal and spatial sampling regimes were employed along Mc-Nutt Creek in Athens, GA, USA. 16S rRNA amplicon libraries were constructed fromsamples collected on a single day from 19 sites spanning the first 16.76 km of thestream. To provide context for this spatial study and evaluate temporal variability,selected sites at the stream’s upper, mid, and lower reaches were sampled daily for5 days preceding and following the spatial study. In a second study, three sites atand near the creek’s headwaters were sampled daily for 11 days to understand initialbacterioplankton community assembly. Both studies revealed decreasing alpha andbeta diversity with increasing downstream distance. These trends were accompaniedby the enrichment of a small fraction of taxa found at low abundance in headwater-proximal sites. Similar sets of taxa consistently increased in relative abundance indownstream samples over time scales ranging from 1 day to 1 year, many of whichbelong to clades known to be abundant in freshwater environments. These resultsunderpin the importance of headwaters as the site of rapid in-stream selection thatresults in the reproducible establishment of a highly stable community of freshwaterriverine bacteria.

IMPORTANCE Headwater streams are critical introduction points of microbial diver-sity for larger connecting rivers and play key roles in the establishment of taxa thatpartake in in-stream nutrient cycling. We examined the microbial community com-position of a first- through third-order stream using fine-scale temporal and spatialregimes. Our results show that the bacterioplankton community develops rapidlyand predictably from the headwater population with increasing total stream length.Along the length of the stream, the microbial community exhibits substantial diver-sity loss and enriches repeatedly for select taxa across days and years, although therelative abundances of individual taxa vary over time and space. This repeated en-richment of a stable stream community likely contributes to the stability and flexibil-ity of downstream communities.

KEYWORDS bacterioplankton, freshwater microbiology

Riverine systems act as an interface between many distinct habitat types, connectinghillslope and bottomland soils to downstream bodies of water and controlling the

flow of bacteria and nutrients from one environment into the next. In many fluvialsystems, alpha and beta diversity both decrease along the length of a stream or streamnetwork (1–11). This suggests that headwater streams are a critical source of microbialbiodiversity. The primary source of microbes entering headwater bacterial communities

Citation Teachey ME, McDonald JM, OttesenEA. 2019. Rapid and stable microbialcommunity assembly in the headwaters of athird-order stream. Appl Environ Microbiol85:e00188-19. https://doi.org/10.1128/AEM.00188-19.

Editor Shuang-Jiang Liu, Chinese Academy ofSciences

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Elizabeth A.Ottesen, ottesen@uga.edu.

Received 22 January 2019Accepted 26 March 2019

Accepted manuscript posted online 5 April2019Published

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appears to be soil and soil waters (1, 2, 8, 12), with pelagic stream community assemblyresulting from the enrichment of bacteria present in these environments at lowabundance. From the headwaters, the bacterioplankton community continues to de-velop as it travels through the stream network (10, 11, 13).

Pelagic stream communities are renewed continuously via the paired forces ofin-stream selection and bulk transport of organisms from upstream and upslopeenvironments. This raises the possibility of rapid and significant shifts in communitycomposition within streams resulting from fluctuations in physicochemical conditions,flow rates, and the composition or origin of microbes entering the stream. Temporaltrends in community profiles have been described in many riverine systems (3, 5–7,12–18). In several of these studies, the same taxa were found over time, varying inabundance based on shifts in physiochemical factors or following landscape-leveldisturbances (5, 10, 13, 17–20). This reoccurrence of taxa has led several groups tosuggest the presence of a core community that is of particular importance in shapingriverine community dynamics (2, 9, 10, 13, 21–23). Conversely, multiple studies havealso found substantial variation in microbial community structure and function overtime in a wide range of study systems (3, 5, 6, 10, 15–18, 20, 22, 24). A temporal studyof headwater streams and an adjoining higher-order stream by Portillo et al. (24)revealed seasonal variation among samples taken from the same site. Despite theirproximity, samples clustered by site regardless of collection day (24). These findingsindicate either that the fluvial core community fluctuates over time or that changingenvironmental conditions lead to alterations in the relative abundance of core versustransient community members.

Few studies have evaluated the extent of short-term variability between the micro-bial communities of headwater streams as well as how daily fluctuations in upstreamcommunity composition may impact downstream community structure and function.Further investigation into the assembly and stability of small stream bacterioplanktonpopulations on both fine temporal (days) and spatial scales is necessary to advance thecurrent understanding of the initial development of freshwater pelagic communities.Fundamental questions remain, including (i) to what extent are fine-scale spatialpatterns of community diversity and composition reproducible over time, and (ii) doin-stream communities exhibit a temporal memory or distance-decay relationship andat what time scale(s)? To directly assess this knowledge gap, two multiday studies ona small creek in Athens, GA, USA, were performed. In the first, we conducted anintensive geographical study consisting of 19 sampling sites along an �16-km transectof the stream to evaluate spatial patterns of community diversity. To evaluate temporalvariability in microbial community assembly, three of these sites (at upper, middle, andlower reaches of the stream) were sampled for the 5 days preceding and following thespatial study. As rapid changes in the community composition were observed in theupper reaches, a second study was conducted in which three headwater-proximal siteswere visited daily for 11 days to more completely characterize temporal variability inthis region of the stream. Three additional sites in this upper section of the creek weresampled at the beginning, middle, and end of the second study to provide a detailedsnap shot of spatial variation.

Our chosen study site, McNutt Creek (Fig. 1A), has consistently demonstrated a lossof diversity with increasing dendritic distance as well as a corresponding increase incommon freshwater taxa across seasons, as seen in larger riverine systems and higher-order streams (25). The sampling sites along the creek capture bacterial communitydevelopment at first- through third-order locations (first order, no confluences withsmaller streams; second order, positions downstream of a confluence with a first-ordertributary; third order, positions downstream of a confluence between two second-orderstreams). These characteristics make McNutt Creek an ideal study system to addressthese questions regarding bacterial community assembly in headwater streams. In thiswork, a rapid rise in dominance of freshwater-associated bacterial taxa along the streamflow path was observed. Community assembly was renewed daily and quickly recov-

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ered following landscape-level disturbance, revealing robust community stability acrossthe stream’s flow path.

RESULTSPhysiochemical results. Two studies of temporal and spatial dynamics in microbial

community composition in McNutt Creek were conducted in 2016 and 2017. The initialstudy (in 2016) examined community progression and temporal stability along thelength of the creek. To capture a high-resolution spatial picture of community pro-gression, 19 sites along the entire creek length were sampled on a single day. Toevaluate the historical context and temporal progression of observed patterns, water

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Cellvibrio; OTU15

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unclass.Betaproteobacteria; OTU7

SBla14; OTU00172

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betIV; OTU11

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unclass. Bacteroidetes; OTU86

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FIG 1 Transitions in bacterial communities from the headwaters to downstream sites on McNutt Creek. (A) McNutt creek is shown in dark blue, and all sampleswere taken along its length (at numbered collection sites). Tributaries are shown in light blue. (B) The relative abundances of OTUs at each location. All OTUsrepresenting 1.5% or more of the community in any sample in the full study (including samples not shown here) are displayed. The legend lists the highesttaxonomic level assigned to each OTU and is sorted and colored by phyla (greens, Actinobacteria; blues, Bacteroidetes; purples, Firmicutes; reds, Proteobacteria;yellows, Verrucomicrobia). OTU numbers are constant across all figures. All taxa present at �1.5% of all samples are grouped into the “other” category (gray).Shannon diversity (C) and OTU richness (D) are plotted against log cumulative dendritic distance. (E) PCoA, with samples colored according to their locationalong the stream path. Inset displays PCoA 1 plotted against log cumulative dendritic distance. Maps created using ArcMap 10.5.

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was also collected at three sites (sites 3, 12, and 18) for the 5 days immediatelypreceding and following the 19-site survey. Table S2 in the supplemental materialpresents environmental metadata associated with the samples, including dissolvedoxygen (DO), water temperature, land use, and land cover parameters. Most measuresdid not show significant trends along the length of the stream, with the exception oftemperature (rho � 0.66, P � 3 � 10�3). There was measurable precipitation on the11th (0.33 cm), 12th (0.31 cm), 14th (1.56 cm), and 17th (0.20 cm) of June for a total of2.39 cm of rain over the course of this study. All samples collected on 14 June weretaken before the rainfall, with the exception of site 4.

A second study was completed in 2017 to investigate community compositionchange at the headwaters and focused on the six upstream-most sites (sites 1 to 6).Sites 1, 3, and 6 were sampled daily for 11 days, and sites 2, 4, and 5 were sampled ondays 1, 6, and 11 to contextualize these results. Average daily air and water tempera-tures were slightly lower than observed during the 2016 study, while total dissolvednitrogen (TDN), total dissolved phosphorus (TDP), and dissolved organic carbon (DOC)were measured at site 3 only and found to be comparable to measurements taken in2016 (Table S2). A total of 3.03 cm of rainfall fell during the 2017 study, with measurableprecipitation occurring on 30 August (0.43 cm) and 31 August (2.59 cm).

Longitudinal study of McNutt creek. In the 2016 longitudinal study, alpha diver-sity (Shannon’s) significantly decreased with increasing cumulative dendritic distance(CDD; P � 1 � 10�4, rho � �0.63) (Fig. 1C). This trend was confirmed using Simpson’sindex (P � 0.02, rho � �0.54). Richness, on the contrary, exhibited no significant trendover the stream length when calculated using total number of unique operationaltaxonomic units (OTUs; in rarefied data) or Chao1 index (Fig. 1D).

To explore changes in microbial community structure and beta diversity, principal-coordinate analysis (PCoA) was completed using rarified samples (Fig. 1E). It is of notethat principal-component analysis (PCA) and nonmetric multidimensional scaling(NMDS) ordinations were also run for all instances in which PCoAs are reported andyielded highly similar results. Log-transformed total CDD correlated strongly with theposition of samples along the first principal coordinate (Fig. 1E, inset) (P � 1 � 10�4,R2 � 0.87). Upstream samples are scattered but downstream sites are clustered moretightly, suggesting a decrease in variance with increasing CDD. The incorporation ofphysiochemical, land use, and nutrient data suggests that tree cover type (deciduousupstream and evergreen downstream), dissolved oxygen levels, water temperature,and urbanization factors (impervious surface level and ground cover type) may influ-ence microbial community structure (see Fig. S1). However, given that only a singlelinked flow path was examined, these relationships are difficult to interpret and may beautocorrelative.

To directly assess distance-decay relationships along the stream length, Bray-Curtisdissimilarity was calculated between each sample site and plotted against the length ofstream between them (Fig. S2). These relationships were calculated in terms of up-stream and downstream proximity. For both weighted and unweighted measures, 12relationships were significant (P � 0.05). All sites downstream of site 13 displayedsignificant dissimilarity between adjacent sites, indicating a downstream increase insimilarity decay.

In addition to the analysis of community-level trends, we examined longitudinaltrends in the abundance of individual taxa (OTUs). Among the 250 most abundant taxa,a statistically significant negative correlation (Spearman’s, false discovery rate [FDR]-adjusted P � 0.05) was found between the relative abundances of 75 OTUs and CDD(Fig. 2). Together, negatively correlated taxa represent 48.83% of sequences in theheadwater site and 6.53% of sequences at site 19 (Fig. 2B). In contrast, a statisticallysignificant positive correlation was found between 35 OTUs and total CDD (Fig. 2A).Positively correlated taxa include OTUs belonging to the freshwater-associated Actino-bacteria clade Luna1-A OTU2, and Bacteroidetes clades bacIII-A OTU3 and bacI-A OTU6(nomenclature as reported by Newton et al. [26]) (Fig. 2A). All positively correlated taxa

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together represent 4.07% of sequences in the furthest upstream site (site 1) and 45.74%of sequences in the furthest downstream site (site 19).

Daily fluctuations in community compositions at the upper, middle, and lowerreaches of McNutt Creek. Daily assessment of sites 3, 12, and 18 during June 2016revealed that the taxonomic profile of the community at each site was relatively stableat all taxonomic levels, including at the level of individual OTUs (Fig. 3A). For site 3, 675OTUs (of 14,860 observed) were shared across the entire 11-day time series, represent-ing an average of 96.11% (range, 95.12% to 97.20%) of recovered sequences. For site12, 513 OTUs (of 15,022) were shared across the sampling period, representing anaverage of 96.75% (range, 95.47% to 97.64%) of recovered sequences. For site 18, 413OTUs (of 15,122) were shared across the 11-day time series, representing an average of95.17% (range, 88.30% to 97.47%) of recovered sequences. Across all dates and times,344 OTUs were shared, representing 93.61% of recovered sequences.

Alpha diversity measurements fluctuated at all sites throughout the study (Fig. 3B).The bacterial community at site 3 was the most diverse (Shannon index, P � 0.01 versussites 12 and 18). When individual days were considered, site 3 had a higher diversity(Shannon) than the other two sites on 9 of 11 sampling dates. The differences inShannon indices between sites 12 and 18 were smaller and not statistically significant.The relationship between these sites on individual days was also less predictable, withsite 18 exhibiting a Shannon of less than 12 for only 4 of 11 days.

Beta diversity analyses suggest that site 3 hosted a distinct community fromdownstream sites. Weighted Bray-Curtis analyses show the median dissimilarities be-tween site 3 and the two downstream sites are markedly higher than the median

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FIG 2 Changes in the abundances of positively and negatively correlated OTUs along the stream length.The relative abundances of all significantly positively (A) or negatively (B) correlated taxa comprising atleast 1% of any sample are displayed in color. All additional taxa below this threshold are reported as“others” in gray, and any taxa that are unclassified beyond the specified level are labeled with theabbreviation “un.” OTU numbers are reported to the right of all taxa for reference.

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dissimilarity between sites 12 and 18. Similarly, a PCoA shows a clear separation of site3 from the downstream samples (Fig. 3C), while samples from sites 12 and 18 clustertogether, which may be driven by differences in water temperature (P � 1 � 10�3).Permutational multivariate analyses of variance (PERMANOVAs) revealed that sites butnot sampling days had a significant effect on community composition (P � 1 � 10�3

and 0.28, respectively) and that sites and days together were also significant(P � 1 � 10�3 and 4 � 10�3, respectively). To assess community progression at indi-vidual sites, Bray-Curtis dissimilarities between samples collected 1 to 5 days apart atthe same site were calculated. These analyses show temporal trends in communitysimilarity over time for abundance-weighted (P � 0.01 for sites 3 and 18) but notunweighted (presence/absence) measures (see Fig. S3). No significance was found foreither measure at site 12.

To understand which taxa consistently increase or decrease downstream, down-stream trends in the abundance of the 250 most-abundant taxa were analyzed. Twohundred thirteen were found to decrease downstream on at least 1 day. ZB2 OTU208,ABY1 OTU280, unclassified Rhodospirillaceae OTU38, and bacII-A OTU4 all decreaseddownstream on 10 of the 11 sampling days. In contrast, 94 of the 250 were identified

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FIG 3 Daily analysis of bacterioplankton communities across the length of the creek. (A) The relative abundances of OTUs present at �1.5% of any one sampleare displayed (all other OTUs are grouped under “other”). (B) Shannon diversity plotted against day of sampling for each of the three selected sites, with sites3, 12, and 18 shown by red, green, and blue dots, respectively. This color scheme was used to show sample ordination in the PCoA plot (C), in which envfitwas used to calculate loadings of the parameters listed in Table S1 in the supplemental material. (D) Bray-Curtis dissimilarity was determined for and betweenall samples taken from each site. Dissimilarity calculated between sites is denoted in the format of site number versus site number, i.e., “3 v 12.” Blue dotsrepresent individual samples and open circles represent outliers. *, significant difference between sample sets.

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as increasing on at least 1 day, although only 12 increased on at least 5 days. BacI-AOTU6 and bacIII-A OTU3 were found to increase between sites 3, 12, and 18 duringmultiple days. Alphaproteobacterium Alf-V OTU193 was the most consistent OTU of thetop 250, increasing down the stream length for 9 of the sampling days. Other organ-isms, such as Luna1-A OTU2, bacV OTU10, betI-A OTU1, and betIV OTU11, that in-creased along the full flow path in the 19-site study only exhibited increases acrossthese three sites for a few of the individual sampling days.

Temporal and spatial trends in community assembly at headwater-proximalsites. The 2017 spatial and temporal study examined community assembly near thestream headwaters in greater temporal and spatial detail. Three longitudinal studies ofthe 6 most uppermost stream sites were performed at 5-day intervals to evaluatespatial and temporal trends in community composition (Fig. 4). Across these threecollections, qualitatively similar trends in taxonomic composition were observed(Fig. 4A). This result was supported by the outcome of PERMANOVA used to test theeffects of site and day on headwater community composition. The effect of site but notcollection day was significant (P � 0.04 and 0.12, respectively), and the effect of site andday together was significant (P � 0.02 and 0.04, respectively). Alpha diversity wassignificantly negatively correlated with log CDD on the second (but not the first orthird) longitudinal sample collection (P � 4.5 � 10�3) (Fig. 4B).

Daily fluctuations in microbial community composition were evaluated at sites 1, 3,and 6 from 24 August to 3 September 2017 (Fig. 5A). Alpha and beta diversity wereconsistently greater at site 1 and lower at sites 3 and 6, in a manner similar to that of

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FIG 4 Community analysis of all headwater site samples collected at three time points in 2017. (A) Therelative abundances of taxa present in each sample are displayed according to the parameters definedin the legend for Fig. 1. (B) Diversity for each sample plotted according to the CDD measurement for eachsite.

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the whole stream (Fig. 1C). Site 1 was found to be significantly more diverse than sites3 and 6 (Shannon, P � 5.7 � 10�3 and �1 � 10�4, respectively), which were notsignificantly different. Day-to-day beta dissimilarity was assessed for each focus site (site1, 3, and 6) to understand community change over time (Fig. 5D). The median weightedBray-Curtis dissimilarity across time was higher at site 1 than either downstream siteand significantly different from site 3 by presence-absence measures (P � 0.02) andfrom site 6 by both measures (P � 3.4 � 10�3 and �1 � 10�4). At site 1, 197 OTUs of15,338 were shared across the entire 11-day time series, representing an average of87.11% (range, 81.83% to 94.35%) of sequences recovered from this site. For site 3, 97(of 15,438) were shared across the sampling period, representing an average of 86.31%(range, 71.42% to 98.70%). This was substantially lower than the 675 OTUs shared atthis site during the 2016 daily study. For site 6, 34 OTUs (of 15,501) were shared acrossthe 11-day time series, representing an average of 91.06% (range, 76.18% to 98.65%) ofrecovered sequences.

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FIG 5 Analysis of daily water samples collected from three headwater-proximal sites across multiple days. (A) Relative abundances of all taxa present in eachsample are displayed according to parameters defined in the legend for Fig. 1 (B) Community diversity observed at each site over the course of the study, withsamples from sites 1, 3, and 6 represented by red, green, and blue points, respectively. (C) Ordination of all daily samples in the resulting PCoA plot. (D)Bray-Curtis dissimilarity was determined for and between all samples taken from each site. Dissimilarity calculated between sites is denoted in the format ofsite number versus site number, i.e., “1 v 3.” Blue dots represent individual samples and open circles represent outliers. *, significant difference between samplesets.

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In a PCoA of these data (Fig. 5C), headwater samples (site 1) clustered separatelyfrom those from sites 3 and 6, with the exception of samples taken after the rain event(days 8 and 9). A PERMANOVA revealed a significant effect of site but not sampling dayon community composition (P � 1 � 10�3 for site and 0.12 for day); however, the effectof sampling day and sites together was significant (P � 0.03). In addition, dissolvedoxygen (DO) content was found to correlate significantly with the PCoA (P � 6 � 10�3

and 0.02 for DO% and DO mg/liter, respectively). No individual sites exhibited signifi-cant temporal distance-decay relationships in Bray-Curtis dissimilarity, with samplestaken 24 h apart showing similar levels of dissimilarity as samples taken up to 5 daysapart (see Fig. S4).

Multiple OTUs were present across the time series. All three sites were consistentlydominated by betI-A OTU1 during the study period, and no consistent trend in itsabundance was observed between sites. However, other Proteobacteria showed posi-tional trends in representation. For example, alfVI OTU26 and betIV OTU11 were foundto increase on 5 days, though they showed decreases on a single study day. Otherproteobacterial OTUs were noted to decrease longitudinally on several days over thestudy period, including Telmatospirillum OTU46, Bdellovibrio OTU17, and GeobacterOTU47. As was observed in 2016, Luna1-A OTU2, and bacIII-A OTU3 increased acrossthe three sites on 8 or more of the study days. Interestingly, bacII-A OTU4 exhibited apositive trend with total CDD on 6 of the 11 sampling days in 2017. These results arein stark contrast to the negative trends OTU4 exhibited for 10 of 11 days in 2016.

The 2.54-cm rain event captured between day 7 and 8 (30 and 31 August) hadvariable effects on the abundances of prominent taxa at each site. As mentioned above,several Proteobacteria, which increased along the sampled flow path on other days,decreased on either day 8 or 9. This was also the case for bacV OTU10, though this OTUhad inconsistencies in trend direction in the 2016 study. Taxa that typically showedpositive trends with CDD (e.g., Luna1-A OTU2, bacIII-A OTU3, and bacII-A OTU4) did noton days 8 or 9 but returned to exhibiting trends in increasing on day 10.

Comparisons of community variation across daily and annual time scales at asingle site. Site 3 was sampled in common across the two studies and used to evaluatecommunity variability at both daily and annual time scales. A clear difference in therelative abundances of multiple bacterial phyla was observed across the two time series(Fig. 4 and 5). In particular, Proteobacteria were found at higher abundances in 2017,while Bacteroidetes and Actinobacteria were found at lower abundances (P � 0.01 for allcomparisons). At the OTU level, 756 of the 770 taxa found in 90% of the 2017 site 3samples were also found in 90% of samples collected in 2016. While only 25 of the8,943 OTUs detected across all samples in 2017 were also present in all samplescollected in 2016, this included 24 of the 100 most abundant OTUs.

The 2017 daily time series exhibited significantly greater day-to-day dissimilarity byboth weighted and unweighted measures than observed in 2016 (t test, P � 0.01 andP � 1 � 10�4, respectively) (Fig. 6). In fact, day-to-day variability in 2017 was similar inscale to cross-year variability between 2016 and 2017. To account for the potentialeffects of the rain event on day 8 of the 2017 study, significance was reevaluated byremoving days 8 and 9 from the data set and rerunning dissimilarity calculations.Weighted dissimilarity was still significantly different although less so (P � 0.03). Un-weighted dissimilarity, however, was no longer significant (P � 0.97). A principal-coordinate analysis showed some degree of clustering within years, with the 2017samples largely separating along dimension 1 and 2016 samples separating alongdimension 2, which may be driven by significant differences in water temperaturebetween the two samples sets (P � 1 � 10�3).

DISCUSSION

This work examined the bacterial community assembly at a daily resolution in athird-order stream located in a temperate urban watershed. While pelagic freshwaterbacterioplankton communities were previously described around the world, the sta-bility and renewal of these populations in headwater streams remains in question. In

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particular, we aimed to elucidate, on fine temporal and spatial scales, the degree towhich lower-order streams mimicked the community assemblages and diversity trendsobserved in higher-order rivers and whole watersheds (1, 2, 4, 8, 9, 11).

Along the length of McNutt Creek, an inverse relationship between alpha diversityand CDD was observed, which was previously reported in other river systems (9, 10, 12,23). The patterns of decreasing alpha diversity with downstream distance traveled weremost apparent across the full stream length (Spearman’s R2 � 0.63, P � 5 � 10�3),however, site-to-site fluctuations were apparent in both daily studies. It is also of notethat alpha diversity calculations for the 2017 study were typically lower than thoserecorded in 2016, although the cause of this diversity loss is unknown.

Downstream trends in site-to-site beta diversity comparisons were present butrelatively weak, primarily exhibiting differences between the headwater and furthestsites downstream. For example, significant unweighted beta diversity relationshipswere found between site 3 and both 12 and 18 in the 2016 study (Fig. 3D) (P� 1 � 10�4

for both). When dissimilarity was assessed along the entire stream length on a site-by-site basis, the majority of sites were found to have a significant positive correlation withdistance, particularly at the extreme ends of the stream (see Fig. S3 in the supplementalmaterial). During 2017, the only significant beta diversity relationships were betweensite 1 and both downstream sites, 3 and 6. These results confirm earlier findings in thebroader Upper Oconee watershed, where three of five seasons exhibited trends in beta

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FIG 6 Comparison of site 3 across years. (A) Resulting ordination plot of PCoA. Samples collected in 2016and 2017 are shown in red and blue, respectively. The envfit function was run with daily metadata, butno values were significant, with the single exception of temperature. (B) Bray-Curtis dissimilarity wascalculated for samples from each year and between years. *, significant difference between sample sets.

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diversity loss with increasing cumulative dendritic distance (10). This pattern of dissim-ilarity decreases with increasing hydrologic distance is not unique to the Upper Oconeewatershed and was also observed across the Ybbs river network in Austria (2).

The effects of time between samples collected on community structure wereapparent at some but not all sites, suggesting a limited impact of the previouscommunity composition on later ones. A similar trend in diversity was recorded in rockbiofilms in a headwater stream, in which weekly samples from the same site were lesssimilar than those taken from other locations along the creek, although site locationswere considerably closer together than in the present work (27). As dispersal in streamsis primarily unilateral as microbes are passively transported, this speaks to the strengthof renewal of these communities and their resilience to immigrant taxa from neigh-boring environments.

Water temperature is the sole environmental factor that was found to have astatistically significant correlation with community composition over both time andspace, suggesting that water temperature may play a key role in shaping streamcommunity composition. None of the other physiochemical parameters measured inthis study showed consistent statistically significant correlations with community pro-gression or composition. These results are not entirely surprising given the results of aprevious study on the greater Upper Oconee watershed, which showed that theposition within the watershed had far more significant impacts on community com-position than individual physicochemical factors (10). In addition, studies in other fluvialand freshwater systems have revealed substantial temporal and spatial variability in therelative importance of different physiochemical parameters in shaping communitycomposition (25, 28). Land use parameters associated with development were signifi-cantly associated with community composition across sites (Fig. S1), and similar rela-tionships were also noted by de Oliveira and Margis (13). However, in this study, theserelationships are more likely to be a coincidental finding due to the fact that down-stream portions of this stream happened to be more highly developed.

During the 2017 sampling period of headwater sites, a storm event resulting in2.54 cm of rainfall occurred. Rainfall was previously shown to introduce taxa intofreshwater systems and increase diversity (22, 29, 30). While our results are consistentwith this, it is interesting that each of the three sites sampled appeared to be affecteddifferently by the influx of rainwater. At site 1, we observed a notable loss of alphadiversity paired with the introduction of Bacteroidetes and Actinobacteria, while site 3exhibited a noticeable increase in total diversity, and site 6 exhibited a decrease indiversity and an increase in the fraction of Proteobacteria (Fig. 2). All sites returned topre-rain community compositions within 48 h (Fig. 4). Several other minor precipitationevents occurred during both studies; however, no strong disturbance was detected inthe community data. While further studies are merited to better understand both theconsistency in disruption and recovery of bacterioplankton populations along theentire stream reach, these data suggest that headwater bacterial communities arehighly resilient to rainwater influx.

Throughout both 2016 and 2017 studies, a small set of taxa found at low abundancein the headwaters of the stream was enriched in downstream sites. This enrichmentcorresponded with the significant decreases of many OTUs prevalent at the headwatersites. These findings support the hypothesis that freshwater streams function as a majorsite of selection and species sorting (2, 8, 10, 12). The set of taxa that were specificallyenriched in downstream environments was relatively consistent over time. MultipleOTUs were identified as significantly positively correlated with stream length acrossdays. Many of these OTUs belonged to well-known “typical” freshwater bacterial clades(10, 23, 25, 26), including Luna1-A (OTU2), bacIII-A (OTU3), bacVI (OTU90), bacV (OTU10),betI-A (OTU1), betIV (OTU11), and bacI-A (OTU6). It is of note that in the 2016 study, ashift in prevalent Bacteroidetes members from bacII-A OTU4 in upstream environmentsto bacI-A OTU6 and bacIII-A OTU3 further downstream occurred, suggesting thatcertain Bacteroidetes clades are better adapted either for life in different regions of thestream or for long-term exposure in freshwater environments.

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These data parallel findings in the existing literature in which a consistent freshwaterstream microbiome has been proposed (2, 9, 10, 13, 19, 21, 31), though the exactdefinition of this population and the nomenclature describing this phenomenon havebeen highly variable. For example, the “core” freshwater community of stream biofilmsand sediment was defined by Besemer et al. as taxa found in at least 50% of all samples(2). A stricter definition was employed in de Oliveira and Margis’ seasonal study of anentire river length, describing the core community as taxa that persisted across allsamples and time points (13). In contrast, Ruiz-Gonzalez et al. (21) defined the “coreseed bank” of bacterial taxa as those that were more abundant in downstream thanupstream sites versus “restricted” taxa that decreased in abundance with increasingdownstream distance. (21). It is clear that a select and consistent set of taxa dominatefreshwater habitats in fluvial systems at all scales (23, 25, 28, 31). However, it is also clearthat the abundances of these microbes can vary substantially, both within and betweenfluvial networks (28). While these variations are often posited to be driven by physi-cochemical variables, the relationships found between specific taxa and differentenvironmental variables have often varied across studies (4, 16) and, in some cases,even across time within a single system (19). With these and other works, it becomesincreasingly apparent that the concept of a “typical” freshwater microbiome (25)extends across all levels of the freshwater continuum, with even a single streamexhibiting strong and reproducible selection for a small subset of microbial taxa thatexhibit variable abundances over time.

Conclusions. It is well documented that, globally, freshwater ecosystems are dom-inated by a consistent set of taxa known as typical freshwater bacteria (26). Headwaterstreams act as a primary entry point for soil and soil water bacteria into river networksand were previously shown to host elevated diversity levels compared to those ofhigher-order water bodies. In this work, we demonstrate temporally stable trends inbacterial diversity and taxonomic representation along the length of the creek. Whilelongitudinal trends in bacterial diversity across watersheds were previously docu-mented, this study demonstrated consistent enrichment of sequences belonging tomultiple OTUs (97% identity sequence clusters) across time scales ranging from days toyears. Furthermore, we found striking community stability at individual stream sites,with little evidence for a temporal distance-decay relationship in community compo-sition at individual sites, and interannual variation that was comparable in scale to dailyvariation. These findings reinforce the hypothesis that entry into freshwater fluvialsystems serves as a strong selective filter resulting in the enrichment of a select set ofbacterial taxa. In addition, this work suggests that downstream ecosystems are stronglystructured by rapid community assembly processes taking place in headwater-proximalstreams.

MATERIALS AND METHODSStudy site description. McNutt Creek is a 20-km-long stream in Athens, GA, USA, that flows through

a mixed land use area, spanning agricultural, residential, and forested sections (Fig. 1A, see also Table S2in the supplemental material). The stream width ranges from 1.10 m (site 1) to 5.18 m (site 19). McNuttCreek is part of the Upper Oconee Watershed, a temperate urban watershed that provides drinking waterfor the city of Athens and the surrounding area.

Sampling schemes. Pelagic water samples were collected from McNutt Creek, Athens, GA, USA(33°55=50.2�N, 83°30=30.91�W). Nineteen sampling locations were selected based on their location in thestream network (headwaters versus main stem) and the ability to access these sites, as some werelocated on private property and required the consent of the owners. Water samples were collected in2016 and 2017 to assess community assembly and stability along the headwater (1st order) to main stem(3rd order) longitudinal gradient. In 2016, three streams (sites 3, 12, and 19) were sampled daily from 9to 19 June. On 14 June, all nineteen sites were sampled within 7 h. Three groups of three to fourvolunteers each were sent out to survey a third of the creek, working from the most upstream site tothose downstream.

At each site, water was collected mid-stream from the water column approximately at mid-depth in4-liter acid-washed Cubitainers. Each container was rinsed with stream water three times before the finalsample was collected. Water was processed either immediately on site or within 1 h of collectionfollowing the sampling methods outlined by Hassell et al. (10). Samples were run through sterilizedtubing with in-line filtration through a 5.0-�m 47-mm-diameter SVPP prefilter (Millipore) to captureparticulate matter. The effluent was then run through a 0.22-�m 2-ml Sterivex filter (Millipore). A total

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of 500 ml of water was filtered for each sample. Filters were preserved at �80°C until DNA extraction. Inaddition to the filtered water samples, a YSI Professional Plus meter was used to measure temperature,pH, dissolved oxygen percentage, and conductivity in the field (Table S2). Dissolved organic carbon(DOC), total dissolved nitrogen (TDN), and total dissolved phosphorus (TDP) were measured for selectedfiltered water samples by the Stable Isotope Ecology Lab at the University of Georgia (Table S2). Daily airtemperature measurements were available through Weather History for Athens (https://www.wunderground.com/history/airport/KAHN/), and daily precipitation measures were available through theNational Weather Service (https://www.weather.gov/ffc/rainfall_scorecard).

In 2017, sampling was focused on headwater-proximal sites (sites 1 to 6) to determine the repro-ducibility of initial community assembly. Sites 1, 3, and 6 were sampled daily from 24 August to 3September. On 24 and 29 August and 3 September, samples were collected from all six uppermost sites(sites 1 to 6). The 2017 samples were collected using the same methods as used in 2016.

Watershed characteristics. Geographic information system (GIS) analysis was used to determinephysical differences between each site’s watershed, drainage network, and land use/land cover. ArcMap10.5 was used to calculate each sample site’s watershed area, total cumulative dendritic distance (CDD),land use/land cover characteristics, and impervious surface area. Each sample point’s watershed wasdelineated using “hydrology” tools in the Spatial Analyst toolbox and the national elevation data set’s30-m digital elevation model (DEM) (32). Flow direction and flow accumulation rasters were created froma hydrologically corrected DEM (“fill” tool was used to correct the DEM). After the sampling points wereassociated with the flow accumulation raster (using the Snap Pour Point tool), each watershed wasdelineated using the “watershed” tool. Total dendritic distance was calculated by using each samplingpoint’s watershed to clip the high-resolution national hydrography data set’s stream layer (33). Similarly,each watershed was used to extract land use/land cover characteristics and impervious surface coverfrom the 2011 national land cover database (34, 35).

DNA extraction. DNA was extracted from the Sterivex filters according to the methods outlined byHassell et al. (10). Filters were thawed, and then 1 ml of lysis buffer (40 mM EDTA, 50 mM Tris [pH 8.3],0.73 M sucrose) and lysosome dissolved in lysis buffer (2.11 mg/ml final concentration) were added andincubated at 37°C for 30 min while rotating. Proteinase K dissolved in lysis buffer (0.79 mg/ml finalconcentration) and 200 �l 10% SDS were added for a second incubation step at 55°C for 2 h. The lysatewas extracted and mixed with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1; pH 8.0).Samples were centrifuged for 5 min at 3,500 � g, and the top phase was saved. Next, 0.04� volume 5 MNaCl and 0.7� volume isopropanol were added, mixed, and incubated at room temperature for 10 min.Samples were centrifuged for 15 min at 17,000 � g, and the supernatant was discarded. DNA wasresuspended in 400 �l elution buffer (Omega Biotek, Norcross, GA, USA) and incubated at 65°C whilerotating for 10 min. DNA was then processed using Omega Bio-tek’s E.Z.N.A. Water DNA kit according tothe manufacturer’s protocol from step 13 through completion (Omega Bio-tek, May 2013 version).

PCR, sequencing, and analysis. According to the methods outlined by Tinker and Ottesen (36), theV4 region of the 16S rRNA gene was amplified in each DNA sample. The resulting library wassubmitted to the Georgia Genomics Facility for sequencing (Illumina MiSeq 250 � 250 bp; Illumina,Inc., San Diego, CA).

Returned reads were processed using the mothur software package (37) according to the MiSeqstandard operating protocol with the following modifications: reads that fell outside 200 to 275 bp wereexcluded from contig generation; SILVA reference database release v123 was used for sequencealignment; primers GTGCCAGCMGCCGCGGTAA and GGACTACHVGGGTWTCTAAT were used to performin silico PCR; the VSEARCH algorithm was used to identify chimeric sequences (38); taxonomic classifi-cation was completed following the TaxAss workflow (39–41), using the FreshTrain data set to firstclassify as many sequences as possible using the freshwater-associated database and then classifyingremaining sequences using the 5 May 2013 release of the Greengenes reference database, version 13_8.During taxonomic assignment, a bootstrap value of 70 was used; an additional remove.lineage commandwas run to ensure that sequences from cyanobacteria, chloroplasts, and unknown taxa were removed.Operational taxonomic units (OTUs) were called at 97% or greater sequence similarity. From the 91samples that were collected from these studies, a total of 6,119,579 16S rRNA gene sequences passedquality filtering steps, resulting in an average of 67,248 sequence reads and 1,562 OTUs per sample (seeTable S1). Rarefaction curves were calculated for each study and are shown in Fig. S5.

Statistical analysis was completed in R using the vegan package (42). When necessary for analysis,samples were rarefied to a depth of 3,291 sequences. The envfit function was used to calculate significant(P � 0.05) physiochemical parameters and watershed characteristics that are displayed as loadings onordination plots. The cor.test function was utilized to test for significance of Spearman’s correlations. ttests were used to determine significance between boxplots. When appropriate, the Benjamini-Hochbergprocedure was used to adjust P values for false discovery rate correction.

Accession number(s). The raw sequences from these experiments are available under the accessionnumbers SRP155540 in the NCBI Sequence Read Archive.

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/AEM

.00188-19.SUPPLEMENTAL FILE 1, PDF file, 2.0 MB.SUPPLEMENTAL FILE 2, XLSX file, 0.1 MB.SUPPLEMENTAL FILE 3, XLSX file, 0.1 MB.

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ACKNOWLEDGMENTSWe thank the following volunteers for their participation in sample collection on 14

June 2016: Kara Tinker, Andi Esterle, Trace Borchardt, Emma Parks, Amy Ward, AlvinSoto, Chris Moxley, and Jason Westrich. We also thank Norman Hassell for his assistancein study preparation and sample collection.

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