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This study of recreation in two geographically distinct regions of the United States indicates that social media can predict visitation at recreation sites on public land. We conclude that social media data can be applied with moderate success to estimate visitation at sites that are unmonitored or otherwise lack on-site counts, even in new regions. A basic visitation model that relies solely on generic predictors (e.g., weather, holidays, and seasonality) is only modestly successful due to regional differences in visitor behavior (Model 1). Performance is improved by including relationships between visitation and social media (Flickr, Instagram, and Twitter), even when these relationships are transferred from a different region (WWA, Model 2). These results are consistent with prior research findings that social media counts are correlated with on-site visitor counts from public lands8,9,24,32, and extend earlier findings by showing the potential for statistical models to estimate absolute numbers of visitors at unmonitored sites with parameters derived from social media. This is evidence of patterns in how visitors use and share social media. Furthermore, it suggests that measurable variables associated with social media use could support transferable models for accurately estimating visitation to public lands across large geographies.
We expected to detect regional differences in social media use given regional distinctions in climate, land management, population density, mobile phone signal coverage, and the demographics of visitors. Contrary to our expectations, we observed that the rate of posting to social media about recreation visits is similar across sites in NNM and WWA, and both regions displayed positive correlations between each of these social media data sources and observed visitation (Fig. 3). Furthermore, a model parameterized with social media use in WWA (Model 2) explains 45% of the variation in visitation across all 13 sites in NNM and 79% of the variability in visitation at the subset of sites that had social media posts. Re-parameterizing Model 2 with a portion of NNM visitation data does not improve its performance (Model 3) until these data are considered at the site level (Model 5). There is a noisy but consistent relationship between a destination’s popularity with visitors and its popularity on social media, regardless of whether the site is in NNM or WWA. We interpret this as evidence that visitors are equally likely to share their recreation experiences in NNM or WWA, despite the regional differences in the types of recreation opportunities and the people who are visiting.
Although there are consistent relationships between social media use and visitation at a regional scale, we see large site-to-site variability in how visitors use social media within both regions. Beyond simple differences in numbers of posts by site, the proportion of people who post about their visits varies by destination, ranging from 7% of visitors to Kasha–Katuwe Tent Rocks National Monument posting on Instagram to zero recorded social media user-days at several trails in the Valles Caldera National Preserve. As a result, models with random effects that allow sites to have unique relationships between social media, weather, and visitation (Models 4–5) perform substantially better than models that assume social media use is consistently related to visitation for all types of sites (Models 1–3). This is especially true at the most and least visited sites (Figs. 4, 5), where visitors may be sharing social media differently than they do at moderately visited sites, and responding differently to other conditions such as weather or holidays. These results indicate that while data on social media use are helpful for predicting visitation with moderate certainty in an otherwise unknown region (Model 1 vs. 2), their utility for estimating visitation is less clear when local data on the effects of environmental and institutional conditions such as weather and holidays are available to parameterize site-specific models.
A primary goal of this study is to test approaches for estimating visitation over relatively small areas in order to explore the limits of the data and methods. We find that six of the 13 sites in NNM—representing individual trails or groups of trails within a larger park—lack social media during the study period. Model 2, which depends on social media data to estimate visitation at unmonitored sites, consequently performs relatively poorly at these six sites (Fig. 4). Generally, sites that have sparse social media data tend to receive few visitors, but there are exceptions. Alcove House in Bandelier National Monument, for instance, is a very highly visited site that lacks Instagram images in our study because the site does not appear as a prescribed location for Instagram users. Our informal observation is that many visitors instead share untagged photographs of Alcove House or assign their images to other relevant place names such as Bandelier National Monument. Clearly, there are thresholds to where and how social media can be leveraged for visitor estimation. Our research suggests that future studies and visitation models could be improved by accounting for the popularity of sites on social media in the study design12. Visitation models that include predictors derived from social media (Models 2, 3, and 5, here) will likely out-perform alternative models for estimating use at popular sites or when longer time series are available. At locations with low or no posting activity, where social media contributes less to visitor estimates, it could be more useful to collect on-site data such as vehicle and pedestrian counts. Further research is necessary to understand what combination of on-site, social media, environmental, and other data is most valuable at different spatial and temporal scales.
These observations suggest that variability in correlations between social media and on-site visitor counts seen here and in previous studies8,12,20 is derived from local factors influencing visitors’ day-to-day decisions about whether and how to share a destination on social media. Choices about whether to post to social media are likely influenced by the characteristics of the local site—perhaps its topography, amenities, predominant activity, or unique natural features—and the ways that people relate to these features33. The characteristics of the visitors and the relative contributions of the natural versus the social experience in the motivation for the trip may play a role. For example, if a given type of site attracts visitors wishing to “unplug” and have a nature-based outing (a calm forest glade, say), there may be fewer posts per visit than for a site attracting visitors who desire a social experience within a natural setting (a famous scenic overlook, for instance). Another possibility is that the prevailing popularity of certain destinations on social media creates a positive feedback, whereby new visitors feel compelled to share content about their visit in response to the posts of others or the local hashtags that may make it easier or more enticing to post. Variability could also arise from the recent trend towards discouraging visitors from posting geolocated content and attracting attention to less popular or back-country sites that are not equipped to sustain higher use, although this is probably of minor importance, currently.
This is the first study to our knowledge that develops and tests models for estimating absolute numbers of visitors at unmonitored recreation sites or times using multiple social media data sources with differential effects. Building on earlier research exploring relationships of park visitation with numbers of posts to multiple social media platforms9,12,20,27, the present study tests whether models with a mixture of predictors to represent varying effects of three online platforms can estimate visitation in novel situations. We find that each social media data source contributes information that explains a statistically significant portion of the variability in visitation and improves the accuracy of the estimate. This is the case not only for Instagram, which captures 3–4% of visitor-days at our research sites in WWA and NNM, but also for Flickr and Twitter, with relatively small amounts of content shared ( More

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Threatened species assessments
We assessed the proportion of the proposed budget allocated to RM for a total of 2328 species, independently managed subspecies, or distinct populations (hereafter species): 700 in NZ, 361 in NSW, and 1267 freshwater and terrestrial species in the U.S. In all jurisdictions this included the most threatened listed species and/or those with recovery plans: species with Threatened and Endangered status in the U.S. with active recovery plans as of January 2017, species that met a series of criteria in NSW as of 2013 (e.g., excluding less threatened species that do not require any active intervention and those with a large geographic range17), and the most threatened species in New Zealand as of 2012, which included all species in the Threatened and At-Risk categories with declining populations42. In all three jurisdictions, species are listed for legal protection if they are at risk of extinction. Once listed, recovery planning (including proposed projects, management tasks, and budgets) documents are developed with the objective of securing species from extinction and recovering populations to a point that they can be de-listed. Although our dataset examining threatened species recovery planning is the most comprehensive to date, our data do not represent all spending on species—there are other activities for both management action and RM that occur at a sub-jurisdictional level or outside of government.
Estimating resources allocated to RM vs action
We gathered information on the planned costs of management tasks necessary to achieve recovery for threatened species from previously published recovery planning databases (details provided in refs. 15,16,43,44 and Supplementary Methods). Briefly, for NZ and NSW, a suite of management tasks had been developed during structured expert elicitation workshops, as part of a systematic prioritization exercise16,17. For the U.S., management tasks and their cost had been extracted from each species’ published recovery plans (Supplementary Methods15). These data represent an evolution of the implementation of a systematic and cost-effective approach to endangered species resource allocation (i.e., the Project Prioritization Protocol), beginning with NZ in 200916, and subsequently applied to NSW in 201317 and the U.S. in 201615.
For each proposed management task we used the methods description to categorize tasks as research and monitoring or action based on the definitions in IUCN classification schemes (https://www.iucnredlist.org/resources/classification-schemes, Supplementary Table 145). For NZ and NSW, using previously published datasets we used a combination of the methods description field and 4 other columns that classified the management task methods into increasingly general categories16,17,43. We used keywords such as survey, monitor, surveillance, develop techniques, inventory, research, and develop plan to search for research and monitoring tasks. We reviewed the management tasks identified by these broad search terms to ensure only research and monitoring tasks were included. We also reviewed the management tasks that were not captured by search terms to ensure no research and monitoring tasks were excluded. For the US, the methods descriptions were too complex for keyword searches. Instead, the first author and a trained technician classified each management task manually. To ensure that management tasks were being classified similarly, the first 200 tasks were classified by both observers and any uncertainty was flagged for review together.
For all jurisdictions, any methods descriptions that were vague, lacked context, or required further assumptions were excluded (2.6% of management tasks, U.S. only). Some management tasks (3.9%) were scored as both action and RM (e.g., translocate birds, action, and monitor the success of the release, RM; weed surveillance, RM, and control, action). For some management tasks, the distinction between action and RM was unclear. These tasks were discussed among the authors and the technician to reach a consensus. For example, ‘standard surveillance to detect invasive mammals’ in NZ could be considered an action, since it is required to detect and subsequently control invasions. However, we assigned it as RM because other management tasks clearly include an action component (e.g., ‘surveillance for invasive species and control if detected’) and other authors have categorized invasive species surveillance as monitoring46. Generally, management tasks to develop conservation plans are distinct from implementing plans and were thus scored as RM (K. Martin pers. comm.). Where we were unable to distinguish between RM and action, we scored as both action and RM.
For a subset of 8050 management tasks (the first 207 species) in U.S. recovery plans, we further categorized the type of RM to explore common RM activities (Supplementary Table 1). Because we found that assigning management tasks into these 17 categories was challenging without making subjective judgement calls, we did not analyze specific tasks further.
We estimated the cost of implementing each management task for each species following similar methods to those previously published, calculating costs over 50 years15, Supplementary Methods16,17. We calculated the proportion of the proposed budget allocated to RM for each species as the total cost of all management tasks scored as research or monitoring divided by the total cost of all management tasks. For management tasks that were scored as both action and RM, we multiplied the cost of the task by the average proportional difference between action and RM for each jurisdiction.
Factors affecting the proportion allocated to RM
We compared the characteristics of each species recovery plan with the proportion of proposed spending designated as RM. Characteristics available in recovery planning databases for all three jurisdictions included taxon, the estimated benefit of implementing all management tasks, and the total budget estimated for each species (Table 1, Supplementary Methods). The most general category shared among all jurisdictions was taxon, resulting in nine categories: amphibians, birds, bryophytes, fishes, fungus, invertebrates, mammals, reptiles, and vascular plants (set as a reference category). Lichens were removed from further analysis because there were only two species. For NZ and NSW, we extracted expert-elicited estimates of the benefit of implementing all management tasks, where experts were asked to consider the probability of species being secure in 50 years with and without the suite of management tasks16,17. Thus, benefit was calculated as the difference between the probability of security with and without the management tasks. For the U.S., in the absence of expert elicitation, the benefit of completing all management tasks in a recovery plan was approximated using information embedded in Recovery Priority Numbers (RPN). RPNs are an 18-category numeric rank for each species based on three categories of threat (high, moderate, and low), high or low recovery potential, and taxonomic distinctness monotypic genus, species, and subspecies,47. The limitations of using RPN to estimate the probability of persistence with or without management are discussed by Gerber et al.15 and Avery-Gomm48. To generate the total budget for each species, we used previously published total costs, which considered actions that benefited more than one species cost as shared among species projects15,16,17. In all further analysis, we removed species with a proposed budget of 0 (23 species in the U.S.) and extinct species (Guam broadbill—Myiagra freycineti and Eastern puma—Puma concolor couguar).
We explored additional characteristics unique to U.S. recovery planning documents, using U.S. data only (Table 1). These included: the federal listing status, the number of species in the recovery plan (66% of plans include multiple species), the priority assigned to each management task (1: emergency measures needed to prevent extinction, 2: measures required to stabilize a species headed for extinction, and 3: needed to delist), the estimated management task duration in years, the fiscal year the management task was implemented, the management task status (ongoing, complete, planned, discontinued), the total estimated time to recovery, and an RPN, which we used to make a new factor called ‘recovery potential’ (one of six scores based on the RPN, where the highest had a high probability of recovery and low degree of threat and the lowest had a low probability of recovery and a high degree of threat). Federal listing status was collapsed from six into three categories: endangered, threatened, and not listed (including candidate species, species removed from ESA due to recovery, or populations considered as ‘non-essential, experimental’). Taxa were assigned to eight categories: amphibians, birds, fishes, invertebrates (set as a reference category), mammals, reptiles, and flowering and non-flowering plants.
Quantitative analysis
To examine what characteristics of recovery plans are associated with the proportion of the budget allocated to RM we used beta regression in the betareg package49 in R version 3.6.150. We fit two models—one including all data, with jurisdiction included as a covariate, and one including a wider suite of covariates only available for the U.S. (Table 1). All continuous covariates were standardized by subtracting the mean and dividing by the standard deviation to ensure the resulting parameter estimates would be comparable51. We standardized the total budget of each jurisdiction separately to account for each countries’ different currency and year the budget was estimated. To improve model fit we removed five species with total budgets over 5 million dollars (five times the median: Barton Springs salamander – Eurycea sosorum, Austin blind salamander—Eurycea waterlooensis, Indiana bat—Myotis sodalis, Bull trout – Salvelinus confluentus, Grizzly Bear—Ursus arctos horribilis). Our results are robust to the inclusion or exclusion of these species.
Categorical covariates were converted to dummy variables. To select a reference category, we ran an initial model, using the category with the lowest mean proportion of budget RM as the reference. In this initial model, we selected the dummy variable with the highest variance inflation factor VIF in the car package52; as the reference in the final model. As a result, all VIF were 3). We excluded correlated covariates in successive models and chose the final model with the lowest Akaike’s Information Criterion (AIC53). The final model excluded the total proposed budget, which was correlated with the number of species in multi-species plans and the first fiscal year of the earliest RM. We consider any covariates where 95% confidence intervals around parameter estimates exclude zero to indicate a significant effect.
Estimating species recovery outcomes
To assess the relationship between the proportion of the budget allocated to RM and species recovery outcomes, we extracted a previously published index of recovery for U.S. listed species2 and developed similar indices based on annual and semi-annual reports from NZ and NSW (Supplementary Methods).
To generate the U.S. recovery index, Gerber2 calculated sums of biennial status data from reports to Congress during 1989–2011 (total of 11 status reports30). For each species, reports included whether their status was extinct, declining (scored as −1), stable (scored as 0), improving (scored as +1), or unknown. These scores were summed, generating values from −11 to 11, indicating whether species are declining or improving more frequently.
To develop recovery indices for NZ and NSW, we used similar reports through the New Zealand Threat Classification System and New South Wales Saving our Species annual report card over 4 and 5 assessment periods respectively. Assessments were annual in NSW and in NZ the periods between reports were on average every 4 years (Supplementary Methods). For each update or report card, we used a similar scoring (−1, 0, and +1) to indicate whether species were declining, stable, or improving between assessments (further details in Supplementary Methods). Note that in this analysis we were limited to a subset of the 2328 threatened species (78.5% of U.S. species, 13.5% of NZ species, and 14.7% of NSW species). Other studies have noted the limitations of recovery assessments28.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article. More

Overall strategy to detect the relevance of Unknown taxa
A pipeline based on network analysis was developed to detect and quantitatively measure the overall and individual impact of Unknown taxa on their environmental communities (Fig. 1). Briefly, Illumina 16S rRNA sequencing fastq files belonging to four distinctive aquatic extreme environments (i.e., hot springs, hypersaline, deep sea, and polar habitats that included both Arctic and Antarctic samples) were collected from public databases and 45 different BioProjects (Fig. 2a and Supplementary Dataset S1). We included different environment types to assess general and environment-specific patterns and chose to use extreme habitats as they comprise some of the harshest and relatively understudied habitats on Earth, and therefore, are likely to contain uncharacterized organisms.
Fig. 1: Overview of the analysis pipeline.

A minimum of 250 samples was retrieved for each of the four different extreme environments—hot springs (red), hypersaline (dark green), deep sea (turquoise), and polar (blue). Sequence reads were quality filtered, assigned to a taxonomy, and clustered to OTUs. At each classification level, any unassigned, ambiguous, or uncultured OTUs were designated as Unknowns, or “microbial dark matter” (MDM). For each environment, at each classification level, the direct co-occurrence relationship between all OTUs was mathematically modeled as a network. Networks were created for each environment, across all taxonomic classification levels, including all OTUs (Original, orange), excluding MDM (Without Unknowns, light green), and excluding an equal number of random Knowns (Bootstrap, blue). Network centrality metrics (i.e., degree, betweenness, and closeness) were calculated for each node, compared, and visualized as boxplots between these network types. Hub scores were calculated for each node in the Original network and networks were recreated, resizing by hub score, where the largest size node indicates a top hub species.

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Fig. 2: Summary of environmental 16S rRNA gene data.

a Map of the sample sites used in this study. Circles symbolize sample locations and are color-coded by environment: hot springs (HS, red), hypersaline (HY, dark green), deep sea (DS, turquoise), and polar (PO, blue). b Summary of data used in this study. OTUs counts are provided at the genus level. c Proportion of “microbial dark matter” (MDM) OTUs for each environment labeled as unassigned (dark blue), uncultured (dark red), and ambiguous (yellow) after SILVA and UCLUST-based taxonomic assignment to OTU. d Venn diagram of shared OTUs in four extreme environments. Each pie chart depicts the proportion of unique OTUs that were Known (lighter shade) and Unknown (darker shade) for each environment, with the bottom-most pie chart showing combined data for all environments. e Prevalence curves indicate the number of unique OTUs consistently present at an increasing number of samples. Dotted lines signify the prevalence of MDM OTUs and solid lines signify the prevalence of Known OTUs.

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After quality filtering, reads were mapped to OTUs and annotated against the SILVA (v128) reference database [38] by an open-reference strategy, i.e., allowing the detection of Unknown OTUs [39]. Over two million Known and novel taxa were observed with the vast majority of the taxa annotated as Bacteria. Only the bacterial taxa or OTUs unclassified at the domain-level were targeted for downstream network analyses to demonstrate the feasibility of this approach across ecosystems. As the term “microbial dark matter” can have a broad meaning, here, we define Unknown taxa as uncultured, unassigned, or ambiguous by the reference database at each taxonomic classification level (e.g., phylum to genus). For each environment, networks reflecting across-samples co-occurrence relationships between all taxa, Known and Unknown, were constructed and referred to as the “Original” networks (Fig. 1). To assess the role of the Unknown taxa on network structure and properties, the Unknown nodes were removed from the ‘Original’ network and a new network, referred to as ‘Without Unknown’ was reconstructed. To ensure that changes in network properties were not caused just by the number of nodes, a third network, referred to as the “Bootstrap” network, was created where a random set of nodes of the same number as the Unknown OTUs was removed. The relevance of the Unknown taxa was assessed by comparing changes in degree, closeness, and betweenness scores between the three network types and by evaluating the frequency of Unknowns as top hubs within each of the “Original” environmental networks.
A similar and significant fraction of Unknown taxa populates diverse environments
To assess whether there were distinctive patterns or trends of Unknown taxa within the four targeted environments, data from each habitat type were mined from several geographical locations across the globe (Fig. 2a and Supplementary Dataset S1). Reads were collected from the online repositories National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) and Joint Genome Institute Genomes Online Database (JGI Gold). For each environment, between 255 and 286 16S rRNA samples and between 51 and 57 million reads were included in the analysis, resulting in a total of 219,980,340 reads from 1086 samples (Fig. 2b).
After processing, quality filtering, and OTU assignment steps were completed, there were 2102,595 unique OTUs totaling 164,896,127 amplicon read counts derived from these samples (Fig. 2b). The relative proportions of Unknown OTUs, which were designated as unassigned, ambiguous, or uncultured by the SILVA database, were compared between environments. Results indicated that all environments showed similar relative contributions of these three Unknown types, with unassigned and uncultured OTUs making up the majority of the Unknown component composition (Fig. 2c). Regardless of environment type, within each of the four habitats, between 25 and 38% of unique OTUs were cataloged as Unknown (Fig. 2b, d). Samples collected from polar habitats were significantly enriched (Fisher’s Exact Test p value < 0.05) in Unknown OTUs despite having the highest total read counts. The higher proportion of Unknown OTUs in the polar samples compared to the other habitats likely reflects the less-well-characterized biological diversity of these Arctic and Antarctic ecosystems. Next, the proportion of shared Known and Unknown OTUs between environments was evaluated. Most OTUs, regardless of assigned or unassigned taxonomic status, were environment-specific, with only 11,318 out of the 2,102,595 OTUs present in all four of the environments (Fig. 2d). The majority of shared OTUs were observed between the hypersaline and polar environments and between hypersaline and hot springs environments, possibly reflecting the widespread distribution of hypersaline habitats across diverse thermal zones. Unsurprisingly, polar and hot springs environmental samples shared the least number of OTUs (Fig. 2d). Given this low common OTU pool across environments, network analyses were applied to each environment independently. Last, the prevalence (i.e., the percentage of samples with nonzero counts in which an OTU was detected) of Known and Unknown OTUs was evaluated at the genus level to assess the consistency of OTU detection within each environment. The OTU matrix was sparse, with the majority of taxa observed in ≤50 (~20%) samples (Fig. 2e). However, prevalence curves were generally very similar for Known and Unknown OTUs in all four environments, indicating that Unknown OTUs are not necessarily rarer than already characterized species (Fig. 2e). Moreover, we confirmed that Unknown OTUs, like Known OTUs, were generally present and consistent across multiple studies within the same environment and did not tend to concentrate in any particular project (Supplementary Figs. S1–4). Consequently, these results indicated that a network analysis of these data would be a reflection of the co-occurrence structure of the community and not of potential compositional bias. Network analysis of OTU abundance at different taxonomic levels reveals the connectivity of unknown microbes Having demonstrated that the Unknown taxa comprise a substantial proportion of unique OTUs and have comparable abundances to Known taxa within a community, network metrics were used to effectively compare the ecological relevance of both Known and Unknown taxa in subsequent networks. Microbial association networks were constructed that featured only significant co-occurrence correlation relationships for OTUs with a notable prevalence in each environment, meaning that any OTUs that were not detected in a sufficient number of samples were removed. To select a suitable prevalence threshold, the proportion of Known and Unknown taxa across a range of sample percentages was evaluated (Supplementary Fig. S5). Across all taxonomic levels, the Known and Unknown taxa of hot springs and polar habitats were more prevalent than those of hypersaline and deep sea communities; therefore, a slightly more stringent prevalence threshold (40%) was chosen for the former, and a lower threshold value (30%) chosen for the latter environments. These thresholds resulted in the retention of a similar fraction of data from the initial OTU count (Table 1), with 102–297 nodes present per environment, making the networks both suitably large and comparable. Table 1 Breakdown of node and edge attributes across extreme environmental networks. Full size table The SpiecEasi Meinshausen–Buhlmann (MB) neighborhood algorithm was then used to construct networks (see “Methods”) that contained at least 100 nodes (i.e., OTUs) per environment and had edge-to-node ratios that varied from 1.9 and 2.9 (Table 1). Although most OTUs that passed the prevalence criteria became elements of the networks, no relationship between the initial data size (i.e., number of samples and taxa) and the interconnectedness (i.e., nodes/edges ratio) of the resulting network was observed (Table 1). The two environments with the highest number of initial OTUs (i.e., hypersaline and deep sea) had the lowest number of prevalent members, 193 and 102, respectively, and very different edge/node ratios, 2.7 and 1.9, respectively, indicating that high prevalence does not necessarily correlate with high co-occurrence. Similarly, the polar and hot springs networks retained a high number of prevalent OTUs but differed in edge number, yet again, indicating that the observed network structures were the result of the intrinsic properties of each environment and were not dependent on the sampling procedure. Interestingly, when evaluating the proportion of Unknown edges and Unknown-Unknown connections at the genus level, similar patterns were observed across environments. Between 45 and 62% of all connected nodes were Unknown OTUs and a higher proportion of Unknown-Unknown versus Known-Known links was present at the genus level (Table 1), for all environments but hot springs, where a higher proportion of Known-Unknown and Known-Known links was observed. The results of this global analysis of network construction and composition demonstrate that although the general community connectivity might be environment-specific, the relative contribution of Known and Unknown taxa to these networks is similar. Once again, network properties were not a direct outcome of sampling biases, but more likely, reflect the biology of their respective ecosystems. Unknown taxa play important roles in interconnectedness and connectivity of extreme environmental microbial networks Next, the position and neighborhood of Unknown nodes were examined. At the phylum level, Unknown taxa were present in the hot spring, hypersaline, and polar environments, but were not found in the deep sea network (Supplementary Fig. S6). The class level was the first taxonomic classification rank in which Unknown taxa were present in all environmental networks. To accurately assess the role of Unknowns, we evaluated class-level networks and observed that the hypersaline and polar Unknown OTUs created distinctive clusters, whereas hot springs and deep sea Unknown OTUs were intermixed with Known taxa (Fig. 3a). Hypersaline and polar Unknowns consistently appeared to be isolated and peripherally located compared to the centrally positioned hot springs and deep sea Unknown nodes across almost all classification ranks (Supplementary Fig. S6). Consequently, these results suggest that the clustering pattern is unrelated to a higher abundance of Unknowns and is more environment dependent. For example, the targeted hot spring environments had the highest number of Unknown OTUs yet showed the most dispersed connections between the these taxa (Fig. 3a). Thus, the inclusion of the Unknown taxa in our environmental networks models was, as anticipated, not solely the consequence of their level of prevalence, but rather a reflection of a particular abundance co-variation pattern. Fig. 3: Analysis of environmental network taxa interconnectedness. a Microbial networks at class taxonomic classification level. Nodes are colored by class assignment, with gray nodes representing Unknown taxa at the class level. b Bar graphs of the co-occurrence relationships (i.e., edges) of Unknown OTUs with other taxa at the class level within each environmental network. Y axis labels and colors signify the different classes with which Unknowns were found to co-occur. Unknown-Unknown relationships are represented in gray. Full size image For all four environments, Unknown OTUs had more frequently shared edges among them than with classified taxa (Fig. 3b). Unknown OTUs were found to frequently co-occur with each other within each environmental network, although the frequency of within-class interactions for Unknowns at the class level was found to be statistically no greater than all other within-class interactions for each environment (Supplementary Fig. S7). In fact, the pattern of a high frequency of shared edges among members of the same class held true for known classes as well (Supplementary Fig. S8). In accordance with other studies, these results demonstrate that OTUs of the same taxonomic classification most frequently co-occur with each other [40, 41]. Furthermore, the high frequency of shared edges between Unknown classes suggests that Unknown OTUs might be taxonomically related. To ensure that the observations found were reproducible, robust, and not biased by earlier steps of the analysis, the diversity and position of Unknown taxa in the networks were examined for several parameters. Although the number of Unknown nodes changed at each level (Supplementary Fig. S6), the environment-specific network patterns observed at the class level (Fig. 3a) were retained at other taxonomic levels. In addition, to determine whether the topology of the network was a direct consequence of our correlation metric of choice or the prevalence threshold, three other network construction approaches were used: SparCC [11], CClasso [42], and Pearson correlation. Network analyses were performed across a range of prevalence thresholds (15–35%, at 5% increments). Again, regardless of the network construction approach or sample percentage applied, network shape and Unknown OTU position remained consistent and each environment exhibited a distinctive pattern of Unknown taxa inclusion. For example, Unknown nodes continued to occupy peripheral positions for hypersaline and polar networks, whereas nodes in hot springs and deep sea environments were more centrally located when applying different correlation metrics (Supplementary Fig. S9). Although networks appeared “noisy” at more lenient prevalence thresholds (15–20%; Supplementary Figs. S10–13), the networks and positioning of Unknowns at higher percent thresholds were similar in appearance to the “Original” networks for all environments. Based on these results, we found that our general analysis strategy was robust across parameter choices and, therefore, these networks captured critical features of the relationships among taxa within each distinctive environment. Microbial dark matter acts as unifiers and frequent hubs within extreme environmental networks Although these results suggest that Unknown taxa were highly interconnected, these observations did not reveal how the presence of Unknowns affected the overall community structure. To more fully understand this role, we analyzed how network properties changed in the presence and absence of Unknown OTU nodes. We evaluated changes in degree, betweenness, and closeness, as different network metrics reveal different aspects of the relevance of nodes within their networks. This approach has the potential to ascertain whether certain Unknown OTUs were more centrally positioned (e.g., due to high closeness scores), more essential for joining other taxa (e.g., high betweenness), or simply more prevalent and likely to co-occur with others (e.g., high degree). To control for the effect of node removal and distinguish effects of Unknown taxa from network size, networks were generated that excluded several randomly picked Known nodes equal to the number of Unknown OTUs. This process was repeated 100 times to create a distribution of “Null” or “Bootstrap” networks for statistical comparisons. Differences in network parameters between networks without Unknown OTUs and the “Original” or “Bootstrap” networks were determined by the Wilcoxon test and p values were adjusted using the Holm method [43]. Strikingly, removal of the Unknown taxa caused a statistically significant impact on all measured network metrics in all four studied environments (Table 2, Fig. 4, and Supplementary Figs. S14–24). In the polar environments, for example, removal of Unknown OTUs caused a significant decrease in degree (p value  More