Research article  Open  Published:
Difficulty in inferring microbial community structure based on cooccurrence network approaches
BMC Bioinformaticsvolume 20, Article number: 329 (2019)
Abstract
Background
Cooccurrence networks—ecological associations between sampled populations of microbial communities inferred from taxonomic composition data obtained from highthroughput sequencing techniques—are widely used in microbial ecology. Several cooccurrence network methods have been proposed. Cooccurrence network methods only infer ecological associations and are often used to discuss species interactions. However, validity of this application of cooccurrence network methods is currently debated. In particular, they simply evaluate using parametric statistical models, even though microbial compositions are determined through population dynamics.
Results
We comprehensively evaluated the validity of common methods for inferring microbial ecological networks through realistic simulations. We evaluated how correctly nine widely used methods describe interaction patterns in ecological communities. Contrary to previous studies, the performance of the cooccurrence network methods on compositional data was almost equal to or less than that of classical methods (e.g., Pearson’s correlation). The methods described the interaction patterns in dense and/or heterogeneous networks rather inadequately. Cooccurrence network performance also depended upon interaction types; specifically, the interaction patterns in competitive communities were relatively accurately predicted while those in predator–prey (parasitic) communities were relatively inadequately predicted.
Conclusions
Our findings indicated that cooccurrence network approaches may be insufficient in interpreting species interactions in microbiome studies. However, the results do not diminish the importance of these approaches. Rather, they highlight the need for further careful evaluation of the validity of these muchused methods and the development of more suitable methods for inferring microbial ecological networks.
Background
Many microbes engage with one another through interspecific interactions (e.g., mutualistic and competitive interactions) to compose ecological communities and interrelate with their surrounding environments (e.g., their hosts) [1]. Investigating such communities is important not only in the context of basic scientific research [2, 3], but also in applied biological research fields, such as in medical [4] and environmental sciences [5]. Remarkable development of highthroughput sequencing techniques—e.g., 16S ribosomal RNA gene sequencing and metagenomics as well as computational pipelines—have provided snapshots of taxonomic compositions in microbial communities across diverse ecosystems [6] and revealed that microbial compositions are associated with human health and ecological environments. For example, microbial composition in the human gut is interrelated with by numerous diseases—such as diabetes and cardiovascular disease—age, diet, and antibiotic use [7, 8]. The composition of soil microbial communities is related to climate, aridity, pH, and plant productivity [9]. However, previous studies have been limited to the context of species composition, and the effect of the structure of microbial communities (microbial ecological networks) on such associations is unclear due to a lack of reliable methods through which real interaction networks can be captured. Thus, cooccurrence networks, which infer ecological associations between sampled populations of microbial communities obtained from highthroughput sequencing techniques, have been attracting attention [10]. Cooccurrence network approaches are also related to weighted correlation network analyses [11,12,13] for inferring molecular networks from highthroughput experimental data, such as gene expression data. A number of methods for inferring microbial association have been proposed.
As a simple metric, Pearson’s correlation coefficient is considered. Additionally, Spearman’s correlation coefficient and maximal information coefficient (MIC) [14] are useful for accurately detecting nonlinear associations. However, these metrics may not be applicable to compositional data because the assumption of independent variables may not be satisfied due to the constant sum constraint [15]. Particularly, spurious correlations may be observed when directly applying these metrics to compositional data. To avoid this limitation, Sparse Correlations for Compositional data (SparCC) [16] has been developed. SparCC is an iterative approximation approach and estimates the correlations between the underlying absolute abundances using the logratio transformation of compositional data under the assumptions that realworld microbial networks are largescale and sparse. However, SparCC is not efficient due to its high computational complexity. Thus, regularized estimation of the basis covariance based on compositional data (REBACCA) [17] and correlation inference for compositional data through Lasso (CCLasso) [18] have been proposed. These methods are considerably faster than SparCC by using the l_{1}norm shrinkage method (i.e., least absolute shrinkage and selection operator; Lasso). SparCC has further limitations, as it does not consider errors in compositional data and the inferred covariance matrix may be not positive definite. To avoid these limitations, CCLasso considers a loss function inspired by the lasso penalized Dtrace loss.
However, correlationbased approaches such as those mentioned above may detect indirect associations. To differentiate direct and indirect interactions in correlation inference, other methods have been developed. In this context, inverse covariance matrixbased approaches are often used because they estimate an underlying graphical model, employing the concept of conditional independence. Typically, Pearson’s and Spearman’s partial correlation coefficients are used [19]; however, they may be not applicable to compositional data because statistical artifacts may occur due to the constant sum constraint. Thus, SParse InversE Covariance Estimation for Ecological ASsociation Inference (SPIECEASI) was proposed [20]. It infers an ecological network (inverse covariance matrix) from compositional data using the logratio transformation and sparse neighborhood selection.
These inference methods have been implemented as software packages and applied in several microbial ecology studies, such as investigations of human [21,22,23,24] and soil microbiomes [25,26,27]. While these methods only infer ecological associations, they are often used for discussing biological insights into interspecies interactions (i.e., microbial ecological networks [28]).
Nevertheless, further careful examination may be required to determine the importance of cooccurrence network approaches. The validity of these inference methods is still debatable [29] because they simply employ parametric statistical models, although microbial abundances are determined through population dynamics [2, 3]. Berry and Widder [30] used a mathematical model to determine population dynamics, generating (relative) abundance data based on population dynamics on an interaction pattern (network structure), and evaluated how correctly correlationbased methods reproduce the original interaction pattern. In particular, detecting interactions was harder for larger and/or more heterogeneous networks. However, they only compared earlier methods (e.g., Pearson’s correlation and SparCC) and not later methods (e.g., CCLasso) and the graphical modelbased methods. In addition, whether further examination and comparison of performance is required remains debatable, since arbitrary thresholds were used to calculate sensitivity and specificity. Moreover, the effects of interaction type, such as mutualism or competition, on cooccurrence network performance were poorly considered, even though pairs of species exhibit welldefined interactions in natural systems [31]. Weiss et al. [10] considered interaction types and evaluated correlationbased methods using a population dynamics model; however, they only examined smallscale (up to six species) networks due to system complexity, although compositionaldata methods (e.g., SparCC) assume largescale networks. Furthermore, graphical modelbased methods were not evaluated.
We comprehensively evaluated the validity of both correlationbased and graphical modelbased methods for inferring microbial ecological networks. In particular, we focused on nine widely used methods. Following previous studies [10, 30], we generated relative abundance (compositional) data using a dynamical model with network structure and evaluated how accurately these methods recapitulate the network structure. We show that the performance of later methods was almost equal to or less than that of classical methods, contrary to previous studies. Moreover, we also demonstrate that cooccurrence network performance depends upon interaction types.
Methods
Generation of relative abundance data using a dynamical model
Following [30], we used the nspecies generalized Lotka–Volterra (GLV) equation to generate abundance data:
where N_{i}(t) and r_{i} correspond to the abundance of species i at time t and the growth rate of species i, respectively. M_{ij} is an interaction matrix and indicates the contribution of species j to the growth of species i. In particular, M_{ij} was determined by considering network structure and interaction types; the diagonal elements M_{ii} in the interaction matrices, representing selfregulation, were set to − 1. Unlike a similar model used in a previous study [30], the carrying capacity of each species is set to be equivalent to its growth rate for simplicity.
To generate M_{ij}, we first produce undirected networks with n nodes and average degree 〈k〉 = 2m/n, where n indicate the number of species and m is the number of edges. This is done by generating adjacency matrices A_{ij} using models for generating networks. Following Layeghifard et al. [28], three types of network structure were considered: random networks, smallworld networks, and scalefree networks. In all cases A_{ij} = 1 if node (species) i interacts with node (species) j and A_{ij} = 0, otherwise, and A_{ij} = A_{ji} to have undirected networks.
The Erdős–Rényi model [32] was used to generate random networks in which the node degree follows a Poisson distribution where the mean is 〈k〉. The model networks are generated by drawing edges between m (=n〈k〉/2) node pairs that were randomly selected from the set of all possible node pairs. Specifically, we used erdos.renyi.game in the igraph package (version 1.2.2) of R (version 3.5.1; www.rproject.org), with the argument type = “gnm”.
However, realworld networks, including microbial ecological networks, are not random; instead, they are clustered (compartmentalized) and heterogeneous [28, 32,33,34].
The Watts–Strogatz model [35] was used to generate smallworld networks whose clustering coefficients are higher than expected and random. The model networks are generated by randomly rewiring ⌊p_{WS}m + 0.5⌋ edges in a onedimensional lattice where p_{WS} corresponds to the rewiring probability (ratio) ranging within [0,1]. Specifically, we used the sample_smallworld function in the igraph package; p_{WS} was set to 0.05.
The Chung–Lu model [36] was used to generate scalefree networks in which the degree distributions are heterogeneous. In the model, m (=n〈k〉/2) edges are drawn between randomly selected nodes according to node weight (i + i_{0} − 1)^{ξ} where ξ ∈ [0, 1] and i denotes the node index (i.e., i = 1, …, n) and the constant i_{0} is considered to eliminate the finitesize effects [37]. A generated network shows that P(k) ∝ k^{−γ}, where γ = 1 + 1/ξ [36, 37] and P(k) is the degree distribution. Specifically, we used the static.power.law.game function in the igraph package with the argument finite.size.correction = TRUE. In this study, we avoided the emergence of selfloops and multiple edges. γ was set to 2.2 because γ in many realworld networks is between 2 and 2.5 [38].
Following the work of Allesina and Tang [31], we considered five types of interaction matrices: random, mutualistic, competitive, predator–prey (parasitic), and a mixture of competition and mutualism interaction matrices. Following simulationbased studies using GLV equations [39,40,41], the (absolute) weights of interactions (i.e., the elements in interaction matrices M_{ij}) were drawn from uniform distributions.
In the random interaction matrices, M_{ij} was drawn from a uniform distribution of [−s_{max}, s_{max}] if A_{ij} = 1, and M_{ij} = 0 otherwise, where s_{max} is the upper (lower) limit for interaction strength. Given the definitions of mutualistic, competitive, and predator–prey (parasitic) interactions (see below for details), the random interaction matrices generated contain a mixture of these interaction types. For large n, in particular, mutualistic, competitive, and predator–prey interactions occur in the ratio of 1:1:2.
A mutualistic interaction between species i and j indicates that M_{ij} > 0 and M_{ji} > 0 because the species positively affect each other’s growth. In mutualistic interaction matrices, M_{ij} was drawn from a uniform distribution of (0, s_{max}] if A_{ij} = 1, and M_{ij} = 0 otherwise. It should be noted that M_{ji} is also positive if A_{ij} = 1 because A_{ij} = A_{ji}, but A_{ij} is independent from M_{ij}.
A competitive interaction between species i and j indicates that M_{ij} < 0 and M_{ji} < 0 because the species negatively affect each other’s growth. In competitive interaction matrices, M_{ij} was drawn from a uniform distribution of [−s_{max}, 0) if A_{ij} = 1, and M_{ij} = 0 otherwise. It should be noted that M_{ji} is also negative if A_{ij} = 1 because A_{ij} = A_{ji}, but A_{ij} is independent from M_{ij}.
Following a previous study [31], we generated interaction matrices consisting of a mixture of mutualistic and competitive interactions. For each species pair (i, j)_{i < j}, we obtained a random value p_{1} from a uniform distribution of [0, 1] if A_{ij} = 1. After, M_{ij} and M_{ji} were independently drawn from a uniform distribution of (0, s_{max}] if p_{1} ≤ p_{C} from a uniform distribution of [−s_{max}, 0) otherwise where p_{C} corresponds to the ratio of competitive interactions to all interactions. It should be noted that M_{ij} = 0 if A_{ij} = 0.
A predator–prey (parasitic) interaction between species i and j indicates that M_{ij} and M_{ji} have opposite signs (e.g., whenever M_{ij} > 0, then M_{ji} < 0) because species i (j) positively contributes to the growth of species j (i), but the growth of species i (j) is negatively affected by species j (i). The predator–prey interaction matrices were generated as follows: for each species pair (i, j)_{i < j}, we obtained a random value p_{2} from a uniform distribution of [0, 1] if A_{ij} = 1. If p_{2} ≤ 0.5, M_{ij} was drawn from a uniform distribution of [−s_{max}, 0) and M_{ji} was drawn from a uniform distribution of (0, s_{max}], while if p_{2} > 0.5 we did the opposite: M_{ij} and M_{ji} were independently drawn from uniform distributions (0, s_{max}] and [−s_{max}, 0), respectively. It should be noted that M_{ij} = 0 if A_{ij} = 0.
To investigate the effect of predator–prey interactions on cooccurrence network performance, we also considered interaction matrices consisting of a mixture of competitive and predator–prey interactions. For each species pair (i, j)_{i < j}, we obtained a random value p_{3} from a uniform distribution of [0, 1] if A_{ij} = 1; then, M_{ij} and M_{ji} were determined based on to the above definition of competitive interactions if p_{3} ≤ p_{C}, otherwise they were determined based on the above definition of predator–prey interactions. It should be noted that M_{ij} = 0 if A_{ij} = 0.
To obtain species abundances using the nspecies GLV equations, we used the generateDataSet function in the R package seqtime (version 0.1.1) [40]; environmental perturbance was excluded for simplicity. Following Faust et al. [40], the GLV equations were numerically solved with initial species abundances that were independently drawn from a Poisson distribution with mean of 100 (i.e., the total number of individuals is 100n). Following previous studies [40, 41], the growth rates of species (r_{i}) were independently drawn from a uniform distribution of (0,1]. Following the default options of the generateDataSet function, species abundances were obtained at the 1000time step. We empirically confirmed that species abundances reached a steady state before the 1000time step (Additional file 1: Figure S1). The absolute abundances were converted into relative values. The relative abundance P_{i} of species i was calculated as \( {N}_i/{\sum}_{j=1}^n{N}_j \) where N_{i} is the absolute abundance of species i at the time step. The resulting absolute and relative abundances were recorded. This process was repeated until the desired number of samples was obtained. The source codes for dataset generation are available in Additional file 2.
Cooccurrence network methods
We evaluated the extent to which the nine cooccurrence network methods decipher original interaction patterns (i.e., adjacency matrix A_{ij}) from the generated relative abundance (compositional) dataset based on associations between species abundances (see Additional file 1: Figure S2). In particular, six correlationbased methods were investigated: Pearson’s correlation (PEA), Spearman’s correlation (SPE), MIC [14], SparCC [16], REBACCA [17], and CCLasso [18]. Moreover, three graphical modelbased methods were also investigated: Pearson’s partial correlation (PPEA), Spearman’s partial correlation (PSPE), and SPIECEASI [20].
The pairwise Pearson’s and Spearman’s correlation matrices were calculated using the cor function in R with the arguments method = “pearson” and method = “spearman”, respectively. The pairwise MICs were determined using the mine function in the R package minerva (version 1.5). We also estimated the ecological microbial networks using the SparCC, REBACCA, and CCLasso algorithms. The SparCC program was downloaded from bitbucket.org/yonatanf/sparcc on November 11, 2018, and it ran under the Python environment (version 2.7.15; www.python.org). The REBACCA program was obtained from faculty.wcas.northwestern.edu/~hji403/REBACCA.htm on November 16, 2018. The CCLasso program was obtained from github.com/huayingfang/CCLasso on November 13, 2018. REBACCA and CCLasso ran under the R environment. We used SparCC, REBACCA, and CCLasso with the default options, but we provided the option pseudo = 1 when using CCLasso for convergence.
The Pearson’s and Spearman’s partial correlation coefficients were calculated using the pcor function in the R package ppcor (version 1.1) with the arguments method = “pearson” and method = “spearman”, respectively. We also obtained the cooccurrence networks using the SPIECEASI algorithm with neighborhood selection. The SPIECEASI program was downloaded from github.com/zdk123/SpiecEasi on November 13, 2018. We used SPIECEASI in the R environment with the default options.
Evaluating cooccurrence network performance
Following previous studies [20], to evaluate cooccurrence network performance (i.e., how well the estimated cooccurrence network describes the original interaction pattern A_{ij}), we obtained the precision–recall (PR) curve based on confidence scores of interactions for each inference result, comparing the lower triangular parts of confidence score matrices and A_{ij} because the matrices were symmetric. It should be noted that the lower triangular parts were vectorized after excluding the diagonal terms. The precision and recall were calculated by binarizing the confidence scores at a threshold. The PR curve was obtained as the relationship between precision and recall for different threshold. We used the absolute correlation coefficients for the Pearson’s correlation, Spearman’s correlation, MIC, Pearson’s partial correlation, Spearman’s partial correlation, SparCC, and CCLasso for the confidence scores. Following previous studies [17, 20], edgewise stability scores were used for REBACCA and SPIECEASI. Furthermore, we summarized the PR curve with the area under the PR curve (AUPR). The AUPR values were averaged over 50 iterations of dataset generation and performance evaluation with randomly assigned parameters for each iteration. The PR curves and AUPR values were obtained using the pr.curve function in the R package PRROC (version 1.3.1). We also computed the baselinecorrected AUPR values because positive and negative ratios affect PR curves. The baselinecorrected AUPR value was defined as (AUPR_{obs} – AUPR_{rand}) / (1 – AUPR_{rand}), where AUPR_{obs} and AUPR_{rand} correspond to the observed AUPR value and the AUPR value obtained from random prediction (i.e., 2m/[n(n − 1)] = 〈k〉/(n − 1)), respectively. The source codes for evaluating cooccurrence network performance are available in Additional file 2.
It is important to mention that the problem of falsenegative interactions may occur when we do performance analysis based on adjacency matrices A_{ij}: negligible interactions (i.e., when both M_{ij} and M_{ji} have very small values) have negligible effects on population dynamics and act as no interaction. It may happen even if the corresponding nodes are connected (i.e., A_{ij} = A_{ji} = 1). However, this problem hardly affects cooccurrence network performance. Supposing such falsenegative interactions occur if M_{ij} < s_{c} and M_{ji} < s_{c} when A_{ij} = A_{ji} = 1 where s_{c} is a small value, the expected ratio of falsenegative interactions to all interacting pairs (edges) is described as (s_{c} / s_{max})^{2} because M_{ij} and M_{ji} are independently drawn from the uniform distribution of (0, s_{max}]. Assuming that s_{max} = 0.5 and s_{c} = 0.01, for example, 0.04% of m edges indicate falsenegative interactions.
Results
Compositionaldata cooccurrence network methods performance did not exceed that of classical methods
We generated relative abundance datasets through population dynamics. In particular, we used the GLV equations with an interaction matrix M_{ij} constructed from an interaction pattern A_{ij} (random, smallworld, or scalefree network structure) by considering types of interaction matrices (random, mutualistic, competitive, predator–prey (parasitic), or mixture of competition and mutualism interaction matrices). We investigated how well cooccurrence network methods decipher interaction patterns from relative abundance data by evaluating the consistency between the confidence score matrices obtained from the methods and A_{ij} based on the (baselinecorrected) AUPR values.
We investigated the case of random interaction matrices constructed based on random network structures (Fig. 1). We found that cooccurrence network performance (AUPR value) was moderate. For example, the AUPR value was at most ~ 0.65 when network size (the number of species) n = 50 and average degree 〈k〉 = 2 (Fig. 1a), and it was at most ~ 0.45 when n = 50 and 〈k〉 = 8 (Fig. 1b). As expected from limitations due to the constant sum constraint, the performance of the classical cooccurrence network methods (e.g., Pearson’s correlation) generally decreased when using compositional data (Additional file 1: Figure S3), and the performance of the partial correlationbased methods declined largely.
More importantly, we found that the performance of the compositionaldata cooccurrence network methods were almost equal to or less than that of classical methods, excluding Spearman’s partial correlationbased method; in particular, the performance of some compositionaldata methods was lower than that of the classical methods. Specifically, the AUPR values of SparCC, an earlier compositionaldata method, were lower than those of Pearson’s correlation [p < 2.2e–16 using ttest when n = 50 and 〈k〉 = 2 (Fig. 1a) and p < 2.2e–16 using ttest when n = 50 and 〈k〉 = 8 (Fig. 1b)]. Moreover, The AUPR values of REBACCA, a later compositionaldata method, were also lower than those of Pearson’s correlation [p < 2.2e–16 using ttest when n = 50 and 〈k〉 = 2 (Fig. 1a) and p < 2.2e–16 using ttest when n = 50 and 〈k〉 = 8 (Fig. 1b)]. For 50node networks, the performance of CCLasso and SPIECEASI was similar to that of classical methods when 〈k〉 = 2 (Fig. 1a) and 〈k〉 = 8 (Fig. 1b). However, the performance of later compositionaldata methods (e.g., CCLasso) was higher than that of the earlier compositionaldata method (i.e., SparCC). Specifically, the AUPR values of CCLasso were lower than those of SparCC [p < 2.2e–16 using ttest when n = 50 and 〈k〉 = 2 (Fig. 1a) and p = 3.2e–7 using ttest when n = 50 and 〈k〉 = 8 (Fig. 1b)].
The graphical modelbased methods were not more efficient than the correlationbased methods. Spearman’s partial correlationbased method was inferior to Pearson’s correlationbased method (p < 2.2e–16 using ttest) and Spearman’s correlationbased method (p < 2.2e–16 using ttest) when n = 50 and 〈k〉 = 2 (Fig. 1a); however, the AUPR value of Spearman’s partial correlationbased method was similar to that of Pearson’s and Spearman’s correlationbased methods when n = 50 and 〈k〉 = 8 (Fig. 1b). Both Pearson’s partial correlationbased method and Pearson’s correlationbased method exhibited similar performance. The performance of the graphical modelbased method for compositional data (SPIECEASI) was similar to that of other correlationbased methods (e.g., Pearson’s correlation), although it was higher than that of the correlationbased methods for compositional data. Specifically, the AUPR values of SPIECEASI were higher than those of SparCC [p < 2.2e–16 using ttest when n = 50 and 〈k〉 = 2 (Fig. 1a) and p < 2.2e–16 using ttest when n = 50 and 〈k〉 = 8 (Fig. 1b)].
Cooccurrence network performance was evaluated when the average degree (Fig. 1a and b) and number of nodes (network size; Fig. 1c and d) varied; moreover, it was also examined for other types of network structure: smallworld networks (Additional file 1: Figure S4) and scalefree networks (Additional file 1: Figure S5).
Interaction patterns in more complex networks are harder to predict
It is noteworthy that network size, average degree, and network type affected cooccurrence network performance. The cooccurrence network performance (baselinecorrected AUPR values) varied with network size in some methods (Fig. 1c and d). In particular, the performance of Spearman’s partial correlationbased method increased with network size in dense networks, while the performance of REBACCA decreased with network size in sparse networks. However, cooccurrence network performance was nearly independent of network size when n > 20 in most methods. The interaction patterns in small networks were poorly predicted; the cooccurrence network methods are not suitable for capturing interaction patterns in small networks. The differences in the performance between the cooccurrence network methods and random predictions were not remarkable because the degree of freedom was low in small networks.
More importantly, the interaction patterns in denser networks generally were more difficult to predict; in particular, we observed general negative correlations between the performance (baselinecorrected AUPR value) and average degree when n = 50 (Fig. 2a) and n = 100 (Fig. 2b). However, the performance of Spearman’s partial correlationbased method (PSPE) increased for 〈k〉 < ~8 and decreased for 〈k〉 ≥ ~8 when n = 50 and 100. This method exhibited the highest performance for dense networks while it exhibited relatively low performance for sparse networks; nonetheless, it should be noted that this method poorly predicted interactions patterns (the baselinecorrected AUPR value was at most ~ 0.4 when 〈k〉 ≥ ~8). The cooccurrence network performance slightly increased when using more samples (Additional file 1: Figure S6); in particular, we investigated cases in which network size (n = 50 and 100) and average degree (〈k〉 = 2 and 8) differed and found that cooccurrence network performance was almost independent of sample number when it exceeds 200 in most methods.
The correlations between the baselinecorrected AUPR values and average degree were also investigated in smallworld networks (Additional file 1: Figure S4 and S7) and scalefree networks (Additional file 1: Figures S5 and S8), and the negative correlations between the baselinecorrected AUPR values and average degree were specifically observed. However, cooccurrence network performance moderately varied according to network type in large and dense networks when focusing on each inference method (Fig. 3). In particular, we investigated Pearson’s correlationbased method (a classical correlationbased method; Fig. 3a and b), Pearson’s partial correlationbased method (a classical graphical modelbased method; Fig. 3c and d), CCLasso (a correlationbased method for compositional data; Fig. 3e and f), and SPEICEASI (a graphical modelbased method for compositional data; Fig. 3g and h). In general, the lowest performance was observed for scalefree networks, while the highest performance was observed for smallworld networks (Fig. 3). Specifically, the baselinecorrected AUPR values for scalefree networks were lower than those for small world networks when n = 100 and 〈k〉 = 8 (p < 2.2e–16 using ttest for Pearson’s correlationbased method; p = 7.7e–5 using ttest for Pearson’s partial correlationbased method; p = 0.027 using ttest for CCLasso; p = 1.9e–13 using ttest for SPEICEASI). Moreover, the baselinecorrected AUPR values for scalefree networks were lower than those for random networks when n = 100 and 〈k〉 = 8 for Pearson’s correlationbased method (p = 2.9e–3 using ttest) and SPEICEASI (p = 7.4e–3 using ttest).
The results indicating that compositionaldata cooccurrence network methods were not more efficient than classical methods and that interaction patterns in more complex networks are more difficult to predict (Figs. 1, 2 and 3) were also generally confirmed in the other types of interactions matrices: competitive (Additional file 1: Figures S9–S11), mutualistic (Additional file 1: Figures S12 and S13), predator–prey (Additional file 1: Figures S14–S16), and mutualismcompetition mixture interaction matrices (Additional file 1: Figures S17–S19).
Predatorprey (parasitic) interactions decrease cooccurrence network performance
The types of interaction matrices notably affected cooccurrence network performance (Fig. 4). Specifically, in most methods, the interaction patterns in predator–prey (parasitic) communities (interaction matrices) were the most difficult to predict, while those in competitive communities were the easiest to predict. Specifically, the AUPR values for predator–prey communities were significantly lower than those for competitive communities for Pearson’s correlationbased method (p < 2.2e–16 using ttest; Fig. 4a), Spearman’s correlationbased method (p < 2.2e–16 using ttest; Fig. 4b), MICbased method (p < 2.2e–16 using ttest; Fig. 4c), SparCC (p < 2.2e–16 using ttest; Fig. 4d), REBACCA (p < 2.2e–16 using ttest; Fig. 4e), CCLasso (p < 2.2e–16 using ttest; Fig. 4f), Pearson’s partial correlationbased method (p < 2.2e–16 using ttest; Fig. 4g), Spearman’s partial correlationbased method (p < 2.2e–16 using ttest; Fig. 4h), and SPEICEASI (p < 2.2e–16 using ttest; Fig. 4i). Additionally, cooccurrence network methods relatively accurately predicted interactions patterns in mutual communities and competition–mutualism mixture communities; however, they described the interaction patterns in random communities poorly. Specifically, the AUPR values for random communities also were significantly lower than those for competitive communities for Pearson’s correlationbased method (p < 2.2e–16 using ttest; Fig. 4a), Spearman’s correlationbased method (p < 2.2e–16 using ttest; Fig. 4b), MICbased method (p < 2.2e–16 using ttest; Fig. 4c), REBACCA (p < 2.2e–16 using ttest; Fig. 4e), CCLasso (p < 2.2e–16 using ttest; Fig. 4f), Pearson’s partial correlationbased method (p < 2.2e–16 using ttest; Fig. 4g), Spearman’s partial correlationbased method (p < 2.2e–16 using ttest; Fig. 4h), and SPEICEASI (p < 2.2e–16 using ttest; Fig. 4i). Similar tendencies of the effect of interaction types on cooccurrence network performance were observed in varying network sizes (i.e., n = 20 and 100; Additional file 1: Figure S20), average degrees (i.e., 〈k〉 = 4 and 8; Additional file 1: Figure S21), and network structures (i.e., smallworld and scalefree network structures; Additional file 1: Figure S22).
We hypothesized that cooccurrence network performance decreases as the ratio of predator–prey (parasitic) interactions increases because the worst performance and second worst performance were observed for predator–prey and random communities, respectively. Note that almost half of the interactions are spontaneously set to predator–prey interactions in random communities (see “Generation of relative abundance data using a dynamical model” section). To test this hypothesis, we considered interaction matrices consisting of a mixture of competitive and predator–prey interactions because cooccurrence network performance was best and worst in competitive and predator–prey (parasitic) communities, respectively. In particular, we considered competition–parasitism mixture communities with the ratio p_{C} of competitive interactions to all interactions and investigated the relationship between the ratio of predator–prey interactions (i.e., 1 − p_{C}) and AUPR values. As representative examples, we investigated Pearson’s correlationbased method (a classical correlationbased method; Fig. 5a), Pearson’s partial correlation method (a classical graphical modelbased method; Fig. 5b), CCLasso (a correlationbased method for compositional data; Fig. 5c), and SPIECEASI (a graphical modelbased for compositional data; Fig. 5d). As expected, we found negative correlations between cooccurrence network performance (AUPR value) and the ratio of predator–prey interactions (Fig. 5). Such negative correlations were also observed in cases with different network sizes (n = 50 and 100) and average degrees (〈k〉 = 2 and 8).
Discussion
Inspired by previous studies [30], we evaluated how well cooccurrence network methods recapitulate microbial ecological networks using a population dynamics model; cooccurrence network methods are often used for discussing species interactions although they only infer ecological associations. We compared wideranging methods using realistic simulations. Our results provide additional and complementary insights into cooccurrence network approaches in microbiome studies.
The results indicate that compositionaldata methods, such as SparCC and SPIECEASI, are less useful in inferring microbial ecological networks than previously thought. As shown in Fig. 1, the performance (AUPR values) of the compositionaldata methods was moderate; furthermore, these compositionaldata methods were not more efficient than the classical methods, such as Pearson’s correlationbased method. This result is inconsistent with previous studies [17, 18, 20]. This discrepancy was mainly due to differences in cooccurrence network method validation between this and previous studies. Specifically, previous studies generated abundance data from a multivariable distribution with a given mean and covariance matrix and examined how accurately cooccurrence network methods describe the original covariance matrix structure. However, this study considered species abundances determined through population dynamics (GLV equations) and examined how accurately the methods reproduced interaction patterns in ecological communities [30].
Population dynamics may lead to more complex associations between species abundances than parametric statistical models due to the nonlinearity of GLV equations. In compositional data cooccurrence network methods, such complex associations were likely difficult to detect because they assumed linear relationships between species abundances. The performance of Spearman’s correlationbased and MICbased methods was almost equal to or higher than those of compositionaldata methods because they can consider nonlinear associations, although such classical methods did not consider the effects of the constant sum constraint in compositional data. However, Pearson’s correlationbased method also exhibited a similar or higher performance than that of the compositionaldata methods (Fig. 1), although it assumes linear relationships between species abundances in addition to the constant sum constraint. This may be due to approximation in the compositionaldata methods, which estimate covariance matrices of the underlying absolute abundances from relative abundances using iterative approximation approaches. Thus, compositionaldata methods may fail to correctly estimate the covariance structure of absolute abundance. According to a previous study [18], such a limitation is present in SparCC. REBACCA is similarly limited because its formalism is comparable to SparCC, although sparse methods are different between SparCC and REBACCA; thus, the performance of SparCC and REBACCA may have been low for similar reasons. On the other hand, CCLasso avoids these limitations [18], performing better than SparCC and REBACCA. However, more improvements may be required for CCLasso. It performed similarly to Pearson’s correlationbased method, which exhibited a higher performance using absolute abundances (particularly in sparse networks; Additional file 1: Figure S3). This indicates that CCLasso did not sufficiently infer the covariance structure of absolute abundances.
The graphical modelbased methods were not more efficient than the correlationbased methods, although they do not consistently detect indirect associations (Fig. 1). In particular, Pearson’s and Spearman’s partial correlationbased (classical graphical modelbased) methods were not more useful for inferring interaction patterns in ecological communities than Pearson’s and Spearman’s correlationbased (classical correlationbased) methods, and Spearman’s partial correlationbased method predicted interaction patterns in ecological communities poorly. This may have occurred due to the effects of the constant sum constraint in compositional data; specifically, these classical graphical modelbased methods exhibited high performance with absolute abundances (Additional file 1: Figure S3). The effects of the constant sum constraint in partial correlationbased may be more significant than those in correlationbased methods, and errors due to the constant sum constraint in pairwise correlations (zero thorder partial correlations) may be amplified when calculating higherorder partial correlations. Thus, classical graphicalbased models may be less useful than classical correlationbased models. The graphical modelbased method for compositional data SPIECEASI has a similar problem. Similar to other correlationbased methods for compositional data (e.g., SparCC), SPIECEASI estimates absolute abundances from relative abundances. The estimated absolute abundances are not entirely accurate, which may be amplified in partial correlation (or regression) coefficients because SPIECEASI calculates coefficients based on the estimated values with the errors as classical partial correlationbased methods. CCLasso considers such errors through a loss function. Thus, CCLasso exhibited performance similar to SPIECEASI, although it did not directly consider avoiding indirect associations.
Interaction patterns in dense networks were difficult to predict (Fig. 2). This is generally because more indirect associations are observed; however, this may be because the assumption of sparsity in addition to errors due to absolute abundance approximation from relative abundances for compositionaldata methods. This assumption is based on observations that realworld networks are very large and sparse [42], and sparsity is achieved through Lasso in the compositionaldata methods. However, these Lassobased methods might have overlooked important associations due to shrinkage and selection; Lasso may pick only one or a few strongly correlated variable pairs and shrink the rest to 0, i.e., no association [43]. To avoid this limitation, for example, we may need to consider the elasticnet and relaxed Lasso to estimate ecological associations under the sparsity assumption.
Additionally, interaction patterns in heterogeneous networks were the most difficult to detect while those in smallworld networks, which are homogenous, were the easiest (Fig. 3). This result indicates that heterogeneity in degree distribution diminishes the performance of cooccurrence networks. This is consistent with the results of a previous study [30] in which it was observed that networks suffer from local hot spots of spurious correlation (indirect association) in the neighborhood of hub species that engage in many interactions. We expected that the graphicalbased cooccurrence network method SPIECEASI avoided this limitation; however, the performance of SPIECEASI was similar to that of the other methods, as mentioned above. This may be due to the nonlinearity of species abundances and errors resulting from absolute abundance approximation from relative abundances. SPIECEASI may need to be improved.
Cooccurrence network performance increased with more samples (Additional file 1: Figure S6). More than 200 samples were required until plateaued performance was obtained. However, experimental studies may be able to consider fewer samples, down to 30 samples or less. Cooccurrence network methods that exhibit high performance with small samples must be developed.
More importantly, we found that interaction types affect cooccurrence network performance (Fig. 4). A previous study [10] also investigated the effects of interaction types. However, it used timeseries data generated from GLV equations and is limited to smallscale networks to avoid system complexity. The behavior of the Lotka–Volterra systems is less understood for systems larger than two species, and small variations in the interaction matrix lead to significantly different abundance patterns. To investigate largescale networks, we used steadystate species abundances generated from GLV equations, inspired by a previous study [30]. The data generation method performed by Berry and Widder [30] and in this study differs from the other previous study [10] although both studies considered GLV equations. Generated datasets are considered as a collection of steadystate snapshots (i.e., crosssectional data) rather than timeseries (longitudinal) data. Moreover, it is reasonable that observed species abundances are considered as crosssectional data rather than longitudinal data in many microbiome studies. Despite the importance of timeseries microbiome analysis [40, 44], time resolutions are still low due to technical limitations. As a result, we found that predator–prey (parasitic) interactions decrease cooccurrence network performance (AUPR values; Fig. 5). This result indicates detecting predator–prey interactions is more difficult than detecting other types of interactions, such as competitive and mutualistic interactions. This may be due to the behavioral complexity of predator–prey systems. The dynamics of predator–prey systems are known to be more complex than those of the other types of systems, even if the systems consist of only two species; in particular, predator–prey systems tend to oscillate [31]. Complex nonlinear associations are observed between the resulting species abundances obtained from predator–prey communities; thus, cooccurrence network methods failed to predict interaction patterns in these communities. This limitation may be important because predator–prey (parasitic) interactions play important roles in microbe–microbe interactions and human–microbiome interactions [45]. To avoid this issue, a compositionaldata version of cooccurrence network methods based on maximal informationbased nonparametric exploration, such as MIC, must be developed as MIC can detect such complex nonlinear associations [14].
However, further careful examination may be required. For example, more realistic dynamical models must be considered. For simplicity, we used classical GLV equations, and the conclusions we reached are limited to the species abundances generated from this classical model. The GLV equations may not reflect realworld microbial ecosystems. Ideally, we should have compared the generated data with realworld data; however, such comparisons were impossible because of a lack of compiled realworld data. The data on species (relative) abundances are available in several databases (e.g., Human Microbiome Project [4] and Earth Microbiome Project [5]); however, the model parameters (growth rate r_{i} and interaction matrix M_{ij}) can be adjusted to nearly fit realworld abundance data using optimization methods. Thus, r_{i} and M_{ij} in realworld microbial ecosystems are needed to evaluate the validity of the generated abundance data; these realworld data are poorly investigated. However, this limitation does pose a significant problem because the main result is the difficulty in inferring microbial ecological networks using cooccurrence network methods. Realworld ecosystems are likely more complex. For example, species abundances, growth rates, and interaction matrices are temporally changed due to environmental perturbations. In this case, inferring ecological associations and interactions may be more difficult. Thus, it is believed that the main result also holds in more realistic ecosystems.
To more accurately detect ecological associations and directly detect species–species interactions, however, alternative methods are also needed. For example, a method grounded in maximum entropy models of statistical physics has been proposed to differentiate direct and indirect associations [46]. The difficulty of interpreting species–species interactions from cooccurrence data has been pointed out in community ecology [47]. To overcome this difficulty, Markov networks (Markov random fields) have been used for inferring species–species interactions from cooccurrence data in community ecology [48]. Dynamics (time series)based methods are also useful. For example, convergent cross mapping [49] may be useful. This method is based on nonlinear statespace reconstruction and can distinguish causality in complex systems from correlation. The sparse Smap method [50] is a dataoriented equationfree modeling approach for multispecies ecological dynamics whose interaction topology is unknown, and it generates a sparse interaction network from a multivariate ecological time series without presuming any mathematical formulation for the underlying microbial processes. Another method, proposed by Xiao et al. [51], is based on Jacobian (community) matrices and can infer network topology and intertaxa interaction types without assuming any particular population dynamics model from steadystate abundance data. Randomly distributed embedding [52] is a modelfree framework that achieves accurate futurestate prediction based on shortterm highdimensional data. However, these methods are not applicable to compositional data and must be improved. Thus, we did not consider these methods in this study.
Conclusions
Our findings indicate that cooccurrence network methods are not efficient in interpreting interspecies interactions in microbiome studies because these methods only infer ecological associations. However, these results do not diminish the importance of cooccurrence network approaches. Cooccurrence network approaches remain a challenging research topic in the postgenomic era due to the importance of human [4] and ecological microbiomes [5]. Our findings highlight the need for further careful investigation of the validity of these widely used methods and development of more suitable approaches for inferring microbial ecological networks.
Availability of data and materials
All data generated and analyzed during this study are included in this published article and its supplementary information files.
Abbreviations
 AUPR:

area under the precision–recall curve
 CCLasso:

correlation inference for compositional data through Lasso
 GLV:

generalized Lotka–Volterra
 Lasso:

least absolute shrinkage and selection operator
 MIC:

maximal information coefficient
 PEA:

Pearson’s correlation
 PPEA:

Pearson’s partial correlation
 PR:

precision–recall
 PSPE:

Spearman’s partial correlation
 REBACCA:

regularized estimation of the basis covariance based on compositional data
 SparCC:

sparse correlations for compositional data
 SPE:

Spearman’s correlation
 SPIECEASI:

sparse inverse covariance estimation for ecological association inference
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Acknowledgments
We would like to thank Editage (www.editage.jp) for English language editing.
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HH and KT conceived and designed the study. HH and KT prepared the source codes for numerical simulations. HH performed numerical simulations. HH and KT interpreted the results. HH and KT drafted the manuscript. All authors read and approved the final manuscript.
This study was supported by JSPS KAKENHI Grant Number JP17H04703. The funding body had no role in the design, collection, analysis, or interpretation of this study.
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Correspondence to Kazuhiro Takemoto.
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Keywords
 Microbiome
 Correlation network analysis
 Microbial ecology
 Complex networks