1
Co-occurring soil bacteria exhibit a robust competitive hierarchy and lack of non-1
transitive interactions 2
Logan M. Higgins
1,2*
, Jonathan Friedman
1,3
, Hao Shen
4
& Jeff Gore
1*
3
1
Physics of Living Systems, Department of Physics, Massachusetts Institute of 4
Technology, Cambridge, Massachusetts 02139, USA 5
2
Microbiology Graduate Program, Massachusetts Institute of Technology, Cambridge, 6
Massachusetts 02139, USA 7
3
Department of Plant Pathology and Microbiology, Robert H. Smith Faculty of 8
Agriculture, Food, and the Environment, Hebrew University of Jerusalem, Rehovot 9
76100, Israel 10
3
School of Life Sciences, Peking University, Beijing 100871, China 11
*
e-mail: loganh@mit.edu; gore@mit.edu 12
13
Microbial communities are typically incredibly diverse, and this diversity is thought 14
to play a key role in community function. However, explaining how this diversity 15
can be maintained is a major challenge in ecology. Temporal fluctuations and 16
spatial structure in the environment likely play a key role, but it has also been 17
suggested that the structure of interactions within the community may act as a 18
stabilizing force for species diversity. In particular, if competitive interactions are 19
non-transitive as in the classic rock-paper-scissors game, they can contribute to the 20
maintenance of species diversity; on the other hand, if they are predominantly 21
hierarchical, any observed diversity must be maintained via other mechanisms. 22
Here, we investigate the network of pairwise competitive interactions in a model 23
community consisting of 20 strains of naturally co-occurring soil bacteria. We find 24
that the interaction network is strongly hierarchical and lacks significant non-25
transitive motifs, a result that is robust across multiple environments. Moreover, in 26
agreement with recently proposed community assembly rules, the full 20-strain 27
competition resulted in extinction of all but three of the most highly competitive 28
strains, indicating that higher order interactions do not play a major role in 29
structuring this community. The lack of non-transitivity and higher order 30
interactions in vitro indicates that other factors, such as temporal or spatial 31
heterogeneity, must be at play in enabling these strains to coexist in nature. 32
33
Despite their small size, microbes play outsized roles at multiple ecosystem scales, from 34
the planetary
1
to that of the human individual
2
. Like their macroscopic counterparts, 35
microbes typically exist in diverse communities whose functions are intimately related to 36
their structure. Diversity impacts an ecological community’s stability, resilience to 37
perturbations, and its ability to provide ecosystem services
3
. Therefore, a long-standing 38
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2
area of interest in microbial ecology has been understanding the factors that give rise to 39
the diversity observed within microbial communities
4,5
. A better understanding of the 40
structure of microbial communities is desirable for both managing existing microbial 41
communities
6
and, eventually, engineering them de novo
7
. 42
Many factors can contribute to the generation and maintenance of diversity in ecological 43
communities. Non-transitivity
8
, bistability
9
, weak interactions
10
, facilitation, multiple 44
limiting factors, and spatial or temporal segregation
11
have all been hypothesized to play 45
a role; however, there is little empirical data regarding the relative importance of each of 46
these factors in actual natural communities. By investigating the network of underlying 47
interactions among the members of a given community, we can better understand each 48
factor’s relative importance in structuring the community
12
. Since interspecific 49
competition is thought to be a dominant factor in determining whether a given species 50
can persist in a community
13,14
, the network of competitive interactions between species 51
may be particularly informative of the structure of the community within which the 52
interaction takes place. Features of competitive interaction networks that could contribute 53
to community diversity can include non-transitive motifs such as the classic rock-paper-54
scissors triad, network modularity
15
, or overall trends towards weak interactions among 55
species 56
While non-transitivity in particular is often cited as a potential driver of interspecies 57
coexistence
16,17,18
, the degree to which it occurs in natural communities remains largely 58
unknown. Indeed, Levine and colleagues recently asserted that despite the theoretical 59
potential of non-transitive interactions to stabilize community structure, there is scant 60
evidence that they are widespread in natural systems, and that further empirical studies 61
are warranted
19
. Recent experimental work using a field-parameterized model of 62
competition in annual plants
20
and naturally co-occurring Streptomyces bacteria
9
suggest 63
that rock-paper-scissors type interactions may be less common in natural communities 64
than we might assume; however, further studies of competitive interaction networks in 65
diverse ecological communities are warranted, particularly among phylogenetically 66
diverse natural assemblages. 67
Here, we add to this small but growing body of research that suggests that non-transitive 68
interactions may play a less significant role in maintaining species diversity than is 69
commonly assumed. We use a model system composed of heterotrophic bacteria isolated 70
from a single soil grain. By competing in all pairwise combinations in laboratory culture, 71
we find that the overarching feature of the resulting interaction network is a strong 72
competitive hierarchy, a feature that is naturally at odds with a high incidence of non-73
transitivity. Therefore, in the natural environment of these bacteria, other factors must be 74
at play that account for their ability to co-occur. 75
Results 76
To probe the network of pairwise interactions in a community of diverse microbes, we 77
isolated a collection 20 strains of naturally co-occurring heterotrophic bacteria from a 78
single grain of soil. This strain collection is phylogenetically diverse and spans 16 species 79
across seven genera and five families (Fig. 1a and Methods). Similar to ref
21
, we co-80
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inoculated all pairwise combinations of the 20 strains at varying initial fractions and 81
propagated them through at least five growth-dilution cycles. During each growth cycle, 82
cells were cultured for 24 hours and then diluted by a factor of 100 into fresh media. The 83
final outcome of competition was determined by plating the cultures on solid agar and 84
counting colonies, which are morphologically distinct (Fig. 1c and Supplementary Fig. 85
1). Plating results were confirmed via next-generation sequencing for a random subset of 86
the pairs (Supplementary Fig. 6). 87
Pairwise competitions resulted in one of three qualitatively different outcomes: exclusion, 88
coexistence, or bistability (Fig. 2a-c and Methods). In 153 of the 190 pairs (81%), only 89
one strain could invade the other and drove it to extinction, an outcome we call exclusion. 90
Nineteen pairs (10%) were mutually invasible, and thus exhibited coexistence over the 91
time span of the experiment. Finally, 15 pairs (8%) were mutually non-invasible, an 92
outcome that we call bistability. In a small number of pairs (3; 2%), we were unable to 93
determine the outcome due to contamination. Due to the high incidence of exclusion and 94
bistable outcomes, we conclude that these strains interact in the experimental 95
environment primarily through competition. 96
To quantify the strains’ overall competitive ability, we define each strain’s competitive 97
score to be its mean final fraction across all pairwise competitions. The competitive 98
scores that we measured spanned nearly the entire possible range, from a low of 0.03 to a 99
high of 0.91 (Fig. 1b and Supplementary Table 1). 100
The strains exhibit a strong competitive hierarchy. Very few strains were able to exclude 101
a strain with a higher competitive score; out of 187 pairwise competitions measured, only 102
five resulted in the lower-ranked strain excluding the higher-ranked one (Fig. 2d). The 103
degree of hierarchy in this interaction network is highly significant when compared to 104
networks with randomized outcomes (p < 10
-19
; Fig. 2e). To assess whether the 105
hierarchical pattern was specific to a particular environment, we repeated the 106
competitions with subsets of the full 20-strain collection in different growth media and 107
with different dilution rates (Supplementary Fig. 2). We found that the resultant 108
interaction networks in these different environments were also highly hierarchical, 109
despite changes in which strains were most competitive (Fig. 2f and Supplementary Fig. 110
3). Thus, we conclude that hierarchy in pairwise competition is a robust feature of this 111
model community. 112
Next, we asked what characteristics of a strain might best predict its performance in 113
competition. We hypothesized that strains that grow well in monoculture will have 114
competitive advantages over strains that grow more poorly. Indeed, we found that 115
exponential growth rate (r) was positively correlated with competitive score (Spearman’s 116
rho = 0.77; p < 10
-4
; Fig. 3a) and that the typical outcome was for the strain with the 117
higher r to exclude the strain with the lower r, which occurred for 67% of pairs (Fig 3c). 118
Carrying capacity (K) in monoculture was less predictive of competitive superiority, but 119
was still significantly correlated (Spearman’s rho = 0.55, p < 0.05; Fig. 3b). In general, 120
the likelihood of outcomes other than the stronger grower outcompeting the weaker 121
grower decreases for large differences in r and K (Supplementary Fig. 4). While 122
differences in these two parameters can be indicators of the likelihood of a given 123
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competitive outcome, there are many exceptions, and, indeed, some of the stronger 124
competitors do not necessarily have correspondingly strong single-species growth 125
parameters. Thus, while the each species’ intrinsic growth ability correlates with 126
competitive ability, the significant number of exceptions indicates that growth ability 127
alone does not fully explain the hierarchical competitive structure that we observe. 128
An important corollary of the high degree of hierarchy we observed in the interaction 129
network is that non-transitive motifs are vanishingly rare. Non-transitive motifs are 130
instances in which a clear competitive hierarchy among members of a sub-group does not 131
exist, the classic example being a rock-paper-scissors (RPS) triad. Of the 987 triads in our 132
collection for which complete pairwise outcome data are available, only three (0.3%) 133
display the RPS topology. This number is significantly less than is found in randomized 134
networks, where on average 14% of triads were RPS (p < 10
15
; Fig. 4). Furthermore, the 135
three triads that we classify as RPS each feature strains that display unusually high 136
variability from experiment to experiment, possibly due to rapid evolution, and further 137
efforts to characterize these triads failed to reproduce the non-transitive network 138
topology. As dictated by its hierarchical structure, our network is also highly enriched for 139
perfectly hierarchical feedforward loops, which were observed in over 50% of triads (Fig. 140
4). Due to the paucity and irreproducibility of observable non-transitive relationships 141
among our strains in vitro, we conclude that such relationships are unlikely to be a 142
significant contributor to their coexistence in a natural environment. 143
Given the hierarchical structure of the pairwise interaction network, we wondered about 144
the potential of higher-order interactions and indirect effects among our strains to give 145
rise to a diverse community. To address this, we inoculated three replicate cultures with 146
equal proportions of all 20 strains and propagated them through five growth-dilution 147
cycles (Fig. 5b). The resulting assemblages were highly replicable, and consisted of three 148
strains representing some of the strongest competitors in pairwise experiments (Fig. 5a,c), 149
all of which were found to coexist with each other in pairwise competition. Notably, this 150
combination of survivors was consistent with the simple community assembly rule we 151
recently developed
21
: namely, that a strain is expected to survive in multispecies 152
competition if and only if it is not excluded by any other surviving species. Since 153
pairwise outcomes alone are sufficient to predict the outcome of multispecies competition 154
in this environment, we conclude that higher-order interactions are unlikely to play a 155
major role in structuring this community. 156
Discussion 157
Many factors can contribute to the generation and maintenance of diversity in ecological 158
communities. Non-transitivity, facilitation, bistability, weak interactions, multiple 159
limiting factors, and spatial or temporal segregation have all been hypothesized to play a 160
role
22
; however, there is little empirical data regarding the relative importance of each of 161
these factors in actual natural communities. Here, we explored one such community. In 162
this work, we explored the network of pairwise interactions for a community of naturally 163
co-occurring bacteria. Our results indicate that diversity in this community is likely 164
maintained primarily due to factors including and spatial or temporal segregation or 165
multiple limiting factors, rather than frequent bistability, non-transitivity, or higher order 166
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interactions, all of which have been hypothesized to play a role in generating and 167
maintaining diversity. Nonetheless, we still do not completely understand the processes 168
that give rise to the diversity we observe in nature. 169
Given that soil is a heterogeneous mixture with a multitude of microhabitats, microbial 170
co-occurrence in soil may be facilitated by niche separation and spatial de-mixing. This 171
would allow the coexistence of strains that display strong inhibitory interactions in well-172
mixed environments. Microbes in soil also experience a strongly fluctuating environment, 173
which can lead to coexistence of multiple strains over time via the soil spore bank. 174
Members of the genus Bacillus are particularly well known for their spore-forming 175
ability, which may allow them to persist in a non-vegetative, and therefore non-176
competitive state, until conditions favor their growth
23
. Finally, our experimental 177
approach clearly requires that the strains to be competed be culturable in the laboratory, 178
so it is impossible for us to exclude the possibility that other strains present within the 179
soil might behave very differently. 180
Simulations of our experimental system using the generalized Lotka-Volterra model 181
(gLV) predicted that, if the underlying ecological interactions among species are assigned 182
at random, the pairwise interaction network should become less hierarchical at lower 183
death rates, corresponding to a lower daily dilution rate in our experimental setup 184
(Supplementary Fig. 5). In order to test this hypothesis, we competed a subset of pairs 185
while experimentally reducing the dilution rate from 1:100 to 1:10 (Fig. 2f). The 186
hierarchical network structure was robust to this manipulation, and remained highly 187
correlated with growth rates in monoculture. While it is possible that reducing the death 188
rate further could weaken the hierarchy by reducing the importance of a growth rate 189
advantage in determining survival, the most straightforward interpretation of our data is 190
that the hierarchy is not simply due to differences in growth rates. 191
This experimental system also gives us the opportunity to test the importance of higher 192
order interactions in shaping communities. Higher order interactions are said to take 193
place when the presence of an additional species changes the interaction between two 194
existing species
24
, and have the potential to contribute to the maintenance of species 195
diversity
25
. In bacterial systems, this can be driven by complex networks of selective 196
antibiotic production and sensitivity
26
. Despite the potential for higher order interactions 197
in our model community, our simple assembly rule
21
, which disregards higher order 198
interactions entirely, accurately predicted the survivors in all-versus-all competition in 199
vitro, suggesting that higher order interactions are not a major driver of community 200
structure in this instance. 201
The observation of high levels of diversity in communities of competing organisms is a 202
long-standing paradox in community ecology
27
. In this work, we showed that a bottom-203
up approach to studying community assembly can be useful in narrowing down the range 204
of possible explanations for the diversity we observe in nature. However, this approach 205
necessitates removing the organisms from their natural environment, including the larger 206
community in which the species of interest are embedded. Future work combining in 207
vitro competition experiments with a more mechanistic understanding of the influence of 208
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