# Evolution in response to an abiotic stress shapes species coexistence

Inês Fragata, Diogo Godinho, Leonor Rodrigues, Miguel Cruz, Flore Zélé, Oscar Godoy, Sara Magalhães

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Inês Fragata, Diogo Godinho, Leonor Rodrigues, Miguel Cruz, Flore Zélé, et al.. Evolution in response to an abiotic stress shapes species coexistence. 2025. hal-04288847v3

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Evolution in response to an abiotic stress shapes species

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coexistence

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Inês Fragata¹*, Diogo P. Godinho¹*, Leonor R. Rodrigues¹, Miguel A. Cruz¹, Flore Zélé²,

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Oscar Godoy³#, Sara Magalhães¹#

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1 - cE3c - Center for Ecology, Evolution and Environmental Changes & CHANGE - Global

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Change and Sustainability Institute, Departamento de Biologia Animal, Faculdade de

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Ciências, Universidade de Lisboa, Portugal.

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2 - Institut des Sciences de l'Evolution de Montpellier (ISEM), Université de Montpellier,

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CNRS, IRD, EPHE, Montpellier, France.

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3 – Estación Biológica de Doñana (EBD-CSIC), Americo Vespucio 26, E-41092 Sevilla,

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Spain. https://orcid.org/0000-0003-4988-6626

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* co-first; # co-last

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ª Current address: Gulbenkian Institute for Molecular Medicine (GIMM), Oeiras, Portugal

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Corresponding author: Inês Fragata (irfragata@ciencias.ulisboa.pt)

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Keywords: Experimental Evolution; Coexistence theory; Competition; Spider mites

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Running title: Evolution shapes biotic interactions

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Authorship Statement: IF, OG and SM conceived the original idea. IF, DG, OG and SM designed the experiments. IF, DG, LRR and MC performed the experiments and collected the data. IF performed the modelling work and analysed data, with advice from FZ and OG. IF produced the figures, with help from LRR. IF, SM and OG wrote the first draft of the manuscript, and all authors contributed substantially to revisions. SM supervised DG, IF,

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24 LRR and MC, FZ supervised MC, OG supervised IF. SM provided the financial support for
25 the experiments, SM and OG for the meetings to analyze the data and write the paper.

26 **Data accessibility statement:** Data and scripts are available in figshare in the link
27 https://figshare.com/s/f001d9f699a4027d7b62, with the doi: 10.6084/m9 figshare.28369472

28 **Type of article:** Letter

29 **Number of words in the abstract:** 150

30 **Number of words in the main document:** 5017

31 **Number of references:** 69

32 **Number of figures:** 4
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**Abstract**

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**Introduction**

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Adaptation to abiotic stresses generally relies on traits that are not independent from those affecting species interactions. Still, the impact of such evolutionary processes on coexistence remains elusive. Here, we studied two spider mite species evolving separately on tomato plants that either hyper-accumulated cadmium, a stressful environment for the mites, or on control plants without cadmium. Through combining experimental evolution and structural stability theory, we found that adaptation to cadmium of both species shifted predictions from exclusion to coexistence. This shift occured due to a simultaneous increase in intra and a decrease in interspecific competition, but only in cadmium environments. These predictions were further confirmed with complementary experiments of population dynamics, underscoring that evolution of single species in a new environment, even in absence of interspecific competitors, shapes species coexistence. Hence, population shifts to novel environments may have unforeseen evolutionary consequences for community composition and the maintenance of species diversity.

Understanding how evolutionary dynamics shape and are shaped by long-term species persistence is key to predict community composition and biodiversity maintenance. Indeed, theory predicts that evolving with competitors may lead to changes in competitive interactions (i.e., the negative per-capita effect of one species on another (Abrams & Matsuda 1994; Bernhardt et al. 2020; Sakarchi & Germain 2023), which in turn can affect species coexistence (Edwards et al. 2018; Germain et al. 2018; Vasseur et al. 2011; Yamamichi & Letten 2021). In line with these predictions, empirical studies have shown that short term evolution in presence of competitors can modify competitive traits (Hart et al. 2019) and
change coexistence patterns (Germain et al. 2020; Hiltunen et al. 2017; Lankau 2011; Sakarchi & Germain 2023; Zhao et al. 2016).

Although the abovementioned studies have demonstrated that short-term evolution is an important force shaping ecological patterns, we may still be under-estimating the potential role of evolution in species coexistence. An important yet overlooked possibility is that traits that affect species coexistence may be selected even in the absence of competitors. For example, species arriving in an environment before their interspecific competitors may rapidly adapt to that environment, thus potentially monopolizing it (Nadeau et al. 2021). Likewise, prior adaptation to a given environment can lead to a lower relative growth rate of late arriving species (Low-Décarie et al. 2011). Moreover, trait evolution in a given environment can affect species community composition in other environments (Fukano et al. 2022; Gallego & Narwani 2022; Limberger & Fussmann 2021). However, to date no study has tested if and how trait evolution in response to an abiotic selection pressure can change intra- and interspecific competition and its impact on species coexistence. Hence, our knowledge of the role of evolution in response to abiotic stresses in shaping coexistence patterns is yet in its infancy.

The recent development of the complementary theoretical frameworks of modern coexistence theory and structural stability (Chesson 2000; Saavedra et al. 2017; Yamamichi et al. 2022) provides clear mechanistic pathways to understand how competing species coexist in ecological time. Both theories agree that species can coexist whenever their niche differences, which arise when species specialize in different resources, overcome their differences in population growth in the presence of competitors (Barabás et al. 2018; Chesson 2000; Godoy et al. 2018), but structural stability allows incorporating stochasticity and interactions other than direct competition (Rohr et al. 2014). This framework defines that species coexistence is possible when the opportunities for species to coexist (measured by the
size of the feasibility domain) can accommodate the differences in intrinsic growth rates
between species(Godoy et al. 2018; Saavedra et al. 2017). To date, this theoretical
framework has been seldom applied to address the role of evolution in shaping coexistence
outcomes.

Here, we assessed how evolution in response to an abiotic stress affects species coexistence by combining two powerful tools: experimental evolution and structural stability.

We used a system composed of two closely-related spider mite species, *Tetranychus urticae* and *T. evansi*, known to engage in competitive interactions when feeding on tomato plants (*Solanum lycopersicum*) (Fragata *et al.* 2022; Sarmento *et al.* 2011). These plants can hyper-accumulate cadmium in their upper parts, which strongly reduces spider mite fecundity and offspring survival, producing a strong selective pressure (Godinho *et al.* 2018, 2023, 2024).

Here, each spider mite species evolved for 42 generations on plants with or without cadmium in absence of interspecific competitors (Fig. S1A). Subsequently, changes in intrinsic growth rates and in the strength of biotic interactions (intra- and interspecific competition) were assessed in both environments using a fully orthogonal experimental design (Fig. S1B). Then, information from this experimental evolution were used to further parameterize a population model, which was coupled with structural stability theory to predict how evolution, of each species or both, in response to an abiotic stress (cadmium) affect competitive outcomes.

Finally, we empirically validated the short-term predictions of our models by conducting a population growth experiment with the same full orthogonal design (Fig. S1C).

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## 103 Material and methods

Details of the maintenance of outbred and experimental evolution populations, common garden procedures and all experiments performed are available in Supplementary Material and Methods.
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108 **Experimental evolution**

109 All experimental evolution populations were initiated by transferring 220 adult mated
110 females of *Tetranychus urticae* or *Tetranychus evansi* from the outbred populations
111 (described in Godinho et al. 2020, 2024) to an experimental box containing four tomato
112 leaves with their stems in water. We established five replicate populations for each selection
113 regime (Fig. S1A): *T. urticae* or *T. evansi*, exposed to leaves from plants watered either with
114 a 2mM cadmium solution (the cadmium selection regime) or with water only (the no-
115 cadmium selection regime). This cadmium concentration is highly detrimental to both spider
116 mite species, resulting in at least a 50% reduction in fecundity relative to mites on metal-free
117 plants (Godinho *et al.* 2018, 2023). The detrimental effect of cadmium was confirmed in our
118 populations, with mites that evolved on plants without cadmium showing a decrease in
119 intrinsic growth rate when feeding on plants with cadmium (see details in Supplementary
120 Material). Every two weeks (circa one mite generation), 220 adult mated females were
121 transferred to a new box containing four new tomato leaves (i.e., populations were
122 maintained in discrete generations). This setup mimics colonization of a new plant, as
123 dispersal is mainly performed by young mated females (Li & Margolies 1993). If the total
124 number of mated females did not reach 220, the transfer was complemented with females
125 taken from the respective T-1 box or from the base population (cf. Godinho et al. 2020, 2024
126 for details). Replicate 2 of the *T. urticae* cadmium selection regime was not tested because
127 not enough females were available. Prior to the experiments, individuals from all regimes
128 were placed in a common garden of cadmium-free tomato leaves for two generations
129 (generation 40 and 42 for the competitive ability and population growth experiments,
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132 **Empirical estimation of competitive abilities and intrinsic growth rate**

133 To test if evolution on plants with or without cadmium affected the probability of coexistence
134 between the two species in these environments, we estimated the intrinsic growth rates and
135 the strength of intra- and interspecific interactions of mites from each selection regime in
136 each environment (Fig. S1B), following the methodology described in (Hart et al. 2018).
137 Briefly, we placed one focal female alone or with 1, 3 or 9 females (i.e., competitors) on leaf
138 disks from plants with or without cadmium. The focal females were exposed to competitor
139 females either from the same (intraspecific) or a different (interspecific) selection regime (full
140 factorial design) with matching population replicates (e.g. replicate 1 of *T. urticae* was
141 exposed to replicate 1 of *T. evansi*, etc). Females were left to oviposit for three days, then
142 killed. Two weeks later, the number of adult females per patch was counted. Ten focal
143 females per intra- or interspecific treatment were done for each of the experimental replicate
144 populations of the four selection regimes for each environment.

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146 **Empirical estimation of population growth rate in presence of interspecific competitors**

147 To test if evolution on plants with or without cadmium affected the population growth rate
148 under competition, we placed six females of the two species from different selection regimes
149 on plants with or without cadmium (full factorial design, i.e., eight experimental conditions,
150 Fig. S1C) and measured the number of adult females of each species produced after two
151 generations. Ten experimental replicates were tested per experimental condition, per replicate
152 population. This data was then compared with model predictions based on parameters
153 estimated from the intrinsic growth rate and competitive abilities (cf. Model validation
154 section below).

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156 **Theoretical estimation of competition and growth parameters**
Data collected in the intrinsic growth rate and competitive ability experiment was used to parameterize the Ricker competition model (Fowler 1981; Ricker 1954), which has been used in a previous study with spider mites (Bisschop et al. 2022). This model allows incorporating positive interactions (i.e., facilitation, Bowler et al. 2022; Buche et al. 2025; Martyn et al. 2021; Ricker 1954; Stouffer 2022), which based on initial data scrutiny, were likely to occur in our system. The Ricker model is described by the following equation:

$$ (1) \frac{N_{i,t+1}}{N_{i,t}} = \lambda_i e^{-\alpha_{ii} N_{i,t} - \alpha_{ij} N_{j,t}} $$

where $N_{i,t+1}$ is the number of individuals of species i in the next generation, $\lambda_i$ the intrinsic growth rate of species i in absence of competitors, $\alpha_{ii}$ the strength of intraspecific competition, $\alpha_{ij}$ the strength of interspecific competition, and $N_{i,t}$, $N_{j,t}$ the number of competitors of species i and j, respectively, in the current generation. We used the “cxr” package (García-Callejas et al. 2020) in R to estimate $\lambda_i$, $\alpha_{ii}$ and $\alpha_{ij}$ for each replicate population separately and with all replicates of the same selection regime pooled (to increase statistical power). To include the filtering effect of the cadmium environment and because the “cxr” package does not allow the inclusion of zeros, we transformed our data by summing one to each of our datapoints (as the logarithm of one is zero). Initial parameters were set at $\lambda_i=1$, $\alpha_{ii}=\alpha_{ij}=0.1$ and confidence intervals were estimated from 1000 bootstrap iterations. Model fitting assessment was done by visual inspection (available in the git repository) and by comparing the euclidian distance between observed and predicted values (Fig. S2), with the crx package. To estimate the relative impact of intra- and interspecific competition on population growth, we simulated offspring production of ten females using only the intrinsic growth rate, the joint effects of intrinsic growth rate and of intraspecific competition and the
joint effects of those traits plus interspecific competition. Confidence intervals were

calculated using the lower and upper estimates of the intrinsic growth rate and intra and

interspecific competition indexes obtained from the crx package (García-Callejas *et al.* 2020).

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184 **Structural stability approach to predict coexistence**

Estimates obtained from the Ricker model (Fig. S3, S4, S5, S6) showed widespread weak
positive interspecific interactions (i.e., facilitation), thus we used the structural stability
framework (Allen-Perkins et al. 2023; Saavedra et al. 2017, 2020) to predict the outcomes of
species interactions as other common approaches to study species coexistence (e.g. Chesson
2000) can not account for positive interactions. The structuralist approach utilizes the
strength and sign of species interactions to estimate the size of the feasibility domain.
Coexistence is possible if the vector containing the intrinsic growth rate of both species falls
within the feasibility domain. In principle, the feasibility domain can include both negative
and positive intrinsic growth rates. However, we restricted coexistence predictions to positive
values (i.e. bounded by the vectors [0, α₂₂] and [0, α₁₁]) to match the information coming
from our experimental system as species can either have zero or positive growth rates.
Finally, to account for uncertainty in the estimation of model parameters, we estimated the
feasibility domain bound by the upper (lower) 95% of the vector [0, α₂₂] and the lower
(upper) boundaries of the [0, α₂₁] to obtain the widest (narrowest) conditions under which
species are able to coexist.

To measure each species' vulnerability to exclusion, we tested how resistant coexistence is
to perturbations. Following a recently-developed tools within the structuralist approach
(Allen-Perkins *et al.* 2023; Medeiros *et al.* 2021), we estimated the species exclusion
distance, which corresponds to the minimal distance between the vector of intrinsic growth
rates (r= [λ₁, λⱼ]) and the edges of the feasibility domain (vectors corresponding to either [0, 
$\frac{\alpha_{22}}{\alpha_{12}}$] or[0, $\frac{\alpha_{21}}{\alpha_{11}}$]) as a proxy for how strong a perturbation must be to change the coexistence
outcome. To calculate the distance between the vector of intrinsic growth rates and the edges
of the feasibility domain, we applied the following formula (Allen-Perkins et al. 2023):

$$
208 \quad (2) \ dist_i = \cos^{-1} \begin{bmatrix} \lambda_i \\ \lambda_j \end{bmatrix} \cdot \begin{bmatrix} \alpha_{21} \\ \alpha_{11} \end{bmatrix}
$$

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210 Model validation

211 We validated our approach by comparing model predictions with the empirical results from
212 the population growth experiment. For that, we used the Ricker model to predict the relative
213 abundance of each species after two generations, starting with the initial conditions of the
214 experiment. Then, we performed a general linear regression with a binomial error distribution
215 to estimate the correlation between the mean observed proportions of each competitor (using
216 the cbind function to join the number of *T. evansi* and *T. urticae* females) in all replicates of
217 all treatments with the model proportion estimates (Number of *T. evansi* females/Number of
218 adult females) obtained for each replicate.
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220 Statistical analyses

221 To test how evolution on plants with cadmium affected population growth and
222 competition in the cadmium environment, we used general linear models with a gamma
223 distribution for the intrinsic growth rate, and with a normal distribution for the intra- and
224 interspecific competition coefficients. All models were applied separately for both species,
225 and normality and dispersion of the residuals was inspected for all models tested. We
226 included the parameters estimated with the crx package for each replicate as a dependent
227 variable and selection regime as fixed factor (with two levels, cadmium and no-cadmium).
For the strength of interspecific competition, the model included the selection regime of the focal and of the competitor species, as well as their interaction. Whenever the interaction term was significant, we applied contrasts for all combinations of focal and competitor selection regimes. Similar models were applied to test the impact of evolution in cadmium on the performance in the no-cadmium environment. Finally, we estimated the likelihood of finding differences between environments/selection regimes using 1000 bootstrap iterations. For that, we randomized (with replacement) the parameter estimates within environments/selection regimes (depending on the model) and applied the same models as described above. Then, we estimated the probability of randomly obtaining a significant p-value (below or equal to 0.05) with our data set by computing the number of times a significant result was obtained.

All analyses were done using the package "glmmTMB" (Brooks et al. 2017) in R 4.2.1 version (R Core Team 2022). Contrasts were performed using the "emmeans" package (Lenth 2024), analyses of residuals were done for each model using the DHARMa package (Hartig 2022), and graphical representation was done using "ggplot2" (Wickham 2016). All data and scripts are available in the git repository https://github.com/irfragata/evolution_cadmium_coexistence.

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## 246 Results

### 247 Evolution in cadmium changes the strength of interspecific competition

After evolution for circa 40 generations in the cadmium environment, the intrinsic growth rate of both populations evolving in that environment was higher than that of populations evolving on plants without cadmium (*T. urticae*: $\chi^2_{1,6}=6.187$, P-value=0.013, *T. evansi*: $Z_{1,7}=4.483$, P-value=0.034, Fig 1, Fig. S7, Table S1). In addition, the strength of intraspecific competition increased for *T. evansi* ($\chi^2_{1,7}=8.570$, P-value=0.003, Fig S3A, Table S1), but not
for *T. urticae* ($\chi^2_{1,6} = 0.131$, P-value=0.717, Fig S3B, Table S1). This resulted in a decreased predicted number of offspring produced by *T. evansi* in competition (relative to when growing alone) but only when *T. evansi* evolved in the cadmium selection regime (Fig. 1A, $\lambda$ vs $\lambda+\alpha_{ii}$ for the yellow and blue vs the green and red dot). Moreover, evolving on plants with cadmium made *T. evansi* more sensitive to competition with *T. urticae* from the no-cadmium selection regime (Fig. S4A, Table S2A, S2B, contrasts: T ratio$_{1,13}$= -0.053, P-value=0.003), reducing the predicted number of offspring produced after one generation of competition (Fig. 1A, $\lambda+\alpha_{ii}+\alpha_{ij}$, comparing yellow and red dots). Conversely, evolution in cadmium did not affect the sensitivity to interspecific competition in *T. urticae* (Fig. S4B, Table S2A). We observed weak positive interactions within and between species in several replicates (i.e., negative alpha values) across selection regimes (Fig S3, S4), which reinforces the added value of using the structural stability framework.

In the no-cadmium environment, there were no significant differences in the intrinsic growth rate (Fig. S8, *T. evansi*: $\chi^2_{1,7}=2.628$, P-value=0.105; *T. urticae*: $\chi^2_{1,6}= 0.121$, P-value=0.728, Table S1), intraspecific competition (Fig. S5, *T. evansi*: $\chi^2_{1,7}= 1.445$, P-value=0.229; *T. urticae*: $\chi^2_{1,6}= 0.003$, P-value=0.998, Table S1) or interspecific competition (Fig. S6, Table S1, S3) between populations evolving in that environment and those evolving in the environment with cadmium. Weak intra- and interspecific facilitation was found for several replicates across selection regimes (Figs. S5, S6), similarly to what happened in the cadmium environment.

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274 Evolution of both competitors increases the probability of long-term coexistence in the
cadmium environment

276 In the cadmium environment, coexistence was more likely when both species had evolved in
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intraspecific competition for the better competitor (*T. evansi*, Fig S3). The prediction of coexistence of both species after evolution in cadmium is robust to parameter variation because it holds even after taking uncertainty (95% CI) into account when estimating the feasibility domain (Fig 2, light blue region), and the differences of species intrinsic growth rates (Fig. 3, S9).

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When only one species evolved in cadmium, coexistence was not possible in that environment (Figs 2, 3). Contrary to our initial expectation that the species that evolved in cadmium would be best suited to the new environment and thus exclude competitors from the no-cadmium selection regimes, we observe that the species that evolved in cadmium was predicted to be excluded (Figs. 2, 3, S9). When *T. evansi* evolved in cadmium, this can be explained by increased intraspecific competition (Fig S3) and increased sensitivity to competition from *T. urticae* of the no-cadmium selection regime (Fig S4). In the reverse case, *T. urticae* that evolved in cadmium was negatively affected by interspecific competition with *T. evansi* from the no-cadmium selection regime but did not affect the competitor (Fig. S4), which led to a prediction of exclusion in the cadmium environment (Fig. 4). Again, these predictions, are robust to variation in parameter estimation when accounting for lower and upper 95% confidence intervals.

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In the no-cadmium environment, the two species were generally predicted to coexist except when *T. urticae* from the cadmium selection regime competed with *T. evansi* from the no-cadmium selection regime, in which case *T. urticae* was predicted to be excluded by *T. evansi* (Fig. 2). However, the minimum distance to the edges of the feasibility domain was short in all cases (Fig. 3, S9), especially when considering the feasibility domains with lower confidence intervals, indicating that small perturbations may shift the prediction from coexistence to exclusion and vice versa. Importantly, there was high heterogeneity among replicates but little effect of the evolutionary history of both species (Fig. 3). Thus, evolving
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in the cadmium environment did not affect the range of opportunities for species to coexist in
the no-cadmium environment (Fig. 2, S9).

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Simulated competitive dynamics partially recover empirical patterns

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To validate our approach, we compared theoretical predictions with a two-generation
population dynamic experiment with the two species from all combinations of selection
regimes. (Fig. S1). We observed a close agreement between model predictions and
experimental observations in terms of proportion of *T. evansi* in the populations (Fig. 4, slope
pooled replicates: 0.707, P-value<0.001), despite some heterogeneity between replicate
populations, especially in the no-cadmium environment (slope with replicates: 0.631, P-
value<0.001, Table S4).

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Discussion

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We used a combination of experimental evolution and structural stability theory to predict the
impact of evolution of two spider mite species in response to an abiotic selection pressure
(cadmium) on coexistence patterns, in an environment either with or without cadmium. To
obtain theoretical predictions, we quantified the intrinsic growth rate of each species and the
strength of their intra- and interspecific competitive interactions across different
environmental and selection scenarios. While evolutionary history had little effect on
coexistence patterns in the environment without cadmium, the independent evolution of both
species in response to cadmium led to a change from competitive exclusion to coexistence in
that stressful environment. This was due to differential changes in intrinsic growth rates,
which equalized fitness between species, coupled with an increase in intraspecific
competition in the superior competitor, which in turn increased structural niche differences,
and therefore the range of fitness differences compatible with persistence of both species. In

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sum, we show that evolution in response to an abiotic selection pressure can change the interactions between and within populations and affect the probability of coexistence, even without a direct selection pressure posed by the presence of competitors as showed previously(Germain *et al.* 2020; Sakarchi & Germain 2023; Vasseur *et al.* 2011; Zhao *et al.* 2016).

In more detail, we found that both species adapted to the cadmium environment within 42 generations of experimental evolution. This is supported by the observed increase in the intrinsic growth rate in the cadmium environment of populations from the cadmium selection regime, when compared to the no-cadmium regime. A previous study reported that these populations of *T. evansi* did not shown signs of adaptation at the 33rd generation of evolution (Godinho *et al.* 2024). Therefore, we believe the adaptation reported here occurred at a later time point. Moreover this previous study revealed lower performance of *T. evansi* populations from the cadmium selection regime on the environment without cadmium, compared to populations evolving in the no-cadmium selection regime (Godinho *et al.* 2024).

We do not recover this result, suggesting that extending time for evolution to operate led to reduced costs of adaptation. This adaptation pattern is compatible with the existence of cryptic genetic variation that is released upon evolution in the cadmium environment (Paaby & Rockman 2014). This is also in line with the absence of genetic variation for traits associated to performance in the cadmium environment, found in the populations used to initiate the experimental evolution. Still, such adaptation may be too slow to enable the establishment of mites in cadmium-contaminated sites. Indeed, assuming equal generation time in the lab and in the field, one tomato growing season roughly corresponds to 15 generations in spider mites, which is not sufficient to lead to genetic changes allowing adaptation to cadmium.
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Evolution of *T. evansi* in cadmium also led to stronger sensitivity to intraspecific competition in that environment, as compared to mites evolving on plants without cadmium. Evidence that intraspecific competitive ability may evolve during adaptation to a novel abiotic environment has been accumulating (Bernhardt *et al.* 2020; Bono *et al.* 2015; Limberger & Fussmann 2021). There are many reasons to expect such evolution (Siepielski *et al.* 2016). For example, individuals may become more efficient at extracting resources when these are limiting (Bernhardt *et al.* 2020), or higher population growth may lead to higher densities being reached earlier, thus increasing intraspecific competition. Measuring intraspecific competition should thus be mainstreamed in experimental evolution studies, which typically measure only individual life-history traits such as fecundity and survival (Kawecki *et al.* 2012).

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An overlooked logical follow-up of our observations is that adaptation to abiotic selection pressures may affect interspecific interactions as well. Here, we shed light on this understudied hypothesis by documenting that, when spider mites evolved in cadmium, they affected their competitors less or equally than mites that evolved on plants without cadmium. This counter-intuitive result may be due to a growth-competition trade-off, given that both species also increased their growth rate on plants with cadmium upon evolution in that environment. Such trade-off has been an assumption of theoretical models (Wittmann & Fukami 2018), and experimentally demonstrated in *Brassica nigra* (Lankau 2008; Lankau & Strauss 2007). For example, algae adapt more rapidly to abiotic selection pressures when they evolve alone vs. when in presence of competitors (Limberger & Fussmann 2021), which is compatible with the existence of such trade-off. In sum, we show that interspecific competitive ability evolves in absence of competitors, a result similar to when competing species coevolve (Fukano *et al.* 2022; Germain *et al.* 2020; Hart *et al.* 2019; Sakarchi & Germain 2023). The fact that we recover such patterns even in the absence of a competitor in
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the environment implies both that causality needs to be scrutinized in eco-evolutionary
studies and that evolution has consequences for community structure that are much more far
reaching than initially thought.

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Many empirical studies have shown that adaptation to an abiotic selection pressure can
change species interactions (Bach *et al.* 2018; Fukano *et al.* 2022; Limberger & Fussmann
2021) but none have addressed how it affects species coexistence. In fact, the only studies
applying coexistence theory to evolutionary data concern adaptation to the presence of a
competitor (Germain *et al.* 2020; Hart *et al.* 2019; Zhao *et al.* 2016). In our study, we found
that, in the cadmium environment, species are only predicted to coexist when both
competitors evolved separately in the presence of cadmium. Such change from competitive
exclusion to coexistence was not due to the large change of a specific parameter but rather a
small but full reorganization of how species grow and interact with each other. Namely,
coexistence was due to a combination of an increase in the intrinsic growth rate of both
species as they adapt to the cadmium environment, with stronger intraspecific competition of
the superior competitor, coupled with a reduced impact of interspecific competition.
Although the ultimate mechanisms of these changes are unclear, we speculate that evolution
improved the ability of spider mites to feed on tomato plants that accumulated cadmium,
likely by increasing resource uptake, which in turn decreased resources available to others
(thus increasing intraspecific competition). This positive covariance between an increase in
fitness and an in self-limitation is congruent with theoretical predictions (Carroll *et al.* 2011),
previously reported in other systems (e.g. Angert *et al.* 2009). Additionally, the observed
reduction in interspecific competition (leading often to reciprocal facilitation) for the two
species could be due to increased benefits from plant defence suppression, documented in
both populations used (Fragata *et al.* 2022). Curiously, studies addressing how evolution in
the presence of a competitor affected species coexistence have not found such a radical effect

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of changing from exclusion to coexistence. Instead, they found that coexistence is maintained when species coevolve, albeit by mechanisms different than those operating in the absence of such evolution (Germain *et al.* 2020; Hart *et al.* 2019; Zhao *et al.* 2016). We thus find that adaptation to an abiotic selection pressure may affect community composition to a higher degree than direct adaptation to the presence of competitors.

We also provide a test for the accuracy of the theoretical predictions, by comparing model predictions based on parameters estimated from competition experiments with a short-term population dynamics experiment. We found a high overlap between predicted and observed relative abundances of the two species. Some mismatch was found when only one of the populations evolved in the cadmium environment. This is probably due to stochasticity, as the experiment started with a small number of individuals (six females of each species). Still, the strong correlation between observed and predicted values suggests that our model parameterization is robust to changes in the number of individuals, resource and space availability, indicating that the combination of theoretical modelling and experimental estimation of competitive interactions has a high predictive power.

Another strength of our study is that, unlike previous studies, we applied a full factorial design, in which we explore how different combinations of evolutionary histories of both competitors affect the probability of coexistence. We find that coexistence in an environment with cadmium is only possible when both species evolved in that environment. Hence, community assembly does not follow a linear path, being contingent on the evolutionary history of the two species. This suggests that asynchronies in the arrival to cadmium-contaminated sites may lead to species exclusion, due to evolution of the species first colonizing that environment. Coexistence is only possible when the two species are both adapted, which can be achieved either if they arrive simultaneously to a site with cadmium, then adapt at a similar pace, or if they are already adapted upon arrival. Hence, as previously
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shown for short-term differences in arrival time (Fragata *et al.* 2022), we here show that
historical contingencies affect species coexistence also via their effect on evolution.

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As found in studies with no evolution, we found that interactions are specific to a given
environment (Grainger et al. 2019; Granjel et al. 2023; Matías et al. 2018; Wainwright et al.
2019). In general, our results align well with previous work showing that stable coexistence is
fostered in stressful environments via a reduction in growth rate that equalize fitness
differences, and by a shift from interspecific to intraspecific competition, which increases the
range of coexistence opportunities (i.e. increases the size of the feasibility domain) (Grainger
et al. 2019; Granjel et al. 2023; Matías et al. 2018; Song et al. 2020b; Wainwright et al.
2019), and stabilize the population dynamics of interacting species. However, our study
tempers this statement by the finding that this is only true when both species have evolved in
that stressful environment. Moreover, the use of the structural stability framework allows
incorporating sensitivity to changes in environmental conditions in these coexistence
predictions(Allen-Perkins et al. 2023; Song et al. 2020a; Tabi et al. 2020). In the environment
without cadmium, we found a small distance to the edge (i.e., low robustness) across all
selection regimes, suggesting that changes in competitive outcomes are likely to occur due to
stochastic events. Instead, in the cadmium environment, the distance to the edge was higher
in all cases, suggesting that communities in that environment are more long-lasting. Thus, our
results suggest that communities in cadmium-free environments will be modulated by small
environmental changes, whereas those in environments with cadmium will be more shaped
by evolution.

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Our study highlights the added value of combining experimental evolution and
controlled experiments with ecological theory, by providing novel insights of how species
adaptation to an abiotic stressor affects their ability to coexist. This work provides significant
advances in both evolutionary and ecological fields. On the one hand, it shows that traits such

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as the strength of intra- and interspecific competition should be incorporated in the
characterization of species adaptation to novel environments. This is particularly true in
experimental evolution studies, which generally quantify evolution by measuring classical
life-history traits of single individuals (Garland & Rose 2009; Kawecki et al. 2012), which
may not reflect the conditions in which populations evolve (Chippindale et al. 2003). On the
other hand, ecological studies benefit from incorporating past evolutionary history, as such
approach has the capacity to refine our understanding of how species interact and coexist
(Leibold et al. 2019; Urban & De Meester 2009; Wittmann & Fukami 2018; Yamamichi et
al. 2020). Thus, we highlight the need to combine ecological and evolutionary perspectives
and methodologies to understand community composition.

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463

464 Acknowledgments:

This work was financed by an ERC (European Research Council) consolidator grant 465 COMPCON, GA 725419 attributed to SM, an ERC starting grant DYNAMICTRIO, GA 466 467 101042392 attributed to IF, and by FCT (Fundação para Ciência e Tecnologia) with the 468 Junior researcher contract (CEECIND/02616/2018) attributed to IF. OG acknowledges 469 financial support provided by the Spanish Ministry of Economy and Competitiveness 470 (MINECO) and by the European Social Fund through the UCCO project (CNS2023-144337).

This is contribution ISEM-2023-XXX of the Institute of Evolutionary Science of Montpellier 471 472 (ISEM). The authors acknowledge stimulating discussion with all members of the Mite 473 Squad and Raul Costa-Pereira, which have significantly improved the experimental design 474 and interpretation of the results, and Marta Artal and Liesbeth de Jong for invaluable 475 infrastructure for meetings in Sevilla. For the purpose of Open Access, a CC-BY 4.0 public 476 copyright licence has been applied by the authors to the present document and will be applied
477 to all subsequent versions up to the Author Accepted Manuscript arising from this

478 submission.

479

480 **Competing interests:** Authors declare no competing interests.

481

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<figure><img src="image_1.png" /><figcaption>Figure 1 – Predicted number of female offspring after one generation for A) *T. evansi* (Te) and B) *T. urticae* (Tu), to estimate the relative effect of competition on the growth rate of the different populations. To represent the cumulative impact of each parameter on offspring production, predictions were done using only the intrinsic growth rate ($\lambda$), the intrinsic growth rate plus intraspecific competition ($\lambda + \alpha_{ii}$) and the latter plus interspecific competition ($\lambda + \alpha_{ii} + \alpha_{ij}$) in an environment with or without cadmium (left and right panels, respectively). Note that, since two of the four treatments share the same focal</figcaption></figure>

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668 selection regime (e.g. for *T. urticae* the orange and red dots correspond to the focal control 669 regime "Tu no-cadmium"), two colours are duplicates for the metrics without interspecific 670 competition (i. e., *λ* and *λ* + *α*<sub>ii</sub>). Starting conditions for simulations: 10 individuals of each 671 species. Treatments correspond to combinations of *T. evansi* (Te) and *T. urticae* (Tu) 672 selection regimes (cf. colour code). Note the different scales on the X-axis between the two 673 species (A vs B). Confidence intervals for each prediction were estimated using the lower and 674 upper estimates of the intrinsic growth rate and intra and interspecific competition indexes 675 obtained from the model. 676

<figure><img src="image_2.png" /></figure>

678 Figure 2 – Feasibility domain predicted for environments with (A) or without (B) cadmium 679 for the different combinations of cadmium and no-cadmium selection regimes in which *T. 680 urticae* (Tu) or *T. evansi* (Te) have evolved. The x and y axis correspond to the positive 681 intrinsic growth rate for *T. urticae* and *T. evansi*, respectively. The arrow represents the 682 vector of the intrinsic growth for each of the two species and the edges of the cone delimiting 683 the area under which the isoclines cross at positive abundances (i.e., the feasibility domain, in 684
which coexistence is possible). Dashed lines surrounding the arrows represent the 95%
confidence interval of the intrinsic growth rate. Arrows that fall outside of the cone indicate
that Te excludes Tu, except in the upper-right panel of figure A (Te cadmium- Tu no-
cadmium), in which Tu excludes Te. The dark grey region indicates the feasibility domain
obtained from the ratio of intra and interspecific competitive abilities estimated with data
from all replicates pooled (see methods). This region has been evaluated only under the
conditions in which both species show positive intrinsic growth rates. The light blue and light
orange regions delimitate the smallest and largest feasibility domains obtained from the lower
and upper 95% confidence interval of the competitive ability parameters. White regions
denote the unfeasibility domain (region for exclusion). Confidence intervals were obtained
from the upper and lower parameters estimates from the data of all experimental replicates
pooled.

697

<figure><img src="image_3.png" /><figcaption>● Te no cadmium:Tu no cadmium
○ Te no cadmium:Tu cadmium
● Te cadmium:Tu no cadmium
○ Te cadmium:Tu cadmium</figcaption></figure>

698
Figure 3 – Distance between the vector of intrinsic growth rates and the closest edge of the feasibility domain in environments with (left panel) or without (right panel) cadmium for the different combinations of no-cadmium or cadmium selection regimes for *T. evansi* (Te) and for *T. urticae* (Tu) (cf. colour code). Positive distances indicate that the vector of growth rates is inside the feasibility domain (i.e., coexistence is possible), and negative distances indicate that the vector is outside the feasibility domain (i.e., exclusion is predicted). Circles correspond to the distance calculated with data from all replicates pooled and triangles to distances calculated per replicate. Confidence intervals were obtained from the lower and upper estimates of the parameters from the data of all experimental replicates pooled.

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

Te no cadmium:Tu no cadmium

<figure><img src="image_6.png" /></figure>

Te no cadmium:Tu cadmium

<figure><img src="image_7.png" /></figure>

Te cadmium:Tu no cadmium

<figure><img src="image_8.png" /></figure>

Te cadmium:Tu cadmium

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Figure 4 – Correlation between predicted and observed relative abundance of *T. evansi* (Number of *T. evansi* females/Number of females of both species) after 2 generations in an independent population growth experiment in which the two species are released at equal densities. Squares indicate data from the cadmium environment and diamonds data from the no-cadmium environment. The full line represents the slope obtained from a general linear model (see methods) and the dashed lines to the standard error obtained from the model. Treatments correspond to combinations of selection regimes to which *T. evansi* (Te) and *T. urticae* (Tu) were exposed (cf. colour code). Predicted error bars were calculated based on the upper and lower parameter estimates of all data pooled and observed error bars were obtained from the standard error of the replicate populations.

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