Synthetic Eco-Evolutionary Dynamics in Simple Molecular Environment

Reviewer #1 (Public Review):

This work describes a new and powerful approach to a central question in ecology: what are the relative contributions of resource utilisation vs interactions between individuals in the shaping of an ecosystem? This approach relies on a very original quantitative experimental set-up whose power lies in its simplicity, allowing an exceptional level of control over ecological parameters and of measurement accuracy.

In this experimental system, the shared resource corresponds to 10^12 copies of a fixed single-stranded target DNA molecule to which 10^15 random single-stranded DNA molecules (the individuals populating the ecosystem) can bind. The binding process is cycled, with a 1000x-PCR amplification step between successive binding steps. The composition of the population is monitored via high-throughput DNA sequencing. Sequence data analysis describes the change in population diversity over cycles. The results are interpreted using estimated binding interactions of individuals with the target resource, as well as estimated binding interactions between individuals and also self-interactions (that can all be directly predicted as they correspond to DNA-DNA interactions). A simple model provides a framework to account for ecosystem dynamics over cycles. Finally, the trajectory of some individuals with high frequency in late cycles is traced back to the earliest cycles at which they are detected by sequencing. Their propensities to bind the resource, to form hairpins, or to form homodimers suggest how different interaction modes shape the composition of the population over cycles.

The authors report a shift from selection for binding to the resource to interactions between individuals and self-interactions over the course of cycles as the main drivers of their ecosystem. The outcome of the experiment is far from trivial as the individual-resource binding energy initially determines the relative enrichment of individuals, and then seems to saturate. The richness of the population dynamics observed with this simple system is thus comparable to that found in some natural ecosystems. The findings obtained with this new approach will likely guide the exploration of natural ecosystems in which parameters and observables are much less accessible.

My review focuses mainly on the experimental aspects of this work given my own expertise. The introduction exposes very convincingly the scientific context of this work, justifying the need for such an approach to address questions pertaining to ecology. The manuscript describes very clearly and rigorously the experimental set-up. The main strengths of this work are (i) the outstanding originality of the experimental approach and (ii) its simplicity. With this setup, central questions in ecology can be addressed in a quantitative manner, including the possibility of running trajectories in parallel to generalize the findings, as reported here. Technical aspects have been carefully implemented, from the design of random individuals bearing flanking regions for PCR amplification, binding selection and (low error) amplification protocols, and sequencing read-out whose depth is sufficient to capture the relevant dynamics. One missing aspect in the data analysis is the quantification of the effect of PCR amplification steps in shaping the ecosystem (to be modeled if significant). In addition, as it stands the current work does not fully harness the power of the approach. For instance, with this setup, one can tune the relative contributions of binding selection vs amplification for instance (to disentangle forces that shape the ecosystem). One can also run cycles with new DNA individuals, designed with arbitrarily chosen resource binding vs self-binding, that are predicted to dominate depending on chosen ecological parameters.

Reviewer #2 (Public Review):

Summary:
In this manuscript, the authors introduced ADSE, a SELEX-based protocol to explore the mechanism of emergency of species. They used DNA hybridization (to the bait pool, "resources") as the driving force for selection and quantitatively investigated the factors that may contribute to the survival during generation evolution (progress of SELEX cycle), revealing that besides individual-resource binding, the inter- and intra-individual interactions were also important features along with mutualism and parasitism.

Strengths:
The design of using pure biochemical affinity assay to study eco-evolution is interesting, providing an important viewpoint to partly explain the molecular mechanism of evolution.

Weaknesses:
Though the evidence of the study is somewhat convincing, some aspects still need to be improved, mostly technical issues.

Author Response

We thank the reviewers for their work, their careful reading of our manuscript, their appreciative evaluation and their comments and suggestions, which we will consider to ameliorate the paper.

For now, we anticipate two short considerations.

We agree that the PCR step in the ADSE evolutive process might introduce a bias in the population and that such effect should be better examined. We have in fact started performing new experiments, among which ADSE evolution cycles without resources. From the elements we currently have, we see the PCR bias effect as minor, not making a significant difference in the emergence and interaction of species we have reported.

ADSE protocol is markedly simpler than any other evolution protocol based on even the most basic cellular processes. However, many are the experimental parameters which can be changed in ADSE: initial DNAi population (level of randomness vs. combination of designed sequences), resource structure (resource sequence and length, bead-resource linker length and type), capture condition (length and concentration of DNAi, pH, temperature, bead density), amplification step (choice of polymerase and rate of mutation, length of primers, thermal protocol). The availability of these parameters is a strength of ASDE, making possible exploring a large variety of evolution condition and to introduce kinetic drifts (e.g. in the resources). At the same time, the variety of parameters prompted us to make choices as discussed in the article and to stick to them in all our experiments. The exploration of the many variants that can be considered, some of them very interesting, and some of which proposed by the reviewers, would require an important experimental work that we are planning to conduct for a few among these possibilities, to be part of future publications.