At the interface between Microbial Ecology and Evolutionary Biology
I began with the idea I would write this great piece that illuminated the interface between microbial ecology and evolution, as one where Darwin’s concepts of evolution via natural selection could be experimentally tested in practical time periods. However, I got stuck very early on – just what is the ‘unit’ that is evolving? Plant and animal ecologists have the biological species concept upon which they can frame their thinking. There is no such concept for species of Bacteria and Archaea. The 16S rDNA gene has provided wonderful insights into the phylogeny of Bacteria and Archaea but even full-length sequencing falls short of 100% accuracy in assigning species. As an ecologist, I am most interested in distributions of ecotypes. Plant and animal ecologists speak of ecotypes as different populations of a species that live in distant and ecologically distinct environments to which they have made ‘hard-wired’ (genetic) adaptations. An important question is the extent to which this holds for Bacteria and Archaea.
Two scientists who work in this area are Frederick Cohan and Kostas Konstantinidis. Fred’s research has been directed to identifying ecotypes in natural systems and elucidating the mechanisms whereby they form and coexist. He has developed theories of how bacteria may speciate. About 20 years ago, Kostas developed the use of Average Nucleotide Identity (ANI) between orthologous genes as a means to determine the relatedness between bacteria. As genomics and bioinformatics techniques have advanced, this approach has led to some interesting findings.
Bacterial species as cohesive units
Cohan argues that species are “cohesive” because there are forces that limit genetic diversification. He posits selection and homologous recombination as those forces. Selection operates via periodic sweeps – an adaptive mutation within an individual cell confers a faster growth rate/reduced loss rate upon it and its progeny such that after enough generations, the pre-existing genetic diversity within the local population is swept away. The idea that recombination is cohesive rather than divisive accrues from the observation that the probability of recombination into the genome is proportional to the homology between the recombining segments – hence primarily incorporating sequences from within the species rather than without.
Konstantinidis’ ANI analyses provide support for the existence of cohesion – he and his colleagues find a gap, with ANI of 90-95% being rare for a pair of genomes. That is, genomes from the same species have ANI>95% whereas comparisons between 2 different species yield ANI < 90%. Interestingly, when a set of genomes from the same species are interrogated, another discontinuity is found between 99.2 and 99.8%. Does this genomic microdiversity correlate with distinct ecotypes in nature?
Ecotypes
Conventional ecological thinking revolves around competition, coexistence, predation, parasitism and metabolic interactions occurring between species. But there is abundant evidence both from natural systems and from laboratory studies that intraspecific diversity exists and is ecologically meaningful. That is to say, microbial communities likely consist not only of diverse persistent species (ANI > 95%) but also another layer of microdiversity. Even if habitats are not ‘distant’ in the sense used for plant and animal ecotypes, might there be ‘distant’ habitats on a microbial scale (e.g., the pore network in an unsaturated soil) in which communities only coalesce at slow rates or ephemeral habitats (caused by the dynamics of physical mixing in aquatic habitats and/or colonizable particles)?
Cohan (2006) has proposed a “stable ecotype model” in which a population of cells shares the same ecological niche and genome sequence divergence is purged by periodic selection events. From experiments with a laboratory microcosm, he found that mutations which led to new ecotypes were just as common as those that improved the fitness of an existing ecotype. For natural communities, Cohan has favored a sequence-first approach that would presume sequence clusters correspond to different ecotypes.
The alternative is a function-first approach: identification of ecologically relevant functional differences among isolated microbes, followed by genome analysis. Examples include the work of the Chisholm lab at MIT on high-light vs low-light adapted ecotypes of Prochlorococcus and that of the Ward lab at Montana State on temperature-adapted Syncechococcus ecotypes along a thermal gradient.
Eco-Evo
Recall that four fundamental processes affect the evolution of organsisms: mutation, gene flow, natural selection and genetic drift. The dilemma here is how to identify the biological population (the fundamental unit of evolution) in prokaryotes – the analog of interbreeding plant or animal individuals. This would be a group that has reasonable gene flow among them (but keep in mind that mechanisms may differ among species, that rates of recombination vary substantially across species, and that the extant environmental conditions (cell density, physical mixing rates, spatial isolation) may impact the rates of gene flow as well as the strength of selection). This is where I got stuck in my thinking about the interface between ecology and evolution.
But I think there is a path forward and work over the past decade illustrates a path. Shotgun metagenomics has been touted as an approach for the analysis of natural communities (Simpson et al., 2025), but I think this misses the mark for reasons they cite as technical limitations – among others Metagenomically Assembled Genomes (MAGs) are composites of individual differences in the biological population such that the information on microdiversity is lost. A better alternative would be to concentrate on single-cell genomics as was done for Prochlorococcus. Kashtan et al. (2014) analyzed >1000 co-occurring cells by flow-sorting and subsequent DNA amplification. ITS ribotyping suggested there could be >>10 co-occurring subpopulations by this criterion. Larger-scale genome sequencing delineated 5 major clades. Each of these has a distinct genomic backbone, with highly conserved core genes but also a smaller set of ‘flexible’ genes. The latter varied substantially between clades. There was also fine-scale variation between core genes and flexible gene content which illustrates astounding microdiversity within this organism in the wild.
Single-cell genomics remains a small endeavor – I find only about 25 papers per year using this approach in the 2020s. It is surprising to me that no one has followed up on the Kashtan et al. (2014) findings. Technical challenges remain but are being addressed (Hosokawa and Nishikawa, 2024); it strikes me that this approach can uniquely provide the data necessary to assess genomic microdiversity in natural populations.
Analyzing systems in which the organism of interest is present in high relative abundance will help. Hence, analysis of microbial primary producers (as in the case of marine Prochlorococcus (Coleman et al., 2007) and thermophilic Synechococcus) are excellent ideas. I would add freshwater planktonic cyanobacteria: taxa such as Oscillatoria, Aphanizomenon and Anabaena are filamentous so that each “unit” will have many cells worth of DNA to isolate and amplify. These organisms can also be found in a set of lakes that differ in their distance from one another, permitting an analysis of biogeography upon their evolution. As an example, Gollnisch et al. (2024) carried out a biogeographical study on an eukaryotic phytoplankter. Among non-photosynthetic prokaryotes, highly abundant taxa such as Pelagibacter (SAR11) would be of interest.
I am optimistic that there can be further breakthroughs in this area and that it can contribute to the interface of ecology and evolution more broadly than just in microbes. There is adequate theory – Cohan’s stable ecotype model (Cohan, 2006) and its alternatives. Further developments in single cell technologies would make tests of theory in natural populations tractable. These could be complemented by laboratory experiments in microbial evolution to investigate mechanisms. Macrobe ecologists think of species as fixed assemblages of genes that determine the species’ phenotype. But the process is really a dynamic one of complex interactions between evolutionary processes and environmental factors that produce an evolutionary trajectory. A working framework is that microbial communities consist of units that can be distinguished by their genome sequence clusters and that these clusters exist both at the species level (ANI > 95%) and also as intraspecies ecotypes. Prokaryotic ecotypes may differ from those in plants or animals in that they are not necessarily geographically isolated but co-occur as a consequence of niches structured either in space or time. Experimentally pursuing the interface between ecology and evolution in prokaryotic populations has advantages due to large population sizes, multiple mechanisms of genetic exchange (even across taxa), and relatively small size of recombined sequences. These can accelerate the formation of both new clades and the pace of improved adaptation to the environment.
Analyzing systems both in the wild and under controlled laboratory conditions can identify principles regarding (i) habitats and/or taxa for which recombination rates greatly exceed mutation rates, (ii) when horizontal gene transfer is an important driving force or (iii) how environmental factors such as cell density (biofilms/GI tract vs the dilute ocean) affect the relative importance of recombination vs mutation in the organisms’ evolution and ecology.
This topic raises several very important philosophical issues:
What is (and how to determine) the fundamental evolutionary unit (the ‘biological population’) for Bacteria and Archaea?
Are the mechanisms of gene flow in prokaryotes analogous enough to those in eukaryotes to make the former relevant to the interface between ecology and evolution of the latter?
What technical approaches are best suited to identify principles at the ecology/evolution interface?
Given that the topic interfaces two silos of biology (ecology & evolution), what funding programs should be supporting research in this area?
References
Allewalt JP et al. (2006). Effect of Temperature and Light on Growth of and Photosynthesis by Synechococcus Isolates Typical of Those Predominating in the Octopus Spring Microbial Mat Community of Yellowstone National Park. Appl Environ Microbiol 72: 544-550. https://doi.org/10.1128/AEM.72.1.544-550.2006
Cohan, F.M. (2006) Towards a conceptual and operational union of bacterial systematics, ecology, and evolution. Philos. Trans. R. Soc.Lond. B Biol. Sci. 361, 1985–1996
Coleman, ML. et al. (2007) Code and context: Prochlorococcus as a model for cross-scale biology. Trends Microbiol 15: 398 - 407
Gollnisch R et al. (2024) Single-cell genomics of a bloom-forming phytoplankton species reveals population genetic structure across continents, ISME Journal 18: wrae045. https://doi.org/10.1093/ismejo/wrae045
Hosokawa, M., Nishikawa, Y. (2024) Tools for microbial single-cell genomics for obtaining uncultured microbial genomes. Biophys Rev 16, 69–77 (2024). https://doi.org/10.1007/s12551-023-01124-y
Kashtan N et al. (2014) Single-Cell Genomics Reveals Hundreds of Coexisting Subpopulations in Wild Prochlorococcus.Science344,416-420. DOI:10.1126/science.1248575
Simpson AMR et al. (2025) Investigating bacterial evolution in nature with metagenomics. Curr Opin Microbiol 87: 102654. https://doi.org/10.1016/j.mib.2025.102654.
Today’s Moment of Zen
I am leaving tomorrow for a 3 week tour of Tibet, Nepal and Bhutan. I am hoping to personally capture some Zen on this journey.
