Reconstructing ancestral character states is an exciting but difficult problem. The fossil record carries a great deal of information, but it is incomplete and not always easy to connect to data from modern species. Alternatively, ancestral states can be estimated by modelling trait evolution across a phylogeny, and fitting to values observed in extant species. This approach, however, is heavily dependent on the underlying assumptions, and typically results in wide confidence intervals.

An alternative approach is to gain information on ancestral character states from DNA sequence data. This can be done directly when the trait of interest is known to be determined by a single, or a small number, of major effect genes. In some of these cases it can even be possible to investigate an ancestral trait of interest by inferring and resurrecting ancestral sequences in the laboratory. Examples where this has been successfully used to address evolutionary questions range from the nocturnality of early mammals [1], to the loss of functional uricases in primates, leading to high rates of gout, obesity and hypertension in present day humans [2]. Another possibility is to rely on correlations between species traits and the genome average substitution rate/process. For instance, it is well established that the ratio of nonsynonymous to synonymous substitution rate, dN/dS, is generally higher in large than in small species of mammals, presumably due to a reduced effective population size in the former. By estimating ancestral dN/dS, one can therefore gain information on ancestral body mass (e.g. [3-4]).

The interesting paper by Wu et al. [5] further develops this second possibility of incorporating information on rate variation derived from genomic data in the estimation of ancestral traits. The authors analyse a large set of 1185 genes in 89 species of mammals, without any prior information on gene function. The substitution rate is estimated for each gene and each branch of the mammalian tree, and taken as an indicator of the selective constraint applying to a specific gene in a specific lineage – more constraint, slower evolution. Rate variation is modelled as resulting from a gene effect, a branch effect, and a gene X branch interaction effect, which captures lineage-specific peculiarities in the distribution of functional constraint across genes. The interaction term in terminal branches is regressed to observed trait values, and the relationship is used to predict ancestral traits from interaction terms in internal branches. The power and accuracy of the estimates are convincingly assessed via cross validation. Using this method, the authors were also able to use an unbiased approach to determine which genes were the main contributors to the evolution of the life-history traits they reconstructed.

The ancestors to current placental mammals are predicted to have been insectivorous - meaning that the estimated distribution of selective constraint across genes in basal branches of the tree resembles that of extant insectivorous taxa - consistent with the mainstream palaeontological hypothesis. Another interesting result is the prediction that only nocturnal lineages have passed the Cretaceous/Tertiary boundary, so that the ancestors of current orders of placentals would all have been nocturnal. This suggests that the so-called "nocturnal bottleneck hypothesis" should probably be amended. Similar reconstructions are achieved for seasonality, sociality and monogamy – with variable levels of uncertainty.

The beauty of the approach is to analyse the variance, not only the mean, of substitution rate across genes, and their methods allow for the identification of the genes contributing to trait evolution without relying on functional annotations. This paper only analyses discrete traits, but the framework can probably be extended to continuous traits as well.

References

[1] Bickelmann C, Morrow JM, Du J, Schott RK, van Hazel I, Lim S, Müller J, Chang BSW, 2015. The molecular origin and evolution of dim-light vision in mammals. Evolution 69: 2995-3003. doi: https://doi.org/10.1111/evo.12794

[2] Kratzer, JT, Lanaspa MA, Murphy MN, Cicerchi C, Graves CL, Tipton PA, Ortlund EA, Johnson RJ, Gaucher EA, 2014. Evolutionary history and metabolic insights of ancient mammalian uricases. Proceedings of the National Academy of Science, USA 111:3763-3768. doi: https://doi.org/10.1073/pnas.1320393111

[3] Lartillot N, Delsuc F. 2012. Joint reconstruction of divergence times and life-history evolution in placental mammals using a phylogenetic covariance model. Evolution 66:1773-1787. doi: https://doi.org/10.1111/j.1558-5646.2011.01558.x

[4] Romiguier J, Ranwez V, Douzery EJ, Galtier N. 2013. Genomic evidence for large, long-lived ancestors to placental mammals. Molecular Biology and Evolution 30:5-13. doi: https://doi.org/10.1093/molbev/mss211

[5] Wu J, Yonezawa T, Kishino H. 2016. Rates of Molecular Evolution Suggest Natural History of Life History Traits and a Post-K-Pg Nocturnal Bottleneck of Placentals. Current Biology 27: 3025-3033. doi: https://doi.org/10.1016/j.cub.2017.08.043

Domestication is a complex process that imprints the demography and the genomes of domesticated populations, enforcing strong selective pressures on traits favourable to humans, e.g. for food production [1]. Domestication has been quite intensely studied in plants and animals, but less so in micro-organisms such as fungi, despite their assets (e.g. their small genomes and tractability in the lab). This elegant study by Bennetot and collaborators [2] on the cheese-making fungus Geotrichum candidum adds to the mounting body of studies in the genomics of fungi, proving they are excellent models in evolutionary biology for studying adaptation and drift in eukaryotes [3].

Bennetot et al. newly showed with whole genome sequences that all G. candidum strains isolated from cheese form a monophyletic clade subdivided into three genetically differentiated populations with several admixed strains, while the wild strains sampled from diverse geographic locations form a sister clade. This suggests the wild progenitor was not sampled in the present study and calls for future exciting work on the domestication history of the G. candidum fungus. The authors scanned the genomes for footprints of adaptation to the cheese environment and identified promising candidates, such as a gene involved in iron uptake (this element is limiting in cheese). Their functional genome analysis also provides evidence for higher contents of transposable elements in cheese-making strains, likely due to relaxed selection during the domestication process.

This paper is particularly impressive in that the authors complemented the population genomic approach with the phenotypic characterization of the strains and tested their ability to outcompete common fungal food spoilers. The authors convincingly showed that cheese-making strains display phenotypic differences relative to wild relatives for multiple traits such as slower growth, lower proteolysis activity and a greater amount of volatiles attractive to consumers, these phenotypes being beneficial for cheese making.

Finally, this work is particularly inspiring because it thoroughly discusses convergent evolution during domestication in different cheese-associated fungi. Indeed, studying populations experiencing similar environmental pressures is fundamental to understanding whether evolution is repeatable [4]. For instance, all three cheese populations of G. candidum exhibit a lower genetic diversity than wild populations. However, only one population displays a stronger domestication syndrome, resembling the Penicillium camemberti situation [5]. Furthermore, different cheese-making practices may have led to varying situations with clonal lineages in non-Roquefort P. roqueforti and P. camemberti [5, 6], while the cheese-making G. candidum populations still harbour some diversity. In a nutshell, Bennetot's study makes an important contribution to evolutionary biology and highlights the value of diversifying our model organisms toward under-represented clades.

REFERENCES

[1] Diamond J (2002) Evolution, consequences and future of plant and animal domestication. Nature 418: 700–707. https://doi.org/10.1038/nature01019

[2] Bennetot B, Vernadet J-P, Perkins V, Hautefeuille S, Rodríguez de la Vega RC, O’Donnell S, Snirc A, Grondin C, Lessard M-H, Peron A-C, Labrie S, Landaud S, Giraud T, Ropars J (2023) Domestication of different varieties in the cheese-making fungus Geotrichum candidum. bioRxiv, 2022.05.17.492043, ver. 4 peer-reviewed and recommended by Peer Community in Evolutionary Biology. https://doi.org/10.1101/2022.05.17.492043 

[3] Gladieux P, Ropars J, Badouin H, Branca A, Aguileta G, de Vienne DM, Rodríguez de la Vega RC, Branco S, Giraud T (2014) Fungal evolutionary genomics provides insight into the mechanisms of adaptive divergence in eukaryotes. Mol. Ecol. 23: 753–773. https://doi.org/10.1111/mec.12631

[4] Bolnick DI, Barrett RD, Oke KB, Rennison DJ, Stuart YE (2018) (Non)Parallel evolution. Ann. Rev. Ecol. Evol. Syst. 49: 303–330. https://doi.org/10.1146/annurev-ecolsys-110617-062240 

[5] Ropars J, Didiot E, Rodríguez de la Vega RC, Bennetot B, Coton M, Poirier E, Coton E, Snirc A, Le Prieur S, Giraud T (2020) Domestication of the Emblematic White Cheese-Making Fungus Penicillium camemberti and Its Diversification into Two Varieties. Current Biol. 30: 4441–4453.e4. https://doi.org/10.1016/j.cub.2020.08.082

[6] Dumas, E, Feurtey, A, Rodríguez de la Vega, RC, Le Prieur S, Snirc A, Coton M, Thierry A, Coton E, Le Piver M, Roueyre D, Ropars J, Branca A, Giraud T (2020) Independent domestication events in the blue-cheese fungus Penicillium roqueforti. Mol Ecol. 29: 2639–2660. https://doi.org/10.1111/mec.15359

Local adaptation, the higher fitness a population achieves in its local “home” environment relative to other environments is a crucial phase in the divergence of populations, and as such both generates and maintains diversity. Local adaptation is enhanced by selection and genetic variation in the relevant traits, and decreased by gene flow and genetic drift.

Demonstrating local adaptation is laborious, and is typically done with a reciprocal transplant design [1], documenting repeated geographic clines [e.g. 2, 3] also provides strong evidence of local adaptation. Even when well documented, it is often unknown which aspects of the environment impose selection. Indeed, differences in environment between different sites that are measured during studies of local adaptation explain little of the variance in the degree of local adaptation [4]. This poses a problem to population management. Given climate change and habitat destruction, understanding the environmental drivers of local adaptation can be crucially important to conducting successful assisted migration or targeted gene flow.

In this manuscript, Macdonald et al. [5] propose a means of identifying which aspects of the environment select for local adaptation without conducting a reciprocal transplant experiment. The idea is that the strength of relationships between traits and environmental variables that are due to plastic responses to the environment will not be influenced by gene flow, but the strength of trait-environment relationships that are due to local adaptation should decrease with gene flow. This then can be used to reduce the somewhat arbitrary list of environmental variables on which data are available down to a targeted list more likely to drive local adaptation in specific traits. To perform such an analysis requires three things: 1) measurements of traits of interest in a species across locations, 2) an estimate of gene flow between locations, which can be replaced with a biologically meaningful estimate of how well connected those locations are from the point of view of the study species, and 3) data on climate and other environmental variables from across a species’ range, many of which are available on line.

Macdonald et al. [5] demonstrate their approach using a skink (Lampropholis coggeri). They collected morphological and physiological data on individuals from multiple populations. They estimated connectivity among those locations using information on habitat suitability and dispersal potential [6], and gleaned climatic data from available databases and the literature. They find that two physiological traits, the critical minimum and maximum temperatures, show the strongest signs of local adaptation, specifically local adaptation to annual mean precipitation, precipitation of the driest quarter, and minimum annual temperature. These are then aspects of skink phenotype and skink habitats that could be explored further, or could be used to provide background information if migration efforts, for example for genetic rescue [7] were initiated. The approach laid out has the potential to spark a novel genre of research on local adaptation. It its simplest form, knowing that local adaptation is eroded by gene flow, it is intuitive to consider that if connectivity reduces the strength of the relationship between an environmental variable and a trait, that the trait might be involved in local adaptation. The approach is less intuitive than that, however – it relies not connectivity per-se, but the interaction between connectivity and different environmental variables and how that interaction alters trait-environment relationships. The authors lay out a number of useful caveats and potential areas that could use further development. It will be interesting to see how the community of evolutionary biologists responds.

References

[1] Blanquart F, Kaltz O, Nuismer SL and Gandon S. 2013. A practical guide to measuring local adaptation. Ecology Letters, 16: 1195-1205. doi: 10.1111/ele.12150

[2] Huey RB, Gilchrist GW, Carlson ML, Berrigan D and Serra L. 2000. Rapid evolution of a geographic cline in size in an introduced fly. Science, 287: 308-309. doi: 10.1126/science.287.5451.308

[3] Milesi P, Lenormand T, Lagneau C, Weill M and Labbé P. 2016. Relating fitness to long-term environmental variations in natura. Molecular Ecology, 25: 5483-5499. doi: 10.1111/mec.13855

[4] Hereford, J. 2009. A quantitative survey of local adaptation and fitness trade-offs. The American Naturalist 173: 579-588. doi: 10.1086/597611

[5] Macdonald SL, Llewelyn J and Phillips BL. 2017. Using connectivity to identify climatic drivers of local adaptation. bioRxiv, ver. 4 of October 4, 2017. doi: 10.1101/145169

[6] Macdonald SL, Llewelyn J, Moritz C and Phillips BL. 2017. Peripheral isolates as sources of adaptive diversity under climate change. Frontiers in Ecology and Evolution, 5:88. doi: 10.3389/fevo.2017.00088

[7] Whiteley AR, Fitzpatrick SW, Funk WC and Tallmon DA. 2015. Genetic rescue to the rescue. Trends in Ecology & Evolution, 30: 42-49. doi: 10.1016/j.tree.2014.10.009

In recent years many studies have documented shifts in phenology in response to climate change, be it in arrival times in migrating birds, budset in trees, adult emergence in butterflies, or flowering time in annual plants (Coen et al. 2018; Piao et al. 2019). While these changes are, in part, explained by phenotypic plasticity, more and more studies find that they involve also genetic changes, that is, they involve evolutionary change (e.g., Metz et al. 2020). Yet, evolutionary change may occur through genetic drift as well as selection. Therefore, in order to demonstrate adaptive evolutionary change in response to climate change, drift has to be excluded as an alternative explanation (Hansen et al. 2012). A new study by Gay et al. (2021) shows just how difficult this can be. 

The authors investigated a recent evolutionary shift in flowering time by in a population an annual plant that reproduces predominantly by self-fertilization. The population has recently been subjected to increased temperatures and reduced rainfalls both of which are believed to select for earlier flowering times. They used a “resurrection” approach (Orsini et al. 2013; Weider et al. 2018): Genotypes from the past (resurrected from seeds) were compared alongside more recent genotypes (from more recently collected seeds) under identical conditions in the greenhouse. Using an experimental design that replicated genotypes, eliminated maternal effects, and controlled for microenvironmental variation, they found said genetic change in flowering times: Genotypes obtained from recently collected seeds flowered significantly (about 2 days) earlier than those obtained 22 generations before. However, neutral markers (microsatellites) also showed strong changes in allele frequencies across the 22 generations, suggesting that effective population size, Ne, was low (i.e., genetic drift was strong), which is typical for highly self-fertilizing populations. In addition, several multilocus genotypes were present at high frequencies and persisted over the 22 generations, almost as in clonal populations (e.g., Schaffner et al. 2019). The challenge was thus to evaluate whether the observed evolutionary change was the result of an adaptive response to selection or may be explained by drift alone. 

Here, Gay et al. (2021) took a particularly careful and thorough approach. First, they carried out a selection gradient analysis, finding that earlier-flowering plants produced more seeds than later-flowering plants. This suggests that, under greenhouse conditions, there was indeed selection for earlier flowering times. Second, investigating other populations from the same region (all populations are located on the Mediterranean island of Corsica, France), they found that a concurrent shift to earlier flowering times occurred also in these populations. Under the hypothesis that the populations can be regarded as independent replicates of the evolutionary process, the observation of concurrent shifts rules out genetic drift (under drift, the direction of change is expected to be random). 

The study may well have stopped here, concluding that there is good evidence for an adaptive response to selection for earlier flowering times in these self-fertilizing plants, at least under the hypothesis that selection gradients estimated in the greenhouse are relevant to field conditions. However, the authors went one step further. They used the change in the frequencies of the multilocus genotypes across the 22 generations as an estimate of realized fitness in the field and compared them to the phenotypic assays from the greenhouse. The results showed a tendency for high-fitness genotypes (positive frequency changes) to flower earlier and to produce more seeds than low-fitness genotypes. However, a simulation model showed that the observed correlations could be explained by drift alone, as long as Ne is lower than ca. 150 individuals. The findings were thus consistent with an adaptive evolutionary change in response to selection, but drift could only be excluded as the sole explanation if the effective population size was large enough. 

The study did provide two estimates of Ne (19 and 136 individuals, based on individual microsatellite loci or multilocus genotypes, respectively), but both are problematic. First, frequency changes over time may be influenced by the presence of a seed bank or by immigration from a genetically dissimilar population, which may lead to an underestimation of Ne (Wang and Whitlock 2003). Indeed, the low effective size inferred from the allele frequency changes at microsatellite loci appears to be inconsistent with levels of genetic diversity found in the population. Moreover, high self-fertilization reduces effective recombination and therefore leads to non-independence among loci. This lowers the precision of the Ne estimates (due to a higher sampling variance) and may also violate the assumption of neutrality due to the possibility of selection (e.g., due to inbreeding depression) at linked loci, which may be anywhere in the genome in case of high degrees of self-fertilization. 

There is thus no definite answer to the question of whether or not the observed changes in flowering time in this population were driven by selection. The study sets high standards for other, similar ones, in terms of thoroughness of the analyses and care in interpreting the findings. It also serves as a very instructive reminder to carefully check the assumptions when estimating neutral expectations, especially when working on species with complicated demographies or non-standard life cycles. Indeed the issues encountered here, in particular the difficulty of establishing neutral expectations in species with low effective recombination, may apply to many other species, including partially or fully asexual ones (Hartfield 2016). Furthermore, they may not be limited to estimating Ne but may also apply, for instance, to the establishment of neutral baselines for outlier analyses in genome scans (see e.g, Orsini et al. 2012). 

References

Cohen JM, Lajeunesse MJ, Rohr JR (2018) A global synthesis of animal phenological responses to climate change. Nature Climate Change, 8, 224–228. https://doi.org/10.1038/s41558-018-0067-3

Gay L, Dhinaut J, Jullien M, Vitalis R, Navascués M, Ranwez V, Ronfort J (2021) Evolution of flowering time in a selfing annual plant: Roles of adaptation and genetic drift. bioRxiv, 2020.08.21.261230, ver. 4 recommended and peer-reviewed by Peer Community in Evolutionary Biology. https://doi.org/10.1101/2020.08.21.261230

Hansen MM, Olivieri I, Waller DM, Nielsen EE (2012) Monitoring adaptive genetic responses to environmental change. Molecular Ecology, 21, 1311–1329. https://doi.org/10.1111/j.1365-294X.2011.05463.x

Hartfield M (2016) Evolutionary genetic consequences of facultative sex and outcrossing. Journal of Evolutionary Biology, 29, 5–22. https://doi.org/10.1111/jeb.12770

Metz J, Lampei C, Bäumler L, Bocherens H, Dittberner H, Henneberg L, Meaux J de, Tielbörger K (2020) Rapid adaptive evolution to drought in a subset of plant traits in a large-scale climate change experiment. Ecology Letters, 23, 1643–1653. https://doi.org/10.1111/ele.13596

Orsini L, Schwenk K, De Meester L, Colbourne JK, Pfrender ME, Weider LJ (2013) The evolutionary time machine: using dormant propagules to forecast how populations can adapt to changing environments. Trends in Ecology & Evolution, 28, 274–282. https://doi.org/10.1016/j.tree.2013.01.009

Orsini L, Spanier KI, Meester LD (2012) Genomic signature of natural and anthropogenic stress in wild populations of the waterflea Daphnia magna: validation in space, time and experimental evolution. Molecular Ecology, 21, 2160–2175. https://doi.org/10.1111/j.1365-294X.2011.05429.x

Piao S, Liu Q, Chen A, Janssens IA, Fu Y, Dai J, Liu L, Lian X, Shen M, Zhu X (2019) Plant phenology and global climate change: Current progresses and challenges. Global Change Biology, 25, 1922–1940. https://doi.org/10.1111/gcb.14619

Schaffner LR, Govaert L, De Meester L, Ellner SP, Fairchild E, Miner BE, Rudstam LG, Spaak P, Hairston NG (2019) Consumer-resource dynamics is an eco-evolutionary process in a natural plankton community. Nature Ecology & Evolution, 3, 1351–1358. https://doi.org/10.1038/s41559-019-0960-9

Wang J, Whitlock MC (2003) Estimating Effective Population Size and Migration Rates From Genetic Samples Over Space and Time. Genetics, 163, 429–446. PMID: 12586728

Weider LJ, Jeyasingh PD, Frisch D (2018) Evolutionary aspects of resurrection ecology: Progress, scope, and applications—An overview. Evolutionary Applications, 11, 3–10. https://doi.org/10.1111/eva.12563

Symbiotic associations with bacterial partners have facilitated important evolutionary transitions in the life histories of eukaryotes. For instance, many insects have established long-term interactions with intracellular bacteria that provide them with essential nutrients lacking in their diet. However, despite the high level of interdependency among organisms involved in endosymbiotic systems, examples of symbiont replacements along the evolutionary history of insect hosts are numerous. In their paper, Husnik and McCutcheon [1] test the stability of symbiotic systems in a particularly imbricated Russian-doll type interaction, where one bacterium lives insides another bacterium, which itself lives inside insect cells. For their study, they chose representative species of mealybugs (Pseudococcidae), a species rich group of sap-feeding insects that hosts diverse and complex symbiotic systems. In species of the subfamily Pseudococcinae, data published so far suggest that the primary symbiont, a ß-proteobacterium named Tremblaya princeps, is supplemented by a second bacterial symbiont (a ϒ-proteobaterium) that lives within its cytoplasm; both participate to the metabolic pathways that provide essential amino acids and vitamins to their hosts. Here, Husnik and McCutcheon generate host and endosymbiont genome data for five phylogenetically divergent species of Pseudococcinae in order to better understand: 1) the evolutionary history of the symbiotic associations; 2) the metabolic roles of each partner, 3) the timing and origin of Horizontal Gene Transfers (HGT) between the hosts and their symbionts.
Their results show that all species harbour the primary and at least one secondary symbiont, whose intra-bacterial localization was verified using fluorescence in situ hybridization. In one species (Pseudococcus longispinus), Tremblaya even hosts two intracellular bacteria each with a large genome (even though it is not entirely clear whether these two symbionts are indeed found within Tremblaya). The obligate presence of an intra-bacterial symbiont is best explained by the loss in Tremblaya princeps of critical genes for translation that require “intracellular complementation”.
The most striking result concerns the identity of the intra-bacterial symbiont: genome sizes and structures of the “secondary” (intra-Tremblaya) symbionts vary drastically according to the host species. Phylogenetic analyses based on 80 conserved proteins, place all these ϒ-proteoacteria (except one of the P. longispinus symbionts) in a Sodalis allied clade. However, their relationships do not mirror the one of their hosts, and some of them show signs of very recent acquisition. Altogether these results provide strong evidence for several independent acquisitions of these highly intra-bacterial integrated symbionts. Scenarios for the history of the symbiosis are clearly laid out and discussed by the authors, and the scenario involving several independent replacements of a the intra-Tremblaya symbiont by diverse Sodalis-like bacteria appears the most likely given the data presented here. Selected biosynthetic functions are then mapped onto the host and symbiont genomes, showing the high level of interdependency of the partners for the synthesis of essential amino acids and vitamin. These maps also identify genes in the host genome that might have been acquired through HGT from bacteria and show that many of them are shared by all mealybug species sequenced so far. HGT events have thus predated the acquisitions of the current “intra-Tremblaya“ symbionts and have probably been acquired from previous symbiont infections.
Overall, this is a thorough study, using a diverse set of data and meticulous analyses that present convincing evidence that replacements of symbionts occurred repeatedly even in an imbricated symbiotic system. The phylogenetic analyses inferring the timing of HGTs also depict a highly dynamic history of gene losses and retentions in both host and symbiont genomes. Altogether these results demonstrate how the chimeric nature of individuals allows shuffling at different levels of organisation: organisms like symbionts can be replaced, genes can be exchanged among the partners of the chimera. The surprise is that constraints arising from interdependencies do not impede these exchanges. In the discussion, the authors present a very interesting parallel with the evolution of organelles: if the story presented here mirrors the one of mitochondria, it supports a gradualist view where mitochondria arrived late in the evolution of eukaryotic cells that already contained many bacterial genes resulting from HGT from previous symbionts.

Reference

[1] Husnik F., McCutcheon JP. 2016. Repeated replacements of an intrabacterial symbiont in the tripartite nested mealybug symbiosis. PNAS 113: E5416-E5424. doi: 10.1073/pnas.1603910113