Nobody can ignore that a full understanding of evolution requires an integrated approach from both conceptual and methodological viewpoints. Although some life-history traits, e.g. flowering time, have long been receiving more attention than others, in many cases because the former are more workable than the latter, we must acknowledge that our comprehension about how evolution works is strongly biased and limited. In the Arabidopsis community, such an integration is making good progress as an increasing number of research groups worldwide are changing the way in which evolution is put to the test.

This manuscript [1] is a good example of that as the authors raise an important issue in evolutionary biology by combining gene expression and flowering time data from different sources. In particular, the authors explore how variation in flowering time, which determines lifespan, and host immunity defenses co-vary, which is interpreted in terms of co-evolution between the two traits. Interestingly, the authors go beyond that pattern by separating lifespan-dependent from lifespan–independent defense genes, and by showing that defense genes with variants known to impact fitness in the field are among the genes whose expression co-varies most strongly with flowering time. Finally, these results are supported by a simple mathematical model indicating that such a relationship can also be expected theoretically.

Overall, the readers will find many conceptual and methodological elements of interest in this manuscript. The idea that evolution is better understood under the scope of life history variation is really exciting and challenging, and in my opinion on the right track for disentangling the inherent complexities of evolutionary research. However, only when we face complexity, we also face its costs and burdens. In this particular case, the well-known co-variation between seed dormancy and flowering time is a missing piece, as well as the identification of (variation in) putative selective pressures accounting for the co-evolution between defense mechanisms and life history (seed dormancy vs. flowering time) along environmental gradients. More intellectual, technical and methodological challenges that with no doubt are totally worth it.

Reference

[1] Glander S, He F, Schmitz G, Witten A, Telschow A, de Meaux J. 2017. Assortment of flowering time and defense alleles in natural Arabidopsis thaliana populations suggests co-evolution between defense and vegetative lifespan strategies. bioRxiv ver.1 of June 19, 2017. doi: 10.1101/131136

It has long been known that most multicellular eukaryotes rely on microbial partners for a variety of functions including nutrition, immune reactions and defence against enemies. Lichens are probably the most popular example of a symbiosis involving a photosynthetic microorganism (an algae, a cyanobacteria or both) living embedded within the filaments of a fungus (usually an ascomycete). The latter is the backbone structure of the lichen, whereas the former provides photosynthetic products. Lichens are unique among symbioses because the structures the fungus and the photosynthetic microorganism form together do not resemble any of the two species living in isolation. Classic textbook examples like lichens are not often challenged and this is what Toby Spribille and his co-authors did with their paper published in July 2016 in Science [1]. This story started with the study of two species of macrolichens from the class of Lecanoromycetes that are commonly found in the mountains of Montana (US): Bryoria fremontii and B. tortuosa. For more than 90 years, these species have been known to differ in their chemical composition and colour, but studies performed so far failed in finding differences at the molecular level in both the mycobiont and the photobiont. These two species were therefore considered as nomenclatural synonyms, and the origin of their differences remained elusive. To solve this mystery, the authors of this work performed a transcriptome-wide analysis that, relative to previous studies, expanded the taxonomic range to all Fungi. This analysis revealed higher abundances of a previously unknown basidiomycete yeast from the genus Cyphobasidium in one of the lichen species, a pattern that was further confirmed by combining microscopy imaging and the fluorescent in situ hybridisation technique (FISH).

Finding out that a previously unknown micro-organism changes the colour and the chemical composition of an organism is surprising but not new. For instance, bacterial symbionts are able to trigger colour changes in some insect species [2], and endophyte fungi are responsible for the production of defensive compounds in the leaves of several grasses [3]. The study by Spribille and his co-authors is fascinating because it demonstrates that Cyphobasidium yeasts have played a key role in the evolution and diversification of Lecanoromycetes, one of the most diverse classes of macrolichens. Indeed these basidiomycete yeasts were not only found in Bryoria but in 52 other lichen genera from all six continents, and these included 42 out of 56 genera in the family Parmeliaceae. Most of these sequences formed a highly supported monophyletic group, and a molecular clock revealed that the origin of many macrolichen groups occurred around the same time Cyphobasidium yeasts split from Cystobasidium, their nearest relatives. This newly discovered passenger is therefore an ancient inhabitant of lichens and has driven the evolution of this emblematic group of organisms.

This study raises an important question on the stability of complex symbiotic partnerships. In intimate obligatory symbioses the evolutionary interests of both partners are often identical and what is good for one is also good for the other. This is the case of several insects that feed on poor diets like phloem and xylem sap, and which carry vertically-transmitted symbionts that provide essential nutrients. Molecular phylogenetic studies have repeatedly shown that in several insect groups transition to phloem or xylem feeding occurred at the same time these nutritional symbionts were acquired [4]. In lichens, an outstanding question is to know what was the key feature Cyphobasidium yeasts brought to the symbiosis. As suggested by the authors, these yeasts are likely to be involved in the production of secondary defensive metabolites and architectural structures, but, are these services enough to explain the diversity found in macrolichens? This paper is an appealing example of a multipartite symbiosis where the different partners share an ancient evolutionary history.

References

[1] Spribille T, Tuovinen V, Resl P, et al. 2016. Basidiomycete yeasts in the cortex of ascomycete macrolichens. Science 353:488–92. doi: 10.1126/science.aaf8287

[2] Tsuchida T, Koga R, Horikawa M, et al. 2010. Symbiotic Bacterium Modifies Aphid Body Color. Science 330:1102–1104. doi: 10.1126/science.1195463

[3] Clay K. 1988. Fungal Endophytes of Grasses: A Defensive Mutualism between Plants and Fungi. Ecology 69:10–16. doi: 10.2307/1943155

[4] Moran NA. 2007. Symbiosis as an adaptive process and source of phenotypic complexity. Proceeding of the National Academy of Science USA 104:8627–8633. doi: 10.1073/pnas.0611659104

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

Many species breed in environments that are unpredictable, for instance in terms of the availability of resources needed to raise the offspring. Organisms might respond to such spatial and temporal unpredictability by adopting plastic responses to adjust their reproductive investment according to perceived cues of environmental quality. Some species such as the amphibians might also face the problem of ephemeral habitats, when the ponds where they breed have a chance of drying up before metamorphosis has occurred. In this case, maximizing long-term fitness might involve a strategy of spreading the risk, even though the reproductive success of a single reproductive bout might be lower. Understanding how animals (and plants) get adapted to stochastic environments is particularly crucial in the current context of rapid environmental changes.

In this article, Jourdan-Pineau et al. report the results of field surveys of the Parsley Frog (Pelodytes punctatus) in Southern France. This frog has peculiar breeding phenology with females breeding in autumn and spring. The authors provide quite an extensive amount of information on the reproductive success of eggs laid in each season and the possible ecological factors accounting for differences between seasons. Although the presence of interspecific competitors and predators does not seem to account for pond-specific reproductive success, the survival of tadpoles hatching from eggs laid in spring is severely impaired when tadpoles from the autumn cohort have managed to survive. This intraspecific competition takes the form of a “priority” effect where tadpoles from the autumn cohort outcompete the smaller larvae from the spring cohort. Given this strong priority effect, one might tentatively predict that females laying in spring should avoid ponds with tadpoles from the autumn cohort. Surprisingly, however, the authors did not find any evidence for such avoidance, which might indicate strong constraints on the availability of ponds where females might possibly lay.

Assuming that each female can indeed lay both in autumn and spring, how is this bimodal phenology maintained? Would not be worthier to allocate all the eggs to the autumn (or the spring) laying season? Eggs laid in autumn and spring have to face different environmental hazards, reducing their hatching success and the probability to produce metamorphs (for instance, tadpoles hatching from eggs laid in autumn have to overwinter which might be a particularly risky phase).             

Jourdan-Pineau and coworkers addressed this question by adapting a bet-hedging model that was initially developed to investigate the strategy of allocation into seed dormancy of annual plants (Cohen 1966) to the case of the bimodal phenology of the Parsley Frog. By feeding the model with the parameter values obtained from the field surveys, they found that the two breeding strategies (laying in autumn and in spring) can coexist as long as the probability of breeding success in the autumn cohort is between 20% and 80% (the range of values allowing the coexistence of a bimodal phenology shrinking a little bit when considering that frogs can reproduce 5 times during their lifespan instead of three times).

This paper provides a very nice illustration of the importance of combining approaches (here field monitoring to gather data that can be used to feed models) to understand the evolution of peculiar breeding strategies. Although future work should attempt to gather individual-based data (in addition to population data), this work shows that spreading the risk can be an adaptive strategy in environments characterized by strong stochastic variation.

References

Cohen D (1966) Optimizing reproduction in a randomly varying environment. Journal of Theoretical Biology, 12, 119–129. https://doi.org/10.1016/0022-5193(66)90188-3

Jourdan-Pineau H., Crochet P.-A., David P. (2022) Bimodal breeding phenology in the Parsley Frog Pelodytes punctatus as a bet-hedging strategy in an unpredictable environment despite strong priority effects. bioRxiv, 2022.02.24.481784, ver. 5 peer-reviewed and recommended by Peer Community in Evolutionary Biology. https://doi.org/10.1101/2022.02.24.481784

Estimating the genetic and environmental components of variation in reproductive success is crucial to understanding the adaptive potential of populations to environmental change. To date, the heritability of lifetime reproductive success (fitness) has been estimated in a handful of wild animal population, mostly in mammals and birds, but has never been estimated for a marine species. The primary reason that such estimates are lacking in marine species is that most marine organisms have a dispersive larval phase, making it extraordinarily difficult to track the fate of offspring from one generation to the next.
In this study, Salles et al. [1] use an unprecedented 10 year data set for a wild population of orange clownfish (Amphiprion percula) to estimate the environmental, maternal and additive genetic components of life time reproductive success for the self-recruiting portion of the local population. Previous studies show that over 50% of juvenile clownfish recruiting to the population of clownfish at Kimbe Island (Kimbe Bay, PNG) are natal to the population. In other words, >50% of the juveniles recruiting to the population at Kimbe Island are offspring of parents from Kimbe Island. The identity and location of every adult clownfish in the Kimbe Island population was tracked over 10 years. At the same time newly recruiting juveniles were collected at regular intervals (biennially) and their parentage assigned with high confidence by 22 polymorphic microsatellite loci. Salles et al. then used a pedigree comprising 1735 individuals from up to 5 generations of clownfish at Kimbe Island to assess the contribution of every breeding pair of clownfish to self-recruitment within the local population. Because clownfish are site attached and live in close association with a host sea anemone, it was also possible to examine the contribution of reef location and host anemones species (either Heteractis magnifica or Stichodactyla gigantea) to reproductive success within the local population.
The study found that breeders from the eastern side of Kimbe Island, and mostly inhabiting S. gigantea sea anemones, produced more juveniles that recruited to the local population than breeders from other location around the island, or inhabiting H. magnifica. In fact, host anemone species and geographic location explained about 97% of the variance in reproductive success within the local population (i.e. excluding successful recruitment to other populations). By contrast, maternal and additive genetic effects explained only 1.9% and 1.3% of the variance, respectively. In other words, reef location and the species of host anemone inhabited had an overwhelming influence on the long-term contribution of breeding pairs of clownfish to replenishment of the local population. This overwhelming effect of the local habitat on reproductive success means that the population is potentially susceptible to rapid environmental changes - for example if S. giganta sea anemones are disproportionately susceptible to global warming, or reef habitats on the eastern side of the island are more susceptible to disturbance. By contrast, the small component of additive genetic variance in local reproductive success translated into low heritability and evolvability of lifetime reproductive success within the local population, as predicted by theory [2] and observed in some terrestrial species. Consequently, fitness would evolve slowly to environmental change.
Establishing the components of variation in fitness in a wild population of marine fishes is an astonishing achievement, made possible by the unprecedented long-term individual-level monitoring of the entire population of clownfish at Kimbe Island. A next step in this research would be to include other clownfish populations that are demographically and genetically connected to the Kimbe Island population through larval dispersal. It would be intriguing to establish the environmental, maternal and additive genetic components of reproductive success in the dispersing part of the Kimbe Island population, to see if this potentially differs among breeders who contribute more or less to replenishment within the local population.

References

[1] Salles, O. C., Almany, G. R., Berumen, M.L., Jones, G. P., Saenz-Agudelo, P., Srinivasan, M., Thorrold, S. R., Pujol, B., Planes, S. (2019). Strong habitat and weak genetic effects shape the lifetime reproductive success in a wild clownfish population. Zenodo, 3476529, ver. 3 peer-reviewed and recommended by Peer Community In Evolutionary Biology. doi: 10.5281/zenodo.3476529
[2] Fisher, R.A. (1930). The genetical theory of natural selection. Clarendon Press, Oxford, U.K.