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

The unique challenges associated with living on an island often result in organisms displaying a specific suite of traits commonly referred to as “island syndrome” (Adler and Levins, 1994; Burns, 2019; Baeckens and Van Damme, 2020). Large phenotypic shifts such as changes in size (e.g. shifts to gigantism or dwarfism, Lomolino, 2005) or coloration (Doutrelant et al., 2016) abound in the literature. However, less obvious phenotypes may also play a key role in adaptation to islands.

One such trait, reduced immune function, has important implications for the future of island populations in the face of anthropogenic-induced changes. Due to lower parasite pressure caused by a less diverse and less virulent parasite population, island hosts may show a decrease in immune defenses (Beadell et al., 2006; Pérez‐Rodríguez et al., 2013). However, this hypothesis has been challenged, as many studies have found ambiguous or conflicting results (Matson, 2006; Illera et al., 2015).

While most previous work has examined various immunological parameters (e.g., antibody concentrations), here, Barthe et al. (2022) take the novel approach of examining molecular signatures of immune genes. Using comparative genomic data from 34 different species of birds the authors examine the ratio of synonymous substitutions (i.e., not changing an amino acid) to non-synonymous substitutions (i.e., changing an amino acid) in innate and acquired immune genes (Pn/Ps ratio). Because population sizes on islands are lower which will affect molecular evolution, they compare these results to data from 97 control genes.  Assuming relaxed selection on islands predicts that the difference between the Pn/Ps ratio of immune genes and of control genes (ΔPn/Ps) is greater in island species compared to mainland ones.

As with previous work the authors found that the results differ depending on the category of immune genes. Both forms of innate defense: beta-defensins and Toll-like receptors did not show higher ΔPn/Ps for island populations. As these genes still have a higher Pn/Ps than control genes, the authors argue these results are in line with these genes being under purifying selection but lacking an “island effect”. Instead, the authors argue that demographic effects (i.e., reductions in Ne) may lead to the decreased immunity documented in other studies. In contrast, there was a reduction in Pn/Ps in MHC II genes, known to be under balancing selection. This reduction was stronger in island species and thus the authors argue that this is the only class of genes where a role for relaxed selection can be invoked. 

Together these results demonstrate that the changes in immunity experienced by island species are complex and that different categories of immune genes can experience different selective pressures. By including control genes in their study, they particularly highlight the importance of accounting for shifts in Ne when examining patterns of island species evolution. Hopefully, this kind of framework will be applied to other taxa to determine if these results are widespread or more specific to birds. 

References

Adler GH, Levins R (1994) The Island Syndrome in Rodent Populations. The Quarterly Review of Biology, 69, 473–490. https://doi.org/10.1086/418744

Baeckens S, Van Damme R (2020) The island syndrome. Current Biology, 30, R338–R339. https://doi.org/10.1016/j.cub.2020.03.029

Barthe M, Doutrelant C, Covas R, Melo M, Illera JC, Tilak M-K, Colombier C, Leroy T, Loiseau C, Nabholz B (2022) Evolution of immune genes in island birds: reduction in population sizes can explain island syndrome. bioRxiv, 2021.11.21.469450, ver. 4 peer-reviewed and recommended by Peer Community in Evolutionary Biology. https://doi.org/10.1101/2021.11.21.469450

Beadell JS, Ishtiaq F, Covas R, Melo M, Warren BH, Atkinson CT, Bensch S, Graves GR, Jhala YV, Peirce MA, Rahmani AR, Fonseca DM, Fleischer RC (2006) Global phylogeographic limits of Hawaii’s avian malaria. Proceedings of the Royal Society B: Biological Sciences, 273, 2935–2944. https://doi.org/10.1098/rspb.2006.3671

Burns KC (2019) Evolution in Isolation: The Search for an Island Syndrome in Plants. Cambridge University Press, Cambridge. https://doi.org/10.1017/9781108379953

Doutrelant C, Paquet M, Renoult JP, Grégoire A, Crochet P-A, Covas R (2016) Worldwide patterns of bird colouration on islands. Ecology Letters, 19, 537–545. https://doi.org/10.1111/ele.12588

Illera JC, Fernández-Álvarez Á, Hernández-Flores CN, Foronda P (2015) Unforeseen biogeographical patterns in a multiple parasite system in Macaronesia. Journal of Biogeography, 42, 1858–1870. https://doi.org/10.1111/jbi.12548

Lomolino MV (2005) Body size evolution in insular vertebrates: generality of the island rule. Journal of Biogeography, 32, 1683–1699. https://doi.org/10.1111/j.1365-2699.2005.01314.x

Matson KD (2006) Are there differences in immune function between continental and insular birds? Proceedings of the Royal Society B: Biological Sciences, 273, 2267–2274. https://doi.org/10.1098/rspb.2006.3590

Pérez-Rodríguez A, Ramírez Á, Richardson DS, Pérez-Tris J (2013) Evolution of parasite island syndromes without long-term host population isolation: parasite dynamics in Macaronesian blackcaps Sylvia atricapilla. Global Ecology and Biogeography, 22, 1272–1281. https://doi.org/10.1111/geb.12084

As plants cannot run from their enemies, natural selection has favoured the evolution of diverse chemical compounds (phytochemicals) to protect them against herbivores and pathogens. This provides an opportunity for plant feeders to exploit these compounds to combat their own enemies. Indeed, it is widely known that herbivores use such compounds as protection against predators [1]. Recently, this reasoning has been extended to pathogens, and elegant studies have shown that some herbivores feed on phytochemical-containing plants reducing both parasite abundance within hosts [2] and their virulence [3].
Suffering less from parasites is clearly beneficial for infected herbivores. Why then, is this behaviour not fixed in nature? There are, at least, two possible explanations. First, feeding on ‘medicinal’, often toxic, plants may impose costs upon uninfected individuals. Second, parasites may evolve resistance to such chemicals. Whereas the first possibility has been explored, and is taken as evidence for ‘self-medication’ [3], the second hypothesis requires investigation. A recent study by Palmer-Young et al. [4] fills this gap. This article reports evolution of resistance to two different phytochemicals, alone and in combination, in the trypanosome Crithidia bombi, a bumble bee (Bombus impatiens) parasite. To show this, the authors experimentally evolved a strain of C. bombi in the presence of thymol, eugenol or both simultaneously. These phytochemicals are commonly found in the nectar of several plant species, in particular those of the Lamiaceae, a family containing several aromatic herbs. Prior to selection both phytochemicals reduced C. bombi growth by about 50%. However, C. bombi rapidly evolved resistance in both single and the double phytochemical treatments. Moreover, no cost of resistance was detected when evolved parasites were placed in the ancestral, phytochemical-free environment. Therefore, resistance to phytochemicals would be expected to spread rapidly in natural populations of C. bombi. Clearly, thus, the herbivore strategy of sequestering plant secondary chemical compounds as a defence against their pathogens should fail. The question then is ‘why do they still do it’? Can self-medication work in the longer-term for bumblebees?
Well, yes. The very fact that resistance evolved shows that resistance is not fixed in natural C. bombi populations. This is surprising considering that resistance is not costly. This might be due to a number of reasons. Firstly, there may be costs of resistance that were not detected in this experiment. Second, it may not be possible to evolve universal resistance to the heterogeneity present in the phytochemical environment. Indeed, phytochemical environments are highly varied in time and space and bumblebees will visit different plants presenting different phytochemical cocktails throughout the season. Furthermore, migration of bees from populations exposed to different phytochemicals may prevent the fixation of one resistance type.
Or, it may be self-medication behaviour itself that prevents the evolution of resistance? Indeed, in the same way that infected bees seek cooler temperatures to slow growth of a parasitoid fly [5], they may also seek a more varied diet with diverse phytochemicals to which the parasite cannot evolve, but which reduces parasite growth. Further understanding of arthropod self-medication may thus be instrumental to prevent the observed worldwide decline of pollinators [6]. Furthermore, it may inform on mechanisms that prevent rapid evolution of drug resistance in other systems.

References

[1] Duffey SS. 1980. Sequestration of plant natural products by insects. Annual Review of Entomology 25: 447-477. doi: 10.1146/annurev.en.25.010180.002311

[2] Richardson LL et al. 2015. Secondary metabolites in floral nectar reduce parasite infections in bumblebees. Proceedings of the Royal Society of London B 282: 20142471. doi: 10.1098/rspb.2014.2471

[3] Lefèvre T et al. 2010. Evidence for trans-generational medication in nature. Ecology Letters 13: 1485-93. doi: 10.1111/j.1461-0248.2010.01537.x

[4] Palmer-Young EC, Sadd BM, Adler LS. 2017. Evolution of resistance to single and combined floral phytochemicals by a bumble bee parasite. Journal of Evolutionary Biology 30: 300-312. doi: 10.1111/jeb.13002

[5] Müller CB, Schmid-Hempel P. 1993. Exploitation of cold temperature as defence against parasitoids in bumblebees. Nature 363: 65-67. doi: 10.1038/363065a0

[6] Potts SG et al. 2010. Global pollinator declines: trends, impacts and drivers. Trends in Ecology and Evolution 25: 345-353. doi: 10.1016/j.tree.2010.01.007

This manuscript [1] investigates the evolutionary history of the DAN gene family—a group of genes important for embryonic development of limbs, kidneys, and left-right axis speciation. This gene family has also been implicated in a number of diseases, including cancer and nephropathies. DAN genes have been associated with the inhibition of the bone morphogenetic protein (BMP) signaling pathway. Despite this detailed biochemical and functional knowledge and clear importance for development and disease, evolution of this gene family has remained understudied. The diversification of this gene family was investigated in all major groups of vertebrates. The monophyly of the gene members belonging to this gene family was confirmed. A total of five clades were delineated, and two novel lineages were discovered. The first lineage was only retained in cephalochordates (amphioxus), whereas the second one (GREM3) was retained by cartilaginous fish, holostean fish, and coelanth. Moreover, the patterns of chromosomal synteny in the chromosomal regions harboring DAN genes were investigated. Additionally, the authors reconstructed the ancestral gene repertoires and studied the differential retention/loss of individual gene members across the phylogeny. They concluded that the ancestor of gnathostome vertebrates possessed eight DAN genes that underwent differential retention during the evolutionary history of this group. During radiation of vertebrates, GREM1, GREM2, SOST, SOSTDC1, and NBL1 were retained in all major vertebrate groups. At the same time, GREM3, CER1, and DAND5 were differentially lost in some vertebrate lineages. At least two DAN genes were present in the common ancestor of vertebrates, and at least three DAN genes were present in the common ancestor of chordates. Therefore the patterns of retention and diversification in this gene family appear to be complex. Evolutionary slowdown for the DAN gene family was observed in mammals, suggesting selective constraints. Overall, this article puts the biomedical importance of the DAN family in the evolutionary perspective.

References

[1] Opazo JC, Hoffmann FG, Zavala K, Edwards SV (2020) Evolution of the DAN gene family in vertebrates. bioRxiv, 794404, ver. 3 peer-reviewed and recommended by PCI Evolutionary Biology. doi: 10.1101/794404

Monnin et al. [1] here studied how Drosophila populations are affected when exposed to a high virulent endosymbiotic wMelPop Wolbachia strain and why virulent vertically transmitting endosymbionts persist in nature. This virulent wMelPop strain has been described to be a blocker of dengue and other arboviral infections in arthropod vector species, such as Aedes aegypti. Whereas it can thus function as a mutualistic symbiont, it here acts as an antagonist along the mutualism-antagonism continuum symbionts operate. The wMelPop strain is not a natural occurring strain in Drosophila melanogaster and thus the start of this experiment can be seen as a novel host-pathogen association. Through experimental evolution of 17 generations, the authors studied how high temperature affects wMelPop Wolbachia virulence and Drosophila melanogaster survival. The authors used Drosophila strains that were selected for late reproduction, given that this should favor evolution to a lower virulence. Assumptions for this hypothesis are not given in the manuscript here, but it can indeed be assumed that energy that is assimilated to symbiont tolerance instead of reproduction may lead to reduced virulence evolution. This has equally been suggested by Reyserhove et al. [2] in a dynamics energy budget model tailored to Daphnia magna virulence evolution upon a viral infection causing White fat Cell disease, reconstructing changing environments through time.
Contrary to their expectations for vertically transmitting symbionts, the authors did not find a reduction in wMelPop Wolbachia virulence during the course of the experimental evolution experiment under high temperature. Important is what this learns for virulence evolution, also for currently horizontal transmitting disease epidemics (such as COVID-19). It mainly reflects that evolution of virulence for new host-pathogen associations is difficult to predict and that it may take multiple generations before optimal levels of virulence are reached [3,4]. These optimal levels of virulence will depend on trade-offs with other life history traits of the symbiont, but also on host demography, host heterogeneity, amongst others [5,6]. Multiple microbial interactions may affect the outcome of virulence evolution [7]. Given that no germ-free individuals were used, it can be expected that other components of the Drosophila microbiome may have played a role in the virulence evolution. In most cases, microbiota have been described as defensive or protective for virulent symbionts [8], but they may also have stimulated the high levels of virulence. Especially, given that upon higher temperatures, Wolbachia growth may have been increased, host metabolic demands increased [9], host immune responses affected and microbial communities changed [10]. This may have resulted in increased competitive interactions to retrieve host resources, sustaining high virulence levels of the symbiont.
A nice asset of this study is that the phenotypic results obtained in the experimental evolution set-up were linked with wMelPop density measurement and octomom copy number quantifications. Octomom is a specific 8-n genes region of the Wolbachia genome responsible for wMelPop virulence, so there is a link between the phenotypic and molecular functions of the involved symbiont. The authors found that density, octomom copy number and virulence were correlated to each other. An important note the authors address in their discussion is that, to exclude the possibility that octomom copy number has an effect on density, and density on virulence, the effect of these variables should be assessed independently of temperature and age. The obtained results are a valuable contribution to the ongoing debate on the relationship between wMelPop octomom copy number, density and virulence.

References

[1] Monnin, D., Kremer, N., Michaud, C., Villa, M., Henri, H., Desouhant, E. and Vavre, F. (2020) Experimental evolution of virulence and associated traits in a Drosophila melanogaster – Wolbachia symbiosis. bioRxiv, 2020.04.26.062265, ver. 4 peer-reviewed and recommended by PCI Evol Biol. doi: https://doi.org/10.1101/2020.04.26.062265
[2] Reyserhove, L., Samaey, G., Muylaert, K., Coppé, V., Van Colen, W., and Decaestecker, E. (2017). A historical perspective of nutrient change impact on an infectious disease in Daphnia. Ecology, 98(11), 2784-2798. doi: https://doi.org/10.1002/ecy.1994
[3] Ebert, D., and Bull, J. J. (2003). Challenging the trade-off model for the evolution of virulence: is virulence management feasible?. Trends in microbiology, 11(1), 15-20. doi: https://doi.org/10.1016/S0966-842X(02)00003-3
[4] Houwenhuyse, S., Macke, E., Reyserhove, L., Bulteel, L., and Decaestecker, E. (2018). Back to the future in a petri dish: Origin and impact of resurrected microbes in natural populations. Evolutionary Applications, 11(1), 29-41. doi: https://doi.org/10.1111/eva.12538
[5] Day, T., and Gandon, S. (2007). Applying population‐genetic models in theoretical evolutionary epidemiology. Ecology Letters, 10(10), 876-888. doi: https://doi.org/10.1111/j.1461-0248.2007.01091.x
[6] Alizon, S., Hurford, A., Mideo, N., and Van Baalen, M. (2009). Virulence evolution and the trade‐off hypothesis: history, current state of affairs and the future. Journal of evolutionary biology, 22(2), 245-259. doi: https://doi.org/10.1111/j.1420-9101.2008.01658.x
[7] Alizon, S., de Roode, J. C., and Michalakis, Y. (2013). Multiple infections and the evolution of virulence. Ecology letters, 16(4), 556-567. doi: https://doi.org/10.1111/ele.12076
[8] Decaestecker, E., and King, K. (2019). Red queen dynamics. Reference module in earth systems and environmental sciences, 3, 185-192. doi: https://doi.org/10.1016/B978-0-12-409548-9.10550-0
[9] Kirk, D., Jones, N., Peacock, S., Phillips, J., Molnár, P. K., Krkošek, M., and Luijckx, P. (2018). Empirical evidence that metabolic theory describes the temperature dependency of within-host parasite dynamics. PLoS biology, 16(2), e2004608. doi: https://doi.org/10.1371/journal.pbio.2004608
[10] Frankel-Bricker, J., Song, M. J., Benner, M. J., and Schaack, S. (2019). Variation in the microbiota associated with Daphnia magna across genotypes, populations, and temperature. Microbial ecology, 1-12. doi: https://doi.org/10.1007/s00248-019-01412-9