Rapid evolution of selfing syndrome in plants studied with resurrection ecology

Overview

The resurrection method is a new ecological approach for the study of rapid adaptation in natural populations. A basic resurrection study is a common garden experiment where ancestral genotypes (preserved as seeds in natural seed banks or seed repositories) are grown side-by-side with their descendant genotypes (sampled in the same site at present). This setting allows for instant comparison between ancestral and descendant genotypes. If phenotypic trait shifts are detected between ancestors and descendants, they can be interpreted as the outcome of rapid evolution.

The resurrection method opens the possibility for the study of rapid evolution in arable weeds, which are exposed to new, intense selective pressures with the development of modern agriculture and global change. In particular, pollinator decline, coupled with phenological mismatches in flowering time due to temperature fluctuations, could lead towards increased self-pollination and a decreased attractivity of plants to pollinators (selfing syndrome). Alternatively, arable weeds could evolve towards increasing their attractivity to pollinators, thus maintaining pollinator interactions despite the reduction in pollination services. Empirical observation of contemporary evolution in arable weeds has produced data in line with both hypotheses, and it is likely that the direction of trait changes is species- and even population-specific.

I used the resurrection method with two self-compatible, insect-pollinated, annual, arable weeds (Matthiola tricuspidata and Centaurium erythraea) to investigate the effects of climate change and pollinator decline on the evolution of their reproductive and pollination strategies. An innovative aspect I implemented is the upgrade of the basic resurrection common garden with other approaches from evolutionary ecology – reciprocal transplant in simulated environments, genomic tools, and ecological epigenetics.

Are observed trait shifts in Matthiola tricuspidata compatible with adaptation to climate change and pollinator decline? A reciprocal “transplant in time” experiment

While common garden resurrection experiments can tell us about phenotypic trait shifts between ancestors and descendants, they cannot directly test whether these shifts are adaptive. I tested the adaptive character of observed phenotypic shifts between ancestors and descendants in the Mediterranean annual, Matthiola tricuspidata, using reciprocal “transplants in time”. To do this, ancestral and descendant genotypes of one natural population were grown in simulated past and present-day environmental conditions in regulated growth chambers. Past conditions were characterized by lower temperature, higher precipitation levels and humidity compared to present-day conditions. In both experimental environments, ancestral and descendant genotypes were significantly different from one another. In both growth chambers, descendants flowered earlier (consistent with adaptation to warmer springs, Figure 1 top), had larger daily floral displays (consistent with adaptation to maintain pollinator interactions via increase attractivity, Figure 1 middle). Plant size was measured as a fitness proxy, showing overall larger plants in the present-day, warmer and drier environment. Ancestors were larger than descendants in the past environment, but no significant differences between ancestors and descendants, suggesting that both ancestral and descendant genotypes are capable of increasing their vegetative growth in warmer and drier environments (Figure 1 bottom).

Adaptation of Centraurium erythraea to pollinator decline and climate change revealed by the resurrection method

Ancestral and descendant seeds from two natural populations of C. erythraea – one from Belgium (ancestors and descendants sampled 24 years apart) and one from Poland (sampled 17 years apart) were grown in a common garden, and phenotypic and genetic diversity was compared to disentangle the effects of random from those of selective evolution.

In both populations, the descendants had larger daily floral displays (Figure 2) and larger petals (Figure 3), whereas the anther-stigma distance remained unchanged (Figure 4). Altogether, the observations show an evolution to increased flower attractiveness that could maintain plant-pollinator interactions.

Pairwise Fst estimates showed low differentiation between ancestors and descendants in Belgium (0.077), and high differentiation in Poland (0.292). This suggests the occurrence of major evolutionary or demographic changes in the Polish population since the sampling of ancestral genotypes (e.g. migration, extinction and recolonization) or possible high sampling bias in the past. This result illustrates a shortcoming of resurrection studies that is often acknowledged but rarely accounted for – with only two temporal points in a single population, it is difficult to disentangle the effects of natural selection from stochastic variation in allelic frequencies due to random evolutionary processes or sampling bias. As returning in the past to resample the population is not an option, testing for different evolutionary scenarios using approximate Bayesian computations (ABC) could bring insight into the population evolutionary history. This nevertheless requires genotyping neighboring populations to be able to infer the probability that Poland the sampled descendant genotypes are direct offspring of the genotypes present in 2003, or were introduced from another population in its vicinity.

Disentangling genetic from hereditary epigenetic variation in the rapid adaptation of Centaurium erythrae. Combining the resurrection method with ecological epigenetics

This ongoing pilot introduces ecological epigenetics within the existing framework of resurrection ecology, for a novel approach of the study of rapid plant adaptation to global change and pollinator decline via transgenerational plasticity. Indeed, it has been shown that transgenerational plasticity can persist in plants for multiple generations, mediated via hereditary epigenetic modifications such as DNA methylation.

To test for potential effects of transgenerational plasticity mediated by hereditary epigenetic variation, I cultivated F1 descendants of the same two populations of C. erythraea as above in a common garden. Half of the plants were subjected to a demethylation treatment by spraying a solution of 5-azacitidine, which should indiscriminately reduce methylation levels in all treated plants. If phenotypic trait shifts are due to hereditary epigenetic differences, I expect the phenotypic differences between ancestors and descendants to be reduced or even cancelled out in the demethylation compared to the control treatment. If the differences between ancestors and descendants are mainly governed by genetic variation, then the demethylation treatment should not have an effect on the overall differences between ancestors and descendants (Figure 5). The study will investigate shifts in life history traits for the entire lifecycle of C. eythraea from juvenile growth rates, through leaf economic spectra, flowering phenology, to plant attractivity and fitness.

LIFE for Minuartia

Minuartia smejkalii is an edaphic perennial plant endemic to the Czech Republic. The species’ already restricted habitat to serpentine soils in two natural reserves south of Prague was further fragmented and degraded by anthropogenic actions since the 1960s. The LIFE for Minuartia project’s main goal was to reinforce extant and restore extinct populations of M. smejkalii via habitat restoration, population demographic and genetic reinforcement from appropriate in situ and ex situ cultivated materials.

I was in charge of the conservation genetics aspect of LIFE for Minuartia which included

• assessing genetic variation and its distribution estimated from anonymous RADSeq markers among populations, regions, as well as among all Minuartia species present in the Czech Republic.
• study the occurrence of inbreeding depression, outbreeding depression and heterosis by comparing the fitness of inbred and outbred offspring produced from controlled pollinations and cultivated under different conditions.

The results showed that M. smejkalii is genetically differentiated from other Minuartia taxa present in the Czech Republic.

Moreover, intrapopulation genetic variation was high and inter-region variation was low, which could suggest gene flow between regions, or, more plausibly, a genetic lag due to the longevity of the species. Although the estimates varied between traits and environments, I detected high inbreeding depression in large populations, high heterosis in small populations, and low to no outbreeding depression. Altogether, these results offer an optimistic perspective for the conservation of the species which still presents significant genetic diversity and heterosis that can be used to introduce genotypes that will increase the adaptive potential of extant populations with minimal risk of breaking down adaptive complexes.

Host race evolution in Phelipanche ramosa

Weedy parasites invade crops often with dire consequences for yield. The devastating effects of weedy parasites are aided by modern agriculture, notably the reduced genetic resources of monoculture crop resistance which are regularly circumvented by the parasite, and the inability to discriminate between crops and weedy parasites for mass eradications. Phelipanche ramosa is a weedy parasite with a broad geographic distribution that attacks a diverse range of dicotyledonous crops. The species has been documented to regularly invade new crops, and quickly overcome defense mechanisms of resistant crop varieties. As a postdoc, I investigated the distribution of genetic diversity and host preference of P. ramosa across the Mediterranean basin with the goal to understand its evolutionary history and identify factors that lead to the emergence of new host races in western France.

To assess the distribution of different genotypes along geographic and host plant gradients of P. ramosa I genotyped over 100 populations of P. ramosa harvested on various crops using SSR markers. I then tested host preference in rhizotrons for a representative core collection of 15 populations. I identified three highly homozygous genetic groups with distinct host preferences and life cycles. Type 1 had restricted distribution in western France and adapted to canola as host by extending its life cycle, so it matches the winter annual cycle of canola. This is the most recently emerged host race, coinciding with the introduction of canola cultures in western France in the 1980s. Type 2a was found mostly in eastern France, with preference for hemp as main host. Type 2b had a broad distribution, with overall good performance on all hosts, but some preference for hosts of the Solanaceae family.

Looking for cowslips

Heterostylous plants such as Primula veris have two floral morphs with different and reciprocate positions of the anther and stigma, which facilitate pollen transfer between morphs, and hinder it within morphs, thus favoring cross-pollination. Cross-pollination is further favored by morph self-incompatibility. In a population at equilibrium, isoplethy (equal frequencies of each morph) is expected, because it maximises average fitness in the population. Disturbance events, such as population fragmentation which can cause stochastic morph frequency variation and thus limit the availability for suitable mating partners in a population. Coupled with effects of climate change that affect plants and pollinators alike, deviations from isoplethy may thus impose an increased threat to plants with complex mating systems, such as heterostyly.

The citizen science campaign Looking for Cowslips gathered data about the distribution of floral morphs in P. veris. Between 2021 and 2022, the campaign gathered data on over 8000 locations across Europe thanks to the contribution of local campaign managers coordinated by the research team of T. Aavik at the University of Tartu, Estonia. The results expectedly showed that deviation from isoplethy is more pronounced in smaller populations that are more susceptible to genetic drift. Surprisingly, the direction of the deviation was not random, and populations with excess of the short-styled morph prevailed across Europe. The deviation in favor of the short-styled morph was more pronounced closer to urban areas, and in populations with higher precipitation. These results open the question about possible fitness advantage of the short styled morph that would favor its distribution, but also about the relationship between P. veris and its pollinators, especially in the context of climate change that would modify precipitation patterns.

I was involved in the campaign as National Coordinator for North Macedonia. All national coordinators were given a starting communication package with social media posts, graphics, and press releases that they could distribute through their own networks, and were offered monthly group consultations with the PR expert of the campaign. The local implementation of the campaign was left at their discretion.

Figure 2. Distribution of cowslip observation from the Looking for Cowslips campaign in 2021 and 2022. Note the high density of observations in N. Macedonia (central part of the Balkan peninsula).

As a national coordinator, I integrated the Looking for Cowslips campaign in the Science for Children education platform. This resulted in a twofold benefit – recruitment of participants was facilitated by reaching out to the followers of the Science for Children platform, and on the other hand, Looking for Cowslips provided educational material (1, 2, 3, 4) about plant reproduction and biodiversity conservation for Science for Children. The 2022 campaign had an even broader span of activities, organized in the scope of the “Spring of cowslips” event. The outcome of this huge communication and education effort was visible in the results of the campaign – North Macedonia, being one of the smallest European countries with a population of 2 million, was among the top 10 contributors of the campaign (Figure 1).