Interview with the authors: Unparallel differentially expressed genes in parallel ecological divergence

In a recent paper in Molecular Ecology, Szukala et al. quantified the degree of gene expression and functional parallelism across polytopic divergence of montane and alpine ecotypes of in Heliosperma pusillum (Caryophyllaceae) and gained insights into the architecture of adaptive traits. They performed RNA-seq analyses of plants grown in a common garden and detected a large proportion of differentially expressed genes in each replicate ecotype pair. Functional enrichment of these genes, however, revealed that the traits affected by significant expression divergence are largely consistent across ecotype pairs, suggesting a polygenic architecture for the diverged adaptive traits and multiple routes for adaptation. A new genome assembly for H. pusillum was also presented in this study.

We sent a number of questions to lead authors of this work, Aglaia Szukala and Ovidiu Paun, to get more detail on this study.

Upper panels: Graphical representations of the alpine (A) and montane (M) ecotypes of Heliosperma pusillum and their ecological niches. Lower panel: Map showing the study sites of ecotype pairs that evolved in parallel.

What led to your interest in this topic / what was the motivation for this study? 

We are fascinated by the concept of parallel evolution and the molecular mechanisms behind this process. Given that drift is a major driver of evolution and due to the traditional focus on mono- or oligogenic traits, parallel evolution has been considered to be a rare process until recently. However, together with the increasing understanding of polygenic adaptation (Barghi et al., 2020), it has become clear that parallel evolution is relatively frequent, with implications across evolutionary biology and ecology. Specifically, for our study, previous works (Trucchi et al., 2017; Bertel et al., 2018) reported some evidence that altitudinal ecotypes in H. pusillum diverged in parallel. We wanted to rigorously test this hypothesis using demographic modeling and understand the level of molecular parallelism, with regard to divergent gene expression and outlier SNPs.

What difficulties did you run into along the way? 

For several reasons related to the planning of field work and the development of the wider project over years, we had to deal with uneven sampling sizes across populations, which needed to be taken into account, especially for the demographic modeling analyses. Fun fact: in reciprocal translocation experiments of a complementary study (Szukala et al., 2022) on the same species, whose data is also included in the present paper, we chose to use alpine microsites to plant our accessions that were fairly flat (in an otherwise steep area) and free of other plants. At the end of the vegetation season, those sites proved to be resting places for chamois which squeezed and munched most of our plants, while overfertilizing them. In previous years, when reciprocal transplantations were performed as preparation for this study, we faced droughts, poor germination and survival rates at some sites, leading to uneven sampling sizes across sites. Take-home message: experiments in the wild are always a challenge.

What is the biggest or most surprising innovation highlighted in this study? 

It is unclear how much overlap of divergence outliers is to be expected across natural evolutionary replicates. Our study showed a surprisingly low amount of shared molecular differentiation, which we did not expect given that the geographic range considered is relatively small and our study system is in a phase of incipient speciation with no reproductive isolation detectable (Bertel et al., 2016). The extremely low sharing of differentially expressed genes and outlier SNPs, but high similarity of GO terms involved across independent divergence events, indicates that the polygenic architecture of traits is relevant for adaptation of these populations to distinct altitudinal zones in the Alps.

Moving forward, what are the next steps in this area of research?

We further investigated the role of phenotypic plasticity for the development of parallel evolution in our system (Szukala et al., 2022) towards a better understanding of the relative role of genetic and environmentally-induced phenotypic variation in such replicated divergence events. We are also interested if other molecular mechanisms, which are sensitive to environmental input, such as epigenetic signals, could play an important role in parallel evolution. Further, we wish to understand how polygenic adaptation affects signatures of parallel evolution. Very interesting is to question if adaptation can use different genes to produce similar outcomes even in very closely related lineages, and how frequent this process takes place compared to the re-use of standing variation.

Alpine (blue) and montane (light orange) ecotypes of Heliosperma pusillum and their environments. In the alpine environment glabrous plants grow on more humid screes and meadows, also in proximity of streams. In the montane environment below the tree line, pubescent plants typically grow under rocks overhangs or on the rock as chasmophytes. Photo credit: Szukala A and Paun O.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology?

Being curious and exploiting the most advanced and newest methods is good, but don´t forget to be robust, careful, and bias-aware when it comes to the interpretation of results.

What have you learned about methods and resource development over the course of this project?

It is often difficult to quantify and describe results relative to expectations in an objective way, because it is hard to formulate objective expectations in natural systems.

Describe the significance of this research for the general scientific community in one sentence.

Repeated evolution of similar phenotypes can involve different sets of genes.

Describe the significance of this research for your scientific community in one sentence.

Polygenic traits offer different genetic substrates for parallel evolution of similar phenotypes.


Barghi N, Hermisson J, Schlötterer C. 2020. Polygenic adaptation: a unifying framework to understand positive selection. Nature Reviews Genetics 21: 769–781.

Bertel C, Hülber K, Frajman B, Schönswetter P. 2016. No evidence of intrinsic reproductive isolation between two reciprocally non-monophyletic, ecologically differentiated mountain plants at an early stage of speciation. Evolutionary Ecology 30: 1031–1042.

Bertel C, Rešetnik I, Frajman B, Erschbamer B, Hülber K, Schönswetter P. 2018. Natural selection drives parallel divergence in the mountain plant Heliosperma pusillum s.l. Oikos 127: 1355–1367.

Trucchi E, Frajman B, Haverkamp THA, Schönswetter P, Paun O. 2017. Genomic analyses suggest parallel ecological divergence in Heliosperma pusillum (Caryophyllaceae). New Phytologist 216: 267–278.

Szukala A, Bertel C, Frajman B, Schönswetter P, Paun O. 2022. Parallel adaptation to lower altitudes is associated with enhanced plasticity in Heliosperma pusillum (Caryophyllaceae). bioRxiv 2022.05.28.493825; doi: 10.1101/2022.05.28.493825.

Featured study

Szukala A, Lovegrove-Walsh J, Luqman H, Fior S, Wolfe TM, Frajman B, Schönswetter P, Paun O. 2022. Polygenic routes lead to parallel altitudinal adaptation in Heliosperma pusillum (Caryophyllaceae). Molecular Ecology.