Intraspecific genomic variability substantially affects a species’ adaptive potential to ecological interactions and climatic conditions (Hirsch et al., 2014; Badet et al., 2020; Resl et al., 2022). Variation in genomic content at the gene-level, i.e., presence/absences of genes, within species are regularly found in bacteria, and are often associated with adaptations to specific environments and antibiotic resistances (Masignani et al., 2005; Heuer et al., 2011). Recent studies suggest that eukaryotic species, like bacterial ones, can have intraspecific variation in genomic content at the gene-level (Hirsch et al., 2014; Badet et al., 2020; Gerdol et al., 2020). To this date, it is largely unknown how relevant such variation in gene content is for eukaryotes, and if it is ecologically important for adaptations to specific environments. Intraspecific variation in gene content contributes to the formation of genetically diverse populations, and therefore its characterization is vital to understand the mechanisms of adaptation to specific environments (Plissonneau et al., 2018; Drott et al., 2021; Merges et al., 2022).

The lichen symbiosis, a stable association of mainly fungal and photobiont partners as well as an associated microbiome, lends itself to the study of environmentally-driven gene content, because many species have remarkable environmental tolerances and maintain populations in different climate zones (Kappen, 2000; Werth and Sork, 2014; Singh et al., 2017; Grimm et al., 2021; Jung et al., 2021; Tanunchai et al., 2022). Previous studies showed that environmental differentiation in lichens can be found at the level of single nucleotide polymorphisms (SNPs), which often significantly correlate with differences in geography and ecology and may thus be involved in environmental specialization (Peksa and Skaloud, 2011; Castillo et al., 2012; Hodkinson et al., 2012). Population genomic analyses based on SNPs suggest the presence of genome-wide differentiation between populations in different climate zones (Dal Grande et al., 2017; Rolshausen et al., 2022). To date, the only study which assesses variation in gene content associated with environmental adaptation in lichens is limited to a single species: Singh et al. (2021) show that some gene clusters associated with natural product biosynthesis in Umbilicaria pustulata have elevation-specific distributions. However, we currently lack information on the extent of parallel evolution in populations of different species that have independently evolved under similar environmental conditions. We do not know, e.g., (1) whether different species of lichen-forming fungi maintain homologous population-specific genes along comparable environmental gradients and (2) whether intraspecific variation in gene content (e.g., abundance patterns of genes) has independently converged on the same altitudinal patterns in different species, and can thus be linked to environmental preferences of lichens.

Modern evolutionary approaches leverage DNA sequencing to infer ecological and evolutionary processes that occur at the population level. Here we combine comparative genomics and population genomics to assess the presence/absence of genes in high elevation and low elevation genomes of two lichenized fungi of the genus Umbilicaria (Umbilicaria phaea and U. pustulata). The two species have evolved on different continents under similar climatic selective pressures: U. pustulata in Europe and U. phaea in North America each occupy the cold temperate as well as the Mediterranean climate zone. We tracked gene content variation along replicated elevation gradients in both species based on a total of 2050 individuals in 26 populations. Specifically, we addressed the following research questions: (a) Which genes are linked to environmental conditions at high elevation (cold temperate climate) and low elevation (Mediterranean climate) across species? To address this question, we assessed which genes are exclusive to climate zones. (b) Do abundances of genes specific to a climate zone co-vary with elevation in populations of U. phaea and U. pustulata? To address the second question, we assessed which genes increase or decline in abundance with increasing elevation.

Although adaptations to environmental gradients may lead to variation in gene content, assessments of gene presence/absence patterns across populations and species of lichenized fungi are still missing. Here we assess signatures of parallel evolution at the level of gene presence and absence in lichenized fungi of the genus Umbilicaria, and trace the discovered genes in lichen populations along five replicated elevation gradients across two continents. While our whole genome comparison based on four de novo sequenced specimen (two per species) suggested up to 29 elevation-specific orthogroups (i.e., 16 low elevation-specific and 13 high elevation-specific orthogroups, Figure 1), the population-level verification approach showed only one gene, putatively encoding a protein kinase (PK), which linearly declined in abundance with increasing elevation, and was truly absent in the highest population. This suggests either high strain-specificity of certain genes, or high false positive recovery of gene presence/absence patterns when relying on comparative genomics approaches based on only a few individuals. Thereby extrapolating the significance of gene content variation assessed only with comparative genomics approaches on a few individuals can be potentially misleading when interpreting the evolutionary significance of the variation at population level. Regarding the PK gene consistently absent in high elevation genomes and populations, we found that the discovered gene declines linearly across all populations, suggesting an evolutionary benefit only at lower altitudes. Alternatively, the loss of the gene at higher elevations might benefit individuals in cold climates. To our knowledge, we report for the first time parallel gene presence and absence patterns correlating with climatic niches in different species of lichenized fungi. However, it remains to be analyzed, if the identified molecular trait is associated to a particular phenotype that can be associated with an adaptive function.

In bacteria variation in gene content is assumed to be driven by selection for environmental conditions that are relatively rare across the entire range of a species (Qi et al., 2017). Recent evidence suggests that specific populations of lichenized fungi may contain unique biosynthetic gene clusters (Singh et al., 2021), and our current findings show that also other genes can be elevation-specific. The gradual gene loss across populations with increasing elevation may suggests a decline of selective benefit and may indicate that certain variations in gene content could be of functional importance for local adaptation and climatic tolerances in lichenized fungi. The conserved domain search revealed a catalytic domain of a PK, a common eukaryotic protein superfamily. PKs selectively modify other proteins by phosphorylation, changing their enzymatic activity, cellular location and association with other proteins (Cheng et al., 2002; Hanks, 2003; Asano et al., 2005; Heinisch and Rodicio, 2018). Within a genome, PKs are encoded by a large multigene family with genes being distributed among multiple chromosomes. Putatively, the high number of PK genes has arisen by genome segmental duplication events (Asano et al., 2005; Heinisch and Rodicio, 2018). In our study, the presence/absence of a single PK gene may suggest a climate-specific ancestral genome segmental duplication event. Across the tree of life, e.g., in bacteria (Christ and Chin, 2008), fungi (James et al., 2008) and plants (DeBolt, 2010; Prunier et al., 2019), organisms subjected to selection under high temperatures show higher probability for genome segmental duplications (Christ and Chin, 2008; James et al., 2008; DeBolt, 2010; Kondrashov, 2012). For example, adaptation to heat stress through gene duplication of stress-related genes has been shown in Escherichia coli (Riehle et al., 2001; Kondrashov, 2012), where an upregulation through gene duplication of genes may play a role in adaptation (Kondrashov, 2012; Kuzmin et al., 2022). However, due to the scarcity of functional annotation of non-model organisms and the resulting lack of in-depth functional annotation of the gene in question, the mechanisms generating such population level diversification and the underlying molecular mechanisms behind such adaptations are yet to be understood.

While environmental adaptations are commonly highly polygenic (Rivas et al., 2018; Barghi et al., 2019; Hartke et al., 2021; Pfenninger et al., 2021), there is increasing evidence of the effect of single gene content variation (Liu et al., 2021). For example, as has been recently shown in agave, where a single gene encoding a phosphoenolpyruvate carboxylase enhances the plant’s climate resilience (Liu et al., 2021). Not only the gain of genes, but also the loss of genes has been associated with adaptive traits, such as the evolution of particular diets in bats (Blumer et al., 2022). Therefore, we consider the parallel loss of a homologous gene in two species and two geographic settings a rare find, and a step forward in understanding the genomic underpinnings of climatic tolerances in lichenized fungi. Future research should address the functional importance of the gene present at low altitude in the Mediterranean climate zone, and specifically explore the effects of variation in gene abundances across populations. Additionally, future research should consider using heterologous expression approaches to reveal whether the gene presence could induce tolerances to warm conditions.

Our study demonstrates how comparative genomics in combination with population genomic data can reveal patterns of gene content variation across climatic gradients. In addition, the tracking of gene content variation across populations provides a widely applicable framework for retrieving meaningful biogeographical determinants of gene presence/absence patterns. We contribute to understanding convergence and parallel evolution at the genomic level, by providing insights into gene content variation of lichenized fungi in relation to climatic gradients. This suggests a promising new research direction with implications for understanding evolutionary trajectories in relation to climatic change.

Raw sequence reads were deposited in the National Centre for Biotechnology Information (NCBI) Sequence Read Archive under the BioProject accession numbers PRJNA693984 and PRJNA820300.

DM and IS conceived the ideas and wrote the manuscript. IS and FD collected the data. DM, GS, and HV performed genome assembly and annotations. DM analyzed the data. FD provided analytical guidance. All authors contributed to the various drafts and gave final approval for publication.

This research has been partly funded by the Hesse’s “state initiative for the development of scientific and economic excellence” (LOEWE initiative) through the LOEWE Center for Translational Biodiversity Genomics (TBG).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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