Evolutionary Forces Shaping Trans-Species Polymorphisms in Genus Cucumis

Abstract

Trans-species polymorphisms (TSPs) are fundamental to preserving ancient genetic diversity, yet the evolutionary forces driving their long-term maintenance remain largely unexplored. Here, we investigate genome-wide TSPs in two Cucumis species, cucumber and melon, using whole-genome sequencing data from over 1200 accessions. A total of 5149 TSPs were identified, which predominantly located in genic and promoter regions. Coalescent analysis indicated that both gene flow and balancing selection have contributed to the persistence of these ancestral alleles. Moreover, among the 99 genes with shared coding-region polymorphisms, two genes that cluster by alleles rather than by species provide evidence of long-term balancing selection. These genes are involved in the immune and stress response processes with pleiotropic effects. Our findings elucidate the complex evolutionary forces driving TSPs in Cucumis, providing mechanistic insights into the maintenance of intraspecific genetic diversity in plants across deep evolutionary timescales.

1. Introduction

Trans-species polymorphisms (TSPs) are ancestral allelic variants that have been maintained across diverging lineages since their split from a common ancestor [1,2]. The persistence of these ancient polymorphisms over deep phylogenetic timescales challenges the theoretical expectation that genetic drift and directional selection would eliminate such variation over evolutionary time. This paradox raises fundamental questions about the mechanisms that allow specific alleles to evade both selective sweeps and purifying selection [3]. Studying TSPs thus offers critical insights into the balance between neutral evolutionary processes and adaptive forces, while also providing a unique window into the long-term maintenance and functional relevance of genetic diversity.

TSPs play a crucial role in adaptive evolution. In plants, for example, TSPs are often associated with resistance (R) genes and self-incompatibility (S) genes, where they are maintained through balancing selection, thus contributing to the species’ resistance to pathogens [4]. Moreover, TSPs are also involved in environmental adaptability. In animals, TSPs in major histocompatibility complex (MHC) genes, for instance, are maintained through negative frequency-dependent selection, allowing organisms to adapt to a constantly changing pathogen environment [5].

The evolutionary origins and maintenance of TSPs involve complex mechanisms. TSPs can easily be confounded with recurrent mutations or introgression, as both processes can lead to shared polymorphisms in closely related species [6]. Recurrent mutations primarily contribute to polymorphism sharing in recently diverged lineages, as the probability of identical mutation events decreases over longer evolutionary periods. Interspecific introgression, on the other hand, typically shows incomplete lineage sorting patterns, which are inconsistent with the retention of ancestral polymorphisms. These mutations tend to be progressively eroded by selection over macro-evolutionary timescales. In contrast, the long-term preservation of TSPs is generally attributed to balancing selection, which sustains allelic diversity through various selective forces, such as negative frequency-dependent selection, heterozygote advantage, and spatiotemporally fluctuating selection [1,7,8]. Balancing selection leaves distinctive genomic signatures, including local increases in nucleotide diversity that exceed neutral expectations, allele frequency spectra skewed toward intermediate frequencies, and accelerated linkage disequilibrium decay around selected loci [9]. In particular, orthologous sequences from different species under strong balancing selection cluster by allele, as tight linkage limits recombination-mediated allele segregation. This contrasts with neutral shared polymorphisms (e.g., recurrent mutations), where phylogenetic signals predominantly follow species divergence patterns. Classical examples, including the major histocompatibility complex in vertebrates (MHC) [8,10] and disease resistance genes in plants [4,11,12,13,14], underscore the adaptive significance of balancing selection. However, current understanding of such evolutionary paradox remains constrained by methodological limitations in distinguishing true TSPs from confounding signals and insufficient systematic analyses across diverse taxonomic groups [7].

The genus Cucumis offers a good system for investigating TSPs, particularly through its two economically significant and phylogenetically divergent species: cucumber (C. sativus) and melon (C. melo) [15]. These two species diverged approximately 4–14 million years ago (MYA) [16,17,18,19,20,21], providing an ideal evolutionary timescale to study ancestral polymorphism retention. Cucumber and melon originated in India and Africa, respectively [22,23]. Both species underwent a domestication process from wild ancestors to cultivated forms, resulting in significant alterations to fruit traits. In cucumber, key selection targets during domestication included fruit length, size, and the loss of bitterness [23]. In melon, substantial changes occurred in fruit size, characteristics, and flavor [22]. The domestication history of melon is particularly complex, involving three independent domestication events in Africa and India, which led to the formation of different cultivated subspecies, such as C. melo ssp. melo and C. melo ssp. agrestis [22]. During domestication, key genes in both cucumber and melon were subjected to strong selective pressures. For instance, in melon, genes associated with fruit quality, size, and bitterness, such as CmBi and CmBt, were fixed during domestication [22]. Similarly, in cucumber, the Bt gene, which is linked to fruit bitterness, also exhibited significant selective signals [23]. These genetic changes not only shaped the phenotypic traits of cultivated varieties but also profoundly influenced their genetic structure. Recent advances in cucurbit genomics have established Cucumis as a model for evolutionary genetics, with chromosome-level reference genomes and extensive population resequencing data available [22,23,24,25,26]. These resources have resolved the phylogenetic divergence of both cucumber and melon clearly. Despite their agricultural importance and well-characterized domestication histories, systematic investigations of TSPs remain absent in these species.

Here, we systematically identify genome-wide TSPs within the genus Cucumis, focusing on two representative species, cucumber and melon. Our analyses reveal that, despite their divergent demographic histories, these species retain a substantial number of shared ancestral polymorphisms. The persistence of these TSPs appears to be shaped by multiple evolutionary forces, with no single mechanism fully explaining their long-term maintenance. Furthermore, genomic regions under balancing selection were significantly enriched for loci associated with stress responses and developmental processes. Notably, we identified two candidate genes under long-term balancing selection that may contribute to adaptive variations and environmental fitness in both species.

2. Materials and Methods

2.1. Species Sampling

Sequencing data for 1280 core accessions of C. sativus L. and C. melo L. were downloaded from NCBI under BioProject PRJNA565104 and the Short Read Archive (SRA) under accession SRA056480.

2.2. Variant Calling

Raw reads from 115 cucumber accessions and 1165 melon accessions were aligned to the C. melo reference genome (DHL92 v4.0) [27] using BWA (version 0.7.15) [28] with default parameters. Duplicate reads were identified and marked using the MarkDuplicates tool from Picard (https://broadinstitute.github.io/picard/, accessed on 12 December 2024). The alignments were subsequently converted to BAM format using SAMtools (version 1.10) [29]. Genome-wide SNPs were called and filtered following the Best Practices pipeline by GATK v4.2.0.0 [30].

2.3. Population Genetic Analysis

Phylogenetic analysis was performed using the cucumber and melon accessions described above, with six watermelon accessions included as the outgroup. The watermelon accessions were retrieved from NCBI under BioProject PRJNA527790, with accession numbers SRR8751612 to SRR8751617. Four-fold degenerate sites were extracted from the whole-genome SNP dataset using an in-house script. These 4D SNPs were then used to construct a phylogenetic tree with IQ-TREE [31], utilizing the ‘-m MFP -B 1000’ parameters. Principal component analysis (PCA) was performed using EIGENSOFT (v6.0.2) [32]. ADMIXTURE (v1.3.0) [33] was employed to estimate the optimal number of genetic clusters (K) among the cucumber and melon accessions.

2.4. Identification and Statistic of Trans-Species Polymorphisms

To ensure the reliability of TSPs, the minor allele frequency (MAF) was initially restricted to >0.05, and the site missing rate was limited to <10% for both cucumber and melon groups, using VCFTOOLS (v0.1.16) [34]. Variants that passed these filters were required to be biallelic SNPs. Regions with high (DP ≥ 35) or low (DP ≤ 8) mean site read depths were excluded, and chromosomal endpoints and large gap regions were removed. Repetitive elements identified in the melon genome were classified using RepeatMasker (v4.0.8) [35], and SNPs located within these regions were discarded. Finally, snpEff (v4.3) [36] was employed to annotate the variations. The distribution of TSPs was calculated using an in-house script. Gene Ontology (GO) enrichment analysis was performed using ClusterProfiler (v4.0) [37].

2.5. Demographic Analysis

To investigate the demographic history of cucumber and melon, demographic histories were then inferred using the continuous-time sequential Markovian coalescent approximation implemented in fastsimcoal2 (v2.7) [38]. Orthologous four-fold degenerate sites were used to generate the joint site frequency spectrum (SFS) for the two species with easySFS.py (https://github.com/isaacovercast/easySFS, accessed on 10 December 2024). fsc27 was applied to estimate the folded SFS under two evolutionary models, including scenarios of population divergence with or without ancient gene flow. For each model, a spontaneous mutation rate of 7 × 10⁻⁹ per base pair per generation was applied [39]. Demographic parameters for each model were optimized through 100 independent runs, each incorporating 100,000 coalescent simulations (-n 100,000) and 40 iterations (-L 40) for likelihood maximization.

2.6. Introgression Analysis

Gene introgression was assessed using Dsuite (v0.5) [40] to calculate Patterson’s D (ABBA-BABA) and f₄-ratio statistics. Introgression regions were detected using the subcommand ‘Dsuite Dinvestigate’ with default parameters.

2.7. Identification of Trans-Species Polymorphisms Under Long-Term Balancing Selection

To investigate trans-species polymorphisms within cucumber and melon, only SNPs located in orthologous genic regions shared by both species were considered. To minimize the potential influence of recurrent mutations among trans-species polymorphisms, genes with more than one TSP and at least one TSP in coding regions were selected. Finally, for each retained TSP, 500 base pairs (bp) of the flanking sequence were extracted using bcftools (v1.8) [41]. Multi-sequence alignment was performed using MAFFT (v7.407) [42], and the aligned sequences were then used to construct phylogenetic trees with IQ-TREE. TSP allele trees discordant with the species tree were selected as candidates under long-term balancing selection.

3. Results

3.1. Trans-Species Polymorphisms Between Cucumber and Melon

Whole-genome sequencing data were collected from 1280 accessions, including 115 cucumber accessions [23] and 1165 melon accessions [22] from around the world, and downloaded from public databases to identify TSPs. High-quality reads were aligned to the melon reference genome DHL92 (v4.0) [27] using a standardized pipeline. Variant calling and stringent filtering resulted in a total of 3,342,859 high-confidence biallelic SNPs (see Section 2).

To assess the reliability of SNP datasets, phylogenetic analysis between cucumber and melon was constructed. A phylogenetic tree constructed from four-fold degenerate SNPs clearly separated cucumber and melon into two distinct clades (Figure 1a). The melon clade resolved into six subclades (wild African, cultivated African, wild agrestis, cultivated agrestis, wild melo, and cultivated melo), and the cucumber clade divided into four subclades (Indian, Xishuangbanna, Eurasian, and East Asian), consistent with prior studies [22,23]. Principal Component Analysis (PCA) further confirmed genetic differentiation between and within species (Figure 1b). Principal Component Analysis (PCA) further confirmed the genetic differentiation between and within species (Figure 1b). The first two principal components distinctly separated the cucumber and melon groups, highlighting species-level divergence and validating the robustness of the dataset.

Following comprehensive filtering, a total of 5149 TSPs (identical allele pairs at specific orthologous loci) were identified between cucumber and melon (Figure 1c). These TSPs were unevenly distributed across all chromosomes, with a significant enrichment in genic and promoter regions (the 3000 bp upstream of the start codon), which together accounted for approximately 60% of the total TSPs (Figure 1d). In contrast, intergenic regions displayed a markedly lower density of TSPs (40%). Furthermore, these TSPs spanned 2816 genes, which were significantly enriched in biological processes such as the cellular response to nitrogen compounds (GO:1901699), the metabolic process of organic hydroxy compounds (GO:1901615), and gene silencing by RNA (GO:0031047) (Figure 1e, Table S1). Taken together, these results highlight the functional significance of TSPs in shaping key adaptive traits, contributing to the evolution of cucumber and melon.

3.2. Demographic History of the Two Species

A comprehensive understanding of demographic history is essential for accurately detecting true TSP signals in cucumber and melon. Given the prolonged divergence time and distinct chromosome numbers (Seven vs. Twelve), these two species are highly unlikely to have experienced recent introgression. Therefore, coalescent simulations were conducted under two distinct models (Figure 2a). Model selection was based on Akaike's Information Criterion (AIC), which revealed that the model incorporating ancient gene flow (M2) provided a better fit, as evidenced by a lower AIC value (36,024.143 vs. 49,999.916). Under model M2, cucumber and melon were estimated to have diverged from a common ancestor approximately 7.3 million years ago, with a 95% confidence interval (CI) of 6.5 MYA to 8.9 MYA, consistent with previous studies. Additionally, an ancient migration rate of 3 × 10⁻⁴ per generation was inferred between the two species. This evidence of ancient gene flow suggests that introgression segments may still persist in their genomes despite the deep evolutionary divergence between cucumber and melon. Furthermore, we identified introgression regions using the ABBA-BABA method (Figure 2b, Table S2). Notably, 250 of the 5149 TSPs were located within these introgression regions, indicating the potential influence of ancient introgression on observed TSPs.

3.3. Trans-Species Polymorphisms Under Balancing Selection

To characterize TSPs under balancing selection in cucumber and melon, we implemented a stepwise filtering strategy (Figure 3a). The analysis focused on 1996 TSPs within 12,626 single-copy orthologous genes, ensuring that the analyzed variants were evolutionarily conserved between the two species. By restricting the dataset to genic regions, which are more likely to be functionally relevant, a total of 824 TSPs were retained for further investigation. To strengthen the evidence for long-term balancing selection, we required the presence of at least two TSPs per genic region, with at least one located within a coding sequence. This filtering process resulted in 99 genes containing 259 TSPs. Consistent with expectations for ancestral polymorphisms maintained by selection, these genes exhibited higher Tajima’s D values compared to genome-wide averages (Figure 3b), indicating a deviation from neutral mutations. Gene ontology analyses revealed that these genes were primarily associated with plant growth and development, as well as response to environmental stress, and cell structure and function (Figure S1, Table S3).

If ancient balanced polymorphisms originated prior to species divergence, allele phylogenies might deviate from the species tree. This discordance could arise when multiple linked polymorphisms cause alleles to cluster by sequence similarity rather than species origin. We reconstructed haplotype phylogenies for all 99 genes with TSPs. Two genes exhibited allele clusters that crossed species boundaries, with each subclade containing haplotypes from distinct lineages (Figure 3c–e). In MELO3C007588 (CsaV3_6G043590), we detected two completely linked polymorphisms (r² = 0.47): a nonsynonymous SNP and a 3′ UTR SNP (Figure 3c,d). This gene encodes a secreted peptide precursor with dual functions in both immune modulation and salt tolerance [43]. It was also priorly identified as a candidate resistance gene against Fusarium oxysporum infection in melon based on transcriptomic analysis [44]. Another gene, MELO3C035245 (CsaV3_6G006620), similarly showed allele-specific clustering patterns across species, driven by two nonsynonymous TSPs (r² = 0.45) (Figure 3e). This gene is a leucine-rich repeat (LRR) receptor-like protein (RLP) belonging to a family known to regulate both developmental processes and defense responses, as evidenced by studies in Arabidopsis thaliana [45]. These results suggest that the two genes have undergone long-term balancing selection in cucumber and melon. Their functional versatility indicates that they could act as evolutionary toggles, maintaining pleiotropic fitness by resolving environmental trade-offs. However, further functional validation is required to unravel the specific molecular mechanisms underlying their pleiotropic effects.

4. Discussion

In this study, a substantial number of TSPs were identified using large-scale whole-genome sequencing data from globally representative accessions of cucumber and melon, which diverged over an extensive evolutionary timescale, providing an exceptional opportunity to explore TSPs. The results indicate that various mechanisms, such as introgression and balancing selection, contribute to the persistence of TSPs across cucumber and melon. Additionally, long-term balancing selection is likely to play a crucial role in the formation of adaptive traits throughout the evolutionary trajectories of both species.

Given the current challenges associated with identifying TSPs [2,9], a rigorous multi-step filtering approach was employed to identify whole-genome TSPs. Findings indicate that TSPs are predominantly situated in functionally significant regions and are closely linked to the formation of adaptive traits. The discovery of TSPs within these regions emphasizes their potential role in regulatory mechanisms that influence key plant traits. TSPs located in promoter regions often affect gene expression by altering transcription factor binding or affecting the chromatin state, which can influence adaptive responses to environmental challenges. Similarly, TSPs within untranslated regions (UTRs) may regulate post-transcriptional processes such as mRNA stability, translation efficiency, and localization, which are crucial for fine-tuning gene expression during stress responses, TSPs in intronic regions can play a role in alternative splicing, potentially generating protein isoforms that may be critical for functional flexibility, particularly in response to pathogens or environmental stressors [46,47]. A recent study on Midas cichlid fishes identified a transposon insertion in an intron region, which was associated with a color polymorphism under long-term balancing selection, reflecting the broader functional implications of TSPs in maintaining adaptive traits across generations [46].

The evolution and maintenance of TSPs can involve various mechanisms [1,47,48]. By evaluating demographic parameters, evidence of ancient gene flow was discovered in both cucumber and melon. Subsequently, introgression regions from cucumber into melon were identified, indicating that introgression contributes to the persistence of TSPs. The significance of balancing selection in maintaining genetic variation has garnered considerable attention in evolutionary biology [7,13]. However, detecting balancing selection signals across genomes poses significant challenges [9]. Consequently, relatively few genes have been identified as being under balancing selection, particularly in plants. To address this gap, a multi-step filtering approach was employed to detect TSPs under long-term balancing selection. After filtering, 99 genes exhibited evidence of being under balancing selection, characterized by higher Tajima’s D values that indicate balancing selection signals. These genes were found to be enriched in pathways related to plant growth and environmental stress responses, which is consistent with previous studies [5,10,11,12,14,47]. Finally, two genes associated with immune modulation and stress tolerance were identified as being under long-term balancing selection, potentially contributing to the adaptability of cucumber and melon in fluctuating environments. These findings suggest that the identified genes may play a crucial role in enhancing the environmental resilience of both species. However, while our analysis indicates functional similarities between the two species, a more detailed comparison of the functional metabolic pathways in cucumber and melon is required to determine any species-specific differences. This comparison could reveal whether these genes have divergent roles in the metabolic processes of each species. Further experimental validation, particularly through metabolic profiling and functional genomics, will be necessary to confirm these differences and fully elucidate the contribution of these genes to their adaptive traits.

The TSPs in Cucumis species are notably enriched in genes involved in immune responses and stress tolerance. This is consistent with findings from other plant studies, where TSPs associated with disease resistance genes or self-incompatibility systems are maintained by balancing selection due to their pleiotropic effects on survival in fluctuating environments [12,13,14]. For instance, ton of TSPs in genes like those regulating plant immune responses, including resistance to pathogens, is often a result of negative frequency-dependent selection, where rare alleles confer a survival advantage against evolving pathogens [49]. Similarly, the adaptive of TSPs in Cucumis species likely contributes to their environmental adaptability, as these polymorphisms may enhance the plant’s ability to withstand various biotic and abiotic stressors.

Our study provides a deeper understanding of the mechanisms maintaining TSPs in Cucumis species, which have significant implications for agricultural production and crop improvement. The TSPs identified in our study, particularly those associated with immune responses and stress tolerance, could inform the development of resistant and resilient crop varieties. For instance, TSPs in genes related to disease resistance could serve as genetic resources for breeding plants with enhanced pathogen resistance. This aligns with findings from other studies on disease resistance in plants, where balancing selection at resistance loci has been critical for maintaining genetic diversity and sustainable resistance to pathogens [4]. The potential to harness these ancient polymorphisms in breeding programs offers a novel approach to addressing challenges in crop production, particularly in the face of climate change and emerging plant diseases. Furthermore, the identification of TSPs in genes involved in stress tolerance may also contribute to improving crop resilience under diverse environmental conditions, such as drought, salinity, or temperature stress. However, the practical application of these findings in crop improvement will require further functional validation of the identified TSPs, as well as genetic mapping to identify the most promising targets for breeding.

However, the focus was placed on orthologous genes with two or more shared SNPs under long-term balancing selection in both species. This approach may have overlooked some non-coding TSPs and sites where only a single TSP was initially targeted by balancing selection prior to species divergence. In addition, to avoid false positives, our filtering strategy may be too stringent.

Loci that are shared between species are critical for understanding adaptive mechanisms in organisms. However, due to limited time, weak selection strength, and other factors [50,51,52], detecting these TSPs and understanding their evolutionary forces remain challenging. Therefore, there is a growing need for more powerful methods to detect TSPs. Moving forward, integrating functional validation, comparative epigenomics, and expanded population sampling will be essential to elucidate further the adaptive significance of these shared polymorphisms and their roles in the evolutionary dynamics of species.

5. Conclusions

This study presents a comprehensive investigation of TSPs in two phylogenetically distinct Cucumis species, cucumber (C. sativus L.) and melon (C. melo L.), utilizing large-scale whole-genome sequencing data. The analysis reveals that a significant number of ancestral polymorphisms are conserved across these species, predominantly within genic and promoter regions, which are integral to the maintenance of adaptive traits. The persistence of these TSPs is driven by complex evolutionary forces, including balancing selection and gene flow, which collectively contribute to the preservation of genetic diversity over extended evolutionary timescales. These findings provide valuable insights into the role of TSPs in the evolutionary dynamics of plant species, particularly their contribution to disease resistance and stress tolerance. Notably, candidate genes under long-term balancing selection were identified, which may play a pivotal role in enhancing the environmental adaptability of both cucumber and melon. These results open promising avenues for breeding programs focused on advancing sustainable agriculture, particularly in improving crop resilience to environmental stresses and pathogen pressures. Although significant insights have been gained regarding the evolutionary significance of TSPs, further research is required to functionally validate the identified polymorphisms and explore their practical applications in crop improvement. Ultimately, the study underscores the critical role of ancient genetic diversity in shaping the evolutionary trajectories of plants and provides a valuable resource for future efforts in genetic conservation and crop breeding strategies.