Repetita iuvant. The extraordinary evolutionary history of the endemic palm Chamaerops humilis L. in the Mediterranean

Repetita iuvant. 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The extraordinary evolutionary history of the endemic palm Chamaerops humilis L. in the Mediterranean Mónica Labella-Ortega, Carmine Guarino, Guido Cipriani, Rosario Schicchi, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6595308/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chamaerops humilis L. is the only Arecaceae species endemic to the western Mediterranean. Here, de novo genome sequencing allowed discovering reasons behind its exclusive persistence within the natural distribution area, although the troubled environmental changes occurred. The genome, estimated at 3.44 Gbp, and its annotation enabled the identification of 60,555 genes and 2.87 Gbp of repetitive regions. Evolutionary analyses revealed a whole-genome duplication event 48.02 mya, shared with other palm species, and a later divergence from Phoenix dactylifera (17 mya). The characterization of gene duplication types, and the distribution of K S values, uncovered multiple recent duplication waves, and the presence of C. humilis -specific duplicated genes involved in responses to stress. Furthermore, expansions of transposable elements were detected and appear to be associated with major paleoclimatic events in the Mediterranean. These genomic features likely contributed to the resilience and long-term survival of C. humilis within the Mediterranean. Biological sciences/Plant sciences/Plant evolution Biological sciences/Evolution/Evolutionary genetics Biological sciences/Genetics/Evolutionary biology Figures Figure 1 Figure 2 Figure 3 1. Introduction Analysis of the current global biodiversity landscape confronts us with the evidence that the sixth mass extinction is underway 1 . This conscious or unconscious awareness, coupled with technological advancement, is strongly directing the studies and project activities of research groups worldwide towards the acquisition of deep genomic data, through whole genome sequencing (WGS), in response to the need to crystallise the molecular basis of the evolution and adaptation of living species to environmental change 2 . The tools deemed most effective in achieving this aim are reference genomes and their systematisation in increasingly comprehensive databases useful for collecting and sharing information for biodiversity assessment, conservation strategies and restoration efforts 3 . In the Tree of Life definition, a gap in sequenced plant genomes, which to date remains largely unexplored, is evident 2 . This shortcoming is probably rooted in the challenges posed by the high complexity of plant genomes sequencing and functional analysis, leading to gaps in research 2 , 4 . Due to their greater plasticity and speed of adaptation and evolution, plant genomes tend to be more diverse and larger than the rest of the eukaryotes 4 . Polyploidy, whole genome duplications (WGD), or partial genome duplications, which occurred over the last 200 million years and are still occurring today, add further complexity to the panorama 5 , 6 . Increasing the amount of reference plant genomes is of fundamental importance for understanding the biological and evolutionary meanings - diverse and always new - defined by the WGD and polyploidy 6 . Genome duplication has definitely led to the flowering of new evolutionarily beneficial traits to plant species or communities, influencing gene expression and function, and improving fitness in harsh environments 6 . Ancestral events of genome duplication have led to changes in the functions of organisms, resulting in new arrangements that selective environmental pressure may have favoured in situations of extreme dynamic conditions 7 . Genome duplication events can increase the vigour of individuals, giving rise to a more probable transgressive segregation, reducing the risk of extinction and increasing the rate of evolution and adaptation 5 . Today, the Mediterranean basin is considered a major biodiversity hotspot, considering the wonderful intra- and interspecific variety of populations that have adapted there 8 . What we can observe and study today is the result of a series of upheavals in this relatively small portion of the globe over geological eras, shaping biodiversity through speciation, adaptation and extinction events caused by the sometimes dramatic fluctuations of the paleoclimatic features 9 . Of all the events that occurred in the Mediterranean, the one that probably influenced the shaping of the biosphere, and the echoes of which still resonate in today's biodiversity, is the Miocene 10 . The global changes caused by tectonic movement in the Cenozoic led to a rearrangement of ocean currents and a series of climate fluctuations culminating in the Miocene, during the course of which the Mediterranean basin underwent profound structural changes 11 . The Mediterranean Basin was to be considered part of the tropical biodiversity hotspot of the Western Tethys before its separation from it, and the Mid-Miocene Climate Optimum (MMCO) certainly represented a peak moment for tropical fauna and flora 10 , 12 . In the Late Miocene, the Mediterranean began the evolutionary process that would lead it to a temporary isolation from the Atlantic Ocean that would culminate in the salinity crisis during the Messinian 13 . This sequence of events will lead the Mediterranean to a rapid desiccation that will only be resolved in the Zanclean when, as a result of the Zanclean Flood and the reopening of the Strait of Gibraltar, the sea level will rise again at significant rate, supposedly up to 10 meters per day 12 . The impacts of these geological changes, the relatively sudden change from a tropical to a cold climate, from a humid to a highly arid and saline environment, on the taxa that inhabited the waters and coastlines of the Mediterranean are still the subject of discussion and speculation, with evidence primarily drawn from Miocene fossil records 12 . The vegetation of the Mediterranean was fully affected by the enormity of these changes and in the transition between the Miocene and Quaternary periods underwent a series of structural and compositional remodeling influenced by complex abiotic factors 14 . Analysing the pollen remains and other biomarkers, it was possible to ascertain the transition from thermophilous and hygrophilous vegetation to sclerophyllous formations adapted to aridity. The transition defined a new vegetation landscape, moving from tropical and subtropical forest systems to shrublands and grasslands rich in sclerophyllous species that have marked and defined Mediterranean vegetation to date 15 . The transition from tropical to current Mediterranean vegetation has left traces that can be seen in the almost total disappearance of some taxonomic groups from the Mediterranean basin, whose ancient presence has been confirmed by the discovery of pollen and macroremains 16 . One of these ecological gaps is to be attributed to the Arecaceae family, dominant in the tropical and subtropical biome, present in the pre-Miocene Mediterranean and today represented only by one narrow endemic species: Chamaerops humilis L., the Mediterranean dwarf palm 17 . Chamaerops humilis is the only species in the Chamaerops L. genus, and is narrow endemic to the western Mediterranean area. In addition, C. humilis is a key indicator of thermo-Mediterranean vegetation, characterizing its early successional stages due to its adaptability. It is a species intimately connected with the history of the civilizations developed along the Mediterranean coasts, having represented and still represents a crucial source of food, fibers, ethnobotanical products and being used for ornamental purposes 18 . The present work aims to reconstruct the evolutionary and adaptive history of C. humilis through sequencing of its genome, highlighting WGD events that occurred over time and studying genomic underpinnings that led this iconic species to diverge within the Arecaceae family and become the only endemic palm of the western Mediterranean. In a broader sense, we believe that the results of this work can form the basis for the identification and future definition of strategies for the conservation of endemic and endangered species, drawing on the information carved into the genome of the species themselves. 2. Results 2.1 Genome annotation A total of 147,287 model genes were predicted, and 60,555 genes were annotated, corresponding to 92,564 proteins, representing 13.01% of the whole genome of C. humilis (Fig. 1 ). From all the genes identified, 92.46% were in chromosomes and 7.54% in chromosome 0. The chromosomes with the highest and lowest number of genes were chromosome 2 and chromosome 18, respectively ( Supplementary Table 1 ). Genome completeness analysis assessed with BUSCO showed that 90.8% of the core reference genes were found in the genome and just 2.1% were missing. From the GO analysis, we observed that the biological processes with the highest representation were DNA Integration, Protein phosphorylation and regulation of DNA-templated transcription. Molecular function was primarily characterized by zinc ion binding, RNA/DNA hybrid ribonuclease and nucleic acid binding; and in cellular processes, membrane, nucleus and cytoplasm were the most prevalent ( Supplementary Fig. 1 ). Transposable elements (TEs) were classified into two major classes according to the mechanism of transposition 19 , Class I Retrotransposons which represent almost 78% of the genome, and Class II DNA transposons, which represent 5.3% of the genome ( Supplementary Table 2 ). Of all, the most abundant superfamily was long terminal repeats (LTR) Copia and Gypsy, with 36.94% and 29.81% of the total genome length. The LTR Assembly Index (LAI) 20 , a metric to evaluate the quality and completeness of genome assembly based on LTR retrotransposons, was 19.86, indicating a high assembly quality through repetitive regions, being higher than in the other palms studied, indicating the high quality of the genome assembly. The TE content in palms varies, being C. humilis and Cocos nucifera the palms with the highest amount of TE (2.20 and 2.19 Gbp respectively) followed by E. guineensis (1.80 Gbp) and being P. dactylifera the palm with less TE (0.40 Gbp) ( Supplementary Table 3 ). Insertion time estimation of LTR retrotransposon revealed an increase in the insertion rate of LTR/Copia around 5.5 million years ago (mya), followed by a gradual decrease and a slight increase of approximately 2 mya. In the case of LTR/Gypsy, a steady increase in the LTR insertion was observed from 9 to 4.5 mya, which was then succeeded by a gradual decline ( Supplementary Fig. 2 ). Full-length LTR were classified into lineages, being in the case con Copia retrotransposon, the most abundant Tork, Angela and SIRE lineage (21.78%, 13.80% and 11.72% respectively) and in the case of Gypsy retrotransposon, the most abundant were CRM, Tat and Athila (16.63%, 13.91% and 6.91%, respectively) ( Supplementary Table 4 ). Previously it has been seen this lineage also abundant in P. dactylifera , C. nucifera , Calamus simplicifolius and Elaeis olifera 21 . 2.2 Gene family evolution All-against-all comparisons were performed to identify single-copy genes of C. humilis with orthologues in 6 monocotyledons ( Ananas comosus , C. nucifera , E. guineensis , Oryza sativa , P. dactylifera and Zea mays ) and 4 dicotyledons ( Arabidopsis thaliana , Coffea arabica , Theobroma cacao and Vitis vinifera ). The resulting phylogenetic tree (Fig. 2 a) shows two main clades, monocotyledons and dicotyledons, that split ~ 150 mya (the late Jurassic), in agreement with prior findings 22 . Following monocot-dicot divergence, Poaceae ( Z. mays and O. sativa ) diverged from the rest of the clade ~ 120 mya 23 . Subsequently, ~ 87 mya Bromeliaceae ( A. comosus ) separated from the Arecaceae followed by the separation of C. humilis and P. dactylifera in one clade ~ 23 mya and the final divergence of C. humilis species ~ 17 mya. Given the close evolutionary relationship between C. humilis with P. dactylifera and E. guineensis , the evaluation of their synteny provided a framework to assess the extent of genomic conservation and the structural rearrangements that have occurred over time, revealing only small syntenic blocks among C. humilis and these species (Fig. 2 b). The genome dynamic nature of C. humilis , reflected in the low number of conserved syntenic blocks, underscores the importance of further exploring the expansion and contraction of gene families as a potential driving force behind these changes, ultimately contributing to the monotypic nature of the genus. Collinearity study between Aceraceae was performed and the proportion of genes observed was 1:1, suggesting that they have experienced the same number of whole genome duplications. Using all annotated protein-coding genes from 7 monocotyledons and 4 dicotyledons, orthofinder identified 32,135 total orthogroups and 9,243 orthogroups with all the species present. To evaluate the overall genomic changes throughout evolution in C. humilis , all gene families were analyzed. Among the findings, 1,008 gene families were shared between C. humilis and the other palms, while 3,135 gene families were unique (Fig. 2 c). Of these unique families, 2663 appear to be expanded and 472 gene families contracted. Subsequently, only rapidly evolving gene families were analysed to focus on genes that have undergone significant changes, potentially influenced by selective pressure. 942 gene families in C. humilis that have rapidly evolved in gene copy number since the last common ancestor were identified, reflecting a gain of 12,428 genes and a loss of 290 genes. Based on Gene Ontology enrichment analysis, of the most enriched biological process terms are related to DNA plasticity processes like transposition, cellular cycle control, and interstrand cross-link repair, as well as terms related to stress response like cellular response to water deprivation and response to salt ( Supplementary Table 5 ). 2.3 Whole genome duplication An analysis of the size and frequency of paralogous gene families accumulated in the C. humilis genome provided a record of the whole genome duplication events that occurred in this species. Based on results obtained in other palm species, we expected to find a common whole genome duplication event that occurs in all the Aceraceae (39.87–55.2 mya) as previously reported 24 . WGD events were analyzed by calculating the whole anchor K S age distribution of C. humilis . The presence of some retained anchor pairs between 0 and 1 suggests a recent WGD event ( Supplementary Fig. 3 ), with the 95% confidence interval of the log-normal distribution indicating a peak between 0.16 and 0.60 ( Supplementary Fig. 4A ). Further phylogenetic dating analysis estimated that this WGD event occurred 48.02 mya ( Supplementary Fig. 4B ). In all palms, the same WGD event was observed 24 . However, since this WGD is common among other palm species, the increase in the genome size in C. humilis might be more influenced by other types of duplications. To further explore the dynamics of genome expansion, the gene duplication modes in two palm species were analyzed and compared with C. humilis , offering valuable insights into the genomic strategies that shape the diversity and adaptability of these plants. Considering the complete gene set of C. humilis , only 13.48% (7,490 genes) were WGD derived genes, while in P. dactylifera and E. guineensis this percentage reached 52.64% and 52.03%, respectively ( Supplementary Fig. 5A ). Although the proportion of tandem (TD) (7.10%) and transposon-derived duplications (TRD) (28.05%) in C. humilis was similar to that observed in other palm species, the total number of genes in these duplication types is twice as high compared to the other species ( Supplementary Fig. 5A ). Additionally, the percentage of genes associated with proximal duplication (PD) (9.92%) is slightly higher than in the other palms. Notably, dispersed duplications (DD) represent the 41.44% of the total amount of duplicated genes in C. humilis , while this percentage is lower in P. dactylifera and E. guineensis (9.20% and 8.00%, respectively) ( Supplementary Fig. 5A ). The K S distribution was analyzed for each type of gene duplication. In the case of WGD genes, a shared peak at 0.27 was detected across the three palm species ( Supplementary Fig. 5B ), consistent with a previously reported duplication event dated to 39.87–55.2 mya. This peak is also observed in TRD, suggesting that this ancient event may have played a role in shaping the genomic architecture of these species. Although WGD genes were more abundant in the other palms, C. humilis showed the highest peak in TRD, highlighting the significant role of transposable elements in reshaping its genome, suggesting that transposition-driven duplication has been a major force in its genome evolution. The major difference is also found in TD, PD, TRD and DD duplications. In C. humilis , all these duplication types exhibit a unique recent peak at 0.05, a pattern not observed in the other palm species ( Supplementary Fig. 5B ). This exclusive signal may be linked to species-specific genome expansion and could partially explain the larger genome size observed in C. humilis . Gene homology matrix dot plot shows the collinearity between chromosomes, where a remnant of gene duplications experienced in C. humilis can be seen in all the chromosomes (Fig. 3 a). Most of the dots show a K S value lower than 2, suggesting that many of the duplications are relatively recent, providing evidence of the lasting impact of these duplication events that continue to influence in the genomic landscape of this species. An intraspecific collinearity plot was generated to illustrate the distribution of varying levels of collinearity in C. humilis (Fig. 3 b), revealing a significant number of multiplicons (homeologous regions within a genome that are present in multiple copies), with some regions exhibiting up to nine copies. This underscores the extensive genomic duplication in C. humilis , especially when compared to other palms like E. guineensis and P. dactylifera , which shows no more than four-fold duplications ( Supplementary Fig. 6 ). A comparison between multiplicons observed in the three palms was performed, and 729 multiplicons were found exclusively duplicated in C. humilis , being 17 of them duplicated at least seven times within C. humilis genome. Multiplicons duplicated at least seven-fold times include a total of 184 genes, which are distributed across all chromosomes except for chromosomes 11, 16, and 18 ( Supplementary Fig. 7 ). Notably, the multiplicons exhibiting up to nine-fold duplication are localized in chromosomes 1, 2, 5, 9, 10, 13, 14, and 15, with the highest concentration observed in chromosome 1 (9 genes) ( Supplementary Fig. 7 ). In all the cases dispersed duplications were the most abundant (58%), followed by transposon derived duplication (24.6%) and whole genome duplication derived (11.2%). From all the genes identified, 74.5% were coding genes and 25.5% were repetitive elements (Fig. 3 c). Several proteins were identified that play essential roles in biotic and abiotic stress responses, particularly to cold and salinity. Among these, aspartic peptidases, such as DDI1-type, are linked to low temperatures 25 , while other members of the aspartic peptidase family are involved in pathogen defense and regulation of responses to both biotic and abiotic stresses (e.g., drought, salinity, and cold) 26 . Additionally, GTP-binding nuclear proteins and RING-type E3 ligases, which are integral to cellular signalling and regulatory processes during stress responses, also play a crucial role in responses to stresses like drought, salinity, and low temperatures 27 . Another significant group includes NAC-transcription factors, such as NST1, which are involved in secondary cell wall formation and responses to abiotic stresses like cold and salinity 28 . Other proteins, such as SUT1, regulate immune responses 29 , and plant LRR proteins are involved in pathogen defence 30 , contributing to the overall stress response mechanism. These duplications suggest that the plant has evolved enhanced adaptability through a more redundant and efficient stress response system. Moreover, retrotransposons, including Gypsy and Copia 31 , as well as Ty3-gypsy retrotransposon 32 , are known to be sensitive to various abiotic stresses such as cold, heat, salinity, and infections. Their duplication could contribute to genomic plasticity, providing the plant with the flexibility needed to adjust to fluctuating environmental conditions. 3. Discussion Across palm family, there had been found 58-fold change range of variation in genome size, from 0.53 Gbp un the diploid Licuala orbicularis and L. sarawakensis , to the 38-ploid Voanioala gerardii with a genome size of 30.63 Gbp 33 . The sequenced genome of C. humilis in this study, falls within the average size range of known palms genomes 33 . In this study we compare C. humilis with P. dactylifera and E. guineensis 34 , 35 , as they are the phylogenetic closest relatives. Additionally, P. dactylifera ancestors share a historical presence in Southern Europe environments, making it a particularly relevant species for understanding the evolutionary adaptations of C. humilis in this region 36 . The genome size variation among C. humilis with P. dactylifera and E. guineensis palms can provide insight into how genomic architecture evolves in response to environmental pressures, leading to genome expansion. In plants, this increase in genome size is primarily driven by polyploidization events and the accumulation of substantial numbers of repetitive elements 34 . The large genome size in C. humilis could represent an evolutionary advantage acquired after its divergence from P. dactylifera . Intense environmental fluctuations, including significant climate changes such as glaciation events, rapid shifts in sea surface temperatures and level, and a sharp drop in global mean surface temperature occurred between 25 and 40 mya 37 , are linked to a wave of paleopolyploidizations 24 . The WGD event observed in C. humilis (48.02 mya) corresponds to the one detected in other palms, indicating that it predates their phylogenetic divergence 24 . After WGD events, rediploidization restores diploid-like inheritance through a combination of genomic reorganization and selective gene retention 38 . In C. humilis , after the WGD event, phylogenetic differentiation allowed the identification of a set of expanded gene families. From all of them, the presence of terms related to DNA regulation, such as transposition, as well as terms related to environmental stress, suggests a connection between genomic plasticity and adaptation to environmental changes 39 . The activation of TE in response to environmental stress generates a high genetic variability 40 that must be controlled, justifying the presence of terms associated with DNA repair. Terms related to response to environmental stress indicate that the availability of water, salt and hormones like brassinosteroids, which have been linked to response to various stresses 41 , play a significant role. These observations reinforce the idea that genomic plasticity is a dynamic and regulated response that allows adaptation to adverse conditions 42 . When the C. humilis genome is compared to other palms phylogenetically closed, low synteny is observed. This statement has been seen previously in other monotypic genera, such as Ginkgo biloba 43 and Amborella trichopoda 44 , which has low synteny and significant genomic rearrangements, that could be linked to long term isolation and adaptation to specific ecological niches. Additionally, certain gene duplications appear to be unique to C. humilis . These duplications may have played a crucial role in the species' adaptation to environments with both biotic and abiotic stresses, aligning with its status as one of the most cold-tolerant palms in the Northern Hemisphere 45 . Similar patterns have been observed in other species, where duplicated genes, particularly tandem duplications, contribute to stress response, similarly to Arabidopsis and Oryza , where they enhance biotic stress resilience 4 . Dispersed duplications were the most abundant type of gene duplication in C. humilis . These duplications may result from larger-scale genomic rearrangements, such as duplications, inversions, and transpositions, which can drive gene family’s expansion, functional diversification, and increased genomic flexibility, potentially linked to evolutionary adaptations 46 . This suggests that the duplications in C. humilis may have similarly contributed to its enhanced resilience to environmental stresses. The accumulation of repetitive elements and TE expansions can drive rapid genome size increases, either independently or in concert with WGD 47 . A striking example is maize, where post-WGD TE expansion has led to a genome composed of 85% transposable elements 48 . Similarly, C. humilis exhibits a genome with 82% TE content, suggesting a major role for TEs in its genomic architecture. Previous studies in palms have shown that repeat expansions can be influenced by environmental factors such as aridity 33 . The expansion and regulation of TEs are tightly controlled through mechanisms that suppress their activity and mitigate their effects when necessary. However, under environmental stress or genomic instability, TEs may escape these controls, leading to bursts of amplification 49 . Events such as polyploidy and hybridization can further weaken TE suppression, facilitating their proliferation 50 . While TE insertions are often considered deleterious, they can also contribute to adaptation by generating genetic diversity and regulatory innovations. The observed fluctuation in Copia and Gypsy transposon activity align with major geological and climatic events in the Mediterranean region, particularly the Messinian Salinity Crisis and the subsequent Zanclean flood. Between 5.33 and 5.97 million years ago, the Mediterranean Sea experienced a dramatic isolation from the Atlantic ocean during the Messinian Salinity Crisis, an extraordinary geological event that triggered a precipitous drop in sea levels and led to the extensive deposition of vast salt layers across the basin 51 . Such stressors may disrupt C. humilis genome, destabilizing epigenetic controls and enabling transposon amplification and insertion 52 . The progressive decline of LTR insertions fit with the end of the Messinian salinity crisis, marked by the cataclysmic Zanclean megaflood 53 . This event imposed strong selective pressures in the Mediterranean, favouring genome expansion mechanisms that may have conferred adaptive advantages, such as the amplification of stress-response genetic elements. More recently, during the Quaternary period, alternating glacial and interglacial cycles led to repeated phases of species contraction and expansion, favouring the extinction of those less adapted to extreme environmental changes 54 . Before the onset of the Quaternary, the Mediterranean flora still contained a considerable number of tropical and subtropical tree taxa 55 . In the case of C. humilis , its present-day distribution could be linked to its ability to persist in climatic refugia during these periods 56 . Chamaerops humilis exemplifies a species that, despite these fluctuations, has persisted and adapted. Fossil evidence from Santorini (Greece) suggests that it was present in the region during the Late Pleistocene but later disappeared from that area, reflecting a contraction in its range 57 . However, rather than facing extinction, C. humilis has survived across fragmented Mediterranean habitats, demonstrating its resilience. Despite the tumultuous geoclimatic evolution of the Mediterranean, which has fluctuated between warmer, subtropical conditions, arid and cooler climate, the species has managed to persist. Its genome is a testament to the species' ability to withstand both environmental stressors and historical climate changes. 4. Materials and methods 4.1. Phylogenetic and Genomic Comparative Analysis To investigate the evolutionary history of C. humilis , gene family construction was performed using OrthoFinder (v2.5.5) 58 . The analysis included additional palms, including palm species P. dactylifera 59 , E. guineensis 60 , and C. nucifera 61 , as well as other monocotyledons including Ananas comosus 62 , O. sativa 63 , and Z. mays 64 , and some dicotyledons plants as outgroups including A. thaliana, C. arabica 65 , V. vinifera 66 and T. cacao 67 . The protein sequences of 70 single-copy orthologs were concatenated and aligned using MAFFT (Katoh et al., 2002) with the L-INS-i iterative refinement method. The most suitable model (JTT + G + F) was identified with ModelGenerator v0.85 ( http://bioinf.nuim.ie/software/modelgenerator/ ). Divergence times between species were estimated with BEAST v2.6.3 68 , utilizing the JTT substitution model, four gamma categories, the Yule model, and an uncorrelated relaxed clock with lognormal distribution. The MCMC chain was run for 10,000,000 generations, recording parameters every 1000 generations. Calibration points for BEAST were obtained from the TimeTree database ( http://www.timetree.org/ ) , C. nucifera versus V. vinifera (145 mya), O. sativa versus Z. mais (60 mya), P. dactylifera versus C. nucifera (70 mya) and A. thaliana versus C. arabica (90 mya). The contraction and expansion of the gene families were observed by comparing the differences in cluster size between C. humilis and each species using CAFE (v.5.1.0) 69 . Contracted and expanded gene families were analyzed, and GO enrichment was conducted using topGO 70 . iTOLs 71 was used to visualize the species tree and annotations. Synteny analysis was performed between the two reference Arecaceae genomes: P. dactylifera and E. guineensis using JCVI (v 1.4.25) 72 . 4.2 Whole genome duplication (WGD) analysis WGD analysis was conducted to determine the presence of lineage-specific and/or shared WD events among Aracaceae, using wgd (v2.0.38) 73 . The whole paranome (complete set of paralogous genes within a genome) was constructed using ‘wgd dmd’ with default parameters. To infer genomic collinearity, ‘wgd syn’ was applied using i-ADHoRe (v3.0.01) 74 . Subsequently, 'wgd ksd' was used to construct K S distributions for both the paranome and anchor pairs (pairs of duplicated genes derived from WGD events which reside in duplicated segments) 73 . The exponential-lognormal mixture model (ELMM) and the log-scale Gaussian mixture model (GMM) were employed within ‘wgd viz’ to identify potential WGD components from the whole paranome and anchor pair K S distributions, respectively, facilitating the effective identification of potential WGD events. With the K S distribution of the anchor pairs, only those within the 95% of confidence level were retained using ‘wgd peak’. Finally, using a phylogenetic tree and fossil calibration of 17 related species previously reported by Chen et al 73 , orthogroups were constructed by combining the anchor pairs with related homologous genes. WGD events were dated with ‘wgd focus’ using mcmctree from PAML (v4.9) 75 . When the paralogs of C. humilis and orthologs with other Aracaceae species were identified, only those exclusive of C. humillis and duplicated at least 7 times were evaluated. A functional analysis (GO analysis and literature review) was conducted to determine their biological significance. Duplicated gene pairs were identified and classified into WGD-derived and SSD-derived gene pairs using the R package doubletrouble 76 . Paralog genes that are used as anchor pairs in syntenic regions are classified as SD, typically originating from whole genome duplications. All other duplicates are classified as SSD. These can be further classified based on their proximity: those adjacent in the genome are termed TD; those separated by only a few genes (default is 10, adjustable) are classified as PD; and all others fall under DD. Additionally, duplicates that arise from transposon-derived duplications are known as TRD. The location of duplicated genes and classification were plotted using chromomap in R 77 . Declarations Funding This work was supported by by the European Union - Next-GenerationEU - National Recovery and Resilience Plan (NRRP) – MISSION 4 COMPONENT 2, INVESTIMENT N. 1.1, project reference: ChAMPION - Chamaerops humilis reference genome sequencing for Active conservation of an endemic Mediterranean Palm Including analyses Of biotic interactions Network, CUP F53D2300817000. Author contributions G. C., Z. D., T. M., P. A conceived, write and revised the article, L-O. M. performed bioinformatic analysis, write and revised the article, T.M., R. M-A and M.M extract nucleic acid material, C. G., R. revised the article, S. R. and B. G provided biological material, C. H contributed with bioinformatic analysis and revised the manuscript. Data availability The genome sequence and annotation data for C. humilis , the sequencing data and the transcriptome data are accessible under NCBI BioProject no. PRJNA1247256. References Theissinger, K. et al. How genomics can help biodiversity conservation. Trends Genet. 39 , 545–559 (2023). Twyford, A. D. The road to 10,000 plant genomes. Nat. Plants 4 , 312–313 (2018). Mc Cartney, A. M. et al. The European Reference Genome Atlas: piloting a decentralised approach to equitable biodiversity genomics. Npj Biodivers. 3 , 1–17 (2024). Panchy, N., Lehti-Shiu, M. & Shiu, S.-H. Evolution of Gene Duplication in Plants. Plant Physiol. 171 , 2294–2316 (2016). Van de Peer, Y., Maere, S. & Meyer, A. The evolutionary significance of ancient genome duplications. Nat. Rev. Genet. 10 , 725–732 (2009). 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CAFE 5 models variation in evolutionary rates among gene families. Bioinformatics 36 , 5516–5518 (2021). Adrian Alexa, J. R. topGO. Bioconductor https://doi.org/10.18129/B9.BIOC.TOPGO (2017). Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49 , W293–W296 (2021). Tang, H. et al. Synteny and Collinearity in Plant Genomes. Science 320 , 486–488 (2008). Chen, H., Zwaenepoel, A. & Van de Peer, Y. wgd v2: a suite of tools to uncover and date ancient polyploidy and whole-genome duplication. Bioinformatics 40 , btae272 (2024). Proost, S. et al. i-ADHoRe 3.0—fast and sensitive detection of genomic homology in extremely large data sets. Nucleic Acids Res. 40 , e11 (2012). Yang, Z. PAML 4: Phylogenetic Analysis by Maximum Likelihood. Mol. Biol. Evol. 24 , 1586–1591 (2007). Almeida-Silva, F. & Van de Peer, Y. doubletrouble: an R/Bioconductor package for the identification, classification, and analysis of gene and genome duplications. Bioinformatics 41 , btaf043 (2025). Anand, L. & Rodriguez Lopez, C. M. ChromoMap: an R package for interactive visualization of multi-omics data and annotation of chromosomes. BMC Bioinformatics 23 , 33 (2022). Additional Declarations There is NO Competing Interest. Supplementary Files RepetitaiuvantSupplementaryTables.xlsx Supplementary Table 5 RepetitaiuvantSupplementaryMaterial.pdf Supplementary material Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6595308","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":452374710,"identity":"0194ec2d-3db7-4cca-bb1a-a0fe199e07a3","order_by":0,"name":"Mónica 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Sannio","correspondingAuthor":false,"prefix":"","firstName":"Antonello","middleName":"","lastName":"Prigioniero","suffix":""}],"badges":[],"createdAt":"2025-05-05 14:25:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6595308/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6595308/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82083005,"identity":"80e60dc7-e3f1-49cb-b586-87769922d7d1","added_by":"auto","created_at":"2025-05-06 14:41:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1578700,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal features, functional annotation, and gene duplication landscape of \u003cem\u003eChamaerops humilis\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003eThe figure illustrates key chromosomal characteristics and duplication patterns in \u003cem\u003eC. humilis\u003c/em\u003e in the 18 principal chromosomes. From the outer to the inner rings, the tracks represent: (\u003cstrong\u003ea\u003c/strong\u003e) chromosome size in megabases (Mb); (\u003cstrong\u003eb\u003c/strong\u003e) positions of duplicated genes, with green indicating genes duplicated 7 to 9 times and red indicating genes duplicated fewer than 6 times; (\u003cstrong\u003ec\u003c/strong\u003e) gene density, shown in blue, calculated as the number of annotated genes per 10-Mb bin; and (\u003cstrong\u003ed\u003c/strong\u003e) transposable element (TE) density per 10-Mb bin, with orange representing the overall density and further differentiation of LTR types: green for Gypsy and yellow for Copia.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6595308/v1/74ee3e66493431bdd1f3ad14.png"},{"id":82083004,"identity":"5cf784f2-9ad6-4f38-83cb-b5e787a5a222","added_by":"auto","created_at":"2025-05-06 14:41:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":481874,"visible":true,"origin":"","legend":"\u003cp\u003eGenome evolution of \u003cem\u003eC. humilis\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ea)\u003c/strong\u003e Phylogenetic tree of monocotyledon and dicotyledon species showing their divergence times in million years, calculated based on orthologs within single gene families. The star marks a genome duplication event in the \u003cem\u003eC. humilis\u003c/em\u003e lineage. Total CAFE detected gene families with significant accelerated gene changes of each species; green and red numbers indicate the number of gene expansion or contraction, respectively. At the bottom of the phylogenetic tree, the geological evolution of the \u003cem\u003eC. humilis\u003c/em\u003e current natural range of distribution, coinciding with the actual western Mediterranean, is represented. (\u003cstrong\u003eb)\u003c/strong\u003e Synteny blocks identified among the chromosomes of \u003cem\u003eC. humilis\u003c/em\u003e, \u003cem\u003eE. guineensis\u003c/em\u003e, and \u003cem\u003eP. dactylifera\u003c/em\u003e. (\u003cstrong\u003ec)\u003c/strong\u003e Venn diagram illustrating the shared gene families changed among three palm species: \u003cem\u003eC. humilis\u003c/em\u003e, \u003cem\u003eE. guineensis\u003c/em\u003e and \u003cem\u003eP. dactylifera\u003c/em\u003e. Each number represents the number of gene families shared within the corresponding intersections, while exclusive numbers indicate families unique to a specific species.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6595308/v1/3b3caf2f41025bff85e0642b.png"},{"id":82083442,"identity":"4efb7f61-74fd-4332-9b62-9265ba7b98fe","added_by":"auto","created_at":"2025-05-06 14:49:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":530579,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic Duplication Profile and Functional Insights in \u003cem\u003eChamaerops humilis\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ea) \u003c/strong\u003eIntraspecific genome homology dot plots for \u003cem\u003eC. humilis\u003c/em\u003e. Anchor pairs (homologous genes residing in homeologous segments) are represented as dots colored by their associated \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e values. Axes show genes on corresponding chromosomes. (\u003cstrong\u003eb)\u003c/strong\u003e Intraspecific homology collinearity levels for \u003cem\u003eC. humilis\u003c/em\u003e . The minimum length for segments is set to 100kb. (\u003cstrong\u003ec)\u003c/strong\u003e Donut plot of the main functions of genes and repetitive elements duplicated exclusively in \u003cem\u003eChamaerops humilis\u003c/em\u003e more than 7 times.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6595308/v1/265ac7ac92036957fa16c8d4.png"},{"id":82197160,"identity":"8bb5d306-3ec8-457d-9d9a-6d69e2ecaa14","added_by":"auto","created_at":"2025-05-07 15:18:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3581297,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6595308/v1/4f9be6e7-f784-4b15-a527-f5780cb586c0.pdf"},{"id":82081695,"identity":"ea96ef2c-839c-400b-898e-db9274d050f3","added_by":"auto","created_at":"2025-05-06 14:33:59","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20927,"visible":true,"origin":"","legend":"Supplementary Table 5","description":"","filename":"RepetitaiuvantSupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6595308/v1/fb4868fe52791efac7a2f477.xlsx"},{"id":82083008,"identity":"45cb5e34-6fba-4437-9c2d-a52c3f3a0b92","added_by":"auto","created_at":"2025-05-06 14:41:59","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1676714,"visible":true,"origin":"","legend":"Supplementary material","description":"","filename":"RepetitaiuvantSupplementaryMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6595308/v1/83c9832a879730797b7fca4f.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Repetita iuvant. The extraordinary evolutionary history of the endemic palm Chamaerops humilis L. in the Mediterranean","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAnalysis of the current global biodiversity landscape confronts us with the evidence that the sixth mass extinction is underway \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This conscious or unconscious awareness, coupled with technological advancement, is strongly directing the studies and project activities of research groups worldwide towards the acquisition of deep genomic data, through whole genome sequencing (WGS), in response to the need to crystallise the molecular basis of the evolution and adaptation of living species to environmental change \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The tools deemed most effective in achieving this aim are reference genomes and their systematisation in increasingly comprehensive databases useful for collecting and sharing information for biodiversity assessment, conservation strategies and restoration efforts \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In the Tree of Life definition, a gap in sequenced plant genomes, which to date remains largely unexplored, is evident \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This shortcoming is probably rooted in the challenges posed by the high complexity of plant genomes sequencing and functional analysis, leading to gaps in research \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Due to their greater plasticity and speed of adaptation and evolution, plant genomes tend to be more diverse and larger than the rest of the eukaryotes \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Polyploidy, whole genome duplications (WGD), or partial genome duplications, which occurred over the last 200\u0026nbsp;million years and are still occurring today, add further complexity to the panorama \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Increasing the amount of reference plant genomes is of fundamental importance for understanding the biological and evolutionary meanings - diverse and always new - defined by the WGD and polyploidy \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Genome duplication has definitely led to the flowering of new evolutionarily beneficial traits to plant species or communities, influencing gene expression and function, and improving fitness in harsh environments \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Ancestral events of genome duplication have led to changes in the functions of organisms, resulting in new arrangements that selective environmental pressure may have favoured in situations of extreme dynamic conditions \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Genome duplication events can increase the vigour of individuals, giving rise to a more probable transgressive segregation, reducing the risk of extinction and increasing the rate of evolution and adaptation \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Today, the Mediterranean basin is considered a major biodiversity hotspot, considering the wonderful intra- and interspecific variety of populations that have adapted there \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. What we can observe and study today is the result of a series of upheavals in this relatively small portion of the globe over geological eras, shaping biodiversity through speciation, adaptation and extinction events caused by the sometimes dramatic fluctuations of the paleoclimatic features \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Of all the events that occurred in the Mediterranean, the one that probably influenced the shaping of the biosphere, and the echoes of which still resonate in today's biodiversity, is the Miocene \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The global changes caused by tectonic movement in the Cenozoic led to a rearrangement of ocean currents and a series of climate fluctuations culminating in the Miocene, during the course of which the Mediterranean basin underwent profound structural changes \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The Mediterranean Basin was to be considered part of the tropical biodiversity hotspot of the Western Tethys before its separation from it, and the Mid-Miocene Climate Optimum (MMCO) certainly represented a peak moment for tropical fauna and flora \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In the Late Miocene, the Mediterranean began the evolutionary process that would lead it to a temporary isolation from the Atlantic Ocean that would culminate in the salinity crisis during the Messinian \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This sequence of events will lead the Mediterranean to a rapid desiccation that will only be resolved in the Zanclean when, as a result of the Zanclean Flood and the reopening of the Strait of Gibraltar, the sea level will rise again at significant rate, supposedly up to 10 meters per day \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The impacts of these geological changes, the relatively sudden change from a tropical to a cold climate, from a humid to a highly arid and saline environment, on the taxa that inhabited the waters and coastlines of the Mediterranean are still the subject of discussion and speculation, with evidence primarily drawn from Miocene fossil records \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe vegetation of the Mediterranean was fully affected by the enormity of these changes and in the transition between the Miocene and Quaternary periods underwent a series of structural and compositional remodeling influenced by complex abiotic factors \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Analysing the pollen remains and other biomarkers, it was possible to ascertain the transition from thermophilous and hygrophilous vegetation to sclerophyllous formations adapted to aridity. The transition defined a new vegetation landscape, moving from tropical and subtropical forest systems to shrublands and grasslands rich in sclerophyllous species that have marked and defined Mediterranean vegetation to date \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The transition from tropical to current Mediterranean vegetation has left traces that can be seen in the almost total disappearance of some taxonomic groups from the Mediterranean basin, whose ancient presence has been confirmed by the discovery of pollen and macroremains \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. One of these ecological gaps is to be attributed to the Arecaceae family, dominant in the tropical and subtropical biome, present in the pre-Miocene Mediterranean and today represented only by one narrow endemic species: \u003cem\u003eChamaerops humilis\u003c/em\u003e L., the Mediterranean dwarf palm \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eChamaerops humilis\u003c/em\u003e is the only species in the \u003cem\u003eChamaerops\u003c/em\u003e L. genus, and is narrow endemic to the western Mediterranean area. In addition, \u003cem\u003eC. humilis\u003c/em\u003e is a key indicator of thermo-Mediterranean vegetation, characterizing its early successional stages due to its adaptability. It is a species intimately connected with the history of the civilizations developed along the Mediterranean coasts, having represented and still represents a crucial source of food, fibers, ethnobotanical products and being used for ornamental purposes \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe present work aims to reconstruct the evolutionary and adaptive history of \u003cem\u003eC. humilis\u003c/em\u003e through sequencing of its genome, highlighting WGD events that occurred over time and studying genomic underpinnings that led this iconic species to diverge within the Arecaceae family and become the only endemic palm of the western Mediterranean. In a broader sense, we believe that the results of this work can form the basis for the identification and future definition of strategies for the conservation of endemic and endangered species, drawing on the information carved into the genome of the species themselves.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Genome annotation\u003c/h2\u003e \u003cp\u003eA total of 147,287 model genes were predicted, and 60,555 genes were annotated, corresponding to 92,564 proteins, representing 13.01% of the whole genome of \u003cem\u003eC. humilis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). From all the genes identified, 92.46% were in chromosomes and 7.54% in chromosome 0. The chromosomes with the highest and lowest number of genes were chromosome 2 and chromosome 18, respectively (\u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e). Genome completeness analysis assessed with BUSCO showed that 90.8% of the core reference genes were found in the genome and just 2.1% were missing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom the GO analysis, we observed that the biological processes with the highest representation were DNA Integration, Protein phosphorylation and regulation of DNA-templated transcription. Molecular function was primarily characterized by zinc ion binding, RNA/DNA hybrid ribonuclease and nucleic acid binding; and in cellular processes, membrane, nucleus and cytoplasm were the most prevalent (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTransposable elements (TEs) were classified into two major classes according to the mechanism of transposition \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, Class I Retrotransposons which represent almost 78% of the genome, and Class II DNA transposons, which represent 5.3% of the genome (\u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e). Of all, the most abundant superfamily was long terminal repeats (LTR) Copia and Gypsy, with 36.94% and 29.81% of the total genome length. The LTR Assembly Index (LAI) \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, a metric to evaluate the quality and completeness of genome assembly based on LTR retrotransposons, was 19.86, indicating a high assembly quality through repetitive regions, being higher than in the other palms studied, indicating the high quality of the genome assembly. The TE content in palms varies, being \u003cem\u003eC. humilis\u003c/em\u003e and \u003cem\u003eCocos nucifera\u003c/em\u003e the palms with the highest amount of TE (2.20 and 2.19 Gbp respectively) followed by \u003cem\u003eE. guineensis\u003c/em\u003e (1.80 Gbp) and being \u003cem\u003eP. dactylifera\u003c/em\u003e the palm with less TE (0.40 Gbp) (\u003cb\u003eSupplementary Table\u0026nbsp;3\u003c/b\u003e). Insertion time estimation of LTR retrotransposon revealed an increase in the insertion rate of LTR/Copia around 5.5\u0026nbsp;million years ago (mya), followed by a gradual decrease and a slight increase of approximately 2 mya. In the case of LTR/Gypsy, a steady increase in the LTR insertion was observed from 9 to 4.5 mya, which was then succeeded by a gradual decline (\u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eFull-length LTR were classified into lineages, being in the case con Copia retrotransposon, the most abundant Tork, Angela and SIRE lineage (21.78%, 13.80% and 11.72% respectively) and in the case of Gypsy retrotransposon, the most abundant were CRM, Tat and Athila (16.63%, 13.91% and 6.91%, respectively) (\u003cb\u003eSupplementary Table\u0026nbsp;4\u003c/b\u003e). Previously it has been seen this lineage also abundant in \u003cem\u003eP. dactylifera\u003c/em\u003e, \u003cem\u003eC. nucifera\u003c/em\u003e, \u003cem\u003eCalamus simplicifolius\u003c/em\u003e and \u003cem\u003eElaeis olifera\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Gene family evolution\u003c/h2\u003e \u003cp\u003eAll-against-all comparisons were performed to identify single-copy genes of \u003cem\u003eC. humilis\u003c/em\u003e with orthologues in 6 monocotyledons (\u003cem\u003eAnanas comosus\u003c/em\u003e, \u003cem\u003eC. nucifera\u003c/em\u003e, \u003cem\u003eE. guineensis\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eP. dactylifera\u003c/em\u003e and \u003cem\u003eZea mays\u003c/em\u003e) and 4 dicotyledons (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eCoffea arabica\u003c/em\u003e, \u003cem\u003eTheobroma cacao\u003c/em\u003e and \u003cem\u003eVitis vinifera\u003c/em\u003e). The resulting phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) shows two main clades, monocotyledons and dicotyledons, that split\u0026thinsp;~\u0026thinsp;150 mya (the late Jurassic), in agreement with prior findings \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Following monocot-dicot divergence, Poaceae (\u003cem\u003eZ. mays\u003c/em\u003e and \u003cem\u003eO. sativa\u003c/em\u003e) diverged from the rest of the clade\u0026thinsp;~\u0026thinsp;120 mya \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Subsequently, ~\u0026thinsp;87 mya Bromeliaceae (\u003cem\u003eA. comosus\u003c/em\u003e) separated from the Arecaceae followed by the separation of \u003cem\u003eC. humilis\u003c/em\u003e and \u003cem\u003eP. dactylifera\u003c/em\u003e in one clade\u0026thinsp;~\u0026thinsp;23 mya and the final divergence of \u003cem\u003eC. humilis\u003c/em\u003e species\u0026thinsp;~\u0026thinsp;17 mya.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven the close evolutionary relationship between \u003cem\u003eC. humilis\u003c/em\u003e with \u003cem\u003eP. dactylifera\u003c/em\u003e and \u003cem\u003eE. guineensis\u003c/em\u003e, the evaluation of their synteny provided a framework to assess the extent of genomic conservation and the structural rearrangements that have occurred over time, revealing only small syntenic blocks among \u003cem\u003eC. humilis\u003c/em\u003e and these species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The genome dynamic nature of \u003cem\u003eC. humilis\u003c/em\u003e, reflected in the low number of conserved syntenic blocks, underscores the importance of further exploring the expansion and contraction of gene families as a potential driving force behind these changes, ultimately contributing to the monotypic nature of the genus. Collinearity study between Aceraceae was performed and the proportion of genes observed was 1:1, suggesting that they have experienced the same number of whole genome duplications.\u003c/p\u003e \u003cp\u003eUsing all annotated protein-coding genes from 7 monocotyledons and 4 dicotyledons, orthofinder identified 32,135 total orthogroups and 9,243 orthogroups with all the species present. To evaluate the overall genomic changes throughout evolution in \u003cem\u003eC. humilis\u003c/em\u003e, all gene families were analyzed. Among the findings, 1,008 gene families were shared between \u003cem\u003eC. humilis\u003c/em\u003e and the other palms, while 3,135 gene families were unique (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Of these unique families, 2663 appear to be expanded and 472 gene families contracted.\u003c/p\u003e \u003cp\u003eSubsequently, only rapidly evolving gene families were analysed to focus on genes that have undergone significant changes, potentially influenced by selective pressure. 942 gene families in \u003cem\u003eC. humilis\u003c/em\u003e that have rapidly evolved in gene copy number since the last common ancestor were identified, reflecting a gain of 12,428 genes and a loss of 290 genes. Based on Gene Ontology enrichment analysis, of the most enriched biological process terms are related to DNA plasticity processes like transposition, cellular cycle control, and interstrand cross-link repair, as well as terms related to stress response like cellular response to water deprivation and response to salt (\u003cb\u003eSupplementary Table\u0026nbsp;5\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Whole genome duplication\u003c/h2\u003e \u003cp\u003eAn analysis of the size and frequency of paralogous gene families accumulated in the \u003cem\u003eC. humilis\u003c/em\u003e genome provided a record of the whole genome duplication events that occurred in this species. Based on results obtained in other palm species, we expected to find a common whole genome duplication event that occurs in all the Aceraceae (39.87\u0026ndash;55.2 mya) as previously reported \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWGD events were analyzed by calculating the whole anchor \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e age distribution of \u003cem\u003eC. humilis\u003c/em\u003e. The presence of some retained anchor pairs between 0 and 1 suggests a recent WGD event (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e), with the 95% confidence interval of the log-normal distribution indicating a peak between 0.16 and 0.60 (\u003cb\u003eSupplementary Fig.\u0026nbsp;4A\u003c/b\u003e). Further phylogenetic dating analysis estimated that this WGD event occurred 48.02 mya (\u003cb\u003eSupplementary Fig.\u0026nbsp;4B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn all palms, the same WGD event was observed \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, since this WGD is common among other palm species, the increase in the genome size in \u003cem\u003eC. humilis\u003c/em\u003e might be more influenced by other types of duplications. To further explore the dynamics of genome expansion, the gene duplication modes in two palm species were analyzed and compared with \u003cem\u003eC. humilis\u003c/em\u003e, offering valuable insights into the genomic strategies that shape the diversity and adaptability of these plants.\u003c/p\u003e \u003cp\u003eConsidering the complete gene set of \u003cem\u003eC. humilis\u003c/em\u003e, only 13.48% (7,490 genes) were WGD derived genes, while in \u003cem\u003eP. dactylifera\u003c/em\u003e and \u003cem\u003eE. guineensis\u003c/em\u003e this percentage reached 52.64% and 52.03%, respectively (\u003cb\u003eSupplementary Fig.\u0026nbsp;5A\u003c/b\u003e). Although the proportion of tandem (TD) (7.10%) and transposon-derived duplications (TRD) (28.05%) in \u003cem\u003eC. humilis\u003c/em\u003e was similar to that observed in other palm species, the total number of genes in these duplication types is twice as high compared to the other species (\u003cb\u003eSupplementary Fig.\u0026nbsp;5A\u003c/b\u003e). Additionally, the percentage of genes associated with proximal duplication (PD) (9.92%) is slightly higher than in the other palms. Notably, dispersed duplications (DD) represent the 41.44% of the total amount of duplicated genes in \u003cem\u003eC. humilis\u003c/em\u003e, while this percentage is lower in \u003cem\u003eP. dactylifera\u003c/em\u003e and \u003cem\u003eE. guineensis\u003c/em\u003e (9.20% and 8.00%, respectively) (\u003cb\u003eSupplementary Fig.\u0026nbsp;5A\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e distribution was analyzed for each type of gene duplication. In the case of WGD genes, a shared peak at 0.27 was detected across the three palm species (\u003cb\u003eSupplementary Fig.\u0026nbsp;5B\u003c/b\u003e), consistent with a previously reported duplication event dated to 39.87\u0026ndash;55.2 mya. This peak is also observed in TRD, suggesting that this ancient event may have played a role in shaping the genomic architecture of these species. Although WGD genes were more abundant in the other palms, \u003cem\u003eC. humilis\u003c/em\u003e showed the highest peak in TRD, highlighting the significant role of transposable elements in reshaping its genome, suggesting that transposition-driven duplication has been a major force in its genome evolution.\u003c/p\u003e \u003cp\u003eThe major difference is also found in TD, PD, TRD and DD duplications. In \u003cem\u003eC. humilis\u003c/em\u003e, all these duplication types exhibit a unique recent peak at 0.05, a pattern not observed in the other palm species (\u003cb\u003eSupplementary Fig.\u0026nbsp;5B\u003c/b\u003e). This exclusive signal may be linked to species-specific genome expansion and could partially explain the larger genome size observed in \u003cem\u003eC. humilis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eGene homology matrix dot plot shows the collinearity between chromosomes, where a remnant of gene duplications experienced in \u003cem\u003eC. humilis\u003c/em\u003e can be seen in all the chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Most of the dots show a \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e value lower than 2, suggesting that many of the duplications are relatively recent, providing evidence of the lasting impact of these duplication events that continue to influence in the genomic landscape of this species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAn intraspecific collinearity plot was generated to illustrate the distribution of varying levels of collinearity in \u003cem\u003eC. humilis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), revealing a significant number of multiplicons (homeologous regions within a genome that are present in multiple copies), with some regions exhibiting up to nine copies. This underscores the extensive genomic duplication in \u003cem\u003eC. humilis\u003c/em\u003e, especially when compared to other palms like \u003cem\u003eE. guineensis\u003c/em\u003e and \u003cem\u003eP. dactylifera\u003c/em\u003e, which shows no more than four-fold duplications (\u003cb\u003eSupplementary Fig.\u0026nbsp;6\u003c/b\u003e). A comparison between multiplicons observed in the three palms was performed, and 729 multiplicons were found exclusively duplicated in \u003cem\u003eC. humilis\u003c/em\u003e, being 17 of them duplicated at least seven times within \u003cem\u003eC. humilis\u003c/em\u003e genome.\u003c/p\u003e \u003cp\u003eMultiplicons duplicated at least seven-fold times include a total of 184 genes, which are distributed across all chromosomes except for chromosomes 11, 16, and 18 (\u003cb\u003eSupplementary Fig.\u0026nbsp;7\u003c/b\u003e). Notably, the multiplicons exhibiting up to nine-fold duplication are localized in chromosomes 1, 2, 5, 9, 10, 13, 14, and 15, with the highest concentration observed in chromosome 1 (9 genes) (\u003cb\u003eSupplementary Fig.\u0026nbsp;7\u003c/b\u003e). In all the cases dispersed duplications were the most abundant (58%), followed by transposon derived duplication (24.6%) and whole genome duplication derived (11.2%).\u003c/p\u003e \u003cp\u003eFrom all the genes identified, 74.5% were coding genes and 25.5% were repetitive elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Several proteins were identified that play essential roles in biotic and abiotic stress responses, particularly to cold and salinity. Among these, aspartic peptidases, such as DDI1-type, are linked to low temperatures \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, while other members of the aspartic peptidase family are involved in pathogen defense and regulation of responses to both biotic and abiotic stresses (e.g., drought, salinity, and cold) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Additionally, GTP-binding nuclear proteins and RING-type E3 ligases, which are integral to cellular signalling and regulatory processes during stress responses, also play a crucial role in responses to stresses like drought, salinity, and low temperatures \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Another significant group includes NAC-transcription factors, such as NST1, which are involved in secondary cell wall formation and responses to abiotic stresses like cold and salinity \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Other proteins, such as SUT1, regulate immune responses \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and plant LRR proteins are involved in pathogen defence \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, contributing to the overall stress response mechanism. These duplications suggest that the plant has evolved enhanced adaptability through a more redundant and efficient stress response system.\u003c/p\u003e \u003cp\u003eMoreover, retrotransposons, including Gypsy and Copia \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, as well as Ty3-gypsy retrotransposon \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, are known to be sensitive to various abiotic stresses such as cold, heat, salinity, and infections. Their duplication could contribute to genomic plasticity, providing the plant with the flexibility needed to adjust to fluctuating environmental conditions.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eAcross palm family, there had been found 58-fold change range of variation in genome size, from 0.53 Gbp un the diploid \u003cem\u003eLicuala orbicularis\u003c/em\u003e and \u003cem\u003eL. sarawakensis\u003c/em\u003e, to the 38-ploid \u003cem\u003eVoanioala gerardii\u003c/em\u003e with a genome size of 30.63 Gbp \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The sequenced genome of \u003cem\u003eC. humilis\u003c/em\u003e in this study, falls within the average size range of known palms genomes \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In this study we compare \u003cem\u003eC. humilis\u003c/em\u003e with \u003cem\u003eP. dactylifera\u003c/em\u003e and \u003cem\u003eE. guineensis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, as they are the phylogenetic closest relatives. Additionally, \u003cem\u003eP. dactylifera\u003c/em\u003e ancestors share a historical presence in Southern Europe environments, making it a particularly relevant species for understanding the evolutionary adaptations of \u003cem\u003eC. humilis\u003c/em\u003e in this region \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe genome size variation among \u003cem\u003eC. humilis\u003c/em\u003e with \u003cem\u003eP. dactylifera\u003c/em\u003e and \u003cem\u003eE. guineensis\u003c/em\u003e palms can provide insight into how genomic architecture evolves in response to environmental pressures, leading to genome expansion. In plants, this increase in genome size is primarily driven by polyploidization events and the accumulation of substantial numbers of repetitive elements \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The large genome size in \u003cem\u003eC. humilis\u003c/em\u003e could represent an evolutionary advantage acquired after its divergence from \u003cem\u003eP. dactylifera\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIntense environmental fluctuations, including significant climate changes such as glaciation events, rapid shifts in sea surface temperatures and level, and a sharp drop in global mean surface temperature occurred between 25 and 40 mya \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, are linked to a wave of paleopolyploidizations \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The WGD event observed in \u003cem\u003eC. humilis\u003c/em\u003e (48.02 mya) corresponds to the one detected in other palms, indicating that it predates their phylogenetic divergence \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter WGD events, rediploidization restores diploid-like inheritance through a combination of genomic reorganization and selective gene retention \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eC. humilis\u003c/em\u003e, after the WGD event, phylogenetic differentiation allowed the identification of a set of expanded gene families. From all of them, the presence of terms related to DNA regulation, such as transposition, as well as terms related to environmental stress, suggests a connection between genomic plasticity and adaptation to environmental changes \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The activation of TE in response to environmental stress generates a high genetic variability \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e that must be controlled, justifying the presence of terms associated with DNA repair. Terms related to response to environmental stress indicate that the availability of water, salt and hormones like brassinosteroids, which have been linked to response to various stresses \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, play a significant role. These observations reinforce the idea that genomic plasticity is a dynamic and regulated response that allows adaptation to adverse conditions \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhen the \u003cem\u003eC. humilis\u003c/em\u003e genome is compared to other palms phylogenetically closed, low synteny is observed. This statement has been seen previously in other monotypic genera, such as \u003cem\u003eGinkgo biloba\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eAmborella trichopoda\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, which has low synteny and significant genomic rearrangements, that could be linked to long term isolation and adaptation to specific ecological niches.\u003c/p\u003e \u003cp\u003eAdditionally, certain gene duplications appear to be unique to \u003cem\u003eC. humilis\u003c/em\u003e. These duplications may have played a crucial role in the species' adaptation to environments with both biotic and abiotic stresses, aligning with its status as one of the most cold-tolerant palms in the Northern Hemisphere \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Similar patterns have been observed in other species, where duplicated genes, particularly tandem duplications, contribute to stress response, similarly to \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003eOryza\u003c/em\u003e, where they enhance biotic stress resilience\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Dispersed duplications were the most abundant type of gene duplication in \u003cem\u003eC. humilis\u003c/em\u003e. These duplications may result from larger-scale genomic rearrangements, such as duplications, inversions, and transpositions, which can drive gene family\u0026rsquo;s expansion, functional diversification, and increased genomic flexibility, potentially linked to evolutionary adaptations \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. This suggests that the duplications in \u003cem\u003eC. humilis\u003c/em\u003e may have similarly contributed to its enhanced resilience to environmental stresses.\u003c/p\u003e \u003cp\u003eThe accumulation of repetitive elements and TE expansions can drive rapid genome size increases, either independently or in concert with WGD \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. A striking example is maize, where post-WGD TE expansion has led to a genome composed of 85% transposable elements \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Similarly, \u003cem\u003eC. humilis\u003c/em\u003e exhibits a genome with 82% TE content, suggesting a major role for TEs in its genomic architecture. Previous studies in palms have shown that repeat expansions can be influenced by environmental factors such as aridity \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The expansion and regulation of TEs are tightly controlled through mechanisms that suppress their activity and mitigate their effects when necessary. However, under environmental stress or genomic instability, TEs may escape these controls, leading to bursts of amplification \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Events such as polyploidy and hybridization can further weaken TE suppression, facilitating their proliferation \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. While TE insertions are often considered deleterious, they can also contribute to adaptation by generating genetic diversity and regulatory innovations.\u003c/p\u003e \u003cp\u003eThe observed fluctuation in Copia and Gypsy transposon activity align with major geological and climatic events in the Mediterranean region, particularly the Messinian Salinity Crisis and the subsequent Zanclean flood. Between 5.33 and 5.97\u0026nbsp;million years ago, the Mediterranean Sea experienced a dramatic isolation from the Atlantic ocean during the Messinian Salinity Crisis, an extraordinary geological event that triggered a precipitous drop in sea levels and led to the extensive deposition of vast salt layers across the basin \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Such stressors may disrupt \u003cem\u003eC. humilis\u003c/em\u003e genome, destabilizing epigenetic controls and enabling transposon amplification and insertion \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The progressive decline of LTR insertions fit with the end of the Messinian salinity crisis, marked by the cataclysmic Zanclean megaflood \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. This event imposed strong selective pressures in the Mediterranean, favouring genome expansion mechanisms that may have conferred adaptive advantages, such as the amplification of stress-response genetic elements.\u003c/p\u003e \u003cp\u003eMore recently, during the Quaternary period, alternating glacial and interglacial cycles led to repeated phases of species contraction and expansion, favouring the extinction of those less adapted to extreme environmental changes \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Before the onset of the Quaternary, the Mediterranean flora still contained a considerable number of tropical and subtropical tree taxa \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. In the case of \u003cem\u003eC. humilis\u003c/em\u003e, its present-day distribution could be linked to its ability to persist in climatic refugia during these periods \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eChamaerops humilis\u003c/em\u003e exemplifies a species that, despite these fluctuations, has persisted and adapted. Fossil evidence from Santorini (Greece) suggests that it was present in the region during the Late Pleistocene but later disappeared from that area, reflecting a contraction in its range \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. However, rather than facing extinction, \u003cem\u003eC. humilis\u003c/em\u003e has survived across fragmented Mediterranean habitats, demonstrating its resilience. Despite the tumultuous geoclimatic evolution of the Mediterranean, which has fluctuated between warmer, subtropical conditions, arid and cooler climate, the species has managed to persist. Its genome is a testament to the species' ability to withstand both environmental stressors and historical climate changes.\u003c/p\u003e"},{"header":"4. Materials and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Phylogenetic and Genomic Comparative Analysis\u003c/h2\u003e \u003cp\u003eTo investigate the evolutionary history of \u003cem\u003eC. humilis\u003c/em\u003e, gene family construction was performed using OrthoFinder (v2.5.5) \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The analysis included additional palms, including palm species \u003cem\u003eP. dactylifera\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eE. guineensis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eC. nucifera\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, as well as other monocotyledons including \u003cem\u003eAnanas comosus\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eO. sativa\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eZ. mays\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, and some dicotyledons plants as outgroups including \u003cem\u003eA. thaliana, C. arabica\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eV. vinifera\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eT. cacao\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. The protein sequences of 70 single-copy orthologs were concatenated and aligned using MAFFT (Katoh et al., 2002) with the L-INS-i iterative refinement method. The most suitable model (JTT\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;F) was identified with ModelGenerator v0.85 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinf.nuim.ie/software/modelgenerator/\u003c/span\u003e\u003cspan address=\"http://bioinf.nuim.ie/software/modelgenerator/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Divergence times between species were estimated with BEAST v2.6.3 \u003csup\u003e68\u003c/sup\u003e, utilizing the JTT substitution model, four gamma categories, the Yule model, and an uncorrelated relaxed clock with lognormal distribution. The MCMC chain was run for 10,000,000 generations, recording parameters every 1000 generations. Calibration points for BEAST were obtained from the TimeTree database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.timetree.org/\u003c/span\u003e\u003cspan address=\"http://www.timetree.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, \u003cem\u003eC. nucifera\u003c/em\u003e versus \u003cem\u003eV. vinifera\u003c/em\u003e (145 mya), \u003cem\u003eO. sativa\u003c/em\u003e versus \u003cem\u003eZ. mais\u003c/em\u003e (60 mya), \u003cem\u003eP. dactylifera\u003c/em\u003e versus \u003cem\u003eC. nucifera\u003c/em\u003e (70 mya) and \u003cem\u003eA. thaliana\u003c/em\u003e versus \u003cem\u003eC. arabica\u003c/em\u003e (90 mya). The contraction and expansion of the gene families were observed by comparing the differences in cluster size between \u003cem\u003eC. humilis\u003c/em\u003e and each species using CAFE (v.5.1.0)\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Contracted and expanded gene families were analyzed, and GO enrichment was conducted using topGO \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. iTOLs \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e was used to visualize the species tree and annotations. Synteny analysis was performed between the two reference Arecaceae genomes: \u003cem\u003eP. dactylifera\u003c/em\u003e and \u003cem\u003eE. guineensis\u003c/em\u003e using JCVI (v 1.4.25)\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Whole genome duplication (WGD) analysis\u003c/h2\u003e \u003cp\u003eWGD analysis was conducted to determine the presence of lineage-specific and/or shared WD events among Aracaceae, using wgd (v2.0.38) \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe whole paranome (complete set of paralogous genes within a genome) was constructed using \u0026lsquo;wgd dmd\u0026rsquo; with default parameters. To infer genomic collinearity, \u0026lsquo;wgd syn\u0026rsquo; was applied using i-ADHoRe (v3.0.01)\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Subsequently, 'wgd ksd' was used to construct \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e distributions for both the paranome and anchor pairs (pairs of duplicated genes derived from WGD events which reside in duplicated segments) \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. The exponential-lognormal mixture model (ELMM) and the log-scale Gaussian mixture model (GMM) were employed within \u0026lsquo;wgd viz\u0026rsquo; to identify potential WGD components from the whole paranome and anchor pair \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e distributions, respectively, facilitating the effective identification of potential WGD events. With the \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e distribution of the anchor pairs, only those within the 95% of confidence level were retained using \u0026lsquo;wgd peak\u0026rsquo;. Finally, using a phylogenetic tree and fossil calibration of 17 related species previously reported by Chen et al \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, orthogroups were constructed by combining the anchor pairs with related homologous genes. WGD events were dated with \u0026lsquo;wgd focus\u0026rsquo; using mcmctree from PAML (v4.9)\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhen the paralogs of \u003cem\u003eC. humilis\u003c/em\u003e and orthologs with other Aracaceae species were identified, only those exclusive of \u003cem\u003eC. humillis\u003c/em\u003e and duplicated at least 7 times were evaluated. A functional analysis (GO analysis and literature review) was conducted to determine their biological significance.\u003c/p\u003e \u003cp\u003eDuplicated gene pairs were identified and classified into WGD-derived and SSD-derived gene pairs using the R package doubletrouble \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Paralog genes that are used as anchor pairs in syntenic regions are classified as SD, typically originating from whole genome duplications. All other duplicates are classified as SSD. These can be further classified based on their proximity: those adjacent in the genome are termed TD; those separated by only a few genes (default is 10, adjustable) are classified as PD; and all others fall under DD. Additionally, duplicates that arise from transposon-derived duplications are known as TRD. The location of duplicated genes and classification were plotted using chromomap in R \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by by the European Union - Next-GenerationEU - National Recovery and Resilience Plan (NRRP) \u0026ndash; MISSION 4 COMPONENT 2, INVESTIMENT N. 1.1, project reference: ChAMPION - \u003cem\u003eChamaerops humilis\u003c/em\u003e reference genome sequencing for Active conservation of an endemic Mediterranean Palm Including analyses Of biotic interactions Network, CUP F53D2300817000.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eG. C., Z. D., T. M., P. A conceived, write and revised the article, L-O. M. performed bioinformatic analysis, write and revised the article, T.M., R. M-A and M.M extract nucleic acid material, C. G., R. revised the article, S. R. and B. G provided biological material, C. H contributed with bioinformatic analysis and revised the manuscript.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe genome sequence and annotation data for \u003cem\u003eC. humilis\u003c/em\u003e, the sequencing data and the transcriptome data are accessible under NCBI BioProject no. PRJNA1247256.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTheissinger, K. \u003cem\u003eet al.\u003c/em\u003e How genomics can help biodiversity conservation. \u003cem\u003eTrends Genet. \u003c/em\u003e\u003cstrong\u003e39\u003c/strong\u003e, 545\u0026ndash;559 (2023).\u003c/li\u003e\n\u003cli\u003eTwyford, A. D. The road to 10,000 plant genomes. \u003cem\u003eNat. Plants \u003c/em\u003e\u003cstrong\u003e4\u003c/strong\u003e, 312\u0026ndash;313 (2018).\u003c/li\u003e\n\u003cli\u003eMc Cartney, A. 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ChromoMap: an R package for interactive visualization of multi-omics data and annotation of chromosomes. \u003cem\u003eBMC Bioinformatics \u003c/em\u003e\u003cstrong\u003e23\u003c/strong\u003e, 33 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6595308/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6595308/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eChamaerops humilis\u003c/em\u003e L. is the only Arecaceae species endemic to the western Mediterranean. Here, de novo genome sequencing allowed discovering reasons behind its exclusive persistence within the natural distribution area, although the troubled environmental changes occurred. The genome, estimated at 3.44 Gbp, and its annotation enabled the identification of 60,555 genes and 2.87 Gbp of repetitive regions. Evolutionary analyses revealed a whole-genome duplication event 48.02 mya, shared with other palm species, and a later divergence from \u003cem\u003ePhoenix dactylifera\u003c/em\u003e (17 mya). The characterization of gene duplication types, and the distribution of \u003cem\u003eK\u003c/em\u003e\u003csub\u003eS\u003c/sub\u003e values, uncovered multiple recent duplication waves, and the presence of \u003cem\u003eC. humilis\u003c/em\u003e-specific duplicated genes involved in responses to stress. Furthermore, expansions of transposable elements were detected and appear to be associated with major paleoclimatic events in the Mediterranean. These genomic features likely contributed to the resilience and long-term survival of \u003cem\u003eC. humilis\u003c/em\u003e within the Mediterranean.\u003c/p\u003e","manuscriptTitle":"Repetita iuvant. The extraordinary evolutionary history of the endemic palm Chamaerops humilis L. in the Mediterranean","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 14:33:54","doi":"10.21203/rs.3.rs-6595308/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c8ab7927-d96d-4473-8d0e-51b4192d1add","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48108389,"name":"Biological sciences/Plant sciences/Plant evolution"},{"id":48108390,"name":"Biological sciences/Evolution/Evolutionary genetics"},{"id":48108391,"name":"Biological sciences/Genetics/Evolutionary biology"}],"tags":[],"updatedAt":"2025-05-07T15:10:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-06 14:33:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6595308","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6595308","identity":"rs-6595308","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}