Comparative genomics of Coffea species highlights expansion of gene families linked to adaptation and secondary metabolism

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Meinhardt This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7032019/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 Coffee is among the most widely consumed beverages worldwide, with diverse Coffea species displaying a broad spectrum of flavors and important agronomic traits. Despite recent advances in genomics and the availability of assembled coffee genomes, comparative genomic analyses remain underutilized for crop improvement. In this study, we conducted comprehensive orthology and gene family evolution analyses across 24 plant species–including three Coffea species ( C. arabica , C. canephora , and C. eugenioides ), twelve Lamiales, and eight Solanales–to identify lineage-specific genomic features associated with adaptation and coffee quality traits. Using OrthoFinder in conjunction with gene family evolution models (CAFE5 and COUNT), we identified 1,552 orthogroups that are either specific to Coffea or have undergone significant expansion or contraction. Disease‑resistance genes were the most prominent among rapidly expanded gene families, reflecting ongoing evolutionary arms race with pathogens. We also observed asymmetric patterns of gene family evolution, including contraction of light and ethylene signaling gene families in C. canephora , contrasted by their expansion in C. arabica and C. eugenioides , which may contribute to their adaptation to shaded, high-altitude environments. Further analysis using COUNT identified twelve Coffea -specific gene family expansions associated with secondary metabolite biosynthesis, including key enzymes for caffeine and chlorogenic acid production. Our comprehensive catalog of lineage-specific orthogroups, supported by functional enrichment and phylogenetic analyses, provides a valuable genomic resource to support breeding programs focused on enhancing stress resilience, disease resistance, and cup quality in cultivated coffee. Coffea arabica Comparative genomics Gene family evolution Orthogroup analysis Secondary metabolism Lineage-specific genes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Coffee ranks among the most widely consumed beverages worldwide, supporting the livelihoods of approximately 100 million small farmers and their families globally (Vegro and de Almeida 2020 ; Siles et al. 2022 ; Freitas et al. 2024 ). Its enduring popularity is driven by its stimulating properties as well as the diverse range of flavors and aromas unique to different coffee varieties (ICO 2022 ; Freitas et al. 2024 ). As global consumption continues to rise—particularly with growing demand for specialty coffees—ensuring a consistent and high-quality supply has become increasingly critical (Davis et al. 2012 ; Merga and Alemayehu 2019 ). However, sustainable coffee production has been threatened by the increasing frequency of extreme weather events, which disrupt yields and drive market volatility (Poltronieri and Rossi 2016 ; Massrie 2025 ). These challenges underscore the need for breeding cultivars with enhanced tolerance to abiotic and biotic stresses (Lahai et al. 2025 ). Coffea arabica , representing 60% of global production, has a narrow genetic base stemming from a single hybridization event, limiting the available genetic variation for breeding programs (Scalabrin et al. 2020 ; Krishnan et al. 2021 ). The genetic diversity of other Coffea species including about 130 wild coffee species could provide potential for the genetic improvement of arabica coffee (Montagnon et al. 2025 ). Conventional breeding programs have seen limited progress, partly due to insufficient genomic resources, hindering the identification of novel alleles associated with desirable traits such as stress tolerance, disease resistance, and quality attributes (dos Santos et al. 2022 ; Ngure and Watanabe 2024 ). Recent advancements in genomics, including high-quality genome assemblies for C. arabica (NCBI BioProject: PRJNA698600), C. eugenioides (BioProject: PRJNA497891), and C. canephora (Denoeud et al. 2014 ), provide opportunities for comparative and evolutionary analyses to uncover the genetic basis of important agronomic and quality traits. C. arabica (2n = 4x = 44) is an allotetraploid species that originated from a natural hybridization event between C. eugenioides and C. canephora approximately 350,000–610,000 years ago (Salojärvi et al. 2024 ). This hybridization resulted in a complex genome shaped by contributions from both parental lineages. Understanding how each progenitor genome influences ecological adaptations in C. arabica –such as traits related to stress tolerance, disease resistance, and secondary metabolism–can guide the development of new varieties with enhanced resilience and productivity. Orthology analyses are critical for coffee improvement efforts, enabling researchers to accurately identify candidate genes associated with desirable traits among diverse coffee species (Varshney and Dubey 2009 ; Shahrajabian et al. 2021 ; Mishra et al. 2022 ). Distinguishing orthologs (genes derived from a common ancestral gene across species) from paralogs (genes duplicated within a species) is essential for reliably transferring functional knowledge from well-studied model plants or closely related species to coffee (Koonin 2005 ). This precision allows more accurate prediction of gene function and facilitate the discovery of targets linked to disease resistance, drought tolerance, yield stability, and cup quality (Marraccini et al. 2011 ; Cacas et al. 2011 ). Comparative genomic analyses across Coffea species can also reveal the evolutionary dynamics of gene families (orthogroups) associated with key traits such as flavor biochemistry, defense, or temperature adaptation (Denoeud et al. 2014 ; Lachica et al. 2025 ). In particular, lineage-specific or rapidly evolving orthogroups, defined as gene families unique to or significantly diverged in a lineage, are of great interest due to their roles in adaptive evolution and functional diversification (Lespinet et al. 2002 ; Park et al. 2023 ). The absence of certain orthogroups in specific lineages may reflect gene loss driven by relaxed selection or functional redundancy, while their expansion may be linked to lineage-specific innovations critical for ecological adaptation or agronomic performance. Expanding comparative analyses beyond C. arabica and its diploid progenitors to include other plant species provides a broader evolutionary perspective. Coffea belongs to the Order Gentianales within the Lamiids clade, which also includes the Orders Lamiales, Solanales, Boraginales, and Vahliales (Stull et al. 2015 ; Deng et al. 2015 ; Alawfi and Alzahrani 2023 ). Comparing coffee genomes to those of related species within these Orders enable the identification of gene families uniquely conserved or diversified in the coffee lineage. Such coffee-specific orthogroups may include genes encoding enzymes involved in secondary metabolism (e.g., alkaloids and phenolics) that contribute to the distinctive aroma and flavor of coffee (Sunarharum et al. 2014 ). For example, lineage-specific expansions of N-methyltransferases involved in caffeine biosynthesis have been identified in coffee, arising through gene duplication events after divergence from a common ancestor (Denoeud et al. 2014 ). Identifying lineage-specific genes or pathways also holds profound implications for managing biotic and abiotic stresses. As the global climate changes and pests and diseases become more prevalent, coffee growers are under pressure to adopt more resilient cultivars (Ahmed et al. 2021 ; Cassamo et al. 2023 ). By uncovering the genetic mechanisms that enable C. canephora to thrive in hotter conditions or C. arabica to endure cooler, high-altitude environments, breeders can integrate these adaptive traits into breeding programs. Ultimately, this approach supports developing varieties with broader environmental tolerance while preserving or even improving cup quality (Ferrão et al. 2024 ). In this study, we conducted an in-depth comparative genomic analysis to investigate orthologous and paralogous relationships among three coffee species: C. arabica , C. canephora , and C. eugenioides . We extended our analysis to 21 additional plant species from Lamiales, Solanales, and Gentianales to identify Coffea -specific genes and gene families. By focusing on lineage-specific and rapidly evolving orthogroups, we aimed to elucidate the genetic and evolutionary basis for coffee-specific traits and adaptations. Functional enrichment and phylogenetic analyses were conducted to evaluate the biological relevance and evolutionary patterns of these gene sets, with a particular emphasis on lineage-specific adaptive evolution in the Coffea genus. Our findings deepen our understanding of coffee biology and provide practical targets to improve crop performance, quality, and sustainability. Methods Genome data retrieval and processing Genomic protein sequences for 24 plant species were retrieved from NCBI Database ( https://www.ncbi.nlm.nih.gov/datasets/genome/ ) (Supplementary Table S1 ). This dataset includes three coffee species ( C. arabica , C. eugenioides , C. canephora ) and 21 additional species chosen based on their evolutionary relationships with Coffea . Coffea resides within the Order Gentianales of the Lamiid clade, closely related to the Orders Lamiales and Solanales (Stull et al. 2015 ; APG IV 2016 ). Thus, twelve species from Lamiales, eight from Solanales, and four from Gentianales (including the three coffee species) with complete genome assemblies and protein annotations were selected. For genes with multiple isoforms, the longest protein isoform was selected as a representative sequence (Park et al. 2020 ). The finalized protein dataset was subsequently used to identify orthologous groups across the species. Orthogroup inference and phylogenetic analysis Orthologous gene identification was conducted using OrthoFinder v2.5.5 (Emms and Kelly 2019 ) with protein-coding sequences from all 24 species as input. The analysis was performed using default parameters, which involve an all-versus-all search using Dimond BLASTP (Buchfink et al. 2021 ) to assess sequence similarity, followed by hierarchical clustering to assign genes into orthogroups. These orthogroups represent gene families derived from a single ancestral gene in the last common ancestor of the species examined. For each orthogroup, multiple sequence alignments were generated using MAFFT (Katoh and Standley 2013 ) and gene trees were inferred using FastTree (Price et al. 2010 ). The species tree was reconstructed using the STAG method (Species Tree from All Genes), and rooting was achieved using the STRIDE method (Species Tree Root Inference from Duplication Events) as implemented in OrthoFinder. Trees were visualized using FigTree v1.4.4 ( http://tree.bio.ed.ac.uk/software/figtree ). Gene family evolution analysis Gene family expansion and contraction were analyzed using Computational Analysis of gene Family Evolution (CAFE) v5.1 (Mendes et al. 2021 ). To minimize biases associated with large variance in gene copy numbers, orthogroups containing at least 100 gene models in any species were excluded from the CAFE analysis. The lambda (λ) parameter, representing the birth and death rate of gene families, was uniformly estimated across the phylogeny under a Poisson rate model. An error rate, accounting for potential inaccuracies in genome annotation (e.g. missing or incorrectly assigned gene copies), was also estimated and incorporated into the analysis. Gene families exhibiting significant expanding or contraction were identified as rapidly evolving if they showed a CAFE family-wide P -value of less than 0.05. Gene family evolution was further analyzed using COUNT software (Csűös 2010 ), which employs a Bayesian posterior probabilistic model to estimate gene family gain, loss, and duplication events along each branch of the phylogenetic tree. COUNT is particularly effective for handling zero-inflated gene family distributions, characterized by the absence of genes (i.e., zero-copy number) in most lineages due to gene loss or limited lineage-specific expansions. By modeling both presence/absence and copy number variation, COUNT enhances accuracy in evolutionary inferences, especially for gene families that are rare or highly specific to particular lineages. Functional annotation and GO enrichment Functional annotation of the entire protein set was preformed using the Trinotate pipeline (Bryant et al. 2017 ), as described by Park et al ( 2020 ). Briefly, genomic protein sequences were first queried against the UniProtKB/Swiss-Prot database using BLASTP with an E-value cutoff of 1e-5. In addition, the protein sequences were analyzed against the Pfam database using the hmmsearch tool of HMMER (Finn et al. 2011 ) to identify conserved domains. Based on sequence similarity, functional and Gene Ontology (GO) annotations were assigned to the proteins from UniProtKB/Swiss-Prot entries. For each orthogroup, a consensus function was derived from the majority annotation among its member proteins. GO enrichment analysis was carried out to identify biological processes significantly overrepresented in orthogroups using the hypergeometric test implemented via the phyper function in R (R core Team 2024 ). A False Discovery Rate (FDR) threshold of 0.05 was applied to determine statistical significance. Chromosomal mapping of candidate genes Chromosomal locations of selected gene families were retrieved from the C. arabica reference genome (NCBI: GCF_003713225.1). Gene loci were visualized using the LinkageMapView package in R (Ouellette et al. 2018 ), enabling interpretation of their distribution across the 22 chromosomes and subgenomes corresponding to C. canephora and C. eugenioides origins. Results Orthologous group construction Coffea belongs to the Order Gentianales, which is closely related to Lamiales, Solanales, Boraginales, and Vahliales, within the Lamiids clade (Stull et al. 2015 ; APG IV 2016 ). To understand the orthologous relationships between Coffea and other species and within Coffea genus, we analyzed genomic data from three Coffea species ( C. arabica , C. canephora , and C. eugenioides ) and 21 additional plant species of the Lamiids clade. Given the availability of complete genome sequences and gene annotations, we selected twelve species from Lamiales, eight species from Solanales, one from Gentianales. None from Boraginales and Vahliales were selected due to lack of species with complete genome. Orthologous gene identification was performed using OrthoFinder (Emms and Kelly 2019 ) on the total 769,395 genes across the 24 species. The analysis identified 30,986 orthogroups, to which 95% (728,690 genes) of the total genes were assigned, while 5% (40,705 genes) remained unassigned. Orthogroup sizes varied considerably: 40% contained five genes or less, 32% had between 6 and 30 genes, 25% had between 31 and 100 genes, and only 2% consisted of more than 100 genes, reaching up to 1,764 gene members (Supplementary Fig. S1 ). The largest orthogroup (OG00000, 1,764 genes) was annotated as a Myb-DNA-binding transcription factor. The second largest (OG00001), with 1,505 was annotated as retrovirus-related Pol polyprotein (Supplementary Table S2 ). Notably, four additional orthogroups among the top ten–such as OG00003 with 1,282, OG00004 with 1,149, OG00005 with 1,117, and OG00007 with 1,038–were also associated with transposon-related functions (Supplementary Table S2 ), suggesting extensive duplication and possibly active transposition events across the species analyzed. Two orthogroups could not be assigned with any functions due to lack of homology to proteins of the UniProtKB/Swiss-Prot database and the remaining two orthogroups were annotated as leucine-rich repeat (LRR) receptor-like kinase and Pentatricopeptide repeat (PPR)-containing protein. Using gene trees derived from all orthogroups, OrthoFinder constructed a species tree that provide foundation to differentiate orthologs from paralogs across the species. The resulting species tree was consistent with established phylogenetic relationships, grouping species according to taxonomic Orders (Fig. 1 ). Lamiales species clustered closer to Solanales than to Gentianales, consistent with previous phylogenetic studies based on plastid sequences (Stull et al. 2015 ). Gene assignments to orthogroups varied among species, ranging from 86% of total genes in Striga asiatica to 99.4% in C. eugenioides (Fig. 2a). C. arabica and C. canephora exhibited assignment rates of 98.6% and 94%, respectively. Gene duplication rate among species also varied considerably, with D. hygrometricum highest (62%) and C. canephora lowest (6.8%) (Fig. 2b). Notably, gene duplication rates exhibited a strong positive correlation with total gene counts, with R 2 of 0.61, suggesting a pivotal role of gene duplication in genome evolution (Fig. S2 ). Particularly, three species showed a strikingly high degree of gene duplication: D. hygrometricum (62%), S. splendens (59%), and C. arabica (38%) (Fig. 2b). These elevated duplication rates could be attributed to whole-genome duplication events, as these three species are known as tetraploids (Xiao et al. 2015 ; Jia et al. 2021 ). Lineage-specific orthogroups and unassigned genes Orthogroup memberships were analyzed to elucidate lineage-specific features. For coffee species within Gentianales, we focused on species-level orthogroups (including the three coffee species and O. corymbose ), while for non-coffee species, we aggregated orthogroup memberships at the Order level (Lamiales and Solanales), resulting in six membership groups (Fig. 3 ). Overall, 37% (11,454) of orthogroups contained at least one gene from these six groups, while 22% (6,956) were exclusive to Lamiales and 14% (4,248) were exclusive to Solanales. Notably, only 1.7% (524) were unique to the three coffee species (Supplementary Table S3). GO enrichment analysis of the Coffea -specific orthogroups highlighted defense/stress-related processes (hypersensitive response, salicylic acid biosynthesis, response to benzoic acid, response to xenobiotic stimulus, and cellular hypotonic response), metabolic pathways (alkaloid, thalianol, and fatty acid beta-oxidation), and protein regulation (SRP-dependent protein targeting, serine/threonine phosphatase activity) (Table 1). Given that alkaloid and thalianol-derived terpenoids can serve as chemical deterrents against pathogens due to their toxicity and antimicrobial properties (Khameneh et al. 2019 ; Yan et al. 2021 ), the enrichments suggested that Coffea species may have evolved unique defense mechanisms including specialized metabolism, contributing to their distinctive flavor profiles. Additional GO terms, while less significantly enriched, further revealed a strong enrichment for plant defense, secondary metabolism, and stress responses. These included systemic acquired resistance (GO:0009627), salicylic acid metabolism and biosynthesis (GO:0009696, GO:0080142), indole alkaloid biosynthesis (GO:0035835), eugenol biosynthesis (GO:0042855), glycyrrhetinate biosynthesis (GO:1902386), pentacyclic triterpenoid metabolism (GO:0019742), catabolism of 11-oxo-β-amyrin (GO:1902382), photorespiration, photooxidative stress, and xenobiotic catabolism. Together, these processes highlighted a lineage-specific adaptation in Coffea involving an intricate interplay of defense, metabolic reprogramming, and stress adaptation (Supplementary Table S4 ). When focusing on species-specific orthogroups within Coffea , 0.7% (n = 205) of orthogroups were specific to C. arabica , 0.5% (n = 163) to C. canephora , and 0.2% (n = 71) to C. eugenioides . GO enrichment analyses revealed species-specific functional signatures (Supplementary Table S5). Orthogroups specific to C. eugenioides exhibited enrichments similar to the Coffea group in defense- and metabolism-related processes, such as nucleotide metabolism (inosine monophosphate (IMP) metabolism), folate metabolism (tetrahydrofolate biosynthesis), and secondary metabolite biosynthesis (coumarin, coniferin). Additionally, developmental and adaptative processes were enriched, including photorespiration, cell growth, floral organ identity, and root morphogenesis (Table 2). In contrast, C. canephora showed enrichments in GO terms associated with diverse biological processes, including cytoskeletal dynamics (actin filament severing and capping, astral microtubule organization, mitotic spindle localization), primary and secondary metabolism (xylulose, thymidine, riboflavin, and neutral amino acid transport, carotenoids, lignans, and isoprenoids) and regulatory and hormonal signaling pathways (photosynthesis, brassinosteroid-mediated signaling, and cellular response to ethylene stimulus) (Table 2). C. arabica- specific orthogroups were enriched in plastid transcription and photoreactive repair, linked to enhanced photosynthesis efficiency (Table 2). These divergent enrichment patterns highlighted species-specific evolutionary adaptations, likely reflecting diverse survival strategies and distinct chemical profiles across species. In addition to the lineage-specific orthogroups, we examined unassigned genes—those not placed in any orthogroup due to lack of detectable homology to genes in other species. These include orphan genes, possibly conferring unique functions to their respective species. We identified 601 unassigned genes in C. arabica , 1,530 genes in C. canephora , and 166 genes for C. eugenioides . While no significant GO term enrichment was found for the unassigned genes in C. arabica and C. canephora , C. eugenioides showed enrichments related to leaf development (adaxial/abaxial pattern specification), defense responses (positive regulation of defense response to bacterium), and metal homeostasis (intracellular sequestering of iron and manganese ion homeostasis) (Supplementary Table S6). Lineage-specific expansion and contraction of gene families in coffee Gene families that undergo rapid expansion (gene duplication/gain) or contraction (gene loss) provide valuable insights into adaptive evolution, functional innovation, and the loss of redundant traits under selective pressures (Koonin 2005 ; Lažetić and Troemel 2021 ; Fang et al. 2022 ). CAFE5 detected significantly expanded (187) and contracted (3) orthogroups in Coffea (Fig. 4 , Supplementary Table S7). Among the most significantly expanded were gene families associated with disease resistance, particularly NBS-LRR containing proteins, suggesting strong pathogen-driven selective pressures (Supplementary Table S7). Enriched GO terms in the expanded families revealed a wide range of biological processes, with a strong emphasis on defense (salicylic acid response, pathogen detection), specialized metabolism (alkaloids, terpenoids, coumarins), hormone signaling, and circadian/light signaling (Supplementary Table S8). The three contracted families involved protein regulation and homeostasis (pectinesterase inhibitor, kinase-associated proteins). (Supplementary Table S7) At the species level, C. arabica showed extensive gene family expansions (367 orthogroups) compared to contractions (20 orthogroups), consistent with its recent allopolyploid origin (Supplementary Table S9). In contrast, the diploid C. canephora exhibited more pronounced gene family contractions (113) than expansions (18) (Supplementary Table S10), while C. eugenioides showed a balanced pattern (60 expansions and 59 contractions) (Supplementary Table S11). Expanded families in C. arabica included transposon-related, signaling (Ras, Zinc finger RICESLEEPER), and metabolic enzymes (Cytochrome P450, pectate lyase) (Supplementary Table S9). Interestingly, many of these expansions appeared to contain genes from C. eugenioides but few genes from C. canephora. For example, orthogroup OG00085 (Cytochrome P450) included only four genes from C. canephora but contained 63 genes of C. eugenioides and 97 of C. arabica . Phylogenic analysis indicated that all coffee genes of OG00085 descended from three ancestral genes, followed by duplications predominantly in C. eugenioides and C. arabica (Supplementary Fig. S3). Contracted orthogroups included several disease resistance-related proteins (salicylic acid signaling components, and receptor-like kinases) and metabolic enzymes, suggesting potential reduction in innate immune or metabolic capacity (Supplementary Table S9). In C. canephora , expansions included multiple components related to plant immune responses, including resistance (R) proteins, receptor-like kinases, and hormone signaling, while contractions were more extensive and spanned diverse functional categories including stress responses, transcriptional regulation, hormone and light signaling, protein degradation, metabolism, and transposable element activity (Supplementary Table S10). Many of these contractions appeared to be lineage-specific to C. canephora and absent from C. eugenioides . For instance, orthogroup OG00337, annotated as Far-Red Impaired response 1 (FAR1) involved in phytochrome-signaling, contained no gene from C. canephora but had 58 genes from C. eugenioides and 46 genes from C. arabica . Interestingly, we found that four additional FAR1-related orthogroups (OG01057, OG01614, OG01227, OG09699) were also contracted specifically in C. canephora (Supplementary Table S10). C. eugenioides showed expansions in gene families associated with disease resistance, transposons, light signaling (FAR1), and protein modification (Dolichol-phosphate mannose synthase and Cytosolic sulfotransferase), while contractions involved diverse biological roles including disease-resistance, hormone and stress signaling, secondary metabolism, transposon, and development (MADS-box) (Supplementary Table S11). Overall, disease-resistance-related gene families were commonly identified as rapidly evolving across all three Coffea species, suggesting dynamic immune adaptation driven by their respective ecological pressures. Furthermore, these patterns suggested that polyploidy in C. arabica facilitated the retention and expansion of gene families, particularly those derived from C. eugenioides , while C. canephora appeared to have undergone more extensive gene family reduction. Coffea -specific expansions in secondary metabolism gene pathways Caffeine is synthesized only a few plant genera– Coffea (coffee), Theobroma (cacao), and Camellia (tea)–and phylogenetic studies indicated that genes involved in caffeine biosynthesis evolved independently through convergent evolution (Denoeud et al. 2014 ). Three paralogous N-methyltransferases catalyze the sequential methylation of xanthosine to caffeine: xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT), and dimethylxanthine methyltransferase (DXMT) (Denoeud et al. 2014 ). Consistent with previous findings, our orthology analysis clustered these methyltransferase paralogs into a single orthogroup (OG04312). This orthogroup included five genes from Solanales species, none from Lamiales, and, within Gentianales, it contained three genes from O. corymbose , nine from C. canephora , 11 from C. eugenioides , and 15 from C. arabica . A phylogenetic reconstruction of OG04312 showed that, apart from two outliers, all Coffea methyltransferases formed a monophyletic, coffee-lineage-specific clade, indicating an expansion event unique to the Coffea lineage (Fig. 5 ). Despite this evident expansion, OG04312 was not identified as “rapidly evolving” by CAFE5. This is likely due to the limitations of single-λ birth–death model in CAFE5, which averages gene gain/loss rates across the entire phylogeny. As a result, gene families that expand predominantly at a single lineage node may not reach statistical significance under this model. To better detect Coffea- lineage-restricted duplications, we applied COUNT Bayesian duplication-gain-loss model (Csűös 2010 ). COUNT estimates the posterior probability of gene gain, duplication, or loss events on a per-branch basis, making it sensitive to lineage-restricted events. Using this approach, OG04312 showed a high posterior probability of expansion (0.84) at the node corresponding to the Order Gentianales. Using a posterior-probability threshold of 0.84, we identified 11 additional orthogroups with putative Coffea -lineage-specific duplications (Table 3 ). Several of these gene families are involved in secondary metabolism, potentially contributing to traits unique to Coffea . For example, orthogroups annotated as caffeic acid 3-O-methyltransferase (COMT) and cinnamoyl-CoA reductase (CAD) play critical roles in the phenylpropanoid pathway and may influence chlorogenic acid biosynthesis, major precursors of phenolic compounds that contribute to coffee bitterness (Campa et al. 2003 ). Orthogroups of strictosidine synthase, UDP-glycosyltransferase, and beta-amyrin monooxygenase genes are involved in alkaloid or terpenoid metabolism, potentially influencing coffee aroma and flavor. Additional lineage-specific duplications were detected in orthogroups associated with broader physiological and stress-related functions: NIN-like protein 4 (NLP4; associated with nitrate signaling), alkenal reductase and citrate-binding protein (detoxification/homeostasis), WAT1-related protein (cell wall development), and leucine-rich repeat receptor kinases (immune signaling). Discussion Lineage-specific gene families play crucial roles in adaptative evolution and functional diversification in plants. When a gene family is unique to, or undergoes rapid expansion in a particular lineage, it often reflects the selective pressures imposed by a specific ecological niche (Lespinet et al. 2002 ; Rensing et al. 2008 ). Identifying lineage-specific gene sets is therefore a powerful strategy for uncovering the genetic mechanisms underlying biologically and agriculturally important traits. In crops, these genes often underlie key domestication traits, enhanced stress tolerance, or the biosynthesis of specialized metabolites (Huang et al. 2012 ; Qin et al. 2014 ). For instance, lineage-specific orthogroups may encode enzymes involved in the production of defensive metabolites, proteins mediating abiotic stress responses, or regulatory factors fine-tuning developmental processes under local environmental conditions. In the Coffea genus, lineage-specific gene family expansions appear central to the extensive diversity observed in agronomic traits, flavor profiles, and aroma characteristics, much of which likely arose through adaptive evolution. A striking illustration is the expansion of the N-methyltransferase (NMT) gene family responsible for caffeine biosynthesis. Phylogenetic evidence clearly indicates that these methyltransferases evolved independently in Coffea , Camellia , and Theobroma through convergent evolution–parallel recruitment of NMT genes for caffeine production in each lineage rather than inheritance from a common caffeinated ancestor. (Denoeud et al. 2014 ; Xia et al. 2017 ). This underscores the value of comparative orthogroup analyses between Coffea and its closely related species in identifying key-trait associated genes, with broad implications for comparative genomics, evolutionary biology, and crop improvement. Our comparative analysis showed that gene duplication rates varied widely even among closely related species. While total gene count and duplication frequency were generally positively correlated (Supplementary Fig. S2 ), several exceptions suggest that gene duplication is not uniformly responsible for genome expansion. For instance, within the Order Gentianales, C. eugenioides , C. canephora , and O. corymbose exhibit similar total gene counts (ranging from 24,917 to 28,952), but show markedly different duplication rates: 26% in O. corymbose , 13% in C. eugenioides , and 7% in C. canephora . These differences imply that gene duplication may be concentrated in specific functional gene families, likely reflecting species-specific adaptive pressures. Indeed, functional enrichment among duplicated genes in C. eugenioides and C. canephora revealed overrepresentation of functions related to defense, signaling, and metabolism, supporting the hypothesis of adaptation-driven gene family expansion (Supplementary Table S12). These findings suggest that duplication patterns are functionally biased rather than random, providing insight into the mechanisms underlying lineage-specific innovation in Coffea species. To further investigate how these duplication patterns contribute to functional divergence among species, we examined rapidly evolving orthogroups that exhibit species-specific expansion or contraction. Interestingly, many of the orthogroups that were rapidly contracting in C. canephora included genes present in both C. eugenioides and C. arabica , with several of these orthogroups identified as rapidly expanding in those species (Supplementary Table S10). This asymmetric pattern aligns with phylogenetic evidence placing C. eugenioides as more closely related to C. arabica , and suggests contrasting evolutionary pressures that led to gene loss in the C. canephora lineage following divergence. Alternatively, this pattern could reflect pre-existing differences between C. eugenioides and C. canephora prior to the hybridization event that gave rise to C. arabica . To evaluate these possibilities, we examined the FAR1 gene family (OG00337) as a case study. Phylogenetic analysis indicated that all members descended from a common ancestral gene, followed by lineage-specific duplications in C. arabica and C. eugenioides , but complete absence in C. canephora (Supplementary Fig. S4). Chromosomal mapping in C. arabica showed that FAR1 genes were distributed across both subgenomes, implying ancestral presence in both parental lineages and suggesting gene loss specifically in C. canephora (Supplementary Fig. S5). FAR1 proteins are integral to Phytochrome A signaling, mediating red/far-red light sensing, shade avoidance, temperature adaptation, and flowering time (Lin and Wang 2004 ; Wang and Wang 2015 ; Lu et al. 2022 ). In Betula pendula (silver birch), natural variation in the FAR1 homologs correlates with latitude, longitude, and local temperatures, reinforcing their role in environmental adaptation (Salojärvi et al. 2017 ). Given the ecological preference of C. arabica and C. eugenioides for shaded, high-altitude habitats versus the warmer, sunnier lowlands favored by C. canephora (Lashermes et al. 1999 ; Davis et al. 2012 ), FAR1 gene expansion may support adaptation to low-light and cooler conditions. Another example of potentially adaptation-driven expansion involves the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene family, which catalyzes the final step of ethylene biosynthesis. Orthogroup OG00545 contains only three ACO genes from C. canephora , compared to 13 genes from C. eugenioides and 16 from C. arabica , identified as rapidly contracting in C. canephora and rapidly expanding in C. arabica (Supplementary Table S9; Supplementary Table S10). The dramatic disparity suggests that C. arabica and C. eugenioides might have expanded their ACO repertories to fine-tune ethylene responses under their native high-altitude, shaded environments. Ethylene regulates a wide range of physiological processes, including seed germination, fruit ripening, abiotic stress responses, often in coordination with abscisic acid (ABA) and light-signaling pathway (Ju and Chang 2015 ). For instance, ethylene can promote cell elongation and regulate leaf morphology, potentially reducing pathogen exposure and limiting water loss. In C. arabica , ACO expression (e.g., CaACO1 ) is downregulated during water deficit and restored upon rehydration, coordinating with ABA to modulate stomatal opening (Lima et al. 2021 ). Ethylene also plays a key role in fruit ripening in coffee, with multiple ACO genes increasing expression in ripening stages, especially early-ripening cultivars (Ságio et al. 2014 ). These findings suggest that ACO gene expansion may confer fine-tuned ethylene-mediated regulation of development and stress responses in C. arabica . CAFE5 models gene family evolution using a single birth-death rate λ applied uniformly across the phylogeny, assessing gene family size changes against the expectations under this constant rate (Csűös 2010 ). However, when a gene family is absent in most species (i.e., zero-inflation), isolated expansions at specific nodes can be statistically masked by the prevalence of zeros (“averaged out” by the zeros), reducing detection sensitivity. Consequently, CAFE5 may fail to detect lineage-restricted expansions or contractions. In contrast, the COUNT tool employs a more flexible gain-loss-duplication (GLD) model with separate parameters, explicitly distinguishing the first “gain” of a family (zero to one) from subsequent duplications (n to n + 1), offering improved resolution in detecting lineage-restricted events (Csűös 2010 ). Using COUNT’s Bayesian framework, we identified 12 gene families that exhibit Coffea -lineage-specific expansions, many of which are involved in secondary metabolism including methyltransferases responsible for caffeine biosynthesis (Table 3 ). These expansions likely reflect evolutionary adaptations for producing coffee-specific compounds such as caffeine and chlorogenic acids, both of which contribute to distinct flavor and chemical defense profiles. Although further phylogenetic and functional validation of these families is needed to elucidate their roles in trait development and adaptation, the lineage specificity and association with sensory and adaptive traits make these gene families promising targets for breeding programs aimed at improving coffee quality. Collectively, our analysis of lineage-specific orthogroups, species-specific orphan genes, and gene duplication dynamics provides critical insights into the unique genomic landscape and adaptive evolution of coffee species. These findings offer a valuable genomic resource for identifying genetic targets that can be leveraged to enhance stress resilience, disease resistance, and cup quality in cultivated coffee, thereby supporting future crop improvement efforts. Declarations Data availability All data generated during this study are provided within the manuscript or supplementary information files. Competing interests The authors declare no competing interests. Funding This research was funded by appropriated funds from the United States Department of Agriculture, Agricultural Research Service (USDA-ARS) (Project Number 8042-21000-303-00D) Author contribution SP conceptualized the study and designed the experiments. SP, DZ, and LWM secured funding for the project. SP and SO performed data analysis with assistance from EA, DZ, and LWM. SP wrote the initial draft, and all authors reviewed and approved the final version. Acknowledgements This research used resources provided by the SCINet project of the USDA Agricultural Research Service (ARS project number: 8042-21000-303-00D). Mention of a trade name, proprietary product, or vendor does not constitute an endorsement, guarantee, or warranty by the USDA and does not imply its approval to the exclusion of other products or vendors that may be suitable. References Ahmed S, Brinkley S, Smith E, et al (2021) Climate Change and Coffee Quality: Systematic Review on the Effects of Environmental and Management Variation on Secondary Metabolites and Sensory Attributes of Coffea arabica and Coffea canephora. Front Plant Sci 12: Alawfi MS, Alzahrani DA (2023) Insights into the phylogenetic relationship of the lamiids orders based on whole chloroplast genome sequencing. 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Antibiotics 10:318. https://doi.org/10.3390/antibiotics10030318 Table 2 and 3 Table 2 and 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigureS15.docx TableS112legends.docx SupplementaryTableS111.xlsx Table2.docx Table3.docx 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7032019","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":483001166,"identity":"483ee9d6-eac5-4f66-94bb-3ab5802e1368","order_by":0,"name":"Sunchung Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYBAC+QYGBmYYh7GhAkgeIKDF4ACKljPEaGFA1tLYRowWidyDnwvb7tg1SDc/+zhz3mF7vuMHmF98bMOtRX5GXrL0zLZnyQ0yx4xnbtx2OHHmmQQ2y5l4tDDcyDFj5m07nMwgkWDM+HDb4QSDAwlsxjxniNKS/pnx4ZzD9gbnHxCnxY5BIseYcWPDYcYNNxKYH/NU4PH+mTfG0jznDiewyZwpZpxxLD1x5o2HbYwz8GiRb88x/MxTdtieX7p9M2NPjbU93/nkwx8+GOBxGBQktknA2YxIbDzAngFJGfMHYrSMglEwCkbBiAEAfTtWQgjZXggAAAAASUVORK5CYII=","orcid":"","institution":"Beltsville Agricultural Research Center","correspondingAuthor":true,"prefix":"","firstName":"Sunchung","middleName":"","lastName":"Park","suffix":""},{"id":483001168,"identity":"97b4fa18-fafd-45b3-af9d-ecccf2873cf8","order_by":1,"name":"Ezekiel Ahn","email":"","orcid":"","institution":"Beltsville Agricultural Research Center","correspondingAuthor":false,"prefix":"","firstName":"Ezekiel","middleName":"","lastName":"Ahn","suffix":""},{"id":483001169,"identity":"42a9b029-bc8e-48df-b0c0-737133dd34e6","order_by":2,"name":"Sookyung Oh","email":"","orcid":"","institution":"Beltsville Agricultural Research Center","correspondingAuthor":false,"prefix":"","firstName":"Sookyung","middleName":"","lastName":"Oh","suffix":""},{"id":483001170,"identity":"c7ef117a-509f-45f2-8315-eb85d342e91f","order_by":3,"name":"Dapeng Zhang","email":"","orcid":"","institution":"Beltsville Agricultural Research Center","correspondingAuthor":false,"prefix":"","firstName":"Dapeng","middleName":"","lastName":"Zhang","suffix":""},{"id":483001171,"identity":"36d2627d-2446-4b61-89a4-18f8cc2a794e","order_by":4,"name":"Lyndel W. Meinhardt","email":"","orcid":"","institution":"Beltsville Agricultural Research Center","correspondingAuthor":false,"prefix":"","firstName":"Lyndel","middleName":"W.","lastName":"Meinhardt","suffix":""}],"badges":[],"createdAt":"2025-07-02 18:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7032019/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7032019/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86432910,"identity":"fef40a89-05c7-470a-acfc-9baf9cba0f18","added_by":"auto","created_at":"2025-07-10 14:58:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":178095,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecies tree inferred using the STAG method. \u003c/strong\u003eThe tree was reconstructed by integrating gene trees from all orthogroups using the Species Tree Inference from All Genes (STAG) method, which identifies the most consistent species relationships supported across gene trees. Branch colors represent different taxonomic Orders: blue for Lamiales, brown for Solanales, and red for Gentianales.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7032019/v1/5c1f8dc5d158cf929fb0b26e.png"},{"id":86432020,"identity":"d76d3036-e94a-4088-8dd7-c24b233c8a7a","added_by":"auto","created_at":"2025-07-10 14:50:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":238106,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummary of orthogroup analysis across 24 plant species \u003c/strong\u003e(a) Proportion of genes assigned to orthogroups, and (b) proportion of gene duplication events, both shown as percentages of the total gene count for each species. (c) Number of genes unassigned to any orthogroup per species. (d) Number of orthogroups specific to each species. Different colors represent the taxonomic Order of each species: blue for Lamiales, brown for Solanales, and red for Gentianales.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7032019/v1/29b4f96d9c5092157216a501.png"},{"id":86432023,"identity":"1ece1775-bc45-48f4-9541-57e7ee9762f4","added_by":"auto","created_at":"2025-07-10 14:50:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":118264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of species membership across orthogroups. \u003c/strong\u003eThe histogram displays the number of orthogroups (y-axis) grouped by the number of species contributing to each orthogroup (x-axis). Species from Lamiales and Solanales are aggregated at the Order level, while \u003cem\u003eCoffea\u003c/em\u003e species are shown individually. Dots below each bar indicate the membership groups (Lamiales, Solanales or individual species) present in the orthogroups, with \u003cem\u003eCoffea\u003c/em\u003e species highlighted in red.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7032019/v1/60a37a4e740c5018a74b723f.png"},{"id":86432027,"identity":"fc2cebfd-e14b-47c5-94d2-94250e945b60","added_by":"auto","created_at":"2025-07-10 14:50:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":78185,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNumber of significantly rapidly contracted (-) or expanded (+) orthogroups across species. \u003c/strong\u003eSpecies from the Orders Lamiales and Solanales are collapsed at the order level, with values representing the median number of rapidly evolving orthogroups for species within each Order. Node-specific contractions and expansions inferred for the \u003cem\u003eCoffea\u003c/em\u003e lineage are shown at the \u003cem\u003eCoffea\u003c/em\u003e node.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7032019/v1/fe00ae08cd0bcf64783d9a57.png"},{"id":86432915,"identity":"bea43747-ea97-4a92-99a5-f0fab4468157","added_by":"auto","created_at":"2025-07-10 14:58:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":193349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of the caffeine synthesis gene family (OG04312)\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eThe tree was constructed using the maximum likelihood method in FastTree, based on multiple protein sequence alignments. Genes are color-coded by taxonomic origin: species-level resolution for \u003cem\u003eCoffea\u003c/em\u003e species and Order-level resolution for species from Solanales and Lamiales. Gene identifiers include abbreviated species names as prefixes: Can,\u003cem\u003e Capsicum annuum\u003c/em\u003e; Ds,\u003cem\u003e Datura stramonium\u003c/em\u003e; Lf,\u003cem\u003e Lycium ferocissimum; \u003c/em\u003eNa, \u003cem\u003eNicotiana attenuata; \u003c/em\u003eOc, \u003cem\u003eO. corymbosa\u003c/em\u003e; Ca, \u003cem\u003eC. arabica\u003c/em\u003e; Cc, \u003cem\u003eC. canephora\u003c/em\u003e; Ce, \u003cem\u003eC. eugenioides\u003c/em\u003e. Three paralogous N-methyltransferases are labeled: xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT), and dimethylxanthine methyltransferase (DXMT).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7032019/v1/0ace32578bd35715d7849008.png"},{"id":88467978,"identity":"c6daffcc-278b-482f-b2e7-04d50f422f81","added_by":"auto","created_at":"2025-08-06 18:01:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1718474,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7032019/v1/fac6d646-b53b-4a5e-9407-98c2949e8dba.pdf"},{"id":86432026,"identity":"82a57ae2-3be4-43cf-a39f-4ab270b025d6","added_by":"auto","created_at":"2025-07-10 14:50:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":352323,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS15.docx","url":"https://assets-eu.researchsquare.com/files/rs-7032019/v1/576077adfca9862790d61e00.docx"},{"id":86432022,"identity":"29e4617c-151d-4915-9520-1cfe98a15b31","added_by":"auto","created_at":"2025-07-10 14:50:34","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14181,"visible":true,"origin":"","legend":"","description":"","filename":"TableS112legends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7032019/v1/d1d5523667323a9b3a03c910.docx"},{"id":86433287,"identity":"b47afeb1-4ee7-4954-bc94-04532a98720e","added_by":"auto","created_at":"2025-07-10 15:06:34","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1244175,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS111.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7032019/v1/4bf8ac04cee5a885f2e66abc.xlsx"},{"id":86433288,"identity":"69b0535a-a925-4cfb-bd5d-3dd658e5c253","added_by":"auto","created_at":"2025-07-10 15:06:34","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":19975,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7032019/v1/74ba0e5ee140fb0d79261410.docx"},{"id":86432031,"identity":"5ba351f1-01fb-4408-bde5-2b93435b0a5f","added_by":"auto","created_at":"2025-07-10 14:50:34","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":20807,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7032019/v1/637a608f030b347f2bc238c5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eComparative genomics of \u003cem\u003eCoffea\u003c/em\u003e species highlights expansion of gene families linked to adaptation and secondary metabolism\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCoffee ranks among the most widely consumed beverages worldwide, supporting the livelihoods of approximately 100\u0026nbsp;million small farmers and their families globally (Vegro and de Almeida \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Siles et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Freitas et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Its enduring popularity is driven by its stimulating properties as well as the diverse range of flavors and aromas unique to different coffee varieties (ICO \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Freitas et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As global consumption continues to rise—particularly with growing demand for specialty coffees—ensuring a consistent and high-quality supply has become increasingly critical (Davis et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Merga and Alemayehu \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, sustainable coffee production has been threatened by the increasing frequency of extreme weather events, which disrupt yields and drive market volatility (Poltronieri and Rossi \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Massrie \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These challenges underscore the need for breeding cultivars with enhanced tolerance to abiotic and biotic stresses (Lahai et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eCoffea arabica\u003c/em\u003e, representing 60% of global production, has a narrow genetic base stemming from a single hybridization event, limiting the available genetic variation for breeding programs (Scalabrin et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Krishnan et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The genetic diversity of other \u003cem\u003eCoffea\u003c/em\u003e species including about 130 wild coffee species could provide potential for the genetic improvement of arabica coffee (Montagnon et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Conventional breeding programs have seen limited progress, partly due to insufficient genomic resources, hindering the identification of novel alleles associated with desirable traits such as stress tolerance, disease resistance, and quality attributes (dos Santos et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ngure and Watanabe \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecent advancements in genomics, including high-quality genome assemblies for \u003cem\u003eC. arabica\u003c/em\u003e (NCBI BioProject: PRJNA698600), \u003cem\u003eC. eugenioides\u003c/em\u003e (BioProject: PRJNA497891), and \u003cem\u003eC. canephora\u003c/em\u003e (Denoeud et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), provide opportunities for comparative and evolutionary analyses to uncover the genetic basis of important agronomic and quality traits. \u003cem\u003eC. arabica\u003c/em\u003e (2n = 4x = 44) is an allotetraploid species that originated from a natural hybridization event between \u003cem\u003eC. eugenioides\u003c/em\u003e and \u003cem\u003eC. canephora\u003c/em\u003e approximately 350,000–610,000 years ago (Salojärvi et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This hybridization resulted in a complex genome shaped by contributions from both parental lineages. Understanding how each progenitor genome influences ecological adaptations in \u003cem\u003eC. arabica\u003c/em\u003e–such as traits related to stress tolerance, disease resistance, and secondary metabolism–can guide the development of new varieties with enhanced resilience and productivity.\u003c/p\u003e\u003cp\u003eOrthology analyses are critical for coffee improvement efforts, enabling researchers to accurately identify candidate genes associated with desirable traits among diverse coffee species (Varshney and Dubey \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Shahrajabian et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mishra et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Distinguishing orthologs (genes derived from a common ancestral gene across species) from paralogs (genes duplicated within a species) is essential for reliably transferring functional knowledge from well-studied model plants or closely related species to coffee (Koonin \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This precision allows more accurate prediction of gene function and facilitate the discovery of targets linked to disease resistance, drought tolerance, yield stability, and cup quality (Marraccini et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Cacas et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eComparative genomic analyses across \u003cem\u003eCoffea\u003c/em\u003e species can also reveal the evolutionary dynamics of gene families (orthogroups) associated with key traits such as flavor biochemistry, defense, or temperature adaptation (Denoeud et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lachica et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In particular, lineage-specific or rapidly evolving orthogroups, defined as gene families unique to or significantly diverged in a lineage, are of great interest due to their roles in adaptive evolution and functional diversification (Lespinet et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The absence of certain orthogroups in specific lineages may reflect gene loss driven by relaxed selection or functional redundancy, while their expansion may be linked to lineage-specific innovations critical for ecological adaptation or agronomic performance.\u003c/p\u003e\u003cp\u003eExpanding comparative analyses beyond \u003cem\u003eC. arabica\u003c/em\u003e and its diploid progenitors to include other plant species provides a broader evolutionary perspective. \u003cem\u003eCoffea\u003c/em\u003e belongs to the Order Gentianales within the Lamiids clade, which also includes the Orders Lamiales, Solanales, Boraginales, and Vahliales (Stull et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Deng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Alawfi and Alzahrani \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Comparing coffee genomes to those of related species within these Orders enable the identification of gene families uniquely conserved or diversified in the coffee lineage. Such coffee-specific orthogroups may include genes encoding enzymes involved in secondary metabolism (e.g., alkaloids and phenolics) that contribute to the distinctive aroma and flavor of coffee (Sunarharum et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For example, lineage-specific expansions of N-methyltransferases involved in caffeine biosynthesis have been identified in coffee, arising through gene duplication events after divergence from a common ancestor (Denoeud et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIdentifying lineage-specific genes or pathways also holds profound implications for managing biotic and abiotic stresses. As the global climate changes and pests and diseases become more prevalent, coffee growers are under pressure to adopt more resilient cultivars (Ahmed et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Cassamo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). By uncovering the genetic mechanisms that enable \u003cem\u003eC. canephora\u003c/em\u003e to thrive in hotter conditions or \u003cem\u003eC. arabica\u003c/em\u003e to endure cooler, high-altitude environments, breeders can integrate these adaptive traits into breeding programs. Ultimately, this approach supports developing varieties with broader environmental tolerance while preserving or even improving cup quality (Ferrão et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we conducted an in-depth comparative genomic analysis to investigate orthologous and paralogous relationships among three coffee species: \u003cem\u003eC. arabica\u003c/em\u003e, \u003cem\u003eC. canephora\u003c/em\u003e, and \u003cem\u003eC. eugenioides\u003c/em\u003e. We extended our analysis to 21 additional plant species from Lamiales, Solanales, and Gentianales to identify \u003cem\u003eCoffea\u003c/em\u003e-specific genes and gene families. By focusing on lineage-specific and rapidly evolving orthogroups, we aimed to elucidate the genetic and evolutionary basis for coffee-specific traits and adaptations. Functional enrichment and phylogenetic analyses were conducted to evaluate the biological relevance and evolutionary patterns of these gene sets, with a particular emphasis on lineage-specific adaptive evolution in the \u003cem\u003eCoffea\u003c/em\u003e genus. Our findings deepen our understanding of coffee biology and provide practical targets to improve crop performance, quality, and sustainability.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eGenome data retrieval and processing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGenomic protein sequences for 24 plant species were retrieved from NCBI Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/datasets/genome/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/datasets/genome/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This dataset includes three coffee species (\u003cem\u003eC. arabica\u003c/em\u003e, \u003cem\u003eC. eugenioides\u003c/em\u003e, \u003cem\u003eC. canephora\u003c/em\u003e) and 21 additional species chosen based on their evolutionary relationships with \u003cem\u003eCoffea\u003c/em\u003e. \u003cem\u003eCoffea\u003c/em\u003e resides within the Order Gentianales of the Lamiid clade, closely related to the Orders Lamiales and Solanales (Stull et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; APG IV \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Thus, twelve species from Lamiales, eight from Solanales, and four from Gentianales (including the three coffee species) with complete genome assemblies and protein annotations were selected. For genes with multiple isoforms, the longest protein isoform was selected as a representative sequence (Park et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The finalized protein dataset was subsequently used to identify orthologous groups across the species.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOrthogroup inference and phylogenetic analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOrthologous gene identification was conducted using OrthoFinder v2.5.5 (Emms and Kelly \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) with protein-coding sequences from all 24 species as input. The analysis was performed using default parameters, which involve an all-versus-all search using Dimond BLASTP (Buchfink et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) to assess sequence similarity, followed by hierarchical clustering to assign genes into orthogroups. These orthogroups represent gene families derived from a single ancestral gene in the last common ancestor of the species examined. For each orthogroup, multiple sequence alignments were generated using MAFFT (Katoh and Standley \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and gene trees were inferred using FastTree (Price et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The species tree was reconstructed using the STAG method (Species Tree from All Genes), and rooting was achieved using the STRIDE method (Species Tree Root Inference from Duplication Events) as implemented in OrthoFinder. Trees were visualized using FigTree v1.4.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree\u003c/span\u003e\u003cspan address=\"http://tree.bio.ed.ac.uk/software/figtree\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene family evolution analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGene family expansion and contraction were analyzed using Computational Analysis of gene Family Evolution (CAFE) v5.1 (Mendes et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To minimize biases associated with large variance in gene copy numbers, orthogroups containing at least 100 gene models in any species were excluded from the CAFE analysis. The lambda (λ) parameter, representing the birth and death rate of gene families, was uniformly estimated across the phylogeny under a Poisson rate model. An error rate, accounting for potential inaccuracies in genome annotation (e.g. missing or incorrectly assigned gene copies), was also estimated and incorporated into the analysis. Gene families exhibiting significant expanding or contraction were identified as rapidly evolving if they showed a CAFE family-wide \u003cem\u003eP\u003c/em\u003e-value of less than 0.05.\u003c/p\u003e\u003cp\u003eGene family evolution was further analyzed using COUNT software (Csűös \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), which employs a Bayesian posterior probabilistic model to estimate gene family gain, loss, and duplication events along each branch of the phylogenetic tree. COUNT is particularly effective for handling zero-inflated gene family distributions, characterized by the absence of genes (i.e., zero-copy number) in most lineages due to gene loss or limited lineage-specific expansions. By modeling both presence/absence and copy number variation, COUNT enhances accuracy in evolutionary inferences, especially for gene families that are rare or highly specific to particular lineages.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFunctional annotation and GO enrichment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFunctional annotation of the entire protein set was preformed using the Trinotate pipeline (Bryant et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), as described by Park et al (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Briefly, genomic protein sequences were first queried against the UniProtKB/Swiss-Prot database using BLASTP with an E-value cutoff of 1e-5. In addition, the protein sequences were analyzed against the Pfam database using the hmmsearch tool of HMMER (Finn et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) to identify conserved domains. Based on sequence similarity, functional and Gene Ontology (GO) annotations were assigned to the proteins from UniProtKB/Swiss-Prot entries. For each orthogroup, a consensus function was derived from the majority annotation among its member proteins. GO enrichment analysis was carried out to identify biological processes significantly overrepresented in orthogroups using the hypergeometric test implemented via the phyper function in R (R core Team \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A False Discovery Rate (FDR) threshold of 0.05 was applied to determine statistical significance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChromosomal mapping of candidate genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChromosomal locations of selected gene families were retrieved from the \u003cem\u003eC. arabica\u003c/em\u003e reference genome (NCBI: GCF_003713225.1). Gene loci were visualized using the LinkageMapView package in R (Ouellette et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), enabling interpretation of their distribution across the 22 chromosomes and subgenomes corresponding to \u003cem\u003eC. canephora\u003c/em\u003e and \u003cem\u003eC. eugenioides\u003c/em\u003e origins.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eOrthologous group construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCoffea\u003c/em\u003e belongs to the Order Gentianales, which is closely related to Lamiales, Solanales, Boraginales, and Vahliales, within the Lamiids clade (Stull et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; APG IV \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). To understand the orthologous relationships between \u003cem\u003eCoffea\u003c/em\u003e and other species and within \u003cem\u003eCoffea\u003c/em\u003e genus, we analyzed genomic data from three \u003cem\u003eCoffea\u003c/em\u003e species (\u003cem\u003eC. arabica\u003c/em\u003e, \u003cem\u003eC. canephora\u003c/em\u003e, and \u003cem\u003eC. eugenioides\u003c/em\u003e) and 21 additional plant species of the Lamiids clade. Given the availability of complete genome sequences and gene annotations, we selected twelve species from Lamiales, eight species from Solanales, one from Gentianales. None from Boraginales and Vahliales were selected due to lack of species with complete genome. Orthologous gene identification was performed using OrthoFinder (Emms and Kelly \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) on the total 769,395 genes across the 24 species. The analysis identified 30,986 orthogroups, to which 95% (728,690 genes) of the total genes were assigned, while 5% (40,705 genes) remained unassigned. Orthogroup sizes varied considerably: 40% contained five genes or less, 32% had between 6 and 30 genes, 25% had between 31 and 100 genes, and only 2% consisted of more than 100 genes, reaching up to 1,764 gene members (Supplementary Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). The largest orthogroup (OG00000, 1,764 genes) was annotated as a Myb-DNA-binding transcription factor. The second largest (OG00001), with 1,505 was annotated as retrovirus-related Pol polyprotein (Supplementary Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). Notably, four additional orthogroups among the top ten\u0026ndash;such as OG00003 with 1,282, OG00004 with 1,149, OG00005 with 1,117, and OG00007 with 1,038\u0026ndash;were also associated with transposon-related functions (Supplementary Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e), suggesting extensive duplication and possibly active transposition events across the species analyzed. Two orthogroups could not be assigned with any functions due to lack of homology to proteins of the UniProtKB/Swiss-Prot database and the remaining two orthogroups were annotated as leucine-rich repeat (LRR) receptor-like kinase and Pentatricopeptide repeat (PPR)-containing protein.\u003c/p\u003e\n\u003cp\u003eUsing gene trees derived from all orthogroups, OrthoFinder constructed a species tree that provide foundation to differentiate orthologs from paralogs across the species. The resulting species tree was consistent with established phylogenetic relationships, grouping species according to taxonomic Orders (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Lamiales species clustered closer to Solanales than to Gentianales, consistent with previous phylogenetic studies based on plastid sequences (Stull et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Gene assignments to orthogroups varied among species, ranging from 86% of total genes in \u003cem\u003eStriga asiatica\u003c/em\u003e to 99.4% in \u003cem\u003eC. eugenioides\u003c/em\u003e (Fig. 2a). \u003cem\u003eC. arabica\u003c/em\u003e and \u003cem\u003eC. canephora\u003c/em\u003e exhibited assignment rates of 98.6% and 94%, respectively. Gene duplication rate among species also varied considerably, with \u003cem\u003eD. hygrometricum\u003c/em\u003e highest (62%) and \u003cem\u003eC. canephora\u003c/em\u003e lowest (6.8%) (Fig. 2b). Notably, gene duplication rates exhibited a strong positive correlation with total gene counts, with R\u003csup\u003e2\u003c/sup\u003e of 0.61, suggesting a pivotal role of gene duplication in genome evolution (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). Particularly, three species showed a strikingly high degree of gene duplication: \u003cem\u003eD. hygrometricum\u003c/em\u003e (62%), \u003cem\u003eS. splendens\u003c/em\u003e (59%), and \u003cem\u003eC. arabica\u003c/em\u003e (38%) (Fig. 2b). These elevated duplication rates could be attributed to whole-genome duplication events, as these three species are known as tetraploids (Xiao et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Jia et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLineage-specific orthogroups and unassigned genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOrthogroup memberships were analyzed to elucidate lineage-specific features. For coffee species within Gentianales, we focused on species-level orthogroups (including the three coffee species and \u003cem\u003eO. corymbose\u003c/em\u003e), while for non-coffee species, we aggregated orthogroup memberships at the Order level (Lamiales and Solanales), resulting in six membership groups (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Overall, 37% (11,454) of orthogroups contained at least one gene from these six groups, while 22% (6,956) were exclusive to Lamiales and 14% (4,248) were exclusive to Solanales. Notably, only 1.7% (524) were unique to the three coffee species (Supplementary Table S3). GO enrichment analysis of the \u003cem\u003eCoffea\u003c/em\u003e-specific orthogroups highlighted defense/stress-related processes (hypersensitive response, salicylic acid biosynthesis, response to benzoic acid, response to xenobiotic stimulus, and cellular hypotonic response), metabolic pathways (alkaloid, thalianol, and fatty acid beta-oxidation), and protein regulation (SRP-dependent protein targeting, serine/threonine phosphatase activity) (Table 1). Given that alkaloid and thalianol-derived terpenoids can serve as chemical deterrents against pathogens due to their toxicity and antimicrobial properties (Khameneh et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yan et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e), the enrichments suggested that \u003cem\u003eCoffea\u003c/em\u003e species may have evolved unique defense mechanisms including specialized metabolism, contributing to their distinctive flavor profiles.\u003c/p\u003e\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003eAdditional GO terms, while less significantly enriched, further revealed a strong enrichment for plant defense, secondary metabolism, and stress responses. These included systemic acquired resistance (GO:0009627), salicylic acid metabolism and biosynthesis (GO:0009696, GO:0080142), indole alkaloid biosynthesis (GO:0035835), eugenol biosynthesis (GO:0042855), glycyrrhetinate biosynthesis (GO:1902386), pentacyclic triterpenoid metabolism (GO:0019742), catabolism of 11-oxo-\u0026beta;-amyrin (GO:1902382), photorespiration, photooxidative stress, and xenobiotic catabolism. Together, these processes highlighted a lineage-specific adaptation in \u003cem\u003eCoffea\u003c/em\u003e involving an intricate interplay of defense, metabolic reprogramming, and stress adaptation (Supplementary Table S4\u003cstrong\u003e).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen focusing on species-specific orthogroups within \u003cem\u003eCoffea\u003c/em\u003e, 0.7% (n\u0026thinsp;=\u0026thinsp;205) of orthogroups were specific to \u003cem\u003eC. arabica\u003c/em\u003e, 0.5% (n\u0026thinsp;=\u0026thinsp;163) to \u003cem\u003eC. canephora\u003c/em\u003e, and 0.2% (n\u0026thinsp;=\u0026thinsp;71) to \u003cem\u003eC. eugenioides\u003c/em\u003e. GO enrichment analyses revealed species-specific functional signatures (Supplementary Table S5). Orthogroups specific to \u003cem\u003eC. eugenioides\u003c/em\u003e exhibited enrichments similar to the \u003cem\u003eCoffea\u003c/em\u003e group in defense- and metabolism-related processes, such as nucleotide metabolism (inosine monophosphate (IMP) metabolism), folate metabolism (tetrahydrofolate biosynthesis), and secondary metabolite biosynthesis (coumarin, coniferin). Additionally, developmental and adaptative processes were enriched, including photorespiration, cell growth, floral organ identity, and root morphogenesis (Table 2).\u003c/p\u003e\n\u003cp\u003eIn contrast, \u003cem\u003eC. canephora\u003c/em\u003e showed enrichments in GO terms associated with diverse biological processes, including cytoskeletal dynamics (actin filament severing and capping, astral microtubule organization, mitotic spindle localization), primary and secondary metabolism (xylulose, thymidine, riboflavin, and neutral amino acid transport, carotenoids, lignans, and isoprenoids) and regulatory and hormonal signaling pathways (photosynthesis, brassinosteroid-mediated signaling, and cellular response to ethylene stimulus) (Table 2). \u003cem\u003eC. arabica-\u003c/em\u003especific orthogroups were enriched in plastid transcription and photoreactive repair, linked to enhanced photosynthesis efficiency (Table\u0026nbsp;2). These divergent enrichment patterns highlighted species-specific evolutionary adaptations, likely reflecting diverse survival strategies and distinct chemical profiles across species.\u003c/p\u003e\n\u003cp\u003eIn addition to the lineage-specific orthogroups, we examined unassigned genes\u0026mdash;those not placed in any orthogroup due to lack of detectable homology to genes in other species. These include orphan genes, possibly conferring unique functions to their respective species. We identified 601 unassigned genes in \u003cem\u003eC. arabica\u003c/em\u003e, 1,530 genes in \u003cem\u003eC. canephora\u003c/em\u003e, and 166 genes for \u003cem\u003eC. eugenioides\u003c/em\u003e. While no significant GO term enrichment was found for the unassigned genes in \u003cem\u003eC. arabica\u003c/em\u003e and \u003cem\u003eC. canephora\u003c/em\u003e, \u003cem\u003eC. eugenioides\u003c/em\u003e showed enrichments related to leaf development (adaxial/abaxial pattern specification), defense responses (positive regulation of defense response to bacterium), and metal homeostasis (intracellular sequestering of iron and manganese ion homeostasis) (Supplementary Table S6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLineage-specific expansion and contraction of gene families in coffee\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene families that undergo rapid expansion (gene duplication/gain) or contraction (gene loss) provide valuable insights into adaptive evolution, functional innovation, and the loss of redundant traits under selective pressures (Koonin \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e; Lažetić and Troemel \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Fang et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). CAFE5 detected significantly expanded (187) and contracted (3) orthogroups in \u003cem\u003eCoffea\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Table S7). Among the most significantly expanded were gene families associated with disease resistance, particularly NBS-LRR containing proteins, suggesting strong pathogen-driven selective pressures (Supplementary Table S7). Enriched GO terms in the expanded families revealed a wide range of biological processes, with a strong emphasis on defense (salicylic acid response, pathogen detection), specialized metabolism (alkaloids, terpenoids, coumarins), hormone signaling, and circadian/light signaling (Supplementary Table S8). The three contracted families involved protein regulation and homeostasis (pectinesterase inhibitor, kinase-associated proteins). (Supplementary Table S7)\u003c/p\u003e\n\u003cp\u003eAt the species level, \u003cem\u003eC. arabica\u003c/em\u003e showed extensive gene family expansions (367 orthogroups) compared to contractions (20 orthogroups), consistent with its recent allopolyploid origin (Supplementary Table S9). In contrast, the diploid \u003cem\u003eC. canephora\u003c/em\u003e exhibited more pronounced gene family contractions (113) than expansions (18) (Supplementary Table S10), while \u003cem\u003eC. eugenioides\u003c/em\u003e showed a balanced pattern (60 expansions and 59 contractions) (Supplementary Table S11).\u003c/p\u003e\n\u003cp\u003eExpanded families in \u003cem\u003eC. arabica\u003c/em\u003e included transposon-related, signaling (Ras, Zinc finger RICESLEEPER), and metabolic enzymes (Cytochrome P450, pectate lyase) (Supplementary Table S9). Interestingly, many of these expansions appeared to contain genes from \u003cem\u003eC. eugenioides\u003c/em\u003e but few genes from \u003cem\u003eC. canephora.\u003c/em\u003e For example, orthogroup OG00085 (Cytochrome P450) included only four genes from \u003cem\u003eC. canephora\u003c/em\u003e but contained 63 genes of \u003cem\u003eC. eugenioides\u003c/em\u003e and 97 of \u003cem\u003eC. arabica\u003c/em\u003e. Phylogenic analysis indicated that all coffee genes of OG00085 descended from three ancestral genes, followed by duplications predominantly in \u003cem\u003eC. eugenioides\u003c/em\u003e and \u003cem\u003eC. arabica\u003c/em\u003e (Supplementary Fig. S3). Contracted orthogroups included several disease resistance-related proteins (salicylic acid signaling components, and receptor-like kinases) and metabolic enzymes, suggesting potential reduction in innate immune or metabolic capacity (Supplementary Table S9).\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eC. canephora\u003c/em\u003e, expansions included multiple components related to plant immune responses, including resistance (R) proteins, receptor-like kinases, and hormone signaling, while contractions were more extensive and spanned diverse functional categories including stress responses, transcriptional regulation, hormone and light signaling, protein degradation, metabolism, and transposable element activity (Supplementary Table S10). Many of these contractions appeared to be lineage-specific to \u003cem\u003eC. canephora\u003c/em\u003e and absent from \u003cem\u003eC. eugenioides\u003c/em\u003e. For instance, orthogroup OG00337, annotated as Far-Red Impaired response 1 (FAR1) involved in phytochrome-signaling, contained no gene from \u003cem\u003eC. canephora\u003c/em\u003e but had 58 genes from \u003cem\u003eC. eugenioides\u003c/em\u003e and 46 genes from \u003cem\u003eC. arabica\u003c/em\u003e. Interestingly, we found that four additional FAR1-related orthogroups (OG01057, OG01614, OG01227, OG09699) were also contracted specifically in C. \u003cem\u003ecanephora\u003c/em\u003e (Supplementary Table S10).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. eugenioides\u003c/em\u003e showed expansions in gene families associated with disease resistance, transposons, light signaling (FAR1), and protein modification (Dolichol-phosphate mannose synthase and Cytosolic sulfotransferase), while contractions involved diverse biological roles including disease-resistance, hormone and stress signaling, secondary metabolism, transposon, and development (MADS-box) (Supplementary Table S11).\u003c/p\u003e\n\u003cp\u003eOverall, disease-resistance-related gene families were commonly identified as rapidly evolving across all three \u003cem\u003eCoffea\u003c/em\u003e species, suggesting dynamic immune adaptation driven by their respective ecological pressures. Furthermore, these patterns suggested that polyploidy in \u003cem\u003eC. arabica\u003c/em\u003e facilitated the retention and expansion of gene families, particularly those derived from \u003cem\u003eC. eugenioides\u003c/em\u003e, while \u003cem\u003eC. canephora\u003c/em\u003e appeared to have undergone more extensive gene family reduction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCoffea\u003c/strong\u003e\u003cstrong\u003e-specific expansions in secondary metabolism gene pathways\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCaffeine is synthesized only a few plant genera\u0026ndash;\u003cem\u003eCoffea\u003c/em\u003e (coffee), \u003cem\u003eTheobroma\u003c/em\u003e (cacao), and \u003cem\u003eCamellia\u003c/em\u003e (tea)\u0026ndash;and phylogenetic studies indicated that genes involved in caffeine biosynthesis evolved independently through convergent evolution (Denoeud et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Three paralogous N-methyltransferases catalyze the sequential methylation of xanthosine to caffeine: xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT), and dimethylxanthine methyltransferase (DXMT) (Denoeud et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eConsistent with previous findings, our orthology analysis clustered these methyltransferase paralogs into a single orthogroup (OG04312). This orthogroup included five genes from Solanales species, none from Lamiales, and, within Gentianales, it contained three genes from \u003cem\u003eO. corymbose\u003c/em\u003e, nine from \u003cem\u003eC. canephora\u003c/em\u003e, 11 from \u003cem\u003eC. eugenioides\u003c/em\u003e, and 15 from \u003cem\u003eC. arabica\u003c/em\u003e. A phylogenetic reconstruction of OG04312 showed that, apart from two outliers, all \u003cem\u003eCoffea\u003c/em\u003e methyltransferases formed a monophyletic, coffee-lineage-specific clade, indicating an expansion event unique to the \u003cem\u003eCoffea\u003c/em\u003e lineage (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eDespite this evident expansion, OG04312 was not identified as \u0026ldquo;rapidly evolving\u0026rdquo; by CAFE5. This is likely due to the limitations of single-\u0026lambda; birth\u0026ndash;death model in CAFE5, which averages gene gain/loss rates across the entire phylogeny. As a result, gene families that expand predominantly at a single lineage node may not reach statistical significance under this model.\u003c/p\u003e\n\u003cp\u003eTo better detect \u003cem\u003eCoffea-\u003c/em\u003elineage-restricted duplications, we applied COUNT Bayesian duplication-gain-loss model (Csű\u0026ouml;s \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). COUNT estimates the posterior probability of gene gain, duplication, or loss events on a per-branch basis, making it sensitive to lineage-restricted events. Using this approach, OG04312 showed a high posterior probability of expansion (0.84) at the node corresponding to the Order Gentianales. Using a posterior-probability threshold of 0.84, we identified 11 additional orthogroups with putative \u003cem\u003eCoffea\u003c/em\u003e-lineage-specific duplications (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Several of these gene families are involved in secondary metabolism, potentially contributing to traits unique to \u003cem\u003eCoffea\u003c/em\u003e. For example, orthogroups annotated as caffeic acid 3-O-methyltransferase (COMT) and cinnamoyl-CoA reductase (CAD) play critical roles in the phenylpropanoid pathway and may influence chlorogenic acid biosynthesis, major precursors of phenolic compounds that contribute to coffee bitterness (Campa et al. \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). Orthogroups of strictosidine synthase, UDP-glycosyltransferase, and beta-amyrin monooxygenase genes are involved in alkaloid or terpenoid metabolism, potentially influencing coffee aroma and flavor.\u003c/p\u003e\n\u003cp\u003eAdditional lineage-specific duplications were detected in orthogroups associated with broader physiological and stress-related functions: NIN-like protein 4 (NLP4; associated with nitrate signaling), alkenal reductase and citrate-binding protein (detoxification/homeostasis), WAT1-related protein (cell wall development), and leucine-rich repeat receptor kinases (immune signaling).\u003c/p\u003e\n"},{"header":"Discussion","content":"\u003cp\u003eLineage-specific gene families play crucial roles in adaptative evolution and functional diversification in plants. When a gene family is unique to, or undergoes rapid expansion in a particular lineage, it often reflects the selective pressures imposed by a specific ecological niche (Lespinet et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Rensing et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Identifying lineage-specific gene sets is therefore a powerful strategy for uncovering the genetic mechanisms underlying biologically and agriculturally important traits. In crops, these genes often underlie key domestication traits, enhanced stress tolerance, or the biosynthesis of specialized metabolites (Huang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Qin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For instance, lineage-specific orthogroups may encode enzymes involved in the production of defensive metabolites, proteins mediating abiotic stress responses, or regulatory factors fine-tuning developmental processes under local environmental conditions.\u003c/p\u003e\u003cp\u003eIn the \u003cem\u003eCoffea\u003c/em\u003e genus, lineage-specific gene family expansions appear central to the extensive diversity observed in agronomic traits, flavor profiles, and aroma characteristics, much of which likely arose through adaptive evolution. A striking illustration is the expansion of the N-methyltransferase (NMT) gene family responsible for caffeine biosynthesis. Phylogenetic evidence clearly indicates that these methyltransferases evolved independently in \u003cem\u003eCoffea\u003c/em\u003e, \u003cem\u003eCamellia\u003c/em\u003e, and \u003cem\u003eTheobroma\u003c/em\u003e through convergent evolution\u0026ndash;parallel recruitment of NMT genes for caffeine production in each lineage rather than inheritance from a common caffeinated ancestor. (Denoeud et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Xia et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This underscores the value of comparative orthogroup analyses between \u003cem\u003eCoffea\u003c/em\u003e and its closely related species in identifying key-trait associated genes, with broad implications for comparative genomics, evolutionary biology, and crop improvement.\u003c/p\u003e\u003cp\u003eOur comparative analysis showed that gene duplication rates varied widely even among closely related species. While total gene count and duplication frequency were generally positively correlated (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), several exceptions suggest that gene duplication is not uniformly responsible for genome expansion. For instance, within the Order Gentianales, \u003cem\u003eC. eugenioides\u003c/em\u003e, \u003cem\u003eC. canephora\u003c/em\u003e, and \u003cem\u003eO. corymbose\u003c/em\u003e exhibit similar total gene counts (ranging from 24,917 to 28,952), but show markedly different duplication rates: 26% in \u003cem\u003eO. corymbose\u003c/em\u003e, 13% in \u003cem\u003eC. eugenioides\u003c/em\u003e, and 7% in \u003cem\u003eC. canephora\u003c/em\u003e. These differences imply that gene duplication may be concentrated in specific functional gene families, likely reflecting species-specific adaptive pressures. Indeed, functional enrichment among duplicated genes in \u003cem\u003eC. eugenioides\u003c/em\u003e and \u003cem\u003eC. canephora\u003c/em\u003e revealed overrepresentation of functions related to defense, signaling, and metabolism, supporting the hypothesis of adaptation-driven gene family expansion (Supplementary Table S12). These findings suggest that duplication patterns are functionally biased rather than random, providing insight into the mechanisms underlying lineage-specific innovation in \u003cem\u003eCoffea\u003c/em\u003e species.\u003c/p\u003e\u003cp\u003eTo further investigate how these duplication patterns contribute to functional divergence among species, we examined rapidly evolving orthogroups that exhibit species-specific expansion or contraction. Interestingly, many of the orthogroups that were rapidly contracting in \u003cem\u003eC. canephora\u003c/em\u003e included genes present in both \u003cem\u003eC. eugenioides\u003c/em\u003e and \u003cem\u003eC. arabica\u003c/em\u003e, with several of these orthogroups identified as rapidly expanding in those species (Supplementary Table S10). This asymmetric pattern aligns with phylogenetic evidence placing \u003cem\u003eC. eugenioides\u003c/em\u003e as more closely related to \u003cem\u003eC. arabica\u003c/em\u003e, and suggests contrasting evolutionary pressures that led to gene loss in the \u003cem\u003eC. canephora\u003c/em\u003e lineage following divergence. Alternatively, this pattern could reflect pre-existing differences between \u003cem\u003eC. eugenioides\u003c/em\u003e and \u003cem\u003eC. canephora\u003c/em\u003e prior to the hybridization event that gave rise to \u003cem\u003eC. arabica\u003c/em\u003e. To evaluate these possibilities, we examined the \u003cem\u003eFAR1\u003c/em\u003e gene family (OG00337) as a case study. Phylogenetic analysis indicated that all members descended from a common ancestral gene, followed by lineage-specific duplications in \u003cem\u003eC. arabica\u003c/em\u003e and \u003cem\u003eC. eugenioides\u003c/em\u003e, but complete absence in \u003cem\u003eC. canephora\u003c/em\u003e (Supplementary Fig. S4). Chromosomal mapping in \u003cem\u003eC. arabica\u003c/em\u003e showed that \u003cem\u003eFAR1\u003c/em\u003e genes were distributed across both subgenomes, implying ancestral presence in both parental lineages and suggesting gene loss specifically in \u003cem\u003eC. canephora\u003c/em\u003e (Supplementary Fig. S5). FAR1 proteins are integral to Phytochrome A signaling, mediating red/far-red light sensing, shade avoidance, temperature adaptation, and flowering time (Lin and Wang \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Wang and Wang \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In \u003cem\u003eBetula pendula\u003c/em\u003e (silver birch), natural variation in the \u003cem\u003eFAR1\u003c/em\u003e homologs correlates with latitude, longitude, and local temperatures, reinforcing their role in environmental adaptation (Saloj\u0026auml;rvi et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Given the ecological preference of \u003cem\u003eC. arabica\u003c/em\u003e and \u003cem\u003eC. eugenioides\u003c/em\u003e for shaded, high-altitude habitats versus the warmer, sunnier lowlands favored by \u003cem\u003eC. canephora\u003c/em\u003e (Lashermes et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Davis et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), \u003cem\u003eFAR1\u003c/em\u003e gene expansion may support adaptation to low-light and cooler conditions.\u003c/p\u003e\u003cp\u003eAnother example of potentially adaptation-driven expansion involves the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene family, which catalyzes the final step of ethylene biosynthesis. Orthogroup OG00545 contains only three \u003cem\u003eACO\u003c/em\u003e genes from \u003cem\u003eC. canephora\u003c/em\u003e, compared to 13 genes from \u003cem\u003eC. eugenioides\u003c/em\u003e and 16 from \u003cem\u003eC. arabica\u003c/em\u003e, identified as rapidly contracting in \u003cem\u003eC. canephora\u003c/em\u003e and rapidly expanding in \u003cem\u003eC. arabica\u003c/em\u003e (Supplementary Table S9; Supplementary Table S10). The dramatic disparity suggests that \u003cem\u003eC. arabica\u003c/em\u003e and \u003cem\u003eC. eugenioides\u003c/em\u003e might have expanded their \u003cem\u003eACO\u003c/em\u003e repertories to fine-tune ethylene responses under their native high-altitude, shaded environments. Ethylene regulates a wide range of physiological processes, including seed germination, fruit ripening, abiotic stress responses, often in coordination with abscisic acid (ABA) and light-signaling pathway (Ju and Chang \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For instance, ethylene can promote cell elongation and regulate leaf morphology, potentially reducing pathogen exposure and limiting water loss. In \u003cem\u003eC. arabica\u003c/em\u003e, \u003cem\u003eACO\u003c/em\u003e expression (e.g., \u003cem\u003eCaACO1\u003c/em\u003e) is downregulated during water deficit and restored upon rehydration, coordinating with ABA to modulate stomatal opening (Lima et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ethylene also plays a key role in fruit ripening in coffee, with multiple \u003cem\u003eACO\u003c/em\u003e genes increasing expression in ripening stages, especially early-ripening cultivars (S\u0026aacute;gio et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These findings suggest that ACO gene expansion may confer fine-tuned ethylene-mediated regulation of development and stress responses in \u003cem\u003eC. arabica\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eCAFE5 models gene family evolution using a single birth-death rate λ applied uniformly across the phylogeny, assessing gene family size changes against the expectations under this constant rate (Csű\u0026ouml;s \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, when a gene family is absent in most species (i.e., zero-inflation), isolated expansions at specific nodes can be statistically masked by the prevalence of zeros (\u0026ldquo;averaged out\u0026rdquo; by the zeros), reducing detection sensitivity. Consequently, CAFE5 may fail to detect lineage-restricted expansions or contractions. In contrast, the COUNT tool employs a more flexible gain-loss-duplication (GLD) model with separate parameters, explicitly distinguishing the first \u0026ldquo;gain\u0026rdquo; of a family (zero to one) from subsequent duplications (n to n\u0026thinsp;+\u0026thinsp;1), offering improved resolution in detecting lineage-restricted events (Csű\u0026ouml;s \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Using COUNT\u0026rsquo;s Bayesian framework, we identified 12 gene families that exhibit \u003cem\u003eCoffea\u003c/em\u003e-lineage-specific expansions, many of which are involved in secondary metabolism including methyltransferases responsible for caffeine biosynthesis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These expansions likely reflect evolutionary adaptations for producing coffee-specific compounds such as caffeine and chlorogenic acids, both of which contribute to distinct flavor and chemical defense profiles. Although further phylogenetic and functional validation of these families is needed to elucidate their roles in trait development and adaptation, the lineage specificity and association with sensory and adaptive traits make these gene families promising targets for breeding programs aimed at improving coffee quality.\u003c/p\u003e\u003cp\u003eCollectively, our analysis of lineage-specific orthogroups, species-specific orphan genes, and gene duplication dynamics provides critical insights into the unique genomic landscape and adaptive evolution of coffee species. These findings offer a valuable genomic resource for identifying genetic targets that can be leveraged to enhance stress resilience, disease resistance, and cup quality in cultivated coffee, thereby supporting future crop improvement efforts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated during this study are\u0026nbsp;provided within the manuscript or supplementary information files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by appropriated funds from the United States Department of Agriculture, Agricultural Research Service (USDA-ARS) (Project Number 8042-21000-303-00D)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSP conceptualized the study and designed the experiments. SP, DZ, and LWM secured funding for the project. SP and SO performed data analysis with assistance from EA, DZ, and LWM.\u0026nbsp;SP wrote the initial draft, and all authors reviewed and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research used resources provided by the SCINet project of the USDA Agricultural Research Service (ARS project number: 8042-21000-303-00D). Mention of a trade name, proprietary product, or vendor does not constitute an endorsement, guarantee, or warranty by the USDA and does not imply its approval to the exclusion of other products or vendors that may be suitable.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmed S, Brinkley S, Smith E, et al (2021) Climate Change and Coffee Quality: Systematic Review on the Effects of Environmental and Management Variation on Secondary Metabolites and Sensory Attributes of Coffea arabica and Coffea canephora. Front Plant Sci 12:\u003c/li\u003e\n\u003cli\u003eAlawfi MS, Alzahrani DA (2023) Insights into the phylogenetic relationship of the lamiids orders based on whole chloroplast genome sequencing. J King Saud Univ - Sci 35:102398. https://doi.org/10.1016/j.jksus.2022.102398\u003c/li\u003e\n\u003cli\u003eAPG IV (2016) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Bot J Linn Soc 181:1\u0026ndash;20. https://doi.org/10.1111/boj.12385\u003c/li\u003e\n\u003cli\u003eBryant DM, Johnson K, DiTommaso T, et al (2017) A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors. Cell Rep 18:762\u0026ndash;776. https://doi.org/10.1016/j.celrep.2016.12.063\u003c/li\u003e\n\u003cli\u003eBuchfink B, Reuter K, Drost H-G (2021) Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods 18:366\u0026ndash;368. https://doi.org/10.1038/s41592-021-01101-x\u003c/li\u003e\n\u003cli\u003eCacas J-L, Petitot A-S, Bernier L, et al (2011) Identification and characterization of the Non-race specific Disease Resistance 1 (NDR1) orthologous protein in coffee. BMC Plant Biol 11:144. https://doi.org/10.1186/1471-2229-11-144\u003c/li\u003e\n\u003cli\u003eCampa C, Noirot M, Bourgeois M, et al (2003) Genetic mapping of a caffeoyl-coenzyme A 3-O-methyltransferase gene in coffee trees. Impact on chlorogenic acid content. Theor Appl Genet 107:751\u0026ndash;756. https://doi.org/10.1007/s00122-003-1310-4\u003c/li\u003e\n\u003cli\u003eCassamo CT, Draper D, Romeiras MM, et al (2023) Impact of climate changes in the suitable areas for \u003cem\u003eCoffea arabica\u003c/em\u003e L. production in Mozambique: Agroforestry as an alternative management system to strengthen crop sustainability. Agric Ecosyst Environ 346:108341. https://doi.org/10.1016/j.agee.2022.108341\u003c/li\u003e\n\u003cli\u003eCsű\u0026ouml;s M (2010) Count: evolutionary analysis of phylogenetic profiles with parsimony and likelihood. Bioinformatics 26:1910\u0026ndash;1912. https://doi.org/10.1093/bioinformatics/btq315\u003c/li\u003e\n\u003cli\u003eDavis AP, Gole TW, Baena S, Moat J (2012) The Impact of Climate Change on Indigenous Arabica Coffee (Coffea arabica): Predicting Future Trends and Identifying Priorities. 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Antibiotics 10:318. https://doi.org/10.3390/antibiotics10030318\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 2 and 3","content":"\u003cp\u003eTable 2 and 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Coffea arabica, Comparative genomics, Gene family evolution, Orthogroup analysis, Secondary metabolism, Lineage-specific genes","lastPublishedDoi":"10.21203/rs.3.rs-7032019/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7032019/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCoffee is among the most widely consumed beverages worldwide, with diverse \u003cem\u003eCoffea\u003c/em\u003e species displaying a broad spectrum of flavors and important agronomic traits. Despite recent advances in genomics and the availability of assembled coffee genomes, comparative genomic analyses remain underutilized for crop improvement. In this study, we conducted comprehensive orthology and gene family evolution analyses across 24 plant species\u0026ndash;including three \u003cem\u003eCoffea\u003c/em\u003e species (\u003cem\u003eC. arabica\u003c/em\u003e, \u003cem\u003eC. canephora\u003c/em\u003e, and \u003cem\u003eC. eugenioides\u003c/em\u003e), twelve Lamiales, and eight Solanales\u0026ndash;to identify lineage-specific genomic features associated with adaptation and coffee quality traits. Using OrthoFinder in conjunction with gene family evolution models (CAFE5 and COUNT), we identified 1,552 orthogroups that are either specific to \u003cem\u003eCoffea\u003c/em\u003e or have undergone significant expansion or contraction. Disease‑resistance genes were the most prominent among rapidly expanded gene families, reflecting ongoing evolutionary arms race with pathogens. We also observed asymmetric patterns of gene family evolution, including contraction of light and ethylene signaling gene families in \u003cem\u003eC. canephora\u003c/em\u003e, contrasted by their expansion in \u003cem\u003eC. arabica\u003c/em\u003e and \u003cem\u003eC. eugenioides\u003c/em\u003e, which may contribute to their adaptation to shaded, high-altitude environments. Further analysis using COUNT identified twelve \u003cem\u003eCoffea\u003c/em\u003e-specific gene family expansions associated with secondary metabolite biosynthesis, including key enzymes for caffeine and chlorogenic acid production. Our comprehensive catalog of lineage-specific orthogroups, supported by functional enrichment and phylogenetic analyses, provides a valuable genomic resource to support breeding programs focused on enhancing stress resilience, disease resistance, and cup quality in cultivated coffee.\u003c/p\u003e","manuscriptTitle":"Comparative genomics of Coffea species highlights expansion of gene families linked to adaptation and secondary metabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 14:50:29","doi":"10.21203/rs.3.rs-7032019/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":"cbc2c391-eb3d-416d-bb76-e7924e915079","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-14T18:08:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-10 14:50:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7032019","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7032019","identity":"rs-7032019","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}