Phylogenomic profile of exon-intron organization across angiosperms, their relationships with protein domains, and functional implications

Domain: chemistry
Published: 2026-01-07
Editorial outcome: minor_revision

Abstract
Eukaryotic genes usually encode proteins and contain exons in different size, including micro and small exon categories. However, genome-wide gene exon-intron organizations and their conservation across angiosperms have not been investigated. Among exons of protein-coding genes from 46 angiosperms and four gymnosperms, ~35% are micro and small exons affecting most genes and the remaining exons are defined here as medium, large, or super exons. Exon-intron structural comparison using our new bioinformatic tool, ExonEvo, reveals that, ~69% of conserved exons in homologous genes are found in two or more major angiosperm subgroups, including ~47% as the seed plant-wide conserved exons that are present in most genes in individual species. An examination of domain-exon correspondence revealed that most conserved protein domains match two or more conserved exon families and genes encoding the same domain family members can have different domain-exon correspondence. Moreover, most flower-preferential genes contain conserved exons and most exons with exon skipping during splicing are shared by seed plants, suggesting that alternative splicing of the exons could be a conserved mechanism for regulating protein structure. Our phylogenomic landscape of exon structure and domain-exon organization supports a model for conservation of exon/domain structure in relation to post-transcriptional regulation of gene/protein function.

Introduction
Most eukaryotic genes have exons and introns and can have diverse exon-intron organization and functions. The introns are removed via splicing to generate mature mRNAs; RNA splicing is essential for proper expression of intron-containing genes and requires conserved spliceosome and splicing factors1–3. Eukaryotic proteins generally have one or more functional domains, with lengths from ~35 to ~250 amino acids4. Domains have diverse activities, such as ligand binding, catalysis, and macro-molecular interaction and often define conserved protein families across animals, fungi, and plants5. Gene family members are thought to be derived from a common ancestral gene and share one or more conserved domains, often with similar domain organizations and similar known or predicted functions. Analyses of genes in human and eight other eukaryotic genomes revealed that exon boundaries often correspond closely to those of protein domains6. In addition, transcripts with different combinations of exons and/or different exon boundaries can be generated from the same gene via alternative splicing, resulting in the production of distinct protein isoforms with diverse domain structures7, including the presence/absence of all/part of domains. Therefore, splicing is a crucial step for regulating gene expression and protein function. Furthermore, during evolution exon shuffling alters the number of exons (and introns) of genes, thereby changing protein sequences and function8. Thus, understanding the patterns of conservation and divergence of exon sizes and their relationships to protein domains is an important aspect of evolutionary analyses of gene structure and function.

Flowering plants (angiosperms) are the most species-rich group of land plants, with over 370,000 species, and are the sister group to gymnosperms (~1000 non-flowering seed plants, such as pine and ginkgo)9–11. Seed plants are also highly complex in structure and function12,13 and are extremely important for the human society, providing foods, clothing, shelter, medicine and many other uses; they are also crucial for the health of diverse ecosystems, supporting many animals, fungi and other organisms14,15. In seed plants, exon lengths range from one bp [e.g., in the Arabidopsis thaliana (Arabidopsis hereafter for brevity) Anaphase Promoting Complex subunit 11 gene16] to >10 kb, with average exon lengths in specific plants spanning from 201 to 319 bp17. The smallest group of exons with fewer than 51 bp has been referred to as micro exon, found in both plants and animals18–21; ~8000 micro exons were detected in >6000 genes with introns in Arabidopsis and ~13,000 micro exons were detected in human genes18,22. More recently, the next shortest exons (with 51~100 bp) have been called small exons and 39,025 small exons were detected in 12,525 Arabidopsis genes22. Micro exons are found in Arabidopsis floral homeotic genes (also called the ABCE genes), such as AG, AP1, AP2, and AP3, and encode portions of DNA-binding or protein-interaction domains22. These floral homeotic genes belong to the MADS-box and AP2 gene families that have significantly higher frequencies of micro-exon-containing genes23. Are the patterns of micro and small exons conserved across flowering plants for floral homeotic genes and other genes? To address the question, genome-wide patterns of the number and size of exons need to be investigated in angiosperms.

Among angiosperms, alternative splicing of specific genes has been described for several species, including Arabidopsis, rice, tomato, soybean, cotton, Populus, and bamboo24–30. A form of alternative splicing, exon skipping, results in the lack of a specific exon in the mature mRNA and lead to the absence or shortening of functional domains or altered protein reading frame31. For example, the cotton Ga05g01029 transcript, including a 45-bp micro exon encoding part of the DNA-binding AP2 domain, was detected in both vegetative and reproductive organs; however, its isoform excluding this micro exon was detected in vegetative organs (cotyledon, stem, root, leaf) but not in floral organs, such as the sepal, petal, stigma, and anther (stamen)32. DNA-binding proteins often contain both DNA-binding and protein-interaction domains. In some DNA-binding protein families, some paralogues are truncated and lack some domain(s) and have been reported to modulate the function of other full-length paralogues33–35. It is possible that isoforms lacking (part of) a domain due to alternative splicing can affect the protein activities. For example, a SEP3 protein (an E-function floral homeotic protein) lacking a portion of the K domain, which is required for the formation of the quartet protein complex, due to the skipping of a micro exon, was reported to affect flower development36–38. In addition, the Arabidopsis GRP20 protein was shown to be required for normal flower development and for proper retention of micro exons in transcripts of several floral homeotic genes, such as AG, AP1, AP2, AP3, and SEP322. Also, the skip of micro exons of these genes was found to cause floral phenotype changes (e.g., SEP3 in ref. 36), showing the importance of micro exons in function of these developmental regulators. Although the micro exons in these floral homeotic genes and other members of the MADS-box and AP2 gene families are conserved in representative angiosperms22,23, genome-wide exploration of conservation pattern of micro exons and exons of other size groups across angiosperms is needed to provide further understanding the evolution of gene structures and their relation to functional domain.

Among protein-coding plant genes, ~62% contain named protein domains; ~65% of these domains belong to protein families that are conserved across land plants and green algae from genomic analyses of seven angiosperms and seven other green plants39. Phylogenetic analyses of specific gene families using plant genomes support general conservation of structures (e.g., refs. 22, 40–47). For example, phylogenetic analysis of 101 genes encoding ICK/KRP kinases from 38 angiosperms and two gymnosperms supports three clades named as A, B, and C groups; all three groups share two exons encoding the conserved CDK/cyclin interacting/inhibiting domain, whereas groups A and B share two additional exons, but group C genes have a distinct long exon45. Also, most members of the cytochrome P450 (CYP) superfamily have similar exon-intron structures40, but in pomegranate some CYP75 paralogues lack an intron and have different expression patterns during fruit development from other paralogues40. However, the variations in exon/intron structures for most angiosperm genes, including those containing micro/small exons, are largely unknown. Furthermore, regarding the relationship between exon-intron structure and their encoded protein domains, a comparison of seven angiosperms and three other land plants identified 35 conserved exon structures, including smaller micro exons of <=15 bp and their flanking exon sequences, and presented information about the corresponding domains such as AP2, peptidase, and gelsolin48. A genome-wide investigation of correspondence between exons and protein domains across angiosperms can further facilitate the understanding of functional similarities and differences among genes and through evolutionary history and is now feasible with the publication of high-quality sequenced angiosperm genomes, including >10 Telomere-to-Telomere (T2T) genomes with assembled chromosomes (e.g., refs. 49–58).

RNA expression of genes provides valuable clues to gene functions, including differential expression patterns related to organs and environmental conditions. Specifically, expression patterns of genes containing micro and small exons might suggest function in various plant organs and can be influenced by various environmental cues. In addition, alternative RNA splicing could affect micro and small exons and support hypotheses about regulation of expression and protein diversity for these genes22. One of major alternative splicing types commonly found in plants, humans and other animals is exon skipping. For example, the skipping of a micro exon in the SEP3 transcript can lead to defects in flower development36,38. Also, a conserved 9-base micro exon in the Arabidopsis WRINKLED1 (WRI1) gene belonging to the AP2 family is important for fatty acid synthesis23,59. In particular, because micro and small exons encode relatively short peptide sequences, protein isoforms related to the presence/absence of micro/small exon-encoded peptide regions might have relatively subtle functional differences, in comparison to those involving larger exons/domains. However, the landscapes of expression patterns of genes containing micro/small exons vs exons of other sizes and alternative splicing involving exons of different sizes are largely unknown in different plant organs.

To address the questions regarding genome-wide conservation and patterns of angiosperm exon sizes, their relationship to protein domains, expression of genes with exons of various sizes and their alternative splicing, we selected genomes from 46 representative angiosperms and four representative gymnosperms and profiled the exon sizes and numbers in protein-coding genes. In addition, we performed phylogenomic analyses to estimate the conserved exons shared by two or more homologous genes, particularly those across seed plants or among angiosperms. Among these conserved exons, their matched domains and the domain-exon correspondence were analyzed. Then, we analyzed public RNA-Seq datasets to examine expression patterns of genes containing conserved exons and to detect alternative splicing of conserved exons. These results present a comprehensive and global angiosperm evolutionary landscape of exon structure and relationship to domain organization, with additional information on expression patterns of gene containing exons of different sizes and provide valuable resources for diverse investigations of gene structure and function.

Results
Landscape of the number and size of exons among angiosperms
Relatively recent high-quality assembly of sequenced plant genomes has provided accurate gene structure annotation for genome-wide comparative examination of exons across angiosperms. To survey the exon size distribution, here we selected 46 angiosperm genomes, including 13 T2T genomes and 33 other high-quality genomes with chromosomal assemblies and four gymnosperm chromosomal genomes for comparison as outgroups (see methods and Supplementary Data 1). We examined a total of 9,188,769 exons of protein-coding genes from these 50 genomes and summarized the number of exons per genome in range from 79,276 to 607,568 (median: 165,147) (Fig. 1a–c, Supplementary Fig. 1; Supplementary Data 2), providing a dataset for large-scale in-depth exon comparison.

To compare the exon length distribution patterns among these genomes, exons were divided into five categories according to their lengths (Fig. 1d and Supplementary Figs. 1 and 2). In addition to the previously defined micro and small exons, for convenience, we referred to the exons with length ranging from 101 to 200 bp as ‘medium exons’, those from 201 to 500 bp as ‘large exons’, and those larger than 500 bp as ‘super exons’. Among 126,433 exons from 24,040 genes that are annotated in the T2T Arabidopsis genome60, 6.2% (7865) were micro exons, 28.3% (35,818) were small exons, 33.3% (42,050) were medium exons, 22.2% (28,015) were large exons, and 10% (12,685) were super exons (Supplementary Fig. 2). In the rice (Oryza sativa cv. japonica) T2T genome54, we detected 15,014 micro exons (6.5% of 230,786 exons), 53,135 (23%) small exons, 65,246 (28.3%) medium exons, 56,350 (24.4%) large exons, and 41,041 (17.8%) super exons (Fig. 1d, Supplementary Fig. 2). Patterns of exon distribution similar to those in Arabidopsis and rice were also detected in other angiosperms, with medium exons being the largest size category (median of ~32%) among the five sizes (Fig. 1d). Across the 50 genomes examined here, genes containing medium exon(s) account for ~58.5% of the protein-coding genes in a genome (Supplementary Fig. 3a, b). Medium exons encode peptides with 33–66 amino acids and preliminary analysis suggested that they might frequently match the size range for angiosperm protein functional units (domains). We examined all annotated domains encoded by conserved exons in 1,528,298 genes from 50 genomes, and found that medium exons represent 32.9% (median value) of exons matching part(s) of and/or whole domains in one of the genomes (Supplementary Fig. 4a, b). Medium exons account for 18.5% of these exons matching >50% of a domain (Supplementary Fig. 4c). Moreover, for 17 to 692 domain families (depending on different angiosperm species), medium exons are enriched (p-value<0.05 under two-sided hypergeometric test; see methods) among all exons encoding all/part of domains; such domain families include the MYB DNA-binding domain (PF13921), RRM 1 (PF00076), and WD40 (PF12894) (Supplementary Fig. 4d, e). For example, GO enrichment analyses of the Arabidopsis genes containing medium exon(s) suggested that these genes are associated with GO terms for growth (e.g., seed growth), response to stresses (e.g., cellular response to heat), regulation of gene expression (e.g., RNA endonuclease activity), and others (Supplementary Data 3). The results imply that the medium exons have a broad range of functions in plant growth and adaptation.

Among the five categories, the micro and small exons together account for ~35% of all exons per genome (Fig. 1d). In particular, the number of small exons per genome (median: 35,024; range from 22,970 to 160,327) is more than 3 times that of micro exons (median: 9757; range from 5174 to 49,288) (Fig. 1e and Supplementary Fig. 2; see the previous paragraph for numbers of exons of different size categories in Arabidopsis and rice). Genes containing micro and/or small exons represent ~53.5% of genes per genome (Fig. 1f), implying that the micro/small exons might be important structure elements of angiosperm genes. GO analyses of the Arabidopsis genes showed that GO terms significantly enriched only in the micro exon containing genes are related to growth (e.g., vegetative growth), regulation of gene expression (e.g., cis-regulatory region sequence-specific DNA binding), post-translational modification (e.g., proteasome binding), transport (e.g., sucrose transport) (Supplementary Data 3). GO terms significantly enriched only in the small exon-containing genes are related to development (e.g., petal development, corolla development, endosperm development), cellular process (e.g., meiosis II cell cycle process), protein modification (e.g., protein methylation), and others (Supplementary Data 3). GO terms significantly enriched in above two gene sets involve nucleosome assembly, protein refolding, vernalization response, histone deacetylase activity, and others (Supplementary Data 3). The GO enrichment results further suggest that micro/small exon-containing genes have diverse and important functions.

Large and super exons account for ~23% of exons per genome (Fig. 1d) and correspond to most (97.4%=35.7%+61.7%) genes without introns (Supplementary Fig. 3c). For the 46 angiosperms analyses here, the genes with a single exon (lacking an intron) account for 4.97% ~40.33% (median value: 21.21%) of all genes in each genome (Supplementary Fig. 3d; Supplementary Data 2). For example, 5482 Arabidopsis genes lack an intron, including 1570 with lengths of 200-500 bp (large exons) and 3592 in the length range of super exons, accounting for ~94% of these single exon genes (Supplementary Fig. 1 and Supplementary Data 2). In rice (Oryza sativa ssp. japonica cv. Nipponbare, T2T genome), 15,136 (26.39%) of 57,359 genes contain a single exon54, with 6233 being large exons and 8176 super exons, accounting for 95% of the rice single-exon genes, indicating that most intronless genes fall into the large and super exon categories. In addition, the Chinese pine (Pinus tabuliformis) genome, known as the largest seed-plant sequenced genome to date17, includes 38,784 singe-exon genes (with 156~6807 bp of exon length) that account for 50.87% of 76,234 genes (Supplementary Fig. 3d; Supplementary Data 2). Of the 38,784 single exons, 37,370 (96%) are either large (19,933) or super (17,437) exons (Supplementary Fig. 1 and Supplementary Data 2). Our results suggest that these long single-exon genes are an important part of the genome. GO terms significantly enriched in genes with a single large/super exon include phloem development, mRNA transcription, signaling receptor binding, ubiquitin-protein transferase activity, response to decreased oxygen levels, and others (Supplementary Data 3). Their lack of introns indicates that they are probably not regulated at the level of splicing, further suggesting relatively uniform functions among organs and growth conditions.

In intron-containing genes (median of ~78.79% of all genes in an angiosperm genome), the exons have a median length of 127 bp (ranging from 110 to 151 bp among angiosperms) (Supplementary Data 2). For example, the eudicot carrot (Daucus carota; T2T genome52) has 28,606 (78.87%) intron-containing genes with a median exon length of 129 bp, including 19,380 genes with micro and/or small exons (Supplementary Data 2). In the triploid banana (Musa acuminata; a monocot) T2T genome61, 24,807 intron-containing genes from the subgenome BXJ2 have a median exon length of 132 bp and 65% of these genes carry micro and/or small exons (Supplementary Data 2). Magnoliids have a pattern similar to eudicots and monocots (Fig. 1b); for instance, 70% of 27,730 avocado (Persea americana) genes62 include micro/small exons (Supplementary Data 2). The above results indicate that most of the protein-coding genes have introns, which depend on proper splicing for function and can be regulated at the splicing level. The possibility of alternative splicing can also increase the protein structural and functional diversity of these genes. To test this, we examined published alternative splicing datasets in Arabidopsis to estimate the number of affected exons in different length categories. Among 1531 exons exhibiting exon-skipping under 72-h heat stress relative to control in Arabidopsis63, 323 (21.1%) are micro exons, 780 (50.95%) small exons, 325 (21.23%) medium exons, 91 (5.94%) large exons, and 12 (0.78%) super exons (Supplementary Data 4). The percentage (50.95%) of small exons among skipped exons is greater than genome-wide percentage (28.32%) of small exons (two-sided hypergeometric test, p-value=1.11E-77; Supplementary Data 4). In another study of poplar root transcriptome under lead stress relative to control24, among 113 skipped exons, 19 (16.81%), 52 (46.02%), 28 (24.78%), 12 (10.62%), and 2 (1.77%) are micro, small, medium, large, and super exons, respectively, suggesting that alternative splicing, including those involving micro and small exons, contributed to transcript/protein diversity in stress response. These cases further suggest that micro, small, and medium exons likely represent frequently skipped exons for increasing functional diversity.

Phylogenomic analyses of conserved exons among angiosperm lineages
The above results of similar distributions among diverse angiosperms of both exon sizes and genes containing either micro/small exons and those containing a single large/super exon suggest possible evolutionary conservation of exon organizations among homologous genes, although these genes could differ in exon numbers and sizes (Fig. 1). To further detect the conservation of individual exons shared by genes of different angiosperm subgroups, we compared the exons among genes in an orthogroup with members related by gene ancestry (Fig. 2). Conserved exons were previously examined by manually comparing exon positions in multiple sequence alignments of the MADS-box22 and CYP7540 gene families; in addition, conserved short micro exons (≤15 bp) were determined along with the conservation of their upstream and downstream exon sequences48. However, an algorithm for genome-wide detection of conserved exons among multiple species is unavailable. To identify conserved exons, we performed phylogenomic analyses for orthogroups with >5 genes from >2 species (including ≥1 angiosperm), generating 16,754 gene trees (see methods). The genes in these gene trees contain a total of 6,347,314 exons, including 443,716 micro exons, 1,822,032 small exons, 2,123,073 medium exons, 1,345,267 large exons, and 613,226 super exons (Supplementary Fig. 3). We define conserved exons between two or more genes in the same orthogroup in three types: (1) highly conserved exons as having at least 50% amino acid sequence similarity; (2) moderately conserved micro exons with 30%~50% amino acid sequence similarity and moderately conserved exons in four other length categories showing at least 20% sequence similarity; and (3) candidate conserved exons as exons if their flanking exons are conserved as defined following criteria 1 or 2 (see methods). For statistics of conserved exons across angiosperms (rather than a species), each conserved exon family containing members from genes of an orthogroup (referred to as “conserved exon” hereafter for brevity, unless otherwise noted) is counted once regardless of the number of genes containing the exon. Following these criteria, we developed a new bioinformatic tool, ExonEvo, to identify conserved exons in annotated genomes using phylogenomics (see methods). We then used ExonEvo to compare genes in the above-mentioned gene trees and identified 141,229 conserved exons (including 122,059 highly conserved exons, 14,548 moderately conserved exons, and 4622 candidate conserved exons; Supplementary Fig. 5), each of which is present in two or more genes from the 50 genomes. In individual species, the conserved exons represent 69.8%~84.9% (median 79.9%) of total annotated exons (Fig. 2a, b). Among these conserved exons, 14,107 are micro exons (Supplementary Data 5), 44,336 small exons (Supplementary Data 6), 45,961 medium exons (Supplementary Data 7), 27,364 large exons (Supplementary Data 8), and 9461 super exons (Supplementary Data 9).

Among the 6347 Arabidopsis micro exons included in the phylogenomic analyses, 70.8% (4496) are conserved; phylogenomic results also indicate 71.1% (7007) of 9861 micro exons in maize (Zea mays) are conserved. Among the plant micro exons examined here by phylogenomics, the conserved micro exons account for 31.6%~74.1% (median: 60.55%) of the detected micro exons per species (Supplementary Fig. 6a, b), suggesting that different lineages might have retained different numbers of micro exons during evolution (see below), although it is possible that some species might also have gained some micro exons. In addition, conserved small exons account for 67.8%~91.4% (median: 85.7%) of the detected small exons per species (Supplementary Fig. 6c). For instance, 89.1% (30,069) of 33,747 detected small exons in tomato (Solanum lycopersicum) are conserved and 91.3% (30,294) of 33,183 detected small exons in barley (Hordeum vulgare) are conserved. The higher percentage of conserved small exons than that of the micro exons suggests that the small exons might have been under greater selection possibly due to their greater structural and functional contribution to the corresponding genes, whereas the higher variability of micro exons among species might allow differential splicing diversity in a species/lineage-dependent manner. Furthermore, the median percentage of the conserved medium, large, and super exons per species was estimated as 82.3%, 70.2%, and 44.9%, respectively (Supplementary Fig. 6d–f). The variability of the percentage of conserved exons per species among five categories suggests evolutionary plasticity of gene structures of different gene orthogroups among angiosperm species.

Among these conserved exons, 14,107 conserved micro exons were detected in 5762 orthogroups (containing a total of 182,094 genes, when counted separately for genes of individual species) from across 50 seed plants included here (Supplementary Fig. 7). Among these 5762 orthogroups, 80.9% (4659) have genes from at least one angiosperm and one gymnosperm and are considered conserved among seed plants. These 4659 seed-plant orthogroups contain 81.5% (11,502) of all conserved micro exons (Supplementary Fig. 7), implying that most conserved micro exons are derived from the most recent common ancestor (MRCA) of seed plants. In addition, conserved small exons (44,336) were detected in 10,345 orthogroups (containing 494,140 genes; Supplementary Fig. 7). A comparison of these 10,345 orthogroups indicated that 76.9% (7953) of these orthogroups have genes from at least one angiosperm and one gymnosperm and that these 7953 orthogroups include 36,399 conserved small exons, accounting for 82.1% of 44,336 conserved small exons, again suggesting that most conserved small exons are derived from the MRCA of angiosperms and gymnosperms. Moreover, conserved medium exons were found in 12,119 orthogroups (containing 613,886 genes); conserved large exons were identified in 12,395 orthogroups (with 553,124 genes); and conserved super exons in 6453 orthogroups (243,260 genes; Supplementary Fig. 7). A comparison of orthogroups including conserved medium, large, and super exons retrieved patterns similar to those for micro and small exons: 9074 seed-plant orthogroups contain 80.2% (36,854) of all conserved medium exons; 8802 seed-plant orthogroups include 74.1% (20,283) of all conserved large exons; and 4,102 seed-plant orthogroups contain 65.3% (6175) of all conserved super exons. Hence, most of the conserved exons are likely derived from the MRCA of seed plants.

To further investigate the conservation patterns of exons, including those that are not found in both angiosperm and gymnosperm genes (as described above), we used ExonEvo to infer the MRCA of the genes containing conserved exons in gene trees and to reconcile the MRCA to the species tree (Supplementary Figs. 8–13). Among the retrieved patterns, the largest group (median of 78.7% among species) of the conserved exons are shared by seed plants (Fig. 2c), supporting the above results of orthogroup analyses. Among the conserved micro exons, 3411 exons were mapped to the MRCA of seed plants, 2353 to the MRCA of angiosperms, 1292 to that of eudicots, 274 to that of monocots, and 10,188 to other phylogenetic positions (Supplementary Fig. 9). The pattern of conserved micro exons shared by seed plants and various other groups implies that the retained exons have experienced diverse retention and loss events across angiosperms since they diverged from gymnosperms. In addition, our ExonEvo analyses of phylogenetic positions of the small exons showed that 22,120 exons are shared by seed plants, 7596 by angiosperms (but not gymnosperms), 3002 by eudicots, 1393 by rosids (124+146+318) or asterids (73+485+247) (both largest eudicot subgroups), 5614 by other groups represented by two or more species here, and 4515 by paralogues within a single species (Supplementary Fig. 10). The diverse conservation landscapes of the small exons across angiosperms suggest that gene structures have changed involving small exons during angiosperm evolution. ExonEvo analyses also suggested that 53.6% (24,634) of all conserved medium exons, 45.8% (12,545) of all conserved large exons, and 38.8% (3675) of all conserved super exons are mapped to the ancestor of angiosperms and gymnosperms (Supplementary Figs. 11–14), implying different retention patterns of these three exon-size categories after angiosperms diverged from gymnosperms. Overall, 66,385 conserved exons were mapped to the MRCA of seed plants, 26,454 to angiosperm ancestor, 1919 to gymnosperm ancestor, 10,160 to eudicots, 2108 to monocots, 271 to magnoliids, 14,118 to different lineages, and 19,814 to other phylogenetic positions (Fig. 3a and Supplementary Figs. 9–13). It seems possible that the ancestral gene structure genes might be differentially retained during evolution, allowing for changes in splicing and protein function.

Among the 50 genomes, 913,316 genes have conserved exons; 7773~36,354 (median 12,989) genes in specific genomes contain seed plant-wide conserved exons, representing 65.2%~95.6% (median 76.7%) of the annotated genes per genome (Fig. 2d). These seed-plant conserved exons affect most genes and, therefore, likely have the greatest importance in gene evolution. For example, 11,355 Arabidopsis genes contain at least one seed-plant conserved exon; among these 11,355 genes, 8090 genes only contain conserved exons that are shared by seed plants, while the remaining 3265 genes also contain conserved exons shared by other groups, such as angiosperms. In rice, 13,021 genes contain at least one seed-plant conserved exon; among these 13,021 genes, 9361 genes only contain seed-plant conserved exons, while 3660 genes also contain conserved exons of other phylogenetic positions. Similar conservation patterns of exons were found in other genomes, with most genes containing conserved exons that are shared by seed plants (Fig. 2d). The results suggest that the ancestral exon structures have been retained in most genes, with likely conservation of possible splicing regulation and protein isoforms.

In addition to the conserved exons, we also surveyed the exons that are merged from two or more conserved exons [due to loss of intron(s)] and retrieved 3236~17,770 (median 5786) merged exons in individual species (Fig. 2e). For example, genes encoding ubiquitin-conjugating enzyme 19 (UBC19) have two conserved micro exons, two conserved small exons, one conserved medium exon, and one conserved large exon (Supplementary Fig. 15). Among these six exons shared by seed plants, five conserved exons were merged into a large exon in the grass UBC19 genes (Supplementary Fig. 15), implying that grasses might have lost the functional plasticity of ancestral UBC19 genes related to transcript/protein isoforms in comparison to other seed plants.

As the seed-plant conserved exons are the largest group, accounting for 47% of all conserved exons (Fig. 3a), we further examined their retention in major groups of angiosperms, including two major subclades of eudicots, rosids (such as Arabidopsis and soybean) and asterids (such as tomato), other eudicots, monocots, magnoliids, and other angiosperms. Among the seed-plant conserved exons, numbers of exons in each of the five size categories shared by various combination of the angiosperm groups are shown in Fig. 3b–e. Specifically, 99% (65,722) of the seed-plant exons were detected in at least one genome of core angiosperms (here including eudicots, monocots, and magnoliids), while 85.3% (56,630) were detected in at least one of the two basal angiosperm genomes (Fig. 3b). Small and medium exons together represent the majority (70.4%; Fig. 3c) of seed-plant conserved exons. Furthermore, 77.3% (51,309) of the seed-plant conserved exons are retained in all six angiosperm groups (Fig. 3d), including 2442 micro, 18,004 small, 19,478 medium, 9129 large, and 2256 super exons (Fig. 3e). Among 3411 micro exons, the largest portion (2442) represents the conserved exons shared by all six angiosperm groups (Fig. 3c–e). For instance, among the seed-plant homologs of the Arabidopsis MKK6 gene (AT5G56580), the conserved micro exon (EXON0044515; Supplementary Data 5) is detected in rosids (such as Arabidopsis and apple) and asterids (such as tomato and kiwifruit), other eudicots (such as Papaver), monocots (such as rice and maize), magnoliids (such as avocado), and other angiosperms (such as Amborella trichopoda). Among the remaining 969 (=3411-2442) micro exons, 287 exons are detected in all but one group. For example, 127 exons are present in core eudicots and other angiosperms but absent in early diverging eudicots (Nelumbo nucifera and Papaver somniferum). Among the 127 exons, 41 were apparently merged with adjacent exon(s) with the loss of the intervening intron, resulting in longer exons in N. nucifera and P. somniferum and retention of the protein coding sequences. For another 42 exons that were absent in N. nucifera and P. somniferum, the entire genes were not detected in these plants. The remaining 44 (=127-41-42) conserved exons were part of members of orthogroups, but the genomic sequences of the corresponding genes in N. nucifera and P. somniferum lacked a detectable similar sequence to the conserved exons, suggesting that the exons are not required for the function of these early diverging eudicot genes. On the other hand, 300 exons are detected in only one angiosperm group; for example, 77 exons present in monocots but absent in other angiosperm groups include 44 exons that were merged into long exons in at least one other angiosperm groups, suggesting specific retention in one group and fusion or losses in others during the angiosperm history. Analyses of these 22,120 seed-plant conserved small exons showed that the largest portion (18,004) represents the small exons shared by all six angiosperm groups, again suggesting angiosperm-wide retention of these small exons. Among 4,116 other small exons, 1412 exons are absent in only one of the groups (including 370 ones merged into other exons in one corresponding group), whereas 1022 exons are present in only one group (543 of them fused into long exons in other groups), showing different retention and fusion patterns of the ancestral exons among angiosperm groups. In addition, analyses of three other exon-size categories revealed that 19,478 medium exons, 9129 large exons, and 2256 super exons are shared by all six angiosperm groups, suggesting that all angiosperm groups retained these exons after their ancestor diverged from gymnosperms. Generally, 77.3% (51,309=2442+18,004+19,478+9129+2256) of the 66,385 seed-plant conserved exons are retained in all angiosperm groups (Fig. 3b, c). Among the remaining 15,076 exons, 4675 seed plant conserved exons are detected in nearly all groups but one group (1313 merged in one corresponding group), while 3716 exons are detected in only one of the groups (1867 merged in other groups). The pattern simply that most of the seed-plant conserved exons have been retained in all angiosperm groups, likely allowing the conservation of possible regulation at the splicing level during the diversification of the vast number of angiosperms.

Diverse patterns of conserved exons corresponding to protein domains
As both exons and protein domains have variable sizes, there could be several possible corresponding relationships between exons and their encoded protein domains. Sequences of two or more exons might encode a single domain, or a single (relatively large) exon could encode an entire domain. Conversely, a single exon can encode portions or all of multiple domains. When a domain is encoded by (parts of) multiple exons, it could have structurally different isoforms due to alternative splicing involving one or more of the corresponding exons, with potentially different functions. On the other hand, a domain encoded by a single exon would have a uniform structure in all transcripts containing the exon with the same function. However, the genome-wide patterns of correspondence between exons and domains have not been reported in part due to the lack of analyses of the conservation of exons. In addition, different gene families can have similar sequences of a shared domain that is characterized by a PFAM profile. We defined a domain family as containing domains detected in one or more orthogroups using the same PFAM profile. The domain from each orthogroup is referred to as a member of the domain family. Whether different members of the same domain family can have different patterns of domain-exon organization is not clear on a genome level across many angiosperms, even though such knowledge can help us understand possible exon splicing patterns concerning domain functions. The above results on angiosperm exon conservation provide an opportunity to explore the evolutionary patterns of domains encoded by conserved exons in angiosperms. We found that 106,809 (=92,868+6400+5087+2454; ~76%) conserved exons have annotated domain information (Fig. 4a), whereas 34,420 (~24%) conserved exons with annotated open reading frames for protein-coding sequences (including 17,288 exons being the first or last exons in genes and 17,132 exons in other positions) do not match annotated domains (not even “functional unknown domains”); these conserved protein-coding regions could be added to future domain databases using updated gene annotation algorithms or supported by new functional studies.

According to the relative positions between exons and their corresponding domain, we assigned exons into four categories of “domain-exon organization” (Fig. 4a; see Supplementary Data 10 for the correspondence in individual conserved exons in 50 genomes). As an example, in seed plant homologs of the MALE STERILITY 2 (MS2)64, a region of ~912-bp encoding a NAD_binding_4 domain (an essential domain for MS2 gene function65) corresponds to 6 conserved exons shared by seed plants (Supplementary Fig. 16). Such a pattern of multiple exons encoding a domain is referred to as category-I here. Among 106,809 conserved exons with annotated domain information, 92,868 exons (including 54,195 exons mapped at the MRCA of seed plants) belong to category-I and represent the largest fraction of all conserved exons that matched domains. These exons match 46,399 domains, which belong to 10,765 domain families according to sequence similarity and Pfam definitions (Fig. 4a). The functions of most domains in angiosperms are probably conserved based on their matched conserved exons. In addition, we defined the pattern of a single exon matching only one whole domain as category-II (Fig. 4a). For example, sequences (138 bp) of a single exon (in median length of 212 bp) shared by angiosperms encodes a MYB DNA-binding domain (PF00249; Supplementary Fig. 17). Category-II includes 6400 conserved exons encoding 6400 domains of 3661 domain families (Fig. 4a), representing the second largest portion of all conserved exons that matched domains. Furthermore, we also observed cases where a single exon matches multiple domains (Fig. 4a). For instance, in angiosperm WRKY37 genes (Supplementary Fig. 18), the WRKY DNA-binding domain (PF03106) is encoded by a portion of a conserved medium exon (138 bp) shared by angiosperms and a portion of a 538-bp super exon, which also includes sequences encoding another domain (similar to Hid1: High-temperature-induced Dauer-formation 1; PF12722) with a signaling function in worms. Such a pattern of an exon encoding at least one whole domain and part of another domain is defined here as category-III (Fig. 4a). To avoid overlapping definitions, domains entirely encoded by category-III exons are referred to as category-III domain(s), whereas the domains partially encoded by category-III exons and partially by additional exon(s) are assigned to category-I. There are 5087 category-III exons and they encode 11,455 domains of 3422 domain families (Fig. 4a). When a single exon encodes multiple whole domains and does not match any part of other domains, the correspondence was referred to as category-IV (Fig. 4a). An example of category-IV can be found in AUXIN RESPONSE FACTOR (ARF) genes (functioning in flora development, such as the ref. 66) with a super exon of 1101 bp conserved among seed plants; regions of this super exon with 306 bp and 252 bp encode, respectively, a B3 DNA-binding domain (PF02362) and an ARF domain (PF06507) (Supplementary Fig. 19). Category-IV has 2454 exons that encode 6692 domains of 2777 domain families (Fig. 4a). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants.

Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore, domains with different domain-exon categories (as described above) can be encoded by homologs and belong to the same domain family. To probe the evolutionary dynamics of exon-intron structure, we compared exon/domain categories among domain families and obtained the overlap between the four sets of domain families. Among 13,463 domain families recognized in the PFAM database and observed here (Fig. 4b), 8495 (~63.1%) have only one category of exon-domain correspondence (Categories-I, II, III, and IV with 6227, 889, 779, and 600 families, respectively); whereas only 524 (~3.9%) domain families have all four categories, with the remaining families having two or three categories (Fig. 4b). Thus, most of domain families have only one category of exon-domain correspondence, suggesting a high degree of evolutionary stability of exon-intron structure. Specifically, 6227 domain families are only found in category-I, such as the NAC domain (PF01849). The domains in these families match multiple exons and, hence, might have evolved to have diverse functions due to possible alternative splicing of exons. Also, 889 families have only category-II domains corresponding to a single exon, implying that these families might have more similar functions in comparison with the families with domains matching multiple exons (category-I). Further, 779 and 600 families were only detected in category-III and category-IV (Fig. 4b). These four categories provide a general portrait of relationships between conserved exons and their encoded domains in seed plants. Homologous genes encoding members of the same domain family can have different exon-intron structures; therefore

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study sets out to better understand the conservation and divergence of exon sizes and their relationships to protein domains. These data should provide important input into evolutionary analyses of gene structure and function. To do this, the authors focus on plants (angiosperms and gymnosperms) and use 50 species with available WGS assemblies and annotations.\nSpecifically, the authors address questions regarding genome-wide conservation and patterns of exon sizes, their relationships to domains, expression of genes with exons of various sizes, and their alternative splicing, within angiosperms using data from the 46 species of angiosperms represented in their 50 species genome data sets. These were used to profile the exon sizes and number of the protein-coding gene, identify and characterize conserved exons, and explore the relationship between conserved exons and protein domains. The authors also analyzed public RNA-Seq datasets to examine expression patterns of genes containing conserved exons and to detect alternative splicing of conserved exons.\nPage 2, line 1 substitute Alternative for Alternatively.\nPage 3 line 39, change require to requires\nPage 3 line 40 “such ligand binding …” -> such as ligand binding …\nPage 4 line 76 you state “some paralogues of DNA-binding proteins lack a region”. What do paralogues refer to Are you referring to specific member genes within gene families that are missing domains, or are you referring to mRNA isoforms produced by AS that may exclude a domain such that expression of both isoforms leads to 2 distinct proteins?\nPage 4 line 95, remove the word “although”\nPage 5 line 123 – matched is misspelled.\nLine 205 – percent -> percentage\nLine 219 was -> were\nI don’t understand the relevance of line 208 and line 209 to the discussion of micro and small exons being disproportionately represented within exons that undergo exon skipping. What point are you trying to make?\nLine 228 is confusing – Alternative what? Alternatively spliced short exons?\nLine 433 belong -> belonging\nLine 438: this sentence is confusing, what are you trying to say??\nLine 458: to -> in\nLine 743: transcript -> transcripts\nLine 754: missing words beteen Angiosperms and gymnosperms\nLine 766 -7: missing word, for example, “some these conserved” -> “some of these conserved\" This sentence needs to be rewritten for clarity.\nLine 777-781: This needs to be re-written.\nMajor questions for revision and comment:\nThe last section of your results is concerned with whether AS affected conserved exons and how AS might affect conserved specific domain families. However, your analysis is completely restricted to Arabidopsis, even though you analyze RNA-Seq data from Arabidopsis, soybean, tomato, rice, and Amborella trichopoda. Why do you only discuss AS relative to Arabidopsis, when you could have used the RNA-Seq data for the other species to examine whether the conserves exons (shared with Arabidopsis) have evidence of alternative splicing in these other plants?\nLines 226-228 – your criteria for identifying conserved exons within an orthogroup seems very relaxed: “conserved exons between genes in an orthogroup as encoding at least 4 contiguous identical or similar amino acids and differing in length by < 20% between any two exons”. What is your rationale for 4aa? Have you run analyses at differing lengths -perhaps a plot of the proportion of aa conservation between all pairs of a set of conserved exons would suffice to illustrate the validity of this conservation (4aa) choice. I would also think you would benefit by considering exon order when identifying sets of conserved exons (like you do for AS exons).\nLine 355: “whereas 87,218 conserved exons do not match annotated domains (not even “functional unknown domains”), indicating that these conserved gene regions have not yet been recognized by gene annotation algorithms or any functional studies.” Do these exons have coding potential? Do they potentially represent 5 and 3’ UTRs? Can you look a their position within genes and coding potential to rule out the possibility that they are non-coding and thus contain no domains?\nPage 5 line 150. You state “It is possible that medium exons might have a frequently suitable size range for functional units (domains) in angiosperm genes.” And follow this up with a discussion of GO analysis in Arabidopsis. You should follow this statement up computationally. Do medium sized exons contain whole domains, what proportion of medium sized exons contain whole domains, and what are these domains – it seems you could do this with a HMMER search of translations of the medium exons, perhaps concentrating on the conserved ones?\nPage 7 line 175 you state “Large and super exons account for ~23% of exons per genome and correspond to most genes without introns” and further state that Chinese pine “ includes 38,784 singe-exon genes (with 156~6,807 bp of exon length) that account for\n184 50.87% of 76,234 genes”. How many of these are conserved, especially those that as single exon genes with super exon sizes? Can you predict if they are functional? Perhaps a substantial portion of these represent processed pseudogenes and need to be identified as such (in all species considered) and removed from analysis.\nAlmost every section of the results ends in a statement that micro or small exons of whichever classification you are discussing could be involved in regulating the gene by splicing, presumably alternative splicing (AS). This is always under the hypothesis that the removal of a small or micro exon would occur via exon skipping (ES), and this would have a small but specific change on the protein product that has functional significance. ES events are very common in animals, but relatively infrequent in plants. Alternative acceptor or alternative donor events are much more common in plants than ES events, and both of these would also result in small changes in the protein product, as long as they preserved the reading frame. None of the alternative splicing mechanisms other than exon-skip are discussed or considered. A statement that conserved micro or small exons (conserved or not) may regulate or change protein product through splicing is true, but obvious. To determine any relationship between AS and conserved exons in regulating protein requires identification of conserved AS events across species, and the correlation of these to conserved exons, which this study does not do.\nAs I mentioned previously, there are several alternative splicing mechanisms other than exon skip, and any of these can impact exons of any size, Subtle shifts in splice site choice (AtlA or Alt D) can affect any exon – for example, a super exon, and also result in a relatively small protein-coding change (as long as the event keeps the exon in frame). Your discussion seems to focus on the alternative splicing of micro/small exons as a mechanism to contribute to the diverse regulatory roles of genes. This is true, but not a necessity, and it’s possible that AS of these exons are a minority of events. To make any substantial claims you need to examine the other AS methods, and do the analysis to determine if your conserved micro and small exons are included in conserved AS events, and to what extent.\nWhat is your rationale for examining expression with transcriptomic data in Arabidopsis, soybean, tomato, rice, and Amborella trichopoda only, while your analyses include many species outside the clades that contain these species?\nYour exon collection for the study is pulled from the largest transcript of each annotated gene in the species you focus on. How confident are you in the annotations?\nIn cases where there exist multiple isoforms in the annotation, perhaps you should be looking at all the alternatively spliced isoforms to identify a non-redundant set of all exons for a given gene rather than only those defined within the longest transcript.\nLines 711-724. Your review of published transcriptomes in Arabidopsis looks at the correlation of exon size with exons that undergo exon skipping, and states that many of the skipped exons are conserved, and that exon skipping may be a conserved regulatory mechanism for these exons. Again, to make any substantive claim with certainty requires you to show that the conserved exons are also subject to skipping in other species.", "reviewer_2": "Zhang et al. conducted an interesting and solid analysis about the phylogenomic profile of exon-intron organization across seed plants, and the relationships between exon-intron organization with protein domains and functions. They examined over 6.3 million exons for 46 angiosperms and 4 gymnosperms and found that the micro and small exons account for ~35% of all exons. They showed that micro and small exons may have important contributions to alternative splicing and resultant flexibility of functions of proteins. This study provided us a big picture for how the exon-intron structures organized among genes across seed plants. Comments are listed below.\nMajor points:\nHow did the authors distinguish exon loss (e.g., Line 315) from potential mis-annotation of gene exon-intron structures in non-model species?\nThe authors emphasized a lot about the relationship between exon size and alternative splicing. But whether the length of introns contribute more to skipping of the exons between them remained also unknown. The single-factor-analysis may lead to bias conclusion. It is also interesting to know whether micro and/or small exons tend to be accompanied by shorter or longer introns than exons with longer length.\nThere were too many numbers of several exon categories in the main text, which is not friendly for the readers to follow the flow of this manuscript. Would suggest the authors to take good advantages of figures to illustrate different numbers in each comparison, and emphasize more about the interesting findings and indications in the main text.\nThe authors took a lot of examples for each types of exons or categories, but sometimes lacked the implications indicated by those examples, such as examples in Lines 551-571 (Fig. 5c).\nIn the section “Preferential expression of genes containing conserved exons among major organs”, especially the last paragraph in Lines 680-697, it sounds like that the expression profiles of genes (root, stem, fruit and other tissues) are correlated with exon-intron organization. However, to my knowledge, the gene expression profiles should be more likely to be regulated by cis-elements. It is reasonable to show that different transcripts of genes, the products of alternative splicing, may have preference for different tissue types. With regard to this, I like the section “Domains in genes contained conserved exons affected by exon skipping and implications in flower development”\nMinor points:\nPlease double check that all the numbers with more than three digits were split with comma.\nLine 15, “with two” may be “including two”?\nLine 23, may define what are conserved exons?\nLines 30-31, lack a transition between “micro and/or small exons” and “alternative splicing”\nLine 37, “and have diverse functions” is kindly of meaningless. May be written as “have diverse exon-intron organization and functions”?\nLine 66, before this sentence, why it is important to study the genome-wide patterns of the number and size of exons was not specified.\nLine 80, may miss a comma between “exon” and “was”\nLines 81-83, lack a transition between “the Arabidopsis GRP20 protein was shown to be required for proper retention of micro exons in transcripts of several floral homeotic genes, such as AG, AP1, AP2, AP3, and SEP3” and “the importance of micro exons in function of these developmental regulators”. The author may want to say the skip of micro exons of these genes lead to obvious phenotype changes?\nLines 145-146, may also provide the proportions of the numbers, for the comparison with those in Arabidopsis.\nLine 148, (~32%) should be moved after “with the most exons”\nLine 204, add proportions for other numbers\nLines 205-207, should specify what statistic test was conducted. In addition, “The percent of both micro and small exons among skipped exons are much greater than genome-wide percentage of these categories” should be tuned down since the percent and number of micro were smaller than those of medium. The same comment for lines 210-212.\nLines 207-209, exons in the example case were not specified to categories, as the cases before and after this.\nLine 221, flanking intron sequences, instead of flanking exon sequences?\nLine 250-251, it’s surprising that the super exons are less conserved than other types of exons.\nLine 305, typo “in in”\nLines 311-314, the numbers are referring to Fig. 3a, but the figure cited before this sentence is Fig. 3b. Did not read in a good flow.\nLine 425, “for protein interaction”, does that mean all the domains listed in Lines 423-425 are all function for protein interaction?\nColors in Fig. 4d are similar to colors in Fig. 4b, which led to misunderstanding at the first glance.\nThe paragraph after Line 534 may be organized as a new section, since all the above paragraphs were talking about the numbers of different types of exons.\nLines 544-550, the types A-D should be marked in the Figure 5b.\nLines 700-709, may move the background part to introduction section.\nLine 721, miss “%” after 70.5."}, "author_response": "Dear Reviewer #1 and #2:\nWe greatly appreciate your positive feedback and very valuable comments and suggestions. For your\nconvenience regarding our revision, we briefly summarized our some of the major revisions below and\nprovided detailed point-by-point responses on the subsequent pages.\nWe define a conserved exon family as those exons that are shared by two or more genes in an orthogroup\nof genes derived from an ancestral gene in the most recent common ancestor of angiosperms; we referred\nto such a conserved exon family as a “conserved exon” for brevity, unless otherwise noted. We have\nimproved the detection of conserved exons with a set of criteria using amino acid sequence similarity and\nposition relative to other conserved exons and classified them in three types: (1) highly conserved exons as\nhaving at least 50% amino acid sequence similarity; (2) moderately conserved micro exons with 30%~50%\namino acid sequence similarity and moderately conserved exons in four other length categories showing at\nleast 20% similarity; and (3) candidate conserved exons as exons if their flanking exons are conserved as\ndefined following criteria 1 or 2. We identified 141,229 conserved exons (slightly fewer than 143,112 ones\nin our original submitted manuscript; <1.4% reduction). In addition, we examined annotated protein\ndomains in all 1,528,298 genes from 50 genomes (instead of the previous analyses of predicted ancestral\ngenes) and investigated individual exons in all conserved family of exons for the corresponding domains.\nWe found that 106,809 (75.6% of 141,229) conserved exons (partially) match known (annotated) domains,\naccounting for 59.3% of all annotated domains in PFAM and InterPro databases. The new results and the\nrelevant subsequent analyses further support our conclusion that most conserved exon families match\nknown domains and that most domain families have only one type of exon-domain\ncorrespondence/organization.\nTo examine the alternative splicing events for conserved exons, we expanded the analyses of transcriptome\ndatasets from the previous single species (Arabidopsis) to a total of 11 angiosperms that have available\ntranscriptome datasets (with three biological replicates) from multiple organs (flower, fruit, and leaf)\nand/or two other organs (stem and root). We detected exon skipping, A3SS, and A5SS events in all 11\nspecies and identified alternative splicing events affecting conserved exons, including those events for\nexons in two or more species that belong to the same conserved exon family. We found that 17,032\nconserved exon families included specific exons that were detected to be skipped in at least two of the\n11 species, 30,339 had exons showing conserved A3SS events, and 12,185 had exons with conserved\nA5SS events. We also found that 22,689, 17,369, and 17,770 conserved exon families have exon\nmembers exhibiting skipping, A3SS, and A5SS events, respectively, in only one of these 11 species.\nThese results suggest that specific forms of alternative splicing of some conserved exons are shared\nby at least some angiosperms. These results on alternatively splicing are presented in the revised\nmanuscript. In addition, these transcriptome datasets were also used for investigation of the conservation\nof expression patterns of genes containing conserved exons.\nIn general, we have revised and improved the results of exon conservation, exon-domain organization,\nexpression of genes containing conserved exon(s), and alternative splicing of conserved exons. To present\nour results, we have added new version of Supplemental Table 1, Supplementary Data 6 and 7,\nSupplementary Tables 9-12, and Supplementary Figures 2-5, 7, 38-40, 45, 48-52, and updated Figures 2-7,\nSupplementary Data 1-5, Supplementary Tables 5-8, Supplementary Figures 6, 9-14, 41.\nAgain, thank you very much for your careful review of the previous manuscript. We hope that these\nchanges have addressed the concerns.\nHong Ma\nThis study sets out to better understand the conservation and divergence of exon sizes and their\nrelationships to protein domains. These data should provide important input into evolutionary analyses of\ngene structure and function. To do this, the authors focus on plants (angiosperms and gymnosperms) and\nuse 50 species with available WGS assemblies and annotations.\nResponse: We thank the reviewer for the positive feedback.\nSpecifically, the authors address questions regarding genome-wide conservation and patterns of exon sizes,\ntheir relationships to domains, expression of genes with exons of various sizes, and their alternative\nsplicing, within angiosperms using data from the 46 species of angiosperms represented in their 50 species\ngenome data sets. These were used to profile the exon sizes and number of the protein-coding gene,\nidentify and characterize conserved exons, and explore the relationship between conserved exons and\nprotein domains. The authors also analyzed public RNA-Seq datasets to examine expression patterns of\ngenes containing conserved exons and to detect alternative splicing of conserved exons.\nResponse: We thank the reviewer for the positive feedback.\nPage 2, line 1 substitute Alternative for Alternatively.\nResponse: Thanks. Done.\nPage 3 line 39, change require to requires\nResponse: Thanks. Done.\nPage 3 line 40 “such ligand binding …” -> such as ligand binding …\nResponse: Thanks. Done.\nPage 4 line 76 you state “some paralogues of DNA-binding proteins lack a region”. What do paralogues\nrefer to Are you referring to specific member genes within gene families that are missing domains, or are\nyou referring to mRNA isoforms produced by AS that may exclude a domain such that expression of both\nisoforms leads to 2 distinct proteins?\nResponse: We are sorry for the unclear statements in the previous manuscript. We have revised the\nsentences in the revised manuscript as shown below.\n“DNA-binding proteins often contain both DNA-binding and protein-interaction domains. In some DNA-\nbinding protein families, some paralogues are truncated and lack some domain(s) and have been reported\nto modulate the function of other full-length paralogues31-33.” (Lines 82-84)\nPage 4 line 95, remove the word “although”\nResponse: Thanks. Done.\nPage 5 line 123 – matched is misspelled.\nResponse: Thanks. Done.\nResponse: Thanks. Done.\nLine 219 was -> were\nResponse: Thanks. Done.\nI don’t understand the relevance of line 208 and line 209 to the discussion of micro and small exons being\ndisproportionately represented within exons that undergo exon skipping. What point are you trying to\nmake?\nResponse: We are sorry that the original sentence was not clear. In the revised manuscript, we have\nexpanded the analyses of alternative splicing to include exon skipping, alternative 5’ splicing site (A5SS),\nand A3SS using transcriptome data from 11 angiosperms (soybean, apple, watermelon, tomato, grape,\nPapaver (poppy), maize, wheat, pineapple, and Amborella trichopoda, in addition to Arabidopsis). In light\nof the expanded analyses and results, we feel that the previous sentence indicated in this comment is not\nnecessary. We have removed the sentence.\nLine 228 is confusing – Alternative what? Alternatively spliced short exons?\nResponse: We are sorry for the unclear description previously. In the originally submitted manuscript, the\nword “alternative” should have been “alternatively”. In the revised manuscript, we have optimized the\ncriteria for defining conserved exons and we have revised the statements as follows:\n“We define conserved exons between two or more genes in the same orthogroup in three types: (1) highly\nconserved exons as having at least 50% amino acid sequence similarity; (2) moderately conserved micro\nexons with 30%~50% amino acid sequence similarity and moderately conserved exons in four other length\ncategories showing at least 20% sequence similarity; and (3) candidate conserved exons as exons if their\nflanking exons are conserved as defined following criteria 1 or 2 (see methods).” (Lines 246-250)\nLine 433 belong -> belonging\nResponse: Thanks. Done.\nLine 438: this sentence is confusing, what are you trying to say??\nResponse: We are sorry for the unclear description. We have revised this sentence as shown below.\n“Protein isoforms that differ due to the presence or absence of relatively short peptides encoded by micro\nor small exons could differ in some functional aspects, but not losing all protein activities.” (Lines 480-\n481)\nLine 458: to -> in\nResponse: Thanks. Done.\nLine 743: transcript -> transcripts\nResponse: Thanks. Done.\nResponse: Thanks; we should have included “and” between “angiosperms” and “gymnosperms”. We\nrevised this sentence in our revised manuscript as follows.\n“Our phylogenomic analyses of exon landscapes across 46 angiosperms and 4 gymnosperms provide\ndistribution profiles in five different length categories, including previously proposed micro and small\nexons, and present their conservation among seed plants, angiosperms, and angiosperm subgroups on gene\nstructure and domain-exon correspondences.” (Lines 805-808)\nLine 766 -7: missing word, for example, “some these conserved” -> “some of these conserved\" This\nsentence needs to be rewritten for clarity.\nResponse: We are sorry for the confusing wording. We have revised this sentence in our newly organized\ndiscussion section as follows.\n“These non-conserved super exons probably resulted from fusion of two (or more) conserved exons via\nloss of intron(s) during angiosperm evolution, suggesting that potential alternative splicing of the ancestral\n(unfused) exons is not part of mechanisms regulating the transcript isoforms for the relevant exon(s).”\n(Lines 832-835)\nLine 777-781: This needs to be re-written.\nResponse: We are sorry that the original sentence was not clear. We revised this sentence in our revised\nmanuscript as follows.\n“For example, among homologues of the MS2 gene, which is required for normal anther development,\nmost seed plant genes have 9 exons, representing a likely ancestral gene structure (Supplementary Fig. 16).\nHowever, in coffee two annotated adjacent genes111 on chromosome 6 are found here to be homologous to\nMS2: one of these (GSCOCT00040587001) has 4 exons corresponding to the four upstream ancestral MS2\nexons and the other (GSCOCT00040586001) has 5 exons corresponding to the five downstream ancestral\nexons. Our results support a likely gene model that combines the two coffee genes into a single gene, with\n9 exons similar to other angiosperm MS2 homologues.” (Lines 911-917)\nMajor questions for revision and comment:\nThe last section of your results is concerned with whether AS affected conserved exons and how AS might\naffect conserved specific domain families. However, your analysis is completely restricted to Arabidopsis,\neven though you analyze RNA-Seq data from Arabidopsis, soybean, tomato, rice, and Amborella\ntrichopoda. Why do you only discuss AS relative to Arabidopsis, when you could have used the RNA-Seq\ndata for the other species to examine whether the conserves exons (shared with Arabidopsis) have evidence\nof alternative splicing in these other plants?\nResponse: We thank the reviewer for this valuable suggestion. In the revised manuscript, besides\nArabidopsis, we have added analyses of AS events using published RNA-Seq datasets from 10 other\nspecies, apple, soybean, watermelon, tomato, grape, Papaver (poppy), maize, wheat, pineapple, and\n(representing 8 orders and 9 families) and due the available high-quality datasets from several major\norgans/tissues, including leaves, stems, flowers, and fruits. Although rice is an important monocot with the\nfirst sequenced genome among monocots (e.g., Goff et al., 2004, Science), much of the differential gene\nexpression studies were performed using microarrays (e.g., Ma et al., 2005, Genome Research). Such data\ncould not be used to detect AS. We searched for recent RNA-seq datasets, but we could not find a study\nwith similar major tissues as other species and enough biological replicates. We provide a revised Figure 7\nand related Supplementary Figures 48-49, Supplementary Tables 7, 9, 10, and Supplementary Data 7. We\nhave revised our manuscript to report and discuss the conserved exons affected by exon skipping across\nthese 11 species as follows.\nLines 717-769:\n“To address these questions, we analyzed alternative splicing events affecting protein-coding exons using\nrMATS (see methods) in comparison with the annotated exon-intron structure, by analyses of RNA-seq\ndatasets of 11 angiosperms (Arabidopsis thaliana, apple, soybean, watermelon, tomato, grape, Papaver\n[poppy], maize, wheat, pineapple, and Amborella trichopoda), which were selected to represent\nangiosperm diversity and for their available high-quality datasets. We found a total of 40,416 exon\nskipping events in root transcripts (10 species), 26,650 in stem transcripts (7 species), 58,224 in the leaf\n(11 species), 52,095 in the flower (11 species), and 47,003 in the fruit (11 species) (Supplementary Fig.\n48). Among these skipped exons, small exons are the largest class and are enriched in each tissue of each\nspecies relative to the proportion of small exons among intron-containing genes from the respective\nspecies (Supplementary Fig. 48). Super exons are the smallest class among skipped exons in all tissues and\nlarge exons are the second-smallest class in most tissues (Fig. 7a, Supplementary Fig. 48); it is possible\nthat the absence of a super exon can cause a severe functional defect of the protein, so such skipping is not\nbeneficial to plant fitness. We also found other types of alternative splicing (A3SS and A5SS,\nSupplementary Fig. 49). However, intron retention results are prone to false positives due to incompletely\nspliced transcripts, thus not examined here.\nWe next investigated the extent of exon skipping that affected members of conserved exon families. In the\nfloral transcriptome of a particular species the 11 angiosperms investigated here, 610~10,957 (median of\n3,850) events affecting conserved exons were detected, with a combined total of 40,732 events\n(Supplementary Table 9). Similarly, thousands of exon skipping events were detected in the other tissues,\nwith a total of 36,367 and 45,748 skipping events, respectively, detected in the fruit and the leaf and\naffecting conserved exons (Supplementary Table 9). The large fractions of conserved exons experiencing\nexon skipping suggest that alternative splicing (especially exon skipping) might be a conserved regulatory\nmechanism for these exons and that proteins encoded by transcripts lacking the exon might have a\ndifferent function from proteins encoded by the full-length transcripts.\nTo identify shared exon skipping among the 11 species, we searched for the conserved exons that were\nskipped in a focal tissue in at least two species and found 6,275 such skipped exons in root, 3,106 in stem,\n10,447 in leaf, 8,977 in flower, and 7,655 in fruit (Fig. 7b; see individual exons in Supplementary Data 7),\n5\nsuggesting that many of the exon skipping events are conserved to some extent. In individual species,\nabout 50.3%~68.8% (median of 60.7%, in a species) of skipping events in the flower affected conserved\nexons in two or more species. In addition, among 36,367 skipping events affecting conserved exons\ndetected in the fruit, 50.9%~64.6% (median of 57.2%, in a species) of them were shared by two or more\nspecies. Among 45,748 skipping events in the leaf affecting conserved exons, 55.8%~68.8% (median of\n65.3%) of events detected in individual species were shared by two or more species. Specifically in the\nArabidopsis flower, 675 skipping events were detected that affected individuals of conserved exons,\nincluding 235 events detected only in Arabidopsis and 440 (65.2%) events shared by Arabidopsis and\nother species. For example, a conserved exon (EXON0070818) in seed-plants homologues of AT1G44760,\nwhich is a proposed target gene of AP1 and AP3102, was found to be skipped in the flower of Arabidopsis\n(Exon 2 in AT1G44760.2), soybean (Exon 2 in GmW82.14G142700.1), and wheat (Exon 2 in\nTraesCS1B03G0773200.1) (Table 1; see affected exons in Supplementary Data 7). Some exon skipping\nevents were also shared by two or more the other 10 species analyzed here, such as the 409 events in\ntomato and other species (Table 1). For instance, members of the conserved exon family (EXON0023600)\nin angiosperm homologues of ARF8 (AT5G37020), which has regulatory role in stamen elongation in\nArabidopsis103, were skipped in the flower of tomato (Exon 2 in Solyc03g031970.4.1) and grape (Exon 2\nin both Vitis12g00136.t01 and Vitis10g01227.t01) (Table 1 and Supplementary Data 7).\nFor exons skipped in the Arabidopsis flower, we found that 126 of 145 micro exons, 376 of 425 small\nexons, 150 of 188 medium exons, 21 of 29 large exons, and 2 of 4 super exons were conserved exons and\nthat the seed-plant wide conserved exons account for 77.8% (98 of 126), 85.4% (321 of 376), 86% (129 of\n150), 85.7% (18 of 21), and 100% (2 of 2) of conserved micro, small, medium, large, and super exons,\nrespectively, that were skipped in the flower (Supplementary Fig. 48). Similar patterns were also found in\nother species. In general, seed-plant wide conserved exons are the majority (> 52%) of micro, small, and\nmedium exons affected by skipping in at least one tissue (Fig. 7a, Supplementary Fig. 48). In each\ntissue >56% of these exons are micro or small exons (Fig. 7b), implying that skipping particularly\ninvolving micro and small exons contributed substantially to transcript/protein diversity. In addition, a total\nof 17,032 conserved exons skipped in at least two species account for ~43% of these 39,721 conserved\nexons with skipping event detected in at least one of these 11 species (Supplementary Table 10),\nsuggesting that skipping of conserved exons are conserved across angiosperms.”\nLines 226-228 – your criteria for identifying conserved exons within an orthogroup seems very relaxed:\n“conserved exons between genes in an orthogroup as encoding at least 4 contiguous identical or similar\namino acids and differing in length by < 20% between any two exons”. What is your rationale for 4aa?\nHave you run analyses at differing lengths -perhaps a plot of the proportion of aa conservation between all\npairs of a set of conserved exons would suffice to illustrate the validity of this conservation (4aa) choice.\nI would also think you would benefit by considering exon order when identifying sets of conserved exons\n(like you do for AS exons).\nResponse: We sincerely thank the reviewer for these comments to help us improve our analyses. We have\nredefined the criteria for conservation of exons using amino acid sequence similarity and other features\n(see below in the quoted texts from the revised manuscript). We performed additional analyses to compare\nthe proportion of aa conservation between all pairs of a set of conserved exons, generating 141,229\nconserved exons (slightly fewer than 143,112 exons in our original submitted manuscript; <1.4%\n6\nreduction). We revised the sections of results and methods in our manuscript and supplied a new\nSupplementary Fig. 5. With information on these conserved exons, we updated the Supplementary Data 1-\n5 and Supplementary Figures. We also revised the numbers related to conserved exons in following\nsections. The revised number also support the conclusions in our originally submitted manuscript.\nFor results:\n“We define conserved exons between two or more genes in the same orthogroup in three types: (1) highly\nconserved exons as having at least 50% amino acid sequence similarity; (2) moderately conserved micro\nexons with 30%~50% amino acid sequence similarity and moderately conserved exons in four other length\ncategories showing at least 20% sequence similarity; and (3) candidate conserved exons as exons if their\nflanking exons are conserved as defined following criteria 1 or 2 (see methods). For statistics of conserved\nexons across angiosperms (rather than a species), each conserved exon family containing members from\ngenes of an orthogroup (referred to as “conserved exon” hereafter for brevity, unless otherwise noted) is\ncounted once regardless of the number of genes containing the exon. Following these criteria, we\ndeveloped a new bioinformatic tool, ExonEvo, to identify conserved exons in annotated genomes using\nphylogenomics (see methods). We then used ExonEvo to compare genes in the above-mentioned gene\ntrees and identified 141,229 conserved exons (including 122,059 highly conserved exons, 14,548\nmoderately conserved exons, and 4,622 candidate conserved exons; Supplementary Fig. 5), each of which\nis present in two or more genes from the 50 genomes. In individual species, the conserved exons represent\n69.8%~84.9% (median 79.9%) of total annotated exons (Fig. 2a, 2b). Among these conserved exons,\n14,107 are micro exons (Supplementary Data 1), 44,336 small exons (Supplementary Data 2), 45,961\nmedium exons (Supplementary Data 3), 27,364 large exons (Supplementary Data 4), and 9,461 super\nexons (Supplementary Data 5).” (Lines 246-260)\na, Bar-plots of counts of highly conserved exons. Color of bars represent different ranges of exon sequence\nsimilarity as indicated on the right.\nb, Bar-plots of counts of likely conserved exons. Color of bars represent different ranges of exon sequence\nsimilarity as indicated on the right.\nc, Bar-plots of counts of candidate conserved exons with flanking conserved exons.\nFor methods:\n“We defined the conservation of two exons according to their amino acid sequence similarity, exon length\nand position. Specifically, the amino acid sequence similarity of any two exons was estimated by amino\nacid substitution matrices in BLOSUM-62163. The sequence similarity of exons A (from gene-1) and B\n(from gene-2) was defined as the ratio of the same or similar amino acids counts between the two exons to\nthe number of amino acids in exon-A that were aligned to the amino acids in exon-B (excluding gaps) in\nthe amino acid alignment among gene-1, gene-2, and other homologous genes in an orthogroup. We\n8\nestimated the similarity between exon-A and exon-B, when there is a difference in length between the two\nexons less than 20% of the longer exon, and defined exons with >50% similarity as highly conserved\nexons. We also compared their flanking closest exons (if occurred) and defined such exons with flanking\nconserved exons as candidate conserved exons. For micro exons, we proposed exons in the similarity\nrange from 30% to 50% as moderately conserved exons. For other exons, we proposed exons in the\nsimilarity range from 20% to 50% as moderately conserved exons.” (Lines 962-972)\nLine 355: “whereas 87,218 conserved exons do not match annotated domains (not even “functional\nunknown domains”), indicating that these conserved gene regions have not yet been recognized by gene\nannotation algorithms or any functional studies.” Do these exons have coding potential? Do they\npotentially represent 5 and 3’ UTRs? Can you look at their position within genes and coding potential to\nrule out the possibility that they are non-coding and thus contain no domains?\nResponse: We thank the reviewer for this valuable comment and we are sorry that we did not describe\nclearly about the domain analyses of conserved exons. The exons included in domain analyses were all\nannotated to encode proteins, as they are from the CDS of protein-coding genes. We examined annotated\nprotein domains in all 1,528,298 genes from 50 genomes (instead of the previous analyses of predicted\nancestral genes) and investigated individual exons in all conserved family of exons for the corresponding\ndomains, resulting in the detection of more correspondence of conserved exons with protein domains. Our\nnew exon conservation and domain analyses found ~76% (106,809) of 141,229 conserved exons matching\nprotein domains. The number of conserved exons that do not match annotated domains was revised to be\n34,420, accounting for ~24% of 141,229 conserved exons, which is less than the portion (61%=87,218 out\nof 143,112) reported in our originally submitted manuscript. Among the 34,420 exons without any\nmatching known domains, 17,288 (50.2%) exons are the first or last exons and 17,132 (49.8%) are exons\nin other positions. We have revised the analyses in the Results and Methods sections of the revised\nmanuscript, as shown below:\nLines 395-400:\n“We found that 106,809 (=92,868+6,400+5,087+2,454; ~76%) conserved exons have annotated domain\ninformation (Fig. 4a), whereas 34,420 (~24%) conserved exons with annotated open reading frames for\nprotein-coding sequences (including 17,288 exons being the first or last exons in genes and 17,132 exons\nin other positions) do not match annotated domains (not even “functional unknown domains”); these\nconserved protein-coding regions could be added to future domain databases using updated gene\nannotation algorithms or supported by new functional studies.”\nLines 1006-1020:\n“Conserved domain analyses\nTo investigate the correspondence of conserved exons with protein domains, we utilized the hmmsearch\ntool in HMMER package v3.4 to align protein sequences against the Pfam database v37.0 (releases/2024-\n9\n04-09; https://ftp.ebi.ac.uk/pub/databases/Pfam/releases/Pfam37.0/Pfam-A.hmm.gz) with the E-value\ncutoff of 0.0001. To reduce computation time, we removed redundant domain names, when two or more\ndomains were annotated for the same sequence region, by selecting the domain with the longest coverage.\nTo include the domains frequently described in literatures and to reduce the number of predictions on a\ngiven sequence region, we compared such domains in same length, retrieved their literature records in the\nEurope PMC (https://europepmc.org/; retrieved on 2025-01-15), and preferred to select domains with\nhigher records other than other domains. We also preferred to use the annotated domain other than the\nunannotated one (DUF). To reduce alternative PFAM annotations that are predicted on different exons\nmatching a conserved exon, we retrieved a total of 22,702 InterPro accessions and their associated 22,950\nPFAM accessions from the InterPro database (releases/2025-02-03;\nhttps://ftp.ebi.ac.uk/pub/databases/interpro/current_release/interpro.xml.gz) and used the unique InterPro\naccession to replace one or more homologues PFAM accessions. Specifically, 162 individuals feature at\nleast two PFAMs per InterPro accession.”\nPage 5 line 150. You state “It is possible that medium exons might have a frequently suitable size range for\nfunctional units (domains) in angiosperm genes.” And follow this up with a discussion of GO analysis in\nArabidopsis. You should follow this statement up computationally. Do medium sized exons contain whole\ndomains, what proportion of medium sized exons contain whole domains, and what are these domains – it\nseems you could do this with a HMMER search of translations of the medium exons, perhaps\nconcentrating on the conserved ones?\nResponse: We thank the reviewer for this valuable suggestion. We have expanded this analysis by\nexamining annotated protein domains in all 1,528,298 genes from 50 genomes and provide a new\nSupplementary Fig 4. We have revised the relevant text in our revised manuscript as shown below.\n“Medium exons encode peptides with 33-66 amino acids and preliminary analysis suggested that they\nmight frequently match the size range for angiosperm protein functional units (domains). We examined all\nannotated domains encoded by conserved exons in 1,528,298 genes from 50 genomes, and found that\nmedium exons represent 32.9% (median value) of exons matching part(s) of and/or whole domains in one\nof the genomes (Supplementary Fig. 4a,b). Medium exons account for 18.5% of these exons\nmatching >50% of a domain (Supplementary Fig. 4c). Moreover, for 17 to 692 domain families\n(depending on different angiosperm species), medium exons are enriched (p-value <0.05 under two-sided\nhypergeometric test; see methods) among all exons encoding all/part of domains; such domain families\ninclude the MYB DNA-binding domain (PF13921), RRM 1 (PF00076), and WD40 (PF12894)\n(Supplementary Fig. 4d,e).” (Lines 163-171)\n1 0\nSupplementary Figure 4 Exons and domain families in each genome\na, A phylogeny of angiosperm species, with the same information as that in Figure 1a.\nb, Bar-plots of percentage of exons matching annotated domains. Colors of bars represent different exon\nlength categories as shown at the bottom.\nc, Bar-plots of percentage of exons matching >50% of a domain. Meaning of bar colors is same as that in\npart b.\nd, Bar-plots of counts (number shown to the right of the bar) of domain families enriched in medium exons\nunder two-sided hypergeometric test.\ne, The top 3 domain families enriched in medium exons.\nPage 7 line 175 you state “Large and super exons account for ~23% of exons per genome and correspond\nto most genes without introns” and further state that Chinese pine “includes 38,784 singe-exon genes (with\n1 1\n156~6,807 bp of exon length) that account for 184 50.87% of 76,234 genes”. How many of these are\nconserved, especially those that as single exon genes with super exon sizes? Can you predict if they are\nfunctional? Perhaps a substantial portion of these represent processed pseudogenes and need to be\nidentified as such (in all species considered) and removed from analysis.\nResponse: We thank the reviewer for this valuable suggestion. We investigated the conservation and\nexpression of the single-exon genes and searched features of predicted processed pseudogenes. We have\nadded a paragraph to discuss it in revised main text as shown below.\nLines 842-860:\n“A relatively large number (246,011 of 928,992 in total) of super exons correspond to intronless genes,\nwhich might be generated via reverse transcription and insertion into the genome and include possible\npseudogenes105-107, which possess sequence features, such as poly-A tracks near the 3’ end and portions\nsimilar to repetitive elements. Among intronless genes, sequence conservation and detectable expression\nsupport functional genes105,108, whereas pseudogenes might have accumulated mutations and sequence\ndivergence. As the Chinese pine genome15 has an unusually large number (17,437) of super intronless\ngenes, we investigated these genes for the properties as possible evidence of pseudogenes and found that\n13,975 (80.1% of 17,437) lacked characteristics of processed pseudogenes, including 913 conserved exons\n(Supplementary Table 11). The remaining 3,462 (19.9%) super intronless genes have features that could be\nassociated with processed pseudogene, including 203 conserved exons/genes. The conservation of these\n203 intronless genes suggests that they might be functional, similar to known functional intronless genes\nfrom genetic studies and supported by our observation that 182 of the 203 conserved intronless genes\nshowed detectable expression in at least one of four organs (megagametophyte, leaf, stem, and needle;\nSupplementary Table 11). The remaining 21 (=203-182) conserved intronless genes did not have detectable\nexpression in the four organs, accounting for ~1.9% of 1,116 (=913+203) conserved super intronless\ngenes, which were included in the subsequent analyses of conserved exons (Supplementary Table 11).\nAmong 3,259 (=3,462-203) non-conserved intronless genes with pseudogene-like features, 2,666 showed\ndetectable expression in at least one organ, whereas the remaining 593 showed no detectable expression in\nany of the four organs and are candidate processed pseudogenes, corresponding to less than 3.4% of\n17,437 super intronless genes in Chinese pine, and require further annotation using additional RNA-Seq\ndatasets in future studies.”\nAlmost every section of the results ends in a statement that micro or small exons of whichever\nclassification you are discussing could be involved in regulating the gene by splicing, presumably\nalternative splicing (AS). This is always under the hypothesis that the removal of a small or micro exon\nwould occur via exon skipping (ES), and this would have a small but specific change on the protein\nproduct that has functional significance. ES events are very common in animals, but relatively infrequent\nin plants. Alternative acceptor or alternative donor events are much more common in plants than ES\nevents, and both of these would also result in small changes in the protein product, as long as they\n1 2\npreserved the reading frame. None of the alternative splicing mechanisms other than exon-skip are\ndiscussed or considered. A statement that conserved micro or small exons (conserved or not) may regulate\nor change protein product through splicing is true, but obvious. To determine any relationship between AS\nand conserved exons in regulating protein requires identification of conserved AS events across species,\nand the correlation of these to conserved exons, which this study does not do.\nResponse: We thank the reviewer for this valuable suggestion. We performed additional analyses to detect\nES, A3SS, and A5SS events using RNA-seq datasets of 11 species. We have described these results for\nconserved ES results in above response. We supplied a new Supplementary Figure 49 and Supplementary\nTable 10 to compare the A3SS/A5SS events commonly detected in at least two species. We show the\nresults for conserved A3SS/A5SS events as follows.\n“Our analyses of alternative splicing found that the conserved exon share by multiple species can be\ncommonly skipped in some of these species. For such conserved exons with skipping in one of five tissues,\nmore than half are micro or small exons (Fig. 7b). Most of those conserved exons match domains that are\nencoded by multiple micro and/or other exons. It is likely that skipping such conserved exons is a common\nmechanism for protein diversity across species. In addition, other alternative splicing mechanisms, such as\nalternative 3’ splice site (A3SS) and alternative 5’ splice site (A5SS), were also found to alter exon size,\nincluding thousands of conserved exons that were shortened by A3SS, including 14,506 and 13,848\nconserved exons in the flower and fruit, respectively, and shared by two or more species (Supplementary\nFig. 49; also for A5SS). In short, 47,708 (~45%) and 29,955 (~28%) of 105,971 conserved exons present\nin at least one of the 11 angiosperms were affected by A3SS and A5SS, respectively (Supplementary Table\n10). These results suggest that A3SS and A5SS events are also conserved alternative splicing mechanisms\ncontributing to protein diversity across species. Further analyses of the conserved exons affected by\nA3SS/A5SS events indicate that nearly half of them have the altered length in MT in at least one species,\nimplying that shortened exon size would not result into reading frame change in downstream domains\n(Supplementary Fig. 49). In general, our exon conservation investigation has a broad evolutionary\nimplication in exon skipping and other alternative splicing mechanisms in plants.” (Lines 862-876)\n1 3\nSupplementary Figure 49 Summary of conserved exons affected by A3SS/A5SS events commonly\ndetected in at least two species.\na, Bar-plots of number of conserved exons that exhibit A3SS (left part) or A5SS (right part) in at least two\nspecies. Colors of bars represent exon categories as shown below. For exons affected by A3SS, 11,224,\n7,322, 13,943, 14,506, and 13,848 conserved exons exhibit same event in two or more species in root,\nstem, leaf, flower, and fruit, respectively. Tissue patterns in exons affected by A5SS are similar to that by\nA3SS, with 4,145 conserved exons in flower as the largest group. In addition, 47,708 conserved exons\nhave A3SS events in at least one of 11 angiosperms, accounting for ~45% of 105,971 conserved exons\nshared by at least one of these 11 angiosperms (Supplementary Table 10). Similarly, 29,955 conserved\nexons have A5SS events in at least one of these angiosperms, accounting for ~28% of these 105,971\nconserved exons (Supplementary Table 10).\nb, Bar-plots of number of conserved exons with altered length in MT in at least two species (light green),\nonly one species (blue) and others (dark green).\nAs I mentioned previously, there are several alternative splicing mechanisms other than exon skip, and any\nof these can impact exons of any size, Subtle shifts in splice site choice (AtlA or Alt D) can affect any exon\n– for example, a super exon, and also result in a relatively small protein-coding change (as long as the\nevent keeps the exon in frame). Your discussion seems to focus on the alternative splicing of micro/small\n1 4\nexons as a mechanism to contribute to the diverse regulatory roles of genes. This is true, but not a\nnecessity, and it’s possible that AS of these exons are a minority of events. To make any substantial claims\nyou need to examine the other AS methods, and do the analysis to determine if your conserved micro and\nsmall exons are included in conserved AS events, and to what extent.\nResponse: We sincerely thank the reviewer for these comments to help us improve our analyses. Please see\nthe above response.\nWhat is your rationale for examining expression with transcriptomic data in Arabidopsis, soybean, tomato,\nrice, and Amborella trichopoda only, while your analyses include many species outside the clades that\ncontain these species?\nResponse: We thank the reviewer for this valuable comments. We added sentences to the method section in\nour new version of manuscript as shown below.\n“To infer functional implications of conserved exons, we analyzed RNA-Seq datasets of 11 species that\nhave publicly available transcriptomic datasets from flower, fruit, and at least one of three other tissues\n(root, stem, and leaf), with each tissue having >=3 replicates released on the same SRA project in NCBI,\ntotaling 178 transcriptomic datasets (see Supplementary Table 7 for accession numbers of transcriptomes\nin NCBI).” (Lines 1030-1033)\nYour exon collection for the study is pulled from the largest transcript of each annotated gene in the\nspecies you focus on. How confident are you in the annotations?\nResponse: We thank the reviewer for this valuable comments. Because we included 50 genomes to\nrepresent the evolutionary range of angiosperms and gymnosperms, the qualities of genome annotations\nare not even for genomes of varying sizes and complexity, even though we tried our best to use the highest\nquality genome for each major group (family or order). However, we do not have the resources and\ninformation to access the accuracy of the annotations for each genome, but assume that relatively small\nfractions of the annotation. To address this valuable comment, we examine the Arabidopsis genome, one of\nthe most accurately annotated genomes with strong support from multiple experimental data, to estimate\nthe possible effects of the longest mRNA on exon conservation and supply a new Supplementary Table 12.\nWe organized a new paragraph to discuss it in our manuscript as follows.\n“Alternative splicing contributes to some genes in a genome with two or more annotated transcripts; to\nestimate possible effect of alternative transcripts on the analyses of exon conservation, we examined the\nArabidopsis genome with highly accurate annotations (Supplementary Table 12). Among the 18,197\nArabidopsis genes (in the 10,216 HOGs) included in our comparative analyses, 8,015 genes have >=2\ntranscripts per gene and include 5,762 genes with a total of 14,150 alternatively spliced exons58. In 3,489\nof these alternatively spliced genes, the longest transcript also has the longest coding region and most\nlonger exons; however, for 2,273 genes, the longest transcript has a shorter coding region with either fewer\n(1,110 genes) or shorter (1,163 genes) exon(s). We examined 4,433 exons from these 2,273 genes,\nincluding 2,672 exons with altered length ranges or intron retention and 1,761 additional exons that did not\n1 5\ncorrespond to exons in the longest transcripts (see details in Supplementary Table 12). We assessed\nsequence similarity between these 4,433 exons and the protein-coding sequences of other genes within\ntheir respective orthogroups (Supplementary Table 12), revealing that 2,585 exons showed no similarity to\nother exons, whereas 952 exons matched individual conserved exons. These two types did not affect the\noverall analyses of conserved exons. In addition, 896 exons each matched two or more exons from\ndifferent conserved exon families, suggesting that these 896 exons might have been the result of intronless\nin Arabidopsis (or a group including Arabidopsis). The analyses using the longest transcript did not\ninclude these 896 exons among the conserved exons, accounting for ~1.1% of 82,271 conserved individual\nexons in the Arabidopsis genome. Because our results on conserved exon families contain members from\nmultiple genes, the annotation differences of this small number of genes do not affect the main conclusions\nof this study.” (Lines 876-893)\n1 6\nSupplementary Table 12 Summary of conserved exons in Arabidopsis genome\nDatasets of Arabidopsis genes Number of genes\nData1: gene included in 10216 orthogroups with >5 genes from >=2 species 18,197\nData2: gene in Data1 with >=2 mRNA 8,015\nData3: gene in Data2 with known alternative splicing events in non-UTR exons 5,762\nData4: gene in Data3 with the longest transcript exhibiting the longest coding region and most longer exons 3,489\nData5: gene in Data3 with the longest transcript exhibiting a shorter coding region with either fewer or shorter exon(s) 2,273\nExon Type Number of exons\nExons in Data1 105,477\nConserved exons in Data1 82,271\nAlternatively spliced exons in Data3 14,150\nAlternatively spliced exons in Data5 4,433\nAmong 4433 exons, exons with altered length ranges or intron retention 2,672\nAmong 4433 exons, newly annotated exons that did not correspond to exons in the longest transcripts 1,761\nAmong 4433 exons, exons showed no similarity to other exons via sequence similarity examination 2,585\nAmong 4433 exons, exons matched individual conserved exons via sequence similarity examination 952\nAmong 4433 exons, exons each matched multiple exons from different conserved exon families via sequence similarity examination 896\nSummary\nPercentage of exons with conservations that might be affected by the investigation in the longest transcripts 1.1% (=896/82,271*100)\nPercentage of exons with conservations that are not affected by the investigation in the longest transcripts 98.9% (=100%-1.1%)\nalternatively spliced isoforms to identify a non-redundant set of all exons for a given gene rather than only\nthose defined within the longest transcript.\nResponse: We thank the reviewer for this valuable comments. We discussed the possible effect in our\nmanuscript as shown above.\nLines 711-724. Your review of published transcriptomes in Arabidopsis looks at the correlation of exon\nsize with exons that undergo exon skipping, and states that many of the skipped exons are conserved, and\nthat exon skipping may be a conserved regulatory mechanism for these exons. Again, to make any\nsubstantive claim with certainty requires you to show that the conserved exons are also subject to skipping\nin other species.\nResponse: We sincerely thank the reviewer for these comments to help us improve our analyses. We have\nincluded 10 additional species as described in responses above and reported the identification of conserved\nexons that were skipped in two or more species.\n(Remarks on code availability)\nI reviewed the code, but did not run it. It is well documented and seems to be in order, but again, I did not\nexplicitly test it.\nResponse: We thank the reviewer for the positive feedback.\n1 8\nReviewer #2 (Remarks to the Author):\nZhang et al. conducted an interesting and solid analysis about the phylogenomic profile of exon-intron\norganization across seed plants, and the relationships between exon-intron organization with protein\ndomains and functions. They examined over 6.3 million exons for 46 angiosperms and 4 gymnosperms\nand found that the micro and small exons account for ~35% of all exons. They showed that micro and\nsmall exons may have important contributions to alternative splicing and resultant flexibility of functions\nof proteins. This study provided us a big picture for how the exon-intron structures organized among genes\nacross seed plants. Comments are listed below.\nResponse: We thank the reviewer for the positive feedback.\nMajor points:\nHow did the authors distinguish exon loss (e.g., Line 315) from potential mis-annotation of gene exon-\nintron structures in non-model species?\nResponse: We thank the reviewer for these valuable comments. We have performed sequence similarity\nanalyses and revised our manuscript as shown below.\n“For example, 127 exons are present in core eudicots and other angiosperms but absent in early diverging\neudicots (Nelumbo nucifera and Papaver somniferum). Among the 127 exons, 41 were apparently merged\nwith adjacent exon(s) with the loss of the intervening intron, resulting in longer exons in N. nucifera and P.\nsomniferum and retention of the protein coding sequences. For another 42 exons that were absent in N.\nnucifera and P. somniferum, the entire genes were not detected in these plants. The remaining 44 (=127-\n41-42) conserved exons were part of members of orthogroups, but the genomic sequences of the\ncorresponding genes in N. nucifera and P. somniferum lacked detectable similar sequence to the conserved\nexons, suggesting that the exons are not required for the function of these early diverging eudicot genes.”\n(Lines 355-362)\nThe authors emphasized a lot about the relationship between exon size and alternative splicing. But\nwhether the length of introns contribute more to skipping of the exons between them remained also\nunknown. The single-factor-analysis may lead to bias conclusion. It is also interesting to know whether\nmicro and/or small exons tend to be accompanied by shorter or longer introns than exons with longer\nlength.\nResponse: We thank the reviewer for these valuable suggestions. We have performed additional analyses,\nsupplied a new supplementary Figure 50, and added a new paragraph to our manuscript as shown below.\nLines 771-780:\n“Splicing can be regulated via cis elements in introns3,104. To gain possible clues about introns, we also\ninvestigated the combined length of two introns flanking an exon affected by skipping and found that the\nmedian value of intron length increased from micro exons to longer ones in each of the 11 species, such as\n1 9\n684 bp, 813 bp, and 1,068 bp, respectively, for median intron length corresponding to micro, small, and\nother conserved exons that were skipped in at least one tissue (Supplementary Fig. 50). In each of five\nexon size categories, the combined length of flanking introns for conserved exons that were skipped in at\nleast one tissue is longer than that for all genome-wide exons (Supplementary Fig. 50), suggesting that\nexons regulated by exon skipping might be regulated by cis elements in flanking introns, similar to the cis-\nelements reported for regulating splicing104. In addition, the shorter intron length flanking conserved micro\nand small exons (Supplementary Fig. 50) suggest that they might be regulated via different mechanisms,\nsuch as cis elements found in exons and specific RNA-binding protein(s), as recently reported20.”\nSupplementary Fig. 50 is very long, thus not included here. Please see it in Supplementary Figures.\nThere were too many numbers of several exon categories in the main text, which is not friendly for the\nreaders to follow the flow of this manuscript. Would suggest the authors to take good advantages of figures\nto illustrate different numbers in each comparison, and emphasize more about the interesting findings and\nindications in the main text.\nResponse: We thank the reviewer for these valuable suggestions. We supplied new supplementary Figures\n2-4, 38-40, 45, 48-51 to illustrate different numbers in each comparison.\nThe authors took a lot of examples for each types of exons or categories, but sometimes lacked the\nimplications indicated by those examples, such as examples in Lines 551-571 (Fig. 5c).\nResponse: We thank the reviewer for these valuable suggestions. We have reduced the description of\nexamples in the revised manuscript (Lines 593-599) compared with the previous manuscript and provided\nsome possible implications in the revised manuscript as shown below.\n“These four groups of domain-exon organizations are found in genes related to regulation of expression\n(Fig. 5c), such as the ATRX7 genes encoding the SET domain (group-A), the RH9 genes for the DEAD\ndomain (group-B), the JANUS genes coding for the RRM_5 domain (group-C), and the UBP12 genes for\nthe UCH domain (group-D). The four groups are also found in genes for development/reproduction, stress\nresponses, immunity, and signaling (Fig. 5c; see other examples in Supplementary Table 6), suggesting\nthat they offer a potentially common modulation of domain-exon structures via splicing in a variety of\nplant genes.” (Lines 593-599)\nIn the section “Preferential expression of genes containing conserved exons among major organs”,\nespecially the last paragraph in Lines 680-697, it sounds like that the expression profiles of genes (root,\nstem, fruit and other tissues) are correlated with exon-intron organization. However, to my knowledge, the\ngene expression profiles should be more likely to be regulated by cis-elements. It is reasonable to show\nthat different transcripts of genes, the products of alternative splicing, may have preference for different\ntissue types. With regard to this, I like the section “Domains in genes contained conserved exons affected\nby exon skipping and implications in flower development”\n2 0\nResponse: We thank the reviewer for these valuable suggestions. We agree with the reviewer’s comment.\nWe revised the title for this section as ‘Expression of genes containing conserved exons among major\norgans’. We also revised the last paragraph in this section as shown below.\n“Auxin is an important plant hormone and regulate multiple developmental processes100. Among PEGs is\nthe AUXIN RESISTANT1 (AUX1) gene encoding an auxin influx transporter with a conserved Aa_trans\ndomain (Figure 6b; Supplementary Fig. 46). The Aa_trans domain matches six internal exons in AUX1\nhomologues in most cases, but in Solanaceae and monocot species there is only one exon corresponding to\nthe last two of the six exons in AUX1 homologues of other species (Supplementary Fig. 46). Among the\nsix exons, the first (3’) and last (5’) are in MT and others in NMT (Fig. 6b). Other PEGs related to\ndevelopment and metabolism with internal exons in MT encode the Pkinase domain, such as the PHOT2\ngenes mentioned in above section (Figure 6b; Supplementary Fig. 32) and the Arabidopsis LUTEIN\nDEFICIENT1 (LUT1) gene encoding a heme-containing monooxygenase with a p450 domain101 (Figure\n6b; Supplementary Fig. 47).” (Lines 698-706)\nMinor points:\nPlease double check that all the numbers with more than three digits were split with comma.\nResponse: We thank the reviewer for these valuable suggestions. We have revised all the numbers with\nmore than three digits with a comma.\nLine 15, “with two” may be “including two”?\nResponse: Thanks. Done.\nLine 23, may define what are conserved exons?\nResponse: Thanks. We revised it in our new version of manuscript as follows.\n“We developed a new bioinformatic tool, ExonEvo, for comparison of the exon-intron structure and found\nthat, among conserved exons (median of ~79.9% of all exons in a species) shared by homologous genes,\nmost (~69%) are found in two or more major angiosperm subgroups, including ~47% that are conserved\nbetween angiosperms and gymnosperms.” (Lines 27-30)\nLines 30-31, lack a transition between “micro and/or small exons” and “alternative splicing”\nResponse: Thanks. We revised it in our new version of manuscript as shown below.\n“Skipping of such conserved exons encoding known domains is a possible conserved mechanism for\nregulation of protein structure.” (Lines 38-39)\nLine 37, “and have diverse functions” is kindly of meaningless. May be written as “have diverse exon-\nintron organization and functions”?\nResponse: Thanks. Done.\n2 1\nLine 66, before this sentence, why it is important to study the genome-wide patterns of the number and\nsize of exons was not specified.\nResponse: Thanks. We revised it in our new version of manuscript as shown below.\n“Are the patterns of micro and small exons conserved across flowering plants for floral homeotic genes\nand other genes? To address the question, genome-wide patterns of the number and size of exons need to\nbe investigated in angiosperms.” (Lines 72-74)\nLine 80, may miss a comma between “exon” and “was”\nResponse: Thanks. Done.\nLines 81-83, lack a transition between “the Arabidopsis GRP20 protein was shown to be required for\nproper retention of micro exons in transcripts of several floral homeotic genes, such as AG, AP1, AP2,\nAP3, and SEP3” and “the importance of micro exons in function of these developmental regulators”. The\nauthor may want to say the skip of micro exons of these genes lead to obvious phenotype changes?\nResponse: Thanks. We revised the sentences as shown below.\n“In addition, the Arabidopsis GRP20 protein was shown to be required for normal flower development and\nfor proper retention of micro exons in transcripts of several floral homeotic genes, such as AG, AP1, AP2,\nAP3, and SEP320. Also, the skip of micro exons of these genes was found to cause floral phenotype\nchanges (e.g., SEP3 in ref34), showing the importance of micro exons in function of these developmental\nregulators.” (Lines 88-91)\nLines 145-146, may also provide the proportions of the numbers, for the comparison with those in\nArabidopsis.\nResponse: Thanks. We revised this sentence as shown below.\n“In the rice (Oryza sativa cv. japonica) T2T genome52, we detected 15,014 micro exons (6.5% of 230,786\nexons), 53,135 (23%) small exons, 65,246 (28.3%) medium exons, 56,350 (24.4%) large exons, and\n41,041 (17.8%) super exons (Fig. 1d, Supplementary Fig. 2).” (Lines 158-160)\nLine 148, (~32%) should be moved after “with the most exons”\nResponse: Thanks. Done.\nLine 204, add proportions for other numbers\nResponse: Thanks. We revised this sentence as shown below.\n“Among 1,531 exons exhibiting exon-skipping under 72-h heat stress relative to control in Arabidopsis61,\n323 (21.1%) are micro exons, 780 (50.95%) small exons, 325 (21.23%) medium exons, 91 (5.94%) large\nexons, and 12 (0.78%) super exons (Supplementary Table 4).” (Lines 222-224)\n2 2\nLines 205-207, should specify what statistic test was conducted. In addition, “The percent of both micro\nand small exons among skipped exons are much greater than genome-wide percentage of these categories”\nshould be tuned down since the percent and number of micro were smaller than those of medium. The\nsame comment for lines 210-212.\nResponse: Thanks. We have revised these sentences in our new version of manuscript as shown below.\n“The percentage (50.95%) of small exons among skipped exons is greater than genome-wide percentage\n(28.32%) of small exons (two-sided hypergeometric test, p-value = 1.11E-77; Supplementary Table 4).”\n(Lines 224-226)\n“In another study of poplar root transcriptome under lead stress relative to control22, among 113 skipped\nexons, 19 (16.81%), 52 (46.02%), 28 (24.78%), 12 (10.62%), and 2 (1.77%) are micro, small, medium,\nlarge, and super exons, respectively, suggesting that alternative splicing, including those involving micro\nand small exons, contributed to transcript/protein diversity in stress response.” (Lines 226-229)\nLines 207-209, exons in the example case were not specified to categories, as the cases before and after\nthis.\nResponse: Thanks. We have removed the sentence (showed in our originally submitted manuscript Lines\n207-209) from our revised manuscript as explained in above response.\nLine 221, flanking intron sequences, instead of flanking exon sequences?\nResponse: Thanks. We revised this sentence as shown below.\n“Conserved exons were previously examined by manually comparing exon positions in multiple sequence\nalignments of the MADS-box20 and CYP7538 gene families; in addition, conserved short micro exons (≤\n15 bp) were determined along with the conservation of their upstream and downstream exon sequences46.”\n(Lines 238-241)\nLines 250-251, it’s surprising that the super exons are less conserved than other types of exons.\nResponse: Thanks. The super exons when merged from other short conserved exons were not included in\nthe conserved super exons across species. To help readers better understanding our exon conservation,\nwe added a new paragraph to discuss it in our revised manuscript as follows.\n“Our comparative analyses of exons across 50 seed plants revealed that a large majority of each of the four\nsmaller size categories are conserved (with median values of 60.55%, 85.7%, 82.3%, and 70.2% for micro,\nsmall, medium, and large exons, respectively). However, only 44.9% of super exons are conserved; this\nrelatively low proportion was puzzling at first and led to further examinations. One contributing factor is\nthat some super exons correspond to two or more smaller exons in other genes of the same orthogroup and\nour definition of conserved exons would exclude such super exons from conserved exons when they only\nappear one species. From 50 genomes we detected 823~4,341 non-conserved exons per species (median of\n2 3\n1,334), which correspond to two or more conserved exons in other genes of the same orthogroup, with a\ncombined total of 76,762 non-conserved super exons for the 50 species. These non-conserved super exons\nprobably resulted from fusion of two (or more) conserved exons via loss of intron(s) during angiosperm\nevolution, suggesting that potential alternative splicing of the ancestral (unfused) exons is not part of\nmechanisms regulating the transcript isoforms for the relevant exon(s). Analyses of all exon size categories\nrevealed that 59,338 conserved exon families contain at least one member that was fusion in one or more\nof the 50 species and also exhibit alternative splicing in one of the 11 species analyzed here\n(Supplementary Fig. 52a-c). In individual species, such fused exons account for ~65% (Supplementary\nFig. 52d) of all exons likely fused from conserved exons (Fig. 2e), again suggesting changes in potential\nalternative splicing during angiosperm evolution for exons of different size.” (Lines 825-840)\nLine 305, typo “in in”\nResponse: Thanks. Done.\nLines 311-314, the numbers are referring to Fig. 3a, but the figure cited before this sentence is Fig. 3b. Did\nnot read in a good flow.\nResponse: Thanks for this comment and we apologize for the unclear description in the original\nmanuscript. We have revised the text along with figure citations as shown below.\n“As the seed-plant conserved exons are the largest group, accounting for 47% of all conserved exons (Fig.\n3a), we further examined their retention in major groups of angiosperms, including two major subclades of\neudicots, rosids (such as Arabidopsis and soybean) and asterids (such as tomato), other eudicots,\nmonocots, magnoliids, and other angiosperms. Among the seed-plant conserved exons, numbers of exons\nin each of the five size categories shared by various combination of the angiosperm groups are shown in\nFig. 3b-e. Specifically, 99% (65,722) of the seed-plant exons were detected in at least one genome of core\nangiosperms (here including eudicots, monocots, and magnoliids), while 85.3% (56,630) were detected in\nat least one of the two basal angiosperm genomes (Fig. 3b). Small and medium exons together represent\nthe majority (70.4%; Fig. 3c) of seed-plant conserved exons. Furthermore, 77.3% (51,309) of the seed-\nplant conserved exons are retained in all six angiosperm groups (Fig. 3d), including 2,442 micro, 18,004\nsmall, 19,478 medium, 9,129 large, and 2,256 super exons (Fig. 3e). Among 3,411 micro exons, the largest\nportion (2,442) represents the conserved exons shared by all six angiosperm groups (Fig. 3c-e). For\ninstance, among the seed-plant homologues of the Arabidopsis MKK6 gene (AT5G56580), the conserved\nmicro exon (EXON0044515; Supplementary Data 1) is detected in rosids (such as Arabidopsis and apple)\nand asterids (such as tomato and kiwifruit), other eudicots (such as Papaver), monocots (such as rice and\nmaize), magnoliids (such as avocado), and other angiosperms (such as Amborella trichopoda).” (Lines\n340-354)\n2 4\nFigure 3 Comparison of the retention patterns of conserved exons among major groups\na, A pie chart of the percentages of the conserved exons mapped at different phylogenetic positions of the\nspecies tree, showing that most of conserved exons are shared by seed plants.\nb, Number of the 66,385 seed-plant conserved exons retained at the MRCA of different angiosperm\ngroups. Numbers above/below a branch represent the number of conserved exons retained in at least one of\nthe descendant groups derived from this branch. The black number represents the total number. Red, blue,\norange, green, and purple numbers represent the number of micro, small, medium, large, and super exons,\nrespectively.\nc, A pie chart of the percentages of the seed-plant conserved exons in different exon length categories.\nd, A pie chart of the percentages of the seed-plant conserved exons with different retention patterns among\nsix angiosperm groups. See these angiosperm groups in part b.\ne, An Up-Set plot of the number of the seed-plant conserved exons retained in different plant groups.\nPoints below bars represent retention of the exon in different groups of seed plants. Lines joining different\npoints represent different retention patterns. The bar-plot shows the number of exons with different\nretention patterns. The largest number of conserved exons mapped at the MRCA of seed plants represent\n2 5\nthese exons retained in all six angiosperm groups, indicating a high degree of conservation. Five colors of\nthe bars represent five exon length categories as shown in the inset box.\nLine 425, “for protein interaction”, does that mean all the domains listed in Lines 423-425 are all function\nfor protein interaction?\nResponse: We are sorry for the unclear description. Because the domains include DNA-binding and other\ndomains, we have revised this sentence, without the words “for protein interaction”, as shown below.\n“We found 522 other domain families with members in each of the four exon-domain categories (Fig. 4b),\nsuch as the MYB-like (PF00249) and helix-loop-helix DNA-binding domains (PF00010), protein kinase\ndomain (PF00069), RNA recognition motif (RRM_1; PF00076), and leucine rich repeat (PF13516).”\n(Lines 469-471)\nColors in Fig. 4d are similar to colors in Fig. 4b, which led to misunderstanding at the first glance.\nResponse: Thanks. We have revised the colors of Fig. 4d as shown below.\nThe paragraph after Line 534 may be organized as a new section, since all the above paragraphs were\ntalking about the numbers of different types of exons.\nResponse: Thanks. We organized it as a new section entitled ‘Examination of conserved exon lengths for\nmultiple of three” (Line 576).\nLines 544-550, the types A-D should be marked in the Figure 5b.\nResponse: Thanks. We have marked the types A-D in Figure 5b as shown below.\n2 6\nLines 700-709, may move the background part to introduction section.\nResponse: Thanks. We have moved some information into introduction section.\nIn introduction:\n“In addition, alternative RNA splicing could affect micro and small exons and support hypotheses about\nregulation of expression and protein diversity for these genes20. One of major alternative splicing types\ncommonly found in plants, humans and other animals is exon skipping. For example, the skipping of a\n2 7\nmicro exon in the SEP3 transcript can lead to defects in flower development34,36. Also, a conserved 9-base\nmicro exon in the Arabidopsis WRINKLED1 (WRI1) gene belonging to the AP2 family is important for\nfatty acid synthesis21,57. In particular, because micro and small exons encode relatively short peptide\nsequences, protein isoforms related to the presence/absence of micro/small exon-encoded peptide regions\nmight have relatively subtle functional differences, in comparison to those involving larger exons/domains.\nHowever, the landscapes of expression patterns of genes containing micro/small exons vs exons of other\nsizes and alternative splicing involving exons of different sizes are largely unknown in different plant\norgans.” (Lines 118-128)\nIn results:\n“Alternative splicing is a crucial mechanism that increases the diversity of protein isoforms in eukaryotic\norganisms7. Genes containing conserved exons and with expression in multiple tissues and organs could\nhave alternatively spliced transcripts, including those due to exon skipping, thereby increasing the\nfunctional diversity of their protein products. The results reported in the above sections indicate that most\nplant genes are conserved across angiosperms and encode protein domains matching multiple exons,\nsuggesting that regulation by alternative splicing could affect their domain structure and function.\nHowever, whether alternative splicing affected conserved exons and patterns of alternative splicing that\nmight affect specific conserved domain families are largely unknown.” (Lines 709-715)\nLine 721, miss “%” after 70.5.\nResponse: Thanks. Done.\nReviewer #2 (Remarks on code availability):\nThe code ExonEvo is much straightforward to use and the input files are resultant files from other\ncommonly used software.\nResponse: We thank the reviewer for the positive feedback.\n2 8\nReviewer #1 (Remarks to the Author):\nThank you for attending to the comments by reviewers. I appreciate the improved clarity of the\nmanuscript, as well as the improved interpretation of your data and the additional analyses you\nperformed.\nResponse: We greatly thank the reviewer for the positive feedback.\nThere remain a few typos. For example:\nLine 822 “domains and they possible regulation”: convert they to their.\nResponse: Thanks. Done.\nLine 830 “only appear one species.”: add the word ‘in’ before the word ‘one’\nResponse: Thanks. Done.\nLine 836 “one member that was fusion in one”: convert fusion to fused.\nResponse: Thanks. Done.\nLine 862 “the conserved exon share by multiple species” should read “the conserved exons shared by\nmultiple species”\nResponse: Thanks. Done.\nI urge you to carefully review your manuscript to catch additional typos that may remain.\nAdditionally, please check that references to supplementary data sets are accurate. Line 198 discusses\nsuper exons, but refers to supplementary data set #2, which contains data on 'conserved small exons'\nResponse: We thank the reviewer for the suggestion. Supplementary Data 2 exhibits the number of\ngenes with/without introns and their exons in five categories. We have revised this sentence in our\nmanuscript as shown below.\n“For example, 5,482 Arabidopsis genes lack an intron, including 1,570 with lengths of 200-500 bp\n(large exons) and 3,592 in the length range of super exons, accounting for ~94% of these single exon\ngenes (Supplementary Fig. 1 and Supplementary Data 2).” (Lines 184-186)\nReviewer #2 (Remarks to the Author):\nThe revised manuscript had addressed all my concerns and I'd suggest it to be published.\nResponse: We greatly thank the reviewer for the positive feedback.\nReviewer #2 (Remarks on code availability):\nThe code ExonEvo is much straightforward to use and the input files are resultant files from other\ncommonly used software.\nResponse: We greatly appreciate this positive comment."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for attending to the comments by reviewers. I appreciate the improved clarity of the manuscript, as well as the improved interpretation of your data and the additional analyses you performed.\nThere remain a few typos. For example:\nLine 822 “domains and they possible regulation”: convert they to their.\nLine 830 “only appear one species.”: add the word ‘in’ before the word ‘one’\nLine 836 “one member that was fusion in one”: convert fusion to fused.\nLine 862 “the conserved exon share by multiple species” should read “the conserved exons shared by multiple species”\nI urge you to carefully review your manuscript to catch additional typos that may remain.\nAdditionally, please check that references to supplementary data sets are accurate. Line 198 discusses super exons, but refers to supplementary data set #2, which contains data on 'conserved small exons'", "reviewer_2": "The revised manuscript had addressed all my concerns and I'd suggest it to be published."}, "author_response": "Response: We thank the reviewer for the suggestion. Supplementary Data 2 exhibits the number of genes with/without introns and their exons in five categories. We have revised this sentence in our manuscript as shown below.\n“For example, 5,482 Arabidopsis genes lack an intron, including 1,570 with lengths of 200-500 bp (large exons) and 3,592 in the length range of super exons, accounting for ~94% of these single exon genes (Supplementary Fig. 1 and Supplementary Data 2).” (Lines 184-186)\nResponse: We greatly thank the reviewer for the positive feedback.\nResponse: We greatly appreciate this positive comment."}]

Decision letter