Evolution of the Expansin Superfamily in Bryophytes and Green Algae

Evolution of the Expansin Superfamily in Bryophytes and Green Algae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evolution of the Expansin Superfamily in Bryophytes and Green Algae Maddisyn E Behney, Tyani Orta, Robert E Carey This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8810289/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Apr, 2026 Read the published version in Protoplasma → Version 1 posted You are reading this latest preprint version Abstract Expansins are a superfamily of non-enzymatic proteins that mediate plant cell wall loosening and play essential roles in growth and development. The expansin superfamily consists of four families: EXPA, EXPB, EXLA, and EXLB. Although the expansin gene families have been well characterized in angiosperms, their evolutionary history in early-diverging land plants and green algae remains incompletely resolved. This study examines expansin superfamilies in two liverworts ( Marchantia polymorpha and Conocephalum conicum ), a hornwort ( Anthoceros agrestis ), a moss ( Ceratodon purpureus ), and two green algal species ( Chara braunii and Spirogloea muscicola ). Expansin genes were identified from these organisms using BLAST searches. These newly assembled gene families were then analyzed to determine the relationships between them and gain insight into early expansin evolution. No EXLA or EXLB genes were found in bryophytes or green algae. Bryophytes contain EXPA genes; however, EXPBs were not detected in liverworts and are present only in mosses and hornworts. Although green algal expansins share key characteristics of both EXPA and EXPB genes, evidence suggests they are not members of either family. The data presented here raises interesting questions about the timing of EXPA and EXPB evolution in land plants. expansin gene family evolution bryophytes green algae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Expansins are a superfamily of non-enzymatic proteins critical for pH-dependent plant cell wall extension (McQueen-Mason 1992). Processes such as fruit ripening, seed germination, pollen tube growth, and general cell growth rely on expansin-mediated cell wall modification (Rose et al. 1997 ; Chen and Bradford 2000 ; Cho and Cosgrove 2000 ; Valdivia et al. 2007 ). Expansins are composed of approximately 270 amino acids, have a cleaved signal peptide, and a two-domain structure, which includes an N-terminal domain and a C-terminal domain (Cosgrove 2024 ). Within each domain, there are conserved amino acids (AAs) and AA motifs that appear important for expansin function (Cosgrove et al. 2002 ). The expansin super-family consists of four families: EXPA, EXPB, EXLA, and EXLB (Sampedro and Cosgrove 2005 ). EXPAs and EXPBs have been experimentally demonstrated to have cell wall loosening activity (Cosgrove 1989 ; Li et al. 1993 ). EXLAs and EXLBs have an unknown function and are included in the superfamily based on sequence similarity (Sampedro and Cosgrove 2005 ). Previous analyses have indicated that the common ancestor of all angiosperms likely had a minimum of 12 EXPAs, 2 EXPBs, 1 EXLA and 2 EXLBs (Sampedro et al. 2005 ). These clades have been shown to be highly conserved amongst all angiosperms studied so far (Sampedro et al. 2006 ; Seader et al. 2016 ; Wang et al. 2024 ). Although lycophytes have conserved EXPA and EXPB sequences, they lack EXLA and EXLB genes. The common ancestor of lycophytes and angiosperms is believed to have a minimum of 2 EXPAs and 1 EXPB (Carey et al. 2013 ). The common ancestor of mosses and angiosperms appears to also have had a minimum of 2 EXPAs and 1 EXPB. Moss expansins are far more divergent when compared to either angiosperm or lycophyte expansins, indicating a more independent evolutionary history. Despite this divergence, moss expansins still share the structure and common motifs associated with expansins (Carey and Cosgrove 2007 ). To further elucidate the history of expansins in early diverging land plant lineages, we have extended these analyses to two liverworts ( Marchantia polymorpha and Conocephalum conicum ), a hornwort ( Anthoceros agrestis ), and an additional moss ( Ceratodon purpureus ). We have also expanded this analysis to two green algae species ( Chara braunii and Spirogloea muscicola ) in hopes of understanding the evolution of the expansin superfamily as plants transitioned to life on land. MATERIALS AND METHODS Identifying expansin superfamilies Marchantia The M. polymorpha expansin superfamily was assembled from MpTak v5.1 of MarpolBase using tBLASTx with known A. thaliana expansin sequences as queries (Sampedro et al. 2005 ; Montgomery et al. 2020 ). Conocephalum The C. conicum expansin superfamily was assembled from genome assembly ASM5008447v1 in NCBI with NCBI tBLASTx using known A. thaliana expansin sequences as queries. (Sampedro et al. 2005 ; Sayers et al. 2025 ). Ceratodon The C. purpureus expansin superfamily was assembled from genome assembly CpurpureusR40_1_0 using Phytozome BLASTn with known P. patens and A. thaliana expansin sequences as queries (Sampedro et al. 2005 ; Carey and Cosgrove 2007 ; Goodstein et al. 2012 ; Carey et al. 2021 ). Additional sequences (CpEXPA22, CpEXPB4, CpEXPB5) were gathered from the genome with NCBI tBLASTx with known P. patens expansin sequences as queries (Sayers et al. 2025 ; Carey and Cosgrove 2007 ). Anthoceros The A. agrestis expansin superfamily was assembled from the [Bonn] genome assembly using A. thaliana as queries (Sampedro et al. 2005 ; Li et al. 2020 .). Blast searches of the A. agrestis genome were performed locally using BLAST + 2.15 (Camacho et al. 2009 ). Chara The C. braunii expansin superfamily was assembled from the Chara braunii S276 genome using PhycoCosm BLASTn with known P. patens expansin sequences as queries (Carey and Cosgrove 2007 ; Nishiyama et al. 2018 ; Grigoriev et al. 2021 ). Spirogloea The S. muscicola expansin superfamily was assembled from the Spirogloea_muscicola _CCAC_0214 genome using PhycoCosm BLASTn with known P. patens expansin sequences as queries (Carey and Cosgrove 2007 ; Cheng et al. 2019 ; Grigoriev et al. 2021 ). Sequence Alignments The protein sequences encoded by the expansin superfamilies of M. polymorpha, C. conicum, A. agrestis, C. purpureus, C. braunii , and S. muscicola were aligned with selected angiosperms, lycophytes, and bryophytes. Three alignments were constructed for phylogenetic analysis. The first alignment contained the EXPA sequences of M. polymorpha , C. conicum, A. agrestis , and C. purpureus , along with known P. patens EXPAs, S. moellendorffii EXPAs, and Arabidopsis -rice EXPA sequences representing EXPA clades I-XII (Carey and Cosgrove 2007 ; Carey et al. 2013 ; Sampedro et al. 2005 ). P. patens PpEXPA20 was excluded from the alignment due to a long c-terminal extension that degrades the alignment. C. braunii EXPs 8, 10, and 11 and PpEXPB1 were also included in the alignment to root the resulting tree. The second alignment included EXPB sequences of A. agrestis and C. purpureus , along with known P. patens EXPBs, S. moellendorffii EXPBs, and representative Arabidopsis -rice sequences of EXPB, EXLA, and EXLB clades (Carey and Cosgrove 2007 ; Carey et al. 2013 ; Sampedro et al. 2005 ). Included in the alignment were C. braunii EXPs 8, 10, and 11 and PpEXPA1 to help root and resolve the tree. The third alignment constructed contained representative bryophyte, lycophyte, and Arabidopsis -rice EXPA and EXPB genes of along with expansin sequences from C. braunii and S. muscicola (Carey and Cosgrove 2007 ; Carey et al. 2013 ; Sampedro et al. 2005 ). Such an alignment was necessary to analyze the relationship between algae expansins and terrestrial plant expansins. Alignments were created using the default parameters of the MUSCLE algorithm within the Unipro UGENE software package (Okonechnikov et al. 2012 ). Alignments were then trimmed at a conserved tryptophan upstream of the expansin HATFYG motif and a conserved phenylalanine near the c-terminus. Phylogenetic Analysis Bayesian Phylogenetic trees were constructed using MrBayes v3.2.5 (Ronquist et al. 2012 ). Priors for the Bayesian trees were as follows: Jones amino acid model, gamma estimation, 7,000,000 generations, two runs of five Markov chains each – burnin as indicated in figure legends. The consensus trees were visualized using Figtree, where the bryophyte EXPA tree was manually rooted at C. braunii sequences CbEXP8, 10, and 11, and the EXPB tree was manually rooted at the C. braunii sequences CbEXP8, 10, and 11 (Rambaut et al. 2018). Although CbEXP8, 10, and 11 may not be the true root for either the EXPA or EXPB tree, such C. braunii sequences were chosen to facilitate comparison between the two trees. The algae EXP tree was left unrooted. Neighbor Joining Neighbor Joining trees were constructed using MEGA11 (Tamura et al. 2021 ). Parameters were as follows: bootstrap 500, Poisson model, pairwise deletion. The bryophyte EXPA tree was manually rooted at CbEXPA10, and the EXPB tree was manually rooted at the C. braunii sequences CbEXP8, 10, and 11. Maximum Likelihood Maximum Likelihood trees were constructed using PhyML 3.0 (Guindon at el. 2010). Trees were constructed with automatic model selection by AIC and bootstrap 100. Poisson Corrected Distances For analysis of algal expansins, an alignment of algae EXPs, bryophyte EXPAs and EXPBs, lycophyte EXPAs and EXPBs, and angiosperm EXPAs and EXPBs was created using the MUSCLE algorithm within Unipro UGENE and was trimmed as was done for phylogenetic tree construction (Okonechnikov et al. 2012 ). PC distances were calculated using MEGA11 with standard error calculated for these values using 500 bootstrap replicates (Tamura et al. 2021 ). Gaps were handled with complete deletion. Intron Patterns When possible, genomic sequences were compared to cDNA sequences to determine intron patterns. If only genomic sequences were available, translated sequences were aligned with related translated cDNA sequences, and intron locations were estimated based on gaps within the alignment along with previous knowledge of expansin intron patterns. Sequence logo Amino acid sequences for algae EXPs and terrestrial plant EXPAs and EXPBs were aligned using the default parameters of the MUSCLE algorithm within the Unipro UGENE software package (Okonechnikov et al. 2012 ). The alignment was trimmed as was done for phylogenetic tree construction. The alignment was then sub-divided into algae EXL and terrestrial plant EXPA and EXPB portions. The sequence logo was generated using WebLogo (Crooks et al. 2004 ). RESULTS The expansin superfamilies of bryophytes and algae As is the case with all terrestrial plants studied thus far, the EXPA families within the bryophytes studied here are larger than the EXPB families. The lone exception to this is A. agrestis , which has more EXPBs than EXPAs (Cosgrove 2024 ). The EXPA families of M. polymorpha and C. conicum appear to be slightly expanded, with M. polymorpha having 38 EXPAs and C. conicum having 32. Interestingly, no EXPBs were found within either liverwort, despite being found in all mosses and hornworts studied so far (Carey and Cosgrove 2007 ). Both C. braunii and S. muscicola contained expansins; however, such expansins were determined to not belong to any of the four expansin families found in terrestrial plants. Such expansins have been labeled as “EXPs”, as they are expansins but do not share all of the same features of the terrestrial plant expansin families (Table 1 ). In agreement with previous studies of bryophytes and algae, none of the selected species contained EXLAs or EXLBs (Vannerum et al. 2011 ; Carey and Cosgrove 2007 ; Wang et al. 2024 ). Studies of expansins within pteridophytes suggest, along with the absence of such families within the early land plants and algae, it is likely that the EXLA and EXLB families emerged after the divergence of pteridophytes (Carey et al. 2013 ; Vannerum et al. 2011 ). In addition to complete sequences, possible pseudogenes were found in C. conicum . CcEXPA3 contains a frameshift near the expansin “VPC” motif, which was fixed by removing the two bp fifteen amino acids downstream of the motif. CcEXPA23 also had two bp removed from its sequence before the “TATN” expansin motif to fix a frameshift. Four C. braunii sequences also had missing data, which makes it difficult to determine if they are pseudogenes or not (Online Resource 1). Table 1 Size and composition of expansin gene families of selected plant species EXPA EXPB EXLA EXLB Total EXP A. thaliana* 26 6 3 1 36 O. sativa* 33 18 4 1 56 S. moellendorffii Δ 15 2 0 0 17 P. patens † 28 7 0 0 35 C. purpureus 22 6 0 0 28 A. agrestis 7 8 0 0 15 M. polymorpha 38 0 0 0 38 C. conicum 32 0 0 0 32 C. braunii 0 0 0 0 22 S. muscicola 0 0 0 0 12 * At time of search (Sampedro et al. 2005 ) Δ At time of search (Carey et al. 2013 ) † At time of search (Carey and Cosgrove 2007 ) Phylogenetic analysis of bryophyte expansins EXPA Most liverwort and moss EXPAs appear to largely group separately from the angiosperm-specific EXPAs. A previous study of P. patens EXPAs divided the family into six clades named A-F (Carey and Cosgrove 2007 ). The addition of C. purpureus EXPA genes has further refined the structure of the moss EXPA family. P. patens clade A can be further divided into five distinct clades, labeled clades A1 through A5. For the liverworts, there appears to be 17 liverwort-specific clades. Clades designated 1 through 15 group sister to P. patens clades B and A1-A5. MpEXPA14 is grouped with liverwort clades 12 and 13 but does not appear to belong to either clade. Liverwort clade 16 is sister to S. moellendorffii clade B, and MpEXPA9 groups with S. moellendorffii clade A. Liverwort clade 17 is sister to P. patens clade E, which appears to be a moss-liverwort specific clade consisting of MpEXPA1, CcEXPA9, PpEXPA1, and CpEXPA10 (Fig. 1 ). A few bryophyte EXPAs group with known Angiosperm clades. S. moellendorffii EXPAs 5 and 6 along with MpEXPA10 and AaEXPA6 belong to Arabidopsis-rice clade X. Previously described P. patens clade F, containing PpEXPA6 and CpEXPA4, branches with Arabidopsis-rice clade XI. A. agrestis EXPAs 1–4, MpEXPA9, CpEXPA9, and PpEXPAs 8 and 13 of P. patens clade D are sister to Arabidopsis-rice clades I-III (Fig. 1 ). Neighbor joining and maximum likelihood trees did not appear to alter any of these placements (data not shown). As noted previously, most A. agrestis EXPAs group with vascular plant EXPAs, with the only exceptions being AaEXPA5 and AaEXPA7, which group with bryophyte-specific EXPA clades. Overall, A. agrestis appears to have more angiosperm-related than bryophyte-specific EXPAs. It is also important to note that the A. agrestis genome is one of the smallest among land plants, and this may contribute to the reduction of bryophyte-specific EXPAs (Li et al. 2020 ). EXPB AaEXPBs 2 through 8 group together forming an Anthoceros-specific clade (Fig. 2 ). Such clade appears sister to all bryophyte and angiosperm EXPB clades (bpp = 0.946). AaEXPB1 groups sister to Arabidopsis-rice clade II (bpp = 0.994). The inclusion of C. purpureus EXPB genes revealed internal structure of the EXPB family within mosses, with all six genes grouping with P. patens . Four moss-specific EXPB clades were determined. Each clade contained at least one P. patens and C. purpureus , and have been named as clades I, II, III, and IV (Fig. 2 ). Neighbor joining and maximum likelihood trees did not appear to alter any of these placements (data not shown). Phylogenetic analysis of algae expansins Phylogenetic analysis of the algae EXPs was performed to aid in the understanding of the algae EXPs’ relationship to terrestrial plant EXPAs and EXPBs. Both S. muscicola and most C. braunii EXPs grouping separately from both EXPA and EXPB clades (Fig. 3 ). Although CbEXPs 8 and 11 group with the terrestrial plant EXPBs, sequence analysis suggests that they are not members of the EXPB family. Distances of algae expansins to terrestrial plant expansins and sequence logo analysis Utilizing MEGA11, Poisson-corrected (PC) amino acid distances were calculated to better understand the relationship of algae EXPs to terrestrial plant EXPAs and EXPBs. Algae EXPs have much higher within-group distances compared to the terrestrial plant expansins, possibly indicating greater genetic variation among algae expansins (Fig. 4 ). Average between-group distances were calculated for algae EXPs to bryophyte, lycophyte, and angiosperm EXPA and EXPB families (Fig. 4 ). Algae EXPs share similar distances to all terrestrial plant EXPA and EXPB families, suggesting that they are not members of either expansin family. This is further supported by sequence logo analysis of algae EXPs and terrestrial plant EXPAs and EXPBs. The highlighted range of amino acid residues 24–29 shows algae EXPs share a similar motif to ‘DASGTM’ found only in EXPAs. Some algae EXPs also contain an insertion located at amino acid residues 116 to 129 similarly found in EXPAs. However, some algae EXPs have indels more similar to EXPBs at residue ranges 79–94, and 151–159. Algae EXP residues that are more similar to EXPAs include residue numbers 17 (F), 136 (H), 230 (W), 284 (W). Algae EXP residues more similar to EXPBs include residue numbers 195 (W) and 258 (P) (Fig. 5 ). Such analysis, along with the phylogenetic and PC-distance data for algae EXPs, suggests that the origin of these EXP genes predates the EXPA-EXPB split, and that they are not members of either family. Bryophyte and algae intron patterns Along with having conserved amino acid sequences, past studies have shown that expansins have conserved intron patterns (Sampedro et al. 2005 ). It is hypothesized that the ancestral intron pattern for EXPAs consisted of introns A and B, while the likely ancestral pattern for EXPBs consisted of introns A, C, B, and F (Sampedro et al. 2005 ). The intron patterns found in M. polymorpha , A. agrestis , C. purpureus , and C. conicum support the hypothesized ancestral EXPA and EXPB patterns. Details on intron patterns in each species can be found in Online Resource 2. Unusual intron patterns were found in CpEXPB6 and CcEXPA15. CpEXPB6 contains introns A, C, and F along with two novel introns. One novel intron is found between intron C and where B is typically found, with the other novel intron being located after intron F. These novel introns have also been found in PpEXPB3, suggesting that PpEXPB3 and CpEXPB6 are orthologs (Carey and Cosgrove 2007 ). CcEXPA15 has an intron located where intron F is typically found, which may indicate intron F is ancestral to the EXPA family but has been lost in most lineages. In an attempt to uncover the origins of expansin intron patterns, a survey of algae expansin intron patterns was undertaken. C. braunii EXPs 8 and 21 contain introns A and B, and CbEXP21 contains just intron B. CbEXP19 contains a novel intron around fifteen amino acids downstream from where intron F is found. Additionally, a novel intron located two amino acids before the highly conserved ‘HFD’ motif was found in fourteen of the C. braunii expansin sequences. Such intron averages 1500bp in length, and we have designated this intron as intron ‘H.’ Six S. muscicola expansin genes contain introns B and F, with half of those six also containing intron A and the other half containing intron C. Details on intron patterns of C. braunii and S. muscicola can be found in Online Resource 2. Because S. muscicola expansin genes contain introns A, B, C, and F, this may indicate that all four of these introns are ancestral to EXPAs and EXPBs. DISCUSSION Gene family compositions of Bryophytes The lack of EXPB genes in M. polymorpha and C. conicum suggests liverworts lack the EXPB gene family. The timing of the origin of EXPBs, given this absence, depends on the evolutionary relationship between bryophytes. There are multiple competing hypotheses for the evolutionary relationships among bryophytes (Qiu and Mishler 2024 ). Of those competing hypotheses, the two with the most support suggest that bryophytes are either a paraphyletic group with hornworts sister to vascular plants or a monophyletic group sister to vascular plants (Liu et al. 2014 ; Gitzendanner et al. 2018 ). If bryophytes are monophyletic, EXPBs are ancestral to all bryophyte lineages but have been lost in liverworts. If bryophytes are a paraphyletic group, EXPBs arose in the common ancestor of mosses, hornworts, and vascular plants, postdating the divergence of liverworts (Fig. 6 ). Because the exact phylogenetic relationships between bryophyte lineages remain unclear, favoring one of the hypotheses for the lack of EXPBs within M. polymorpha is challenging. Ancestral Genes The internal structure within liverwort gene family suggests that there are at least 19 EXPA genes in the common ancestor of liverworts. Analysis of the mosses reveals a minimum of 10 ancestral EXPA genes and 4 ancestral EXPB genes. Based on A. agrestis’s structure, there are at least 5 ancestral EXPA genes and 2 ancestral EXPB genes. When considering all bryophytes and their internal structure within the EXPA phylogenetic tree, there appears to be 7 ancestral EXPA genes in the common ancestor of all bryophytes. Given the uncertainty in the evolutionary relationships between bryophytes, determining the number of EXPB ancestral genes in the common ancestor is challenging. If their relationship is paraphyletic, then the ancestor of all bryophytes had 0 EXPB genes. However, if bryophytes are monophyletic, the common ancestor would have 2 EXPB genes. As for the common ancestor of mosses and hornworts, if their relationship is paraphyletic, their common ancestor likely had 1 EXPB gene. If they are monophyletic, it seems more likely that the ancestor had 2 EXPB genes. Algae Phylogenetics, Distances, & Sequence Logo The algae EXP genes contain all the characteristics common to expansins, including a cleaved signal peptide at the amino terminus, a series of cysteine residues within patterned motifs of domain 1, an “HFD” motif, and a series of aromatic and tryptophan residues within patterned motifs of domain 2 (Cosgrove et al. 2002 ). However, despite being expansins, they do not clearly meet the criteria to be characterized as either EXPAs or EXPBs. Phylogenetic analysis reveals that both C. braunii and S. muscicola branch separately from EXPA and EXPB families, with the exception of CbEXPs 8 and 11, which branch at the base of the EXPB family (Fig. 3 ). As previously discussed, sequence analysis of algae EXPs indicates similarities to EXPAs and EXPBs, but they do not have all the characteristics of either EXPAs or EXPBs (Fig. 5 ). CbEXPs 8 and 11 share an insertion typically found within EXPBs at residues 151–159; however, the insertion found in the CbEXPs has a divergent sequence compared to EXPBs. This, together with their location in the phylogenetic tree, may suggest that they are evolving characteristics typical of EXPBs, but still lack a complete set of characteristics associated with the EXPB family. The average PC distance between algae EXPs and land plant EXPAs is not much less than the average distance between algae EXPs and land plant EXPBs. The PC distance data seem to confirm what was observed in the phylogenetic and sequence analyses, that algae EXPs are neither EXPAs nor EXPBs. CONCLUSION The absence of EXPBs in M. polymorpha and C. conicum suggests that the EXPB gene family is absent in all liverworts. The most parsimonious explanation of this observation suggests that the origin of the EXPB family postdates the divergence of liverworts from other bryophytes. Investigation of green algae expansins reveals a diverse gene family that, although it shares the common characteristics of expansins, lacks a complete set of characteristic features from either EXPAs or EXPBs. The exact timing of the origination of EXPAs remains unclear. To improve this analysis and resolve the evolutionary history of early land plant expansins, additional genomes should be analyzed. These might include additional green algae, additional bryophyte species, additional lycophyte, and pteridophyte species. Investigations of gene collinearity amongst bryophytes might also help clarify the phylogeny of early land plant expansins. To make more concrete connections between angiosperm and early land plant expansins, gymnosperm species should be investigated and included in the phylogenies. Declarations All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. ACKNOWLEDGEMENTS Lebanon Valley College’s Wolf Research Fund and High Impact Fund both provided funding to MEB and REC LEGEND TO ELECTRONIC SUPPLEMENTARY MATERIAL ESM 1 provides the accession numbers and ranges or chromosome coordinates for each expansin. ESM 2 contains information on the intron patterns found within each expansin. References Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. BMC Bioinformatics 10: 421. doi: 10.1186/1471-2105-10-421 Carey RE, Cosgrove DJ (2007) Portrait of the expansin superfamily in Physcomitrella patens : comparisons with angiosperm expansins. 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Rensing SA, Lang D, Zimmer AD, Terry A, Salamov A, Shapiro H, Nishiyama T, Perroud P-F, Lindquist EA, Kamisugi Y, Tanahashi T, Sakakibara K, Fujita T, Oishi K, Shin-I T, Kuroki Y, Toyoda A, Suzuki Y, Hashimoto S, Yamaguchi K, Sugano S, Kohara Y, Fujiyama A, Anterola A, Aoki S, Ashton N, Barbazuk WB, Barker E, Bennetzen JL, Blankenship R, Cho SH, Dutcher SK, Estelle M, Fawcett JA, Gundlach H, Hanada K, Heyl A, Hicks KA, Hughes J, Lohr M, Mayer K, Melkozernov A, Murata T, Nelson DR, Pils B, Prigge M, Reiss B, Renner T, Rombauts S, Rushton PJ, Sanderfoot A, Schween G, Shiu S-H, Stueber K, Theodoulou FL, Tu H, Peer YV, Verrier PJ, Waters E, Wood A, Yang L, Cove D, Cuming AC, Hasebe M, Lucas S, Mishler BD, Reski R, Grigoriev IV, Quatrano RS, Boore JL (2008) The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants. Science 319(5859): 64–69. doi: 10.1126/science.1150646 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61(3): 539-42. doi: 10.1093/sysbio/sys029 Rose JKC, Lee HH, and Bennett AB (1997) Expression of a divergent expansin gene is fruit-specific and ripening-regulated. Proc Natl Acad Sci USA. 94(11): 5955-5960. doi: 10.1073/pnas.94.11.5955 Sampedro J, Cosgrove DJ (2005) The expansin superfamily. Genome Biol 6(12): 242. doi: 10.1186/gb-2005-6-12-242. Sampedro J, Lee Y, Carey RE, DePamphilis C, Cosgrove DJ (2005) Use of genomic history to improve phylogeny and understanding of births and deaths in a gene family. The Plant Journal 44: 409-419. doi: 10.1111/j.1365-313X.2005.02540 Sampedro J, Carey RE, Cosgrove DJ (2006) Genome histories clarify evolution of the expansin superfamily: new insights from the poplar genome and pine ESTs. J Plant Res. 119(1): 11-21. doi: 10.1007/s10265-005-0253-z Sayers EW, Beck J, Bolton EE, Brister JR, Chan J, Connor R, Feldgarden M, Fine AM, Funk K, Hoffman J, Kannan S, Kelly C, Klimke W, Kim S, Lathrop S, Marchler-Bauer A, Murphy TD, O'Sullivan C, Schmieder E, Skripchenko Y, Stine A, Thibaud-Nissen F, Wang J, Ye J, Zellers E, Schneider VA, Pruitt KD (2025) Database resources of the National Center for Biotechnology Information in 2025. Nucleic Acids Res 53(D1): D20-D29. doi: 10.1093/nar/gkae979 Seader VH, Thornsberry JM, Carey, RE (2016) Utility of the Amborella trichopoda expansin superfamily in elucidating the history of angiosperm expansins. J Plant Res 129: 199–207. doi: 10.1007/s10265-015-0772-1 Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular evolutionary genetics analysis version 11. Mol Biol Evol 38(7): 3022–3027. doi: 10.1093/molbev/msab120 Valdivia ER, Wu Y, Li L-C, Cosgrove DJ, Stephenson AG (2007) A group-1 grass pollen allergen influences the outcome of pollen competition in maize. PLOS ONE 2(1): e154. doi: 10.1371/journal.pone.0000154 Vannerum K, Huysman MJ, Rycke RD, Vuylsteke M, Leliaert F, Pollier J, Lütz-Meindl U, Gillard J, Veylder LD, Goossens A, Inzé D, Vyverman W (2011) Transcriptional analysis of cell growth and morphogenesis in the unicellular green alga Micrasterias (Streptophyta), with emphasis on the role of expansin. BMC Plant Biol 128. doi: 10.1186/1471-2229-11-128 Wang Z, Cao J, Lin N, Li J, Wang Y, Liu W, Yao W, Li Y (2024) Origin, evolution, and diversification of the expansin family in plants. Int J Mol Sci 25(21): 11814. doi: 10.3390/ijms252111814 Additional Declarations No competing interests reported. Supplementary Files ESM1.xlsx ESM2.xlsx Cite Share Download PDF Status: Published Journal Publication published 23 Apr, 2026 Read the published version in Protoplasma → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8810289","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594045004,"identity":"4103eb8a-a7bd-47ba-bf67-755da4f59fb9","order_by":0,"name":"Maddisyn E Behney","email":"","orcid":"","institution":"Lebanon Valley College","correspondingAuthor":false,"prefix":"","firstName":"Maddisyn","middleName":"E","lastName":"Behney","suffix":""},{"id":594045005,"identity":"374de3c9-4b1d-4dac-95af-7e8c320ff1f0","order_by":1,"name":"Tyani Orta","email":"","orcid":"","institution":"Lebanon Valley College","correspondingAuthor":false,"prefix":"","firstName":"Tyani","middleName":"","lastName":"Orta","suffix":""},{"id":594045007,"identity":"3025d95d-fae8-4050-b940-4c562dbad2f0","order_by":2,"name":"Robert E Carey","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYPACGxkwxUOCljQekrUcJkGLefvhZx8+tp3nMTh+gPHB2zYitMicSTOeObPtNo/BmQRmw7nEaJGQYDBm5gVqkZzBwCbNS5wW9s/Mf9vOgbSw/yZSC48xM2PbAR5+CQY2ZuK08OQUM/acS+bh50lslpxzjhgt7Mc3M/wos5NjYz988MObMiK0gAEjG5hsIFY9CPwhRfEoGAWjYBSMOAAAajor+xaOiYwAAAAASUVORK5CYII=","orcid":"","institution":"Lebanon Valley College","correspondingAuthor":true,"prefix":"","firstName":"Robert","middleName":"E","lastName":"Carey","suffix":""}],"badges":[],"createdAt":"2026-02-06 19:09:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8810289/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8810289/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00709-026-02200-2","type":"published","date":"2026-04-23T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":103064799,"identity":"9b754abc-4cc8-46d3-a4ed-3c822363e332","added_by":"auto","created_at":"2026-02-20 11:01:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":868161,"visible":true,"origin":"","legend":"\u003cp\u003eBayesian tree for the \u003cem\u003eM. polymorpha, C. conicum, A. agrestis\u003c/em\u003e and \u003cem\u003eC. purpureus\u003c/em\u003eEXPA families with selected \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eO. sativa\u003c/em\u003e, \u003cem\u003eC. braunii\u003c/em\u003e, \u003cem\u003eS. moellendorffii\u003c/em\u003e, and \u003cem\u003eP. patens\u003c/em\u003e sequences. The tree was constructed from 7,000,000 generations and the burnin was set to 25%. The tree was manually rooted at the \u003cem\u003eC. braunii\u003c/em\u003e sequences CbEXP8, 11 and 10\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8810289/v1/e062749b881a8c66fe23b425.png"},{"id":103064785,"identity":"1fe69920-5d11-4978-b94f-528dcda6b0e2","added_by":"auto","created_at":"2026-02-20 11:01:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":269725,"visible":true,"origin":"","legend":"\u003cp\u003eBayesian tree for the \u003cem\u003eA. agrestis\u003c/em\u003e and \u003cem\u003eC. purpureus\u003c/em\u003e EXPB families with selected \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eO. sativa\u003c/em\u003e, \u003cem\u003eC. braunii\u003c/em\u003e, \u003cem\u003eS. moellendorffii\u003c/em\u003e, and \u003cem\u003eP. patens\u003c/em\u003e sequences. The tree was constructed from 7,000,000 generations and the burnin was set to 25%. The tree was manually rooted at the \u003cem\u003eC. braunii\u003c/em\u003e sequences CbEXP8, 11 and 10\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8810289/v1/70e27dd8ade46b0e53cc1362.png"},{"id":103064789,"identity":"fc74f80f-44c4-4959-b13f-a97a9216f902","added_by":"auto","created_at":"2026-02-20 11:01:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":165911,"visible":true,"origin":"","legend":"\u003cp\u003eUnrooted Bayesian tree for the \u003cem\u003eC. braunii\u003c/em\u003e and \u003cem\u003eS. muscicola\u003c/em\u003e EXP families with selected bryophyte, lycophyte, and angiosperm EXPA and EXPB sequences. The tree was constructed from 7,000,000 generations and the burnin was set to 25%. Confidence levels are Bayesian posterior probabilities\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8810289/v1/5a44de05df20ed1947c3dac3.png"},{"id":103064796,"identity":"3a68c8b1-84ab-4bdd-97fe-4c98e9de3c7f","added_by":"auto","created_at":"2026-02-20 11:01:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":308028,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Between-group average distances of algae EXP genes to terrestrial plant EXPA and EXPB genes. Values indicated are the average Poisson-corrected amino acid distance to the algae EXP family. Error bars are standard errors based on 500 bootstrap replicates. (B) Within-group average distances for algae EXPs and terrestrial plant EXPAs and EXPBs. Values indicated are the average Poisson-corrected amino acid distance to the algae EXP family. Error bars are standard errors based on 500 bootstrap replicates\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8810289/v1/38ba8e7203f956a02f511e36.png"},{"id":103064790,"identity":"c4741502-705a-46f7-95b2-0678654453b4","added_by":"auto","created_at":"2026-02-20 11:01:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":703575,"visible":true,"origin":"","legend":"\u003cp\u003eSeqLogo of the Algae EXP, land plant EXPA, and land plant EXPB families. Each row draws comparisons for each family: the top lines are land plant EXPA sequences, the middle lines are algae EXP sequences, and the bottom lines are land plant EXPB sequences. The height of the letters indicates the frequency of the amino acid in that group of sequences. Highlighted residues represent conserved motifs or indels of the expansin families. Those highlighted in orange are within domain 1 of the expansin protein, and those highlighted in blue are within domain 2\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8810289/v1/d07c0fd18a3bc2a8d9ff6a42.png"},{"id":103064788,"identity":"687b3799-7ea2-4d0e-9beb-e0f5821bbfd4","added_by":"auto","created_at":"2026-02-20 11:01:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":136452,"visible":true,"origin":"","legend":"\u003cp\u003eTwo possible arrangements of the three bryophyte lineages, green algae, and vascular plants. (A) Shows the bryophytes as a paraphyletic group, with liverworts diverging first, followed by mosses then hornworts. (B) Shows bryophytes as a monophyletic group, with liverworts and mosses sharing a more recent common ancestor than with hornworts. Hypothetical origins of EXPAs and EXPBs are indicated\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8810289/v1/3dc5759f3aa8521719b91bd7.png"},{"id":107929113,"identity":"800f13a3-68fc-4803-9a88-87ef7f4effd5","added_by":"auto","created_at":"2026-04-27 16:13:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2792126,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8810289/v1/ad1ab728-6087-4fa1-a8fb-ebe06d5b39dd.pdf"},{"id":103064787,"identity":"1f762b80-8eb2-43a6-bc3c-25266393b0fa","added_by":"auto","created_at":"2026-02-20 11:01:26","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21927,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8810289/v1/23f51f16537f85294c0451cb.xlsx"},{"id":103064798,"identity":"647e1f7e-563f-4644-bc8c-2e9b7e4ae915","added_by":"auto","created_at":"2026-02-20 11:01:30","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21023,"visible":true,"origin":"","legend":"","description":"","filename":"ESM2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8810289/v1/07ceb8e4d1b8664f76d13d93.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evolution of the Expansin Superfamily in Bryophytes and Green Algae","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eExpansins are a superfamily of non-enzymatic proteins critical for pH-dependent plant cell wall extension (McQueen-Mason 1992). Processes such as fruit ripening, seed germination, pollen tube growth, and general cell growth rely on expansin-mediated cell wall modification (Rose et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Chen and Bradford \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Cho and Cosgrove \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Valdivia et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Expansins are composed of approximately 270 amino acids, have a cleaved signal peptide, and a two-domain structure, which includes an N-terminal domain and a C-terminal domain (Cosgrove \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Within each domain, there are conserved amino acids (AAs) and AA motifs that appear important for expansin function (Cosgrove et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe expansin super-family consists of four families: EXPA, EXPB, EXLA, and EXLB (Sampedro and Cosgrove \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). EXPAs and EXPBs have been experimentally demonstrated to have cell wall loosening activity (Cosgrove \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). EXLAs and EXLBs have an unknown function and are included in the superfamily based on sequence similarity (Sampedro and Cosgrove \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Previous analyses have indicated that the common ancestor of all angiosperms likely had a minimum of 12 EXPAs, 2 EXPBs, 1 EXLA and 2 EXLBs (Sampedro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). These clades have been shown to be highly conserved amongst all angiosperms studied so far (Sampedro et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Seader et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough lycophytes have conserved EXPA and EXPB sequences, they lack EXLA and EXLB genes. The common ancestor of lycophytes and angiosperms is believed to have a minimum of 2 EXPAs and 1 EXPB (Carey et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe common ancestor of mosses and angiosperms appears to also have had a minimum of 2 EXPAs and 1 EXPB. Moss expansins are far more divergent when compared to either angiosperm or lycophyte expansins, indicating a more independent evolutionary history. Despite this divergence, moss expansins still share the structure and common motifs associated with expansins (Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo further elucidate the history of expansins in early diverging land plant lineages, we have extended these analyses to two liverworts (\u003cem\u003eMarchantia polymorpha\u003c/em\u003e and \u003cem\u003eConocephalum conicum\u003c/em\u003e), a hornwort (\u003cem\u003eAnthoceros agrestis\u003c/em\u003e), and an additional moss (\u003cem\u003eCeratodon purpureus\u003c/em\u003e). We have also expanded this analysis to two green algae species (\u003cem\u003eChara braunii\u003c/em\u003e and \u003cem\u003eSpirogloea muscicola\u003c/em\u003e) in hopes of understanding the evolution of the expansin superfamily as plants transitioned to life on land.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentifying expansin superfamilies\u003c/h2\u003e \u003cp\u003eMarchantia\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eM. polymorpha\u003c/em\u003e expansin superfamily was assembled from MpTak v5.1 of MarpolBase using tBLASTx with known \u003cem\u003eA. thaliana\u003c/em\u003e expansin sequences as queries (Sampedro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Montgomery et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConocephalum\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eC. conicum\u003c/em\u003e expansin superfamily was assembled from genome assembly ASM5008447v1 in NCBI with NCBI tBLASTx using known \u003cem\u003eA. thaliana\u003c/em\u003e expansin sequences as queries. (Sampedro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sayers et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCeratodon\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eC. purpureus\u003c/em\u003e expansin superfamily was assembled from genome assembly CpurpureusR40_1_0 using Phytozome BLASTn with known \u003cem\u003eP. patens\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e expansin sequences as queries (Sampedro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Goodstein et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Carey et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additional sequences (CpEXPA22, CpEXPB4, CpEXPB5) were gathered from the genome with NCBI tBLASTx with known \u003cem\u003eP. patens\u003c/em\u003e expansin sequences as queries (Sayers et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnthoceros\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eA. agrestis\u003c/em\u003e expansin superfamily was assembled from the [Bonn] genome assembly using \u003cem\u003eA. thaliana\u003c/em\u003e as queries (Sampedro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e.). Blast searches of the \u003cem\u003eA. agrestis\u003c/em\u003e genome were performed locally using BLAST\u0026thinsp;+\u0026thinsp;2.15 (Camacho et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChara\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eC. braunii\u003c/em\u003e expansin superfamily was assembled from the \u003cem\u003eChara braunii\u003c/em\u003e S276 genome using PhycoCosm BLASTn with known \u003cem\u003eP. patens\u003c/em\u003e expansin sequences as queries (Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Nishiyama et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Grigoriev et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSpirogloea\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eS. muscicola\u003c/em\u003e expansin superfamily was assembled from the \u003cem\u003eSpirogloea_muscicola\u003c/em\u003e_CCAC_0214 genome using PhycoCosm BLASTn with known \u003cem\u003eP. patens\u003c/em\u003e expansin sequences as queries (Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Grigoriev et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSequence Alignments\u003c/h3\u003e\n\u003cp\u003eThe protein sequences encoded by the expansin superfamilies of \u003cem\u003eM. polymorpha, C. conicum, A. agrestis, C. purpureus, C. braunii\u003c/em\u003e, and \u003cem\u003eS. muscicola\u003c/em\u003e were aligned with selected angiosperms, lycophytes, and bryophytes. Three alignments were constructed for phylogenetic analysis. The first alignment contained the EXPA sequences of \u003cem\u003eM. polymorpha\u003c/em\u003e, \u003cem\u003eC. conicum, A. agrestis\u003c/em\u003e, and \u003cem\u003eC. purpureus\u003c/em\u003e, along with known \u003cem\u003eP. patens\u003c/em\u003e EXPAs, \u003cem\u003eS. moellendorffii\u003c/em\u003e EXPAs, and \u003cem\u003eArabidopsis\u003c/em\u003e-rice EXPA sequences representing EXPA clades I-XII (Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Carey et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sampedro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). \u003cem\u003eP. patens\u003c/em\u003e PpEXPA20 was excluded from the alignment due to a long c-terminal extension that degrades the alignment. \u003cem\u003eC. braunii\u003c/em\u003e EXPs 8, 10, and 11 and PpEXPB1 were also included in the alignment to root the resulting tree. The second alignment included EXPB sequences of \u003cem\u003eA. agrestis\u003c/em\u003e and \u003cem\u003eC. purpureus\u003c/em\u003e, along with known \u003cem\u003eP. patens\u003c/em\u003e EXPBs, \u003cem\u003eS. moellendorffii\u003c/em\u003e EXPBs, and representative \u003cem\u003eArabidopsis\u003c/em\u003e-rice sequences of EXPB, EXLA, and EXLB clades (Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Carey et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sampedro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Included in the alignment were \u003cem\u003eC. braunii\u003c/em\u003e EXPs 8, 10, and 11 and PpEXPA1 to help root and resolve the tree. The third alignment constructed contained representative bryophyte, lycophyte, and \u003cem\u003eArabidopsis\u003c/em\u003e-rice EXPA and EXPB genes of along with expansin sequences from \u003cem\u003eC. braunii\u003c/em\u003e and \u003cem\u003eS. muscicola\u003c/em\u003e (Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Carey et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sampedro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Such an alignment was necessary to analyze the relationship between algae expansins and terrestrial plant expansins. Alignments were created using the default parameters of the MUSCLE algorithm within the Unipro UGENE software package (Okonechnikov et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Alignments were then trimmed at a conserved tryptophan upstream of the expansin HATFYG motif and a conserved phenylalanine near the c-terminus.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic Analysis\u003c/h3\u003e\n\u003cp\u003eBayesian\u003c/p\u003e \u003cp\u003ePhylogenetic trees were constructed using MrBayes v3.2.5 (Ronquist et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Priors for the Bayesian trees were as follows: Jones amino acid model, gamma estimation, 7,000,000 generations, two runs of five Markov chains each \u0026ndash; burnin as indicated in figure legends. The consensus trees were visualized using Figtree, where the bryophyte EXPA tree was manually rooted at \u003cem\u003eC. braunii\u003c/em\u003e sequences CbEXP8, 10, and 11, and the EXPB tree was manually rooted at the \u003cem\u003eC. braunii\u003c/em\u003e sequences CbEXP8, 10, and 11 (Rambaut \u003cem\u003eet al.\u003c/em\u003e 2018). Although CbEXP8, 10, and 11 may not be the true root for either the EXPA or EXPB tree, such \u003cem\u003eC. braunii\u003c/em\u003e sequences were chosen to facilitate comparison between the two trees. The algae EXP tree was left unrooted.\u003c/p\u003e \u003cp\u003eNeighbor Joining\u003c/p\u003e \u003cp\u003eNeighbor Joining trees were constructed using MEGA11 (Tamura et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Parameters were as follows: bootstrap 500, Poisson model, pairwise deletion. The bryophyte EXPA tree was manually rooted at CbEXPA10, and the EXPB tree was manually rooted at the \u003cem\u003eC. braunii\u003c/em\u003e sequences CbEXP8, 10, and 11.\u003c/p\u003e \u003cp\u003eMaximum Likelihood\u003c/p\u003e \u003cp\u003eMaximum Likelihood trees were constructed using PhyML 3.0 (Guindon at el. 2010). Trees were constructed with automatic model selection by AIC and bootstrap 100.\u003c/p\u003e\n\u003ch3\u003ePoisson Corrected Distances\u003c/h3\u003e\n\u003cp\u003eFor analysis of algal expansins, an alignment of algae EXPs, bryophyte EXPAs and EXPBs, lycophyte EXPAs and EXPBs, and angiosperm EXPAs and EXPBs was created using the MUSCLE algorithm within Unipro UGENE and was trimmed as was done for phylogenetic tree construction (Okonechnikov et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). PC distances were calculated using MEGA11 with standard error calculated for these values using 500 bootstrap replicates (Tamura et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Gaps were handled with complete deletion.\u003c/p\u003e\n\u003ch3\u003eIntron Patterns\u003c/h3\u003e\n\u003cp\u003eWhen possible, genomic sequences were compared to cDNA sequences to determine intron patterns. If only genomic sequences were available, translated sequences were aligned with related translated cDNA sequences, and intron locations were estimated based on gaps within the alignment along with previous knowledge of expansin intron patterns.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSequence logo\u003c/h2\u003e \u003cp\u003eAmino acid sequences for algae EXPs and terrestrial plant EXPAs and EXPBs were aligned using the default parameters of the MUSCLE algorithm within the Unipro UGENE software package (Okonechnikov et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The alignment was trimmed as was done for phylogenetic tree construction. The alignment was then sub-divided into algae EXL and terrestrial plant EXPA and EXPB portions. The sequence logo was generated using WebLogo (Crooks et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe expansin superfamilies of bryophytes and algae\u003c/h2\u003e \u003cp\u003eAs is the case with all terrestrial plants studied thus far, the EXPA families within the bryophytes studied here are larger than the EXPB families. The lone exception to this is \u003cem\u003eA. agrestis\u003c/em\u003e, which has more EXPBs than EXPAs (Cosgrove \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The EXPA families of \u003cem\u003eM. polymorpha\u003c/em\u003e and \u003cem\u003eC. conicum\u003c/em\u003e appear to be slightly expanded, with \u003cem\u003eM. polymorpha\u003c/em\u003e having 38 EXPAs and \u003cem\u003eC. conicum\u003c/em\u003e having 32. Interestingly, no EXPBs were found within either liverwort, despite being found in all mosses and hornworts studied so far (Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Both \u003cem\u003eC. braunii\u003c/em\u003e and \u003cem\u003eS. muscicola\u003c/em\u003e contained expansins; however, such expansins were determined to not belong to any of the four expansin families found in terrestrial plants. Such expansins have been labeled as \u0026ldquo;EXPs\u0026rdquo;, as they are expansins but do not share all of the same features of the terrestrial plant expansin families (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn agreement with previous studies of bryophytes and algae, none of the selected species contained EXLAs or EXLBs (Vannerum et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Studies of expansins within pteridophytes suggest, along with the absence of such families within the early land plants and algae, it is likely that the EXLA and EXLB families emerged after the divergence of pteridophytes (Carey et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Vannerum et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to complete sequences, possible pseudogenes were found in \u003cem\u003eC. conicum\u003c/em\u003e. CcEXPA3 contains a frameshift near the expansin \u0026ldquo;VPC\u0026rdquo; motif, which was fixed by removing the two bp fifteen amino acids downstream of the motif. CcEXPA23 also had two bp removed from its sequence before the \u0026ldquo;TATN\u0026rdquo; expansin motif to fix a frameshift. Four \u003cem\u003eC. braunii\u003c/em\u003e sequences also had missing data, which makes it difficult to determine if they are pseudogenes or not (Online Resource 1).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSize and composition of expansin gene families of selected plant species\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEXPA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEXPB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEXLA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEXLB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTotal EXP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. thaliana*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eO. sativa*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. moellendorffii\u003c/em\u003e\u003csup\u003eΔ\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. patens\u003c/em\u003e\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. purpureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. agrestis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM. polymorpha\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. conicum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. braunii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. muscicola\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003e*\u003c/em\u003e At time of search (Sampedro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003csup\u003eΔ\u003c/sup\u003e At time of search (Carey et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003csup\u003e\u0026dagger;\u003c/sup\u003e At time of search (Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis of bryophyte expansins\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eEXPA\u003c/h2\u003e \u003cp\u003eMost liverwort and moss EXPAs appear to largely group separately from the angiosperm-specific EXPAs. A previous study of \u003cem\u003eP. patens\u003c/em\u003e EXPAs divided the family into six clades named A-F (Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The addition of \u003cem\u003eC. purpureus\u003c/em\u003e EXPA genes has further refined the structure of the moss EXPA family. \u003cem\u003eP. patens\u003c/em\u003e clade A can be further divided into five distinct clades, labeled clades A1 through A5. For the liverworts, there appears to be 17 liverwort-specific clades. Clades designated 1 through 15 group sister to \u003cem\u003eP. patens\u003c/em\u003e clades B and A1-A5. MpEXPA14 is grouped with liverwort clades 12 and 13 but does not appear to belong to either clade. Liverwort clade 16 is sister to \u003cem\u003eS. moellendorffii\u003c/em\u003e clade B, and MpEXPA9 groups with \u003cem\u003eS. moellendorffii\u003c/em\u003e clade A. Liverwort clade 17 is sister to \u003cem\u003eP. patens\u003c/em\u003e clade E, which appears to be a moss-liverwort specific clade consisting of MpEXPA1, CcEXPA9, PpEXPA1, and CpEXPA10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA few bryophyte EXPAs group with known Angiosperm clades. \u003cem\u003eS. moellendorffii\u003c/em\u003e EXPAs 5 and 6 along with MpEXPA10 and AaEXPA6 belong to Arabidopsis-rice clade X. Previously described \u003cem\u003eP. patens\u003c/em\u003e clade F, containing PpEXPA6 and CpEXPA4, branches with Arabidopsis-rice clade XI. \u003cem\u003eA. agrestis\u003c/em\u003e EXPAs 1\u0026ndash;4, MpEXPA9, CpEXPA9, and PpEXPAs 8 and 13 of \u003cem\u003eP. patens\u003c/em\u003e clade D are sister to Arabidopsis-rice clades I-III (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Neighbor joining and maximum likelihood trees did not appear to alter any of these placements (data not shown).\u003c/p\u003e \u003cp\u003eAs noted previously, most \u003cem\u003eA. agrestis\u003c/em\u003e EXPAs group with vascular plant EXPAs, with the only exceptions being AaEXPA5 and AaEXPA7, which group with bryophyte-specific EXPA clades. Overall, \u003cem\u003eA. agrestis\u003c/em\u003e appears to have more angiosperm-related than bryophyte-specific EXPAs. It is also important to note that the \u003cem\u003eA. agrestis\u003c/em\u003e genome is one of the smallest among land plants, and this may contribute to the reduction of bryophyte-specific EXPAs (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEXPB\u003c/h2\u003e \u003cp\u003eAaEXPBs 2 through 8 group together forming an Anthoceros-specific clade (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Such clade appears sister to all bryophyte and angiosperm EXPB clades (bpp\u0026thinsp;=\u0026thinsp;0.946). AaEXPB1 groups sister to Arabidopsis-rice clade II (bpp\u0026thinsp;=\u0026thinsp;0.994).\u003c/p\u003e \u003cp\u003eThe inclusion of \u003cem\u003eC. purpureus\u003c/em\u003e EXPB genes revealed internal structure of the EXPB family within mosses, with all six genes grouping with \u003cem\u003eP. patens\u003c/em\u003e. Four moss-specific EXPB clades were determined. Each clade contained at least one \u003cem\u003eP. patens\u003c/em\u003e and \u003cem\u003eC. purpureus\u003c/em\u003e, and have been named as clades I, II, III, and IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Neighbor joining and maximum likelihood trees did not appear to alter any of these placements (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis of algae expansins\u003c/h2\u003e \u003cp\u003ePhylogenetic analysis of the algae EXPs was performed to aid in the understanding of the algae EXPs\u0026rsquo; relationship to terrestrial plant EXPAs and EXPBs. Both \u003cem\u003eS. muscicola\u003c/em\u003e and most \u003cem\u003eC. braunii\u003c/em\u003e EXPs grouping separately from both EXPA and EXPB clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although CbEXPs 8 and 11 group with the terrestrial plant EXPBs, sequence analysis suggests that they are not members of the EXPB family.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDistances of algae expansins to terrestrial plant expansins and sequence logo analysis\u003c/h2\u003e \u003cp\u003eUtilizing MEGA11, Poisson-corrected (PC) amino acid distances were calculated to better understand the relationship of algae EXPs to terrestrial plant EXPAs and EXPBs. Algae EXPs have much higher within-group distances compared to the terrestrial plant expansins, possibly indicating greater genetic variation among algae expansins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAverage between-group distances were calculated for algae EXPs to bryophyte, lycophyte, and angiosperm EXPA and EXPB families (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Algae EXPs share similar distances to all terrestrial plant EXPA and EXPB families, suggesting that they are not members of either expansin family. This is further supported by sequence logo analysis of algae EXPs and terrestrial plant EXPAs and EXPBs. The highlighted range of amino acid residues 24\u0026ndash;29 shows algae EXPs share a similar motif to \u0026lsquo;DASGTM\u0026rsquo; found only in EXPAs. Some algae EXPs also contain an insertion located at amino acid residues 116 to 129 similarly found in EXPAs. However, some algae EXPs have indels more similar to EXPBs at residue ranges 79\u0026ndash;94, and 151\u0026ndash;159. Algae EXP residues that are more similar to EXPAs include residue numbers 17 (F), 136 (H), 230 (W), 284 (W). Algae EXP residues more similar to EXPBs include residue numbers 195 (W) and 258 (P) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Such analysis, along with the phylogenetic and PC-distance data for algae EXPs, suggests that the origin of these EXP genes predates the EXPA-EXPB split, and that they are not members of either family.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBryophyte and algae intron patterns\u003c/h2\u003e \u003cp\u003eAlong with having conserved amino acid sequences, past studies have shown that expansins have conserved intron patterns (Sampedro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). It is hypothesized that the ancestral intron pattern for EXPAs consisted of introns A and B, while the likely ancestral pattern for EXPBs consisted of introns A, C, B, and F (Sampedro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The intron patterns found in \u003cem\u003eM. polymorpha\u003c/em\u003e, \u003cem\u003eA. agrestis\u003c/em\u003e, \u003cem\u003eC. purpureus\u003c/em\u003e, and \u003cem\u003eC. conicum\u003c/em\u003e support the hypothesized ancestral EXPA and EXPB patterns. Details on intron patterns in each species can be found in Online Resource 2.\u003c/p\u003e \u003cp\u003eUnusual intron patterns were found in CpEXPB6 and CcEXPA15. CpEXPB6 contains introns A, C, and F along with two novel introns. One novel intron is found between intron C and where B is typically found, with the other novel intron being located after intron F. These novel introns have also been found in PpEXPB3, suggesting that PpEXPB3 and CpEXPB6 are orthologs (Carey and Cosgrove \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). CcEXPA15 has an intron located where intron F is typically found, which may indicate intron F is ancestral to the EXPA family but has been lost in most lineages.\u003c/p\u003e \u003cp\u003eIn an attempt to uncover the origins of expansin intron patterns, a survey of algae expansin intron patterns was undertaken. \u003cem\u003eC. braunii\u003c/em\u003e EXPs 8 and 21 contain introns A and B, and CbEXP21 contains just intron B. CbEXP19 contains a novel intron around fifteen amino acids downstream from where intron F is found. Additionally, a novel intron located two amino acids before the highly conserved \u0026lsquo;HFD\u0026rsquo; motif was found in fourteen of the \u003cem\u003eC. braunii\u003c/em\u003e expansin sequences. Such intron averages 1500bp in length, and we have designated this intron as intron \u0026lsquo;H.\u0026rsquo; Six \u003cem\u003eS. muscicola\u003c/em\u003e expansin genes contain introns B and F, with half of those six also containing intron A and the other half containing intron C. Details on intron patterns of \u003cem\u003eC. braunii\u003c/em\u003e and \u003cem\u003eS. muscicola\u003c/em\u003e can be found in Online Resource 2. Because \u003cem\u003eS. muscicola\u003c/em\u003e expansin genes contain introns A, B, C, and F, this may indicate that all four of these introns are ancestral to EXPAs and EXPBs.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGene family compositions of Bryophytes\u003c/h2\u003e \u003cp\u003eThe lack of EXPB genes in \u003cem\u003eM. polymorpha\u003c/em\u003e and \u003cem\u003eC. conicum\u003c/em\u003e suggests liverworts lack the EXPB gene family. The timing of the origin of EXPBs, given this absence, depends on the evolutionary relationship between bryophytes. There are multiple competing hypotheses for the evolutionary relationships among bryophytes (Qiu and Mishler \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Of those competing hypotheses, the two with the most support suggest that bryophytes are either a paraphyletic group with hornworts sister to vascular plants or a monophyletic group sister to vascular plants (Liu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gitzendanner et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). If bryophytes are monophyletic, EXPBs are ancestral to all bryophyte lineages but have been lost in liverworts. If bryophytes are a paraphyletic group, EXPBs arose in the common ancestor of mosses, hornworts, and vascular plants, postdating the divergence of liverworts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Because the exact phylogenetic relationships between bryophyte lineages remain unclear, favoring one of the hypotheses for the lack of EXPBs within \u003cem\u003eM. polymorpha\u003c/em\u003e is challenging.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAncestral Genes\u003c/h2\u003e \u003cp\u003eThe internal structure within liverwort gene family suggests that there are at least 19 EXPA genes in the common ancestor of liverworts. Analysis of the mosses reveals a minimum of 10 ancestral EXPA genes and 4 ancestral EXPB genes. Based on \u003cem\u003eA. agrestis\u0026rsquo;s\u003c/em\u003e structure, there are at least 5 ancestral EXPA genes and 2 ancestral EXPB genes.\u003c/p\u003e \u003cp\u003eWhen considering all bryophytes and their internal structure within the EXPA phylogenetic tree, there appears to be 7 ancestral EXPA genes in the common ancestor of all bryophytes. Given the uncertainty in the evolutionary relationships between bryophytes, determining the number of EXPB ancestral genes in the common ancestor is challenging. If their relationship is paraphyletic, then the ancestor of all bryophytes had 0 EXPB genes. However, if bryophytes are monophyletic, the common ancestor would have 2 EXPB genes. As for the common ancestor of mosses and hornworts, if their relationship is paraphyletic, their common ancestor likely had 1 EXPB gene. If they are monophyletic, it seems more likely that the ancestor had 2 EXPB genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAlgae Phylogenetics, Distances, \u0026amp; Sequence Logo\u003c/h2\u003e \u003cp\u003eThe algae EXP genes contain all the characteristics common to expansins, including a cleaved signal peptide at the amino terminus, a series of cysteine residues within patterned motifs of domain 1, an \u0026ldquo;HFD\u0026rdquo; motif, and a series of aromatic and tryptophan residues within patterned motifs of domain 2 (Cosgrove et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, despite being expansins, they do not clearly meet the criteria to be characterized as either EXPAs or EXPBs.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis reveals that both \u003cem\u003eC. braunii\u003c/em\u003e and \u003cem\u003eS. muscicola\u003c/em\u003e branch separately from EXPA and EXPB families, with the exception of CbEXPs 8 and 11, which branch at the base of the EXPB family (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As previously discussed, sequence analysis of algae EXPs indicates similarities to EXPAs and EXPBs, but they do not have all the characteristics of either EXPAs or EXPBs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). CbEXPs 8 and 11 share an insertion typically found within EXPBs at residues 151\u0026ndash;159; however, the insertion found in the CbEXPs has a divergent sequence compared to EXPBs. This, together with their location in the phylogenetic tree, may suggest that they are evolving characteristics typical of EXPBs, but still lack a complete set of characteristics associated with the EXPB family.\u003c/p\u003e \u003cp\u003eThe average PC distance between algae EXPs and land plant EXPAs is not much less than the average distance between algae EXPs and land plant EXPBs. The PC distance data seem to confirm what was observed in the phylogenetic and sequence analyses, that algae EXPs are neither EXPAs nor EXPBs.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe absence of EXPBs in \u003cem\u003eM. polymorpha\u003c/em\u003e and \u003cem\u003eC. conicum\u003c/em\u003e suggests that the EXPB gene family is absent in all liverworts. The most parsimonious explanation of this observation suggests that the origin of the EXPB family postdates the divergence of liverworts from other bryophytes. Investigation of green algae expansins reveals a diverse gene family that, although it shares the common characteristics of expansins, lacks a complete set of characteristic features from either EXPAs or EXPBs. The exact timing of the origination of EXPAs remains unclear.\u003c/p\u003e \u003cp\u003eTo improve this analysis and resolve the evolutionary history of early land plant expansins, additional genomes should be analyzed. These might include additional green algae, additional bryophyte species, additional lycophyte, and pteridophyte species. Investigations of gene collinearity amongst bryophytes might also help clarify the phylogeny of early land plant expansins. To make more concrete connections between angiosperm and early land plant expansins, gymnosperm species should be investigated and included in the phylogenies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLebanon Valley College\u0026rsquo;s Wolf Research Fund and High Impact Fund both provided funding to MEB and REC\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLEGEND TO ELECTRONIC SUPPLEMENTARY MATERIAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eESM 1 provides the accession numbers and ranges or chromosome coordinates for each expansin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eESM 2 contains information on the intron patterns found within each expansin.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCamacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. 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Int J Mol Sci 25(21): 11814. doi: 10.3390/ijms252111814\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"expansin, gene family evolution, bryophytes, green algae","lastPublishedDoi":"10.21203/rs.3.rs-8810289/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8810289/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExpansins are a superfamily of non-enzymatic proteins that mediate plant cell wall loosening and play essential roles in growth and development. The expansin superfamily consists of four families: EXPA, EXPB, EXLA, and EXLB. Although the expansin gene families have been well characterized in angiosperms, their evolutionary history in early-diverging land plants and green algae remains incompletely resolved. This study examines expansin superfamilies in two liverworts (\u003cem\u003eMarchantia polymorpha\u003c/em\u003e and \u003cem\u003eConocephalum conicum\u003c/em\u003e), a hornwort (\u003cem\u003eAnthoceros agrestis\u003c/em\u003e), a moss (\u003cem\u003eCeratodon purpureus\u003c/em\u003e), and two green algal species (\u003cem\u003eChara braunii\u003c/em\u003e and \u003cem\u003eSpirogloea muscicola\u003c/em\u003e). Expansin genes were identified from these organisms using BLAST searches. These newly assembled gene families were then analyzed to determine the relationships between them and gain insight into early expansin evolution. No EXLA or EXLB genes were found in bryophytes or green algae. Bryophytes contain EXPA genes; however, EXPBs were not detected in liverworts and are present only in mosses and hornworts. Although green algal expansins share key characteristics of both EXPA and EXPB genes, evidence suggests they are not members of either family. The data presented here raises interesting questions about the timing of EXPA and EXPB evolution in land plants.\u003c/p\u003e","manuscriptTitle":"Evolution of the Expansin Superfamily in Bryophytes and Green Algae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-20 11:01:01","doi":"10.21203/rs.3.rs-8810289/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c3a96744-723e-40ad-ae38-50d4be99b324","owner":[],"postedDate":"February 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:12:14+00:00","versionOfRecord":{"articleIdentity":"rs-8810289","link":"https://doi.org/10.1007/s00709-026-02200-2","journal":{"identity":"protoplasma","isVorOnly":false,"title":"Protoplasma"},"publishedOn":"2026-04-23 15:57:35","publishedOnDateReadable":"April 23rd, 2026"},"versionCreatedAt":"2026-02-20 11:01:01","video":"","vorDoi":"10.1007/s00709-026-02200-2","vorDoiUrl":"https://doi.org/10.1007/s00709-026-02200-2","workflowStages":[]},"version":"v1","identity":"rs-8810289","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8810289","identity":"rs-8810289","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}