{"paper_id":"0c711ab6-d47d-4b97-a3b8-99c883a5ae69","body_text":"A conserved minimal core and modular extensions make the fungal flagellum | 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 Article A conserved minimal core and modular extensions make the fungal flagellum Aleksander Kossakowski, Anna Muszewska This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7887758/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Flagellated fungi are pivotal for understanding the evolution of eukaryotic motility, yet knowledge of their flagellar proteins remains fragmentary. We assembled a cross-phyla atlas by mapping 303 reference flagellar proteins on 184 fungal proteomes. Out of 303 orthogroups, 231 orthogroups have fungal homologs, of which 128 orthogroups are specific to flagellated fungi. From these, we propose a 97-orthogroup minimal flagellum encompassing proteins shared by flagellated fungi. This minimal toolkit retains the axonemal core (tubulins, dynein arms), beat regulators (nexin–dynein regulatory complex, radial spokes), and essential intraflagellar transport (core IFT-B with key IFT-A and kinesin-2), plus a pared foundation for basal-body duplication and targeted trafficking (exocyst/TRAPP). Fungal lineages differ in the composition of flagellar modules. Neocallimastigomycota stand out as the most diverged among flagellated lineages with the broadest flagellar toolkit, including a complete BBSome, yet exhibit reductions in centriole-associated modules. Finally, we annotate two Pfam domains of unknown function DUF4470 & DUF4471 within DNAAF3 protein as a single Rossmann-like fold. We delineate a lean, conserved engine for fungal motility and a modular, lineage-specific layer that broadens regulation and sensory capacity. The conservation of flagellar proteins between fungi and animals opens up possibilities of experimental functional validation in models simpler than animals. Biological sciences/Cell biology Biological sciences/Microbiology fungi motility flagellum Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Fungi are one of the most diverse and complex kingdoms on Earth, with an estimated number of species reaching up to thirteen million 1 . These organisms occupy a wide range of ecological niches, including soil, water, air and form a plethora of relationships with other living forms 2 . Within the multiplex fungal tree of life, there are two main fungal supergroups - Dikarya and non-Dikarya, which encompass many fungal phyla - from symbiotic Glomeromycota to obligatory parasitic Microsporidia 3 . Some non-Dikarya fungi have a single or multiple posterior flagella on their zoospores. They are flagellated fungi, which currently consists of: Blastocladiomycota, Chytridiomycota (chytrids), Neocallimastigomycota, Olpidiomycota, Monoblepharidiomycota and Rozella allomycis (Rozellomycota) 4 . A complex motility machinery allowed flagellated fungi to colonize substrates and grow in aquatic environments 5 , 6 . The function of appendices such as flagellum required the orchestration of the cytoskeletal components 7 . It is currently acknowledged that flagellated fungi harbor cytoskeletal components related to their animal counterparts 8 . Flagellar movement requires many cytoskeletal proteins, some of which have been characterized in metazoans. For instance, Prostak et al. in 2021 suggested that Batrachochytrium dendrobatidis builds dynamic actin networks similar to those of animal cells, revealing that actin regulators and myosin motors found in multiple chytrids are also present in animals 9 . Additionally, comparative analyses have shown that Chytridiomycota retains genes associated with centriole duplication, ciliary membrane docking, and basal body organization. This highlights the conservation of key centrosomal and basal body components in chytrids, many of which are shared with metazoans 10 . It is now widely recognized that many motility-related genes were lost in Dikarya but retained in a majority of the remaining Opisthokonta lineages 2 , 11 . The investigation of flagellar proteins has been primarily focused on animal systems. Consequently, fungal phylogenomic studies often characterize flagellar proteins by reference to well-studied animal homologs, including mouse, rat, fruit fly, and human. These organisms are commonly used as models for human infertility 10 , 12 . The limited research on fungal flagella highlights the need for comprehensive studies, built on well-characterized animal references. At the core of the flagellum are microtubules, which are tubular structures composed of subunits known as tubulins. Microtubules form the axoneme of flagellum, providing structural support and stability. The axoneme typically consists of a \"9 + 2\" arrangement, where nine outer microtubule doublets surround a central pair, which is characteristic of motile flagellum in eukaryotic organisms 13 . Motor proteins, particularly dyneins and kinesins, are crucial in the movement of flagellum. Dyneins are responsible for generating the bending motion of microtubules. There are several types of dyneins, but axonemal dyneins are the ones that drive the sliding motion between microtubule doublets, resulting in the beating pattern of flagellum. Kinesins, on the other hand, aid in intracellular transport and are involved in the positioning and assembly of flagellar structures 13 , 14 . Radial spokes are protein complexes that extend from the microtubule doublets to the central pair, serving as structural connectors and playing a role in regulating dynein activity. Nexin linkers cross-link the neighboring microtubule doublets, limiting their sliding motion and maintaining the overall alignment of flagellum 15 . In addition to the structural elements of flagellum, there are a plethora of proteins involved in motility processes, such as sensory signal transduction, ciliary membrane biogenesis, and the regulation of flagellar length and movement. The set of flagellar proteins is not clearly defined and many are poorly characterized in terms of function and structure, particularly those determined by Domains of Unknown Function (DUFs), which are defined and conserved regions of proteins without described functions 16 . Here we present an expanded and refined flagellar protein atlas across fungi. We denote differences between flagellated fungal lineages and conservation of some of the flagellar structures regardless of taxonomic positioning. We extend the list of known flagellar complexes in fungi and describe functional properties of so far uncharacterized proteins. Results Dataset construction and orthogroup identification To identify the set of flagellar proteins in fungi, we scanned 184 fungal proteomes from all fungal phyla for the presence of flagellar proteins from a reference dataset (Supplementary File S1). We created this reference set of 303 flagellar proteins by combining data from Complex Portal 17 (n = 479), CORUM 18 (n = 285), as well as from the works of Pazour et al. 2005 19 , (n = 87) and Long et al. 2025 10 , (n = 62). We collected the orthologs of these 303 proteins creating 303 orthogroups, which allowed us to gather additional reference proteins that would bridge the animal and fungal evolutionary gap. Mapping of the reference flagellar orthogroups on fungal proteomes showed no homologs for 72 orthogroups, suggesting their absence in fungi. From those 72 orthogroups, 36 are retained in Chlamydomonas reinhardtii , in addition to other opisthokonts, for example the large outer-dynein auxiliary chains ODA4/5/14 or kinesin-like FLA10 (Supplementary File S1). Those missing homologs in fungi could be explained by evolutionary distance between algae and fungi. Most of the orthogroups absent from fungi (42/72) have a disordered region equal or greater than 30% of the protein sequence. Many of these proteins have Metazoa specific domains, for instance SPATA7 with HSD3 domain (PF15244) or KIAA0753 with ‘protein moonraker’ (PF15718) domain. On the other hand, 10/72 orthogroups do not have a Pfam domain (Supplementary File S1). The limited taxonomic distribution of aforementioned proteins and their domains suggests that at least some of the orthogroups without fungal homologs could be animal specific (instead of being lost in fungi). In addition, the high level of disordered regions complicates the process of finding homologs. From the remaining 231 orthogroups which have fungal homologs, less than half (n = 103) occur also in non-flagellated fungi, while the majority of orthogroups (n = 128) occur only in flagellated fungal phyla (Blastocladiomycota, Chytridiomycota, Monoblepharidomycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota, Fig. 1 ). We define flagellated-specific orthogroups as occurring in flagellated fungal species within the aforementioned phyla, but with a possible one residual protein (or incomplete loss) within non-flagellated organisms. Proteins in flagellated-fungi specific orthogroups are enriched in coiled-coil segments, low-complexity regions, intrinsic disordered regions and trans-membrane helices, and they often carry repeat-type scaffolding domains such as WD40 (PF00400), LRR (CL0022) and Armadillo (PF00514, Table 1 ). These traits point to flexible components that assemble and dock the flagellum’s axoneme, motor arms and ion-channel complexes. By contrast, orthogroups not specific to flagellated fungi are dominated by Pkinase (PF00069), Tubulin (PF00091) and other housekeeping protein domains. They show lower levels of coiled-coils, disordered and transmembrane regions (Table 1 , Supplementary File S1), reflecting broadly conserved cytoskeletal, signalling and vesicle-trafficking roles that every fungus needs, whether or not it bears a flagellum. In short, the flagellated set is specialised for building and regulating the flagellum, whereas the non-specific set encodes the universal cellular machinery that is shared across the fungal kingdom. Table 1 Feature comparison between 128 flagellated-fungi specific orthogroups and 103 orthogroups shared between fungi with and without flagellum. Orthogroups Specific to flagellated fungi Non-specific to flagellated fungi Number of fungi 21 184 Number of orthogroups 128 103 Total proteins 3 275 11 546 Orthogroups in which every protein contains ≥ 1 coiled-coil 57 / 128 39 / 103 Orthogroups in which every protein contains ≥ 1 low-complexity region 115 / 128 67 / 103 Orthogroups in which every protein contains a signal peptide 27 / 128 35 / 103 Average fraction of intrinsic disorder per protein 22% 20% Proteins with ≥ 1 trans-membrane helix 299 / 3 275 58 / 11 546 Most common protein domains 512 / 3 275 proteins carry WD40, LRR or Armadillo repeats 1 064 / 11 546 proteins carry Pkinase domain; 574 / 11 546 carry Tubulin domain The number of flagellum-specific orthogroups retained in individual fungal isolates varies, however all three top scoring organisms in terms of number of orthogroups retained belong to Neocallimastigomycota. Out of 128 orthogroups present only in flagellated organisms, Neocallimastix lanti (BUSCO score 85.8) retained 109, while Piromyces finnis (BUSCO score 84.7) and Neocallimastix californiae (BUSCO score 84.7) have retained 107. On the other extreme, Olpidium bornovanus retained 51 of 128 orthogroups. However it should be noted that the BUSCO score for this species genome is low (27) and the relatively low number of flagellum-specific orthogroups could also be explained by the species parasitic genome reduction. The next species with the lowest number of flagellated fungi-specific orthogroups is C aulochytrium protostelioides (BUSCO score 67.7) with 65 of 128 orthogroups retained (Fig. 2 , Supplementary File S1, Supplementary Figure S1). Taken together, the patterns of protein retention point to the existence of a subset of core proteins which are maintained across flagellated fungi. Identifying these conserved components can provide insights into the minimal requirements for constructing and operating a functional fungal flagellum. Minimal flagellum proteome From the 231 flagellar orthogroups identified in fungi, 97 occur in at least 18 flagellated fungal species ( > = 85% of taxa), which has been taken as a minimal criterion to consider a given orthogroup as constituting a potential minimal flagellum (Supplementary File S1). A minimal flagellum is the smallest, self-sufficient set of flagellar modules that still supports axoneme assembly and rhythmic beating. In our dataset, this minimalist toolkit retains (i) the structural core of the 9 + 2 axoneme (tubulins and central-pair components), (ii) force-generating and beat-shaping elements (outer/inner dynein arms with the nexin–dynein regulatory complex and radial spokes), (iii) enough intraflagellar transport (IFT) machinery to build and service the organelle (core IFT-B with essential IFT-A and the kinesin-2 motor), and (iv) a pared-down basal foundation (centriole duplication factors, a reduced transition-zone subset, and basic exocyst/TRAPP trafficking to the base, Table 2 ). What is missing, relative to the non-minimal set, are whole accessory systems that expand cargo control and signalling: the BBSome cargo adaptor, the CatSper Ca²⁺channel cassette and its partners, most distal-appendage and pericentriolar scaffolds, multiple additional transition-zone subunits, a suite of ciliogenesis/motility kinases, extra IFT and kinesin components (Supplementary File S1). There are 53 flagellated-fungi specific groups in the minimal flagellar set, out of 128 total flagellated-fungi specific orthogroups. Table 2 Comparison of flagellated-fungi specific orthogroups occurring in minimal flagellum and orthogroups in non-minimal set. Minimal flagellum (97/231) Non-minimal flagellum (134/231) Number of orthogroups 53 75 Total fungal proteins 1 797 1 478 Orthogroups in which every protein contains ≥ 1 coiled-coil 29 / 53 28 / 75 Orthogroups in which every protein contains ≥ 1 low-complexity region (LCR) 51 / 53 64 / 75 Proteins with ≥ 1 trans-membrane helix 32 / 1 797 267 / 1 478 Domain enrichment in proteins 399 / 1 797 proteins with WD40, LRR or Armadillo repeats 104 / 1 478 Ion_trans domain; 77 / 1 478 Pkinase The 53 minimal-flagellum orthogroups specific to flagellated fungi are strongly biased toward coiled-coil regions, low-complexity regions and WD40 (PF00400), LRR (CL0022) and Armadillo (PF00514) scaffolding repeats, while being depleted in transmembrane helices. This points to flexible adapters that link and stabilize the axoneme and its motors without relying on membrane trafficking (Table 2 , Fig. 3 , Supplementary File S1). In addition, we observe a significant increase in the number of copies of DAAM1/2 homologs in Neocallimastigomycota. In our data, we can observe that Neocallimastigomycota have slightly higher copy numbers of homologs than other phyla. This is especially visible for DAAM1/2, with the number of copies, ranging from six up to ten copies per organism (Fig. 3 , Supplementary Figure S2). Dishevelled-associated activator of morphogenesis 1 and 2 (DAAM1/2) bind regulatory GTPases and contain tetratricopeptide(TPR)-repeats. DAAM1/2 promotes actin filament nucleation and elongation, contributes to ciliogenesis, and is required for myocardial maturation and sarcomere assembly. It may also regulate RHOA activation, thereby influencing mitotic spindle orientation and chromosome segregation 21 . The 75 non-minimal flagellated-fungi specific orthogroups are rich in transmembrane regions and favour Ion_trans (PF00520) and Pkinase (PF00069) domains (Table 2 ). These patterns are compatible with functions linked to membrane transport and signal transduction. In addition, CatSper channel complex and BBSome tethering complex are entirely present in non-minimal flagellum orthogroups (Supplementary File S1). Domains of Unknown Functions We annotated two DUFs that are occurring in flagellated fungi - DUF4470 (PF14737) and DUF4471 (PF14740). Both DUFs occur predominantly in Dynein assembly factor 3 axonemal (DNAAF3), a protein involved in preassembly of dyneins into complexes 22 . DUF4470 (PF14737) and DUF4471 (PF14740), both present in protein A0A1Y1V9Y8 (AlphaFold2: AF-A0A1Y1V9Y8-F1-v6 23 ) contain the Rossmann-like fold, confirmed by mapping to Chemotaxis protein methyltransferase (cheR, P07801, pdb: 1AF7 24 ) of Salmonella typhimurium which has a defined Rossmann fold 24 (Fig. 4 , Supplementary Figure S3). Phylum based results While a conserved core of flagellar proteins can be defined across fungi, closer inspection reveals clear phylum-level differences in the retention and composition of individual complexes (Fig. 5 ). One such example can be the BBSome (octameric coat complex acting as membrane-cargo adaptor 25 ) is universally retained in Neocallimastigomycota (P. finnis, Piromyces sp. E2, A. robustus, N. lanti and N. californiae ), encoding all eight components. In contrast, Chytridiomycota exhibit a fragmented BBSome, with only a subset of subunits present, most of which observed in C. confervae which retains five subunits (BBS1, BBS4, BBS5, BBS7, TTC8), R. globosum and B. helicus retain four, C. protostelioides - one, while the remaining chytrids lack the complex entirely. No BBSome proteins are found in the other fungal phyla proteomes used in this study. Aside from BBsome being a major differentiator between Neocallimastigomycota and the remaining fungal phyla, we can observe phyla-specific differences within orthogroups. Although there are no more phyla-specific complexes other than BBSome that are conserved entirely in one phylum, we observe 39 orthogroups with differing phyla protein distributions. One of the notable observations, is that Chytridiomycota entirely miss three out of ten CatSper channel complex components (CATSPERD, CATSPERB, CATSPERG2), which are auxiliary elements of the complex 26 , and are present in at least one representatives of the other major phyla (Blastocladiomycota, Neocallimastigomycota) of flagellated fungi (Fig. 5 ). Neocallimastigomycota lacks 24 out of 39 fungal orthogroups, of which 11 are associated with centrioles. In addition, there are six groups occurring only in Chytridiomycota and three that occur only in Blastocladiomycota. Discussion We performed a comprehensive search for flagellar proteins across diverse fungal representatives which enabled us to explore the evolutionary trajectories of flagellum building blocks. We identified a clear split between flagellated-fungi specific orthogroups (n = 128) and those shared with non-flagellates (n = 103) which mirrors the pruning of the ancestral opisthokont ciliary toolkit. Accordingly, gene families tied to axonemal assembly, IFT scaffolding, and membrane docking (enrichment in coiled-coils, low-complexity/disorder, and repeat scaffolds such as WD40/LRR/ARM) are preferentially retained in flagellated lineages, whereas broadly useful modules (kinases, tubulins) persist across fungi regardless of flagellation. This pattern is consistent with the “delayed loss of ancestral gene families” model inferred from comparative genomics across some of the non-Dikarya 11 , 27 , where traits are shed piecemental and lineage-specifically rather than in a single step 11 . Conversely, enrichment in kinases/tubulins shared between flagellated and non-flagellated fungi is consistent with the description of flagella evolution in literature 11 . Moreover, it opens up the possibility of moonlighting, non-flagellar roles for many signalling and cytoskeletal proteins 19 . As shown in this study, the number of orthogroups retained in individual isolates varies, however all three top scoring organisms in terms of number of orthogroups retained belong to Neocallimastigomycota. This could result from many reasons, one of the most probable would be multiflagellated zoospores of some of those fungi. While generally, flagellated fungi possess a single posterior flagellum, Neocallimastix spp. produces polyflagellate zoospores with a brief motile window before rapid encystment in the viscous rumen, which could lead to a more conserved and robust flagellar toolkit needed for their habitation of the demanding, dense rumen environment 28 . Upon a more detailed inspection, Neocallimastigomycota harbour the broadest and most intact flagellar toolkit, most notably a universally retained, complete BBSome complex functioning as membrane-cargo adaptor 29 , despite concurrent reductions in centriole-associated modules. While currently, there are no experimental studies explaining the specific ecological role of BBSome in fungi (Neocallimastigomycota specifically), Rossier et al. (2023) show retention of two BBSome elements in Piromyces spp . and loss in A. macrogynus, B. dendrobatidi s and R. allomycis , which aligns with our results 29 . Concurrently, despite broad axonemal/IFT retention, Neocallimastigomycota in our dataset show a marked reduction of centriole-associated modules, particularly pericentriolar material and satellites. These modules are known to fine-tune basal-body maturation, protein targeting and ciliary gating rather than build the axoneme itself 30 . Moreover, we observe a high copy number of DAAM1/2 orthologs in Neocallimastigomycota. This could be explained by unusually large, AT-rich, repeat-laden Neocallimastigomycota genomes with extensive gene duplication and horizontal gene transfer. While best documented for CAZymes, the same duplication-prone architecture can be observed for other families, including flagellar modules 31 . DAAM1/2 and their paralogs represent TPR-like repeat proteins composed of multiple units with substantial divergence among their repeat modules. Their specific role in Neocallimastigomycota remains unclear and is not trivial to decipher. In Homo sapiens , DAAM1/2 functions as a central component of diverse protein complexes, not exclusively linked to filament formation. One possible scenario is that Neocallimastigomycota DAAM1/2 paralogs mediate a particularly refined regulation of actin filament formation. Alternatively, some paralogs may have undergone neofunctionalization, acquiring distinct regulatory functions in other cellular compartments. One of the goals of our study was to determine which proteins of the flagellum contribute to its functional core. In consequence, we defined a minimal set of flagellum proteins conserved across flagellated taxa. This enabled us to observe that the minimal flagellum relies more on long cytosolic scaffolds than on membrane complexes: vesicles can still dock and deliver building blocks to the base, IFT trains can move them along the axoneme, and dynein arms can generate beat, but membrane trafficking, ion-channel mediated signalling and regulatory plasticity are limited. In practical terms, this minimal organelle is motile and maintainable, yet optimized for core locomotion with simplified gating and reduced sensory/trafficking capacity. The minimal flagellum can function because of the components it retains – axonemal tubulins, dynein motors, radial-spoke/nexin regulators and the core IFT-B/kinesin-2 train – these are the ones that physically build and bend the “9 + 2” scaffold. In contrast, most proteins lost act upstream or peripherally: the BBSome and several auxiliary IFT or kinesin modules that mainly recycle signalling receptors and membrane proteins. For example, both Trypanosoma brucei and Chlamydomonas mutants lacking a complete BBSome retain normal beat frequency and waveform 32 , 33 . Similarly, mammals lacking CatSper channel subunits retain progressive, low-amplitude beating but cannot hyperactivate, showing that the Ca²⁺-gated CatSper channel complex is a performance enhancer rather than an architectural necessity 34 . Likewise, comparative work on IFT shows that the 11-subunit core IFT-B platform is obligatory for axoneme assembly, whereas many accessory IFT-A subunits are repeatedly lost in organisms whose flagellum (or cilium) serve mainly locomotion rather than signalling 35 . The minimal flagellum set of proteins can be a shortlist for experimental studies on flagellar function and the role of individual proteins in locomotion. Moreover, the conservation of the core flagellar elements between flagellated fungi and animals puts forward the possibility of experimentation in simple models like saprotrophic Chytidiomycota. Conclusions We provide a curated, cross-phyla atlas of fungal flagellar proteins that clearly separates a flagellate-specific, scaffold-rich layer from broadly conserved housekeeping modules. From this, we propose a 97-orthogroup “minimal flagellum” capturing the axonemal core, dynein regulation, and pared IFT, while a non-minimal layer adds membrane channels, cargo adaptors, and signalling capacity. Comparisons across fungal lineages indicate substantial variation in flagellar composition, with Neocallimastigomycota retaining the broadest toolkit and showing select expansions amid centriole reductions, pointing to multiple evolutionary trajectories toward motility. We also annotate the fold for two DUFs occurring in a protein functioning within the flagellum. Methods Using several sources, we built a comprehensive collection of flagellar proteins by integrating datasets from publicly available databases and literature sources. After removing redundant entries, we performed homology searches in a defined set of model organisms to find more reference proteins. We then combined those proteins into separate protein homology groups and screened them against fungal proteomes. Obtained fungal homologs were then manually cleaned to remove potential false positive hits and narrow-down the results. Additionally, we predict the fold for two DUFs potentially related to flagellum, using distant homology methods. Our primary reference dataset comprised proteins from the Complex Portal 17 (n = 479), retrieved using the keyword “cilium,” and from CORUM 18 (n = 285), selected under the predefined categories “cilium assembly” and “cilium movement”. In parallel, we incorporated 87 proteins linked to flagellum in Chlamydomonas reinhardtii , as reported by Pazour et al. (2005) 19 , and 62 proteins from the list of centriole associated proteins defined by Long et al. (2025) 10 . Redundant entries across these datasets were manually removed and unified, creating a set of 303 homolog groups (Supplementary File S1). The 303 orthogroups were mapped on a set of outgroup taxa using blastp (version 2.13.0+; e-value threshold of 1e-3) 36 . The outgroup set included model animals Mus musculus (GCA_000001635.9), Homo sapiens (GCA_000001405.29), Drosophila melanogaster (GCA_000001215.4), and Caenorhabditis elegans (GCA_000002985.3), along with basal Opisthokonta representatives: Monosiga brevicollis (GCF_000002865.3), Capsaspora owczarzaki (GCF_000151315.2), Salpingoeca rosetta (GCF_000188695.1), Fonticula alba (GCF_000388065.1), and Sphaeroforma arctica (GCF_001186125.1), as well as C. reinhardtii (GCA_000002595.3). Homology searches in fungi were performed using 184 fungal proteomes (Supplementary File S1). By proteomes, we define the complete sets of all proteins coded by genes in 184 genome assemblies downloaded from NCBI in May 2022 37 . These comprise all then-available non-Dikarya proteomes and proteomes of representatives of Dikarya classes. To ensure that for each reference protein, sets of reliable fungal homologs were obtained, we checked the conservation of Pfam (database v. 36) protein domains by scanning them with pfamscan.pl v1.6 with its default settings 38 . Each homology group was noted as present or absent from fungi. Additionally, we used tblastn v. 2.13.0 + 36 with an e-value threshold of 1e-5 (against 184 fungal genomes) to eliminate potential false negative results resulting from possible incomplete protein sets in proteomes. For large homology groups resulting from initial blastp scan, we used CLANS software (version 2.0) 39 to narrow-down the results to potential true homologs for each reference query. In addition, we combined homolog groups which contained the same Pfam families. We further used CLANS software to find unique, non-redundant hits for each individual reference query. This approach allowed us to find unique homologs for each query without redundancy to other highly similar queries (e.g. tubulins or kinases). Protein group categorization has been made based on Long et al. (2025) 10 and Merényi et al. (2023) 11 categories as well as on CORUM and ComplexPortal annotations, in addition to UniProt 40 annotations. Fungal homolog groups were characterized using diverse sequence annotation tools and structural comparisons. For the identification of transmembrane helices, TMHMM (version 2.0) 41 was utilized. Sequence disorder predictions were carried out using IUPred (version 2.0) 42 . SEG (version 1.0) 43 was employed for the prediction of low-complexity regions. All of the above-mentioned software was used with the default settings. Multiple sequence alignments were constructed via MAFFT (version 7.475) 44 , employing parameters --localpair and --maxiterate 100. Distant homology searches for DUFs were conducted using MAFFT-obtained MSAs via HHpred on the MPI Toolkit website (version 3.3.0) 45 with target databases of: TIGFAMs 46 , ECOD (version 2022) 47 and RCSB (updated 2022) 48 . Three-dimensional visualization of protein structures was achieved using PyMOL (version 2.5) 49 . The phylogenetic tree was constructed with IQ-TREE (v1.6.9) 50 using maximum likelihood method with automated model selection. The trees were visualized and represented using the iTOL online tool 51 . Figures were created using Python and Matplotlib 52 . Declarations Acknowledgements We are grateful for insightful comments and suggestions by Drishtee Barua. Authors' contributions A.K. and A.M. designed the study and performed analyses. A.K. and A.M. drafted the manuscript. A.K. prepared the figures, A.M. conceptualized the project. Data availability statement All metadata processed in this study are deposited in zenodo: 10.5281/zenodo.17376122 All protein identifiers and genomic assemblies are listed in Supplementary file S1. Funding This work was supported by National Science Centre grant (#2021/41/B/NZ2/02426 to A.M.) Competing interests The author(s) declare that they have no competing interests. References Hyde, K. D. The numbers of fungi. Fungal Divers. 114 , 1–1 (2022). Heitman, J. et al. The Fungal Kingdom . (John Wiley & Sons, 2020). Bridge, P., Smith, D. & Stackebrandt, E. Trends in the Systematics of Bacteria and Fungi . (CABI Publishing, Wallingford, England, 2020). Wijayawardene, N. N. et al. Classes and phyla of the kingdom Fungi. Fungal Divers. 128 , 1–165 (2024). Voigt, K. et al. Early-diverging fungal phyla: taxonomy, species concept, ecology, distribution, anthropogenic impact, and novel phylogenetic proposals. Fungal Divers. 109 , 59–98 (2021). Reich, M. et al. 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IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37 , 1530–1534 (2020). Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49 , W293–W296 (2021). Python Programming Language . https://www.python.org/ (2023). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 25 Nov, 2025 Reviews received at journal 22 Nov, 2025 Reviews received at journal 19 Nov, 2025 Reviews received at journal 10 Nov, 2025 Reviewers agreed at journal 10 Nov, 2025 Reviewers agreed at journal 07 Nov, 2025 Reviewers agreed at journal 05 Nov, 2025 Reviewers invited by journal 03 Nov, 2025 Editor invited by journal 30 Oct, 2025 Editor assigned by journal 21 Oct, 2025 Submission checks completed at journal 21 Oct, 2025 First submitted to journal 17 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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06:33:46\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":53284,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDistribution of 303 flagellar orthogroups across 184 fungi. Orange - orthogroups without fungal homologs, blue - orthogroups with fungal homologs, red - orthogroups occurring both in flagellated and non-flagellated fungi, green - orthogroups specific to flagellated fungi.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7887758/v1/09a20f6be323b9dee4e5bcd5.png\"},{\"id\":95789950,\"identity\":\"1db80923-c8bc-419b-b497-2abe15c29d85\",\"added_by\":\"auto\",\"created_at\":\"2025-11-13 06:33:46\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":189508,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePer-species retention of flagellated-fungi–specific orthogroups, that include at least one protein from each flagellated fungal species (orange), with BUSCO score for each specimen (blue). Taxonomic nomenclature based on Wijayawardene et al. (2024) \\u003ca href=\\\"https://paperpile.com/c/fn8sWH/KYaz\\\"\\u003e\\u003csup\\u003e4\\u003c/sup\\u003e\\u003c/a\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7887758/v1/7212d99f0e9088178511b85f.png\"},{\"id\":95802410,\"identity\":\"795c97e0-f651-4fb4-8412-fa1850ee13d0\",\"added_by\":\"auto\",\"created_at\":\"2025-11-13 08:27:34\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":401958,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTaxonomic distribution of 53 flagellated-fungi specific orthogroups\\u0026nbsp; within the minimal flagellum set, categorized into complexes based on the works of Merényi et al. (2023) \\u003ca href=\\\"https://paperpile.com/c/fn8sWH/dqARD\\\"\\u003e\\u003csup\\u003e11\\u003c/sup\\u003e\\u003c/a\\u003e and Long et al. (2025)\\u003ca href=\\\"https://paperpile.com/c/fn8sWH/zAFM\\\"\\u003e\\u003csup\\u003e10\\u003c/sup\\u003e\\u003c/a\\u003e as well as CORUM and ComplexPortal; Centriole, PCMS - Pericentriolar Material and Satellites. Taxonomic order based on Wijayawardene et al. (2024) \\u003ca href=\\\"https://paperpile.com/c/fn8sWH/KYaz\\\"\\u003e\\u003csup\\u003e4\\u003c/sup\\u003e\\u003c/a\\u003e and Liu et al. (2024)\\u003ca href=\\\"https://paperpile.com/c/fn8sWH/dxxL\\\"\\u003e\\u003csup\\u003e20\\u003c/sup\\u003e\\u003c/a\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7887758/v1/c44ca382d62d17d13c479042.png\"},{\"id\":95789945,\"identity\":\"06829a29-e62c-4a66-b41d-ec1e998d54dc\",\"added_by\":\"auto\",\"created_at\":\"2025-11-13 06:33:46\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":41326,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSecondary-structure topology of the Rossmann fold in DNAAF3 (AF-A0A1Y1V9Y8-F1-v6). DUF4470 (PF14737) - blue, DUF4471 (PF14740) - orange. Dotted lines indicate insertions to fold. Beta-sheets are indicated as arrows, alpha-helices as cylinders.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7887758/v1/0ee1ee0002e5e4c7b78e9db7.png\"},{\"id\":95789951,\"identity\":\"5082b508-9541-4e51-9933-ebeda0c071cf\",\"added_by\":\"auto\",\"created_at\":\"2025-11-13 06:33:46\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":64215,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDifferences in the conservation of flagellar modules between major flagellated fungal phyla (Blastocladiomycota, Neocallimastigomycota, Chytridiomycota). Presence is denoted in blue, absence in orange.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7887758/v1/a2d0a70d0ad946a8eaccdcb4.png\"},{\"id\":95805473,\"identity\":\"bd348aa9-8bb9-4710-ac84-0eeee1432dfa\",\"added_by\":\"auto\",\"created_at\":\"2025-11-13 08:41:25\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1239779,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7887758/v1/66d29f2a-569f-461e-b8c5-57569bef96b6.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"A conserved minimal core and modular extensions make the fungal flagellum\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eFungi are one of the most diverse and complex kingdoms on Earth, with an estimated number of species reaching up to thirteen million \\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. These organisms occupy a wide range of ecological niches, including soil, water, air and form a plethora of relationships with other living forms \\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. Within the multiplex fungal tree of life, there are two main fungal supergroups - Dikarya and non-Dikarya, which encompass many fungal phyla - from symbiotic Glomeromycota to obligatory parasitic Microsporidia \\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e. Some non-Dikarya fungi have a single or multiple posterior flagella on their zoospores. They are flagellated fungi, which currently consists of: Blastocladiomycota, Chytridiomycota (chytrids), Neocallimastigomycota, Olpidiomycota, Monoblepharidiomycota \\u003cem\\u003eand Rozella allomycis\\u003c/em\\u003e (Rozellomycota) \\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. A complex motility machinery allowed flagellated fungi to colonize substrates and grow in aquatic environments \\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. The function of appendices such as flagellum required the orchestration of the cytoskeletal components \\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e. It is currently acknowledged that flagellated fungi harbor cytoskeletal components related to their animal counterparts \\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003eFlagellar movement requires many cytoskeletal proteins, some of which have been characterized in metazoans. For instance, Prostak et al. in 2021 suggested that \\u003cem\\u003eBatrachochytrium dendrobatidis\\u003c/em\\u003e builds dynamic actin networks similar to those of animal cells, revealing that actin regulators and myosin motors found in multiple chytrids are also present in animals \\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e. Additionally, comparative analyses have shown that Chytridiomycota retains genes associated with centriole duplication, ciliary membrane docking, and basal body organization. This highlights the conservation of key centrosomal and basal body components in chytrids, many of which are shared with metazoans \\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003eIt is now widely recognized that many motility-related genes were lost in Dikarya but retained in a majority of the remaining Opisthokonta lineages \\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. The investigation of flagellar proteins has been primarily focused on animal systems. Consequently, fungal phylogenomic studies often characterize flagellar proteins by reference to well-studied animal homologs, including mouse, rat, fruit fly, and human. These organisms are commonly used as models for human infertility \\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e. The limited research on fungal flagella highlights the need for comprehensive studies, built on well-characterized animal references.\\u003c/p\\u003e\\u003cp\\u003eAt the core of the flagellum are microtubules, which are tubular structures composed of subunits known as tubulins. Microtubules form the axoneme of flagellum, providing structural support and stability. The axoneme typically consists of a \\\"9\\u0026thinsp;+\\u0026thinsp;2\\\" arrangement, where nine outer microtubule doublets surround a central pair, which is characteristic of motile flagellum in eukaryotic organisms \\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e. Motor proteins, particularly dyneins and kinesins, are crucial in the movement of flagellum. Dyneins are responsible for generating the bending motion of microtubules. There are several types of dyneins, but axonemal dyneins are the ones that drive the sliding motion between microtubule doublets, resulting in the beating pattern of flagellum. Kinesins, on the other hand, aid in intracellular transport and are involved in the positioning and assembly of flagellar structures \\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e. Radial spokes are protein complexes that extend from the microtubule doublets to the central pair, serving as structural connectors and playing a role in regulating dynein activity. Nexin linkers cross-link the neighboring microtubule doublets, limiting their sliding motion and maintaining the overall alignment of flagellum \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e. In addition to the structural elements of flagellum, there are a plethora of proteins involved in motility processes, such as sensory signal transduction, ciliary membrane biogenesis, and the regulation of flagellar length and movement. The set of flagellar proteins is not clearly defined and many are poorly characterized in terms of function and structure, particularly those determined by Domains of Unknown Function (DUFs), which are defined and conserved regions of proteins without described functions \\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003eHere we present an expanded and refined flagellar protein atlas across fungi. We denote differences between flagellated fungal lineages and conservation of some of the flagellar structures regardless of taxonomic positioning. We extend the list of known flagellar complexes in fungi and describe functional properties of so far uncharacterized proteins.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003eDataset construction and orthogroup identification\\u003c/p\\u003e\\u003cp\\u003eTo identify the set of flagellar proteins in fungi, we scanned 184 fungal proteomes from all fungal phyla for the presence of flagellar proteins from a reference dataset (Supplementary File S1). We created this reference set of 303 flagellar proteins by combining data from Complex Portal \\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e (n\\u0026thinsp;=\\u0026thinsp;479), CORUM \\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e (n\\u0026thinsp;=\\u0026thinsp;285), as well as from the works of Pazour et al. 2005\\u003csup\\u003e19\\u003c/sup\\u003e, (n\\u0026thinsp;=\\u0026thinsp;87) and Long et al. 2025 \\u003csup\\u003e10\\u003c/sup\\u003e, (n\\u0026thinsp;=\\u0026thinsp;62). We collected the orthologs of these 303 proteins creating 303 orthogroups, which allowed us to gather additional reference proteins that would bridge the animal and fungal evolutionary gap. Mapping of the reference flagellar orthogroups on fungal proteomes showed no homologs for 72 orthogroups, suggesting their absence in fungi. From those 72 orthogroups, 36 are retained in \\u003cem\\u003eChlamydomonas reinhardtii\\u003c/em\\u003e, in addition to other opisthokonts, for example the large outer-dynein auxiliary chains ODA4/5/14 or kinesin-like FLA10 (Supplementary File S1). Those missing homologs in fungi could be explained by evolutionary distance between algae and fungi. Most of the orthogroups absent from fungi (42/72) have a disordered region equal or greater than 30% of the protein sequence. Many of these proteins have Metazoa specific domains, for instance SPATA7 with HSD3 domain (PF15244) or KIAA0753 with \\u0026lsquo;protein moonraker\\u0026rsquo; (PF15718) domain. On the other hand, 10/72 orthogroups do not have a Pfam domain (Supplementary File S1). The limited taxonomic distribution of aforementioned proteins and their domains suggests that at least some of the orthogroups without fungal homologs could be animal specific (instead of being lost in fungi). In addition, the high level of disordered regions complicates the process of finding homologs.\\u003c/p\\u003e\\u003cp\\u003eFrom the remaining 231 orthogroups which have fungal homologs, less than half (n\\u0026thinsp;=\\u0026thinsp;103) occur also in non-flagellated fungi, while the majority of orthogroups (n\\u0026thinsp;=\\u0026thinsp;128) occur only in flagellated fungal phyla (Blastocladiomycota, Chytridiomycota, Monoblepharidomycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). We define flagellated-specific orthogroups as occurring in flagellated fungal species within the aforementioned phyla, but with a possible one residual protein (or incomplete loss) within non-flagellated organisms.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eProteins in flagellated-fungi specific orthogroups are enriched in coiled-coil segments, low-complexity regions, intrinsic disordered regions and trans-membrane helices, and they often carry repeat-type scaffolding domains such as WD40 (PF00400), LRR (CL0022) and Armadillo (PF00514, Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). These traits point to flexible components that assemble and dock the flagellum\\u0026rsquo;s axoneme, motor arms and ion-channel complexes. By contrast, orthogroups not specific to flagellated fungi are dominated by Pkinase (PF00069), Tubulin (PF00091) and other housekeeping protein domains. They show lower levels of coiled-coils, disordered and transmembrane regions (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, Supplementary File S1), reflecting broadly conserved cytoskeletal, signalling and vesicle-trafficking roles that every fungus needs, whether or not it bears a flagellum. In short, the flagellated set is specialised for building and regulating the flagellum, whereas the non-specific set encodes the universal cellular machinery that is shared across the fungal kingdom.\\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\\u003eFeature comparison between 128 flagellated-fungi specific orthogroups and 103 orthogroups shared between fungi with and without flagellum.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"3\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eOrthogroups\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eSpecific to flagellated fungi\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eNon-specific to flagellated fungi\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eNumber of fungi\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e21\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e184\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eNumber of orthogroups\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e128\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e103\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eTotal proteins\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e3 275\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e11 546\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eOrthogroups in which every protein contains\\u0026thinsp;\\u0026ge;\\u0026thinsp;1 coiled-coil\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e57 / 128\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e39 / 103\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eOrthogroups in which every protein contains\\u0026thinsp;\\u0026ge;\\u0026thinsp;1 low-complexity region\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e115 / 128\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e67 / 103\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eOrthogroups in which every protein contains a signal peptide\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e27 / 128\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e35 / 103\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAverage fraction of intrinsic disorder per protein\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e22%\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e20%\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eProteins with \\u0026ge;\\u0026thinsp;1 trans-membrane helix\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e299 / 3 275\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e58 / 11 546\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eMost common protein domains\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e512 / 3 275 proteins carry WD40, LRR or Armadillo repeats\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1 064 / 11 546 proteins carry Pkinase domain; 574 / 11 546 carry Tubulin domain\\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\\u003eThe number of flagellum-specific orthogroups retained in individual fungal isolates varies, however all three top scoring organisms in terms of number of orthogroups retained belong to Neocallimastigomycota. Out of 128 orthogroups present only in flagellated organisms, \\u003cem\\u003eNeocallimastix lanti\\u003c/em\\u003e (BUSCO score 85.8) retained 109, while \\u003cem\\u003ePiromyces finnis\\u003c/em\\u003e (BUSCO score 84.7) and \\u003cem\\u003eNeocallimastix californiae\\u003c/em\\u003e (BUSCO score 84.7) have retained 107. On the other extreme, \\u003cem\\u003eOlpidium bornovanus\\u003c/em\\u003e retained 51 of 128 orthogroups. However it should be noted that the BUSCO score for this species genome is low (27) and the relatively low number of flagellum-specific orthogroups could also be explained by the species parasitic genome reduction. The next species with the lowest number of flagellated fungi-specific orthogroups is C\\u003cem\\u003eaulochytrium protostelioides\\u003c/em\\u003e (BUSCO score 67.7) with 65 of 128 orthogroups retained (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, Supplementary File S1, Supplementary Figure S1).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eTaken together, the patterns of protein retention point to the existence of a subset of core proteins which are maintained across flagellated fungi. Identifying these conserved components can provide insights into the minimal requirements for constructing and operating a functional fungal flagellum.\\u003c/p\\u003e\\u003cp\\u003eMinimal flagellum proteome\\u003c/p\\u003e\\u003cp\\u003eFrom the 231 flagellar orthogroups identified in fungi, 97 occur in at least 18 flagellated fungal species (\\u0026thinsp;\\u0026gt;\\u0026thinsp;=\\u0026thinsp;85% of taxa), which has been taken as a minimal criterion to consider a given orthogroup as constituting a potential minimal flagellum (Supplementary File S1). A minimal flagellum is the smallest, self-sufficient set of flagellar modules that still supports axoneme assembly and rhythmic beating. In our dataset, this minimalist toolkit retains (i) the structural core of the 9\\u0026thinsp;+\\u0026thinsp;2 axoneme (tubulins and central-pair components), (ii) force-generating and beat-shaping elements (outer/inner dynein arms with the nexin\\u0026ndash;dynein regulatory complex and radial spokes), (iii) enough intraflagellar transport (IFT) machinery to build and service the organelle (core IFT-B with essential IFT-A and the kinesin-2 motor), and (iv) a pared-down basal foundation (centriole duplication factors, a reduced transition-zone subset, and basic exocyst/TRAPP trafficking to the base, Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). What is missing, relative to the non-minimal set, are whole accessory systems that expand cargo control and signalling: the BBSome cargo adaptor, the CatSper Ca\\u0026sup2;⁺channel cassette and its partners, most distal-appendage and pericentriolar scaffolds, multiple additional transition-zone subunits, a suite of ciliogenesis/motility kinases, extra IFT and kinesin components (Supplementary File S1).\\u003c/p\\u003e\\u003cp\\u003eThere are 53 flagellated-fungi specific groups in the minimal flagellar set, out of 128 total flagellated-fungi specific orthogroups.\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eComparison of flagellated-fungi specific orthogroups occurring in minimal flagellum and orthogroups in non-minimal set.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"3\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\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\\u003eMinimal flagellum (97/231)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eNon-minimal flagellum (134/231)\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eNumber of orthogroups\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e53\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e75\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eTotal fungal proteins\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1 797\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1 478\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eOrthogroups in which every protein contains\\u0026thinsp;\\u0026ge;\\u0026thinsp;1 coiled-coil\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e29 / 53\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e28 / 75\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eOrthogroups in which every protein contains\\u0026thinsp;\\u0026ge;\\u0026thinsp;1 low-complexity region (LCR)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e51 / 53\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e64 / 75\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eProteins with \\u0026ge;\\u0026thinsp;1 trans-membrane helix\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e32 / 1 797\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e267 / 1 478\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eDomain enrichment in proteins\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e399 / 1 797 proteins with WD40, LRR or Armadillo repeats\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e104 / 1 478 Ion_trans domain; 77 / 1 478 Pkinase\\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\\u003eThe 53 minimal-flagellum orthogroups specific to flagellated fungi are strongly biased toward coiled-coil regions, low-complexity regions and WD40 (PF00400), LRR (CL0022) and Armadillo (PF00514) scaffolding repeats, while being depleted in transmembrane helices. This points to flexible adapters that link and stabilize the axoneme and its motors without relying on membrane trafficking (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, Supplementary File S1).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eIn addition, we observe a significant increase in the number of copies of DAAM1/2 homologs in Neocallimastigomycota. In our data, we can observe that Neocallimastigomycota have slightly higher copy numbers of homologs than other phyla. This is especially visible for DAAM1/2, with the number of copies, ranging from six up to ten copies per organism (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, Supplementary Figure S2). Dishevelled-associated activator of morphogenesis 1 and 2 (DAAM1/2) bind regulatory GTPases and contain tetratricopeptide(TPR)-repeats. DAAM1/2 promotes actin filament nucleation and elongation, contributes to ciliogenesis, and is required for myocardial maturation and sarcomere assembly. It may also regulate RHOA activation, thereby influencing mitotic spindle orientation and chromosome segregation \\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003eThe 75 non-minimal flagellated-fungi specific orthogroups are rich in transmembrane regions and favour Ion_trans (PF00520) and Pkinase (PF00069) domains (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). These patterns are compatible with functions linked to membrane transport and signal transduction. In addition, CatSper channel complex and BBSome tethering complex are entirely present in non-minimal flagellum orthogroups (Supplementary File S1).\\u003c/p\\u003e\\u003cp\\u003eDomains of Unknown Functions\\u003c/p\\u003e\\u003cp\\u003eWe annotated two DUFs that are occurring in flagellated fungi - DUF4470 (PF14737) and DUF4471 (PF14740). Both DUFs occur predominantly in Dynein assembly factor 3 axonemal (DNAAF3), a protein involved in preassembly of dyneins into complexes \\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u003c/sup\\u003e. DUF4470 (PF14737) and DUF4471 (PF14740), both present in protein A0A1Y1V9Y8 (AlphaFold2: AF-A0A1Y1V9Y8-F1-v6 \\u003csup\\u003e23\\u003c/sup\\u003e) contain the Rossmann-like fold, confirmed by mapping to Chemotaxis protein methyltransferase (cheR, P07801, pdb: 1AF7 \\u003csup\\u003e24\\u003c/sup\\u003e) of \\u003cem\\u003eSalmonella typhimurium\\u003c/em\\u003e which has a defined Rossmann fold \\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supplementary Figure S3).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003ePhylum based results\\u003c/p\\u003e\\u003cp\\u003eWhile a conserved core of flagellar proteins can be defined across fungi, closer inspection reveals clear phylum-level differences in the retention and composition of individual complexes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). One such example can be the BBSome (octameric coat complex acting as membrane-cargo adaptor \\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e) is universally retained in Neocallimastigomycota (P. \\u003cem\\u003efinnis, Piromyces sp. E2, A. robustus, N. lanti\\u003c/em\\u003e and \\u003cem\\u003eN. californiae\\u003c/em\\u003e), encoding all eight components. In contrast, Chytridiomycota exhibit a fragmented BBSome, with only a subset of subunits present, most of which observed in \\u003cem\\u003eC. confervae\\u003c/em\\u003e which retains five subunits (BBS1, BBS4, BBS5, BBS7, TTC8), \\u003cem\\u003eR. globosum\\u003c/em\\u003e and \\u003cem\\u003eB. helicus\\u003c/em\\u003e retain four, \\u003cem\\u003eC. protostelioides\\u003c/em\\u003e - one, while the remaining chytrids lack the complex entirely. No BBSome proteins are found in the other fungal phyla proteomes used in this study.\\u003c/p\\u003e\\u003cp\\u003eAside from BBsome being a major differentiator between Neocallimastigomycota and the remaining fungal phyla, we can observe phyla-specific differences within orthogroups. Although there are no more phyla-specific complexes other than BBSome that are conserved entirely in one phylum, we observe 39 orthogroups with differing phyla protein distributions. One of the notable observations, is that Chytridiomycota entirely miss three out of ten CatSper channel complex components (CATSPERD, CATSPERB, CATSPERG2), which are auxiliary elements of the complex \\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e, and are present in at least one representatives of the other major phyla (Blastocladiomycota, Neocallimastigomycota) of flagellated fungi (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eNeocallimastigomycota lacks 24 out of 39 fungal orthogroups, of which 11 are associated with centrioles. In addition, there are six groups occurring only in Chytridiomycota and three that occur only in Blastocladiomycota.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eWe performed a comprehensive search for flagellar proteins across diverse fungal representatives which enabled us to explore the evolutionary trajectories of flagellum building blocks.\\u003c/p\\u003e\\u003cp\\u003eWe identified a clear split between flagellated-fungi specific orthogroups (n\\u0026thinsp;=\\u0026thinsp;128) and those shared with non-flagellates (n\\u0026thinsp;=\\u0026thinsp;103) which mirrors the pruning of the ancestral opisthokont ciliary toolkit. Accordingly, gene families tied to axonemal assembly, IFT scaffolding, and membrane docking (enrichment in coiled-coils, low-complexity/disorder, and repeat scaffolds such as WD40/LRR/ARM) are preferentially retained in flagellated lineages, whereas broadly useful modules (kinases, tubulins) persist across fungi regardless of flagellation. This pattern is consistent with the \\u0026ldquo;delayed loss of ancestral gene families\\u0026rdquo; model inferred from comparative genomics across some of the non-Dikarya \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e, where traits are shed piecemental and lineage-specifically rather than in a single step \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. Conversely, enrichment in kinases/tubulins shared between flagellated and non-flagellated fungi is consistent with the description of flagella evolution in literature \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. Moreover, it opens up the possibility of moonlighting, non-flagellar roles for many signalling and cytoskeletal proteins \\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003eAs shown in this study, the number of orthogroups retained in individual isolates varies, however all three top scoring organisms in terms of number of orthogroups retained belong to Neocallimastigomycota. This could result from many reasons, one of the most probable would be multiflagellated zoospores of some of those fungi. While generally, flagellated fungi possess a single posterior flagellum, \\u003cem\\u003eNeocallimastix spp.\\u003c/em\\u003e produces polyflagellate zoospores with a brief motile window before rapid encystment in the viscous rumen, which could lead to a more conserved and robust flagellar toolkit needed for their habitation of the demanding, dense rumen environment \\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003eUpon a more detailed inspection, Neocallimastigomycota harbour the broadest and most intact flagellar toolkit, most notably a universally retained, complete BBSome complex functioning as membrane-cargo adaptor \\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e, despite concurrent reductions in centriole-associated modules.\\u003c/p\\u003e\\u003cp\\u003eWhile currently, there are no experimental studies explaining the specific ecological role of BBSome in fungi (Neocallimastigomycota specifically), Rossier et al. (2023) show retention of two BBSome elements in \\u003cem\\u003ePiromyces spp\\u003c/em\\u003e. and loss in \\u003cem\\u003eA. macrogynus, B. dendrobatidi\\u003c/em\\u003es and \\u003cem\\u003eR. allomycis\\u003c/em\\u003e, which aligns with our results\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e. Concurrently, despite broad axonemal/IFT retention, Neocallimastigomycota in our dataset show a marked reduction of centriole-associated modules, particularly pericentriolar material and satellites. These modules are known to fine-tune basal-body maturation, protein targeting and ciliary gating rather than build the axoneme itself \\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e. Moreover, we observe a high copy number of DAAM1/2 orthologs in Neocallimastigomycota. This could be explained by unusually large, AT-rich, repeat-laden Neocallimastigomycota genomes with extensive gene duplication and horizontal gene transfer. While best documented for CAZymes, the same duplication-prone architecture can be observed for other families, including flagellar modules \\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e. DAAM1/2 and their paralogs represent TPR-like repeat proteins composed of multiple units with substantial divergence among their repeat modules. Their specific role in Neocallimastigomycota remains unclear and is not trivial to decipher. In \\u003cem\\u003eHomo sapiens\\u003c/em\\u003e, DAAM1/2 functions as a central component of diverse protein complexes, not exclusively linked to filament formation. One possible scenario is that Neocallimastigomycota DAAM1/2 paralogs mediate a particularly refined regulation of actin filament formation. Alternatively, some paralogs may have undergone neofunctionalization, acquiring distinct regulatory functions in other cellular compartments.\\u003c/p\\u003e\\u003cp\\u003eOne of the goals of our study was to determine which proteins of the flagellum contribute to its functional core. In consequence, we defined a minimal set of flagellum proteins conserved across flagellated taxa. This enabled us to observe that the minimal flagellum relies more on long cytosolic scaffolds than on membrane complexes: vesicles can still dock and deliver building blocks to the base, IFT trains can move them along the axoneme, and dynein arms can generate beat, but membrane trafficking, ion-channel mediated signalling and regulatory plasticity are limited. In practical terms, this minimal organelle is motile and maintainable, yet optimized for core locomotion with simplified gating and reduced sensory/trafficking capacity. The minimal flagellum can function because of the components it retains \\u0026ndash; axonemal tubulins, dynein motors, radial-spoke/nexin regulators and the core IFT-B/kinesin-2 train \\u0026ndash; these are the ones that physically build and bend the \\u0026ldquo;9\\u0026thinsp;+\\u0026thinsp;2\\u0026rdquo; scaffold. In contrast, most proteins lost act upstream or peripherally: the BBSome and several auxiliary IFT or kinesin modules that mainly recycle signalling receptors and membrane proteins. For example, both \\u003cem\\u003eTrypanosoma brucei\\u003c/em\\u003e and \\u003cem\\u003eChlamydomonas\\u003c/em\\u003e mutants lacking a complete BBSome retain normal beat frequency and waveform \\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e. Similarly, mammals lacking CatSper channel subunits retain progressive, low-amplitude beating but cannot hyperactivate, showing that the Ca\\u0026sup2;⁺-gated CatSper channel complex is a performance enhancer rather than an architectural necessity \\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e. Likewise, comparative work on IFT shows that the 11-subunit core IFT-B platform is obligatory for axoneme assembly, whereas many accessory IFT-A subunits are repeatedly lost in organisms whose flagellum (or cilium) serve mainly locomotion rather than signalling \\u003csup\\u003e\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e. The minimal flagellum set of proteins can be a shortlist for experimental studies on flagellar function and the role of individual proteins in locomotion. Moreover, the conservation of the core flagellar elements between flagellated fungi and animals puts forward the possibility of experimentation in simple models like saprotrophic Chytidiomycota.\\u003c/p\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eWe provide a curated, cross-phyla atlas of fungal flagellar proteins that clearly separates a flagellate-specific, scaffold-rich layer from broadly conserved housekeeping modules. From this, we propose a 97-orthogroup “minimal flagellum” capturing the axonemal core, dynein regulation, and pared IFT, while a non-minimal layer adds membrane channels, cargo adaptors, and signalling capacity. Comparisons across fungal lineages indicate substantial variation in flagellar composition, with Neocallimastigomycota retaining the broadest toolkit and showing select expansions amid centriole reductions, pointing to multiple evolutionary trajectories toward motility. We also annotate the fold for two DUFs occurring in a protein functioning within the flagellum.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003eUsing several sources, we built a comprehensive collection of flagellar proteins by integrating datasets from publicly available databases and literature sources. After removing redundant entries, we performed homology searches in a defined set of model organisms to find more reference proteins. We then combined those proteins into separate protein homology groups and screened them against fungal proteomes. Obtained fungal homologs were then manually cleaned to remove potential false positive hits and narrow-down the results. Additionally, we predict the fold for two DUFs potentially related to flagellum, using distant homology methods.\\u003c/p\\u003e\\u003cp\\u003eOur primary reference dataset comprised proteins from the Complex Portal \\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e (n = 479), retrieved using the keyword “cilium,” and from CORUM \\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e (n = 285), selected under the predefined categories “cilium assembly” and “cilium movement”. In parallel, we incorporated 87 proteins linked to flagellum in \\u003cem\\u003eChlamydomonas reinhardtii\\u003c/em\\u003e, as reported by Pazour et al. (2005) \\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e, and 62 proteins from the list of centriole associated proteins defined by Long et al. (2025) \\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e. Redundant entries across these datasets were manually removed and unified, creating a set of 303 homolog groups (Supplementary File S1). The 303 orthogroups were mapped on a set of outgroup taxa using blastp (version 2.13.0+; e-value threshold of 1e-3)\\u003csup\\u003e36\\u003c/sup\\u003e. The outgroup set included model animals \\u003cem\\u003eMus musculus\\u003c/em\\u003e (GCA_000001635.9), \\u003cem\\u003eHomo sapiens\\u003c/em\\u003e (GCA_000001405.29), \\u003cem\\u003eDrosophila melanogaster\\u003c/em\\u003e (GCA_000001215.4), and \\u003cem\\u003eCaenorhabditis elegans\\u003c/em\\u003e (GCA_000002985.3), along with basal \\u003cem\\u003eOpisthokonta\\u003c/em\\u003e representatives: \\u003cem\\u003eMonosiga brevicollis\\u003c/em\\u003e (GCF_000002865.3), \\u003cem\\u003eCapsaspora owczarzaki\\u003c/em\\u003e (GCF_000151315.2), \\u003cem\\u003eSalpingoeca rosetta\\u003c/em\\u003e (GCF_000188695.1), \\u003cem\\u003eFonticula alba\\u003c/em\\u003e (GCF_000388065.1), and \\u003cem\\u003eSphaeroforma arctica\\u003c/em\\u003e (GCF_001186125.1), as well as \\u003cem\\u003eC. reinhardtii\\u003c/em\\u003e (GCA_000002595.3).\\u003c/p\\u003e\\u003cp\\u003eHomology searches in fungi were performed using 184 fungal proteomes (Supplementary File S1). By proteomes, we define the complete sets of all proteins coded by genes in 184 genome assemblies downloaded from NCBI in May 2022 \\u003csup\\u003e37\\u003c/sup\\u003e. These comprise all then-available non-Dikarya proteomes and proteomes of representatives of \\u003cem\\u003eDikarya\\u003c/em\\u003e classes. To ensure that for each reference protein, sets of reliable fungal homologs were obtained, we checked the conservation of Pfam (database v. 36) protein domains by scanning them with pfamscan.pl v1.6 with its default settings \\u003csup\\u003e\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e. Each homology group was noted as present or absent from fungi. Additionally, we used tblastn v. 2.13.0 + \\u003csup\\u003e36\\u003c/sup\\u003e with an e-value threshold of 1e-5 (against 184 fungal genomes) to eliminate potential false negative results resulting from possible incomplete protein sets in proteomes.\\u003c/p\\u003e\\u003cp\\u003eFor large homology groups resulting from initial blastp scan, we used CLANS software (version 2.0) \\u003csup\\u003e39\\u003c/sup\\u003e to narrow-down the results to potential true homologs for each reference query. In addition, we combined homolog groups which contained the same Pfam families. We further used CLANS software to find unique, non-redundant hits for each individual reference query. This approach allowed us to find unique homologs for each query without redundancy to other highly similar queries (e.g. tubulins or kinases).\\u003c/p\\u003e\\u003cp\\u003eProtein group categorization has been made based on Long et al. (2025) \\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e and Merényi et al. (2023) \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e categories as well as on CORUM and ComplexPortal annotations, in addition to UniProt \\u003csup\\u003e\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e\\u003c/sup\\u003e annotations. Fungal homolog groups were characterized using diverse sequence annotation tools and structural comparisons. For the identification of transmembrane helices, TMHMM (version 2.0) \\u003csup\\u003e41\\u003c/sup\\u003e was utilized. Sequence disorder predictions were carried out using IUPred (version 2.0) \\u003csup\\u003e42\\u003c/sup\\u003e. SEG (version 1.0) \\u003csup\\u003e43\\u003c/sup\\u003e was employed for the prediction of low-complexity regions. All of the above-mentioned software was used with the default settings. Multiple sequence alignments were constructed via MAFFT (version 7.475) \\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e, employing parameters --localpair and --maxiterate 100. Distant homology searches for DUFs were conducted using MAFFT-obtained MSAs via HHpred on the MPI Toolkit website (version 3.3.0) \\u003csup\\u003e45\\u003c/sup\\u003e with target databases of: TIGFAMs \\u003csup\\u003e\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e\\u003c/sup\\u003e, ECOD (version 2022) \\u003csup\\u003e\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e\\u003c/sup\\u003e and RCSB (updated 2022) \\u003csup\\u003e\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e\\u003c/sup\\u003e. Three-dimensional visualization of protein structures was achieved using PyMOL (version 2.5) \\u003csup\\u003e49\\u003c/sup\\u003e. The phylogenetic tree was constructed with IQ-TREE (v1.6.9) \\u003csup\\u003e50\\u003c/sup\\u003e using maximum likelihood method with automated model selection. The trees were visualized and represented using the iTOL online tool \\u003csup\\u003e\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e\\u003c/sup\\u003e. Figures were created using Python and Matplotlib \\u003csup\\u003e\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eAcknowledgements\\u003c/h2\\u003e\\n\\u003cp\\u003eWe are grateful for insightful comments and suggestions by \\u0026nbsp;Drishtee Barua.\\u003c/p\\u003e\\n\\u003ch2\\u003eAuthors' contributions\\u003c/h2\\u003e\\n\\u003cp\\u003eA.K. and A.M. designed the study and performed analyses. A.K. and A.M. drafted the manuscript. A.K. prepared the figures, A.M. conceptualized the project.\\u003c/p\\u003e\\n\\u003ch2\\u003eData availability statement\\u003c/h2\\u003e\\n\\u003cp\\u003eAll metadata processed in this study are deposited in zenodo:\\u0026nbsp;\\u003cstrong\\u003e10.5281/zenodo.17376122\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll protein identifiers and genomic assemblies are listed in Supplementary file S1. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003ch2\\u003eFunding\\u003c/h2\\u003e\\n\\u003cp\\u003eThis work was supported by National Science Centre grant (#2021/41/B/NZ2/02426 to A.M.)\\u003c/p\\u003e\\n\\u003ch2\\u003eCompeting interests\\u003c/h2\\u003e\\n\\u003cp\\u003eThe author(s) declare that they have no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eHyde, K. D. 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The PyMOL Molecular Graphics System, Version 1.8. Preprint at (2015).\\u003c/li\\u003e\\n \\u003cli\\u003eMinh, B. Q. \\u003cem\\u003eet al.\\u003c/em\\u003e IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. \\u003cem\\u003eMol. Biol. Evol.\\u003c/em\\u003e \\u003cstrong\\u003e37\\u003c/strong\\u003e, 1530\\u0026ndash;1534 (2020).\\u003c/li\\u003e\\n \\u003cli\\u003eLetunic, I. \\u0026amp; Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. \\u003cem\\u003eNucleic Acids Res.\\u003c/em\\u003e \\u003cstrong\\u003e49\\u003c/strong\\u003e, W293\\u0026ndash;W296 (2021).\\u003c/li\\u003e\\n \\u003cli\\u003e\\u003cem\\u003ePython Programming Language\\u003c/em\\u003e. https://www.python.org/ (2023).\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"fungi, motility, flagellum\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7887758/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7887758/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eFlagellated fungi are pivotal for understanding the evolution of eukaryotic motility, yet knowledge of their flagellar proteins remains fragmentary. We assembled a cross-phyla atlas by mapping 303 reference flagellar proteins on 184 fungal proteomes. Out of 303 orthogroups, 231 orthogroups have fungal homologs, of which 128 orthogroups are specific to flagellated fungi. From these, we propose a 97-orthogroup minimal flagellum encompassing proteins shared by flagellated fungi. This minimal toolkit retains the axonemal core (tubulins, dynein arms), beat regulators (nexin\\u0026ndash;dynein regulatory complex, radial spokes), and essential intraflagellar transport (core IFT-B with key IFT-A and kinesin-2), plus a pared foundation for basal-body duplication and targeted trafficking (exocyst/TRAPP). Fungal lineages differ in the composition of flagellar modules. Neocallimastigomycota stand out as the most diverged among flagellated lineages with the broadest flagellar toolkit, including a complete BBSome, yet exhibit reductions in centriole-associated modules. Finally, we annotate two Pfam domains of unknown function DUF4470 \\u0026amp; DUF4471 within DNAAF3 protein as a single Rossmann-like fold. We delineate a lean, conserved engine for fungal motility and a modular, lineage-specific layer that broadens regulation and sensory capacity. The conservation of flagellar proteins between fungi and animals opens up possibilities of experimental functional validation in models simpler than animals.\\u003c/p\\u003e\",\"manuscriptTitle\":\"A conserved minimal core and modular extensions make the fungal flagellum\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-11-13 06:33:41\",\"doi\":\"10.21203/rs.3.rs-7887758/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-11-25T06:55:51+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-11-23T03:12:31+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-11-19T15:25:28+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-11-10T11:17:13+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"226322952626479402844147692118747450484\",\"date\":\"2025-11-10T09:37:48+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"63870227669715825615422576892363483370\",\"date\":\"2025-11-08T01:19:49+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"289597361307817378025088480649796851508\",\"date\":\"2025-11-05T14:50:00+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-11-03T10:10:33+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2025-10-30T13:29:11+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-10-21T10:07:53+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-10-21T10:07:03+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2025-10-17T14:36:50+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"0e9540ae-9884-4531-95a6-2b762818bb4c\",\"owner\":[],\"postedDate\":\"November 13th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[{\"id\":57724794,\"name\":\"Biological sciences/Cell biology\"},{\"id\":57724795,\"name\":\"Biological sciences/Microbiology\"}],\"tags\":[],\"updatedAt\":\"2026-05-06T14:39:35+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-11-13 06:33:41\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7887758\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7887758\",\"identity\":\"rs-7887758\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}