Ying Chang

Zygomycete fungi were classified as a single phylum, Zygomycota, based on sexual reproduction by zygospores, frequent asexual reproduction by sporangia, absence of multicellular sporocarps, and production of coenocytic hyphae, all with some exceptions. Molecular phylogenies based on one or a few genes did not support the monophyly of the phylum, however, and the phylum was subsequently abandoned. Here we present phylogenetic analyses of a genome-scale data set for 46 taxa, including 25 zygomycetes and 192 proteins, and we demonstrate that zygomycetes comprise two major clades that form a paraphyletic grade. A formal phylogenetic classification is proposed herein and includes two phyla, six subphyla, four classes and 16 orders. On the basis of these results, the phyla Mucoromycota and Zoopagomycota are circumscribed. Zoopagomycota comprises Entomophtoromycotina, Kickxellomycotina and Zoopagomycotina; it constitutes the earliest diverging lineage of zygomycetes and contains species that are primarily parasites and pathogens of small animals (e.g. amoeba, insects, etc.) and other fungi, i.e. mycoparasites. Mucoromycota comprises Glomeromycotina, Mortierellomycotina, and Mucoromycotina and is sister to Dikarya. It is the more derived clade of zygomycetes and mainly consists of mycorrhizal fungi, root endophytes, and decomposers of plant material. Evolution of trophic modes, morphology, and analysis of genome-scale data are discussed.

Phylogenomic studies using genome-scale amounts of data have greatly improved understanding of the tree of life. Despite the diversity, ecological significance, and biomedical and industrial importance of fungi, evolutionary relationships among several major lineages remain poorly resolved, especially those near the base of the fungal phylogeny. To examine poorly resolved relationships and assess progress toward a genome-scale phylogeny of the fungal kingdom, we compiled a phylogenomic data matrix of 290 genes from the genomes of 1,644 species that includes representatives from most major fungal lineages. We also compiled 11 data matrices by subsampling genes or taxa from the full data matrix based on filtering criteria previously shown to improve phylogenomic inference. Analyses of these 12 data matrices using concatenation- and coalescent-based approaches yielded a robust phylogeny of the fungal kingdom, in which ∼85% of internal branches were congruent across data matrices and approaches used. We found support for several historically poorly resolved relationships as well as evidence for polytomies likely stemming from episodes of ancient diversification. By examining the relative evolutionary divergence of taxonomic groups of equivalent rank, we found that fungal taxonomy is broadly aligned with both genome sequence divergence and divergence time but also identified lineages where current taxonomic circumscription does not reflect their levels of evolutionary divergence. Our results provide a robust phylogenomic framework to explore the tempo and mode of fungal evolution and offer directions for future fungal phylogenetic and taxonomic studies.

Single-repeat R3 MYB transcription factors are critical components of the lateral inhibition machinery that mediates epidermal cell patterning in plants. Sequence analysis of the Arabidopsis genome using the BLAST program reveals that there are a total of six genes, including TRIPTYCHON (TRY), CAPRICE (CPC), TRICHOMELESS1 (TCL1), and ENHANCER of TRY and CPC 1, 2, and 3 (ETC1, ETC2 and ETC3) encoding single-repeat R3 MYB transcription factors that are approximately 50% identical to one another at the amino acid level. Previous studies indicate that these single-repeat R3 MYBs regulate epidermal cell patterning. However, each of the previous studies of these single-repeat R3 MYBs has been limited to an analysis of only a subset of these six genes, and furthermore, they have limited their attention to epidermal development in only one or two of the organs. In addition, the transcriptional regulation of these single-repeat R3 MYB genes remains largely unknown.By analyzing multiple mutant lines, we report here that TCL1 functions redundantly with other single-repeat R3 MYB transcription factors to control both leaf trichome and root hair formation. On the other hand, ETC1 and ETC3 participate in controlling trichome formation on inflorescence stems and pedicles. Further, we discovered that single-repeat R3 MYBs suppress trichome formation on cotyledons and siliques, organs that normally do not bear any trichomes. By using Arabidopsis protoplast transfection assays, we found that all single-repeat R3 MYBs examined interact with GL3, and that GL1 or WER and GL3 or EGL3 are required and sufficient to activate the transcription of TRY, CPC, ETC1 and ETC3, but not TCL1 and ETC2. Furthermore, only ETC1's transcription was greatly reduced in the gl3 egl3 double mutants.Our comprehensive analysis enables us to draw broader conclusions about the role of single-repeat R3 MYB gene family than were possible in the earlier studies, and reveals the genetic basis of organ-specific control of trichome formation. Our findings imply the presence of multiple mechanisms regulating the transcription of single-repeat R3 MYB genes, and provide new insight into the lateral inhibition mechanism that mediates epidermal cell patterning.

Investigating the early diversification of major clades requires well-corroborated and accurate phylogenetic inferences. We examined the performance of a large set of plastid genes for inferring the broad phylogenetic backbone of mosses-the second largest major clade of land plants-and their nearest relatives.We surveyed 14-17 plastid genes from a broadly representative taxonomic sampling of the major bryophyte lineages, including all major lines of non-peristomate mosses. We examined how well these new data corroborated or contradicted the findings of other studies, and investigated the effect of removing rapidly evolving characters. KEY RESULT: We inferred major clades with at least as strong support as other studies that used more taxa. We corroborated current views of overall embryophyte relationships, i.e., (liverworts, (mosses, (hornworts, tracheophytes))), with strong maximum likelihood (ML) bootstrap support, and also placed Zygnematales as the sister group of embryophytes with moderate ML bootstrap support. Within mosses, we confirmed Oedipodiaceae as the sister group of the large clade of peristomate taxa. Likelihood analysis also firmly placed Takakiaceae as the sister group of all other mosses, a strong conflict with parsimony results. Parsimony converged on the Takakia-sister result when rapidly evolving characters were removed, depending on the tree used to classify the site rates.Our findings broadly support the utility of a 14-gene set from the plastome for future, more densely sampled phylogenetic studies of mosses and relatives, potentially complementing anticipated whole-plastome studies. Likelihood and parsimony conflicts flag possible instances of long-branch attraction, including one involving the earliest split in moss phylogeny.

Endogonales (Mucoromycotina), composed of Endogonaceae and Densosporaceae, is the only known non-Dikarya order with ectomycorrhizal members. They also form mycorrhizal-like association with some nonspermatophyte plants. It has been recently proposed that Endogonales were among the earliest mycorrhizal partners with land plants. It remains unknown whether Endogonales possess genomes with mycorrhizal-lifestyle signatures and whether Endogonales originated around the same time as land plants did. We sampled sporocarp tissue from four Endogonaceae collections and performed shotgun genome sequencing. After binning the metagenome data, we assembled and annotated the Endogonaceae genomes. We performed comparative analysis on plant-cell-wall-degrading enzymes (PCWDEs) and small secreted proteins (SSPs). We inferred phylogenetic placement of Endogonaceae and estimated the ages of Endogonaceae and Endogonales with expanded taxon sampling. Endogonaceae have large genomes with high repeat content, low diversity of PCWDEs, but without elevated SSP/secretome ratios. Dating analysis estimated that Endogonaceae originated in the Permian-Triassic boundary and Endogonales originated in the mid-late Silurian. Mycoplasma-related endobacterium sequences were identified in three Endogonaceae genomes. Endogonaceae genomes possess typical signatures of mycorrhizal lifestyle. The early origin of Endogonales suggests that the mycorrhizal association between Endogonales and plants might have played an important role during the colonization of land by plants.

The diploid wild cotton species Gossypium australe possesses excellent traits including resistance to disease and delayed gland morphogenesis, and has been successfully used for distant breeding programmes to incorporate disease resistance traits into domesticated cotton. Here, we sequenced the G. australe genome by integrating PacBio, Illumina short read, BioNano (DLS) and Hi-C technologies, and acquired a high-quality reference genome with a contig N50 of 1.83 Mb and a scaffold N50 of 143.60 Mb. We found that 73.5% of the G. australe genome is composed of various repeat sequences, differing from those of G. arboreum (85.39%), G. hirsutum (69.86%) and G. barbadense (69.83%). The G. australe genome showed closer collinear relationships with the genome of G. arboreum than G. raimondii and has undergone less extensive genome reorganization than the G. arboreum genome. Selection signature and transcriptomics analyses implicated multiple genes in disease resistance responses, including GauCCD7 and GauCBP1, and experiments revealed induction of both genes by Verticillium dahliae and by the plant hormones strigolactone (GR24), salicylic acid (SA) and methyl jasmonate (MeJA). Experiments using a Verticillium-resistant domesticated G. barbadense cultivar confirmed that knockdown of the homologues of these genes caused a significant reduction in resistance against Verticillium dahliae. Moreover, knockdown of a newly identified gland-associated gene GauGRAS1 caused a glandless phenotype in partial tissues using G. australe. The G. australe genome represents a valuable resource for cotton research and distant relative breeding as well as for understanding the evolutionary history of crop genomes.

Most of the described species in kingdom Fungi are contained in two phyla, the Ascomycota and the Basidiomycota (subkingdom Dikarya). As a result, our understanding of the biology of the kingdom is heavily influenced by traits observed in Dikarya, such as aerial spore dispersal and life cycles dominated by mitosis of haploid nuclei. We now appreciate that Fungi comprises numerous phylum-level lineages in addition to those of Dikarya, but the phylogeny and genetic characteristics of most of these lineages are poorly understood due to limited genome sampling. Here, we addressed major evolutionary trends in the non-Dikarya fungi by phylogenomic analysis of 69 newly generated draft genome sequences of the zoosporic (flagellated) lineages of true fungi. Our phylogeny indicated five lineages of zoosporic fungi and placed Blastocladiomycota, which has an alternation of haploid and diploid generations, as branching closer to the Dikarya than to the Chytridiomyceta. Our estimates of heterozygosity based on genome sequence data indicate that the zoosporic lineages plus the Zoopagomycota are frequently characterized by diploid-dominant life cycles. We mapped additional traits, such as ancestral cell-cycle regulators, cell-membrane- and cell-wall-associated genes, and the use of the amino acid selenocysteine on the phylogeny and found that these ancestral traits that are shared with Metazoa have been subject to extensive parallel loss across zoosporic lineages. Together, our results indicate a gradual transition in the genetics and cell biology of fungi from their ancestor and caution against assuming that traits measured in Dikarya are typical of other fungal lineages.

Phloem loading, as the first step of transporting photoassimilates from mesophyll cells to sieve element-companion cell complex, creates a driving force for long-distance nutrient transport. Three loading strategies have been proposed: passive symplastic loading, apoplastic loading and symplastic transfer followed by polymer-trapping of stachyose and raffinose. Although individual species are generally referred to as using a single phloem loading mechanism, it has been suggested that some plants may use more than one, i.e. 'mixed loading'. Here, by using a combination of electron microscopy, reverse genetics and 14 C labeling, loading strategies were studied in cucumber, a polymer-trapping loading species. The results indicate that intermediary cells (ICs), which mediate polymer-trapping, and ordinary companion cells, which mediate apoplastic loading, were mainly found in the fifth and third order veins, respectively. Accordingly, a cucumber galactinol synthase gene (CsGolS1) and a sucrose transporter gene (CsSUT2) were expressed mainly in the fifth/third and the third order veins, respectively. Immunolocalization analysis indicated that CsGolS1 was localized in companion cells (CCs) while CsSUT2 was in CCs and sieve elements (SEs). Suppressing CsGolS1 significantly decreased the stachyose level and increased sucrose content, while suppressing CsSUT2 decreased the sucrose level and increased the stachyose content in leaves. After 14 CO2 labeling, [14 C]sucrose export increased and [14 C]stachyose export reduced from petioles in CsGolS1i plants, but [14 C]sucrose export decreased and [14 C]stachyose export increased into petioles in CsSUT2i plants. Similar results were also observed after pre-treating the CsGolS1i leaves with PCMBS (transporter inhibitor). These results demonstrate that cucumber phloem loading depends on both polymer-trapping and apoplastic loading strategies.

Viruses are key drivers of evolution and ecosystem function and are increasingly recognized as symbionts of fungi. Fungi in early-diverging lineages are widespread, ecologically important, and comprise the majority of the phylogenetic diversity of the kingdom. Viruses infecting early-diverging lineages of fungi have been almost entirely unstudied. In this study, we screened fungi for viruses by two alternative approaches: a classic culture-based method and by transcriptome-mining. The results of our large-scale survey demonstrate that early-diverging lineages have higher infection rates than have been previously reported in other fungal taxa and that laboratory strains worldwide are host to infections, the implications of which are unknown. The function and diversity of mycoviruses found in these basal fungal lineages will help guide future studies into mycovirus origins and their evolutionary ramifications and ecological impacts.

Abstract Fungi have evolved over millions of years and their species diversity is predicted to be the second largest on the earth. Fungi have cross-kingdom interactions with many organisms that have mutually shaped their evolutionary trajectories. Zygomycete fungi hold a pivotal position in the fungal tree of life and provide important perspectives on the early evolution of fungi from aquatic to terrestrial environments. Phylogenomic analyses have found that zygomycete fungi diversified into two separate clades, the Mucoromycota which are frequently associated with plants and Zoopagomycota that are commonly animal-associated fungi. Genetic elements that contributed to the fitness and divergence of these lineages may have been shaped by the varied interactions these fungi have had with plants, animals, bacteria, and other microbes. To investigate this, we performed comparative genomic analyses of the two clades of zygomycetes in the context of Kingdom Fungi, benefiting from our generation of a new collection of zygomycete genomes, including nine produced for this study. We identified lineage-specific genomic content that may contribute to the disparate biology observed in these zygomycetes. Our findings include the discovery of undescribed diversity in CotH, a Mucormycosis pathogenicity factor, which was found in a broad set of zygomycetes. Reconciliation analysis identified multiple duplication events and an expansion of CotH copies throughout the Mucoromycotina, Mortierellomycotina, Neocallimastigomycota, and Basidiobolus lineages. A kingdom-level phylogenomic analysis also identified new evolutionary relationships within the subphyla of Mucoromycota and Zoopagomycota, including supporting the sister-clade relationship between Glomeromycotina and Mortierellomycotina and the placement of Basidiobolus as sister to other Zoopagomycota lineages.

Phylogenetic trees of bryophytes provide important evolutionary context for land plants. However, published inferences of overall embryophyte relationships vary considerably. We performed phylogenomic analyses of bryophytes and relatives using both mitochondrial and plastid gene sets, and investigated bryophyte plastome evolution.We employed diverse likelihood-based analyses to infer large-scale bryophyte phylogeny for mitochondrial and plastid data sets. We tested for changes in purifying selection in plastid genes of a mycoheterotrophic liverwort (Aneura mirabilis) and a putatively mycoheterotrophic moss (Buxbaumia), and compared 15 bryophyte plastomes for major structural rearrangements.Overall land-plant relationships conflict across analyses, generally weakly. However, an underlying (unrooted) four-taxon tree is consistent across most analyses and published studies. Despite gene coverage patchiness, relationships within mosses, liverworts, and hornworts are largely congruent with previous studies, with plastid results generally better supported. Exclusion of RNA edit sites restores cases of unexpected non-monophyly to monophyly for Takakia and two hornwort genera. Relaxed purifying selection affects multiple plastid genes in mycoheterotrophic Aneura but not Buxbaumia. Plastid genome structure is nearly invariant across bryophytes, but the tufA locus, presumed lost in embryophytes, is unexpectedly retained in several mosses.A common unrooted tree underlies embryophyte phylogeny, [(liverworts, mosses), (hornworts, vascular plants)]; rooting inconsistency across studies likely reflects substantial distance to algal outgroups. Analyses combining genomic and transcriptomic data may be misled locally for heavily RNA-edited taxa. The Buxbaumia plastome lacks hallmarks of relaxed selection found in mycoheterotrophic Aneura. Autotrophic bryophyte plastomes, including Buxbaumia, hardly vary in overall structure.

Summary Mitogen‐activated protein kinase (MAPK) cascades play vital roles in regulating plant growth, development, and stress responses. MAPK‐like (MPKL) proteins are a group of kinases containing the MAPK signature TxY motif and showing sequence similarity to MAPKs. However, the functions of plant MPKL proteins are currently unknown. The maize ( Zea mays ) genome contains four genes encoding MPKL proteins, here named ZmMPKL1 to ZmMPKL4 . In this study, we show that ZmMPKL1 possesses kinase activity and that drought‐induced ZmMPKL1 expression, ZmMPKL1 overexpression and knockout maize seedlings exhibited no visible morphological difference from wild‐type B73 seedlings when grown under normal conditions. By contrast, under drought conditions, ZmMPKL1‐ overexpressing seedlings showed increased stomatal aperture, water loss, and leaf wilting and knockout seedlings showed the opposite phenotypes. Moreover, these drought‐sensitive phenotypes in ZmMPKL1‐ overexpressing seedlings were restored by exogenous abscisic acid (ABA). ZmMPKL1 overexpression reduced drought‐induced ABA production in seedlings and the knockout showed enhanced ABA production. Drought‐induced transcription of ABA biosynthetic genes were suppressed and ABA catabolic genes were enhanced in ZmMPKL1‐ overexpressing seedlings, while their transcription were reversely regulated in knockout seedlings. These results suggest that ZmMPKL1 positively regulates seedlings drought sensitivity by altering the transcription of ABA biosynthetic and catabolic genes, and ABA homeostasis.

The zoosporic obligate endoparasites, Olpidium, hold a pivotal position to the reconstruction of the flagellum loss in fungi, one of the key morphological transitions associated with the colonization of land by the early fungi. We generated genome and transcriptome data from non-axenic zoospores of Olpidium bornovanus and used a metagenome approach to extract phylogenetically informative fungal markers. Our phylogenetic reconstruction strongly supported Olpidium as the closest zoosporic relative of the non-flagellated terrestrial fungi. Super-alignment analyses resolved Olpidium as sister to the non-flagellated terrestrial fungi, whereas a super-tree approach recovered different placements of Olpidium, but without strong support. Further investigations detected little conflicting signal among the sampled markers but revealed a potential polytomy in early fungal evolution associated with the branching order among Olpidium, Zoopagomycota and Mucoromycota. The branches defining the evolutionary relationships of these lineages were characterized by short branch lengths and low phylogenetic content and received equivocal support for alternative phylogenetic hypotheses from individual markers. These nodes were marked by important morphological innovations, including the transition to hyphal growth and the loss of flagellum, which enabled early fungi to explore new niches and resulted in rapid and temporally concurrent Precambrian diversifications of the ancestors of several phyla of fungi.

<h2>Summary</h2> Fungi survive in diverse ecological niches by secreting proteins and other molecules into the environment to acquire food and interact with various biotic and abiotic stressors. Fungal secretome content is, therefore, believed to be tightly linked to fungal ecologies. We sampled 132 genomes from the early-diverging terrestrial fungal lineage zygomycetes (Mucoromycota and Zoopagomycota) and characterized their secretome composition. Our analyses revealed that phylogeny played an important role in shaping the secretome composition of zygomycete fungi with trophic mode contributing a smaller amount. Reconstruction of the evolution of secreted digestive enzymes revealed lineage-specific expansions, indicating that Mucoromycota and Zoopagomycota followed different trajectories early in their evolutionary history. We identified the presence of multiple pathogenicity-related proteins in the lineages known as saprotrophs, suggesting that either the ecologies of these fungi are incompletely known, and/or that these pathogenicity-related proteins have important functions associated with saprotrophic ecologies, both of which invite further investigation.

Abstract Verticillium wilt caused by Verticillium dahliae is a serious vascular disease in cotton (Gossypium spp.). V. dahliae induces the expression of the CAROTENOID CLEAVAGE DIOXYGENASE 7 (GauCCD7) gene involved in strigolactone (SL) biosynthesis in Gossypium australe, suggesting a role for SLs in Verticillium wilt resistance. We found that the SL analog rac-GR24 enhanced while the SL biosynthesis inhibitor TIS108 decreased cotton resistance to Verticillium wilt. Knock-down of GbCCD7 and GbCCD8b genes in island cotton (Gossypium barbadense) decreased resistance, whereas overexpression of GbCCD8b in upland cotton (Gossypium hirsutum) increased resistance to Verticillium wilt. Additionally, Arabidopsis (Arabidopsis thaliana) SL mutants defective in CCD7 and CCD8 putative orthologs were susceptible, whereas both Arabidopsis GbCCD7- and GbCCD8b-overexpressing plants were more resistant to Verticillium wilt than wild-type (WT) plants. Transcriptome analyses showed that several genes related to the jasmonic acid (JA)- and abscisic acid (ABA)-signaling pathways, such as MYELOCYTOMATOSIS 2 (GbMYC2) and ABA-INSENSITIVE 5, respectively, were upregulated in the roots of WT cotton plants in responses to rac-GR24 and V. dahliae infection but downregulated in the roots of both GbCCD7- and GbCCD8b-silenced cotton plants. Furthermore, GbMYC2 suppressed the expression of GbCCD7 and GbCCD8b by binding to their promoters, which might regulate the homeostasis of SLs in cotton through a negative feedback loop. We also found that GbCCD7- and GbCCD8b-silenced cotton plants were impaired in V. dahliae-induced reactive oxygen species (ROS) accumulation. Taken together, our results suggest that SLs positively regulate cotton resistance to Verticillium wilt through crosstalk with the JA- and ABA-signaling pathways and by inducing ROS accumulation.

Previous genome-scale phylogenetic analyses of Fungi have under sampled taxa from Zoopagales; this order contains many predacious or parasitic genera, and most have never been grown in pure culture. We sequenced the genomes of 4 zoopagalean taxa that are predators of amoebae, nematodes, or rotifers and the genome of one taxon that is a parasite of amoebae using single cell sequencing methods with whole genome amplification. Each genome was a metagenome, which was assembled and binned using multiple techniques to identify the target genomes. We inferred phylogenies with both super matrix and coalescent approaches using 192 conserved proteins mined from the target genomes and performed ancestral state reconstructions to determine the ancestral trophic lifestyle of the clade. Our results indicate that Zoopagales is monophyletic. Ancestral state reconstructions provide moderate support for mycoparasitism being the ancestral state of the clade.

Abstract Research into secondary metabolism (SM) production by fungi has resulted in the discovery of diverse, biologically active compounds with significant medicinal applications. The fungi rich in SM production are taxonomically concentrated in the subkingdom Dikarya, which comprises the phyla Ascomycota and Basidiomycota. Here, we explore the potential for SM production in Mucoromycota and Zoopagomycota, two phyla of nonflagellated fungi that are not members of Dikarya, by predicting and identifying core genes and gene clusters involved in SM. The majority of non-Dikarya have few genes and gene clusters involved in SM production except for the amphibian gut symbionts in the genus Basidiobolus. Basidiobolus genomes exhibit an enrichment of SM genes involved in siderophore, surfactin-like, and terpene cyclase production, all these with evidence of constitutive gene expression. Gene expression and chemical assays also confirm that Basidiobolus has significant siderophore activity. The expansion of SMs in Basidiobolus are partially due to horizontal gene transfer from bacteria, likely as a consequence of its ecology as an amphibian gut endosymbiont.

Respiratory burst oxidase homologs (Rbohs) play a predominant role in ROS production, which is crucial in plants growth, differentiation, as well as their responses to biotic and abiotic stresses. To date, however, there is little knowledge about the function of cotton Rboh genes. Here, we identified a total of 87 Rbohs from five sequenced Gossypium species (the diploids G. arboretum, G. raimondii, and G. australe, and the allotetraploids G. hirsutum and G. barbadense) via BLAST searching their genomes. Phylogenetic analysis of the putative 87 cotton Rbohs revealed that they were divided into seven clades. All members within the same clade are generally similar to each other in terms of gene structure and conserved domain arrangement. In G. barbadense, expression levels of GbRbohs in the CladeD were induced in response to a fungal pathogen and hormones (i.e., jasmonic acid and abscisic acid), based upon which the main functional member in CladeD was discerned to be GbRboh5/18. Further functional and physiological analyses showed that the knock-down of GbRboh5/18 expression attenuates plant resistance to Verticillium dahliae infection. Combined with the molecular and biochemical analyses, we found less ROS accumulation in GbRboh5/18-VIGS plants than in control plants after V. dahliae infection. Over-expression of GbRboh5/18 in G. barbadense resulted in more ROS accumulation than in control plants. These results suggest that GbRboh5/18 enhances cotton plants’ resistance against V. dahliae by elevating the levels of ROS accumulation. By integrating phylogenetic, molecular, and biochemical approaches, this comprehensive study provided a detailed overview of the number, phylogeny, and evolution of the Rboh gene family from five sequenced Gossypium species, as well as elucidating the function of GbRboh5/18 for plant resistance against V. dahliae. This study sheds fresh light on the molecular evolutionary properties and function of Rboh genes in cotton, and provides a reference for improving cotton’s response to the pathogen V. dahliae.

Cotton architecture is partly determined by shoot branching and flowering patterns. GhBRC1 was previously identified by RNA-seq analysis of nulliplex-branching and normal-branching cotton. However, the roles of GhBRC1 in cotton remain unclear. In the present study, investigations of nuclear localization and transcriptional activity indicated that GhBRC1 has characteristics typical of transcription factors. Gene expression analysis showed that GhBRC1 was highly expressed in axillary buds but displayed different expression patterns between the two branching types. Overexpression of GhBRC1 in Arabidopsis significantly inhibited the number of branches and promoted flowering. In contrast, silencing GhBRC1 in cotton significantly promoted seedling growth. GhBRC1 was induced by multiple hormones, including strigolactones, which promoted seedling growth and seed germination of Arabidopsis plants overexpressing GhBRC1. Consistent with these findings, RNA-seq analysis of virus-induced gene silencing treated cotton revealed that a large number of genes were differentially expressed between GhBRC1-silenced and control plants, and these genes were significantly enriched in plant hormone signalling pathways. Together, our data indicates that GhBRC1 regulates plant branching and flowering through multiple regulatory pathways, especially those regulating plant hormones, with functions partly differing from those of Arabidopsis BRC1. These results provide insights into the molecular mechanisms controlling plant architecture, which is important for breeding cotton with ideal plant architecture and high yield.

The first genome sequenced of a eukaryotic organism was for Saccharomyces cerevisiae, as reported in 1996, but it was more than 10 years before any of the zygomycete fungi, which are the early-diverging terrestrial fungi currently placed in the phyla Mucoromycota and Zoopagomycota, were sequenced. The genome for Rhizopus delemar was completed in 2008; currently, more than 1000 zygomycete genomes have been sequenced. Genomic data from these early-diverging terrestrial fungi revealed deep phylogenetic separation of the two major clades—primarily plant—associated saprotrophic and mycorrhizal Mucoromycota versus the primarily mycoparasitic or animal-associated parasites and commensals in the Zoopagomycota. Genomic studies provide many valuable insights into how these fungi evolved in response to the challenges of living on land, including adaptations to sensing light and gravity, development of hyphal growth, and co-existence with the first terrestrial plants. Genome sequence data have facilitated studies of genome architecture, including a history of genome duplications and horizontal gene transfer events, distribution and organization of mating type loci, rDNA genes and transposable elements, methylation processes, and genes useful for various industrial applications. Pathogenicity genes and specialized secondary metabolites have also been detected in soil saprobes and pathogenic fungi. Novel endosymbiotic bacteria and viruses have been discovered during several zygomycete genome projects. Overall, genomic information has helped to resolve a plethora of research questions, from the placement of zygomycetes on the evolutionary tree of life and in natural ecosystems, to the applied biotechnological and medical questions.

Leaf senescence is the final leaf developmental process that is regulated by both intracellular factors and environmental conditions. The mitogen-activated protein kinase (MAPK) signaling cascades have been shown to play important roles in regulating leaf senescence; however, the component(s) downstream of the MAPK cascades in regulating leaf senescence are not fully understood. Here we showed that the transcriptions of ZmMEK1, ZmSIMK1, and ZmMPK3 were induced during dark-induced maize leaf senescence. Furthermore, in-gel kinase analysis revealed the 42 kDa MAPK was activated. ZmMEK1 interacted with ZmSIMK1 in yeast and maize mesophyll protoplasts and ZmSIMK1 was activated by ZmMEK1 in vitro. Expression of a dominant negative mutant of ZmMEK1 in Arabidopsis transgenic plants induced salicylic acid (SA) accumulation and SA-dependent leaf senescence. ZmMEK1 interacted with Arabidopsis MPK4 in yeast and activated MPK4 in vitro. SA treatment accelerated dark-induced maize leaf senescence. Moreover, blockage of MAPK signaling increased endogenous SA accumulation in maize leaves. These findings suggest that ZmMEK1-ZmSIMK1 cascade and its modulating SA levels play important roles in regulating leaf senescence.

The Lanyu pig is an indigenous breed from Lanyu Islet, located south-east of Taiwan, with phenotypic characteristics distinctive from other pig breeds in Asia and Europe. Based on geographic considerations, the Lanyu pig may have originated from mainland China, Austronesia or the Ryukyu Islands. In the present study, polymorphism of the mitochondrial DNA control region sequence was used to clarify phylogenetic relationships among two herds of Lanyu pigs imported before 1980 from Lanyu Islet into Taiwan and reared in isolation on two different farms. Two distinct mitochondrial control region haplotypes were found. The type I Lanyu sequence appeared independently as a unique clade different from Asian and European pig sequences, while the type II Lanyu sequence was clustered within the major Asian clade. The pairwise distances between the major Asian clade vs. the type I Lanyu and European clades were 0.01726 +/- 0.00275 and 0.01975 +/- 0.00212 changes per site respectively. Estimates of divergence time suggest that the type I Lanyu sequence split from the major Asian pig clade in prehistoric times. The type II Lanyu mtDNA shares a close genetic lineage with Japanese Satsuma and New Zealand Kune Kune mtDNA with pairwise distances of 0.00095 +/- 0.00000 and 0.00192 +/- 0.00000 respectively, indicating gene flow between Lanyu Islet, Japan and Oceania in recent times. Together these results indicate that the type I Lanyu pig has a genetic lineage separate from Asian-type pigs, while the type II Lanyu sequence may represent a more recent introgression of modern Asian pigs.

Relationships among the major branches of moss phylogeny are understudied compared with other major land-plant groups. We addressed this by surveying 14-17 plastid genes from taxa representing the major lineages, using different phylogenetic methods (parsimony, likelihood) and codon- and gene-based data partitioning schemes (likelihood). Our phylogenetic inferences generally corroborated the best supported clades across multiple recent studies, with comparable or higher levels of clade support here. We resolved persistent ambiguities with strong to moderate support across analyses, including several early nodes in subclass Dicranidae, and relationships among other subclasses of peristomate mosses. In particular, we resolved a sister-group relationship between Bryidae and Dicranidae, between these subclasses and Timiidae, and between this entire clade and Funariidae. We consistently recovered Tetraphidopsida (a nematodontous class) as the sister group of arthrodontous mosses (Bryopsida), although with only weak support. Strongly conflicting arrangements at the base of moss phylogeny concerning Takakiopsida and Sphagnopsida, two non-peristomate moss lineages, were inferred in parsimony and likelihood analysis, but this depended on how base-frequency parameters were estimated and how data were partitioned in likelihood analysis. Relationships inferred for the remaining peristomate and non-peristomate moss clades, and their associated support values, were otherwise broadly congruent across analyses.

Abstract Phylogenomic studies based on genome-scale amounts of data have greatly improved understanding of the tree of life. Despite their diversity, ecological significance, and biomedical and industrial importance, large-scale phylogenomic studies of Fungi are lacking. Furthermore, several evolutionary relationships among major fungal lineages remain controversial, especially those at the base of the fungal phylogeny. To begin filling these gaps and assess progress toward a genome-scale phylogeny of the entire fungal kingdom, we compiled a phylogenomic data matrix of 290 genes from the genomes of 1,644 fungal species that includes representatives from most major fungal lineages; we also compiled 11 additional data matrices by subsampling genes or taxa based on filtering criteria previously shown to improve phylogenomic inference. Analyses of these 12 data matrices using concatenation- and coalescent-based approaches yielded a robust phylogeny of the kingdom in which ∼85% of internal branches were congruent across data matrices and approaches used. We found support for several relationships that have been historically contentious (e.g., for the placement of Wallemiomycotina (Basidiomycota), as sister to Agaricomycotina), as well as evidence for polytomies likely stemming from episodes of ancient diversification (e.g., at the base of Basidiomycota). By examining the relative evolutionary divergence of taxonomic groups of equivalent rank, we found that fungal taxonomy is broadly aligned with genome sequence divergence, but also identified lineages, such as the subphylum Saccharomycotina, where current taxonomic circumscription does not fully account for their high levels of evolutionary divergence. Our results provide a robust phylogenomic framework to explore the tempo and mode of fungal evolution and directions for future fungal phylogenetic and taxonomic studies.

The Luxi gamecock developed very unique morphological and behavioral features under the special artificial selection of the most famous Chinese gamecocks. There are very few research studies on the genetics and selection of the Luxi gamecock. We used six methods (Fst, Tajima's D, hapFLK, iHS, XP-EHH, and Runs of homozygosity) to detect selective sweeps in whole-genome resequencing data of 19 Luxi gamecocks compared to other Chinese indigenous chickens. Eleven genes that were highly related to nervous system development (CDH18, SLITRK1, SLITRK6, NDST3, ATP23, LRIG3, IL1RAPL1, GADL1, C5orf22, UGT8, WISP1, and WNT9A) appeared in at least four methods and were regarded as the most significant genes under selection. Differentially expressed gene (DEG) analysis based on the RNA sequencing data of the cerebral cortex and middle brain between six Luxi gamecocks, Tibetan chickens, and white leghorns found that most differentially expressed genes were enriched in pathways with nervous system functions. Genes associated with aggressiveness-related neurotransmitters (SLC4A2, DRD1, DRD2, ADRA2A, and ADRA2B) showed differential expression rates in Luxi gamecocks as well. Combined results showed that most genes in selective sweep regions were also differentially expressed in Luxi gamecocks including the most significant genes (SLITRK6, IL1RAPL1, GADL1, WISP1, and LRIG3). This study provides more insight into molecular mechanisms of the aggressiveness of gamecocks and aims to promote further studies on animal and human aggression.

Guppies are small tropical fish with brightly colored bodies and variable tail shapes. There are two phenotypes of domestic guppy eye color: red and black. The wild type is black-eyed. The main object of this study was to identify candidate genes for the red-eyed phenotype in domestic guppies. We hope to provide molecular genetic information for the development of new domestic guppy strains. Additionally, the results also contribute to basic research concerning guppies. In this study, 121 domestic guppies were used for genomic analysis (GWAS), and 44 genes were identified. Furthermore, 21 domestic guppies were used for transcriptomic analysis, and 874 differentially expressed genes (DEGs) were identified, including 357 upregulated and 517 downregulated genes. Through GO and KEGG enrichment, we identified some important terms or pathways mainly related to melanin biosynthesis and ion transport. qRT-PCR was also performed to verify the differential expression levels of four important candidate genes (TYR, OCA2, SLC45A2, and SLC24A5) between red-eyed and black-eyed guppies. Based on the results of genomic and transcriptomic analyses, we propose that OCA2 is the most important candidate gene for the red-eyed phenotype in guppies.

<h2>ABSTRACT</h2> Hematological parameters refer to the assessment of changes in the number and distribution of blood cells, including leukocytes (<b>LES</b>), erythrocytes (<b>ERS</b>), and platelets (<b>PLS</b>), which are essential for the early diagnosis of hematological system disorders and other systemic diseases in livestock. In this context, the primary objectives of this study were to investigate the genomic background of 19 hematological parameters in Holstein cattle, focusing on LES, ERS, and PLS blood components. Genetic and phenotypic (co)variances of hematological parameters were calculated based on the Average Information Restricted Maximum Likelihood (AIREML) method and 1,610 genotyped individuals and 5,499 hematological parameter records from 4,543 cows. Furthermore, we assessed the genetic relationship between these hematological parameters and other economically important traits in dairy cattle breeding programs. We also carried out genome-wide association studies and candidate gene analyses. Blood samples from 21 primiparous cows were used to identify candidate genes further through RNA sequencing (<b>RNA-seq</b>) analyses. Hematological parameters generally exhibited low-to-moderate heritabilities ranging from 0.01 to 0.29, with genetic correlations between them ranging from −0.88 ± 0.09 (between mononuclear cell ratio and lymphocyte cell ratio) to 0.99 ± 0.01 (between white blood cell count and granulocyte cell count). Furthermore, low to moderate approximate genetic correlations between hematological parameters with one longevity, 4 fertility, and 5 health traits were observed. One-hundred-and-99 significant single nucleotide polymorphisms (<b>SNP</b>) located primarily on the <i>Bos taurus</i> autosomes (<b>BTA</b>) BTA4, BTA6, and BTA8 were associated with 16 hematological parameters. Based on the RNA-seq analyses, 6,687 genes were significantly downregulated and 4,119 genes were upregulated when comparing 2 groups of cows with high and low phenotypic values. By integrating genome-wide association studies (<b>GWAS</b>), RNA-seq, and previously published results, the main candidate genes associated with hematological parameters in Holstein cattle were <i>ACRBP, ADAMTS3, CANT1, CCM2L, CNN3, CPLANE1, GPAT3, GRIP2, PLAGL2, RTL6, SOX4, WDFY3,</i> and <i>ZNF614</i>. Hematological parameters are heritable and moderately to highly genetically correlated among themselves. The large number of candidate genes identified based on GWAS and RNA-seq indicate the polygenic nature and complex genetic determinism of hematological parameters in Holstein cattle.

Maize (Zea mays) seeds are the major source of starch all over the world and the excellent model for researching starch synthesis. Seed starch content is a typical quantitative phenotype and many reports revealed that the glycolytic enzymes are involved in regulating starch synthesis, however the regulatory mechanism is still unclear. Here, we present a comparative phosphoproteomic study of three maize inbred lines with different seed starch content. It reveals that abundances of 62 proteins and 63 phosphoproteins were regulated during maize seed development. Dynamics of 17 enzymes related to glycolysis and starch synthesis were used to construct a phosphorylation regulatory network of starch synthesis. It shows that starch synthesis and glycolysis in maize seeds utilize the same hexose phosphates pool coming from sorbitol and sucrose as carbon source, and phosphorylation of ZmENO1 are suggested to contribute to increase starch content, because it is positively related to seed starch content in different developmental stages and different lines, and the phosphor-mimic mutant (ZmENO1S43D) damaged its enzyme activity which is vital in glycolysis. Our results provide a new sight into regulatory process of seed starch synthesis and can be used in maize breeding for high starch content.

Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant growth, development and stress responses. Here, we report that ZmMKK10, a maize MAP kinase kinase, positively regulates cell death. Sequence comparison to Arabidopsis MKKs has led to ZmMKK10 being classified as a group D MKK. Kinase activity analysis of recombinant ZmMKK10 showed that the Mg2+ ion was required for its kinase activity. Transient expression of ZmMKK10WT or ZmMKK10DD (the active form of ZmMKK10) in maize mesophyll protoplast significantly increased the cell death rate. Inducible expression of ZmMKK10WT or ZmMKK10DD in Arabidopsis transgenic plants caused rapid HR-like cell death, whereas induction of ZmMKK10KR (the inactive form of ZmMKK10) expression in transgenic plants did not yield the same phenotype. Genetic and pharmacological analysis revealed that ZmMKK10-induced cell death in transgenic plants requires the activation of Arabidopsis MPK3 and MPK6 and that it partially depended on ethylene biosynthesis. ZmMPK3 and ZmMPK7, the orthologues of Arabidopsis MPK3 and MPK6, interacted with ZmMKK10 in yeast and ZmMKK10 phosphorylated them both in vitro. Our results demonstrate that ZmMKK10 induces cell death in an ethylene-dependent manner. Furthermore, ZmMPK3 and ZmMPK7 may be the downstream MAPKs in this process.

Fungi survive in diverse ecological niches by secreting proteins and other molecules to the environment to acquire food, compete with other microorganisms, and defend against various biotic and abiotic stresses. Fungal secretome content is therefore believed to be tightly linked to fungal ecologies. The zygomycete fungi (Mucoromycota and Zoopagomycota) represent the earliest branches of terrestrial fungi, many of which remain understudied. Here we sampled 132 zygomycete fungal genomes and characterized their secretome composition, with a focus on digestive enzymes, putative small secreted proteins (SSPs), and other proteins hypothesized to function in the environment. Our analyses revealed that phylogeny played an important role in shaping the secretome composition of zygomycete fungi with trophic mode contributing a smaller amount. Reconstruction of the evolution of secreted digestive enzymes revealed lineage-specific expansions, including the diversification of plant-cell-wall-degrading-enzymes (PCWDEs) in Mucoromycota, and independent expansions of chitinases and proteases within Zoopagomycota, suggesting the two lineages followed different trajectories early in their evolutionary history. We identified the presence of multiple pathogenicity-related proteins in the lineages known as saprotrophs, suggesting that either the ecologies of these fungi are incompletely known, and/or that these pathogenicity-related proteins have important functions associated with saprotrophic ecologies, both of which invite further investigation.

Clastobryopsis imbricata sp. nov. (Pylaisiadelphaceae, Musci) is described on the basis of specimens collected at middle elevations of Doi (Mt.) Inthanon, northern Thailand. The species is characterized by strongly imbricate and spiral foliation of short ascending branches, caudate branch tips bearing smooth, filamentous gemmae in leaf axils, carinate leaves with distinct spine(s) at the base of the dorsal side of the keel, thin-walled, quadrate alar cells arranged in a scalariform manner, narrowly cylindrical capsules with perforate exostome teeth and reduced endostome, and non-collenchymatous exothecial cells. The multistratose alar cells, smooth filamentous gemmae, and non-collenchymatous exothecial cells, as well as phylogenetic inferences based on rbcL sequences, suggest that the species belongs in the genus Clastobryopsis (Pylaisiadelphaceae).

Yakushimabryum longissimum gen. & sp. nov. (Pylaisiadelphaceae, Musci), is described from Yakushima Island, Japan, on the basis of morphology and rbcL data. It is characterized by more or less glossy, yellowish green living plants, naked dormant branch buds, absence of costa in the leaves, smooth and linear leaf lamina cells, alar groups composed of a number of quadrate cells arranged in a scalariform manner, ascending branches that are often elongate into slender tips, and filamentous gemmae consisting of smooth, long-rectangular cells. Molecular phylogenetic analyses suggest an affinity neither to Clastobryopsis nor Gammiella, both of which grow in similar habitats and share alar morphology and shape of propagules, but to Isocladiella and Isopterygium, both of which have filamentous pseudoparaphyllia and different types of propagules. A key to the Japanese genera of Pylaisiadelphaceae is provided.

Abstract The polyketide griseofulvin is a natural antifungal compound and research in griseofulvin has been key in establishing our current understanding of polyketide biosynthesis. Nevertheless, the griseofulvin gsf biosynthetic gene cluster (BGC) remains poorly understood in most fungal species, including Penicillium griseofulvum where griseofulvin was first isolated. To elucidate essential genes involved in griseofulvin biosynthesis, we performed third-generation sequencing to obtain the genome of P. griseofulvum strain D-756. Furthermore, we gathered publicly available genome of 11 other fungal species in which gsf gene cluster was identified. In a comparative genome analysis, we annotated and compared the gsf BGC of all 12 fungal genomes. Our findings show no gene rearrangements at the gsf BGC. Furthermore, seven gsf genes are conserved by most genomes surveyed whereas the remaining six were poorly conserved. This study provides new insights into differences between gsf BGC and suggests that seven gsf genes are essential in griseofulvin production.

• Premise of the study : We present here new bryophyte‐specific primers that permit retrieval of 17 slowly evolving plastid genes and their associated introns and intergenic spacers. These regions were chosen to facilitate accurate phylogenetic inference across a broad range of mosses and other bryophytes. • Methods and Results: We developed 78 new primers for the targeted regions using an initial sampling of exemplar bryophytes and other green plants, to complement those used in vascular plants. We assessed the ability of the new primers to amplify and sequence these regions using a test set of 11 additional exemplar bryophytes. • Conclusions: We show that the newly designed primers facilitate ready retrieval of 14 of 17 targeted regions from a broad range of bryophyte taxa. These primers should prove useful for future studies of bryophyte phylogeny.

Author(s): Myers, JM; Bonds, AE; Clemons, RA; Thapa, NA; Simmons, DR; Carter-House, D; Ortanez, J; Liu, P; Miralles-Duran, A; Desiro, A; Longcore, JE; Bonito, G; Stajich, JE; Spatafora, JW; Chang, Y; Corrochano, LM; Gryganskyi, A; Grigoriev, IV; James, TY

The systematic position of Pterogonidium pulchellum (W.J. Hooker) Muell. Hal. in the Sematophyllaceae and Piloecium pseudorufescens (Hampe) Muell. Hal. in the Myuriaceae is reexamined with new evidence derived from rbcL gene sequences. A total of 18 taxa from Sematophyllaceae, Hypnaceae, Myuriaceae, and Hookeriaceae were included, 11 of which were newly sequenced for the rbcL gene. Analyses were done using maximum parsimony, maximum likelihood (TrN + Γ + I model and GTR + Γ + I model), and Bayesian inference (GTR + Γ + I model). The results show that Pterogonidium pulchellum has a closer relationship with members of Hypnaceae than with Sematophyllaceae, while Piloecium pseudorufescens forms a clade with the Sematophyllaceae in all phylogenetic analyses, with robust branch support. Two SOWH tests were carried out and the results show that it is not appropriate to classify Pterogonidium pulchellum in Sematophyllaceae and it is also not appropriate to classify Piloecium pseudorufescens in Myuriaceae.

Research into secondary metabolism (SM) production by fungi has resulted in the discovery of diverse, biologically active compounds with significant medicinal applications. However, the fungi rich in SM production are taxonomically restricted to Dikarya, two phyla of Kingdom Fungi, Ascomycota and Basidiomycota. Here, we explore the potential for SM production in Mucoromycota and Zoopagomycota, two phyla of nonflagellated fungi that are not members of Dikarya, by predicting and identifying core genes and gene clusters involved in SM. The majority of non-Dikarya have few genes and gene clusters involved in SM production except for the amphibian gut symbionts in the genus Basidiobolus . Basidiobolus genomes exhibit an enrichment of SM genes involved in siderophore, surfactin-like, and terpene cyclase production, all these with evidence of constitutive gene expression. Gene expression and chemical assays confirm that Basidiobolus has significant siderophore activity. The expansion of SMs in Basidiobolus are partially due to horizontal gene transfer from bacteria, likely as a consequence of its ecology as an amphibian gut endosymbiont.

Understanding the underlying pleiotropic relationships among growth and body size traits is important for refining breeding strategies in dairy cattle for optimal body size and growth rate. Therefore, we performed single-trait genome-wide association studies (GWAS) for monthly-recorded body weight (BW), hip height (HH), body length (BL) and chest girth (CG) from birth to 12 mo of age in Holstein animals, followed by stepwise multiple regression of independent or lowly-linked markers from GWAS loci using conditional and joint association analyses (COJO). Subsequently, we conducted a multi-trait meta-analysis to detect pleiotropic markers. Based on the single-trait GWAS, we identified 170 significant single nucleotide polymorphisms (SNPs), in which 59 of them remained significant after the COJO analyses. The most significant SNP, located at BTA7:3,676,741, explained 2.93% of the total phenotypic variance for BW6 (body weight at 6 mo of age). We identified 17 SNPs with potential pleiotropic effects based on the multi-trait meta-analyses, which resulted in 3 additional SNPs in comparison to those detected based on the single-trait GWAS. The identified quantitative trait loci (QTL) regions overlap with genes known to influence human growth-related traits. According to positional and functional analyses, we proposed HMGA2, HNF4G, MED13L, BHLHE40, FRZB, DMP1, TRIB3, and GATAD2A as important candidate genes influencing the studied traits. The combination of single-trait GWAS and meta-analyses of GWAS results improved the efficiency of detecting associated SNPs, and provided new insights into the genetic mechanisms of growth and development in Holstein cattle.

ABSTRACT Fungi have evolved over millions of years and their species diversity is predicted to be the second largest on the earth. Fungi have cross-kingdom interactions with many organisms which have mutually shaped their evolutionary trajectories. Zygomycete fungi hold a pivotal position in the fungal tree of life and provide important perspectives on the early evolution of fungi from aquatic to terrestrial environments. Phylogenomic analyses have found that zygomycete fungi diversified into two separate clades, the Mucoromycota which are frequently associated with plants and Zoopagomycota that are commonly animal-associated fungi. Genetic elements that contributed to the fitness and divergence of these lineages may have been shaped by the varied interactions these fungi have had with plants, animals, bacteria and other microbes. To investigate this, we performed comparative genomic analyses of the two clades in the context of Kingdom Fungi, benefiting from our generation of a new collection of zygomycete genomes. We identified lineage-specific genomic content which may contribute to the disparate biology observed in these zygomycetes. Our findings include the discovery of undescribed diversity in CotH, a Mucormycosis pathogenicity factor, which was found in a broad set of zygomycetes. Reconciliation analysis identified multiple duplication events and an expansion of CotH copies throughout Mucoromycotina, Mortierellomycotina, Neocallimastigomycota, and Basidiobolus lineages. A kingdom-level phylogenomic analysis also identified new evolutionary relationships within the sub-phyla of Mucoromycota and Zoopagomycota.

Additional file 2: Table S1. Differential gene expression between non-heat stress group and heat stress group.

Abstract The zoosporic obligate endoparasites, Olpidium , hold a pivotal position to the reconstruction of the flagellum loss in fungi, one of the key morphological transitions associated with the colonization of land by the early fungi. We generated genome and transcriptome data from non-axenic zoospores of Olpidium bornovanus and used a metagenome approach to extract phylogenetically informative fungal markers. Our phylogenetic reconstruction strongly supported Olpidium as the closest zoosporic relative of the non-flagellated terrestrial fungi. Super-alignment analyses resolved Olpidium as sister to the non-flagellated terrestrial fungi, whereas a super-tree approach recovered different placements of Olpidium , but without strong support. Further investigations detected little conflicting signal among the sampled markers but revealed a potential polytomy in early fungal evolution associated with the branching order among Olpidium , Zoopagomycota and Mucoromycota. The branches defining the evolutionary relationships of these lineages were characterized by short branch lengths and low phylogenetic content and received equivocal support for alternative phylogenetic hypotheses from individual markers. These nodes were marked by important morphological innovations, including the transition to hyphal growth and the loss of flagellum, which enabled early fungi to explore new niches and resulted in rapid and temporally concurrent Precambrian diversifications of the ancestors of several phyla of fungi.