Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=umyc20 Mycologia ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/umyc20 Whole genome sequencing and phylogenomic analysis show support for the splitting of genus Pythium Hai D. T. Nguyen, Annette Dodge, Kasia Dadej, Tara L. Rintoul, Ekaterina Ponomareva, Frank N. Martin, Arthur W. A. M. de Cock, C. André Lévesque, Scott A. Redhead & Christoffel F. J. Spies To cite this article: Hai D. T. Nguyen, Annette Dodge, Kasia Dadej, Tara L. Rintoul, Ekaterina Ponomareva, Frank N. Martin, Arthur W. A. M. de Cock, C. André Lévesque, Scott A. Redhead & Christoffel F. J. Spies (2022) Whole genome sequencing and phylogenomic analysis show support for the splitting of genus Pythium, Mycologia, 114:3, 501-515, DOI: 10.1080/00275514.2022.2045116 To link to this article: https://doi.org/10.1080/00275514.2022.2045116 © 2022 Copyright of the Crown in Canada. Published with license by Taylor & Francis Group, LLC. View supplementary material Published online: 06 May 2022. Submit your article to this journal Article views: 4721 View related articles View Crossmark data Citing articles: 21 View citing articles https://www.tandfonline.com/action/journalInformation?journalCode=umyc20 https://www.tandfonline.com/journals/umyc20?src=pdf https://www.tandfonline.com/action/showCitFormats?doi=10.1080/00275514.2022.2045116 https://doi.org/10.1080/00275514.2022.2045116 https://www.tandfonline.com/doi/suppl/10.1080/00275514.2022.2045116 https://www.tandfonline.com/doi/suppl/10.1080/00275514.2022.2045116 https://www.tandfonline.com/action/authorSubmission?journalCode=umyc20&show=instructions&src=pdf https://www.tandfonline.com/action/authorSubmission?journalCode=umyc20&show=instructions&src=pdf https://www.tandfonline.com/doi/mlt/10.1080/00275514.2022.2045116?src=pdf https://www.tandfonline.com/doi/mlt/10.1080/00275514.2022.2045116?src=pdf http://crossmark.crossref.org/dialog/?doi=10.1080/00275514.2022.2045116&domain=pdf&date_stamp=06 May 2022 http://crossmark.crossref.org/dialog/?doi=10.1080/00275514.2022.2045116&domain=pdf&date_stamp=06 May 2022 https://www.tandfonline.com/doi/citedby/10.1080/00275514.2022.2045116?src=pdf https://www.tandfonline.com/doi/citedby/10.1080/00275514.2022.2045116?src=pdf Whole genome sequencing and phylogenomic analysis show support for the splitting of genus Pythium Hai D. T. Nguyena, Annette Dodgea, Kasia Dadeja, Tara L. Rintoula, Ekaterina Ponomarevaa, Frank N. Martinb, Arthur W. A. M. de Cockc, C. André Lévesquea, Scott A. Redheada, and Christoffel F. J. Spiesd aOttawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, Ontario, K1A 0C6 Canada; bCrop Improvement and Protection Research, Agricultural Research Service, United States Department of Agriculture, Salinas, California 93905, USA; cWesterdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; dPlant Microbiology, Agricultural Research Council - Plant Health and Protection, Private Bag X5017, Stellenbosch, 7599, South Africa ABSTRACT The genus Pythium (nom. cons.) sensu lato (s.l.) is composed of many important species of plant pathogens. Early molecular phylogenetic studies suggested paraphyly of Pythium, which led to a formal proposal by Uzuhashi and colleagues in 2010 to split the genus into Pythium sensu stricto (s.s.), Elongisporangium, Globisporangium, Ovatisporangium (= Phytopythium), and Pilasporangium using morphological characters and phylogenies of the mt cytochrome c oxidase subunit 2 (cox2) and D1–D2 domains of nuc 28S rDNA. Although the split was fairly justified by the delineating morphological characters, there were weaknesses in the molecular analyses, which created reluc- tance in the scientific community to adopt these new genera for the description of new species. In this study, this issue was addressed using phylogenomics. Whole genomes of 109 strains of Pythium and close relatives were sequenced, assembled, and annotated. These data were combined with 10 genomes sequenced in previous studies. Phylogenomic analyses were performed with 148 single- copy genes represented in at least 90% of the taxa in the data set. The results showed support for the division of Pythium s.l. The status of alternative generic names that have been used for species of Pythium in the past (e.g., Artotrogus, Cystosiphon, Eupythium, Nematosporangium, Rheosporangium, Sphaerosporangium) was investigated. Based on our molecular analyses and review of the Pythium generic concepts, we urge the scientific community to adopt the generic names Pythium, Elongisporangium, Globisporangium, and their concepts as proposed by Uzuhashi and colleagues in 2010 in their work going forward. In order to consolidate the taxonomy of these genera, some of the recently described Pythium spp. are transferred to Elongisporangium and Globisporangium. ARTICLE HISTORY Received 13 September 2021 Accepted 18 February 2022 KEYWORDS Illumina sequencing; oomycetes; 21 new taxa INTRODUCTION The genus Pythium Pringsheim, nom. cons., sensu lato (s.l.), non Pythium Nees was described in 1858. Today, it is considered to belong to the order Peronosporales, class Peronosporomycetes, phylum Oomycota, and kingdom Straminipila (Beakes and Thines 2017). Members of this genus are primarily known as patho- gens that can infect a wide variety of plant hosts, algae, fungi, other oomycetes, as well as nematodes, insects, crustaceans, and fish. Pythium species are well known to plant pathologists because many infect below-ground plant parts such as fine roots or germinating seeds, resulting in seedling damping-off and root rot, subse- quently affecting crop yields. Pringsheim (1858) included two species in his origi- nal description of Pythium: P. monospermum, the con- served type species of the genus, and P. entophytum, now recognized as a holocarpic oomycete currently classified as a species of Aphanomycopsis. The main distinguishing feature of Pythium was considered to be the extraspor- angial differentiation of zoospores. Consequently, spe- cies with extrasporangial zoospore differentiation, but highly variable sporangial shapes, were added to this genus in subsequent years, leading to more than 200 species being classified in Pythium to date. Early taxo- nomic studies on the generic and subgeneric classifica- tion of Pythium recognized sporangial characteristics as important diagnostic features. Attempts were made to divide the genus based on sporangial shape, with names such as Nematosporangium introduced for species with filamentous sporangia and Sphaerosporangium or Eupythium for species with globose, subglobose, or citri- form sporangia (Fischer 1892; Nieuwland 1916; Schröter 1893; Sparrow 1931). However, these names are either CONTACT Hai D. T. Nguyen hai.nguyen2@agr.gc.ca; Christoffel F. J. Spies SpiesC@arc.agric.za, Supplemental data for this article can be accessed on the publisher’s Web site. MYCOLOGIA 2022, VOL. 114, NO. 3, 501–515 https://doi.org/10.1080/00275514.2022.2045116 © 2022 Copyright of the Crown in Canada. Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc- nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. Published online 06 May 2022 https://doi.org/10.1080/00275514.2022.2045116 http://www.tandfonline.com https://crossmark.crossref.org/dialog/?doi=10.1080/00275514.2022.2045116&domain=pdf&date_stamp=2022-06-02 invalid or superfluous as defined by the International Code of Nomenclature for algae, fungi and plants (Shenzhen Code; Turland et al. 2018) as discussed in Nomenclature and Taxonomy below; consequently, Pythium remained the current name for the genus. More recently, the use of DNA sequencing and mole- cular phylogenetics became popular and required for biosystematics studies. Early molecular studies revealed the paraphyletic nature of Pythium, and suggestions to split the genus re-emerged (Briard et al. 1995; Cooke et al. 2000). Based on the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) and D1–D3 domains of nuc 28S rDNA phylogenies, Lévesque and de Cock (2004) divided Pythium into 11 clades (A to K) comprising three major groups: species with filamentous sporangia (clades A to C), species with contiguous spor- angia (clade D), and species with globose or ovoid spor- angia (clades E to K). Members of clade K were placed in a newly created genus called Phytopythium (Bala et al. 2010b; de Cock et al. 2015). Molecular phylogenies of the nuc 28S rRNA, ITS, mt cytochrome c oxidase sub- units 1 and 2 (cox1 and cox2), and nuc β-tubulin regions suggested that the remaining 10 clades (A to J) could be divided into two groups: species with filamentous or contiguous zoosporangia (clades A to D) and species with globose zoosporangia (clades E to J) (Hulvey et al. 2010; Lévesque and de Cock 2004; Martin 2000; Riethmüller et al. 2002; Robideau et al. 2011; Villa et al. 2006). However, phylogenetic delineation of genera was complicated by incongruence of the different gene regions and low support for internal nodes. Despite the shortcomings of these early molecular phylogenetic results, Uzuhashi et al. (2010) used D1–D2 domains of nuc 28S rDNA and mt cox2 phylogenies to divide Pythium s.l. into five genera: Pythium sensu stricto (s.s.) (clades A–D), Globisporangium (clades E–G, I, and J), Elongisporangium (clade H), Ovatisporangium (clade K, synonym Phytopythium), and Pilasporangium (distinct from the 11 lettered clades). Uzuhashi et al. (2010) char- acterized Pythium species by filamentous (both nonin- flated and inflated) sporangia. Sporangia in species of the remaining genera are ovoid or pyriform in Ovatisporangium (= Phytopythium), clavate or elongated in Elongisporangium, globose and proliferating in Globisporangium, and globose but not proliferating in Pilasporangium. This proposal was sound morphologically, but it was problematic from a molecular perspective, as there was a lack of significant statistical support for the Globisporangium clade in the nuc 28S rDNA and mt cox2 phylogenies and for the Pythium clade in the mt cox2 phylogeny (see figs. 1 and 2 in Uzuhashi et al. (2010)). Another problem with these phylogenies was that Albugo was placed inside Pythium s.l. on a long branch (fig. 1 in Uzuhashi et al. 2010). Other phylogenetic analyses including additional conventional phylogenetic markers such as β-tubulin, cox1, and ITS revealed that the genera Pythiogeton and Lagena were situated within or closely related to Pythium s.s. (Hyde et al. 2014; Spies et al. 2016), whereas the lack of statistical support for the Globisporangium clade persisted (Hyde et al. 2014). These are some of the reasons researchers have generally been slow to adopt the newly proposed genera. If the backbone relationships could be resolved with single-copy genes extracted from whole-genome sequences and used in phylogenomic analyses, the phy- logenetic issues partly responsible for the reluctance to accept the newly proposed genera in Pythium s.l. could be addressed. The current low cost of next-generation sequencing (NGS) has allowed for affordable whole- genome sequencing. This was less feasible in the early 2000s when most of the key phylogenetic papers on Pythium were published. In the research presented here, the lack of phylogenetic support for the genera intro- duced by Uzuhashi et al. (2010) is addressed using a phylogenomic approach. Draft genome assemblies were generated and annotated for a collection of 109 strains representative of Pythium s.l. This collection included mostly authenticated and type strains studied by van der Plaats-Niterink (1981a) in the various clades designated by Lévesque and de Cock (2004), as well as type strains of several more recently described species. Phylogenetic analyses were subsequently conducted with over a hundred single-copy genes to resolve the uncertain relationships and low support that have thus far drawn the Pythium split proposed by Uzuhashi et al. (2010) into doubt. Additionally, the historical nomenclature of Pythium is reconsidered in light of the revisions proposed by Uzuhashi et al. (2010) and new combinations made where necessary to consolidate generic concepts. Materials and methods Selection of strains and species for sequencing.—A total of 109 strains were sequenced for this study (see SUPPLEMENTARY TABLE 1). These included species of Pythium s.l., Phytopythium, Halophytophthora, and other miscellaneous species that were found to group within Pythium s.l. or were closely related in previous studies, including Salisapilia sapeloensis, Pilasporangium apinafur- cum, and Lagenidium sp. (PWL-2010h). Phytophthora was considered to be out of scope for this study. Among the 109 sequenced isolates, 65 are ex-types and two were strains used for description in the monograph by van der Plaats- Niterink (1981a) (see SUPPLEMENTARY TABLE 1). A total of 90 Pythium s.l. genomes were sequenced, of 502 NGUYEN ET AL.: PYTHIUM PHYLOGENOMICS which 86 represent species that had no prior genome data published. Four species were sequenced in previous studies: Pythium irregulare (Adhikari et al. 2013), Pythium oligan- drum (Kushwaha et al. 2017a), Pythium periplocum (Kushwaha et al. 2017b), and Pythium ultimum var. ulti- mum (Lévesque et al. 2010). An additional 14 genomes of Phytopythium spp. were sequenced, of which 13 had no published whole-genome sequences. In terms of Pythium as classified by Uzuhashi et al. (2010), this collection included 37 genomes of Pythium s.s., 48 genomes of Globisporangium, and five genomes of Elongisporangium. When considering the older lettered clade designations from Lévesque and de Cock (2004), at least four represen- tatives from each clade were included, except for clade C, which had only two species described. An additional 10 genomes were downloaded from the National Center for Biotechnology Information (NCBI): Paralagenidium kar- lingii, Phytopythium vexans (Adhikari et al. 2013), Pilasporangium apinafurcum, and several species of Pythium including Pythium insidiosum (Rujirawat et al. 2015). Two strains for which genome data were down- loaded from NCBI were also resequenced in the current study: Pythium irregulare CBS 250.28 (= DAOM BR 486) and Pilasporangium apinafurcum JCM 30513 (= DAOMC 242887). The entire data set was composed of 119 genomes for phylogenetic analysis, providing enough taxonomic breadth to capture the diversity in Pythium s.l. appropriately. DNA extraction and sequencing.—Mycelia from 10– 14-d-old liquid cultures grown in 2% V8 broth at room temperature were harvested. DNA was extracted follow- ing the protocol of Möller et al. (1992) with a modification to the tissue lysis step. Instead of grinding mycelia in liquid nitrogen, mycelia were placed in 2-mL screw cap tubes containing 0.5-mm glass beads (Precellys VK05 lysing kit; Bertin, Rockville, Maryland), along with TES buffer (100 mM Tris pH 8.0, 10 mM EDTA [ethylenedia- minetetraacetic acid], 2% SDS [sodium dodecyl sulfate]), RNase A/T1 cocktail (Thermo Fisher Scientific, Waltham, Massachusetts), and proteinase K. Lysis was achieved by shaking tubes in a Precellys24 tissue homogenizer (Bertin) for 40 s at a speed of 6000 rpm. Tubes were incubated at 65 C for 1 h, and subsequent steps were performed following the original protocol. At the final step, the DNA pellet was resuspended in 0.1× TE buffer (1× TE =10 mM Tris ph 8.0, 1 mM EDTA [ethylenedia- minetetraacetic acid] diluted to 0.1× by adding 1ml I× TE in 9 ml sterile distilled water) containing 50 μg/mL RNase A, and tubes were incubated at 65 C for 10 min. Prior to NGS, sample identity was verified by DNA barcode sequencing and analysis of ITS and cox1 following proto- cols of Robideau et al. (2011) (data not shown). For each sample (from sequencing batches 0, 1, 2, and 3 in SUPPLEMENTARY TABLE 1), 300 ng of genomic DNA was sheared to 300 or 350 bp with the 8 microTUBE-15 Strip V2 using Covaris LE220 Focused- ultrasonicator (Covaris, Woburn, Massachusetts) fol- lowing the manufacturer’s protocols. The obtained insert fragments were used as a template to construct polymerase chain reaction (PCR) free libraries for dual indexing with NxSeq AmpFREE Low DNA Library Kit (LGC, Biosearch Technologies, Middleton, Wisconsin) and with IDT for Illumina TruSeq UD indexes or TruSeq DNA CD indexes (96 indexes) (Illumina, San Diego, California) following the LGC’s library protocol. Paired-end sequencing was performed on an Illumina NextSeq instrument at the Molecular Technologies Laboratory (Ottawa Research and Development Center, Agriculture and Agri-Food Canada). For some of the Pythium and Phytopythium species (sequencing batch FM in SUPPLEMENTARY TABLE 1), TruSeq Nano DNA libraries (Illumina) were made with ca. 400 bp inserts, and Illumina sequencing (150 bp paired-end) was done at the Genomics Core Facility at Michigan State University (East Lansing, Michigan). Genome assembly and genome annotation.—The bbduk.sh program from BBTOOLS 38.22 (https://jgi.doe. gov/data-and-tools/bbtools/) was used to trim the raw reads and remove adapters (bbduk.sh ref=adapters qtrim=rl trimq=20 minlength=36 ktrim=r forcetrimleft=10 forcetrim- right2=10 tossjunk=t). The quality of the raw and trimmed reads was assessed with FASTQC 0.11.8 (https://www.bioin formatics.babraham.ac.uk/projects/fastqc/). Genome assembly was performed with MEGAHIT 1.1.4 (Li et al. 2015, 2016) with default parameters (k = 21, 29, 39, 59, 79, 99, 119, 141). Contigs were re-ordered from longest to shortest, and contigs shorter than 1000 bp were discarded. Genome assembly statistics were obtained with QUAST 5.0.2 (Gurevich et al. 2013). To evaluate the completeness of the assemblies, BUSCO (Benchmarked Universal Single Copy Orthologs) analyses, using the eukaryota_odb9 and stra- menopiles_odb10 databases, were conducted with BUSCO 3.0.2 (Waterhouse et al. 2018). Sequencing coverage was estimated by mapping the reads back to the assembly with the bbmap.sh program from BBTOOLS with default para- meters, and the resulting alignment (as sorted bam files) were loaded into QUALIMAP 2.2.2 (Okonechnikov et al. 2016). MYCOLOGIA 503 https://jgi.doe.gov/data-and-tools/bbtools/ https://jgi.doe.gov/data-and-tools/bbtools/ https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ Draft genome annotation was performed with the FUNANNOTATE 1.5.2 (https://github.com/nextgenusfs/ funannotate) pipeline following the standard procedure outlined in the “Genome assembly only” tutorial (https://funannotate.readthedocs.io/en/latest/tutorials. html). Briefly, assemblies were repeat-masked with the funannotate mask command, followed by ab initio gene prediction with the funannotate predict command. The funannotate predict command first uses DIAMOND 0.9.21 (Buchfink et al. 2021) and EXONERATE 2.4.0 (Slater and Birney 2005) to map a set of pre-downloaded proteins from UniProtKB/Swiss-Prot database (release Feb 2019) (Bateman 2019) to the input masked genome assembly. It then runs GENEMARK-ES 4.33 (Borodovsky and Lomsadze 2011) to generate one set of gene models, followed by training of AUGUSTUS 3.3.1 (Stanke et al. 2008) with BUSCO data leading to the generation of a second set of gene models. EVIDENCEMODELER (EVM) 1.1.1 (Haas et al. 2008) is used to combine the sets of gene models from ab initio gene predictions to give a final set of gene models. The funannotate predict command was run with these options: –busco_db eukar- yota_odb9–busco_seed_species toxoplasma –organism other. This step produced protein FASTA files of the gene predicted for each genome, which were used in the phylogenomic analyses described below. All genome statistics are summarized in SUPPLEMENTARY TABLE 1. Raw sequence data and assemblies were uploaded to NCBI under BioProject PRJNA601986. Contact the corresponding author for the larger draft annotation files or visit Data Dryad (https://datadryad.org/stash) to download them. Phylogenomic analysis and characterization of genes/gene trees.—Using the protein sequences deter- mined from the genome annotation step above, ortholo- gous group analysis was performed with ORTHOFINDER 2.5.2 (Emms and Kelly 2019) on 119 genomes with default settings. Only 18 single-copy genes were found to be present in all 119 genomes. Since the whole-genome sequencing and annotation are drafts, a 90% taxa thresh- old approach was taken. This is where a single-copy gene would be considered for subsequent phylogenomic ana- lysis only if it is present in ≥107 genomes (i.e., 90% of 119 genomes) and does not appear twice in a given genome (i.e., single copy), resulting in 148 genes retained for phylogenomic analysis. The selected 148 single-copy loci were analyzed with INTERPROSCAN 5.50-84.0 (Jones et al. 2014), and results were tabulated in SUPPLEMENTARY TABLE 2. A 70% taxa threshold approach was also taken for comparison, resulting in 193 genes shown to be single copy in ≥83 genomes. Phylogenomic analysis was performed following a similar methodology from Spatafora et al. (2016) and Nguyen et al. (2019). Briefly, amino acid sequences were aligned with MUSCLE 3.8.1551 (Edgar 2004) and automati- cally trimmed with TRIMAL 1.4.rev15 (Capella-Gutierrez et al. 2009) using the -automated1 option. Maximum like- lihood trees with fast bootstrapping were calculated with RAXML 8.2.12 (Stamatakis 2014) with options -m PROTGAMMAAUTO -x 121 -f a -p 123 -N 100. Using the bipartition trees of individual genes and their respective bootstrapping trees, a multilocus bootstrapping analysis was performed with ASTRAL-III 5.7.4 (Zhang et al. 2018) to obtain the greedy consensus tree as a cladogram (https://github.com/smirarab/ASTRAL/blob/master/ astral-tutorial.md#multi-locus-bootstrapping). A concate- nated tree was also generated using the 148 single-copy genes (90% taxa threshold). Briefly, alignments were con- catenated with the catfasta2phyml.pl script (https://github. com/nylander/catfasta2phyml), and a maximum likelihood analysis was performed with RAxML as described above. Amino acid alignments and generated trees are provided as a packaged SUPPLEMENTARY FILE 1. Trimmed alignment summary statistics were calcu- lated with AMAS (Borowiec 2016). The AfterPhylo.pl script (https://github.com/qiyunzhu/AfterPhylo) was used to calculate the average bootstrap support of each tree. The topological distance (RF distance) between each tree and the 90% threshold ASTRAL-III greedy consensus tree was calculated using the ETE3 python library (http://etetoolkit. org/documentation/ete-compare/). These metrics are summarized in SUPPLEMENTARY TABLE 3. RESULTS Genome statistics.—The summary of the important genome statistics from our 109 draft genomes are shown in TABLE 1 and FIG. 1. The average estimated coverage was ~34× (median = ~28×), giving BUSCO completeness scores of >90% for both the Eukaryota and stramenopiles analyses, with BUSCO duplication of <2% on average. This suggests that an adequate amount of sequencing data was generated to capture most of the gene space, as genomes with higher coverage also return near-perfect BUSCO completeness scores as the ones with lower coverage (FIG. 1A). The average assembly size was ~41 Mb (median = ~40.6 Mb). Genomes with larger assembly sizes tended to have higher number of contigs. The genomes with higher number of contigs tended to have lower N50 scores, and larger assembly sizes tended to have lower N50 scores as well (FIG. 1B). This indicates that there is room for improving the contiguity of the assemblies with long-read sequencing technologies such as PacBio 504 NGUYEN ET AL.: PYTHIUM PHYLOGENOMICS https://github.com/nextgenusfs/funannotate https://github.com/nextgenusfs/funannotate https://funannotate.readthedocs.io/en/latest/tutorials.html https://funannotate.readthedocs.io/en/latest/tutorials.html https://datadryad.org/stash https://github.com/smirarab/ASTRAL/blob/master/astral-tutorial.md#multi-locus-bootstrapping https://github.com/smirarab/ASTRAL/blob/master/astral-tutorial.md#multi-locus-bootstrapping https://github.com/nylander/catfasta2phyml https://github.com/nylander/catfasta2phyml https://github.com/qiyunzhu/AfterPhylo http://etetoolkit.org/documentation/ete-compare/ http://etetoolkit.org/documentation/ete-compare/ or Oxford Nanopore. The average number of gene mod- els found was 15 144 (median = 14 562). The number of gene models was higher in larger assembly sizes, as expected. However, the number of gene models did not necessarily increase with higher sequencing cover- age, reiterating that the amount of sequencing was Table 1. Summary of important genome statistics of the 109 sequenced genomes and 10 genomes downloaded from NCBI. Statistic Coverage (x) Number of contigs Assembly size (bp) GC (%) N50 (bp) BUSCO completeness (eukaryota/stramenopiles) No. gene models predicted Average 34 5843 41 386 811 56.4 20 913 90%/97% 15 144 Median 28 4991 40 610 102 56.3 17 505 90%/98% 14 562 Maximum 195 21 010 67 182 254 63.1 60 250 94%/100% 33 613 Minimum 13 1365 22 437 313 45.7 3366 77%/86% 9119 Figure 1. Correlation between gene space completeness (BUSCO), coverage, number of contigs, N50, assembly size, and number of gene models for all genomes sequenced. A. Scatterplot illustrating the correlation between BUSCO completeness scores and estimated coverage. B. Scatterplot showing the correlation between number of contigs/N50 and assembly size, as well as between N50 and the number of contigs. C. Scatterplot showing the relationship between assembly size/coverage and the number of gene models. MYCOLOGIA 505 adequate to capture most of the possible genes for phy- logenomic analysis (FIG. 1C). Taken together, the data quality and quantity were sufficient for phylogenomic analyses. Phylogenomic analyses and characterization of genes and gene trees.—The amino acid sequences of single-copy genes represented by at least 90% of the genomes in the data set were extracted and analyzed (SUPPLEMENTARY TABLE 3). Initially, 148 single- copy loci were considered for the main phylogenomic analysis. The trimmed alignments had an average length of 238 sites (median = 194 sites) where ~60% of sites were variable on average. Maximum likelihood analyses with bootstrapping were performed on the individual protein alignments. To obtain the overall signal and find nodes that repre- sent genealogical concordance, a greedy consensus cla- dogram was generated based on analyses of the 148 single-copy orthologous genes shared between the 119 genomes (FIG. 2). In this analysis, the lettered clades by Lévesque and de Cock (2004) were monophyletic and well supported (99–100% bootstrap support), with the exception of clade A where P. aphanidermatum was distinct from the remaining clade A species and clade C where P. grandisporangium and P. insidiosum were not grouped monophyletically. For comparison, the same analysis was performed at a 70% taxa threshold approach (193 genes that showed to be single copy in ≥83 genomes). The greedy consen- sus cladogram was nearly identical to the 90% taxa threshold tree (SUPPLEMENTARY FIG. 1). The ETE3 analysis reported the topology of the two trees to be 98% identical. The main difference between these two trees was that in the 90% threshold tree, P. grandisporangium grouped monophyletically with Pythium species from clade D, whereas in the 70% threshold tree it occupied a basal position to other isolates of Pythium s.s. The 90% taxa threshold consensus tree was also compared with the concatenated tree (SUPPLEMENTARY FIG. 2), and the topology of those two trees were 95% identical. The notable differences here were that Salisapilia sapeloensis CBS 127946 was sister to Globisporangium in the ASTRAL consensus tree but sister to all other taxa in the concatenated tree and Lagenidium sp. (PWL-2010 h) CBS 127285 was sister to Pythium clades A and B in the ASTRAL consensus tree but sister to the P. insidiosum (clade C) in the concatenated tree. However, the impor- tant nodes that represented the new genera proposed by Uzuhashi et al. (2010) remained well supported throughout all analyses. When considering the individual trees that made up the 90% taxa threshold phylogenetic analysis, their average bootstrap was only 47%, but each tree resembled the final consensus tree (%ref_br) by 74% on average (SUPPLEMENTARY TABLE 3). Despite the overall low average bootstrap values of individual trees, the nodes that represent the split proposed by Uzuhashi et al. (2010) are still well supported in the final greedy consensus tree, which suggests a strong signal for divergence from a common ancestor at those nodes (FIG. 2). NOMENCLATURE AND TAXONOMY Notes on the nomenclatural history of Pythium.— Pythium Pringsh. 1858 is a conserved name over the unty- pified name Pythium Nees 1823, as proposed by Waterhouse (1968). Historically, three different alternative generic names have been used for species of Pythium (equating to clades A to D sensu Lévesque and de Cock 2004). Artotrogus Montagne 1849 (type species A. hydnosporus = P. hydnosporum, clade D) antedates Pythium Pringsh., but the latter has been conserved against the former (Korf 1988; van der Plaats-Niterink 1981b). Nematosporangium was introduced at subgenus level by Fischer (1892) and raised to genus level by Schröter (1893). Morphologically, this genus included Pythium species with filamentous sporangia delimited from vegetative hyphae by a septum. Schröter (1893) included the conserved type species of Pythium (P. monospermum) in Nematosporangium, thereby making the otherwise valid and legitimate name superfluous. The remaining name is Rheosporangium, introduced by Edson (1915) for descrip- tion of R. aphanidermatum (= P. aphanidermatum). This species was recognized as a species of Pythium by Fitzpatrick (1923) and shown to form part of clade A (Lévesque and de Cock 2004), i.e., phylogenetically Rheosporangium is a taxonomic synonym of Pythium. For these reasons, Pythium as treated by Uzuhashi et al. (2010) currently remains the available valid name of species in clades A to D sensu Lévesque and de Cock (2004). Several alternative generic names for Pythium species with globose to elongate sporangia (i.e., clades E–K) have also been used in the past. Roze and Cornu (1869) intro- duced the genus Cystosiphon for a new species (C. pythioides) with globose sporangia, Pythium-like zoos- pore discharge, and reticulate oospores. This species was transferred to Pythium as P. cystosiphon by Lindstedt (1872) and later corrected as P. pythioides by Ramsbottom (1916). However, since reticulate oospores are not known in any species included in the traditional concept of Pythium, Dick (2001) treated this as a separate 506 NGUYEN ET AL.: PYTHIUM PHYLOGENOMICS Figure 2. ASTRAL greedy consensus cladogram based on analyses of individual bootstrap trees of the 148 single-copy orthologous genes shared between the 119 genomes. Support values show the percentage of bootstrap replicates that contain that branch. The tree was rooted to Paralagenidium karlingii 1391. Both Lévesque and de Cock (2004) lettered clade and Uzuhashi et al. (2010) new genera are labeled on the side. Ex-types are indicated by (T). MYCOLOGIA 507 genus. In the absence of molecular data or other evidence to suggest otherwise, Cystosiphon remains classified as a distinct genus. Schröter (1893), who treated species with filamentous sporangia as Nematosporangium, con- sidered Pythium to include only species with globose to lemon-shaped sporangia and introduced the subgenera Eupythium (species with smooth-walled oogonia) and Artotrogus (species with spiny oogonia). Nieuwland (1916) raised Eupythium to genus level but included Pythium as a synonym of his genus. However, since Pythium Pringsh. is a now conserved valid name that antedates Eupythium, the latter is unavailable. Fischer (1892) introduced Pythium subgenus Sphaerosporangium for species with globose or ellipsoi- dal sporangia. Sparrow (1931) was hesitant to make a decision regarding the classification of Pythium spe- cies with spherical or subspherical sporangia but sug- gested that Sphaerosporangium could be raised to generic level for these, or that they should be included in Phytophthora. He then proceeded to introduce Sphaerosporangium as a new genus, but the generic description provided is for “Phytophthora or Sphaerosporangium n. gen.” and the sporangial charac- teristics given would include Phytophthora as well as Pythium species from clades E to K. No new combina- tions were made, and no species that should be included were mentioned by name. The name was not validly published because it was proposed in anticipa- tion of its future acceptance (Art. 36.1). Furthermore, the original concept of Sphaerosporangium was of a now known paraphyletic group of taxa, whether con- sidering it as a genus (Sparrow 1931), which includes at least Phytophthora and Pythium clades E to K, or as a subgenus (Fischer 1892), which includes Cystosiphon and Pythium clades D to K. Although some other names were introduced for species of Pythium s.l. at the subgeneric level as sections (e.g., sect. Aplerospora, sect. Plerospora, sect. Metasporangium, sect. Orthosporangium) or subgenera (e.g., subg. Aphragmium, subg. Piatyphalla, subg. Stenophalla), these have no standing at generic level and would not compete with any of the generic names that have subsequently been used for this genus. Consequently, the names introduced by Uzuhashi et al. (2010) are legitimate and valid, with the exception of Ovatisporangium, which is a later synonym of Phytopythium Abad et al. (Bala et al. 2010b; de Cock et al. 2015). Changes in taxonomy.—Twenty species phylogeneti- cally grouping within Globisporangium (Abrinbana et al. 2016; Badali et al. 2020; Bahramisharif et al. 2013; Bala et al. 2010a; Bouket et al. 2015; Chen et al. 2021; Ellis et al. 2012; Karaca et al. 2009; Long et al. 2014, 2012; Paul et al. 2008; Rahman et al. 2015; Tojo et al. 2012; Ueta and Tojo 2016; Veterano et al. 2018) and one species group- ing within Elongisporangium (Senda et al. 2009) have been described as Pythium since the taxonomic revisions of Uzuhashi et al. (2010). These species are transferred to their respective genera below. Eight of these were included in our phylogenomic analysis (FIG. 2), whereas the remaining 13 were shown to form part of Globisporangium in their original publications or phy- logenies published by Hyde et al. (2014) or Jayawardena et al. (2020). Two other species, P. longandrum (Paul 2001) and P. paddicum (Hirane 1960), were transferred to Globisporangium by Uzuhashi et al. (2010), but we note that these are invalid: P. paddicum (Art. 37); P. longandrum (Art. 40.1). Additionally, the name P. kandovanense is invalid because the authors who described it did not designate a holotype in a single institute (Art. 40.7). We validated this species name in Globisporangium as a new species and designated a holotype to fix this issue. These taxonomic changes and notes are summarized in SUPPLEMENTARY TABLE 4. Elongisporangium Uzuhashi, Tojo & Kakish., Mycoscience 51:363. 2010. = Pythium Pringsh., Jahrbuchücher für wis- senschaftliche Botanik 1:304. 1858. pro parte, excl. typus (clade H sensu Lévesque and de Cock 2004). = Eupythium Nieuwl., The American Midland Naturalist 4:384. 2016. pro parte. = Sphaerosporangium Sparrow, Science 73:42. 1931. pro parte, nom. invalid. Type species: Elongisporangium anandrum (Drechsler) Uzuhashi, Tojo & Kakish. Elongisporangium senticosum (Senda & Kageyama) H. D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840708 Basionym: Pythium senticosum Senda & Kageyama, Mycologia 101:443. 2009. Typus: JAPAN. GIFU: Takayama, from 50-y-old deciduous broadleaf forest soil, CBS 122490 (ex-type strain), NBRC 104223 (holotype). Globisporangium Uzuhashi, Tojo & Kakish., Mycoscience 51:360. 2010. = Pythium Pringsh., Jahrbuchücher für wissenschaf- tliche Botanik 1:304. 1858. pro parte, excl. typus (clades E, F, G, I, and J sensu Lévesque and de Cock 2004). = Eupythium Nieuwl., The American Midland Naturalist 4:384. 1916. pro parte. 508 NGUYEN ET AL.: PYTHIUM PHYLOGENOMICS = Sphaerosporangium Sparrow, Science 73:42. 1931. pro parte, nom. invalid. Type species: Globisporangium paroecandrum (Drechsler) Uzuhashi, Tojo & Kakish. Globisporangium alternatum (M.Z. Rahman, H.M.A. Abdelzaher & K. Kageyama) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840709 Basionym: Pythium alternatum M.Z. Rahman, H.M. A. Abdelzaher & K. Kageyama, FEMS Microbiology Letters 362:6. 2015. Typus: JAPAN. HOKKAIDO: Rishiri Island, from soil, CBS 139279 (ex-type strain), NBRC H-13257 (holotype). Globisporangium baisense (Y.Y. Long, J.G. Wei & L.D. Guo) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840716 Basionym: Pythium baisense Y.Y. Long, J.G. Wei & L. D. Guo, Mycological Progress 11:691. 2012. Typus: CHINA. GUANGXI: Baise, from soil of lawn, QBS123 (ex-type strain), HMAS242232 (holotype). Globisporangium barbulae (S. Ueta & M. Tojo) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840717 Basionym: Pythium barbulae S. Ueta & M. Tojo, Mycoscience 57:14. 2016. Typus: JAPAN. OSAKA PREFECTURE: Sakai city, from stem-leaf of Barbula unguiculata, MAFF 245167, NBRC 111015, CBS 139569, and OPU1628 (ex-type strains), TNS-F-61716 (holotype). Globisporangium breve (Y.Y. Long, J.G. Wei & L.D. Guo) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840718 Basionym: Pythium breve Y.Y. Long, J.G. Wei & L.D. Guo, Mycological Progress 11:691. 2012. Typus: CHINA. GUANGXI: Nanning, from soil of lawn, CNN213 (ex-type strain), HMAS242231 (holotype). Globisporangium cederbergense (Bahramisharif, Botha & Lamprecht) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840722 Basionym: Pythium cederbergense Bahramisharif, Botha & Lamprecht, Mycologia 105:1184. 2013. Typus: SOUTH AFRICA. WESTERN CAPE PROVINCE: Clanwilliam, from roots of a Aspalathus lin- earis seedling, CBS 133716 (ex-type strain and holotype). Globisporangium emineosum (Bala, de Cock & Lévesque) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840723 Basionym: Pythium emineosum Bala, de Cock & Lévesque, Persoonia 25:25. 2010. Typus: CANADA. BRITISH COLUMBIA: Surrey, juniper (Juniperus communis) roots exhibiting rot, CBS 124057 (ex-type strain), DAOM BR 479 (holotype). Globisporangium ershadii (Badali, Abrinbana & Abdollahz.) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840725 Basionym: Pythium ershadii Badali, Abrinbana & Abdollahz., Mycologia 108:1183. 2016. Typus: IRAN. EAST AZARBAIJAN PROVINCE: Islami Island, from uncultivated soil, IRAN 2379 (ex- type strain), IRAN 16693 F (holotype). Globisporangium huanghuaiense (Jia J. Chen & X.B. Zheng) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840726 Basionym: Pythium huanghuaiense Jia J. Chen & X.B. Zheng, Biodiversity Data Journal 9:e65227. 2021. Typus: CHINA. JIANGSU PROVINCE: Nanjing, from seedlings of Glycine max, Chen94 (ex-type strain), BJFC-C 1993 (holotype). Globisporangium iranense (Badali, Abrinbana & Abdollahz.) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840727 Basionym: Pythium iranense Badali, Abrinbana & Abdollahz., Cryptogamie, Mycologie 41:185. 2020. Typus: IRAN. WEST AZARBAIJAN PROVINCE: Maku, from soil under Prunus armeniaca, IRAN 2386 C (ex-type strain), IRAN 16697 F (holotype). Globisporangium kandovanense H.D.T. Nguyen & C.F. J. Spies, sp. nov. MycoBank MB840728 Description: As “Pythium kandovanense A. Chenari Bouket, M. Arzanlou, M. Tojo & A. Babai-Ahari” nom. invalid. (Art 40.7), International Journal of Systematic and Evolutionary Microbiology 65:2505. 2015. Typus: IRAN. EAST-AZARBAIJAN PROVINCE: Kandovan, from leaves of snow-covered Lolium perenne (Poaceae), CBS 139567 (cryopreserved, holotype desig- nated here). Ex-type strains: CCTU 1813, OPU 1626. Notes: No holotype in a single institute was desig- nated by the original authors; thus, the name “Pythium kandovanense” was not validly published (Art 40.7). We hereby validate that species name in the genus Globisporangium recognized by us and designated CBS 139567 [cryopreserved] as the holotype. Globisporangium monoclinum (Abrinbana, Abdollahz. & Badali) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840733 MYCOLOGIA 509 Basionym: Pythium monoclinum Abrinbana, Abdollahz. & Badali, Cryptogamie, Mycologie 41:185. 2020. Typus: IRAN. EAST AZARBAIJAN PROVINCE: Islami Island, from uncultivated soil, IRAN 2421 C (ex- type strain), IRAN 16695 F (holotype). Globisporangium polare (Tojo, Van West & Hoshino) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840735 Basionym: Pythium polare Tojo, Van West & Hoshino, Fungal Biology 116:762. 2012. Typus: NORWAY. Spitsbergen Island, from Sanionia uncinata, CBS 118203 (holotype and ex-type strain), CBS 118202 (paratype). Globisporangium pyrioosporum (Abdollahz., Badali & Abrinbana) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840737 Basionym: Pythium pyrioosporum Abdollahz., Badali & Abrinbana, Mycologia 108:1183. 2016. Typus: IRAN. WEST AZARBAIJAN PROVINCE: Urmia, from soil under Capsicum annuum, IRAN 2382 (ex-type strain), IRAN 16692 F (holotype). Globisporangium schmitthenneri (M.L. Ellis, Broders & Dorrance) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840739 Basionym: Pythium schmitthenneri M.L. Ellis, Broders & Dorrance, Mycologia 104:481. 2012. Typus: USA. OHIO: Darke County, from soybean (Glycine max) root tissue with a soil-baiting procedure from agronomic soil, CBS 129726 (ex-type strain), Darke1611 (holotype). Globisporangium selbyi (M.L. Ellis, Broders & Dorrance) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840740 Basionym: Pythium selbyi M.L. Ellis, Broders & Dorrance, Mycologia 104:482. 2012. Typus: USA, OHIO: Preble County, from corn (Zea mays) root tissue with a soil-baiting procedure from agronomic soil, CBS 129728 (ex-type strain), CBS H-20615 (holotype). Globisporangium stipitatum (G. Karaca & B. Paul) H.D. T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840741 Basionym: Pythium stipitatum G. Karaca & B. Paul, FEMS Microbiology Letters 295:165. 2009. Typus: FRANCE. From soil, F-1516 (holotype). Globisporangium urmianum (Abrinbana, Badali & Abdollahz.) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840742 Basionym: Pythium urmianum Abrinbana, Badali & Abdollahz., Mycologia 108:1185. 2016. Typus: IRAN. WEST AZARBAIJAN PROVINCE: Urmia, from soil under Prunus amygdalus, IRAN 2376 (ex-type strain), IRAN 16691 F (holotype). Globisporangium viniferum (B. Paul) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840743 Basionym: Pythium viniferum B. Paul, Fungal Diversity 28:57. 2008. Typus: FRANCE. BURGUNDY: Dijon, from soil from vineyards, F-1201 (holotype). Notes: Although not mentioned in the original publication by Paul et al. (2008), the ex-type strain is CBS 119168 according to Robideau et al. (2011). Paul et al. (2008) found the ITS region of Pythium viniferum F-1201 (= CBS 119168) to be highly simi- lar to that of Pythium debaryanum CBS 752.96 (which is not the type strain of Pythium debarya- num) but still described his strain as a new species based mainly on morphological differences. Robideau et al. (2011) found these two strains to be indistinguishable with either cox1 or ITS. A more in-depth investigation into the type of P. debaryanum is needed, but from the molecular data it seems P. viniferum may be a synonym of P. debaryanum. Globisporangium wuhanense (Y.Y. Long, J.G. Wei & L. D. Guo) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840744 Basionym: Pythium wuhanense Y.Y. Long, J.G. Wei & L.D. Guo, Mycological Progress 13:148. 2014. Typus: CHINA. HUBEI PROVINCE: Wuhan, from soil of a paddy field, CGMCC3.15149 (ex-type strain), HMAS243737 (holotype). Globisporangium yorkense (J.E. Blair) H.D.T. Nguyen & C.F.J. Spies, comb. nov. MycoBank MB840745 Basionym: Pythium yorkense [as ‘yorkensis’] J.E. Blair, Plant Pathology 67:623. 2018. Typus: USA. PENNSYLVANIA: York County, soy- bean field soil, CBS 142324 (ex-type strain), C12-118 (holotype). Notes: J.E. Blair originally described this species as Pythium yorkensis (MB821223), but because Pythium and Globisporangium are gender neutral, the suffix should be “-ense” and not “-ensis.” Phytopythium Abad, de Cock, Bala, Robideau, Lodhi & Lévesque, Persoonia (Fungal Planet) 24:137. 2010. 510 NGUYEN ET AL.: PYTHIUM PHYLOGENOMICS = Pythium Pringsh., Jahrbuchücher für wissenschaf- tliche Botanik 1:304. 1858. pro parte, excl. typus (clade K sensu Lévesque and de Cock 2004). = Sphaerosporangium Sparrow, Science 73:42. 1931. pro parte, nom. invalid. = Ovatisporangium Uzuhashi, Tojo & Kakish., Mycoscience 51: 360. 2010. Type species: Phytopythium sindhum Lohdi, Shahzad & Lévesque, Persoonia (Fungal Planet) 24:137. 2010. Pythium Pringsh. emend. Uzuhashi, Tojo & Kakish., Mycoscience 51: 358. 2010. = Artotrogus Mont., Annales des Sciences Naturelles Botanique 11:56. 1849. = Nematosporangium Schr., Die natürlichen Pflanzenfamilien nebst ihren Gattungen und wichtige- ren Arten insbesondere den Nutzplanze, unter Mitwirkung zahlreicher hervorragender Fachgelehrten begründet von A. Engler und K. Prantl. 1:104. 1897. = Rheosporangium Edson., Journal of Agricultural Research Mycologia 4:291. 1915. = Eupythium Nieuwl., The American Midland Naturalist 4:384. 1916. pro parte. Type species: Pythium monospermum Pringsh., Jahrbuchücher für wissenschaftliche Botanik 2:288. 1858. DISCUSSION Molecular phylogenetic studies demonstrated that Pythium s.l. is paraphyletic and reignited an old interest in splitting the genus. That split was proposed formally by Uzuhashi et al. (2010) using morphological data and phylogenies of cox2 and 28S rDNA regions. Although the split was supported morphologically, the oomycete taxonomy community was reluctant to adopt the new genera due to the weak support in their molecular ana- lyses, especially for Globisporangium. Although published phylogenomic analyses of oomycetes supported Globisporangium as a separate genus, these analyses included very limited numbers of Pythium species that did not fully represent the known diversity in the genus (McCarthy and Fitzpatrick 2017; McGowan and Fitzpatrick 2020). In the current study, over a hundred single-copy loci were extracted from whole genomes of 119 strains representing the known diversity of Pythium s. l., and subsequent phylogenomic analyses revealed strongly supported clades for all the genera proposed by Uzuhashi et al. (2010) (FIG. 2, SUPPLEMENTARY FIGS. 1 and 2). Splitting Pythium s.l. in this way resolves the paraphyletic issue noted during early molecular studies of the genus and also corresponds to some degree with early studies considering the generic division of Pythium Pringsh. based on sporangial morphology (Fischer 1892; Schröter 1893; Sparrow 1931). However, the nomencla- tural historical review in this study demonstrated that all the names proposed earlier were not valid. Large genera established prior to the molecular age are often identified as non-monophyletic once sub- jected to sequence-based phylogenetic analyses. This is due to challenges in working strictly with morpho- logical characters or host associations to define a species or genus, especially where convergent evo- lution has led to shared characteristics between dis- tantly related groups. One of the mechanisms available to taxonomists to resolve a large non-mono- phyletic genus is to split it into smaller genera, as was done in genera such as Phoma (e.g., Aveskamp et al. 2010; Chen et al. 2015; de Gruyter et al. 2010), Fusarium (Gräfenhan et al. 2011; Lombard et al. 2015; Schroers et al. 2011), and, of course, also Pythium by Uzuhashi et al. (2010), which is central to this study. Although this makes sense from a taxonomic perspective, the introduction of new names for well-known organisms (e.g., plant or human pathogens) can have far-reaching conse- quences and is often met with considerable resistance from researchers, diagnosticians, practitioners, legis- lators, and crop producers. The contested paraphyly and proposed generic split of Fusarium is a good example of this (Crous et al. 2021; Geiser et al. 2013, 2021; Gräfenhan et al. 2011; O’Donnell et al. 2020; Sandoval-Denis et al. 2019; Schroers et al. 2011; Summerell 2019). The Shenzhen Code (Turland et al. 2018) does make provision for the “retention of names that best serve the stability of nomenclature” through conservation (Art. 14). However, in cases of proven paraphyly, such as Pythium s.l., a stable nomenclature is better served by dividing the genus as supported by phylogenetic evidence. The prolonged reluctance of the scientific community to accept the new names introduced by Uzuhashi et al. (2010) led to increased uncertainty concerning their status, and which names to use. The phylogenomic data presented here confirm the paraphyly of the tradi- tional concept of Pythium Pringsh. and provide convin- cing support for the revisions imposed by Uzuhashi et al. (2010). Furthermore, none of the older generic names used for species of Pythium Pringsh. can compete with those introduced by Uzuhashi et al. (2010). We urge the scientific community to implement these names in order to facilitate their widespread acceptance and reduce the current uncertainty regarding the classification of spe- cies traditionally considered to be Pythium. In order to consolidate the new genera, 21 new combinations were made in this study. MYCOLOGIA 511 One of the questions that remain unanswered is the paraphyly of Pythium with regard to Lagena and possibly Pythiogeton. Phylogenies using conventional markers have suggested that Lagena is related to the clade C species, P. grandisporangium and P. insidiosum, and several unidentified “Lagenidium” species (Hyde et al. 2014; Spies et al. 2016). These “Lagenidium” species, including Lagenidium sp. PWL-2010h that was included in our analyses, were shown to be distinct from Lagenidium s.s. and should perhaps be reclassified as Pythium or Lagena (see supplementary fig. S1 in Spies et al. 2016). Published phylogenies have also resolved Pythiogeton either as a sister clade to Pythium (Huang et al. 2013; Hyde et al. 2014) or as among the unresolved taxa related to clade C (see supplementary fig. S1 in Spies et al. 2016). These published phylogenies revealed poor support for the relationships among these taxa. Similarly, in the 70% and 90% taxa threshold greedy consensus ASTRAL trees presented here, the internal nodes indicat- ing the relationships of P. grandisporangium and P. insidiosum to the remainder of the species in Pythium had low support (≤46%; FIG. 2, SUPPLEMENTARY FIG. 1). Although the concatenated phylogeny provided good support for almost all nodes in Pythium, the clade C species and Lagenidium sp. PWL-2010h were posi- tioned on long branches, indicating considerable differ- ences between these taxa and their closest relatives (SUPPLEMENTARY FIG. 2). Several strains of Pythiogeton were initially considered for inclusion in this study; however, all had lost their viability during storage and could consequently not be sequenced. Phylogenomic analyses that include these and additional related taxa are likely to improve the phylogenetic resolu- tion among clade C Pythium species, Lagena, and Pythiogeton and provide further insights into their generic classification. The emphasis of this study was on the application of genomic data to resolve taxonomic issues in Pythium; however, the data generated has broad relevance to biolo- gical studies involving oomycetes. Microbial metagenomic sequence data from total DNA or RNA, when analyzed with incomplete reference databases, leads to higher false positives, and this phenomenon is particularly significant in eukaryotes (Marcelino et al. 2020). The comprehensive reference data set of whole genomes from our study includ- ing draft annotations is taxonomically verified, making it a solid publicly available resource for environmental shot- gun DNA metagenomic or RNA-seq metatranscriptomic studies that include oomycetes. In conclusion, phylogenomic analyses finally provide convincing molecular phylogenetic support for the divi- sion of Pythium proposed by Uzuhashi et al. (2010) and new combinations have been provided to consolidate the taxonomy of these genera. Based on this evidence, the scientific community should no longer be reluctant to adopt these new generic names in their work going forward. Although there is some possibility of future revisions within Pythium as recognized here, such revi- sions would affect only a few currently known taxa and should only be undertaken once the relationships among the relevant taxa have been resolved. ACKNOWLEDGMENTS We thank the Molecular Technologies Laboratory (MTL) for generating the sequencing data and the Biological Centre of Excellence (BiCoE) for maintaining the high-performance com- puting services that enabled us to perform bioinformatic analyses. DISCLOSURE STATEMENT No potential conflict of interest was reported by the author(s). FUNDING This study was funded by Agriculture and Agri-Food Canada (AAFC) grants J-002272 and J-001564. LITERATURE CITED Abrinbana M, Badali F, Abdollahzadeh J. 2016. Molecular and morphological characterization of three new species of Pythium from Iran: p. ershadii. P. ershadii, P. pyrioosporum, and P. urmianum. Mycologia. 108:1175–88. Adhikari BN, Hamilton JP, Zerillo MM, Tisserat N, Lévesque CA, Buell CR. 2013. Comparative genomics reveals insight into virulence strategies of plant pathogenic oomycetes. PLoS One. 8:e75072. Aveskamp MM, de Gruyter J, Woudenberg JH, Verkley GJ, Crous PW. 2010. 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MYCOLOGIA 515 Abstract INTRODUCTION Materials and methods Selection of strains and species for sequencing.— DNA extraction and sequencing.— Genome assembly and genome annotation.— Phylogenomic analysis and characterization of genes/gene trees.— RESULTS Genome statistics.— Phylogenomic analyses and characterization of genes and gene trees.— NOMENCLATURE AND TAXONOMY Notes on the nomenclatural history of Pythium.— Changes in taxonomy.— DISCUSSION ACKNOWLEDGMENTS DISCLOSURE STATEMENT Funding LITERATURE CITED