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Mycologia
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/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.
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Whole genome sequencing and phylogenomic analysis show support for the
splitting of genus Pythium
Hai D. T. Nguyen
a
, Annette Dodge
a
, Kasia Dadej
a
, Tara L. Rintoul
a
, Ekaterina Ponomareva
a
, Frank N. Martin
b
,
Arthur W. A. M. de Cock
c
, C. André Lévesque
a
, Scott A. Redhead
a
, and Christoel F. J. Spies
d
a
Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, Ontario, K1A 0C6 Canada;
b
Crop
Improvement and Protection Research, Agricultural Research Service, United States Department of Agriculture, Salinas, California 93905, USA;
c
Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands;
d
Plant 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 justied by the delineating
morphological characters, there were weaknesses in the molecular analyses, which created reluc-
tance in the scientic 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 scientic 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
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
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
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.
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