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Comparative Analyses of Complete Peronosporaceae
(Oomycota) Mitogenome Sequences—Insights into
Structural Evolution and Phylogeny
Richard C. Winkworth
1,2,
*, Grace Neal
2
, Raeya A. Ogas
2
, Briana C. W. Nelson
2
,
Patricia A. McLenachan
2
, Stanley E. Bellgard
2
, and Peter J. Lockhart
1,2
1
Bio-Protection Research Centre, Massey University, Palmerston North, New Zealand
2
School of Natural Sciences, Massey University, Palmerston North, New Zealand
*Corresponding author: E-mail: r.c.winkworth@massey.ac.nz.
Accepted: 25 March 2022
Abstract
Members of the Peronosporaceae (Oomycota, Chromista), which currently consists of 25 genera and approximately 1,000
recognized species, are responsible for disease on a wide range of plant hosts. Molecular phylogenetic analyses over the last
two decades have improved our understanding of evolutionary relationships within Peronosporaceae. To date, 16 numbered
and three named clades have been recognized; it is clear from these studies that the current taxonomy does not reflect evo-
lutionary relationships. Whole organelle genome sequences are an increasingly important source of phylogenetic informa-
tion, and in this study, we present comparative and phylogenetic analyses of mitogenome sequences from 15 of the 19
currently recognized clades of Peronosporaceae, including 44 newly assembled sequences. Our analyses suggest strong con-
servation of mitogenome size and gene content across Peronosporaceae but, as previous studies have suggested, limited
conservation of synteny. Specifically, we identified 28 distinct syntenies amongst the 71 examined isolates. Moreover, 19
of the isolates contained inverted or direct repeats, suggesting repeated sequences may be more common than previously
thought. In terms of phylogenetic relationships, our analyses of 34 concatenated mitochondrial gene sequences resulted in a
topology that was broadly consistent with previous studies. However, unlike previous studies concatenated mitochondrial
sequences provided strong support for higher-level relationships within the family.
Key words: genome rearrangements, inverted repeats, mitochondrial genome, Peronosporales, sequence evolution, struc-
tural diversity, synteny.
Introduction
The Peronosporaceae (Oomycota, Chromista) is a mono-
phyletic group of biotrophic and hemibiotrophic phyto-
pathogens (Thines and Choi 2016). Most of the
approximately 1,000 species currently placed in the family
belong to either Peronospora (500 species) or
Phytophthora (200 species) with the remainder divided
amongst 23 smaller genera including Bremia,
Significance
Mitochondrial sequences are commonly used for distinguishing amongst species and reconstructing evolutionary rela-
tionships. However, for many lineages, mitochondrial genomes are poorly characterized. We explored gene content,
gene order, and evolutionary relationships using 44 newly assembled and 27 publicly available mitogenome sequences
for Peronosporaceae. Our results provide new insights into genome and lineage evolution in this economically important
group of plant pathogens.
© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/),which permits unrestrictedreuse,
distribution, and reproduction in any medium, provided the original work is properly cited.
GBE
Genome Biol. Evol. 14(4) https://doi.org/10.1093/gbe/evac049 Advance Access publication 14 April 2022 1
Hyaloperonospora,andPlasmopara (Thines 2014;Thines
and Choi 2016). As a group, the Peronosporaceae infect a
wide array of host plants, impacting both agricultural and
natural ecosystems (e.g., Hansen et al. 2012). Well-known
diseases attributed to members of the family include lettuce
downy mildew (Bremia lactucae), grape downy mildew
(Plasmopara viticola) potato late blight (Phytophthora infes-
tans), and sudden oak death (Phytophthora ramorum)
(Thines 2014;Thines and Choi 2016).
Our understanding of evolutionary relationships within
Peronosporaceae has improved dramatically over the last
two decades. To date much of the focus has been on
Phytophthora (e.g., Cooke et al. 2000,2002;Kroon et al.
2004;Blair et al. 2008;Martin et al. 2014). In their study,
Cooke et al. (2000) analysed nuclear ribosomal internal
transcribed spacer sequences from 47 Phytophthora spe-
cies and identified 10 clades. Subsequent studies (e.g.,
Blair et al. 2008;Martin et al. 2014) have included larger
samples of both taxa and molecular loci. In addition to pro-
viding further support for the groups identified by Cooke
et al. (2000), these studies have identified additional clades
(e.g., Jung et al. 2017a;Bourret et al. 2018). For example,
Bourret et al. (2018) included 135 taxa representing 22
genera of the Peronosporaceae and recognized 16 num-
bered clades, only one of which did not contain a member
of Phytophthora (fig. 1). In addition to numbered
clades, three named lineages have been recognized.
Specifically, clades corresponding to Halophytophthora,
Nothphytophthora, and Phytopythium have been identified
as early-diverging within Peronosporaceae (Jung et al.
2017b;fig. 1). Despite an improved understanding of the
major groupings within Peronosporaceae, relationships
among them have remained uncertain. For example, the
analyses of Blair et al. (2008),Martin et al. (2014), and
Bourret et al. (2018) do not consistently resolve relation-
ships amongst the numbered clades.
Our understanding of species diversity within
Peronosporaceae has also dramatically improved over the
last two decades. In Phytophthora alone, the number of
formally recognized taxa has grown from 60 in 1996
(Erwin and Ribeiro 1996) to in excess of 150 (Thines and
Choi 2016;Yang et al. 2017). For example, Yang et al.
(2017) included 142 formally described and 43 yet to be de-
scribed Phytophthora entities in their phylogenetic ana-
lyses. Rapid growth in the size of the family reflects
increased attention on understanding the diversity in nat-
ural ecosystems such as forests (e.g., Vettraino et al.
2002;Jung et al. 2017a) and streams (e.g., Reeser et al.
2007;Yang et al. 2016;Brazee et al. 2017) as well as in re-
gions such as Asia and South America (e.g., Webber et al.
2011;Lee et al. 2017;Jung et al. 2020;Legeay et al. 2020).
Mitochondria maintain a circular, extra-nuclear genome.
In metazoans, this genome is typically uniparentally
inherited, structurally conserved, and evolves rapidly at
the nucleotide sequence level. As a consequence, the mito-
chondrial genome has become an important source of se-
quence data for resolving evolutionary relationships in
animals (Curole and Kocher 1999;Sullivan et al. 2017). In
contrast, the mitogenomes of many other groups are
more poorly characterized. At least in part, this is due to
marked differences in the size and complexity of mitogen-
omes within and between groups. For example, fungal mi-
togenomes vary from approximately 20 kb to more than
235 kb in size (Sandor et al. 2018).
Complete mitogenome sequences are available for spe-
cies representing eight oomycete genera. Specifically, se-
quences are available for Achlya (O’Brien et al. 2014),
Bremia,Peronospora (Derevnina et al. 2015;Fletcher
et al. 2018), Phytophthora (e.g., Avila-Adame et al. 2006;
Martin et al. 2007;Lassiter et al. 2015;Cai and Scofield
2020;McGowan et al. 2020), Pseudoperonospora
(Rahman et al. 2019), Pythium (Lévesque et al. 2010;
Tangphatsornruang et al. 2016), Saprolegnia (Grayburn
et al. 2004), and Thraustotheca (O’Brien et al. 2014). In
most cases, mitochondrial genome sequences have been
reported for just one (e.g., Bremia,Saprolegnia) or two
(e.g, Pseudoperonospora) taxa. However, sequences have
been reported for 17 members of Phytophthora.
Currently, nine of the numbered clades recognized by
Bourret et al. (2018) are represented by one or more com-
plete mitogenome sequences. Comparative analyses of the
available Phytophthora mitochondrial genome sequences
Phytophthora clade 1
Phytophthora clade 14
Phytophthora clade 13
Phytophthora clade 12
Phytophthora clade 5
Phytophthora clade 4
Phytophthora clade 3
Phytophthora clade 2
Phytophthora clade 11
Phytophthora clade 10
Phytophthora clade 9
Phytophthora clade 8
Phytophthora clade 7
Phytophthora clade 6
Downy mildew clade 16
Phytophthora/Downy mildew clade 15
Nothophytophthora
Halophytophthora
Phytopythium
Pythium (outgroup)
FIG.1.—Schematic diagram summarizing our current understanding
of evolutionary relationships within Peronosporaceae. Included are 14
numbered clades comprised exclusively of Phytophthora, two numbered
clades comprised exclusively (Clade 16) or almost so (Clade 15) of represen-
tatives of the 21 downy mildew genera and three early-diverging clades
broadly corresponding to Halophytophthora,Nothophytophthora,and
Phytopythium. The cladogram is based on the results of Jung et al.
(2017a,2017b), Yang et al. (2017),andBourret et al. (2018).
Winkworth et al. GBE
2Genome Biol. Evol. 14(4) https://doi.org/10.1093/gbe/evac049 Advance Access publication 14 April 2022
have indicated that despite broad conservation of gene
content, gene order varies both within and between taxa
(e.g., Martin et al. 2007;Yuan et al. 2017;McGowan
et al. 2020). Such analyses also suggest that unlike the mi-
togenomes of Pythiaceae and Saprolegniaceae, those of
Phytophthora typically lack inverted repeats.
In this study, we present comparative and phylogenetic
analyses of mitogenomes from a broad sample of
Peronosporaceae. We include 44 newly assembled mito-
chondrial genome sequences representing 34 members of
Peronosporaceae; for all but one of these taxa mitogenome
sequences have not previously been reported. Overall, our
sample includes 15 of the 19 currently recognized clades
of Peronosporaceae and, based on Yang et al. (2017),ap-
proximately 25% (45 taxa) of recognized Phytophthora di-
versity. We discuss our results with respect to the evolution
of mitochondrial genome diversity in Peronosporaceae.
Results
We assembled complete mitochondrial genome sequences
from 44 representatives of the Peronosporaceae.
Mitogenomes were considered complete when the ends
of linear drafts were circularized, and no inconsistencies
(e.g., gaps or misalignments) were observed when the ori-
ginal sequence reads were mapped to the draft. Average
read coverage for the 44 mitogenomes ranged from
528.8 to 38,991.5 reads (table 1).
Genome Size
The 71 complete Peronosporaceae mitochondrial genomes
ranged in size from 36,826 base pair (bp) for Phytophthora
agathidicida to 61,242 bp for Phytopythium vexans.
However, 76.1% (56/71) were ,40,000 bp in length and
only 5.6% (4/71) were .45,000 bp (table 1). A Wilcoxon
rank-sum test comparing mitogenome lengths for our
sample to publicly available sequences for other oomycetes
(e.g., Lévesque et al. 2010;O’Brien et al. 2014;
Tangphatsornruang et al. 2016) indicated that those of the
Peronosporaceae were significantly smaller (n
Peronosporaceae
=71, n
oomycetes
=8, W=13, P=0.000012).
Large (i.e., 7,772–21,950 bp) inverted repeats are a dom-
inant feature of publicly available sequences for other oomy-
cetes (e.g., Lévesque et al. 2010;O’Brien et al. 2014;
Tangphatsornruang et al. 2016). The mitogenomes of
25.5% (15/55) of the sampled taxa contained inverted re-
peats, including representatives of Peronospora,
Phytophthora,Plasmopara,andPhytopythium. Direct repeats
were identified in the B. lactucae,P. polonica,P. sansomeana,
P. sojae,andPe. tabacina mitogenomes. With just one
exception repeated sequences in Peronosporaceae mitogen-
omes were shorter and their contribution to the overall
size of the mitogenome was less than for the other oomy-
cetes. Specifically, in Peronosporaceae the repeats were
.5,442 bp in length and represented 1.2–21.3% of the cor-
responding genome length. The exception was Ph. vexans.In
this case, the inverted repeats were 24,191 bp in length and
accounted for 79.0% of the overall genome length.
Our sample of mitogenome sequences contained 14 taxa
represented by sequences from two isolates and for P. infes-
tans we included sequences representing each of the four
haplotypes described by Avila-Adame et al. (2006). For three
taxa—P. chlamydospora,P. kernoviae,andP. lateralis—both
mitogenome sequences were the same length. Differences
in mitogenome length were ,200 bp for a further 10 taxa
(e.g., P. agathidicida,P. capsici,andP. multivora) but for
amongst isolates of P. infestans and P. palmivora were
1,948 and 2,058 bp, respectively (table 1).
Gene Content and Order
Coding sequences comprised 66.8–92.7% of the sampled
Peronosporaceae mitogenomes; protein coding, tRNA, and
rRNA genes accounted for 55.0–76.3%, 3.7–5.4%, and
8.1–14.0%, respectively. The remaining 7.3–33.2% of
these sequences were noncoding. No introns were identi-
fied, in all cases the entire noncoding component was
intergenic.
For 66.2% (47/71) of the sampled Peronosporaceae
mitogenomes, the gene content included 39 protein
encoding, 2 rRNA, and 25 tRNA genes. A further 15.5%
(11/71) had 39 protein encoding and 2 rRNA genes but
26–27 tRNA genes. All but one of the remaining
Peronosporaceae mitogenomes (16.9%, 12/71) had 38,
40, or 43 protein encoding, 2 rRNA, and 25–27 tRNA genes
(fig. 2). The exception was the Ph. vexans mitogenome,
which contained 65 protein encoding, 4 rRNA, and 42
tRNA genes. Differences in gene content were associated
with inverted repeats (e.g., Ph. vexans and P. alni) or with
a reduced number of hypothetical protein genes (i.e.,
ymf98,ymf99,ymf100, and ymf101) identified.
Specifically, mitogenomes containing 39 protein-encoding
genes typically had four ymf genes whereas the closely re-
lated clade 1 species P. andina,P. ipomoeae,P. mirabilis,
and P. phaseoli all lacked the ymf101 locus and as a result
had 38 protein-encoding genes.
In contrast to the strong conservation of gene content,
our analyses of gene order and orientation indicated that
synteny is poorly conserved in Peronosporaceae mitogen-
omes, although several gene blocks are maintained. Our
sample of 71 Peronosporaceae mitogenomes contained
28 distinct gene arrangements (fig. 2). Isolates representing
the same taxon always shared synteny and, less frequently,
synteny was also shared between taxa. Of the 28 gene ar-
rangements recovered, nine were shared between taxa. In
most cases, taxa that shared the same gene arrangement
belonged to the same numbered clade (e.g., arrangements
I and IV). There were three exceptions, two syntenies were
Comparative Analyses of Peronosporaceae Mitogenomes GBE
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Table 1
Summary Statistics for the Mitochondrial Genome Sequences of the Included Peronosporaceae
Species Genome Proportion of genome Gene numbers
Size Mean read depth Coding Noncoding Protein rRNA tRNA
Clade 1
Phytophthora aleatoria 38,936 4,417.2 0.88 0.12 39 2 25
Phytophthora andina 37,874 –0.9 0.1 38 2 25
Phytophthora cactorum 38,068 23,066 0.9 0.1 39 2 25
Phytophthora infestans 37,922, 37,957, 39,840, 39,870 –0.86–0.90 0.10–0.14 39 2 25
Phytophthora ipomoeae 37,872 –0.9 0.1 39 2 25
Phytophthora mirabilis 37,779 –0.9 0.1 38 2 25
Phytophthora nicotianae 37,673, 37,749 4,180.1–14,772.4 0.90–0.91 0.09–0.10 39 2 25
Phytophthora phaseoli 37,914 –0.9 0.1 38 2 25
Clade 2
Phytophthora capsici 38,418, 38,427 5,258.3–10,992.9 0.89 0.11 39 2 25
Phytophthora colocasiae. 41,297, 41,367 7,474.4–12,996.5 0.82–0.83 0.17–0.18 39 2 25
Phytophthora multivora 37,992, 38,032 1,016.6–1,724.3 0.9 0.1 39 2 25
Phytophthora plurivora 38,082 7,334.1 0.9 0.1 39 2 25
Phytophthora sp. subnubulis 37,854 10,810.8 0.9 0.1 39 2 25
Phytophthora tropicalis 37,047 8,908.8 0.92 0.08 39 2 25
Clade 3
Phytophthora pseudosyringae 39,143 –0.87 0.13 39 2 25
Phytophthora pluvialis 39,325, 39,327 2,163.9–2,175.2 0.87 0.13 39 2 25
Clade 4
Phytophthora litchii 37,950 940.8 0.9 0.1 39 2 25
Phytophthora megakarya 39,277 6,648 0.87 0.13 39 2 25
Phytophthora palmivora 38,741, 40,799 1,081.6–3,269.6 0.84–0.88 0.12–0.16 39 2 25
Clade 5
Phytophthora agathidicida 36,826, 36,844 2,492.0–4,458.4 0.93 0.07 39 2 25
Phytophthora castaneae 37,083 3,535.3 0.92 0.08 39 2 25
Phytophthora cocois 37,078, 37,125 1,904.2–6,857.7 0.92 0.08 39 2 25
Phytophthora heveae 37,150 4,165.4 0.92 0.08 39 2 25
Phytophthora sp. novaeguineae 37,072 5,056 0.92 0.08 39 2 25
Clade 6
Phytophthora chlamydospora 38,329 4,694.7 0.89 0.11 39 2 25
Phytophthora gonapodyides 43,974 –0.78 0.22 39 2 25
Phytophthora pinifolia 43,061 19,135.3 0.88 0.12 43 2 27
Clade 7
Phytophthora ×alni 45,343 8,717.9 0.77 0.23 40 2 25
Phytophthora ×cambivora 51,184 6,800.8 0.67 0.33 39 2 25
Phytophthora cinnamomi 39,225, 39,230 923.6–8,044.3 0.87 0.13 39 2 25
Phytophthora fragariae 44,706 3,548.9 0.76 0.24 39 2 25
Phytophthora rubi 45,523 7,993.4 0.76 0.24 40 2 25
Phytophthora sojae 42,977 –0.8 0.2 40 2 25
Clade 8
Phytophthora cryptogea 38,163 6,829.4 0.89 0.11 39 2 25
Phytophthora lateralis 38,507 5,606.1–38,991.5 0.89 0.11 39 2 26
Phytophthora ramorum 39,314, 39,494 –0.87–0.88 0.12–0.13 40 2 26
Phytophthora sansomeana 39,618 –0.87 0.13 40 2 25
Clade 9
Phytophthora capitosa 44,662 3,126 0.76 0.24 39 2 25
Phytophthora fallax 43,681 5,049 0.78 0.22 39 2 25
Phytophthora polonica 40,467 –0.84 0.16 39 2 25
Clade 10
Phytophthora kernoviae 37,467 528.8–1,357.8 0.91 0.09 39 2 27
Clade 12
Phytophthora quercina 38,471 10,559.3 0.89 0.11 39 2 25
(continued)
Winkworth et al. GBE
4Genome Biol. Evol. 14(4) https://doi.org/10.1093/gbe/evac049 Advance Access publication 14 April 2022
shared by members of clades 1 and 4 (i.e., arrangements XX
and XXI) with a third shared by members of clades 7, 9, 12,
and 15 (i.e., arrangement XXII) (fig. 2).
The extent to which syntenies differ from one another
is broadly consistent with the relatedness of the isolates
being compared. For isolates representing the same tax-
on, common interval distances were always the max-
imum value for the taxon (e.g., 4,284 for P.
agathidicida,P. infestans,andP. kernoviae) whereas
breakpoint and reversal distances were always minimum
values (e.g., both were 0 for P. agathidicida,P. infestans,
and P. kernoviae). In contrast, average common interval
distances were lower (i.e., 382.34–2,639.23) and aver-
age breakpoint (i.e., 3.10–8.99) and reversal (i.e., 1.97–
6.64) distances higher for between species comparisons.
Moreover, average values for between species compari-
sons within a numbered clade were more similar to those
for isolates representing the same taxon than between
clade comparisons (table 2). Similar patterns were also
observed at the level of sequence
divergence (supplementary fig. S1, Supplementary
Material online). Specifically, sequence divergence was
moderately correlated with common interval and break-
point distances (Kendall’sτ=0.205 and 0.251, respect-
ively) whereas the correlation to reversal distances was
strong (Kendall’sτ=0.301); in all three cases the rela-
tionships were statistically significant (P,0.001).
Phylogenetic Analyses
Our final data matrix contained 34 gene partitions from 72
isolates representing 54 members of Peronosporaceae and
one of Pythiaceae (supplementary table S2, Supplementary
Material online). The matrix was 25,443 aligned nucleotide
positions in length, of which 11,225 (44.1%) were variable
and 14,218 (55.9%) invariant. Gapped characters com-
posed 0.03% of the matrix (609/1,831,896 characters);
14 of the 34 gene partitions (41.2%) contain gaps, with
more than 50% (339/609) of the gaps falling within three
partitions (i.e., rps7,rps10, and rps11).
Our IQTREE analysis resulted in well-resolved and gener-
ally well-supported topology (fig. 3). Specifically, of the 69
internal edges in the topology all but 17 were supported by
bootstrap support values of 100% and just one was not
strongly supported (i.e., bootstrap support ,80% boot-
strap). Specifically, the pairing of P. megakarya and P. pal-
mivora (clade 4) received 68% bootstrap support (fig. 3).
All the numbered clades recognized by Bourret et al.
(2018) and represented in our sample were very strongly
supported as monophyletic (bs .98%). The relationships
amongst the numbered clades were also strongly sup-
ported (bs =82–100%; fig. 3). Relationships within the
numbered clades were consistent with those recovered by
Bourret et al. (2018) and when multiple isolates of a species
were included (e.g., P. agathidicida,P. multivora) these
were consistently recovered and strongly supported (all
bs =100%) as sister.
The sampled pool for each of the replicate Bayesian runs
included 60,002 trees with the 95% credible tree set for
each including five trees. The topology of the majority-rule
consensus for the combined pool of sampled Bayesian trees
was the same as that of the maximum likelihood tree. Again
this topology was consistent with that of Bourret et al.
(2018) with respect to the numbered clades and
Table 1 Continued
Species Genome Proportion of genome Gene numbers
Size Mean read depth Coding Noncoding Protein rRNA tRNA
Phytophthora tubulina 38,065 4,667 0.9 0.1 39 2 25
Phytophthora versiformis 38,011 2,937.7 0.9 0.1 39 2 25
Clade 15
Hyaloperonospora arabidopsidis 38,797 3,648.9 0.88 0.12 39 2 25
Peronospora belbahrii 40,063 5,035 0.85 0.15 39 2 25
Peronospora effusa 41,318 –0.83 0.17 39 2 25
Peronospora tabacina 43,225 –0.79 0.21 39 2 25
Phytophthora podocarpi 38,130, 38,136 1,934.9–3,009.7 0.9 0.1 39 2 26
Pseudoperonospora humuli 39,087 –0.87 0.13 39 2 25
Clade 16
Bremia lactucae 39,302 –0.87 0.13 39 2 26
Plasmopara halstedii 38,944 6,619.1 0.88 0.12 39 2 27
Nothophytophthora
Nothophytophthora sp. 38,518 6,444.8 0.89 0.11 39 2 27
Phytopythium
Phytopythium vexans 61,242 5,231.9 0.91 0.09 65 4 42
Pythiaceae
Pythium ultimum Trow 59,689 –0.89 0.11 63 4 42
Comparative Analyses of Peronosporaceae Mitogenomes GBE
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relationships within them. Our Bayesian analysis also pro-
vided very strong support for almost all of the suggested re-
lationships. In this case, all the numbered clades and the
relationships amongst them were supported by posterior
probabilities of 1.00. Moreover, all but four of the suggested
relationships within the numbered clades were also sup-
ported by posterior probabilities of 1.00. The exceptions
were the P. andina-ipomoeae,P. megakarya-palmivora,P.
tubulina-vesiformis,andPeronospora-Pseudoperonospora
clades for which posterior probabilities were 0.93, 0.66,
0.98, and 0.99, respectively (fig. 3).
Concordance factor values (i.e., gCF and sCF) estimated
using IQTREE were typically lower than the bootstrap values
for the corresponding branches. The range of values for
gCF and sCF were 2.9–100.0 and 33.4–100.0, respectively.
In both cases more than half of the values were 50.0 or
above; specifically, 59.4% (41/69) for gCF and 58.0%
(40/69) for sCF. Indeed, the values of gCF and sCF appear
to be strongly correlated (Kendall’sτ=0.765, P,0.001).
Both gCF and sCF are also strongly correlated with both
branch length and bootstrap values (Kendall’sτ=0.432–
0.572, P,0.001) and moderately correlated with posterior
probabilities (Kendall’sτ=0.217–0.268, P,0.031)
(supplementary fig. S2, Supplementary Material online).
Finally, an evaluation of gene order evolution based on
the mitochondrial phylogeny suggested that 48 events
were required to explain our sample of Peronosporaceae mi-
togenomes. These included 38 structural rearrangements
(e.g., inversions, transpositions, inverted transpositions), 6
gains and 4 losses (supplementary fig. S3, Supplementary
Material online). For structural rearrangements, which
were inferred using TreeREx (Bernt et al. 2008), confidence
was high for 74.3% (52/70) and low for 11.4% (8/70) of
nodes. Consistent with the number and diversity of syntenies
we recovered (fig. 2) inferred events were broadly distribu-
ted on the phylogeny. Specifically, events were inferred to
have occurred on 26.6% (37/139) of the branches with a sin-
gle event inferred on 78.4% (29/37) branches and between
two and four events inferred on the remaining 21.6% (8/37)
branches. The 13 numbered clades were each associated
with between one (e.g., clades 5 and 12) and seven (e.g.,
clades 7 and 15) events.
Discussion
We compiled a data set containing complete mitochondrial
genome sequences for 71 members of Peronosporaceae in-
cluding 44 newly assembled genomes. Our sample in-
creases both the number of recognized clades for which
complete mitochondrial genomes are available (i.e., from
9 to 13) and sampling within those clades for which com-
plete mitochondrial genome sequences were already avail-
able (e.g., from one to seven genomes for clade 7). This
much larger sample provides new insights into the struc-
tural diversity of mitogenomes in Peronosporaceae as well
as a large data set for phylogenetic analyses.
Genome Size and Gene Content
Comparisons of the sampled Peronosporaceae mitochon-
drial genomes suggest that they are similar. For example,
with few exceptions both genome size and gene content
were comparable across our sample (table 1). However,
our analyses indicate a statistically significant difference in
mitogenome size between the Peronosporaceae and other
oomycete families. Specifically, the Peronosporaceae mito-
genomes were smaller (Wilcoxon rank-sum test, W=13,
P=0.000012). Despite this overall trend there are excep-
tions. The Phytophthora ×cambivora mitochondrial gen-
ome is comparable in size to those available for
Pythiaceae (i.e., 51,184 bp compared with 54,989–
59,689 bp) and, at 61,242 bp, the mitochondrial genome
of Ph. vexans is the largest yet reported for oomycetes.
Mitochondrial gene content is strongly conserved across
Peronosporaceae and in almost all cases the mitogenomes
are compact with little noncoding sequence (table 1).
Typically, the sampled Peronosporaceae mitogenomes con-
tained 39 protein coding and 2 rRNA genes as well as 25
tRNA genes specifying 19 amino acids. However, in seven
cases mitochondrial gene content has been expanded by
between 1 and 46 genes (fig. 2). For example, the P. kerno-
viae mitogenome contained two additional tRNA genes
(i.e., arrangement XXVI) and for P. pinifolia there were six
additional genes, four protein encoding and two tRNA
genes (i.e., arrangement XIV). The largest increase was
for Ph. vexans (i.e., arrangement XXVIII) where there were
Genomes are oriented relative to the large ribosomal subunit with protein encoding and rRNA genes represented by colored rectangles labeled with standard
gene abbreviations and tRNA genes represented by thick black lines labeled with the one-letter code for the corresponding amino acid. Different syntenies are
labeled with Roman numerals I, P. andina,P. ipomoeae,P. mirabilis,andP. phaseoli; II, B. lactucae; III, Pl. halstedii;IV,P. megakarya;V,P. pseudosyringae and P.
pluvialis;VI,P. capsici,P. multivora,P. plurivora,andP. tropicalis; VII, P. colocasiae and P. sp. subnubulis; VIII, P. agathidicida,P. castaneae,P. cocois,P. heveae,
and P. sp. novaeguineae; IX, H. arabidopsidis;X,Pe. belbahrii;XI,P. podocarpi; XII, Ps. humuli; XIII, P. chlamydospora and P. gonapodyides;XIV,P. pinifolia; XV,
P. alni; XVI, P. cambivora; XVII, P. cinnamomi; XVIII, P. fragariae;XIX,P. rubi; XX, P. infestans and P. palmivora; XXI, P. aleatoria,P. cactorum,P. nicotianae,and
P. litchii; XXII, Pe. effusa,Pe. tabacina,P. polonica,P. quercina,P. sojae,P. tubulina,andP.versiformis; XXIII, P. cryptogea and P. sansomeana; XXIV, P. lateralis
and P. ramorum; XXV, P. captiosa and P. fallax; XXVI, P. kernoviae; XXVII, Nothophytophthora sp.; XXVIII, Ph. vexans. Broadly, genomes are ordered based on
the arrangement of clades in Fig. 3.
Comparative Analyses of Peronosporaceae Mitogenomes GBE
Genome Biol. Evol. 14(4) https://doi.org/10.1093/gbe/evac049 Advance Access publication 14 April 2022 7
27 additional protein encoding, 2 additional rRNA, and 17
additional tRNA genes. Increased protein-encoding gene
content was associated with larger genome sizes.
Specifically, all four Peronosporaceae mitogenomes that
contained more than 39 protein-coding genes were more
than 43,000 bp in length (i.e., P. alni,P. pinifolia,P.rubi,
and Ph. vexans). In some cases, however, larger genome
size was not associated with expanded gene content. For
example, the P. ×cambivora mitochondrial genome had
the typical 39 protein coding, 2 rRNA, and 25 tRNA genes
but was more than 5,000 bp larger than all but one (i.e.,
Ph. vexans) of the mitogenomes in our data set. The re-
peated DNA sequences responsible for the larger size of
the genome were entirely noncoding.
Our analyses included 15 taxa for which mitogenome se-
quences from two or more isolates were included. For three
of these taxa, both sequences were the same length, while
for the remaining 12 taxa the sequences differed in length
by between 2 and 2,058 bp. The majority of the length dif-
ferences were explained by multiple indels, just three cases
involved single indels (e.g., a 180 bp indel in P. ramorum). In
addition to different numbers of indels (i.e., one to many),
their size (i.e., a single nucleotide to .50 nucleotides [nt]),
distribution (i.e., distributed throughout the genome or
clustered), and underlying sequence (i.e., unique or re-
peated sequences) also varied. These observations suggest
that a combination of molecular evolutionary processes
contributes to mitogenome size variation within taxa.
Genome Structure
Previous studies have suggested that although inverted re-
peats are an important feature of both Pythiales and
Saprolegniales mitogenomes they are not typical of
Phytophthora mitogenomes. For example, of the nine
Phytophthora mitochondrial genomes compared by Yuan
et al. (2017) only that of P. ramorum contained inverted re-
peats. However, in our data set inverted repeats longer
than 150 bp were a feature of approximately one-quarter
of the taxa, including 11 members of Phytophthora.
Smaller inverted repeats (i.e., Hyaloperonospora) and direct
repeats (e.g., B. lactucae,Pe. tabacina) were also identified.
In our analyses, the distribution of inverted repeats was
nonrandom with respect to genome size. Mitogenomes
shorter than 38,500 bp in length did not contain inverted
repeats, whereas more than half (56.8%) of those longer
than 38,500 bp and all but one of those longer than
43,000 bp contained them. The distribution of inverted re-
peats also appears to be nonrandom with respect to the
phylogeny. Specifically, with a couple of exceptions, clades
in which inverted repeats were absent (i.e., clades 1–5, 12
and Nothophytophthora) formed a monophyletic group.
The exceptions were clade 16, which despite containing
species that possessed mitogenomes with inverted repeats
was placed within the group lacking them and
Nothophytophthora that falls outside this same group.
Further work is needed but given the similarity of
the inverted repeats in Phytopythium and Pythium,it
is conceivable that the mitogenome of the earliest
Peronosporaceae possessed large inverted repeats.
Despite this, inverted repeats were uncommon amongst
the Peronosporaceae mitogenomes examined and, even
when present, were much smaller than those of
Phytopythium or Pythium (i.e., .5,442 bp vs. 21,950–
24,191). We suggest that large inverted repeats were lost
early in the evolution of Peronosporaceae and that smaller
Table 2
Summary of Between Species Comparisons of Genome Structure for the Sampled Peronosporaceae
Clade Common interval distances Breakpoint distance Reversal distance
Range Means Range Means Range Means
Within clade Between clades Within clade Between clades Within clade Between clades
Clade 1 378–4,284 3,467.78 2,408.76 0–7 0.89 3.59 0–5 0.44 2.33
Clade 2 416–4,284 3,374.20 2,224.44 0–8 1.03 4.27 0–6 0.53 2.97
Clade 3 416–4,284 4,284.00 2,023.26 0–8 0.00 4.38 0–6 0.00 2.90
Clade 4 378–4,284 3,072.00 2,463.11 0–8 1.75 3.49 0–5 1.13 2.29
Clade 5 378–4,284 4,284.00 1,812.97 0–9 0.00 4.86 0–6 0.00 3.02
Clade 6 270–4,284 3,955.88 1,997.70 0–8 0.00 4.27 0–5 0.00 2.86
Clade 7 214–4,284 2,885.71 1,958.55 0–10 3.02 5.54 0–7 1.55 3.55
Clade 8 378–4,416 4,230.67 2,528.61 0–7 0.89 3.71 0–5 0.44 2.37
Clade 9 378–4,284 4,063.56 2,517.81 0–6 1.33 3.71 0–5 0.89 2.53
Clade 10 382–4,284 4,284.00 1,678.29 0–9 0.00 6.07 0–7 0.00 4.68
Clade 12 378–4,284 4,284.00 2,626.62 0–6 0.00 3.38 0–5 0.00 2.12
Clade 15 378–4,416 2,777.88 2,209.62 0–9 3.18 4.70 0–7 2.29 3.23
Clade 16 458–4,416 4,317.00 1,980.16 2–10 1.00 6.44 1–7 0.50 4.24
Nothophytophthora 344–1,394 –1,281.06 3–
12 –8.99 2–9–6.64
Phytopythium 214–458 –382.34 2–6–3.10 1–4–2.37
Winkworth et al. GBE
10 Genome Biol. Evol. 14(4) https://doi.org/10.1093/gbe/evac049 Advance Access publication 14 April 2022
87
0.99
84
1.0
P. infestansa
P. infestansb
P. infestansd
P. infestansc
P. litchii
P. colocasiae
a
P. colocasiae
b
P. pseudosyringae
P. sp. novaeguineae
P. tubulina
P. versiformis
P. quercina
Pseudoperonospora humuli
Peronospora effusa
Peronospora tabacina
P. chlamydosporaa
P. gonapodyides
P. x cambivora
P. x alni
Nothophytophthora sp.
P. r am or um a
P. sansomeana
P. cactorum
P. palmivoraa
P. palmivorab
P. pluvialisb
P. pluvialisa
P. multivora
a
P. multivora
b
P. cinnamomib
P. cinnamomia
P. castaneae
Plasmopara halstedii
P. capsici
a
P. capsici
b
P. cocoisb
P. cocoisa
P. heveae
P. agathidicidaa
P. agathidicidab
P. nicotianaeb
P. nicotianaea
P. lateralisa
P. lateralisb
P. kernoviaea
P. kernoviaeb
P. podocarpib
P. podocarpia
P. cryptogea
P. chlamydosporab
P. fallax
P. capitosa
P. pinifolia
P. r am or um b
P. sojae
P. phaseoli
P. andina
P. ipomoeae
P. mirabilis
Bremia lactucae
P. polonica
P. tropicalis
P. sp. subnubulis
Haloperonospora arabidopsidis
Peronospora belbahrii
P. rubi
P. fragariae
Clade 1
Clade 16
Clade 4
Clade 2
Clade 3
Clade 12
Clade 9
Clade 10
Clade 5
Clade 7
Clade 15
Clade 6
Clade 8
P. plurivora
P. megakarya
P. aleatoria
68
0.66
0.06
94
1.0
99
1.0
88
1.0
92
1.0
99
1.0
82
1.0
92
0.98
90
1.0
86
1.0
83
0.93
Phytopythium vexans
Pythium ultimum
98
1.0
98
1.0
87
1.0
99
1.0
FIG.3.—Phylogenetic relationships within Peronosporaceae based on analyses of the combined 34-gene mitochondrial sequence matrix. The topology is
that recovered by Bayesian analyses with branch lengths proportional to the mean of the corresponding posterior probability density. Values associated with
Comparative Analyses of Peronosporaceae Mitogenomes GBE
Genome Biol. Evol. 14(4) https://doi.org/10.1093/gbe/evac049 Advance Access publication 14 April 2022 11
repeats have subsequently arisen in multiple clades. An al-
ternative explanation is that smaller repeats arose repeated-
ly via progressive reduction of the larger ancestral repeats.
However, in at least some cases (i.e., arrangements XIV and
XV) the smaller inverted repeats contained genes not found
in the larger repeats of Phytopythium or Pythium, making
this explanation less likely.
Our analyses suggest structural rearrangements have
played an important role in the evolution of
Peronosporaceae mitochondrial genomes. Specifically,
based on gene order comparisons we identified 28 struc-
turally distinct mitogenomes among the 55 taxa we
sampled. Each of the 15 named or numbered clades that
we sampled was represented by at least one, and typically
two or more, unique arrangements. For example, all five
sampled clade 5 taxa shared the same gene order whereas
each of the six sampled clade 7 species had different gene
orders. Most commonly gene orders were restricted to spe-
cific named or numbered clades (e.g., arrangements I–V).
However, we identified three arrangements shared by
two or more of the numbered clades (i.e., arrangements
XX–XXII). For example, arrangement XXII was shared by
P. sojae (clade 7), P. polonica (clade 9), all members of clade
12, and 2 of 3 Peronospora species (clade 15).
Pairwise comparisons of the 28 structurally distinct synte-
nies suggestthat the degree of difference between syntenies
varies. Some mitogenomes differed by a single change. For
example, a single inversion (e.g., arrangements V and VII dif-
fer by an inversion of nad5–nad6–tRNA-Arg), loss (e.g., loss
of ymf101 in I relative to XX), or gain (e.g., gain of a second
nad4L copy in XIX relative to XVIII). In others, several changes
are needed to explain the differences. For example, XVIII and
XXII appear to differ on the basis of two overlapping inver-
sions; a large one involving a segment containing 25 genes
(i.e., tRNA-Arg to cob) and a smaller one involving a 10
gene segment (i.e., rps7 to nad7). The most distinctive mito-
genomes were arrangements XI, XXVI, XXVII, and XXVIII. In
most Peronosporaceae the two mitochondrial rRNA genes
group together with rpl5,rpl14, and eight tRNA genes.
However, in these four mitogenome arrangements the two
rRNA genes were separated by 28–37 protein coding and
tRNA genes. In Nothophytophthora sp. (XXVII) and Ph. vex-
ans (XXVIII), the two rpl genes fell next to the ssRNA gene
whereas in P. kernoviae (XXVI) and P. podocarpi (XI), they
are well separated (fig. 2). The distinctiveness of P. kernoviae,
P. podocarpi,andNothophytophthora sp. is consistent with
comparatively low average common interval distances and
comparatively high average breakpoint and reversal dis-
tances for these three species. In contrast, although average
common interval distances were very low for Ph. vexans
(i.e., 214–458 cf., 1,096–4,416), average breakpoint and re-
versal distances were more similar to those of the other species
(i.e., 2–6cf.,0–12 and 1–4cf.,0–9, respectively). This likely re-
flects the presence of large inverted repeat in Ph. vexans.
Further sampling is required to fully characterize the
structural diversity of Peronosporaceae mitogenomes.
However, our analyses suggest several insights. First, differ-
ent evolutionary processes appear to be acting on mitogen-
ome structure within and between taxa. Specifically, within
taxa we only observed nucleotide substitutions and length
differences whereas between taxa structural rearrange-
ments were also observed. Second, the rearrangements
that underpin differences in mitogenome structure be-
tween taxa are not uniformly distributed across the gen-
ome. Instead, the Peronosporaceae mitogenome appears
to be composed of two distinct segments. One, containing
30 protein encoding and ribosomal genes, was stable with
respect to gene content and order. The other varied mark-
edly in both gene content and order. Containing 35–81
genes all of the structural rearrangements that differen-
tiated the 28 observed syntenies were located within this
segment. Despite the variability within this block, all the ex-
amined mitogenomes shared several features. For example,
typically the tRNA genes have maintained their positions
relative to their immediate neighbors (e.g., all the mitogen-
omes contained the gene blocks rps10–tRNA-Arg–
tRNA-Gln–tRNA-Ile–tRNA-Val–rps12 and atp6–tRNA-Asp–
nad3). This implies rearrangements have more commonly
occurred between protein encoding than ribosomal genes.
Finally, our results are not consistent with the suggestion
that inverted repeats stabilize organellar genomes against
intramolecular homologous recombination (e.g., Adelberg
and Bergquist 1972;Hudspeth et al. 1983). Specifically,
we recovered between two and five syntenies from clades
containing mitogenome sequences with inverted repeats
but three or fewer arrangements from those in which mito-
genomes did not contain inverted repeats (clade 10 and
Phytopythium for which we had only one representative
were not considered). Further work is needed but there
are several possible explanations. One possibility is that in-
verted repeats act as rearrangement “hotspots”(e.g.,
Martin et al. 2007). In this case, mitogenomes containing in-
verted repeats may retain the potential for structural
change. Alternatively, genome size may determine whether
rearrangements are likely. For example, loss of the inverted
repeats could lead to genomes that are so compact that re-
arrangements become unlikely. Time may also play a role.
That is, clades with smaller numbers of genome arrange-
ments may have been diversifying for a shorter period of
time than those with larger numbers.
branches are maximum likelihood bootstrap support (upper) and Bayesian posterior probabilities (lower); values are only reported where bootstrap support
was ,100%. Superscript letters distinguish isolates of the same species (see supplementary table S1, Supplementary Material online for details) and the major
clades recognized by Bourret et al. (2018) are indicated on the right.
Winkworth et al. GBE
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Phylogenetic Analyses
The phylogenetic relationships suggested by our analyses of
combined mitochondrial protein-coding gene sequences
were broadly consistent with those reported previously
(e.g., Blair et al. 2008;Yang et al. 2017;Bourret et al.
2018). Specifically, we sampled 13 of the 16 numbered clades
recognized by Bourret et al. (2018) and in our phylogenetic
analyses, each was recovered with very strong support (i.e.,
posterior probabilities =1.0, bootstrap values .98%).
Relationships within the numbered clades were also consist-
ent with those reported by Bourret et al. (2018).Moreover,
all but one of these relationships, the pairing of P. megakarya-
palmivora (posterior probability =0.66, bootstrap value =
68%) received moderate to very strong support in both ana-
lyses. The placements of Nothophytophthora sp. and Ph. vex-
ans were also consistent with previous analyses (e.g., Jung
et al. 2017b). Specifically, Phytopythium branched first with
Nothophytophthora sister to the clade containing
Phytophthora and the downy mildews.
Although previous analyses have provided strong sup-
port for the named and numbered clades, relationships
among them have not been consistently resolved or well
supported. For example, Martin et al. (2014) conducted
analyses of mitochondrial, nuclear, and concatenated ma-
trices finding four different resolutions of relationships for
clade 2. At least, two of these had been recovered in previ-
ous analyses (e.g., Blair et al. 2008;Yang et al. 2017).
Bourret et al. (2018) highlighted hybridization, incomplete
lineage sorting and analytical errors as potential explana-
tions for discordance between mitochondrial and nuclear
topologies. Our analyses suggest an additional insight.
Despite strong support for relationships amongst the
named and numbered clades (i.e., posterior probabilities
=1.0, bootstrap values =82–100%), concordance factor
values for all but two of these relationships (i.e., those in-
volving Phytopythium and Nothophytophthora) fell below
50%. Short branches can contribute to low concordance
factor values and we found strong correlations between
each of the concordance factors and branch length. More
specifically, 45.5% (10/22) of branches representing rela-
tionships between the numbered clades were also amongst
the shortest 50% of internal branches in our topology and
for 80.0% (8/10) of these, the value of one or both con-
cordance factors fell below 50%. Although further analyses
are needed this observation implies that the numbered
clades arose over a relatively short period early in the evolu-
tion of Peronosporaceae. If so, then even in the absence of
hybridization or incomplete lineage sorting these relation-
ships are likely to be difficult to reconstruct, especially
with relatively limited data (Hendy and Penny 1989).
An analysis of genome rearrangements based on our top-
ology is consistent with a complex history of structural evolution
for Peronosporaceae mitogenomes. Our analysis suggested 48
events with up to four on a single branch (i.e., that leading to
Nothophytophthora sp.) and seven within a single clade (i.e.,
clades 7 and 15). However, our taxon sampling is limited and
we remain cautious about inferring ancestral gene order.
Although further sampling is needed similarities between the
mitogenomes of Nothophytophthora,Phytopythium,andPy.
ultimum (Pythiaceae) may provide some insights. Specifically,
all three of these taxa share a gene order in which the ssRNA,
rpl5,rpl14, and several tRNA genes are well separated from
the lsRNA gene. Additionally, Phytopythium has a large in-
verted repeat similar, although not identical in gene content,
to that of Py. ultimum. In both cases, none of the other sampled
Peronosporaceae have equivalent features (e.g., inverted re-
peats of other Peronosporaceae were much smaller and con-
tained fewer genes). A working hypothesis is that the
mitogenomes of the earliest Peronosporaceae possessed these
two features.
Our phylogenetic analyses combined broad samples of
taxa and mitochondrial protein-coding sequences.
Although the resulting phylogeny provides evolutionary in-
sights it is unlikely to fully describe the underlying species
tree. However, a robust mitochondrial phylogeny could
provide a framework for further analyses. One possible
use would be in formal tests of hybridization (e.g., Joly
et al. 2009;Joly 2012). There are numerous examples of
both natural (e.g., Bonants et al. 2000;Nirenberg et al.
2009;Goss et al. 2011) and synthetic (e.g., Goodwin and
Fry 1994;Donahoo et al. 2008)Phytophthora hybrids.
Moreover, in this genus hybridization is associated with
host range shifts (e.g., Ersek et al. 1995;Man in ‘t Veld
et al. 2006) and a role for hybridization in species diversifi-
cation has been hypothesized (Bertier et al. 2013). Testing
for hybridization involves gene tree incongruence, which
could be evaluated using phylogenies based on mitochon-
drial genome sequences. Mitogenome sequences might
also be used to estimate the timeframe over which
Peronosporaceae have evolved. Matari and Blair (2013)
used a multilocus nuclear data set to estimate divergence
times for the oomycetes and complete mitogenome se-
quences have been used for age estimation in
Phytophthora (e.g., Martin et al. 2016;Winkworth et al.
2021). Matrices of mitogenome sequences are relatively
long and this is encouraging in terms of resolving the time-
frame over which the Peronosporaceae have evolved.
Materials and Methods
Fungal Culture and DNA Extraction
We obtained single isolates of Nothophytophthora sp., P.
captiosa,P. chlamydospora, and P. fallax as well as two of
P. palmivora from the International Collection of
Microorganisms from Plants (ICMP) culture collection
(supplementary table S1, Supplementary Material online).
Comparative Analyses of Peronosporaceae Mitogenomes GBE
Genome Biol. Evol. 14(4) https://doi.org/10.1093/gbe/evac049 Advance Access publication 14 April 2022 13
Isolates were cultured on Phytophthora-selective media
(Jeffers and Martin 1986) in the dark at 18°C for up to 10
days. Agar plugs were excised from each plate and incu-
bated overnight at 56°C with 180 µl ATL buffer (Qiagen,
Hilden, Germany) and 20 µl proteinase K (20 mg/ml;
Qiagen). The tubes were then centrifuged with genomic
DNA extracted from the supernatant using the QIAcube®
instrument and the QIAamp® DNA mini QIAcube kit
(Qiagen).
Library Preparation and Genome Sequencing
Shotgun sequencing libraries were prepared from each of
the DNA extracts by the Massey Genome Service
(Palmerston North, New Zealand) using Illumina Nextera
DNA library preparation kits (Illumina, Inc., San Diego,
CA, USA). The Massey Genome Service also performed 2
×250 base pair (bp), paired-end DNA sequencing on
Illumina MiSeq instruments and quality assessed reads
using a combination of SolexaQA (Cox et al. 2010) and
FastQC (Andrews 2010).
Mitochondrial Genome Assembly and Annotation
We assembled complete mitochondrial genome sequences
for 44 isolates representing 34 members of the
Peronosporaceae. In addition to the six newly sequenced
isolates, we assembled 38 genomes using data available
from the NCBI Sequence Read Archive (supplementary
table S1, Supplementary Material online).
For all 44 isolates, we conducted de novo assemblies of
quality-assessed sequence reads using idba_ud (Peng et al.
2012). The resulting contigs were then filtered against a li-
brary of publicly available Peronosporaceae mitochondrial
genome sequences (supplementary table S1,
Supplementary Material online) using BLAST (Altschul
et al. 1997). Draft genomes were assembled from this mito-
chondrial subset using the assembly tools implemented in
Geneious R9 (Kearse et al. 2012). To evaluate drafts, we
used BWA (Li and Durbin 2009) to map the original
quality-assessed sequence reads to the corresponding draft
genome. Inconsistencies in read coverage were interpreted
as assembly errors and draft sequences revised as
appropriate.
The final mitochondrial genome sequences were anno-
tated on the basis of sequence similarity using a combin-
ation of Geneious and DOGMA (Wyman et al. 2004). For
each genome we also identified repeated sequences using
the Repeat Finder tool in Geneious; for these searches we
considered only perfect repeats with a minimum repeat
length of 150 nt.
Data Matrices and Phylogenetic Analyses
For phylogenetic analyses, we assembled multiple se-
quence alignments for the 39 common protein-coding
loci. Initial alignments were generated using ClustalO
(Sievers et al. 2011) and edited in Mesquite v3 (Maddison
and Maddison 2019) to remove portions where ,50% of
the sequences were represented or where alignments
were otherwise ambiguous. For phylogenetic analyses, a
concatenated matrix containing 34 loci was compiled.
Due to high levels of length and sequence variation we ex-
cluded alignments for the four hypothetical proteins (i.e.,
ymf98,ymf99,ymf100, and ymf100) and the
sec-independent transporter protein from further analyses.
Prior to maximum likelihood and Bayesian analyses, we
first used the Bayesian information criterion (BIC; Schwarz
1978) as implemented in jModelTest 2.2 (Guindon and
Gascuel 2003;Posada 2008) to identify best-fit substitution
models for each gene partition. We then performed a parti-
tioned maximum likelihood search using IQ-TREE v2.1.3
(Minh et al. 2020a;Chernomor et al. 2016). For this search,
we applied best-fit substitution models and support for re-
lationships was evaluated using 1,000 bootstrap replicates.
Bayesian searches were performed using MrBayes 3.2
(Ronquist and Huelsenbeck 2003). In this case, we con-
ducted two identical searches, each 2.5 ×10
7
generations
in length and sampled every 1.0 ×10
3
generations. For
each run, we determined burn-in using convergence diag-
nostics and samples drawn prior to stationarity discarded.
Posterior distributions of parameters were examined using
Tracer v1.7 (Rambaut et al. 2018) and consensus trees vi-
sualized with FigTree v1.4.
To examine conflict within our data set, we calculated a
pair of concordance factors as implemented in IQ-TREE
(Minh et al. 2020b). For any given branch in a reference top-
ology (e.g., from an analysis of concatenated sequences in
a phylogenomic study), the gene concordance factor (gCF)
is the percentage of gene trees (e.g., from analyses of the
individual sequence partitions) that contain the same
branch and the site concordance factor (sCF) is the percent-
age of informative alignment positions across the concate-
nated matrix that support the branch. Our analysis made
use of the topology from the partitioned maximum likeli-
hood search as well as those from similar searches for
each of the 34 mitochondrial gene partitions. We used
Kendall’sτ, as implemented in R (R Core Team 2021), to
evaluate whether the two concordance factors were corre-
lated as well as whether they were correlated with the
length of and support values for the corresponding
branches.
Comparisons of Genome Structure
We evaluated the overall structure of the Peronosporaceae
mitochondrial genomes in terms of gene order, gene orien-
tation and repeats. To facilitate these analyses, we first
standardized the annotations for all 71 included mitogen-
ome sequences. We considered a core set of 39 protein
Winkworth et al. GBE
14 Genome Biol. Evol. 14(4) https://doi.org/10.1093/gbe/evac049 Advance Access publication 14 April 2022
coding, 25 tRNA, and 2 rRNA genes common to the major-
ity of the Peronosporaceae mitogenome sequences. In
most cases, departures from this core set involved duplica-
tions of one or more genes or inclusion of open reading
frames. Duplicated core genes and those open reading
frames with high similarity to one of the core genes (e.g.,
the P. infestans orf217 was similar to ymf99) were retained.
We excluded gene annotations not amongst the core set
(e.g., ymf96) and open reading frames not similar to one
of the core genes. We also reoriented all the genomes so
that the large ribosomal RNA subunit was the forward
orientation and that the first residue of the genome corre-
sponded to the first residue of this gene.
To compare the overall structural similarity of
Peronosporaceae mitochondrial genomes we examined
gene order and orientation using the common interval ap-
proach (Uno and Yagiura 2000;Heber and Stoye 2001)as
implemented in CREx (Bernt et al. 2007). CREx does not al-
low for duplicated genes and we used two approaches to
address multiple gene copies. For genes that existed as mul-
tiple copies in all the sampled genomes (e.g., tRNA-Arg and
tRNA-Ser) we labeled each copy with a unique identifier, in
all these cases we were able to distinguish copies based on
their relative position in the genome. For genes duplicated
as part of a repeat in one or a few mitogenomes (e.g., atp6,
tRNA-Asp,nad3,nad5,nad6, and tRNA-Arg in P.pinifolia)
we removed one of the repeats prior to analysis. In each
case, we retained the copy of the repeat that minimized
the number of required changes. For this analysis, we
used default parameters and compared all genome pairs
based on common interval, breakpoint and reversal dis-
tances. We used Kendall’sτ, as implemented in R (R Core
Team 2021), to evaluate whether common interval, break-
point, and reversal distances between pairs of accessions
were correlated with sequence divergence (i.e., p
distances).
We also performed an ancestral character state recon-
struction of gene order using TreeREx (Bernt et al. 2008).
In this case, we used our maximum likelihood tree top-
ology, the same gene orders as for CREx and default
parameters.
Supplementary Material
Supplementary data are available at Genome Biology and
Evolution online.
Acknowledgements
Funding for this research was provided to RCW and PJL by
the BioProtection Research Centre and Massey University. A
New Zealand Ministry of Business, Innovation, and
Employment Strategic Science Investment Fund grant to
SEB also supported this research.
Data Availability
The data underlying this article are available in the GenBank
Nucleotide Database at https://www.ncbi.nlm.nih.gov/
nucleotide, and can be accessed using the accession num-
bers listed in Supplementary material, Supplementary
Material online.
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Associate editor: Liliana Milani
Comparative Analyses of Peronosporaceae Mitogenomes GBE
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