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\begin{document}

\title{Tar spot of maize in the Americas is caused by a complex of closely
related Phyllachora species which vary in their host and geographic
range}



\author[1]{Kirk Broders}%
\author[2]{Gloria Iriarte}%
\author[3]{Gary Bergstrom}%
\author[4]{Emmanuel Byamukama}%
\author[5]{Martin Chilvers}%
\author[6]{Christian Cruz}%
\author[7]{Felipe Dalla Lana}%
\author[8]{Dean Malvick}%
\author[9]{Darren Mueller}%
\author[10]{Pierce  Paul}%
\author[11]{Diana Plewa }%
\author[12]{Richard  Raid}%
\author[9]{Alison Robertson}%
\author[1]{Catalina Salgado}%
\author[13]{Damon Smith}%
\author[6]{Darcy Telenko}%
\author[11]{Nathan Kleczewski}%
\author[11]{Katherine VanEtten}%
\author[11]{Zachary Duray}%
\affil[1]{USDA-ARS}%
\affil[2]{Independent Data Analyst}%
\affil[3]{Cornell University}%
\affil[4]{South Dakota State University}%
\affil[5]{Michigan State University}%
\affil[6]{Purdue University}%
\affil[7]{Pennsylvania State University}%
\affil[8]{University of Minnesota Twin Cities}%
\affil[9]{Iowa State University}%
\affil[10]{The Ohio State University}%
\affil[11]{University of Illinois at Urbana-Champaign}%
\affil[12]{University of Florida}%
\affil[13]{University of Wisconsin-Madison}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
The genus Phyllachora contains numerous obligate fungal parasites that
produce raised, melanized structures called stromata on their plant
hosts. Most members of this genus are not of significant economic
concern, with the exception of P. maydis, the causal agent of tar spot
of maize (Zea mays). Tar spot of maize has emerged as a major threat to
maize production throughout the Americas and continues to spread
throughout North America. To date, species designations for Phyllachora
have been based on host associations and morphology, and the origin and
diversity of the pathogen that causes tar spot is unknown. We assessed
the sequence diversity of 186 single stroma isolates collected from 16
hosts representing 15 countries by amplification of the ITS and LSU gene
regions. Samples included both herbarium and contemporary strains that
covered a temporal range from 1905-2019. These 186 isolates were grouped
into 5 distinct species with strong bootstrap support. We found three
closely related, but genetically distinct groups of Phyllachora are
capable of infecting maize in the United States, we refer to these as
the P. maydis species complex. Based on herbarium species, we
hypothesize that these three groups in the P. maydis species complex
originated from Central America, Mexico and the Caribbean. Although two
of these groups were only found on maize, the third and largest group
contained contemporary strains found on maize and other grass hosts, as
well as herbarium specimens from maize and other grasses that include 10
species of Phyllachora. The herbarium specimens were identified based on
morphology and host association, but our data indicates there may be
significant synonymy in the Phyllachora genus and additional work on
species delineation and host specificity should be considered.%
\end{abstract}%



\sloppy


\textbf{Tar spot of maize in the Americas is caused by a complex of
closely related \emph{Phyllachora} species which vary in their host and
geographic range}

Kirk Broders*\textsuperscript{1}, Gloria
Iriarte-Broders*\textsuperscript{2}, Gary C.
Bergstrom\textsuperscript{3}, Emmanuel Byamukama\textsuperscript{4},
Martin Chilvers\textsuperscript{5}, Christian Cruz\textsuperscript{6},
Felipe Dalla-Lana\textsuperscript{7}, Zachary Duray\textsuperscript{8},
Dean Malvick\textsuperscript{9}, Daren Mueller\textsuperscript{10},
Pierce Paul\textsuperscript{11}, Diane Plewa\textsuperscript{8}, Richard
Raid\textsuperscript{12}, Alison E. Robertson\textsuperscript{10},
Catalina Salgado-Salazar\textsuperscript{13}, Damon
Smith\textsuperscript{14}, Darcy Telenko\textsuperscript{7}, Katherine
VanEtten\textsuperscript{8}, Nathan M Kleczewski*\textsuperscript{8}

\textsuperscript{1} USDA, Agricultural Research Service, National Center
for Agricultural Utilization Research, Mycotoxin Prevention and Applied
Microbiology Research Unit. 1815 N. University, Peoria, IL. 61604, USA

\textsuperscript{2} Independent Data Analyst, Dunlap, IL 61525

\textsuperscript{3} Cornell University, Plant Pathology and
Plant-Microbe Biology Section, School of Integrative Plant Science,
Ithaca, NY 14850

\textsuperscript{4} South Dakota State University, Department of
Agronomy, Horticulture, and Plant Science, Box 2108 University Station,
Brookings, SD, 57007

\textsuperscript{5} Michigan State University, Department of Plant,
Soil, and Microbial Sciences, 578 Wilson Road, East Lansing, MI, 48824

\textsuperscript{6} Purdue University, Department of Botany and Plant
Pathology, 915 W. State Street, West Lafayette, IN 47907

\textsuperscript{7} Pennsylvania State University, Department of Plant
Pathology and Environmental Microbiology, Southeast Agricultural
Research \& Extension Center, 1446 Auction Road, Manheim, PA 17545

\textsuperscript{8} University of Illinois, Department of Crop Sciences,
1101 W Peabody Drive, Urbana, IL

\textsuperscript{9} University of Minnesota, Department of Plant
Pathology, 1991 Upper Buford Circle, St Paul, MN 55108

\textsuperscript{10} Iowa State University, Department of Plant
Pathology and Microbiology, Iowa State University, 2213 Pammel Drive,
Ames, IA, 50010

\textsuperscript{11} The Ohio State University, Department of Plant
Pathology 1680 Madison Avenue, Wooster, OH, 44691

\textsuperscript{12} University of Florida, Department of Plant
Pathology, 2550 Hull Road, Gainesville, FL, 32611

\textsuperscript{13} USDA, Agricultural Research Service, Mycology and
Nematology, Genetic Diversity, and Biology Laboratory, 10300 Baltimore
Avenue, Beltsville MD, 20705

\textsuperscript{14} University of Wisconsin-Madison, Department of
Plant Pathology, 1630 Linden Drive, Madison, WI, 53706

*These authors contributed equally to this manuscript

Corresponding author kirk.broders@usda.gov

Keywords: Phyllachorales, biotrophs, pathogen diversity, tar spot,
phylogeny, sympatric speciation

\textbf{Abstract:}

The genus \emph{Phyllachora} contains numerous obligate fungal parasites
that produce raised, melanized structures called stromata on their plant
hosts. Most members of this genus are not of significant economic
concern, with the exception of \emph{P. maydis} , the causal agent of
tar spot of maize (\emph{Zea mays} ). Tar spot of maize has emerged as a
major threat to maize production throughout the Americas and continues
to spread throughout North America. To date, species designations
for\emph{Phyllachora} have been based on host associations and
morphology, and the origin and diversity of the pathogen that causes tar
spot is unknown. We assessed the sequence diversity of 186 single stroma
isolates collected from 16 hosts representing 15 countries by
amplification of the ITS and LSU gene regions. Samples included both
herbarium and contemporary strains that covered a temporal range from
1905-2019. These 186 isolates were grouped into 5 distinct species with
strong bootstrap support. We found three closely related, but
genetically distinct groups of \emph{Phyllachora} are capable of
infecting maize in the United States, we refer to these as the \emph{P.
maydis} species complex. Based on herbarium species, we hypothesize that
these three groups in the \emph{P. maydis} species complex originated
from Central America, Mexico and the Caribbean. Although two of these
groups were only found on maize, the third and largest group contained
contemporary strains found on maize and other grass hosts, as well as
herbarium specimens from maize and other grasses that include 10 species
of \emph{Phyllachora} . The herbarium specimens were identified based on
morphology and host association, but our data indicates there may be
significant synonymy in the \emph{Phyllachora} genus and additional work
on species delineation and host specificity should be considered.

\textbf{Introduction}

Phyllachorales is a monophyletic order of biotrophic fungi comprised of
approximately 1,226 recognized species (Maharachchikumbura et al. 2016,
Mardones et al. 2017), but global estimates of species within this order
approach 160,000 (Cannon 1997). The Phyllachorales largely contain plant
parasitic fungi and are commonly associated with monocotyledonous plants
across a range of habitats. These fungi are often referred to as ``tar
spot'' fungi due to the production of stromata on plant hosts that
resemble black flecks of tar (Fig 1) (Mardones et al. 2017).

Tar spot of maize (Figure 1 A-C), caused by the fungus \emph{Phyllachora
maydis} , emerged in the United States (U.S.) in 2015, with the disease
expanding each year since the initial report and continuing to have a
significant economic impact on maize across many production regions in
the U.S. (Kleczewski et al. 2020a, Valle-Torres et al. 2020). Since
first being identified in North America in 2015, \emph{P. maydis} has
spread rapidly throughout the U.S. and Canada (Kleczewski and Bowman
2020, Kleczewski et al. 2020), and resulted in yield losses exceeding
\$US 658 million in 2018 (Mueller et al. 2020). \emph{Phyllachora
maydis} caused significant losses again in 2021, appearing to be on par
or greater than 2018 levels (authors personal observations). Although
tar spot symptoms caused by members of the genus \emph{Phyllachora} have
been commonly observed on a number of grasses (Figure 1 D-F) and shrub
species throughout North, Central and South America, historically the
fungus has rarely been known to cause significant plant damage. However,
tar spot has been occasionally reported to cause severe damage to maize
in Mexico, Central America and several Caribbean Islands (Valle-Torres
et al. 2020).

The origin of \emph{P. maydis} within the U.S. is not currently known,
although the presence of two distinct epicenters of maize tar spot in
the Midwest and Southeast indicates at least two separate emergence
events. While tar spot is a new disease on maize in the U.S. and Canada,
it has been present in Mexico, several Caribbean islands including
Puerto Rico, Cuba and the Dominican Republic as well as Central American
Countries, such as Guatemala, Honduras, Nicaragua and Costa Rica for the
last century but only caused limited damage. In addition, tar spot signs
and symptoms caused by \emph{Phyllachora} species are common on several
native and weedy grass species in North America (Figure 1 D-F) (Orton
1944). The monographic work by Orton (1944) was completed solely by
morphological identification and host affinity. Given our understanding
of phenotypic plasticity of many fungi and the ability of biotrophic
pathogens to infect multiple hosts (Morris and Moury 2019), it is
possible that cryptic species or species complexes may be present.

Species definitions within the Phyllachorales have historically been
based largely on morphological characteristics and assumption of high
host specificity, due to their presumed biotrophic nature. However,
there are examples in the genus where this assumption of host
specificity does not hold true (Cannon 1991, Cannon 1997). Furthermore,
species designations based on host specificity are highly dependent on
accurate identification of the host species, which may be difficult or
impossible in some instances. For example, \emph{P. graminis} (Pers.)
Fuckel is considered a ``dustbin'' species where many specimens of
isolates infecting grasses are deposited with the host not often
identified to species (Parbery 1967). Furthermore, factors such as
nutrients available to the fungus, temperature, light quality, light
cycles, substrate type, host, and epigenetic factors may also result in
alterations in fungal morphology that may result in inaccurate species
designations (Slepecky and Starmer 2009, Stockinger et al. 2009, Money
2013, Francisco et al. 2019). Thus, our current understanding of the
genetic diversity, host range and species delimitation within the
genus\emph{Phyllachora} is relatively limited and requires reevaluation.

The recent emergence of \emph{P. maydis} in the U.S. and Canada may also
be associated with the ability of the fungus to better persist and
spread than previously thought. Once established, the fungus can survive
at least one winter at subzero temperatures on corn residue as
ascospores within stromata, which are believed to be the main inoculum
source the following season (Kleczewski et al. 2019, Groves et al.
2020). Under periods of moderate temperatures and wet weather it is
believed that ascospores are dispersed by wind and rain-splash where
they land on the foliage, stalks, and husks of corn. After spore
germination and infection of the host, the fungus remains dormant for at
least 2 weeks after which stromata, and associated spermatia and
ascospores, are produced. Data from Central America indicated a
relatively steep dispersal curve of \emph{P. maydis} ascospores from a
source (Hock et al. 1995). However, the rapid spread of this fungus
throughout the Midwest, coupled with observations of ``top down''
infestations in fields with no history of disease and observations of
infestations of isolated plots located 1,200 m from potential inoculum
sources, indicate that the pathogen can travel much further across
local/regional topographies than estimated previously (Kleczewski et al.
2020).

Based on this information, the emergence of \emph{P. maydis} on corn in
the U.S. and Canada could have been the result of many factors including
the introduction of the fungus on infected plant material, natural
northern dispersal through wind, establishment in the U.S. favored by
climate change, changes in hybrid genetics, a host-jump from a grass
species, or a combination of any of these four. In this study we use DNA
sequence data to understand the genetic diversity of \emph{P.
maydis}populations in contemporary maize production regions in the U.S.,
and compare this to historical specimens of \emph{P. maydis} from
herbarium samples from Mexico, Central and South America, the Caribbean,
and contemporary and herbarium species of \emph{Phyllachora} species
associated with grass hosts in the U.S. The goal of this study is to
understand the genetic diversity and population dynamics within
the\emph{P. maydis} population infecting corn in North America.
Differences in genetic backgrounds may imply dissimilar biology, and
potentially interactions with the host. Furthermore, understanding the
overall phylogenetic diversity and the potential host and geographic
range of\emph{Phyllachora} populations associated with maize and other
grasses in the Americas will help to infer the potential evolutionary
origins and speciation patterns in this genus.

\textbf{MATERIAL AND METHODS}

\textbf{2.1 Sample Collection.}

Samples of maize and wild grasses with characteristic stromata
of\emph{Phyllachora spp} . (Figure 1 A-F)) were collected from across
North America and Mexico in 2018 and 2019 (Table 1). Field specimens of
infested maize and other grasses were collected by numerous individuals
from the agricultural community as described in Kleczewski et al (2020).
Samples were pressed, dried at room temperature, and stored at 20\selectlanguage{ngerman}°C in
manilla envelopes until processed. Herbarium specimens were obtained
from the U.S. National Fungus Collection (BPI, Beltsville, Maryland) and
the University of Illinois Herbarium (Urbana, Illinois), which included
specimens on maize and other grasses from additional hosts, countries,
and years (Table 1). A total of 186 samples from 16 hosts, and 15
countries collected from 1905-2019 were included in the analyses.

\textbf{2.2 DNA extraction, PCR amplification and sequencing of stroma
from leaf tissue.}

The DNA of individual stroma not surrounded by a necrotic halo were
extracted using the X-Tract-N-AMP kit following manufacturer protocols
(Sigma). The complete internal transcribed spacer region of ribosomal
DNA (ITS1-5.8S-ITS2) with primers ITS1f and ITS4 (White et al. 1990).
Stroma without necrotic halos were selected to reduce the potential for
contamination by saprophytic fungi that may be present on necrotic
tissue within these lesions.

The ITS gene region was amplified from DNA extracted from each stroma
using the primer pair ITS1f and ITS4 (White 1990, Bruns and Gardes 1993)
with 35 cycles of the following: 95°C 5 min, 94°C 30s 52°C 30s, 72°C 1
min followed by 72°C for 8 min and a final hold at 4°C in a Thermo
Fisher SimpliAmp thermocycler (Thermo Fisher Scientific, Waltham, WA).
Individual PCR products from corresponding DNA extractions were loaded
into 2\% agarose gels and separated via electrophoresis for 40 minutes
at 110V. All gels contained a \emph{P. maydis} positive control,
a\emph{Fusarium graminearum} positive control, and a negative buffer
control for quality assurance. Bands on gels were visualized using an
Axygen gel imaging station (Axygen, Inc, Union City, CA). Stroma
of\emph{Phyllachora spp.} can be colonized by or associated with several
other fungal species (Hock et al. 1992, Hock et al. 1995, McCoy et al.
2019). Consequently, samples returning a single band between 300-500 bp
were considered free of additional fungal contaminants and used in
subsequent analyses.

DNA from samples returning a single ITS band were subject to
amplification of the Large Ribosomal Subunit (LSU) region using the
primer pair LROR and LR5 (Dayarathne et al. 2017) using the
aforementioned thermocycler conditions. All PCR products were purified
using QIAquick PCR kits (Quiagen, Inc., Hilden, Germany), and the ITS
and LSU amplicons for all samples were sequenced in the forward and
reverse directions at the University of Illinois Core DNA Sequencing
Facility (Urbana, Illinois).

\textbf{2.3 Sequence alignment, phylogenetic analysis and molecular
identification}

Sequences generated from this study were combined with sequences
obtained from GenBank. \emph{Exserohilum turcicum} and \emph{Cocoicola
californica} were selected as the outgroups. Sequence data was aligned
and concatenated using MAFFT v.7
(mafft.cbrc.jp/alignment/server/index/html) using the G-INS-I model and
manually inspected. The best fit partitioning schemes were determined
using PartitionFinder (Lanfear et al. 2017) and used to build the
phylogenies. Both single gene and concatenated gene sets were analyzed
using a maximum likelihood (ML) analysis. The ML phylogenies were
generated by RaxML under GTR model with gamma distributed rate
heterogeneity with 1000 bootstrap replicates. Resulting trees were
visualized with iTOL (Interactive Tree of Life) v.6
(\emph{https://itol.embl.de/}) or MEGA. Sequences generated in this
study were deposited in GenBank (Table 1)

\textbf{RESULTS}

\textbf{3.1 DNA extraction and PCR amplification from herbarium and
contemporary samples}

A total of 186 samples from twelve states in the U.S. (n=130), four
states in Mexico (n=13), three Central American countries (n=13), four
South American countries (n=6), four Caribbean Islands (n=16), Germany
(n=3), India (n=3) and The Philippines (n=2) were sequenced and analyzed
as noted above. There were varying levels of success for the
amplification of each genetic locus among the samples. This was
particularly the case for many of the herbarium samples, some of which
were more than 100 years old. The ITS region was the most successfully
amplified and sequenced, with 168 sequences generated. Whereas 91
sequences were generated for the LSU locus (Table 1).

\textbf{3.2 Phylogenetic diversity of \emph{Phyllachora} isolates
infecting maize and grasses}

Based on both the ITS + LSU (Figure 2) and ITS (Figure S1) phylogenies,
we observed five genetically distinct groups that represent individual
species of \emph{Phyllachora} with strong bootstrap support
(\textgreater{}70\%). The results suggest that tar spot on maize in the
U.S. is caused by three closely related species of
\emph{Phyllachora}(Figure 2). In all, four species were found on maize
but only\emph{Phyllachora} \emph{sp. 1} , \emph{P. sp.} 2, and \emph{P.
sp} .\emph{3} were recovered from contemporary maize in the U.S.,
while\emph{P. sp. 4} was recovered from herbarium samples collected in
Guatemala and Venezuela (Table 1).

Samples of \emph{Phyllachora} \emph{sp. 3} represent the broadest
geographic and host range and was also the most frequently recovered
species associated with \emph{Phyllachora} sp. stroma on maize from both
herbarium and contemporary specimens representing a span of time from
1905-2019 (Table 1). Samples of \emph{P. sp. 3} on maize were reported
and recovered from herbarium samples throughout the Americas including
Bolivia, Colombia, Costa Rica, Cuba, Dominican Republic, Guatemala,
Mexico, Nicaragua, Puerto Rico, and Trinidad and Tobago prior to the
first report of tar spot on maize in the U.S. Importantly the type
specimen of \emph{P. maydis} (BPI638553) collected in Mexico in 1977 and
the \emph{P. maydis} isolate (BPI893226) used in the first report of tar
spot in the U.S. in 2015 are both part of \emph{P. sp. 3} and isolates
of this species have since been recorded in Illinois, Indiana, Iowa,
Michigan, Minnesota and Wisconsin. This represents the widest geographic
range of the maize infecting \emph{Phyllachora} species in the U.S.
among the samples included in this study. However, isolates of \emph{P.
sp. 3} were also recovered from another 10 host species including
monocots and dicots, with a global distribution including 12 countries
across South, Central and North America and the Caribbean, as well as
Germany, India and the Philippines (Table 1; Figure 3). The herbarium
samples associated with each of the 10 host species represented
morphologically recognized species of \emph{Phyllachora}
including\emph{P. graminis} , \emph{P. heraclei, P. junci, P.
chaetochloae, P. diplocarpa, P. epicampis, P. euphorbiaceae, P.
rottboelliae, P. sylvatica, and P. vulgata} .

The other two contemporary maize-infecting species, \emph{P.} \emph{sp.
1} and \emph{P.} \emph{sp. 2} , have a more limited observed host and
geographic range. Both species were only recovered on
maize.\emph{Phyllachora sp. 1} was only recovered from contemporary
maize samples from Indiana and Ohio, whereas \emph{P. sp. 2} was found
on herbarium specimens from Colombia and Puerto Rico and contemporary
specimens from Puerto Rico, Mexico (Guerrero, Oaxaca, Puebla, Veracruz),
and the U.S. (Florida, Illinois and Michigan).

The other species recovered from maize was \emph{Phyllachora sp.
4.}However, samples only included herbarium specimens from Guatemala and
Venezuela and did not include any contemporary maize specimens.
Interestingly, \emph{P. sp. 4} was commonly found among grasses in the
U.S. that are found in proximity to maize production fields in Illinois,
South Dakota and New York (Table 1). Isolates of \emph{P. sp. 4} were
recovered from 6 grass species in 4 tribes in the U.S. representing a
broad host range across a breadth of genetically diverse grass
species.\emph{Phyllachora} \emph{sp. 5} was the only species not
recovered from maize but was found on many of the same grass species as
\emph{P. sp. 4} , including rye, triticale and fall panicum (Table 1).

While there was limited \emph{Phyllachora} sequence data in Genbank, we
were able to include the ITS sequence of 19 isolates representing six
recognized species of \emph{Phyllachora} to determine any relationship
between the isolates used in this study to those submitted previously to
Genbank (Figure 3). In the case of \emph{P. sp. 4} , two isolates
referred to as \emph{P. graminis,} one from \emph{Hordelymus
europaeus}in Germany and one of unknown origin, as well as isolates
of\emph{Phyllachora} on \emph{Elymus kamoji} and \emph{Roegneria sp} .
from China grouped together with strong boostrap support (99\%). There
was also an isolate of \emph{P. graminis} from an unknown grass in
Canada that grouped together with \emph{P. sp. 5} , and the herbarium
specimen of \emph{P. graminis} from \emph{Agropyron repens} in Germany
from this study grouped in \emph{P. sp. 3} (Figure 3). Our results
support the findings of previous observations that \emph{P. graminis} is
a poorly defined polyphyletic species, that has often been assigned to
tar spot symptom on a variety of grass hosts.

\textbf{Discussion}

Since \emph{P. graminis} was described by Nitschke 1870 (Fuckel 1870),
over 300 species have been recorded on graminaceous hosts, and many more
on non-grass hosts. However, Parbery recognized that there are fewer
species associated with grasses and established that there were 95 valid
graminicolous \emph{Phyllachora} species world-wide based on
morphological characteristics (Parbery 1967). In the most complete study
of \emph{Phyllachora} species in North America, Orton (1944) identified
45 morphological species from more than 100 host species (Orton 1944).
While this likely represents a significant overestimation of the true
number of species in North and Central America, it does demonstrate the
vast number of hosts on which \emph{Phyllachora} species have been
reported. Our results based on both herbarium and contemporary samples
of infected hosts indicate that there are far fewer species
of\emph{Phyllachora} in the Americas than indicated by Orton (1944) and
Parbery (1967), and the species that are present have a greater host
range than previously thought. The predominant species in this
study,\emph{P. sp. 3} , has a broad geographic and host range with the
capacity to infect maize throughout South, Central and North America as
well as seven grass species and two dicot species. This phylogenetic
species also includes isolates of 11 morphologically determined species
of\emph{Phyllachora} (\emph{P. chaetochloae, P. diplocarpa, P.
epicampsis, P. euphorbiaceae, P. graminis, P. heraclei, P. junci, P.
maydis, P. rottboelliae, P. sylvatica,} and \emph{P. vulgata} ) from
herbarium samples collected in the Dominican Republic, Germany, India,
Mexico, the Philippines, Trinidad and Tobago and the U.S., indicating
global distribution of this species. This expanded host range also now
complicates the taxonomic status and the name to be retained by this
genetic group. An isolate of \emph{P. graminis} collected
from\emph{Agropyron repens} from Germany was designated as the lectotype
specimen for the genus (Clements and Shear 1931), and the isolate
of\emph{P. graminis} examined in this study was also collected
from\emph{A. repens} in Germany, indicating that \emph{P. graminis} may
have precedence for the species name of \emph{P. sp. 3} . This would
have ramifications for \emph{P. maydis} as well as several
other\emph{Phyllachora} species in \emph{P. sp. 3} (Table 1; Figure 3)
that appear to be synonyms of \emph{P. graminis} . This is based solely
on sequence data from the ITS and/or LSU region and further multi-gene
phylogenetic studies of a larger representation of type material from
herbaria and contemporary \emph{Phyllachora} samples from additional
hosts is needed for a thorough taxonomic assessment of this genus.

The three maize-infecting species, \emph{P. sp. 1, P. sp. 2} and\emph{P.
sp. 3} , have overlapping geographic and host ranges, providing the
opportunity for co-infection and genetic exchange. Co-infection on the
same leaf tissue by \emph{P. sp. 3} and \emph{P. sp. 1} was observed on
four occasions with herbarium samples (BPI893232\_1 and BPI893232\_2 ,
BPI893231\_1 and BPI893231\_2, BPI893226\_1 and BPI893226\_2,
BPI893230\_1 and BPI893230\_2) from 3 counties in Indiana. A recent
fungal community analysis of tar spot lesions on maize found a similar
trend with two distinct OTUs occurring on 21 of 22 maize leaf samples
from Michigan (McCoy et al. 2019). A similar phenomenon has also been
observed in \emph{Albugo candida} , another biotrophic pathogen with a
broad host range (McMullan et al. 2015). Races of \emph{A. candida} were
not able to infect a host on their own but were able to co-infect with a
race-specific isolate that suppressed host immunity in that host. The
offspring of any genetic introgression or recombination resulted in a
race with an expanded host range able to infect both plants infected by
the parental strains of \emph{A. candida} . A whole genome comparison of
these \emph{A. candida} races found a mosaic-like genome structure with
large portions conserved between races, as well as regions with only
89\% sequence similarity. This scenario may explain the wide host range
and variation in morphology between hosts in \emph{Phyllachora} species.
Sexual reproduction in \emph{P. maydis} followed by discharge of
infective ascospores commonly occurs on corn leaves annually in maize
producing regions of the U.S. (Kleczewski et al. 2019, Groves et al.
2020b). The presence of multiple maize infecting species in the
midwestern US, and even on a single infected leaf, combined with
frequent sexual recombination, ascospore release and infection, could
result in novel populations and/or species of \emph{Phyllachora} that
are more virulent on maize or that have an expanded host range. This may
also explain why \emph{P. sp. 3} has such a broad host range
whereas\emph{P. sp. 1} and \emph{P. sp. 2} were only found on maize.
Individual populations may gain the ability to infect a new host but are
still able to sexually recombine with the rest of the population on the
original host species. Given the geographic overlap of many grass
species in Central, South and North America, small populations of
\emph{P. sp. 3}may have adapted to infect a novel grass species, while
maintaining the ability to recombine with the larger \emph{P. sp. 3}
complex, resulting in the expansion of the host range without
specialization and speciation.

Speciation has likely occurred in instances where geographic isolation
of a new host prevented further introgression with the original
population. As maize is commonly grown from Argentina to Canada, it
represents a common host for which distinct
\emph{Phyllachora}populations may infect and recombine resulting in
potentially new and more virulent populations that are still part of the
same species. It is unclear if geographic or genetic barriers lead to
speciation between the closely related \emph{P. sp. 1, P. sp. 2} and
\emph{P. sp. 3} , but the significant overlap in host and geography
would indicate a genetic barrier. While \emph{P. sp. 1} and \emph{P. sp.
2} were only recovered from maize, our sampling scheme was strongly
biased towards maize. It is possible that \emph{P. sp. 1} and \emph{P.
sp. 2} are present on other grass and non-grass hosts in Central and
North America and were not sampled in this study. These non-sampled
hosts, if only infected by one of the \emph{Phyllachora} species, may
represent the isolation that led to adaptation and speciation.

For now the name \emph{Phyllachora maydis} will be retained by \emph{P.
sp. 3} as the \emph{P. maydis} type material (BPI638553) clustered with
this group. However, the presence of three maize-infecting species, the
lack of type material of \emph{P. graminis} , and the potential
taxonomic synonymy with \emph{P. graminis} and several other
\emph{Phyllachora}species makes it difficult to determine which of the
maize infecting species will retain the name \emph{P. maydis} .
Therefore, we recommend referring to \emph{P. sp. 1, P. sp. 2} and
\emph{P. sp. 3} as the\emph{Phyllachora maydis} species complex until
further morphological and multi-gene phylogenetic studies can properly
delineate these species.

In this work, we conducted the most comprehensive assessment
of\emph{Phyllachora maydis} reported to date and provided evidence that
our understanding of this species and genera is limited and requires
significant attention. The reasons for the emergence of tar spot, caused
by three different species of \emph{Phyllachora} that have been present
in Central America, Mexico and the Caribbean for over 75 years is still
unclear. Several scenarios may explain the recent emergence and severity
of tar spot caused by \emph{Phyllachora} \emph{spp} . in the upper
Midwest of the U.S. While \emph{P. sp. 2} and \emph{P. sp. 3} have been
present in both Mexico and Puerto Rico for the last century, it is
possible that when the fungus was able to be dispersed via wind and rain
to the U.S. it could not overwinter in colder climates and the disease
could not get established. In fact, according to the herbarium
specimens, \emph{P. sp. 3} has been present in the U.S. since the 1940s
in California and Arizona on native grasses but not maize. However,
recent studies have demonstrated that \emph{Phyllachora} spp. can
overwinter in Illinois (Kleczewski et al. 2019). Shorter and warmer
winters due to climate change could be playing a role in the ability
of\emph{Phyllachora} spp. to survive further north in the U.S. Changes
in climate patterns during the growing season may also have an impact on
this disease as increased temperature and precipitation may promote
epidemics of this disease. Finally, a change in maize genetics may also
play a role in the increased severity of tar spot. Since maize breeding
programs were not selecting for resistance to tar spot, any partial
resistance that may have been present in U.S. germplasm may have been
lost through genetic drift. The loss of this resistance may not have
been noticed until \emph{Phyllachora} spp. arrived in the primary maize
growing region of the U.S. The disease remains of minor importance in
Mexico and Central American maize production, as resistance to this
disease would be selected for in breeding programs. The most likely
scenario for the emergence of tar spot in the U.S includes a combination
of these factors: 1) introduction of multiple species
of\emph{Phyllachora} from Mexico, Puerto Rico or other Central American
countries through movement of infected plant tissue or possible
long-distance movement via wind, rain, hurricane/tropical storm system,
etc., 2) change in climate in the Midwestern maize growing region more
hospitable to the growth, reproduction and survival of \emph{Phyllachora
spp.} ; and 3) lack of resistance in maize germplasm grown in the
Midwestern U.S.

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\textbf{Table 1.} The sample ID, genetic cluster, geographic and host
origin, year collected and source of the 186 \emph{Phyllachora}
specimens used in this study.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
\textbf{SampleID} & \textbf{Genetic Cluster\textsuperscript{a}} &
\textbf{Species\textsuperscript{b}} & \textbf{State} & \textbf{Country}
& \textbf{Host} & \textbf{Year Collected} & \textbf{Source} &
\textbf{GenBank Accessions} & \textbf{GenBank Accessions}\tabularnewline
& & & & & & & & \textbf{ITS} & \textbf{LSU}\tabularnewline
BPI893226\_2 & 1 & \emph{Phyllachora maydis} & Indiana & USA & \emph{Zea
mays} & 2015 & Field collection & OL342800 &\tabularnewline
BPI893227\_2 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} &
2015 & Field collection & OL342801 &\tabularnewline
BPI893229\_2 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} &
2015 & Field collection & OL342802 &\tabularnewline
BPI893230\_2 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} &
2015 & Field collection & OL342803 &\tabularnewline
BPI893231\_2 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} &
2015 & Field collection & OL342804 &\tabularnewline
BPI893232\_2 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} &
2015 & Field collection & OL342805 &\tabularnewline
C18001-2 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2018
& Field collection & OL342781 &\tabularnewline
C18001-3 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2018
& Field collection & OL342782 &\tabularnewline
C18003-1 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2018
& Field collection & OL342783 &\tabularnewline
C18003-2 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2018
& Field collection & & OL314402\tabularnewline
C18003-3 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2018
& Field collection & OL342784 &\tabularnewline
C18009-1 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2018
& Field collection & OL342785 &\tabularnewline
C18009-2 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2018
& Field collection & OL342786 &\tabularnewline
C18011-3 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2018
& Field collection & OL342787 & OL314403\tabularnewline
C18024-1 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2018
& Field collection & OL342788 &\tabularnewline
C18024-2 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2018
& Field collection & OL342789 &\tabularnewline
C18024-3 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2018
& Field collection & OL342790 &\tabularnewline
C18161-1 & 1 & \emph{P. maydis} & Ohio & USA & \emph{Zea mays} & 2018 &
Field collection & OL342791 & OL314404\tabularnewline
C18161-2 & 1 & \emph{P. maydis} & Ohio & USA & \emph{Zea mays} & 2018 &
Field collection & OL342792 & OL314405\tabularnewline
C18161-3 & 1 & \emph{P. maydis} & Ohio & USA & \emph{Zea mays} & 2018 &
Field collection & & OL314406\tabularnewline
C18162-1 & 1 & \emph{P. maydis} & Ohio & USA & \emph{Zea mays} & 2018 &
Field collection & OL342793 & OL314407\tabularnewline
C18162-3 & 1 & \emph{P. maydis} & Ohio & USA & \emph{Zea mays} & 2018 &
Field collection & OL342794 & OL314408\tabularnewline
C18164-1 & 1 & \emph{P. maydis} & Ohio & USA & \emph{Zea mays} & 2018 &
Field collection & OL342795 &\tabularnewline
C18164-2 & 1 & \emph{P. maydis} & Ohio & USA & \emph{Zea mays} & 2018 &
Field collection & OL342796 & OL314409\tabularnewline
C18164-3 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2018
& Field collection & OL342797 &\tabularnewline
C19043-1 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2019
& Field collection & OL342798 &\tabularnewline
C19043-3 & 1 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2019
& Field collection & OL342799 &\tabularnewline
BPI638548\_1 & 2 & \emph{P. maydis} & Cundinamarca & Colombia &
\emph{Zea mays} & 1940 & USDA Herbarium & OL342824 &\tabularnewline
BPI638554\_1 & 2 & \emph{P. maydis} & A\selectlanguage{ngerman}ñasco & Puerto Rico & \emph{Zea
mays} & 1917 & USDA Herbarium & OL342825 &\tabularnewline
BPI638578\_1 & 2 & \emph{P. maydis} & Vega Baja & Puerto Rico &
\emph{Zea mays} & 1916 & USDA Herbarium & OL342826 &\tabularnewline
BPI910562\_1 & 2 & \emph{P. maydis} & Michigan & USA & \emph{Zea mays} &
2017 & USDA Herbarium & OL342827 &\tabularnewline
C18026-3 & 2 & \emph{P. maydis} & Puebla & Mexico & \emph{Zea mays} &
2018 & Field collection & OL342806 &\tabularnewline
C18030-1 & 2 & \emph{P. maydis} & Guerrero & Mexico & \emph{Zea mays} &
2018 & Field collection & OL342807 & OL314410\tabularnewline
C18030-2 & 2 & \emph{P. maydis} & Guerrero & Mexico & \emph{Zea mays} &
2018 & Field collection & OL342808 & OL314411\tabularnewline
C18030-3 & 2 & \emph{P. maydis} & Guerrero & Mexico & \emph{Zea mays} &
2018 & Field collection & OL342809 & OL314412\tabularnewline
C18031-1 & 2 & \emph{P. maydis} & Veracruz & Mexico & \emph{Zea mays} &
2018 & Field collection & OL342810 & OL314413\tabularnewline
C18031-2 & 2 & \emph{P. maydis} & Veracruz & Mexico & \emph{Zea mays} &
2018 & Field collection & OL342811 & OL314414\tabularnewline
C18031-3 & 2 & \emph{P. maydis} & Veracruz & Mexico & \emph{Zea mays} &
2018 & Field collection & OL342812 & OL314415\tabularnewline
C18033-2 & 2 & \emph{P. maydis} & Oaxaca & Mexico & \emph{Zea mays} &
2018 & Field collection & OL342813 & OL314416\tabularnewline
C18033-3 & 2 & \emph{P. maydis} & Oaxaca & Mexico & \emph{Zea mays} &
2018 & Field collection & OL342814 & OL314417\tabularnewline
C18038-3 & 2 & \emph{P. maydis} & Guerrero & Mexico & \emph{Zea mays} &
2018 & Field collection & OL342815 &\tabularnewline
C18040-1 & 2 & \emph{P. maydis} & Florida & USA & \emph{Zea mays} & 2018
& Field collection & OL342816 &\tabularnewline
C18040-2 & 2 & \emph{P. maydis} & Florida & USA & \emph{Zea mays} & 2018
& Field collection & OL342817 &\tabularnewline
C18040-3 & 2 & \emph{P. maydis} & Florida & USA & \emph{Zea mays} & 2018
& Field collection & OL342818 &\tabularnewline
C18069-1 & 2 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2018 & Field collection & OL342819 & OL314418\tabularnewline
C18069-2 & 2 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2018 & Field collection & OL342820 & OL314419\tabularnewline
C18069-3 & 2 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2018 & Field collection & & OL314420\tabularnewline
C19001-1 & 2 & \emph{P. maydis} & Florida & USA & \emph{Zea mays} & 2019
& Field collection & OL342821 &\tabularnewline
C19001-2 & 2 & \emph{P. maydis} & Florida & USA & \emph{Zea mays} & 2019
& Field collection & OL342822 &\tabularnewline
C19001-3 & 2 & \emph{P. maydis} & Florida & USA & \emph{Zea mays} & 2019
& Field collection & OL342823 &\tabularnewline
92794-1 & 3 & \emph{P. chaetochloae} & Santiago & Dominican Republic &
\emph{Setaria sp.} & 1931 & UIUC Herbarium & OL342860 &\tabularnewline
92794-2 & 3 & \emph{P. chaetochloae} & Santiago & Dominican Republic &
\emph{Setaria sp.} & 1931 & UIUC Herbarium & OL342861 &\tabularnewline
92794-3 & 3 & \emph{P. chaetochloae} & Santiago & Dominican Republic &
\emph{Setaria sp.} & 1931 & UIUC Herbarium & OL342862 &\tabularnewline
92812-1 & 3 & \emph{P. diplocarpa} & California & USA &
\emph{Distichilis spicata} & 1942 & UIUC Herbarium & OL342863
&\tabularnewline
92821-1 & 3 & \emph{P. epicampsis} & Arizona & USA & \emph{Muhlenbergia
emersleyi} & 1948 & UIUC Herbarium & OL342864 &\tabularnewline
92821-2 & 3 & \emph{P. epicampsis} & Arizona & USA & \emph{Muhlenbergia
emersleyi} & 1948 & UIUC Herbarium & OL342865 &\tabularnewline
92821-3 & 3 & \emph{P. epicampsis} & Arizona & USA & \emph{Muhlenbergia
emersleyi} & 1948 & UIUC Herbarium & OL342866 &\tabularnewline
92825-1 & 3 & \emph{P. euphorbiaceae} & Mumbai & India & \emph{Euphorbia
sp.} & 1932 & UIUC Herbarium & OL342867 &\tabularnewline
92825-2 & 3 & \emph{P. euphorbiaceae} & Mumbai & India & \emph{Euphorbia
sp.} & 1932 & UIUC Herbarium & OL342868 &\tabularnewline
92825-3 & 3 & \emph{P. euphorbiaceae} & Mumbai & India & \emph{Euphorbia
sp.} & 1932 & UIUC Herbarium & OL342869 &\tabularnewline
92845-3 & 3 & \emph{P. graminis} & Mittelfranken & Germany &
\emph{Agropyron repens} & 1946 & UIUC Herbarium & OL342870
&\tabularnewline
92922-3 & 3 & \emph{P. heraclei} & Hessen & Germany & \emph{Heracleum
spondylium} & 1977 & UIUC Herbarium & OL342871 &\tabularnewline
92925-2 & 3 & \emph{P. junci} & Holstein & Germany & \emph{Juncus
effusus} & 1946 & UIUC Herbarium & OL342872 &\tabularnewline
92938-1 & 3 & \emph{P. maydis} & Arecibo & Puerto Rico & \emph{Zea mays}
& 1917 & UIUC Herbarium & OL342873 &\tabularnewline
92938-2 & 3 & \emph{P. maydis} & Arecibo & Puerto Rico & \emph{Zea mays}
& 1917 & UIUC Herbarium & OL342874 &\tabularnewline
92938-3 & 3 & \emph{P. maydis} & Arecibo & Puerto Rico & \emph{Zea mays}
& 1917 & UIUC Herbarium & OL342875 &\tabularnewline
92940-2 & 3 & \emph{P. maydis} & Arecibo & Puerto Rico & \emph{Zea mays}
& 1917 & UIUC Herbarium & OL342876 &\tabularnewline
92940-3 & 3 & \emph{P. maydis} & Arecibo & Puerto Rico & \emph{Zea mays}
& 1917 & UIUC Herbarium & OL342877 &\tabularnewline
93013-1 & 3 & \emph{P. rottboelliae} & Luzon & Philippines &
\emph{Rottboellia} & 1931 & UIUC Herbarium & OL342878 &\tabularnewline
93013-2 & 3 & \emph{P. rottboelliae} & Luzon & Philippines &
\emph{Rottboellia} & 1931 & UIUC Herbarium & OL342879 &\tabularnewline
93064-1 & 3 & \emph{P. sylvatica} & California & USA & \emph{Festuca
idahoensis} & 1941 & UIUC Herbarium & OL342880 &\tabularnewline
93064-2 & 3 & \emph{P. sylvatica} & California & USA & \emph{Festuca
idahoensis} & 1941 & UIUC Herbarium & OL342881 &\tabularnewline
93126-2 & 3 & \emph{P. vulgata} & Arizona & USA & \emph{Muhlenbergia
glauca} & 1948 & UIUC Herbarium & OL342882 &\tabularnewline
93126-3 & 3 & \emph{P. vulgata} & Arizona & USA & \emph{Muhlenbergia
glauca} & 1948 & UIUC Herbarium & OL342883 &\tabularnewline
BPI638546\_1 & 3 & \emph{P. maydis} & Maracas Valley & Trinidad and
Tobago & \emph{Zea mays} & 1945 & USDA Herbarium & OL342884
&\tabularnewline
BPI638553\_1 & 3 & \emph{P. maydis} & & Mexico & \emph{Zea mays} & 1977
& USDA Herbarium & OL342885 &\tabularnewline
BPI638556\_1 & 3 & \emph{P. maydis} & Valle del cauca & Colombia &
\emph{Zea mays} & 1929 & USDA Herbarium & OL342886 &\tabularnewline
BPI638558\_1 & 3 & \emph{P. maydis} & Antigua & Guatemala & \emph{Zea
mays} & 1905 & USDA Herbarium & OL342887 &\tabularnewline
BPI638559\_1 & 3 & \emph{P. maydis} & Matagalpa & Nicaragua & \emph{Zea
mays} & 1956 & USDA Herbarium & OL342888 &\tabularnewline
BPI638561\_1 & 3 & \emph{P. maydis} & Veracruz & Mexico & \emph{Zea
mays} & 1932 & USDA Herbarium & OL342889 &\tabularnewline
BPI638564\_1 & 3 & \emph{P. maydis} & & Mexico & \emph{Zea mays} & 1977
& USDA Herbarium & OL342890 &\tabularnewline
BPI638567\_1 & 3 & \emph{P. maydis} & Havana & Cuba & \emph{Zea mays} &
1918 & USDA Herbarium & OL342891 &\tabularnewline
BPI638568\_1 & 3 & \emph{P. maydis} & Alajuela & Costa Rica & \emph{Zea
mays} & 1947 & USDA Herbarium & OL342892 &\tabularnewline
BPI638570\_1 & 3 & \emph{P. maydis} & Vega Baja & Puerto Rico &
\emph{Zea mays} & 1916 & USDA Herbarium & OL342893 &\tabularnewline
BPI638571\_1 & 3 & \emph{P. maydis} & Turrialba & Costa Rica & \emph{Zea
mays} & 1949 & USDA Herbarium & OL342894 &\tabularnewline
BPI638572\_1 & 3 & \emph{P. maydis} & Chimaltenanco & Guatemala &
\emph{Zea mays} & 1940 & USDA Herbarium & OL342895 &\tabularnewline
BPI638574\_1 & 3 & \emph{P. maydis} & Arecibo & Puerto Rico & \emph{Zea
mays} & 1917 & USDA Herbarium & OL342896 &\tabularnewline
BPI638575\_1 & 3 & \emph{P. maydis} & Chimaltenanco & Guatemala &
\emph{Zea mays} & 1942 & USDA Herbarium & OL342897 &\tabularnewline
BPI638577\_1 & 3 & \emph{P. maydis} & Nor Yungas & Bolivia & \emph{Zea
mays} & 1943 & USDA Herbarium & OL342898 &\tabularnewline
BPI638579\_1 & 3 & \emph{P. maydis} & & Guatemala & \emph{Zea mays} &
1941 & USDA Herbarium & OL342899 &\tabularnewline
BPI638580\_1 & 3 & \emph{P. maydis} & Santander & Colombia & \emph{Zea
mays} & 1936 & USDA Herbarium & OL342900 &\tabularnewline
BPI638581\_1 & 3 & \emph{P. maydis} & Vega Baja & Puerto Rico &
\emph{Zea mays} & 1916 & USDA Herbarium & OL342901 &\tabularnewline
BPI638582\_1 & 3 & \emph{P. maydis} & Guatemala & Guatemala & \emph{Zea
mays} & 1905 & USDA Herbarium & OL342902 &\tabularnewline
BPI638584\_1 & 3 & \emph{P. maydis} & Antigua & Guatemala & \emph{Zea
mays} & 1905 & USDA Herbarium & OL342903 &\tabularnewline
BPI638585\_1 & 3 & \emph{P. maydis} & & Guatemala & \emph{Zea mays} &
1906 & USDA Herbarium & OL342904 &\tabularnewline
BPI638586\_1 & 3 & \emph{P. maydis} & Lima & Peru & \emph{Zea mays} &
1929 & USDA Herbarium & OL342905 &\tabularnewline
BPI638587\_1 & 3 & \emph{P. maydis} & La Vega & Dominican Republic &
\emph{Zea mays} & 1930 & USDA Herbarium & OL342906 &\tabularnewline
BPI638588\_1 & 3 & \emph{P. maydis} & & Guatemala & \emph{Zea mays} &
1906 & USDA Herbarium & OL342907 &\tabularnewline
BPI893226\_1 & 3 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} &
2015 & USDA Herbarium & OL342908 &\tabularnewline
BPI893228\_1 & 3 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} &
2015 & USDA Herbarium & OL342909 &\tabularnewline
BPI893230\_1 & 3 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} &
2015 & USDA Herbarium & OL342910 &\tabularnewline
BPI893231\_1 & 3 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} &
2015 & USDA Herbarium & OL342911 &\tabularnewline
BPI893232\_1 & 3 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} &
2015 & USDA Herbarium & OL342912 &\tabularnewline
BPI893233\_1 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2015 & USDA Herbarium & OL342913 & OL314452\tabularnewline
BPI893234\_1 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2015 & USDA Herbarium & OL342914 & OL314453\tabularnewline
BPI910560\_1 & 3 & \emph{P. maydis} & Wisconsin & USA & \emph{Zea mays}
& 2017 & USDA Herbarium & OL342915 &\tabularnewline
BPI910561\_1 & 3 & \emph{P. maydis} & Iowa & USA & \emph{Zea mays} &
2016 & USDA Herbarium & OL342916 &\tabularnewline
C18046-1 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2018 & Field collection & OL342829 & OL314422\tabularnewline
C18046-2 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2018 & Field collection & & OL314421\tabularnewline
C18046-3 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2018 & Field collection & & OL314423\tabularnewline
C18047-1 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2018 & Field collection & OL342830 & OL314424\tabularnewline
C18047-2 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2018 & Field collection & & OL314425\tabularnewline
C18047-3 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2018 & Field collection & & OL314426\tabularnewline
C18049-1 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2018 & Field collection & & OL314427\tabularnewline
C18049-2 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2018 & Field collection & & OL314428\tabularnewline
C18050-2 & 3 & \emph{P. maydis} & Wisconsin & USA & \emph{Zea mays} &
2018 & Field collection & OL342831 &\tabularnewline
C18075-3 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2018 & Field collection & OL342832 & OL314429\tabularnewline
C18119-2 & 3 & \emph{P. maydis} & Wisconsin & USA & \emph{Zea mays} &
2018 & Field collection & & OL314430\tabularnewline
C18119-3 & 3 & \emph{P. maydis} & Wisconsin & USA & \emph{Zea mays} &
2018 & Field collection & & OL314431\tabularnewline
C18136-1 & 3 & \emph{P. maydis} & Wisconsin & USA & \emph{Zea mays} &
2018 & Field collection & & OL314432\tabularnewline
C18148-1 & 3 & \emph{P. maydis} & Iowa & USA & \emph{Zea mays} & 2018 &
Field collection & OL342833 & OL314433\tabularnewline
C18148-2 & 3 & \emph{P. maydis} & Iowa & USA & \emph{Zea mays} & 2018 &
Field collection & OL342834 & OL314434\tabularnewline
C18148-3 & 3 & \emph{P. maydis} & Iowa & USA & \emph{Zea mays} & 2018 &
Field collection & OL342835 & OL314435\tabularnewline
C18149-1 & 3 & \emph{P. maydis} & Iowa & USA & \emph{Zea mays} & 2018 &
Field collection & OL342836 & OL314436\tabularnewline
C18149-2 & 3 & \emph{P. maydis} & Iowa & USA & \emph{Zea mays} & 2018 &
Field collection & OL342837 & OL314437\tabularnewline
C18153-1 & 3 & \emph{P. maydis} & Wisconsin & USA & \emph{Zea mays} &
2018 & Field collection & OL342838 & OL314438\tabularnewline
C18153-2 & 3 & \emph{P. maydis} & Wisconsin & USA & \emph{Zea mays} &
2018 & Field collection & & OL314439\tabularnewline
C19007-1 & 3 & \emph{P. maydis} & Iowa & USA & \emph{Zea mays} & 2019 &
Field collection & OL342839 & OL314440\tabularnewline
C19007-2 & 3 & \emph{P. maydis} & Iowa & USA & \emph{Zea mays} & 2019 &
Field collection & OL342840 & OL314461\tabularnewline
C19007-3 & 3 & \emph{P. maydis} & Iowa & USA & \emph{Zea mays} & 2019 &
Field collection & OL342841 & OL314441\tabularnewline
C19008-1 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2019 & Field collection & OL342842 & OL314442\tabularnewline
C19008-2 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2019 & Field collection & OL342843 & OL314459\tabularnewline
C19008-3 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2019 & Field collection & & OL314443\tabularnewline
C19012-2 & 3 & \emph{P. maydis} & Minnesota & USA & \emph{Zea mays} &
2019 & Field collection & OL342844 & OL314444\tabularnewline
C19012-3 & 3 & \emph{P. maydis} & Minnesota & USA & \emph{Zea mays} &
2019 & Field collection & OL342845 & OL314445\tabularnewline
C19022-2 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2019 & Field collection & OL342846 & OL314460\tabularnewline
C19022-3 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2019 & Field collection & OL342847 & OL314446\tabularnewline
C19025-1 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2019 & Field collection & OL342848 &\tabularnewline
C19025-2 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2019 & Field collection & OL342849 &\tabularnewline
C19025-3 & 3 & \emph{P. maydis} & Illinois & USA & \emph{Zea mays} &
2019 & Field collection & OL342850 &\tabularnewline
C19040-1 & 3 & \emph{P. maydis} & Michigan & USA & \emph{Zea mays} &
2019 & Field collection & OL342851 & OL314447\tabularnewline
C19040-2 & 3 & \emph{P. maydis} & Michigan & USA & \emph{Zea mays} &
2019 & Field collection & OL342852 & OL314456\tabularnewline
C19040-3 & 3 & \emph{P. maydis} & Michigan & USA & \emph{Zea mays} &
2019 & Field collection & OL342853 & OL314448\tabularnewline
C19043-2 & 3 & \emph{P. maydis} & Indiana & USA & \emph{Zea mays} & 2019
& Field collection & OL342854 &\tabularnewline
C19072-1 & 3 & \emph{P. maydis} & Wisconsin & USA & \emph{Zea mays} &
2019 & Field collection & OL342855 & OL314449\tabularnewline
C19072-2 & 3 & \emph{P. maydis} & Wisconsin & USA & \emph{Zea mays} &
2019 & Field collection & & OL314450\tabularnewline
C19072-3 & 3 & \emph{P. maydis} & Wisconsin & USA & \emph{Zea mays} &
2019 & Field collection & OL342856 & OL314451\tabularnewline
C19106-1 & 3 & \emph{P. maydis} & Iowa & USA & \emph{Zea mays} & 2019 &
Field collection & OL342857 & OL314457\tabularnewline
C19106-2 & 3 & \emph{P. maydis} & Iowa & USA & \emph{Zea mays} & 2019 &
Field collection & OL342858 & OL314458\tabularnewline
C19106-3 & 3 & \emph{P. maydis} & Iowa & USA & \emph{Zea mays} & 2019 &
Field collection & OL342859 & OL314462\tabularnewline
BPI638565 & 4 & \emph{P. maydis} & & Venezuela & \emph{Zea mays} & 1957
& USDA Herbarium & OL342920 &\tabularnewline
BPI638576 & 4 & \emph{P. maydis} & Mazatenango & Guatemala & \emph{Zea
mays} & 1906 & USDA Herbarium & OL342921 &\tabularnewline
BPI638583 & 4 & \emph{P. maydis} & & Guatemala & \emph{Zea mays} & 1907
& USDA Herbarium & OL342922 &\tabularnewline
NC19004-1 & 4 & \emph{Phyllachora sp.} & New York & USA &
\emph{Thinopyrum intermedium} & 2019 & Field collection & OL342923 &
OL314463\tabularnewline
NC19004-2 & 4 & \emph{Phyllachora sp.} & New York & USA &
\emph{Thinopyrum intermedium} & 2019 & Field collection & &
OL314464\tabularnewline
NC19004-3 & 4 & \emph{Phyllachora sp.} & New York & USA &
\emph{Thinopyrum intermedium} & 2019 & Field collection & OL342924 &
OL314465\tabularnewline
NC19026-1 & 4 & \emph{Phyllachora sp.} & Illinois & USA &
\emph{triticale} & 2019 & Field collection & OL342917 &
OL314466\tabularnewline
NC19026-2 & 4 & \emph{Phyllachora sp.} & Illinois & USA &
\emph{triticale} & 2019 & Field collection & OL342918 &
OL314467\tabularnewline
NC19026-3 & 4 & \emph{Phyllachora sp.} & Illinois & USA &
\emph{triticale} & 2019 & Field collection & OL342919 &\tabularnewline
NC19029-1 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{unknown}
& 2019 & Field collection & OL342925 & OL314468\tabularnewline
NC19029-2 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{unknown}
& 2019 & Field collection & OL342926 & OL314469\tabularnewline
NC19029-3 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{unknown}
& 2019 & Field collection & & OL314470\tabularnewline
NC19030-1 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{unknown}
& 2019 & Field collection & OL342927 & OL314471\tabularnewline
NC19030-2 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{unknown}
& 2019 & Field collection & OL342928 & OL314472\tabularnewline
NC19030-3 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{unknown}
& 2019 & Field collection & OL342929 & OL314473\tabularnewline
NC19032-1 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{fall
panicum} & 2019 & Field collection & OL342930 & OL314474\tabularnewline
NC19032-2 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{fall
panicum} & 2019 & Field collection & OL342931 & OL314475\tabularnewline
NC19032-3 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{fall
panicum} & 2019 & Field collection & OL342932 & OL314476\tabularnewline
NC19034-1 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{fescue}
& 2019 & Field collection & OL342933 & OL314477\tabularnewline
NC19034-2 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{fescue}
& 2019 & Field collection & OL342934 & OL314478\tabularnewline
NC19034-3 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{fescue}
& 2019 & Field collection & OL342935 & OL314479\tabularnewline
NC19035-1 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{rye} &
2019 & Field collection & OL342936 & OL314480\tabularnewline
NC19035-2 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{rye} &
2019 & Field collection & & OL314481\tabularnewline
NC19035-3 & 4 & \emph{Phyllachora sp.} & Illinois & USA & \emph{rye} &
2019 & Field collection & OL342937 & OL314482\tabularnewline
NC19111-1 & 4 & \emph{Phyllachora sp.} & South Dakota & USA &
\emph{brome grass} & 2019 & Field collection & OL342938 &
OL314483\tabularnewline
NC19111-2 & 4 & \emph{Phyllachora sp.} & South Dakota & USA &
\emph{brome grass} & 2019 & Field collection & OL342939 &
OL314484\tabularnewline
NC19111-3 & 4 & \emph{Phyllachora sp.} & South Dakota & USA &
\emph{brome grass} & 2019 & Field collection & OL342940 &
OL314485\tabularnewline
NC19027-1 & 5 & \emph{Phyllachora sp.} & Illinois & USA &
\emph{triticale} & 2019 & Field collection & OL342941 &
OL314486\tabularnewline
NC19027-3 & 5 & \emph{Phyllachora sp.} & Illinois & USA &
\emph{triticale} & 2019 & Field collection & OL342942 &
OL314487\tabularnewline
NC19028-1 & 5 & \emph{Phyllachora sp.} & Illinois & USA & \emph{unknown}
& 2019 & Field collection & OL342943 & OL314488\tabularnewline
NC19028-2 & 5 & \emph{Phyllachora sp.} & Illinois & USA & \emph{unknown}
& 2019 & Field collection & OL342944 & OL314489\tabularnewline
NC19028-3 & 5 & \emph{Phyllachora sp.} & Illinois & USA & \emph{unknown}
& 2019 & Field collection & OL342945 & OL314490\tabularnewline
NC19033-2 & 5 & \emph{Phyllachora sp.} & Illinois & USA & \emph{fall
panicum} & 2019 & Field collection & OL342946 & OL314491\tabularnewline
NC19037-1 & 5 & \emph{Phyllachora sp.} & Illinois & USA & \emph{rye} &
2019 & Field collection & OL342947 & OL314492\tabularnewline
NC19037-2 & 5 & \emph{Phyllachora sp.} & Illinois & USA & \emph{rye} &
2019 & Field collection & OL342948 & OL314493\tabularnewline
NC19037-3 & 5 & \emph{Phyllachora sp.} & Illinois & USA & \emph{rye} &
2019 & Field collection & OL342949 & OL314494\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a} The genetic cluster was determine as a result of the
phylogenetic analysis of the combined DNA sequences from the ITS and LSU
regions. These are displayed in Figure 2, 3 and Suppl. Figure 1

\textsuperscript{b} For contemporary material collected from field
samples during this study specimens of \emph{Phyllachora} from maize
were assumed to be \emph{P. maydis} and specimens from grass species
were treated as unknown \emph{Phyllachora} sp. For herbarium specimens
we included the species name from the herbarium label.

\textbf{Figure Legend}

\textbf{Figure 1.} Signs and symptoms of \emph{Phyllachora spp} . on
grasses. \emph{P. maydis} on maize at severe levels (A); with ascospores
being extruded from stroma (B) and showing characteristic tapering ends
of mature stromata (C). \emph{Phyllachora spp} . on \emph{Elymus} in
Michigan (D), Fall Ryegrass in Illinois \textbackslash{}euro, and an
unidentified grass in Indiana (F). Photo credit N. Kleczewski

\textbf{Figure 2} . Maximum likelihood phylogenetic tree based on
combined ITS and LSU sequence data from the stroma of
76\emph{Phyllachora} isolates from herbarium and contemporary samples of
infected maize and other grass hosts. \emph{Exserohilum turcicum} was
used as the outgroup.

\textbf{Figure 3.} Maximum likelihood phylogenetic tree based on ITS
sequence data from geographically representative isolates of the 5
genetic groups of maize and grass infecting \emph{Phyllachora} from this
study and \emph{Phyllachora} species available from GenBank. Specimens
highlighted in blue a contemporary isolates collect after 2015 and those
highlighted in orange are herbarium samples collected between 1905-1977.

\textbf{Suppl. Figure 1} . Maximum likelihood phylogenetic tree based on
the ITS sequence data from the stroma of 169 \emph{Phyllachora} isolates
from herbarium and contemporary samples of infected maize and other
grass hosts. \emph{Exserohilum turcicum} was used as the outgroup.

\textbf{Data Accessibility Statement.} All DNA sequence data generated
by this project were deposited in Genbank as accessions
OL314402-OL314494 and OL342781-OL342949.

\textbf{Acknowledgements} . This research was supported in part by the
U.S. Department of Agriculture, Agricultural Research Service. FFAR-ROAR
grant to NK, CC, DT, MC, and DS with matching funds from Pioneer, The
National Corn Growers Association, The Illinois Corn Growers Improvement
Board, and Purdue University. Mention of trade names or commercial
products in this publication is solely for the purpose of providing
specific information and does not imply recommendation or endorsement by
the U.S. Department of Agriculture. USDA is an equal opportunity
provider and employer.

\textbf{Author Contribution}

NK designed experiments and oversaw data collection; ZD and KV conducted
DNA extractions, sequencing and data generation, GIB and KB analyzed
data and generated figures. KB, GIB and NK wrote and prepared the
manuscript. All other authors oversaw sample collection and
distribution. All authors assisted in editing and revising the
manuscript.

\textbf{Competing Interest}

The authors have declared that no competing interests exist. Mention of
trade names or commercial products in this publication is solely for the
purpose of providing specific information and does not imply
recommendation or endorsement by the U.S. Department of Agriculture. The
U.S. Department of Agriculture prohibits discrimination in all its
programs and activities on the basis of race, color, national origin,
age, disability, and where applicable, sex, marital status, familial
status, parental status, religion, sexual orientation, genetic
information, political beliefs, reprisal, or because all or part of an
individual's income is derived from any public assistance program. (Not
all prohibited bases apply to all programs.) Persons with disabilities
who require alternative means for communication of program information
(Braille, large print, audiotape, etc.) should contact USDA's TARGET
Center at (202) 720-2600 (voice and TDD). To file a complaint of
discrimination, write to USDA, Director, Office of Civil Rights, 1400
Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800)
795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal opportunity
provider and employer.\selectlanguage{english}
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