Discussion
Since 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 Phyllachora species world-wide based on
morphological characteristics (Parbery 1967). In the most complete study
of 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 Phyllachora species have been
reported. Our results based on both herbarium and contemporary samples
of infected hosts indicate that there are far fewer species ofPhyllachora 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,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 ofPhyllachora (P. chaetochloae, P. diplocarpa, P. epicampsis,
P. euphorbiaceae, P. graminis, P. heraclei, P. junci, P. maydis, P.
rottboelliae, P. sylvatica, and 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 P. graminis collected fromAgropyron repens from Germany was designated as the lectotype
specimen for the genus (Clements and Shear 1931), and the isolate ofP. graminis examined in this study was also collected fromA. repens in Germany, indicating that P. graminis may have
precedence for the species name of P. sp. 3 . This would have
ramifications for P. maydis as well as several otherPhyllachora species in P. sp. 3 (Table 1; Figure 3) that
appear to be synonyms of 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 Phyllachora samples from additional
hosts is needed for a thorough taxonomic assessment of this genus.
The three maize-infecting species, P. sp. 1, P. sp. 2 andP. 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 P. sp. 3 and 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 Albugo candida , another biotrophic pathogen with a
broad host range (McMullan et al. 2015). Races of 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 A. candida . A whole genome comparison of
these 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 Phyllachora species.
Sexual reproduction in 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 Phyllachora that
are more virulent on maize or that have an expanded host range. This may
also explain why P. sp. 3 has such a broad host range whereasP. sp. 1 and 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 P. sp. 3may have adapted to infect a novel grass species, while maintaining the
ability to recombine with the larger 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 Phyllachorapopulations 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 P. sp. 1, P. sp. 2 and P. sp. 3 , but the
significant overlap in host and geography would indicate a genetic
barrier. While P. sp. 1 and P. sp. 2 were only recovered
from maize, our sampling scheme was strongly biased towards maize. It is
possible that P. sp. 1 and 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 Phyllachora species, may represent the isolation that led
to adaptation and speciation.
For now the name Phyllachora maydis will be retained by P.
sp. 3 as the P. maydis type material (BPI638553) clustered with
this group. However, the presence of three maize-infecting species, the
lack of type material of P. graminis , and the potential taxonomic
synonymy with P. graminis and several other Phyllachoraspecies makes it difficult to determine which of the maize infecting
species will retain the name P. maydis . Therefore, we recommend
referring to P. sp. 1, P. sp. 2 and P. sp. 3 as thePhyllachora 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 ofPhyllachora 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 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 Phyllachora spp . in the upper
Midwest of the U.S. While P. sp. 2 and 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, 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 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 ofPhyllachora 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 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 ofPhyllachora 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 Phyllachora
spp. ; and 3) lack of resistance in maize germplasm grown in the
Midwestern U.S.