Phyllachora species infecting maize and other grass species in the Americas represents a complex of closely related species.

The genus Phyllachora contains numerous obligate fungal parasites that produce raised, melanized structures called stromata on their plant hosts referred to as tar spot. Members of this genus are known to infect many grass species but generally do not cause significant damage or defoliation, with the exception of P. maydis which has emerged as an important pathogen of maize throughout the Americas, but the origin of this pathogen remains unknown. To date, species designations for Phyllachora have been based on host associations and morphology, and most species are assumed to be host specific. We assessed the sequence diversity of 186 single stroma isolates collected from 16 hosts representing 15 countries. Samples included both herbarium and contemporary strains that covered a temporal range from 1905 to 2019. These 186 isolates were grouped into five 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 specimens, 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 previously identified based on morphology and host association. This work represents the first attempt at molecular characterization of Phyllachora species infecting grass hosts and indicates some Phyllachora species can infect a broad range of host species and there may be significant synonymy in the Phyllachora genus. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Ecology and Evolution published by John Wiley & Sons Ltd.

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 (Figure 1) (Mardones et al., 2017).

FIGURE 1

Signs and symptoms of Phyllachora spp. on grasses. Phyllachora maydis on maize at severe levels (a); with ascospores being extruded from stroma (b) and showing characteristic tapering ends of mature stromata (c). Phyllachora spp. on Elymus in Michigan (d), Fall Ryegrass in Illinois, and an unidentified grass in Indiana (f). Photo credit N. Kleczewski

Tar spot of maize (Figure 1a–c), caused by the fungus Phyllachora maydis, emerged in the United States 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 United States (Kleczewski et al., 2020; Valle‐Torres et al., 2020). Since first identified in North America in 2015, P. maydis has spread rapidly throughout the United States and Canada (Kleczewski & Bowman, 2020; Kleczewski et al., 2020), and resulted in yield losses exceeding $US 658 million in 2018 (Mueller et al., 2020). Although tar spot symptoms caused by members of the genus Phyllachora have been commonly observed on a number of grasses (Figure 1d–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 P. maydis within the United States 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 United States 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 Phyllachora species are common on several native and weedy grass species in North America (Figure 1d–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 & 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, 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, 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 (Francisco et al., 2019; Money, 2013; Slepecky & Starmer, 2009; Stockinger et al., 2009). Thus, our current understanding of the genetic diversity, host range, and species delimitation within the genus Phyllachora is relatively limited and requires reevaluation.

The recent emergence of P. maydis in the United States 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 (Groves et al., 2020; Kleczewski et al., 2019). 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 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 P. maydis on corn in the United States 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 United States favored by climate change, changes in hybrid genetics, a host jump from a grass species, or a combination of any of these four. This study represents the first attempt to extract and sequence DNA from Phyllachora stroma on fresh and herbarium‐infected grass specimens. The goal of this study is to understand the genetic diversity of Phyllachora species causing tar spot disease in contemporary corn production regions in the United States, and compare this to historical specimens of Phyllachora species from herbarium samples of maize and other grasses from Mexico, Central, and South America, the Caribbean, and Europe, as well as contemporary and herbarium species of Phyllachora species associated with grass hosts in the United States. This represents the first attempt at genetic characterization of Phyllachora maydis, Phyllachora graminis, and other grass infecting Phyllachora species. The objectives of this study are to: (1) determine if a single species of Phyllachora is responsible for tar spot disease of maize throughout its range in the Americas over the last century or if distinct genetic groups are responsible for these symptoms; and (2) determine if Phyllachora species infecting native and weedy grasses in close proximity to maize production fields are the same species as those infecting maize. Understanding the phylogenetic diversity and the potential host and geographic range of Phyllachora species associated with maize and other grasses in the Americas will also help to infer the potential evolutionary origins and speciation patterns in this genus.

MATERIAL AND METHODS

Sample collection

Samples of maize and wild grasses with characteristic stromata of Phyllachora spp. (Figure 1a–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°C in manila 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 to 2019, were included in the analyses.

TABLE 1

The sample ID, genetic cluster, geographic and host origin, year collected, and source of the 186 Phyllachora specimens used in this study

Sample ID	Genetic cluster a	Species b	State	Country	Host	Year collected	Source	GenBank accessions
								ITS	LSU
BPI893226_2	1	Phyllachora maydis	Indiana	USA	Zea mays	2015	Field collection	OL342800
BPI893227_2	1	P. maydis	Indiana	USA	Zea mays	2015	Field collection	OL342801
BPI893229_2	1	P. maydis	Indiana	USA	Zea mays	2015	Field collection	OL342802
BPI893230_2	1	P. maydis	Indiana	USA	Zea mays	2015	Field collection	OL342803
BPI893231_2	1	P. maydis	Indiana	USA	Zea mays	2015	Field collection	OL342804
BPI893232_2	1	P. maydis	Indiana	USA	Zea mays	2015	Field collection	OL342805
C18001‐2	1	P. maydis	Indiana	USA	Zea mays	2018	Field collection	OL342781
C18001‐3	1	P. maydis	Indiana	USA	Zea mays	2018	Field collection	OL342782
C18003‐1	1	P. maydis	Indiana	USA	Zea mays	2018	Field collection	OL342783
C18003‐2	1	P. maydis	Indiana	USA	Zea mays	2018	Field collection		OL314402
C18003‐3	1	P. maydis	Indiana	USA	Zea mays	2018	Field collection	OL342784
C18009‐1	1	P. maydis	Indiana	USA	Zea mays	2018	Field collection	OL342785
C18009‐2	1	P. maydis	Indiana	USA	Zea mays	2018	Field collection	OL342786
C18011‐3	1	P. maydis	Indiana	USA	Zea mays	2018	Field collection	OL342787	OL314403
C18024‐1	1	P. maydis	Indiana	USA	Zea mays	2018	Field collection	OL342788
C18024‐2	1	P. maydis	Indiana	USA	Zea mays	2018	Field collection	OL342789
C18024‐3	1	P. maydis	Indiana	USA	Zea mays	2018	Field collection	OL342790
C18161‐1	1	P. maydis	Ohio	USA	Zea mays	2018	Field collection	OL342791	OL314404
C18161‐2	1	P. maydis	Ohio	USA	Zea mays	2018	Field collection	OL342792	OL314405
C18161‐3	1	P. maydis	Ohio	USA	Zea mays	2018	Field collection		OL314406
C18162‐1	1	P. maydis	Ohio	USA	Zea mays	2018	Field collection	OL342793	OL314407
C18162‐3	1	P. maydis	Ohio	USA	Zea mays	2018	Field collection	OL342794	OL314408
C18164‐1	1	P. maydis	Ohio	USA	Zea mays	2018	Field collection	OL342795
C18164‐2	1	P. maydis	Ohio	USA	Zea mays	2018	Field collection	OL342796	OL314409
C18164‐3	1	P. maydis	Indiana	USA	Zea mays	2018	Field collection	OL342797
C19043‐1	1	P. maydis	Indiana	USA	Zea mays	2019	Field collection	OL342798
C19043‐3	1	P. maydis	Indiana	USA	Zea mays	2019	Field collection	OL342799
BPI638548_1	2	P. maydis	Cundinamarca	Colombia	Zea mays	1940	USDA Herbarium	OL342824
BPI638554_1	2	P. maydis	Añasco	Puerto Rico	Zea mays	1917	USDA Herbarium	OL342825
BPI638578_1	2	P. maydis	Vega Baja	Puerto Rico	Zea mays	1916	USDA Herbarium	OL342826
BPI910562_1	2	P. maydis	Michigan	USA	Zea mays	2017	USDA Herbarium	OL342827
C18026‐3	2	P. maydis	Puebla	Mexico	Zea mays	2018	Field collection	OL342806
C18030‐1	2	P. maydis	Guerrero	Mexico	Zea mays	2018	Field collection	OL342807	OL314410
C18030‐2	2	P. maydis	Guerrero	Mexico	Zea mays	2018	Field collection	OL342808	OL314411
C18030‐3	2	P. maydis	Guerrero	Mexico	Zea mays	2018	Field collection	OL342809	OL314412
C18031‐1	2	P. maydis	Veracruz	Mexico	Zea mays	2018	Field collection	OL342810	OL314413
C18031‐2	2	P. maydis	Veracruz	Mexico	Zea mays	2018	Field collection	OL342811	OL314414
C18031‐3	2	P. maydis	Veracruz	Mexico	Zea mays	2018	Field collection	OL342812	OL314415
C18033‐2	2	P. maydis	Oaxaca	Mexico	Zea mays	2018	Field collection	OL342813	OL314416
C18033‐3	2	P. maydis	Oaxaca	Mexico	Zea mays	2018	Field collection	OL342814	OL314417
C18038‐3	2	P. maydis	Guerrero	Mexico	Zea mays	2018	Field collection	OL342815
C18040‐1	2	P. maydis	Florida	USA	Zea mays	2018	Field collection	OL342816
C18040‐2	2	P. maydis	Florida	USA	Zea mays	2018	Field collection	OL342817
C18040‐3	2	P. maydis	Florida	USA	Zea mays	2018	Field collection	OL342818
C18069‐1	2	P. maydis	Illinois	USA	Zea mays	2018	Field collection	OL342819	OL314418
C18069‐2	2	P. maydis	Illinois	USA	Zea mays	2018	Field collection	OL342820	OL314419
C18069‐3	2	P. maydis	Illinois	USA	Zea mays	2018	Field collection		OL314420
C19001‐1	2	P. maydis	Florida	USA	Zea mays	2019	Field collection	OL342821
C19001‐2	2	P. maydis	Florida	USA	Zea mays	2019	Field collection	OL342822
C19001‐3	2	P. maydis	Florida	USA	Zea mays	2019	Field collection	OL342823
92794‐1	3	P. chaetochloae	Santiago	Dominican Republic	Setaria sp.	1931	UIUC Herbarium	OL342860
92794‐2	3	P. chaetochloae	Santiago	Dominican Republic	Setaria sp.	1931	UIUC Herbarium	OL342861
92794‐3	3	P. chaetochloae	Santiago	Dominican Republic	Setaria sp.	1931	UIUC Herbarium	OL342862
92812‐1	3	P. diplocarpa	California	USA	Distichilis spicata	1942	UIUC Herbarium	OL342863
92821‐1	3	P. epicampsis	Arizona	USA	Muhlenbergia emersleyi	1948	UIUC Herbarium	OL342864
92821‐2	3	P. epicampsis	Arizona	USA	Muhlenbergia emersleyi	1948	UIUC Herbarium	OL342865
92821‐3	3	P. epicampsis	Arizona	USA	Muhlenbergia emersleyi	1948	UIUC Herbarium	OL342866
92825‐1	3	P. euphorbiaceae	Mumbai	India	Euphorbia sp.	1932	UIUC Herbarium	OL342867
92825‐2	3	P. euphorbiaceae	Mumbai	India	Euphorbia sp.	1932	UIUC Herbarium	OL342868
92825‐3	3	P. euphorbiaceae	Mumbai	India	Euphorbia sp.	1932	UIUC Herbarium	OL342869
92845‐3	3	P. graminis	Mittelfranken	Germany	Agropyron repens	1946	UIUC Herbarium	OL342870
92922‐3	3	P. heraclei	Hessen	Germany	Heracleum spondylium	1977	UIUC Herbarium	OL342871
92925‐2	3	P. junci	Holstein	Germany	Juncus effusus	1946	UIUC Herbarium	OL342872
92938‐1	3	P. maydis	Arecibo	Puerto Rico	Zea mays	1917	UIUC Herbarium	OL342873
92938‐2	3	P. maydis	Arecibo	Puerto Rico	Zea mays	1917	UIUC Herbarium	OL342874
92938‐3	3	P. maydis	Arecibo	Puerto Rico	Zea mays	1917	UIUC Herbarium	OL342875
92940‐2	3	P. maydis	Arecibo	Puerto Rico	Zea mays	1917	UIUC Herbarium	OL342876
92940‐3	3	P. maydis	Arecibo	Puerto Rico	Zea mays	1917	UIUC Herbarium	OL342877
93013‐1	3	P. rottboelliae	Luzon	Philippines	Rottboellia	1931	UIUC Herbarium	OL342878
93013‐2	3	P. rottboelliae	Luzon	Philippines	Rottboellia	1931	UIUC Herbarium	OL342879
93064‐1	3	P. sylvatica	California	USA	Festuca idahoensis	1941	UIUC Herbarium	OL342880
93064‐2	3	P. sylvatica	California	USA	Festuca idahoensis	1941	UIUC Herbarium	OL342881
93126‐2	3	P. vulgata	Arizona	USA	Muhlenbergia glauca	1948	UIUC Herbarium	OL342882
93126‐3	3	P. vulgata	Arizona	USA	Muhlenbergia glauca	1948	UIUC Herbarium	OL342883
BPI638546_1	3	P. maydis	Maracas Valley	Trinidad and Tobago	Zea mays	1945	USDA Herbarium	OL342884
BPI638553_1	3	P. maydis		Mexico	Zea mays	1904	USDA Herbarium	OL342885
BPI638556_1	3	P. maydis	Valle del Cauca	Colombia	Zea mays	1929	USDA Herbarium	OL342886
BPI638558_1	3	P. maydis	Antigua	Guatemala	Zea mays	1905	USDA Herbarium	OL342887
BPI638559_1	3	P. maydis	Matagalpa	Nicaragua	Zea mays	1956	USDA Herbarium	OL342888
BPI638561_1	3	P. maydis	Veracruz	Mexico	Zea mays	1932	USDA Herbarium	OL342889
BPI638564_1	3	P. maydis		Mexico	Zea mays	1977	USDA Herbarium	OL342890
BPI638567_1	3	P. maydis	Havana	Cuba	Zea mays	1918	USDA Herbarium	OL342891
BPI638568_1	3	P. maydis	Alajuela	Costa Rica	Zea mays	1947	USDA Herbarium	OL342892
BPI638570_1	3	P. maydis	Vega Baja	Puerto Rico	Zea mays	1916	USDA Herbarium	OL342893
BPI638571_1	3	P. maydis	Turrialba	Costa Rica	Zea mays	1949	USDA Herbarium	OL342894
BPI638572_1	3	P. maydis	Chimaltenanco	Guatemala	Zea mays	1940	USDA Herbarium	OL342895
BPI638574_1	3	P. maydis	Arecibo	Puerto Rico	Zea mays	1917	USDA Herbarium	OL342896
BPI638575_1	3	P. maydis	Chimaltenanco	Guatemala	Zea mays	1942	USDA Herbarium	OL342897
BPI638577_1	3	P. maydis	Nor Yungas	Bolivia	Zea mays	1943	USDA Herbarium	OL342898
BPI638579_1	3	P. maydis		Guatemala	Zea mays	1941	USDA Herbarium	OL342899
BPI638580_1	3	P. maydis	Santander	Colombia	Zea mays	1936	USDA Herbarium	OL342900
BPI638581_1	3	P. maydis	Vega Baja	Puerto Rico	Zea mays	1916	USDA Herbarium	OL342901
BPI638582_1	3	P. maydis	Guatemala	Guatemala	Zea mays	1905	USDA Herbarium	OL342902
BPI638584_1	3	P. maydis	Antigua	Guatemala	Zea mays	1905	USDA Herbarium	OL342903
BPI638585_1	3	P. maydis		Guatemala	Zea mays	1906	USDA Herbarium	OL342904
BPI638586_1	3	P. maydis	Lima	Peru	Zea mays	1929	USDA Herbarium	OL342905
BPI638587_1	3	P. maydis	La Vega	Dominican Republic	Zea mays	1930	USDA Herbarium	OL342906
BPI638588_1	3	P. maydis		Guatemala	Zea mays	1906	USDA Herbarium	OL342907
BPI893226_1	3	P. maydis	Indiana	USA	Zea mays	2015	USDA Herbarium	OL342908
BPI893228_1	3	P. maydis	Indiana	USA	Zea mays	2015	USDA Herbarium	OL342909
BPI893230_1	3	P. maydis	Indiana	USA	Zea mays	2015	USDA Herbarium	OL342910
BPI893231_1	3	P. maydis	Indiana	USA	Zea mays	2015	USDA Herbarium	OL342911
BPI893232_1	3	P. maydis	Indiana	USA	Zea mays	2015	USDA Herbarium	OL342912
BPI893233_1	3	P. maydis	Illinois	USA	Zea mays	2015	USDA Herbarium	OL342913	OL314452
BPI893234_1	3	P. maydis	Illinois	USA	Zea mays	2015	USDA Herbarium	OL342914	OL314453
BPI910560_1	3	P. maydis	Wisconsin	USA	Zea mays	2017	USDA Herbarium	OL342915
BPI910561_1	3	P. maydis	Iowa	USA	Zea mays	2016	USDA Herbarium	OL342916
C18046‐1	3	P. maydis	Illinois	USA	Zea mays	2018	Field collection	OL342829	OL314422
C18046‐2	3	P. maydis	Illinois	USA	Zea mays	2018	Field collection		OL314421
C18046‐3	3	P. maydis	Illinois	USA	Zea mays	2018	Field collection		OL314423
C18047‐1	3	P. maydis	Illinois	USA	Zea mays	2018	Field collection	OL342830	OL314424
C18047‐2	3	P. maydis	Illinois	USA	Zea mays	2018	Field collection		OL314425
C18047‐3	3	P. maydis	Illinois	USA	Zea mays	2018	Field collection		OL314426
C18049‐1	3	P. maydis	Illinois	USA	Zea mays	2018	Field collection		OL314427
C18049‐2	3	P. maydis	Illinois	USA	Zea mays	2018	Field collection		OL314428
C18050‐2	3	P. maydis	Wisconsin	USA	Zea mays	2018	Field collection	OL342831
C18075‐3	3	P. maydis	Illinois	USA	Zea mays	2018	Field collection	OL342832	OL314429
C18119‐2	3	P. maydis	Wisconsin	USA	Zea mays	2018	Field collection		OL314430
C18119‐3	3	P. maydis	Wisconsin	USA	Zea mays	2018	Field collection		OL314431
C18136‐1	3	P. maydis	Wisconsin	USA	Zea mays	2018	Field collection		OL314432
C18148‐1	3	P. maydis	Iowa	USA	Zea mays	2018	Field collection	OL342833	OL314433
C18148‐2	3	P. maydis	Iowa	USA	Zea mays	2018	Field collection	OL342834	OL314434
C18148‐3	3	P. maydis	Iowa	USA	Zea mays	2018	Field collection	OL342835	OL314435
C18149‐1	3	P. maydis	Iowa	USA	Zea mays	2018	Field collection	OL342836	OL314436
C18149‐2	3	P. maydis	Iowa	USA	Zea mays	2018	Field collection	OL342837	OL314437
C18153‐1	3	P. maydis	Wisconsin	USA	Zea mays	2018	Field collection	OL342838	OL314438
C18153‐2	3	P. maydis	Wisconsin	USA	Zea mays	2018	Field collection		OL314439
C19007‐1	3	P. maydis	Iowa	USA	Zea mays	2019	Field collection	OL342839	OL314440
C19007‐2	3	P. maydis	Iowa	USA	Zea mays	2019	Field collection	OL342840	OL314461
C19007‐3	3	P. maydis	Iowa	USA	Zea mays	2019	Field collection	OL342841	OL314441
C19008‐1	3	P. maydis	Illinois	USA	Zea mays	2019	Field collection	OL342842	OL314442
C19008‐2	3	P. maydis	Illinois	USA	Zea mays	2019	Field collection	OL342843	OL314459
C19008‐3	3	P. maydis	Illinois	USA	Zea mays	2019	Field collection		OL314443
C19012‐2	3	P. maydis	Minnesota	USA	Zea mays	2019	Field collection	OL342844	OL314444
C19012‐3	3	P. maydis	Minnesota	USA	Zea mays	2019	Field collection	OL342845	OL314445
C19022‐2	3	P. maydis	Illinois	USA	Zea mays	2019	Field collection	OL342846	OL314460
C19022‐3	3	P. maydis	Illinois	USA	Zea mays	2019	Field collection	OL342847	OL314446
C19025‐1	3	P. maydis	Illinois	USA	Zea mays	2019	Field collection	OL342848
C19025‐2	3	P. maydis	Illinois	USA	Zea mays	2019	Field collection	OL342849
C19025‐3	3	P. maydis	Illinois	USA	Zea mays	2019	Field collection	OL342850
C19040‐1	3	P. maydis	Michigan	USA	Zea mays	2019	Field collection	OL342851	OL314447
C19040‐2	3	P. maydis	Michigan	USA	Zea mays	2019	Field collection	OL342852	OL314456
C19040‐3	3	P. maydis	Michigan	USA	Zea mays	2019	Field collection	OL342853	OL314448
C19043‐2	3	P. maydis	Indiana	USA	Zea mays	2019	Field collection	OL342854
C19072‐1	3	P. maydis	Wisconsin	USA	Zea mays	2019	Field collection	OL342855	OL314449
C19072‐2	3	P. maydis	Wisconsin	USA	Zea mays	2019	Field collection		OL314450
C19072‐3	3	P. maydis	Wisconsin	USA	Zea mays	2019	Field collection	OL342856	OL314451
C19106‐1	3	P. maydis	Iowa	USA	Zea mays	2019	Field collection	OL342857	OL314457
C19106‐2	3	P. maydis	Iowa	USA	Zea mays	2019	Field collection	OL342858	OL314458
C19106‐3	3	P. maydis	Iowa	USA	Zea mays	2019	Field collection	OL342859	OL314462
BPI638565	4	P. maydis		Venezuela	Zea mays	1957	USDA Herbarium	OL342920
BPI638576	4	P. maydis	Mazatenango	Guatemala	Zea mays	1906	USDA Herbarium	OL342921
BPI638583	4	P. maydis		Guatemala	Zea mays	1907	USDA Herbarium	OL342922
NC19004‐1	4	Phyllachora sp.	New York	USA	Thinopyrum intermedium	2019	Field collection	OL342923	OL314463
NC19004‐2	4	Phyllachora sp.	New York	USA	Thinopyrum intermedium	2019	Field collection		OL314464
NC19004‐3	4	Phyllachora sp.	New York	USA	Thinopyrum intermedium	2019	Field collection	OL342924	OL314465
NC19026‐1	4	Phyllachora sp.	Illinois	USA	Triticale	2019	Field collection	OL342917	OL314466
NC19026‐2	4	Phyllachora sp.	Illinois	USA	Triticale	2019	Field collection	OL342918	OL314467
NC19026‐3	4	Phyllachora sp.	Illinois	USA	Triticale	2019	Field collection	OL342919
NC19029‐1	4	Phyllachora sp.	Illinois	USA	Unknown	2019	Field collection	OL342925	OL314468
NC19029‐2	4	Phyllachora sp.	Illinois	USA	Unknown	2019	Field collection	OL342926	OL314469
NC19029‐3	4	Phyllachora sp.	Illinois	USA	Unknown	2019	Field collection		OL314470
NC19030‐1	4	Phyllachora sp.	Illinois	USA	Unknown	2019	Field collection	OL342927	OL314471
NC19030‐2	4	Phyllachora sp.	Illinois	USA	Unknown	2019	Field collection	OL342928	OL314472
NC19030‐3	4	Phyllachora sp.	Illinois	USA	Unknown	2019	Field collection	OL342929	OL314473
NC19032‐1	4	Phyllachora sp.	Illinois	USA	Fall panicum	2019	Field collection	OL342930	OL314474
NC19032‐2	4	Phyllachora sp.	Illinois	USA	Fall panicum	2019	Field collection	OL342931	OL314475
NC19032‐3	4	Phyllachora sp.	Illinois	USA	Fall panicum	2019	Field collection	OL342932	OL314476
NC19034‐1	4	Phyllachora sp.	Illinois	USA	Fescue	2019	Field collection	OL342933	OL314477
NC19034‐2	4	Phyllachora sp.	Illinois	USA	Fescue	2019	Field collection	OL342934	OL314478
NC19034‐3	4	Phyllachora sp.	Illinois	USA	Fescue	2019	Field collection	OL342935	OL314479
NC19035‐1	4	Phyllachora sp.	Illinois	USA	Rye	2019	Field collection	OL342936	OL314480
NC19035‐2	4	Phyllachora sp.	Illinois	USA	Rye	2019	Field collection		OL314481
NC19035‐3	4	Phyllachora sp.	Illinois	USA	Rye	2019	Field collection	OL342937	OL314482
NC19111‐1	4	Phyllachora sp.	South Dakota	USA	Brome grass	2019	Field collection	OL342938	OL314483
NC19111‐2	4	Phyllachora sp.	South Dakota	USA	Brome grass	2019	Field collection	OL342939	OL314484
NC19111‐3	4	Phyllachora sp.	South Dakota	USA	Brome grass	2019	Field collection	OL342940	OL314485
NC19027‐1	5	Phyllachora sp.	Illinois	USA	Triticale	2019	Field collection	OL342941	OL314486
NC19027‐3	5	Phyllachora sp.	Illinois	USA	Triticale	2019	Field collection	OL342942	OL314487
NC19028‐1	5	Phyllachora sp.	Illinois	USA	Unknown	2019	Field collection	OL342943	OL314488
NC19028‐2	5	Phyllachora sp.	Illinois	USA	Unknown	2019	Field collection	OL342944	OL314489
NC19028‐3	5	Phyllachora sp.	Illinois	USA	Unknown	2019	Field collection	OL342945	OL314490
NC19033‐2	5	Phyllachora sp.	Illinois	USA	Fall panicum	2019	Field collection	OL342946	OL314491
NC19037‐1	5	Phyllachora sp.	Illinois	USA	Rye	2019	Field collection	OL342947	OL314492
NC19037‐2	5	Phyllachora sp.	Illinois	USA	Rye	2019	Field collection	OL342948	OL314493
NC19037‐3	5	Phyllachora sp.	Illinois	USA	Rye	2019	Field collection	OL342949	OL314494

The genetic cluster was determined as a result of the phylogenetic analysis of the combined DNA sequences from the ITS and LSU regions. These are displayed in Figures 2, 3, and Figure S1.

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

FIGURE 2

Maximum likelihood phylogenetic tree based on combined ITS and LSU sequence data from the stroma of 76 Phyllachora isolates from herbarium and contemporary samples of infected maize and other grass hosts. Values of Bayesian PP > 0.70 or ML BS > 70% are given at nodes at the first and second positions, respectively. Exserohilum turcicum CBS 690.71 was used as the outgroup

FIGURE 3

Maximum likelihood phylogenetic tree based on ITS sequence data from geographically representative isolates of the five genetic groups of maize and grass‐infecting Phyllachora from this study and Phyllachora species available from GenBank. Values of Bayesian PP > 0.70 or ML BS > 70% are given at nodes at the first and second positions, respectively. Isolates sequenced in this project are denoted in bold. Information on host plant is indicated in blue text and geographic origin in red text

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 was 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 (Bruns & Gardes, 1993; White et al., 1990) 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 min at 110V. All gels contained a P. maydis‐positive control, a 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 Phyllachora spp. can be colonized by or associated with several other fungal species (Hock et al., 1992, 1995; McCoy et al., 2019). Consequently, samples returning a single band between 300 and 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 LR0R 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).

Sequence alignment, phylogenetic analysis, and molecular identification

Sequences generated from this study were combined with sequences obtained from GenBank. Exserohilum turcicum and Cocoicola californica were selected as the outgroups. Sequence data were aligned and concatenated using MAFFT v.7 (www.mafft.cbrc.jp/alignment/server/) 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 using RaxML and Bayesian inference with MrBayes. The ML phylogenies were generated by RaxML (Stamatakis, 2014) under GTR model with gamma distributed rate heterogeneity with 1000 bootstrap replicates. For the Bayesian inference, we used MrBayes v. 3.2.6 (Ronquist et al., 2012) using the general time reversible (GTR) model selected for the entire unpartitioned alignment, with the likelihood parameters setting (lset) number of substitution types (nst) = 6, with a proportion of sites invariable and the rest drawn from the gamma distribution (rate = invgamma). Four independent analyses, each starting from a random tree, were run under the same conditions for the combined gene alignment. Three hot and one cold chains of Markov Chain Monte Carlo iterations were performed. Analyses were run with 1,000,000 generations with sampling every 100 generations. The first 250,000 generations were discarded as the chains were converging (burnin period). Resulting trees were visualized with iTOL (Interactive Tree of Life) v.6 (https://itol.embl.de/) or MEGA. Sequences generated in this study were deposited in GenBank (Table 1).

RESULTS

DNA extraction and PCR amplification from herbarium and contemporary samples

A total of 186 samples from 12 states in the United States (n = 130), 4 states in Mexico (n = 13), 3 Central American countries (n = 13), 4 South American countries (n = 6), 4 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).

Phylogenetic diversity of Phyllachora isolates infecting maize and grasses

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

Samples of Phyllachora sp. 3 represent the broadest geographic and host range and was also the most frequently recovered species associated with Phyllachora sp. stroma on maize from both herbarium and contemporary specimens representing a span of time from 1904 to 2019 (Table 1). Samples of Phyllachora 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 United States. Importantly, the isosyntype specimen of P. maydis (BPI638553) collected in Mexico in 1904 and the P. maydis isolate (BPI893226) used in the first report of tar spot in the United States in 2015 are both part of Phyllachora 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 Phyllachora species in the United States among the samples included in this study. However, isolates of Phyllachora 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 Phyllachora including P. graminis, 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, Phyllachora sp. 1 and Phyllachora sp. 2, have a more limited observed host and geographic range. Both species were only recovered on maize. Phyllachora sp. 1 was only recovered from contemporary maize samples from Indiana and Ohio, whereas Phyllachora sp. 2 was found on herbarium specimens from Colombia and Puerto Rico and contemporary specimens from Puerto Rico, Mexico (Guerrero, Oaxaca, Puebla, and Veracruz), and the United States (Florida, Illinois, and Michigan).

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

While there was limited Phyllachora sequence data in GenBank, we were able to include the ITS sequence of 19 isolates representing six recognized species of Phyllachora to determine any relationship between the isolates used in this study and those submitted previously to GenBank (Figure 3). In the case of Phyllachora sp. 4, two isolates referred to as P. graminis, one from Hordelymus europaeus in Germany, and one of unknown origin, as well as isolates of Phyllachora on Elymus kamoji and Roegneria sp. from China grouped together with strong Boostrap support (90%) and posterior probability (0.99). There was also an isolate of P. graminis from an unknown grass in Canada that grouped together with Phyllachora sp. 5, and the herbarium specimen of P. graminis from Agropyron repens in Germany from this study grouped in Phyllachora sp. 3 (Figure 3). Our results support the findings of previous observations that P. graminis is a poorly defined polyphyletic species that has often been assigned to tar spot symptom on a variety of grass hosts.

DISCUSSION

Since P. graminis was described by Persoon in 1785 as Sphaeria graminis and then transferred to the genus Phyllachora by Fuckel (1870), over 300 species have been recorded on graminaceous hosts, and many more on non‐grass hosts. However, Parbery (1967) 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. 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 of 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, Phyllachora 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 Phyllachora (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 United States, 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 from Agropyron repens from Germany was designated as the lectotype specimen for the genus (Clements & Shear, 1931), and the isolate of P. graminis examined in this study, while not the lectotype was collected from A. repens in Germany, indicating that P. graminis may have precedence for the species name of Phyllachora sp. 3. This would have ramifications for P. maydis as well as several other Phyllachora species in Phyllachora sp. 3 (Table 1; Figure 3) that appear to be synonyms of P. graminis. This is based 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, Phyllachora sp. 1, Phyllachora sp. 2, and Phyllachora 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 Phyllachora sp. 3 and Phyllachora 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 three 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 United States (Groves et al., 2020; Kleczewski et al., 2019). The presence of multiple maize‐infecting species in the mid‐western United States, 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 Phyllachora sp. 3 has such a broad host range, whereas Phyllachora sp. 1 and Phyllachora 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 Phyllachora sp. 3 may have adapted to infect a novel grass species, while maintaining the ability to recombine with the larger Phyllachora 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 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 Phyllachora sp. 1, Phyllachora sp. 2, and Phyllachora sp. 3, but the significant overlap in host and geography would indicate a genetic barrier. While Phyllachora sp. 1 and Phyllachora sp. 2 were only recovered from maize, our sampling scheme was strongly biased toward maize. It is possible that Phyllachora sp. 1 and Phyllachora 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 Phyllachora sp. 3 as the P. maydis isosyntype 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 Phyllachora species makes it difficult to determine which of the maize‐infecting species will retain the name P. maydis. Therefore, we recommend referring to Phyllachora sp. 1, Phyllachora sp. 2, and Phyllachora sp. 3 as the 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 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 species of Phyllachora that have been present in Central America, Mexico, and the Caribbean for over 75 years are still unclear. Several scenarios may explain the recent emergence and severity of tar spot caused by Phyllachora species in the upper Midwest of the United States. While Phyllachora sp. 2 and Phyllachora 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 United States, it could not overwinter in colder climates and the disease could not become established. In fact, according to the herbarium specimens, Phyllachora sp. 3 has been present in the United States 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 of Phyllachora species to survive further north in the United States. 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 species arrived in the primary maize‐growing region of the United States. 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 United States includes a combination of these factors: (1) introduction of multiple species of 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 Phyllachora spp.; and (3) lack of resistance in maize germplasm grown in the Midwestern United States.

CONFLICT OF 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, and 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.

AUTHOR CONTRIBUTIONS

Kirk Broders: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Methodology (equal); Project administration (equal); Visualization (equal); Writing – original draft (lead); Writing – review  & editing (equal). Gloria Iriarte‐Broders: Data curation (equal); Formal analysis (equal); Methodology (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal). Gary C. Bergstrom: Resources (equal); Writing – review & editing (equal). Emmanuel Byamukama: Resources (equal); Writing – review & editing (equal). Martin Chilvers: Resources (equal); Writing – review & editing (equal). Christian Cruz: Resources (equal); Writing – review & editing (equal). Felipe Dalla‐Lana: Resources (equal); Writing – review & editing (equal). Zachary Duray: Data curation (equal); Formal analysis (supporting); Investigation (supporting). Dean Malvick: Resources (equal); Writing – review & editing (equal). Darren Mueller: Resources (equal); Writing – review & editing (equal). Pierce Paul: Resources (equal); Writing – review & editing (equal). Diana Plewa: Resources (equal); Writing – review & editing (equal). Richard Raid: Resources (equal); Writing – review & editing (equal). Alison E. Robertson: Resources (equal); Writing – review & editing (equal). Catalina Salgado‐Salazar: Formal analysis (supporting); Methodology (supporting); Resources (equal); Writing – original draft (supporting); Writing – review & editing (equal). Damon Smith: Resources (equal); Writing – review & editing (equal). Darcy Telenko: Resources (equal); Writing – review & editing (equal). Katherine VanEtten: Data curation (supporting); Formal analysis (supporting); Investigation (supporting); Resources (equal). Nathan M. Kleczewski: Conceptualization (lead); Data curation (supporting); Formal analysis (supporting); Funding acquisition (lead); Methodology (equal); Project administration (equal); Resources (lead); Supervision (equal); Writing – original draft (equal); Writing – review & editing (equal).

Fig S1

Click here for additional data file.