3.2 Phylogenetic diversity of 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 Phyllachora with strong bootstrap support (>70%). The results suggest that tar spot on maize in the U.S. is caused by three closely related species of Phyllachora(Figure 2). In all, four species were found on maize but onlyPhyllachora sp. 1 , P. sp. 2, and P. sp .3 were recovered from contemporary maize in the U.S., whileP. 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 1905-2019 (Table 1). Samples of 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 P. maydis (BPI638553) collected in Mexico in 1977 and the P. maydis isolate (BPI893226) used in the first report of tar spot in the U.S. in 2015 are both part of 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 Phyllachora species in the U.S. among the samples included in this study. However, isolates of 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 Phyllachora includingP. 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, P. sp. 1 and P. 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 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 Phyllachora sp. 4.However, samples only included herbarium specimens from Guatemala and Venezuela and did not include any contemporary maize specimens. Interestingly, 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 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.Phyllachora sp. 5 was the only species not recovered from maize but was found on many of the same grass species as P. 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 to those submitted previously to Genbank (Figure 3). In the case of P. sp. 4 , two isolates referred to as P. graminis, one from Hordelymus europaeusin Germany and one of unknown origin, as well as isolates ofPhyllachora on Elymus kamoji and Roegneria sp . from China grouped together with strong boostrap support (99%). There was also an isolate of P. graminis from an unknown grass in Canada that grouped together with P. sp. 5 , and the herbarium specimen of P. graminis from Agropyron repens in Germany from this study grouped in P. 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.