Table S4.

Patterns of panicoid rDNA distribution in relation to host phylogeny and speciation

Genus and clade no.	No. of species*/accessions	Description	Interpretation/potential scenario
Panicum 1	11/21 (+ 4 Elymus repens)	All Central Asian and 8 of 10 South American Hordeum species share this ITS type. Sequence variation is very low. Nevertheless, Central Asian species and two Hordeum cordobense accessions form well-supported subclades. One accession (Hordeum patagonicum BCC2064) harbors two ITS subtypes. The South American species Panicum bergii is sister to this clade.	Because of low sequence variation and sharing of this ITS type across a large range of Hordeum species with distribution areas that do not overlap, the most parsimonious explanation is a single transfer from a species closely related to P. bergii predating the diversification of Hordeum section Stenostachys. The transfer presumably occurred in Central Asia before Hordeum colonized the New World in a timeframe between ∼5 and 1.7 Mya inferred from Hordeum phylogeny. In some host species (e.g., Central Asian taxa, H. cordobense), group- or species-specific sequence evolution has occurred after the transfer.
Panicum 2	4/10	This ITS type is shared by four Hordeum species from southern South America. Two subtypes shared by three accessions each can be distinguished. The clade is sister to the Australian species Panicum queenslandicum.	Nine of 10 individuals showing this type also harbor the Panicum 1 ITS type suggesting a subsequent acquisition restricted to a few South American species from a Panicum donor related but not identical to P. queenslandicum. The event has likely happened in South America and either predated the speciation of the respective South American taxa (∼1 Mya) or spread among them later by introgression.
Setaria 3	4/7	The monophyletic Setaria clade is composed of several panicoid genera, including two species of Setaria. None of the groups have the captured ITS type, which occurs only in South American Hordeum species and shows relatively high sequence diversity. Four of the accessions show two to three divergent subtypes (e.g., Set-a, Set-b), which are shared between species.	No closely related panicoid species is known/available; therefore, the donor species or even genus remains unclear in this case but likely has South American origin. Different ITS subtypes within several Hordeum individuals that are shared with other accessions/species suggest that they are paralogs that may have resulted from duplications of the ITS region after acquisition by their hosts. This scenario would argue for a transfer predating their speciation. However, the high sequence diversity within this ITS type may also suggest multiple independent transfers, even from different donor species.
Setaria 4	3/4	One Central Asian Hordeum and two Pseudoroegneria species show this type. Hordeum brevisubulatum is basal to three Pseudoroegneria sequences, which show relatively high ITS sequence divergence. Setaria chondrachne is sister to this clade.	Only Central Asian and North American pooid species show this type, indicating acquisition by the Asian species S. chondrachne or a closely related taxon in Central Asia. Pseudoroegneria species may have obtained it by hybridization with H. brevisubulatum, after which it underwent additional sequence diversification and reached North America along with Ps. spicata, the only diploid New World taxon of this genus.
Paspalum 5	9/16	Nine of 10 South American Hordeum species share this ITS type. All sequences within this clade are very similar. No particular species of Paspalum clusters with this type.	This is the second largest assemblage of species harboring a particular nonnative ITS type. Their high sequence similarity suggests a single transfer event that predated speciation of the South American Hordeum species (∼1 Mya). Multiple independent acquisitions or extensive hybridization are rather unlikely explanations in this case.
Paspalum 6	7/11	This clade shows relatively high sequence diversity. Exclusively New World species harbor this type, among them two North American Hordeum species that occur in derived positions and do not possess any other panicoid ITS types. No particular species of Paspalum matches this type.	High sequence diversity implies high rates of molecular evolution after acquisition or reflects at least two independent events. Sequences of two North American Hordeum taxa are nested within South American species. This pattern is in accordance with their derived phylogenetic position and an inferred long-distance dispersal event from South America to North America. Here, the panicoid ITS, acquired from an unknown Paspalum species donor, seems to reflect the speciation patterns of the host.
Paspalum 7	2/2	Panicoid ITS sequences of one North American and one South American Hordeum species cluster with Paspalum dilatatum, which is native to Brazil and Argentina but was introduced worldwide.	In this case, a particular panicoid species, Pa. dilatatum, can be identified as the likely donor of the exotic ITS type. The two species have nonoverlapping distribution, indicating either two independent transfers from the same donor species or one transfer to their common ancestor, with differential loss of this variant in all other species.
Euclasta 8	7/12	Six South American and one North American Hordeum species show this ITS type. One accession (Hordeum stenostachys BCC2021) possesses two subtypes. Relatively high sequence divergence is observed in this clade, which is sister to E. condylotricha.	The geographically widespread occurrence of this type is in keeping with an acquisition that predated the speciation of the New World species (∼1.46 Mya). E. condylotricha has a wide distribution in the African and Asian tropics as well as southern North America and northern South America. Only two species of genus Euclasta are known (the second one does not occur in the New World); therefore, we assume that E. condylotricha was possibly the actual rDNA donor.
Arundinella 9	2/3	Two Argentinian species have this rare ITS type with sequence that corresponds to A. hispida, a species occurring in tropical Asia, Central America, and South America.	This type occurs in only two species with overlapping distribution. All three accessions also contain Panicum and Paspalum types, which suggests a rather recent acquisition that has happened after the other events; this pattern is in keeping with ITS matching a particular donor species.

Nine clades of panicoid rDNA sequences present in pooid grasses as depicted in Fig. 1A are described and interpreted in the context of host evolution and biogeographic patterns (Fig. 1B) (25, 28, 56, 57). Briefly summarized, three Western Asian and Mediterranean sections of Hordeum constitute the oldest lineages of the genus. The youngest section, Stenostachys, in which the panicoid rDNA is found, split from them ∼5 Mya. The barley genus then spread into Central Asia, and eventually, one lineage split from the Central Asian species ∼1.7 Mya and reached the New World via Beringia. The onset of speciation in the New World clade occurred ∼1.46 Mya. Long-distance dispersal brought the barleys to South America, where they underwent a rapid divergence (∼1 Mya) accompanied by incomplete lineage sorting, which results in allele and haplotype sharing across species. Two North American species, Hordeum pusillum and Hordeum intercedens, originate from a southern lineage that migrated back north again.

^*Different subspecies of H. patagonicum are not distinguished.