Table S1. Epichloë sylvatica immunoblot assays and seed collection sites.
Source / Host Grass / Range / Population / Location / N / % Pos / Datewild collected / B. sylvaticum / Invaded-USA / Bald Hill / 44°34'23.0"N, 123°19'48.5"W / 30 / 0 / 20-Jun-10
wild collected / B. sylvaticum / Invaded-USA / Bald Hill / 44°34'23.0"N, 123°19'48.5"W / 30 / 0 / 26-Aug-10
wild collected / B. sylvaticum / Invaded-USA / Jasper / 44° 0'43.6"N, 122°53'34.1"W / 30 / 0 / 20-Jun-10
wild collected / B. sylvaticum / Invaded-USA / Jasper / 44° 0'43.6"N, 122°53'34.1"W / 30 / 0 / 4-Aug-10
wild collected / B. sylvaticum / Invaded-USA / Jasper Park / 43° 99'31.4"N, 122°89'66"W / 30 / 0 / 4-Aug-10
wild collected / B. sylvaticum / Invaded-USA / Sweet Home / 44°25'18.7"N, 122°40'32.1"W / 30 / 0 / 20-Jun-10
wild collected / B. sylvaticum / Invaded-USA / Sweet Home / 44°25'18.7"N, 122°40'32.1"W / 30 / 0 / 26-Aug-10
wild collected / B. sylvaticum / Invaded-USA / Pisgah / 44° 0'9.80"N, 122°56'59.2"W / 30 / 0 / 20-Jun-10
wild collected / B. sylvaticum / Invaded-USA / Pisgah / 44° 0'9.80"N, 122°56'59.2"W / 30 / 0 / 26-Aug-10
wild collected / B. sylvaticum / Invaded-USA / Pisgah-BPAa / 44° 0'9.80"N, 122°56'59.2"W / 10 / 0 / 26-Aug-10
planted 2008 / B. sylvaticum / Invaded-USA / MacForest / 44°38'42.7"N, 123°18'37.9"W / 1 / 0 / 4-Aug-10
planted 2008 / B. sylvaticum / Invaded-USA / Sweet Home / 44°25'18.7"N, 122°40'32.1"W / 1 / 0 / 4-Aug-10
planted 2008 / B. sylvaticum / Invaded-USA / Fisherman / 43°58'19.5"N, 122°39'37.7"W / 1 / 100 / 4-Aug-10
wild collected / B. sylvaticum / Invaded-USA / Fisherman / 43°58'19.5"N, 122°39'37.7"W / 41 / 100 / 29-Aug-11
wild collected / B. sylvaticum / Invaded-USA / Mill City / 44°45'6.4"N, 122°29'56.7"W / 30 / 0 / 29-Aug-11
planted 2008 / B. sylvaticum / Invaded-USA / Owl / 43° 59'51.6"N, 123°05'72.3"W / 1 / 0 / 4-Aug-10
planted 2008 / B. sylvaticum / Invaded-USA / Jasper / 44° 0'43.6"N, 122°53'34.1"W / 2 / 0 / 4-Aug-10
planted 2008 / B. sylvaticum / Invaded-USA / Pisgah / 44° 0'9.80"N, 122°56'59.2"W / 1 / 0 / 4-Aug-10
planted 2008 / B. sylvaticum / Native-CH / Albisguetli / 47°21'47.4"N, 008°29'98.0"E / 2 / 100 / 4-Aug-10
planted 2008 / B. sylvaticum / Native-CH / Albisguetli / 47°21'47.4"N, 008°29'98.0"E / 2 / 100 / 26-Aug-10
planted 2008 / B. sylvaticum / Native-CH / Flaach I / 47°35'36.4"N, 008°36'27.4"E / 2 / 100 / 4-Aug-10
planted 2008 / B. sylvaticum / Native-CH / Flaach II / 47°59'04.1"N, 008°60'74.0"E / 2 / 100 / 4-Aug-10
planted 2008 / B. sylvaticum / Native-CH / Truebbach I / 47°04'04.1"N, 009°28'48.0"E / 1 / 100 / 26-Aug-10
planted 2008 / B. sylvaticum / Native-CH / Truebbach I / 47°04'04.1"N, 009°28'48.0"E / 1 / 100 / 4-Aug-10
planted 2008 / B. sylvaticum / Native-CH / Truebbach II / 47°04'12.2"N, 009°29'04.8"E / 1 / 100 / 4-Aug-10
wild collected / D. glomeratab / Invaded-USA / UO campus / 44°3'3.21"N, 123°4'21.8"W / 1 / 100 / 20-Jun-10
wild collected / D. glomerata / Invaded-USA / UO campus / 44°3'3.21"N, 123°4'21.8"W / 3 / 100 / 4-Aug-10
wild collected / D. glomerata / Invaded-USA / Bald Hill / 44°34'23.0"N, 123°19'48.5"W / 3 / 100 / 26-Aug-10
a These ten plants were collected about 50 m away from the original population because the original had been sprayed by an herbicide, glyphosate, since we last collected in June. We were still able to collect live material at the original site, however, to be sure the herbicide had not altered infection rates, we also collected unsprayed plants from nearby.
b The Dactylis glomerata were not randomly sampled. They were obviously infected (showing symptoms of choke disease), and were used as positive controls.
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Supplementary Methods:
Infection Rates
To determine whether the fungus was present in the invaded range we first used the standard morphological screen in which leaf sheaths are stained to make the fungal hyphae more evident (Latch & Christensen 1985). There were no differences among US plants, which we initially interpreted as indicative of 100% infected, like the European populations. To confirm, we screened the same populations with the Agrinostics Field Tiller immunoblot kit (Agrinostics Ltd. Co., Watkinsville, GA, USA).
We isolated E. sylvatica from seed with slightly modified version of the fungal culture method outlined by Mirlohi et al. (2006). Briefly, the seeds were deglumed by hand, rinsed in 100% ethanol for 30 seconds, rinsed in sterile water then sterilized by soaking in 30% hydrogen peroxide for 20 minutes, rinsed in sterile water and plated on PDA. Due to contamination, only one culture yielded Epichloë (from Flaesch, Switzerland 47°01'21.1" N, 009°30'03.6" E).
To verify the identity of the isolated endophyte, we sequenced the internal transcribed spacer (ITS) II region of the nuclear ribosomal DNA operon. DNA extraction was according to Winton et al. (2002), using 0.1% DDT in the buffer solution instead of 0.1% 2-mercaptoethanol. We used the standard ITS3 and ITS4 primers (White et al. 1990), with Sanger sequencing carried out by the University of Oregon Sequencing Facility. The resultant sequence (KF225579) has 99.7% sequence similarity with the published Epichloë sylvatica sequence (L78304).
For the PCR screen, DNA was extracted from tillers using two different methods. We initially followed Sullivan and Faeth (2004), but shifted to a more efficient method developed for mushrooms (Dentinger et al. 2010), which yielded better quality fungal DNA. Briefly, a tiller base including a leaf was smashed into a DNA absorbing Whatman FTA card and extracted with the Sigma Extract-N-Amp Plant PCR Kit.
We used Neotyphodium-specific primers (IS-RS-5’ and IS-NS-3’) to amplify the intron region of the ß-tubulin (tub2) gene (Dombrowski et al. 2006). The protocol of Dombrowski et al. (2006) was developed for a different set of Epichloë/host species combinations, and required minor optimization to work well with E. sylvatica in B. sylvaticum. We utilized Sigma JumpStart Taq ReadyMix for our PCR reactions according to the manufacturer’s instructions, with a slightly modified PCR mixture: 5µL JumpStart Taq ReadyMix, 0.4µL MgCl2 (25mM), 0.2µL forward primer, 0.2µL reverse primer, 2.2µL water and 2µL DNA template in a 1:1 dilution. The positive control for these reactions was the E. sylvatica endophyte isolated from Swiss seed, described above. PCR conditions were as follows: Initial denaturation at 94˚C for one minute, then 18 cycles at 94˚C for 25 s, a touchdown annealing temperature starting at 73˚C and decreasing by 0.8˚C per cycle, followed by 72˚C for 3 min. The remainder of the PCR reaction was 32 cycles of 94˚C for 25 s, 58˚C for 1 min, and 72˚C for 2 min, followed by a final extension at 73˚C for 15 min.
Germination rates
It should be noted that this experiment only tests range origin of the maternal genotype. We did not control pollination in the greenhouse, so it is possible that up to fifty percent of the genetic compliment of the seeds may be from the other range, and that there may be effects of hybrid vigor acting to increase observed germination rates. About half of field collected B. sylvaticum appear to be selfed (Rosenthal et al. 2008), but selfing is more common under greenhouse conditions unless pollen is deliberately circulated with fans (M. Cruzan, pers. comm.). If there had been no admixture, we would expect steeper slopes and greater differences, increasing the significance of our results.
Literature Cited in Supplementary Methods
Dentinger, B.T.M., Margaritescu, S. & Moncalvo, J.-M. (2010). Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms. Mol Ecol Resour, 10, 628–633.
Dombrowski, J.E., Baldwin, J.C., Azevedo, M.D. & Banowetz, G.M. (2006). A sensitive PCR-based assay to detect fungi in seed and plant tissue of tall fescue and ryegrass species. Crop Science, 46, 1064.
Latch, G. & Christensen, M.J. (1985). Artificial infection of grasses with endophytes. Annals of Applied Biology, 107, 17–24.
Mirlohi, A., Sabzalian, M.R., Sharifnabi, B. & Nekoui, M.K. (2006). Widespread occurrence of Neotyphodium-like endophyte in populations of Bromus tomentellus Boiss. in Iran. FEMS Microbiology Letters, 256, 126–131.
Rosenthal, D.M., Ramakrishnan, A.P. & Cruzan, M.B. (2008). Evidence for multiple sources of invasion and intraspecific hybridization in Brachypodium sylvaticum (Hudson) Beauv. in North America. Molecular Ecology, 17, 4657–4669.
Sullivan, T.J. & Faeth, S.H. (2004). Gene flow in the endophyte Neotyphodium and implications for coevolution with Festuca arizonica. Molecular Ecology, 13, 649–656.
White, T.J., Bruns, T., Lee, S. & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications, 18, 315–322.
Winton, L.M., Stone, J.K., Watrud, L.S. & Hansen, E.M. (2002). Simultaneous one-tube quantification of host and pathogen DNA with real-time polymerase chain reaction. Phytopathology, 92, 112–116.
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