Extant (fresh) and historic (herbaria) samples of the Phalaris species were used for genomic DNA extraction. A total of n=52 historic specimens were collected from the Bell Museum Herbarium, University of Minnesota, St. Paul, MN (MIN; n = 9; Table 1) and the Ada Hayden Herbarium, Iowa State University, Ames, IA (ISC; n = 43; Table 1). Destructive sampling of ~2.5 x 0.63 cm of leaf tissue was performed on each herbarium specimen for leaf samples positioned in the back of the specimen to not decrease their visual integrity, similar to our previous methodology . Permission for destructive tissue sampling was obtained from each herbarium curator in advance. Specimen notes were added to delineate the specimen-specific sampling. This herbarium collection represents a range of North American Phalaris herbarium specimens with collection dates ranging from 1882 (P. minor Retz.; ISC-V-0021344) to 2001 (P. arundinacea L.; 484712; Table1). Selection of the P. arundinacea herbaria samples were already tested for SNP genetic variation and, provided sufficient and high quality nuclear DNA could be extracted, were determined to be most likely native North American genotypes [29,36].
For additional sampling purposes, seeds were obtained from the U.S. Department of Agriculture Germplasm Resources Information Network (USDA GRIN; https://www.ars-grin.gov/). Germinated seedlings were used as extant specimens of the Phalaris species. Fresh samples served as an amplification and SNP detection validation. Extant specimens included three P. aquatica (PI 476287; PI 476288; PI 303825), two P. arundinacea (PI 241065; PI 422030), and two P. canariensis (PI 578800; PI 578798; Table 2). Seeds were sown in 10 cm square pots filled with Sungro Professional Growing Mix (Sun Gro Horticulture; SKU:5105, Agawam, MA) and placed in a mist house (greenhouse with an intermittent mist system). Once the seedlings germinated and true leaves were developed, plants were moved to the greenhouse for continued growth and leaf harvest. Environmental conditions in both greenhouses were 24.4±3.0/18.3±1.5°C day/night daily integral and a 16 hr long day photoperiod (0600–2200 HR) lighting (400 w high pressure sodium high intensity discharge lamps, HPS-HID) at a minimum of 150 μmol m-2 s-1. Plants were fertilized twice daily, between 0700-0800 and 1600-1700, using a constant liquid feed (CLF) of 125 ppm N from water-soluble 20N–4.4P–16.6K (Scotts, Marysville, OH). Fungicide drenches were applied in monthly rotations. Leaf tissue of matured, fully-expanded leaf trips were harvested and stored at -20°C in sealed plastic bags.
NCBI ITS Resources
National Center for Biotechnology Information (NCBI) database searches were performed to collect existing ITS region sequences for comparative purposes from the twelve Phalaris species included in this study (Table 1). In this research, only Phalaris ITS sequences that contained full ITS1 and ITS2 regions were included and partial sequences were not compared. Partial sequences were not included because missing sequences could contain SNP polymorphism(s) that would contribute to less accurate phylogenetic analysis. Multiple ITS sequences from twelve Phalaris species were found (n = 68, full sequences): P. angusta (KX873129.1, KF753774.1, JF951055.1, JF51054.1), P. aquatica (KU883516.1, KF753775.1, JF951056.1, KF753776.1, KC512901.1, JF951076.1, KX873130.1), P. arundinacea (KF753779.1, JF951077.1, KF713257.1, FJ766174.1, KF713256.1, KU883517.1, KF713255.1, HQ600518.1, KP711073.1, KF713254.1, KF713253.1, FJ821785.1, KF713251.1, HF564628.1, KF713250.1, KF753778.1), P. brachystachys (KC512902.1, KF753780.1, JF951057.1), P. californica (JF951078.1, JF951064.1, KF753781.1), P. canarensis (KX147547.1, DQ539580.1, FJ178782.1, JF951058.1, KX147537.1, KP296086.1), P. caroliniana (JF951065.1, JF951080.1, JF951079.1), P. coerulescens (JF951081.1, JF951066.1, DQ539581.1, KC512900.1, HE802172.1, KF753782.1), P. lemmonii (MF964010.1, JF951082.1), P. minor (JF907187.1, JF951084.1, JF951069.1, JF907187.1, KX873131.1, KU883518.1, JF951086.1), P. paradoxa (JF951070.1, JF951071.1 KX873133.1, KX873132.1, JF951088.1, KF753783.1, JF951089.1, KC512899.1), and P. truncata (L36522.1, KC512903.1, JF951059.1). The geographic source of the P. arundinacea specimens were recorded if information was available on NCBI site or associated published work.
Nuclear DNA was extracted from historic and extant Phalaris samples using Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics Laboratory, Lebanon, NJ) with minimal adjustments to the protocol . Tissue was loaded using scissors and forceps that were washed in soapy water, rinsed twice in distilled water and dried with paper towels. Extant samples, unlike historic ones, were kept cool on dry ice throughout the loading process. Samples were ground for a total of 15 minutes at 1,500 rpm in homogenizer (Geno/Grinder; SPEX SamplePrep, Metuchen, NsJ). Once purified, DNA was suspended in molecular grade water and stored at -20°C. DNA quality and quantity were checked with a spectrophotometer (NanoDrop 2000; Thermo Scientific, Waltham, MA). Quality guidelines were followed as recommended by Thermo Fisher Scientific as ~1.8 O 260/280 and 1.8-2.2 O 260/23 considered as “pure” DNA (NanoDrop 2000/2000c Spectrophotometer V1.0 User Manual, 2009). Genomic DNA was visualized on 1% (w/v) agarose gels (1 × Tris-acetate-EDTA buffer) and stained with ethidium bromide, 6X loading dye (New England BioLabs; Ipswich MA), and a DNA ladder (FullRanger 1kb; Norgen Biotek Corp, Thorold, ON, Canada). Only herbarium specimens with the highest quantity of DNA were used to visualize degradation (200 ng / lane on an agarose gel) due to the limited DNA.
To calculate correlation between herbarium tissue age and the concentration of genomic DNA obtained and to the PCR amplification success from herbarium tissue Microsoft Excel 365 (Microsoft, Redmond, WA) with Correlation function was used to estimate p-value.
Polymerase chain reaction (PCR) was performed using 10 μM of ITS-P5 and ITS-U4 primers . PCR master mix (GoTaq Green Master Mix, M712; Promega, Madison, WI) plus 1 μL of DNA (with a minimum concentration >50ng/μL) or DNA volume was adjusted to obtain 50 ng total DNA. The PCR protocol followed previous methodology : 94°C for 4 min, then 34 cycles of 30 sec at 94°C, 40 sec at 55°C and 1 min at 72°C, finishing with 10 min at 72°C. PCR reactions were visualized using electrophoresis on a 1% (w/v) agarose gel (1 x Tris-acetate-EDTA buffer) with ethidium bromide and a DNA ladder. Expected DNA amplification product of ITS-P5 and ITS-U4 primers was 757 ± 140 bp . If a single amplification product was observed, amplified products were directly purified using PureLink™ Quick PCR Purification Kit (Thermo Fisher Scientific, Waltham, MA) and prepared for Sanger sequencing at the University of Minnesota Genomics Center, following UMGC sample requirements (University of Minnesota Genomics Center – Sanger Sequencing Classic, http://genomics.umn.edu/sanger-sequencing-classic.php). If the purified PCR reaction yielded less than the total DNA required for Sanger sequencing (25 ng/µL), the PCR product was diluted in water (1/50) and 1 ul of diluted product was used and re-amplified following the same procedure (ITS-P5 and ITS-U4, primer set, PCR reaction composition, PCR program).
Sequencing results were edited and quality-checked using 4Peaks software (Nucleobytes, Alsmeer, Netherlands; http://nucleobytes.com/4peaks/) with additional manual sequence trimming. Sequence editing, alignments, annotations and manipulations were done using Geneious 11.1.5 software (Biomatters, Ltd., New Zealand; https://www.geneious.com) . The Arabidopsis thaliana (x52320.1) ITS sequence was initially used to annotate full ITS regions of the newly obtained (n = 36) Phalaris DNA sequences (Table 1, Table 2). Genetic distance among Phalaris species was inferred using the Neighbor-Joining method  and a bootstrap test was performed for each tree (with 100 replicates) among newly obtained and NCBI ITS sequence collection. A multiple sequence alignment was performed with use of MUSCLE alignment . Diagnostic, species-specific SNPs that differentiate among Phalaris species were determined based on multiple alignment of full-length ITS (ITS1 and ITS2 regions) sequences with exclusion of 5.8S ribosomal subunit. Sequences obtained in this research were deposited into the NCBI database, accession numbers can be found in Tables 1 and 2.