Biodiversity Data Journal 11: e101257 CO) doi: 10.3897/BDJ.11.e101257 open access Data Paper Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea (Poaceae) in western Canada and Alaska Diana M. Percy*, Quentin C. B. Cronk? + Department of Botany, University of British Columbia, Vancouver, Canada § Beaty Biodiversity Museum, University of British Columbia, Vancouver, Canada Corresponding author: Diana M. Percy (diana.percy@ubc.ca) Academic editor: Marcin Nobis Received: 31 Jan 2023 | Accepted: 28 Mar 2023 | Published: 11 Apr 2023 Citation: Percy DM, Cronk QCB (2023) Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea (Poaceae) in western Canada and Alaska. Biodiversity Data Journal 11: e101257. https://doi.org/10.3897/BDJ.11.e101257 Abstract Background Phalaris arundinacea L. (reed canary grass) is a widely occurring grass throughout the Northern Hemisphere. In North America, it is thought to consist of introduced agricultural forms from Europe as well as native populations. New information During a survey of Phalaris arundinacea in western Canada, we discovered two distinct ribotypes in the sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA: one full length (ITS-long) and one with a seven base pair deletion (ITS-short). In addition, ITS-long plants have fixed heterozygosity indicating possible polyploidy. Phylogenetic analysis reveals that ITS-short is a unique ribotype that characterises an intraspecific clade. We designed an efficient PCR-based assay that allows sizing of a © Percy D, Cronk Q. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Percy D, Cronk Q 238/245 base pair fragment in a capillary sequencer. This approach provides a novel marker that could be useful in future surveys of Phalaris arundinacea. Keywords internal transcribed spacer, invasive plant, Phalaris, reed canary grass Introduction Phalaris arundinacea L., commonly called reed canary grass (RCG), is a Eurasian and North American perennial grass, with many uses in agriculture (Jakubowski et al. 2011) and biomass energy (Lewandowski et al. 2003). In North America, native populations are considered under threat from invasion and replacement by vigorous introduced genotypes of P arundinacea that have now become a significant invader of wetland and riparian habitats in North America (Lavergne and Molofsky 2004) with considerable ecological impacts (Spyreas et al. 2010). The distribution of Phalaris arundinacea in North America, based on databased herbarium specimens, is shown in Fig. 1. Figure 1. EES Map of North American Phalaris arundinacea herbarium specimens from the Global Biodiversity Information Facility (GBIF; accessed October 2021). Red = 1822-1940; blue = 1941-2018. The dotted line marks the boundary of the western cordilleras. Molecular methods have often been used to distinguish populations of RCG, including isozymes (Gifford et al. 2002), AFLP (Casler et al. 2009), SSR (Jakubowski et al. 2013, Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... 3 Jakubowski et al. 2014, Kettenring et al. 2019), ISSR (Anderson et al. 2016), DartSeq (Noyszewski et al. 2019, Noyszewski et al. 2021) and ITS sequencing (Graper et al. 2021). However, there is still much uncertainty and, in some cases, disagreement, regarding the extent of distribution and location of present day native versus introduced RCG populations in North America (Jakubowski et al. 2013). The aim of this note is to detail an easily scored novel genetic marker that may be of use in future surveys of RCG. Sampling methods Description: Sources of material - herbarium and field A total of 86 samples of Phalaris arundinacea were obtained from herbarium material and additional targeted sampling carried out for this study (Tables 1, 2, Suppl. materials 1, 3). Herbarium samples, from modern to 130 years old and in relatively good condition, were selected for sampling from the University of British Columbia Herbarium (UBC) and the Herbarium of the Bell Museum, University of Minnesota (MIN). Further dried leaf samples (used in a previous study; Kettenring et al. (2019)) were kindly provided by Professor Karen Mock of Utah State University. In addition, extensive field sampling was carried out in Elk Island National Park, Alberta, where park authorities were concerned about the ecologically harmful spread of, as well as appropriate control methods for, Phalaris arundinacea. Further recent samples were sourced from Greater Vancouver. Voucher specimens are deposited in UBC. As outgroups for the phylogenetic analyses, we used eight individuals obtained from herbarium samples of P. aquatica Guss., P canariensis L., P. caroliniana Walter, P coerulescens Desf. and P. paradoxa L. (Suppl. material 2). Table 1. Herbarium specimens identified as ITS-short: determined by sequencing or sizing assay to have a 7 bp deletion in ITS2. An asterisk indicates one individual identified as ITS-short in assay data, but putative hybrid in sequence data; and [] indicates the only sequence found on GenBank with the ITS-short genotype. Region abbreviations: AB Alberta, AK Alaska, BC British Columbia, NWT Northwest Territories. Accession no. Herb. Date Locality Region Habitat V100979 UBC 1950 Chilcotin, Madden Lake BC not recorded V101113 UBC 1950 Chilcotin, Meldrum Creek BC marsh *V152455 UBC 1974 near Shamrock, ca. 30 miles northwest BC in post-glacial bed of the Stuart of Prince George River V162493 UBC 1975 Beaver Lake, Wilson Creek Road, nr. BC swampy lake edge Slocan Lake V27934 UBC 1950 S. of Ft Smith NWT scattered in clumps along dried-up slough V67193 UBC 1957 Kootenay District, Flathead, Procter BC in 2 ft (60 cm) of water at lake Lake edge Accession no. V67205 V67206 V88503 V111611 V242084 V119175 [KF753778] V20064 Table 2. Herb. Date UBC 1957 UBC 1957 UBC 1958 UBC 1955 UBC 2014 ALA 1994 UBC 1945 Percy D, Cronk Q Locality Kootenay District, Sage Creek Lodge. Flathead valley, Marl Lake Kootenay, Nakusp, Wilson Lake. 5 mi (7.5 km) southeast of Fort Simpson, Yoho National Park, Hoodoo Creek Campground area Cook Inlet lowlands, Otter Creek at Loop Road Just east of Fort Saskatchewan Region Habitat BC wet edge of slough BC wet edge of lake BC in peat bog NWT rare in moist black ground in Carex meadow BC somewhat calcareous swampy lakeshore AK herbaceous border of ponded creek AB creek bottom Herbarium specimens identified as ITS-long: determined by sequencing or sizing assay to lack the 7 bp deletion in ITS2. Region abbreviations: BC British Columbia, MB Manitoba, MN Minnesota, NC North Carolina, WA Washington, YT Yukon Territory. Accession no. V226522 V67194 V96302 V97215 V233698 V122558 V228430 V195542 V240112 V227503 V237734 UTC00019311 MN71158 MN71175 Herb. Date UBC 2007 UBC 1957 UBC 1950 UBC 1962 UBC 2007 UBC 1968 UBC 2006 UBC 1979 UBC 2010 UBC 2008 UBC 2007 USU 1935 MIN #1891 MIN 1891 Locality Alaska Highway km 1016 Sage Creek, Flathead Salmon Arm Thompson-Nicola Regional District, Tranquille Greater Vancouver, Delta, Westham Island Avery County, Elk River at Heaton Osoyoos, Haynes Point Provincial Park Pencil Lake, Riding Mountain National Park Whitehorse Vancouver Island, Duncan, Somenos Marsh Vancouver Island, Cumberland Palouse River, Pullman St Anthony Park, Ramsey Ramsey Region Habitat YT apparently seeded along highway BC grassy meadow BC not recorded BC wet meadow BC tidal shore (var. picta) NC marsh BC meadow beside wetland MB road allowance, jet ski trail YT sewage treatment facility BC thick grassy marsh margin BC roadside with introduced grasses WA shallow pools of drying streambed MN see Noyszewski et al. (2021) MN see Noyszewski et al. (2021) Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... 5 Step description: DNA extraction, PCR and sequencing Dried leaf material was ground to a slurry in liquid nitrogen and the DNA extracted using a modified CTAB method (Doyle and Doyle 1987). Full length PCR (ITS1-5.8S-ITS2) was performed using primers ITS-A (forward) (Blattner 1999) and ITS4 (reverse) (White et al. 1990) and PCR conditions 94°C/4 min, followed by 30 cycles of 94°C/30 sec, 50°C/1 min, 72°C/1 min and final extension of 72°C/10min. In cases of highly degraded DNA from older herbarium specimens, ITS1 and ITS2 were amplified separately using primers ITS3P (forward) (Moller and Cronk 1997) and the reverse complement ITS2P (reverse). Bidirectional Sanger sequencing was performed by Eurofins (Kentucky, USA) and sequences were checked using Sequencher version 4.8 (Gene Codes). Sequence alignment and phylogenetic analysis Sequences of 60 individuals were aligned manually using Sequencher and Se-Al (Rambaut 2002). Subunit boundaries follow those determined for Oryza (Takaiwa et al. 1985, Yokota et al. 1989) as follows: 18S/ITS1 CATTG/TCGTG; ITS1/5.8S AAATC/ CACAC; 5.8S/ITS2 CACGC/CAAAA; ITS2/26S GGACC/GCGAC (an example of a full Oryza sequence for location is GenBank accession MF029734). Eight putative hybrids (between the different ribotypes) were excluded from the phylogenetic analysis due to sequence superposition. We included one sample from GenBank (KF 753778) as the only previously databased sequence with the ITS-short genotype. Phylogenetic analysis was performed using three approaches: a Neighbour-joining (NJ) analysis with uncorrected (p) distances and 1000 bootstrap replicates, a Maximum Parsimony (MP) analysis with heuristic search (random addition of taxa and TBR branch swapping), both methods being performed in PAUP* (Swofford 2003); and a Maximum Likelihood (ML) analysis using RAXML (v. 8.2.4) with GTRCAT, 1000 rapid bootstraps and Gamma optimisation of tree space run on the CIPRES Science Gateway (Miller et al. 2010, Stamatakis 2014). The MP analysis also included a gap code matrix (for nine gaps: three in P. arundinacea and six in outgroup taxa). Sequences are deposited in GenBank under accession numbers: 0Q740187-0Q740255. Structural analysis of ITS2 Structural analyses were performed using the ITS2 database (Ankenbrand et al. 2015). We used the Phalaris arundinacea |TS2 structure of GenBank accessions FJ821785 (MFE -66.8 kcal/mol) in the ITS2 database for homology modelling (Wolf et al. 2005) of our common variant (ITS-long) as it had a near identical sequence. As homology modelling of the rare variant (ITS-short) fails on FJ821785, alternative templates for homology modelling were investigated. Plausible configurations for ITS2-short were obtained using Arctagrostis latifolia (EU792351) and Phalaris canariensis (FJ377670) as templates. Capillary sizing assay A primer was designed using the NCBI Primer-BLAST tool (Ye et al. 2012) ITS2AindelR: 5’- GCAGCCATATCTTCGGC-3’ for use in conjunction with ITS primer ITS3P to allow an accurate sizing assay on an ABI 3730 automated DNA Sequencer (Applied Biosystems). 6 Percy D, Cronk Q The primer was combined with a M13 tail (5'-TGTAAAACGACGGCCAGT-3') on the forward primer to facilitate fluorescent dye labelling and a further PIG tail (5°-GTTTCTT-3’) on the reverse primer to promote terminal adenylation. We used a hot start touchdown PCR protocol with 95°C/3 min, followed by 10 cycles of 94°C/30 sec, 65°C/30 sec (-1°C per cycle, R 3°C/sec), 72°C/45 sec, followed by a further 30 cycles of 94°C/30 sec, 55°C/ 30 sec, 72°C/45 sec and a final extension at 72°C/4 min. PCR products were loaded into the capillary machine at 1:30 dilution and traces read using the programme Geneious 8.1.9 (Biomatters Ltd.). The PCR assay was designed to give products of 238 or 245 bp depending on the presence of the 7 bp deletion. A sequencing survey and phylogenetic analysis reveals intraspecific divergence in ITS including a 7 bp deletion Initial results of an ITS sequencing survey of Phalaris arundinacea from western Canada revealed two distinctive sequences. One is full length with fixed heterozygosity characteristic of polyploids; the other is shorter, with a 7 bp deletion in ITS2 and with no fixed heterozygous base positions. The differences are summarised in Table 3. The tree topologies recovered from the different phylogenetic approaches were nearly identical. The matrix length was 603 bp (612 characters with gap coding) and the MP search recovered two trees with length 117 (Cl: 0.93, Rl: 0.98); we present the strict consensus topology in Fig. 2 showing majority rule consensus values as well as NJ and ML bootstrap support values. The best ML model fit for the data (AIC) was GTR+G (-InL 1611.45). Use of outgroups showed that the full length sequence (which we call ITS-long) was likely the ancestral one and the deletion (ITS-short) is a putatively-derived character so far known only from plants in north-western North America (Fig. 3). When compared with all available world-wide sequences from GenBank (including Asia, Europe, North and South America), only one sequence was found to have the ITS-short genotype (KF 753778) from Cook Inlet, Alaska; all other GenBank samples are the ITS-long genotype and ITS-long sequences found in North America are highly similar or identical to European genotypes. Tables 1, 2 show to which clade (ITS-long/-short) historical herbarium specimens can be assigned. Table 3. Molecular characteristics of the 7 bp deletion clade (ITS-short) in comparison to the full length variant (ITS-long). Length variation in Phalaris arundinacea is caused by one 7 bp deletion and a 1 bp homopolymer indel, giving a combined length difference of 6 bp. The aligned sequence length for 52 Phalaris arundinacea individuals using the ITS1-5.8S-ITS2 subunit boundaries following Takaiwa et al. (1985) and Yokota et al. (1989) is 600 bp and, including six outgroup taxa (eight individuals), it is 603 bp. Ambiguity codes (Y, R, S) are given for heterozygotes. Sites homozygous, but polymorphic between different individuals, are given as C/T etc. Individuals that were interpreted as putative hybrids are given in Suppl. material 1. Feature ITS-long ITS-short Outgroups No. of individuals 37 15 8 Total sequence length, ITS1-5.8S- 599 (no variation) 593 (no variation) 598-600 ITS2 (bp) Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... Feature ITS-long ITS-short ITS1 length 219 219 5.8S length 164 164 ITS2 length 216 210 Fixed heterozygosity in ITS1-long Y(30), Y(181), ¥(193), C(30), C(181), C(193), (aligned position) S(208) C(208) Fixed heterozygosity in 5.8S-long Y(345), Y(359) C(345), C(359) (aligned position) Fixed heterozygosity in ITS2-long Y(421), R(489), C(421), A/R(489), (aligned position) Y(587) C(587) Fixed SNPs between groups inITS1 —A(7), C(60), C(195) C7), T(60), Y/T(195) (aligned position) Fixed SNPs between groups inITS2 = 1(413), C(493), T(628) C(413), T/Y(493),C/ (aligned position) Y(528) SSR in ITS2 (aligned position) C5(404-408) C5(404-409) UTCO0019311 *USU 1935, WA B5337 2010, USDA "PIBOsite75" BRCO1 2021, BC Cang-e1 modern, Europe MN71175 *MIN 1891, MN MN71158 *MIN $1891, MN P. arundinacea ITS-long 100 78/99 *UBC 1979, MB *UBC 2007, YT *UBC 2008, BC *UBC 2006, BC *UBC 2007, BC *UBC 2007, BC *UBC 2010, YT V67194 *UBC 1957, BC V96302 *UBC 1950, BC DPQC10A 2021, AB 100/100 P. arundinacea *UBC 1955, NWT ITS-short V162493 *UBC 1975, BC *UBC 2014, BC *UBC 1950, NWT *UBC 1957, BC *UBC 1957, BC *UBC 1957, BC V88503 *UBC 1958, BC V119175 *ALA 1994, AK [KF753778] V128867 *UBC 1970 P. caroliniana, LA V106316 *UBC 1954 P. aquatica, OR V196075 *UBC 1983 P. aquatica, OR V106656 *UBC 1950 P. paradoxa, CA V222762 *UBC 1992 P. coerulescens, Portugal L12636 *UBC 2021 P. canariensis, BC L12638 *UBC 2021 P. canariensis, BC V195437 *UBC 1988 P. canariensis, BC 100/100 700/100 99/100 100 100/100 = 1 change Figure 2. EESl Outgroups 219-222 164 213-216 T/C(30), C(181), T/ C(193), C(208) C(345), C/T(359) C/T(421), G(489), C(587) C(7), C/T(60), C(195) T(413), C(493), T(528) C?-6(404-409) Phylogeny of 53 individuals of Phalaris arundinacea and five outgroup taxa, based on ITS variation. Included are 60 individuals sampled for this study as well as the only GenBank sample of P arundinacea found with the ITS-short sequence [KF753778]. Asterisks indicate samples obtained from herbarium material. The tree is a strict consensus from the MP analysis with Majority Rule consensus values above nodes and NJ/ML bootstrap support values below nodes. Two clades can be seen: the deletion clade (ITS-short) and the full length ITS clade (ITS-long). Sample details are given in Tables 1, 2 and Suppl. materials 1, 2, 3. 8 Percy D, Cronk Q 65 60 55 50 45 -160 -150 -140 -130 -120 -110 -100 Figure 3. EE) Map of western Canada showing the locations of 51 genotyped samples of Phalaris arundinacea. Dotted line indicates the Province of British Columbia. Red dots show the locations of the “short” ribotypes (n = 13); blue “long” (n = 32) and orange putative hybrids (n = 6). Only three placeholder specimens are given for Elk Island National Park (arrowed; see additional map Suppl. material 4). Sample details are given in Tables 1, 2 and Suppl. materials ta 2 52 The 7 bp deletion alters the secondary structure of helix | of ITS2. The predicted secondary structure of the common variant (ITS2-long) of Phalaris arundinacea |TS2 is the usual eukaryotic four helix model (Fig. 4). Homology modelling of the structure of the ITS2-short sequence against this structure fails, as helix |, which has the 7 bp deletion, is not a suitable model. However, homology modelling with a related grass of similar ITS2 sequence suggests a plausible model for helix | despite the deletion (Fig. 4). A PCR-based capillary sizing assay allows rapid detection of the 7 bp deletion clade In order to genotype individuals without sequencing, we developed a primer that amplifies a 238 vs. 245 bp amplicon (short enough to size accurately to a single base pair on a capillary machine). ITS-long gave a clear peak at 245 bp and a complete absence of a peak at 238 bp. Despite using a design to promote terminal adenylation (see Methods), if there is a large amount of starting DNA, this peak was split, showing a peak or shoulder at 244 bp. However, in all cases, the fully adenylated peak was unambiguous and as strong or stronger than the unadenylated peak. ITS-short samples gave a strong, unambiguous peak at 238 bp. Product without terminal adenylation sometimes showed as a shoulder, but Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... 9 never a separate peak. ITS-short samples sometimes showed a small peak at 245 bp, but the 238 peak was, in all cases, much stronger. A total of 68 individuals were sized with this method, providing clade (ITS-long/-short/hybrid) affiliation for an additional 34 individuals. Putative hybrids (10 individuals) were identified either by both sequencing and sizing assay (seven individuals), sequence data only (one individual) and two specimens identified as ITS-short in the length assay, but were determined as a putative hybrid with sequence data (Suppl. material 1). ITS-long ITS-long Figure 4. EES] Secondary structure consequences of the deletion in ITS2. A) Predicted secondary structure of Phalaris arundinacea ITS2, based on the common variant (ITS2-long). B) Detail of helix |; DEL = the bases (GGGATGC) deleted in the ITS2-short variant; HR — C5 = 5 cytosine homopolymer repeat; asterisk T — position of the T/C single nucleotide polymorphism (aligned position 413). C) Possible alternative structure of helix | in the ITS2-short variant, based on homology modelling using Arctagrostis latifolia as the template; the cytosine homopolymer repeat is now C6 (6 cytosines); the arrow shows the position of the deleted sequence. A survey of Elk Island National Park, Alberta, reveals presence of both ITS ribotypes Using the molecular tools detailed above, we were able to conduct extensive sampling of Elk Island National Park (EINP), Alberta. Phalaris arundinacea is extremely abundant at EINP and the material in the Park tends to be strongly spreading-rhizomatous and invasive. EINP is bisected into a northern and southern portion by the east-west highway 16. These portions have different management histories, with the northern portion experiencing much greater public access and road development. We refer to these portions as north EINP and south EINP. In all sampled localities of north EINP, ITS-long was the only genotype detected (except a few possible hybrids at Tawayik Lake). In south EINP the situation is very different. Of the 12 individuals genotyped from south EINP, five were ITS-short (DPQC10A and DPQC11A-D). 10 Percy D, Cronk Q Geographic coverage Description: North-western North America Taxonomic coverage Description: Phalaris arundinacea, P. aquatica, P. canariensis, P. caroliniana, P. coerulescens and P. paradoxa. Usage licence Usage licence: Creative Commons Public Domain Waiver (CC-Zero) Data resources Data package title: Specimen details for all 94 samples genotyped (86 Phalaris arundinacea and eight outgroup taxa sampled). Number of data sets: 1 Data set name: Specimen details for all 94 samples genotyped (86 Phalaris arundinacea and eight outgroup taxa sampled). Description: Suppl. material 3 contains specimen details for all 94 samples genotyped (86 Phalaris arundinacea and eight outgroup taxa sampled). Column label Column description occurrence|ID Specimen Code identifier for the Occurrence. basisOfRecord Specimen type as the specific nature of the data record. eventDate Date of specimen collection. eventRemarks Note of incomplete date information. decimalLatitude The geographic latitude (in decimal degrees, using the spatial reference system given in geodeticDatum) of the geographic centre of a Location. decimalLongitude The geographic longitude (in decimal degrees, using the spatial reference system given in geodeticDatum) of the geographic centre of a Location. geodeticDatum The ellipsoid, geodetic datum or spatial reference system (SRS) upon which the geographic coordinates given in decimalLatitude and decimalLongitude are based. eventRemarks Ribotype of ITS sequence. country The name of the country or major administrative unit in which the Location occurs. locality The specific description of the place. Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... 11 verbatimLocality The original textual description of the place. scientificName The full scientific name, with authorship. identificationQualifier Qualifier on current identification. taxonRank The taxonomic rank of the most specific name in the scientificName. institutionCode The name (or acronym) in use by the Herbarium institution having custody of the object(s) or information referred to in the record. collectionCode The name, acronym, coden or initialism identifying the collection or dataset from which the record was derived. Additional information Implications of two highly divergent intraspecific ribotypes The making of a ribosome is a complex process: it involves multiple steps and over 200 biogenesis factors (Saez-Vasquez and Delseny 2019). In this process, ITS2 plays an important role. The excision of ITS2 from the pre-ribosomal RNA is essential to generate mature 25/26S and 5.8S and the secondary structure of ITS2 is important for this process (Schultz et al. 2005). Embryophytes have four helices (numbered I-IV) arising from a central ring. These helices require complementary base pairing to form (and be stable). They are, therefore, generally quite conserved in sequence, with mutations only surviving if they preserve the pairing energetics of the helix (Zhang et al. 2020). For this reason, it is surprising to see an intraspecific seven base-pair deletion in helix |. In addition, this helix carries an SNP and an extra cytosine in a cytosine repeat sequence. There is an energetically plausible alternative structure for this helix, but it still represents a marked change in helix pairing structure. Furthermore, there are seven SNPs in helix III (although these do not markedly impact helix structure). Given this, it is evident that there are two distinctive ITS2 ribotypes in north-western Canada, being distinguished by two indel events, one with a major impact on helix nucleotide pairing and five SNPs. The ITS-long sequence was highly similar or identical to sequences of known European genotypes obtained from GenBank. In contrast, the ITS-short individuals are often from non-agricultural and remote localities, (e.g. Yoho NP and Cook Inlet Lowlands of Alaska and North West Territory) or from older herbarium specimens (e.g. a 1945 specimen from Fort Saskatchewan, AB). These ITS-short genotypes are almost uniformly from riparian and lacustrine habitats and never grassland. This genotype is currently unknown outside north-western North America. Of historical and previously studied samples, the late 19! century samples (1891), obtained from mid-western North America, Minnesota and proposed as native genotypes in that region by Noyszewski et al. (2021), had the ITS-long genotype in our study; a 1935 specimen from Pullman, Washington, proposed to be an early European introduction by Kettenring et al. (2019), also had the ITS-long genotype; and a modern (2010) specimen from remote northern BC (Kitimat), interpreted as native by Jakubowski et al. (2014), but of mixed heritage by Kettenring et al. (2019), also had ITS- 12 Percy D, Cronk Q long in our study. In summary therefore, across North America, the ITS-long genotype may be present in both native and introduced RCG, whereas the ITS-short genotype appears to be a localised variant in the Pacific northwest. The existence of distinctive North American genotype(s) (e.g. Noyszewski et al. (2021)) suggests that RCG was widespread in North America prior to the massive seeding of introduced agronomic genotypes in forage and revegetation seed mixes (Merigliano and Lesica 1998). However, there is still much uncertainty and, in some cases, disagreement, regarding the extent of distribution and location of present-day native RCG populations in North America (Jakubowski et al. 2013). One potential use of our relatively easily scored genetic marker would be to establish representation and association of the different ribotypes in native populations. Preliminary observations of the growth forms of our sampled RCG suggests that specimens with the ITS-short ribotype tended to be smaller, less strongly rhizomatous and were not noted to be invasive. However, there is no morphologically reliable method of distinguishing native from invasive RCG (Kettenring et al. 2019). The main indicators are vigour of growth and rhizomatous spread, but the usefulness of these indicators is uncertain and subject to environmental variation. Presently, molecular markers will likely remain the primary means of making broad surveys of RCG, to determine the geographical and ecological patterns of native persistence and to identify cryptic invasions of RCG across North America and the potential signature of intraspecific hybrids. Acknowledgements We thank Karin Kettenring, Karen Mock, Jim Walton (Utah State University) and Adam Pidwerberski (Prince Albert National Park, SK) for material of RCG; Frank Lomer for material of Phalaris canariensis; Timothy Whitfeld (Collections Manager) and staff of the University of Minnesota Herbarium, Bell Museum (MIN) for loan of specimens; Spencer Goyette and Linda Jennings (UBC Herbarium) for assistance; Hanna Schoenberg and Pinette Robinson for assistance with work at Elk Island National Park (contract no: 085-5P426); Edward Sun (UBC) for assistance in the laboratory; and Neil Anderson (University of Minnesota) for advice. We are grateful for a permit (no: 21-362) to collect at Miquelon Lake issued by Alberta Environment and Sustainable Resource Development. Lastly, we are grateful for reviews by Jeffrey Saarela, Neil Anderson and one annonymous reviewer that helped improve the manuscript. Author contributions QCBC planned the research; DMP conducted the lab work; both authors collected and analysed the data and both authors wrote the manuscript. Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... 13 References ° Anderson N, Kavova T, Bastlova D, Curn V, Kubatova B, Edwards K, Janué V, Kvét J (2016) Phenotypic and genotypic variation in Czech forage, ornamental and wild populations of reed canarygrass. Crop Science 56 (5): 2421-2435. https://doi.org/ 10.2135/cropsci2015.11.0705 ° Ankenbrand M, Keller A, Wolf M, Schultz J, Forster F (2015) ITS2 Database V: Twice as much: Table 1. 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Journal of Molecular Evolution 29 (4): 294-301. https://doi.org/10.1007/ bf02103617 Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... 15 ° Zhang W, Tian W, Gao Z, Wang G, Zhao H (2020) Phylogenetic utility of rRNA ITS2 sequence-structure under functional constraint. International Journal of Molecular Sciences 21 (17). https://doi.org/10.3390/ijms21176395 Supplementary materials Suppl. material 1: Ten putative hybrids between ITS-long and ITS-short clades. [doi | Authors: Diana M. Percy, Quentin C. B. Cronk Data type: occurrences Brief description: Ten putative hybrids between ITS-long and ITS-short clades. Seven based on both sequence and assay data, one based on sequence data only (marked “) and two samples which appeared hybrid in sequence data, but ITS-short in sizing assay data (marked with *). Region abbreviations: AB Alberta, BC British Columbia. Download file (24.88 kb) Suppl. material 2: Herbarium specimens used as outgroups. EE Authors: Diana M. Percy, Quentin C. B. Cronk Data type: occurrences Brief description: Herbarium specimens used as outgroups. Download file (24.08 kb) Suppl. material 3: Specimen details for all 94 samples genotyped. EE Authors: Diana M. Percy, Quentin C. B. Cronk Data type: occurrences Brief description: Specimen details for all 94 samples genotyped (86 Phalaris arundinacea and eight outgroup taxa sampled). Download file (15.38 kb) Suppl. material 4: Map of Elk Island National Park with the locations of 38 genotyped samples marked. EI Authors: Diana M. Percy, Quentin C. B. Cronk Data type: occurrences Brief description: Map of Elk Island National Park with the locations of 38 genotyped samples marked. Red crosses show the locations of the “short” ribotypes (n = 4); blue crosses “long” (n = 29), and orange circles putative hybrids (n = 5). Download file (45.26 kb)