PhytoKeys 36- 1-26 (2014) A peer-reviewe d open-access journal 1] & doi: 10.3897/phytokeys.36.6784 46Ph y toKe y www.p hyto keys .com Launched to accelerate biodiversity research Crataegus Xninae-celottiae and C. Xcogswellii (Rosaceae, Maleae), two spontaneously formed intersectional nothospecies Knud Ib Christensen'!, Mehdi Zarrei*, Maria Kuzmina’, Nadia Talent*, Charlotte Lin*®, Timothy A. Dickinson*® I Assoc. Prof. M.Sc. Ph.D. Knud Ib Christensen (born 13 October 1955, deceased 16 January 2012), formerly at the Botanical Garden, Natural History Museum of Denmark, University of Copenhagen 2. The Centre for Applied Genomics (ITCAG), The Hospital for Sick Children, Peter Gilgan Centre for Research and Learning, Rm. 139715, 686 Bay St., Toronto, Ontario, M5G 0A4 3 Department of Botany, MRC-166 National Mu- seum of Natural History Smithsonian Institution Rm W106 Washington, DC 20013-7012 USA 4 Green Plant Herbarium (TRT), Department of Natural History, Royal Ontario Museum, 100 Queen’ Park, To- ronto, Ontario Canada M5S 2C6 5 School of Education, University of Stirling, Scotland UK FK9 4LA 6 Department of Ecology & Evolutionary Biology, University of Toronto, 25 Willcocks St., Toronto, Ontario, Canada M5S 3B2 Corresponding author: Timothy A. Dickinson (tim.dickinson@utoronto.ca) Academic editor: A. Sennikov | Received 10 December 2013 | Accepted 17 February 2014 | Published 9 April 2014 Citation: Christensen KI, Zarrei M, Kuzmina M, Talent N, Lin C, Dickinson TA (2014) Crataegus xninae-celottiae and C. xcogswellii (Rosaceae, Maleae), two spontaneously formed intersectional nothospecies. PhytoKeys 36: 1-26. doi: 10.3897/phytokeys.36.6784 Abstract Crataegus monogyna Jacq. is naturalized in North America, where it has hybridized with native diploid hawthorns at least twice. We provide names for the two nothospecies (as well as for the corresponding nothosections and nothoseries), referring to existing documentation in the literature for nothosp. nov. Crataegus xninae-celottiae K.I. Chr. & T.A. Dickinson (C. monogyna x C. punctata Jacq.). New data are provided to further document nothosp. nov. Crataegus xcogswellii K.1. Chr. & T.A. Dickinson (C. mo- nogyna x C. suksdorfii (Sarg.) Kruschke). In both cases, the striking differences in leaf shape between most New World hawthorns and Old World section Crataegus, and the intermediacy of the hybrids, account for the relative ease with which these hybrids can be recognized. Finally, new sequence data from ITS2 and chloroplast DNA barcoding loci confirm the genetic relationships between the two nothospecies and their respective parents. Copyright K.I. Christensen et al. 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 Knud Ib Christensen et al. / PhytoKeys 36: 1-26 (2014) Keywords North America, hawthorn, hybridization, diploid, leaf shape, ITS2, DNA barcodes Introduction Crataegus monogyna Jacq. is a widespread species of Crataegus sect. Crataegus that occurs in much of Europe, northern Africa and western Asia. Within the area of its natural distribution it hybridizes with several other species of sect. Crataegus, e.g., C. laevigata (Poir.) DC., C. rhipidophylla Gand., C. meyeri Pojark., C. pentagyna Waldst. & Kit. ex Willd., C. orientalis M. Bieb., and C. azarolus L., as well as C. nigra Waldst. & Kit. of sect. Sanguineae (Albarouki and Peterson 2007; Byatt 1975; Christensen 1983; Chris- tensen 1992a, b, 1994; Christensen and Zielinski 2008; Donmez 2004). In fact, Chris- tensen (1992) applied the term “compilospecies” to C. monogyna. This term, coined by Harlan and DeWet (1963), describes species that aggressively acquire genes from other species by introgressive hybridization, potentially explaining the “...great variability of C. monogyna and also its wide distribution” in the Old World (Christensen 1992a). Crataegus monogyna was introduced to the U.S.A. and Canada by the early European settlers (Billings 1862; Douglas 1914; Kirk 1819; Provancher 1863). It has often es- caped from cultivation and, e.g., in abandoned fields and woodlands with extensive hawthorn colonization, it may hybridize with native diploid species of Crataegus such as C. punctata Jacq. (sect. Coccineae Loudon; Phipps pers. comm.; Wells and Phipps 1989) and C. suksdorfii (Sarg.) Kruschke (sect. Douglasia Loudon; Dickinson et al. 2008; Love and Feigen 1978; Talent and Dickinson 2005). Because of the striking contrast in leaf shape between members of C. sect. Crataegus and most North American Crataegus species, these hybrids are currently the best-known examples of diploid-dip- loid hybridization in the North American Crataegus flora. We provide names for these two nothospecies (as well as for the corresponding nothosections and nothoseries), referring to existing documentation in the literature for Crataegus xninae-celottiae K.I. Chr. & T.A. Dickinson (C. monogyna x C. punctata Jacq.; Wells and Phipps 1989). We also document variation in leaf shape for the second hybrid, Crataegus xcogswellii KI. Chr. & T.A. Dickinson (Crataegus suksdorfii x C. monogyna), and provide new sequence data from ITS2 and chloroplast DNA barcoding loci that confirm the genetic relationships between the two nothospecies and their respective parents. Methods Sampling. Because the occurrence of C. monogyna and its hybrids is sporadic, most of our samples are non-random, and merely attempt to document the co-occurrence of the parental species and (or) their hybrids (Table 1). Only in the case of the hybrid swarm found at the Cogswell-Foster Preserve in Linn Co., Oregon (site OR1), have we used either the throw of a pair of dice or ignorant person sampling (Ward 1974) in or- Crataegus xninae-celottiae and C. xcogswellii (Rosaceae, Maleae)... 3 Table |. Sites in Canada and the United States at which collections of native and naturalized diploid (unless indicated otherwise) Crataegus were made as vouchers for morphological, chemical, and molecular (boldface) observations (Fig. 1-3; Tables 2-4). Sampled individuals are listed by their collector and col- lection number; principal collector is T. A. Dickinson (D) unless indicated otherwise, as follows: JC, J. Coughlan; CAR, Rebecca Dotterer; EH, E. Harris; EL, E. Y. Y. Lo; RML, R. M. Love; MP, M. A. Purich; Z, P. Zika. State/Province Location Taxon Individuals British Columbia Central Kootenay R.D., Robson, Broadwa- Rene ter Road, Broadwater Road S side Cee na Central Kootenay R.D., Winlaw, next to Winlaw general store (10 miles S of Slocan)| C. suksdorfii BY on bank of small creek (tributary of Slocan | Probably polyploid ae River). California Humboldt Co., Hwy 36, 6.8 air km W of CA11 Bridgeville C. monogyna JCoo1 CAR4 __|rinity Co., T37N R7W S17 lg CAR042 Polyploid? Siskiyou Co., flood plain of the Scott R., 2006-16, 2006-18, CAR5 N side of Fay Lane, between jct. Hwy 3 C. suksdorpfii 2006-19, 2006-22, and bridge CAR044 CAR7 {Siskiyou Co., T26N R11W S17 pee CAR048 Polyploid? CRRRO1 |Sonoma Co., Ragle Ranch, W of Sebas- Cunonbesit jCo03 topol Idaho Benewah Co., T44N R1W S8, Soldier soiladorbi ID10 Creek, W side of Hwy 3 just N of RR Pr ‘i a ‘“ fA loid D1608 crossing and St. Mary’s R. ar se in Montana MT1 Powell Co., Dry Creek, N side, edge of fae woogie D1614, D1619 meadow and gallery forest Ontario C. punctata MP71 TON2 ity of T ; ial Park, Etobicok NTON23 |City of Toronto, Centennial Park, Etobicoke C. xninae-celowtiae |MP24, MP73 Bruce Co., Eastnor Twp., Barrow Bay, E Dickinson & Nguyen ee side Hwy 9 at S.R. 15 we te BB4 Middlesex Co., Ilderton, SE corner Denfield EH52, MP56, MP61, Durham R.M., Bowmanville, floodplain of Side Road and IIderton Road (Hwy 16) PUTER 2003-79 City of Toronto, Ashbridges Bay Park Cshath MP35 C. monogyna MP82, MP83, MP98 2002-13, MP84, MP85, OINA Bowmanville Creek as a MP86 C. punctata MP81 ON4G Perth Co., E side Thames R. North Branch Cinta 2008-724 2 km S of Motherwell Knud Ib Christensen et al. / PhytoKeys 36: 1-26 (2014) State/Province Location Taxon Individuals Oregon EL74, EL78, EL80, C. monogyna EL83, OR1-5, OR1-8, (diploid) OR1-9, OR1-10, ORI- 11, OR1-12, OR1-16 Triploid RML C-2003-25 C. monogyna 99FW7-1, 99FW7-2, 99FW7-3, IDFW7-6, 99FW7-7, I9FW7-8, 99FW7-9, 2009-36, ORI Linn Co., Willamette Valley, Cogswell- EL68, EL71, EL73, Foster Preserve R Lane Co, City of Eugene al C. xcogswellii EL76, EL77, EL79, EL81, EL82, EL84, EL85, OR1-2, OR1-3, OR1-4, OR1-6, ORI- 7, OR1-13, OR1-14, ORI1-15, OR1-17, OR1- 18, OR1-19, OR1-20, RML8718 C. suksdorpii C. xcogswellii EL68, EL69, EL72, EL75, OR1-1, RML8709 RML C-2003-12, RML C-2003-13, RML9304 Forest, Upper Goose Creek Meadow Probably polyploid ORA Douglas Co., Upper Elk Meadow, 28 miles | C. suksdorfii RML8758, RML8767, SSE Cottage Grove Probably polyploid | RML8768 Columbia Co., Sauvie Island, Willow Park |C. monogyna EL108 Island, Willow Bar Islands beach, just N C. xcoeswellii 7.18482 OR11 eS eee of Columbia-Multnomah county line, on yanikeot Cokanbik Rice C. suksdorpii JC117, JC118, JC119 Jackson Co., Rogue River, Old Stage Rd. oes 80 m NE of Ro aa River Hwy/ as eae a Linn Co., Corvallis, KOA Campground, OR22 440 m from hwy 34 on Oakville Rd. SW. | C. suksdorfii JC060 specimen 150 m SE of camp entrance Skamania Co., Cascade Locks, 110 m N of OE? Cascade Locks Rd., on N side of Forest Ln. Sau oh Jeo Multnomah Co., Columbia River Gorge OR37 National Scenic Area, 1.5 km NE of Trout- | C. suksdorfii JC098, JC102 dale Columbia Co., Diblee Pt., Site 350 m N OR38 of Dike Rd., 1.8 km WNW of Lewis and | C. suksdorfii JC136 Clark Bridge Washington Clark Co. S of mouth of Lewis River, ca. pe 1.5 air miles NNW of Ridgefield Com Le Skamania Co., Gifford Pinchot National Csabsdorfi WA8 Forest, Zig Zag Lake, 9 mi NW of Wind Pr Be ie hyakeal Stat Brooks s.n. R robably polyploi Skamania Co., Gifford Pinchot National | C. suksdorfii RML8909 Crataegus xninae-celottiae and C. xcogswellii (Rosaceae, Maleae)... e) der to draw more unbiased samples, with the inevitable consequence that these samples reflect the greater frequency of the introduced species and its hybrids. To mitigate this, we have included individuals of mostly diploid C. suksdorfii from other sites in order to reflect the variation found in this taxon. Note that we distinguish the taxon referred to here as C. suksdorfii from the other western North American black-fruited hawthorn with 20 stamens per flower, C. gay- lussacia A. Heller. This is because these two taxa are allopatric (Coughlan 2012 and unpubl. data), and differ in morphology and cytotype. Crataegus gaylussacia has shorter petioles and thorns that are thicker at their base than is the case with diploid C. suks- dorfii (Dickinson unpubl. data). Molecular data are consistent with C. gaylussacia be- ing an autotriploid derivative of diploid C. suksdorfii (Zarrei et al. http://2012.botany- conference.org/engine/search/index.php?func=detail&aid=536 and unpubl. data; see also Lo et al. 2009). In contrast, the C. suksdorfii complex has been shown to comprise, in addition to diploids, allotriploids and allotetraploids (Zarrei et al. http://2012.bot- anyconference.org/engine/search/index.php?func=detail&caid=536 and unpubl. data). In order to increase our sample for molecular studies we have supplemented field collections of leaf tissue and herbarium vouchers with tissue removed from existing specimens in the ROM Green Plant Herbarium. Historical records of the distribu- tion of C. monogyna were collected from five herbaria across Canada (TRT, MTMG, MT, QFA and UBC). Online databases of Canadian and U.S. herbaria used included ACAD, the Invader Database System of the University of Montana (which contains information for five northwestern states: Idaho, Montana, Oregon, Washington, Wy- oming), OSC, and WTU. Distribution maps were prepared from specimen locality data using SimpleMappr (Shorthouse 2010). Names of Crataegus sections and series used here follow those published by VASCAN (Brouillet et al. 2010), and are accepted names sensu FNA Ed. Comm. in prep. Morphology. For this study we concentrated on capturing and analyzing leaf shape data, as described elsewhere (Dickinson et al. 2008). Many previous studies of hybridization involving C. monogyna (Bradshaw 1953; Byatt 1975; Love and Feigen 1978), and of leaf shape variation in Crataegus generally (e.g. El-Gazzar 1980; Phipps and O’Kennon 2007), have attempted to quantify leaf lobing by means of a ratio of two measurements, x and y, where x is the distance from the tip of a lobe (usually the most basal one) to the deepest point of the sinus between that lobe and the adjacent one above it, and y is a measure of leaf size, usually the parallel distance from the tip of the lobe to the midrib. This approach can be effective when comparisons involve only leaves that have some degree of lobing (e.g. studies of hybridization between C. monogyna and C. laevigata in Europe, or of the lobed leaves of many species belonging to North American C. sect. Coccineae, such as C. punctata). However, when lobing is absent altogether the necessary landmarks (lobe tip, deepest point of the sinus) are absent, and the distance x is undefined or is set to zero (Love and Feigen 1978). In this case, a better approach is to carry out multivariate analyses of additional measurements of leaves and other organs (Wells and Phipps 1989), or to quantify variation in the leaf outline as a whole. Elliptic Fourier coefficients obtained from digitized leaf outlines 6 Knud Ib Christensen et al. / PhytoKeys 36: 1-26 (2014) captured using MorphoSys (Meacham and Duncan 1991), or the Fourier amplitudes derived from them, provide a useful method for doing just this (Dickinson et al. 2008; McLellan and Endler 1998; Rohlf and Archie 1984). Leaf outline data were collected from two overlapping samples: (1) short shoot leaf spectra (Dickinson and Phipps 1984) collected from a random sample of individuals at the Cogswell-Foster Preserve (comprising one C. suksdorfii, seven C. monogyna, and 12 putative hybrids), and (2) leaves on herbarium specimens from the Cogswell-Foster Pre- serve and other locations in the Pacific Northwest. In the latter the attempt was made to sample the leaf shape variation seen in C. suksdorfii as widely as possible. In both cases, var- iation in the shape of the leaf blade (i.e. excluding the petiole) was summarized by means of 39 Fourier amplitudes, and displayed by means of principal components analysis. For each leaf outline we also obtained the area (A) and perimeter (P), so as to cal- culate the inverse of the dissection index described by Kincaid and Schneider (1978), ie. inv(D.L.) = 2(An)”/P, a parameter that has an upper bound of one for a perfect circle regardless of size, and approaches zero as the length of the perimeter increases with increased lobing of the outline (Dickinson 2003; Dorken and Barrett 2004). In addition to outline data we made linear measurements with which to index overall leaf shape: X, leaf blade length above the widest point; Y, leaf width; and Z, leaf blade length below the widest point (Marshall 1978). On some of the flowering specimens in our sample we collected additional data on stamen number, style length and style number (in fruiting specimens, equivalently, pyrene number), and stigma width, in order to compare these with data collected by others from the introduced species and C. punctata. After transformation to a common [0,1] range these data were also sum- marized using principal components analysis. Analyses of variance were carried out on selected measurements. All data analyses described above were carried out using the R environment for statistical computing (R Core Team 2013). Significance of individual principal component axes was evaluated using the broken-stick criterion (Frontier 1976) with the help of R function evp/ot (Borcard et al. 2011). Molecular methods. Four DNA barcodes (rbcL, matk, trnH-psbA, and ITS2; CBOL Plant Working Group 2009; Chase et al. 2007; Hollingsworth et al. 2011) were generated directly from genomic DNA for a worldwide sample of Crataegus (Dickinson et al. http://2011.botanyconference.org/engine/search/720.html; Zarrei et al. unpubl. data). The plastid origin of the markers was used to establish the maternal parentage of the hybrids. DNA was extracted and amplified from leaf tissue of indi- viduals representing the two hybrids and their parent species (Table 2) using Canadian Centre for DNA Barcoding (CCDB) protocols (Ivanova et al. 2011; Kuzmina and Ivanova 201 la, b). This sample overlapped partially with the cloned ITS2 one (below), and provided an additional two C. suksdorfii, 10 C. monogyna, and five C. punctata individuals, as well as one more of each of the two hybrids (Table 2). We also analyzed data from another project (Zarrei et al. http://2012.botany- conference.org/engine/search/index.php?func=detail&aid=536 and unpubl. data) in which ITS2 was cloned for a sample of individuals that included 14 C. suksdorfii, four C. monogyna, three C. punctata and two each of the two hybrids (Table 3). Meth- Crataegus xninae-celottiae and C. xcogswellii (Rosaceae, Maleae)... 7 Table 2. Results of Neighbor-joining clustering of sequence data for chloroplast DNA barcode loci. GenBank accession numbers indicate cluster afhliation (Cluster 1 or 2) for Crataegus species and their putative hybrids. Details of the BOLD data can be found at dx.doi.org/10.5883/DS-CRATMONO. See Table 1 for sites and collectors; eight-digit ROM Green Plant Herbarium (TRT) accession numbers identify vouchers. Cluster 1 — sections Coccineae ClaseBiic acca Chetacas and Douglasia Taxon /site /BOLD /tree / TRT rbcL-a trnH-psbA trnH-psbA Crataegus punctata NTON23 TRT103 MP71 TRT00002237 | KC251377 | KC251652 | ON31 TRT096 MP61 TRT00002228 KC251375 | KC251650 — ON31 TRT105 MP56 TRT00002223 KC251372_| KC251647 ON40 TRT101 MP35 TRT00002203 KC251374 | Kc251649 | ON45 TRT104 MP81 TRT000047 KC251378 | KC251653 ON46 TRT210 2008-72A TRT00000908 | KC251373_ | _KC251648 Crataegus xninae-celottiae NTON23 TRT106 MP24 TRT00002199 KC251651 NTON23 TRT203 MP73 TRT00002239 | KC251350 | KC251624 esa ON45 TRT201 MP85 TRT00002250 KC251348_ | KC251622 ON45 TRT202 MP86 TRT00002251 KC251625 ON45 TRT 204 MP84 TRT00002249 KC251349_| KC251623 Crataegus monogyna BC16 TRT209 2008-26 TRT00002452 KC251343_ | KC251617 CA11 TRT274 JC001 TRT00020101 KC251612 CRRRO1 TRT275 JC003 TRT00020102 KC251341 | KC251615 ON31 TRT109 2003-79 TRT00000395 KC251340_ | KC251614 ON45 TRT108 MP82 KC251342_ | KC251616 ON45 TRT190 MP83 TRT00002248 KC251613 ON45 TRT211 MP98 TRT00029476 KC251336 | KC251610 OR1 TRT005 EL80 TRT00000413 KC251347_ | KC251621 OR1 TRT006 EL83 TRT00000415 KC251620 OR1 TRT007 EL74 TRT00000416 KC251344 | KC251618 ened ee Sena? KC251337 | KC251611 OR11 TRT143 EL108 TRT00000417 KC251345 | KC251619 Crataegus xcogswellii OR1 TRT206 EL71 TRT00002650 KC251627 OR1 TRT207 EL85 TRT00002654 KC251626 OR1 TRT208 EL79 TRT00002657 KC251352 Crataegus suksdorfii CARS TRT129 2006-19 TRT00001569 | KC251419 | KC251692 | CARS TRT133 2006-22 TRT00001563__ | KC251418 | KC251691 CARS TRT140 2006-16 TRT00001567 | KC251417 | KC251690 —_ CARS TRT141 2006-18 TRT00001568 | KC251416 | KC251689 ORI TRT205 EL68 TRT00001724 KC251424 | KC251699 | WA TRT146 218485 TRT00001805 KC251415 | KC251688 | 8 Knud Ib Christensen et al. / PhytoKeys 36: 1-26 (2014) Table 3. Voucher specimens for cloned ITS2 data, listing site number (Table 1), collection number, ROM Green Plant Herbarium (TRT) accession numbers, and the GenBank accession numbers for indi- vidual clones. Taxa Voucher GenBank accession number OR18 Coughlan, Zarrei, and Shaw JCO39 | KC173887, KC173888, KC173889, KC173890, (TRT00020137) KC173891, KC173892, KC173893 OR22 Coughlan, Zarrei, and Shaw JC60 KC173587, KC173588, KC173589, KC173590, (TRT00020146) KC173591, KC173592 OR35 Coughlan, Zarrei, and Shaw JCO92 | KC173957, KC173958, KC173959, KC173960, (TRT00020153) KC173961, KC173962, KC173963, KC173964 KC173595, KC173596, KC173597, KC173598, KC173599, KC173600, KC173601, KC173602, KC173603, KC173604 OR37 Coughlan, Zarrei, and Shaw JC102 | KC174113, KC174114, KC174115, KC174116, OR37 Coughlan, Zarrei, and Shaw JC98 (TRT00020159) (TRT00020163) KC174117 OR11 Coughlan, Zarrei, and Shaw JC117 (TRT00020172) KC174118, KC174119 OR11 Coughlan, Zarrei, and Shaw JC118 —|KC174178, KC174179, KC174180, KC174181, C: sikeclerfi (TRT00020232) KC174182, KC174183 OR11 Coughlan, Zarrei, and Shaw JC119 |KC174144, KC174145, KC174146, KC174147, (TRT00020234) KC174148, KC174149, KC174150 OR38 Coughlan, Zarrei, and Shaw JC136 | KC173605, KC173606, KC173607, KC173608, (TRT00020242) KC173609 CARS Dickinson and Lo 2006-16 KC173531, KC173532, KC173533, KC173534, (TRT00001567) KC173535, KC173536, KC173537, KC173538 KC173522, KC173523, KC173524, KC173525, KG173526, KG173527,-KC1 73528, KCT73529, KC173530 KG173577,-KC173578, KE173579, KE173580; KC173581, KC173582, KC173583, KC173584, KC173585, KC173586 KC173513, KC173514, KC173515, KC173516, KC173517, KC173518, KC173519, KC173520, CARS Lo and Dickinson 2006-22 (TRT00001563) ORI Lo, Dickinson, and Nguyen EL-68 (TRT00001724) WA Zika 18485 (=18430, 18417; TRT00001805) KC173521 ORI Lo, Dickinson, and Nguyen EL-71 KC173663, KC173664, KC173665, KC173666, (TRT00002650) KC173667, KC173668 ORI Lo, Dickinson, and Nguyen EL-79 KC173682, KC173683, KC173684, KC173685, (TRT00002657) KC173686, KC173687 C. xcogswellii KC173669, KC173670, KC173671, KC173672, ORI Lo, Dickinson, and Nguyen EL-85 KC173673, KC173674, KC173675, KC173676, (TRT00002654) KC173677, KC173678, KC173679, KC173680, KC173681 ORI Lo, Dickinson, and Nguyen EL-74 KC173650, KC173651, KC173652, KC173653, (TRT00000416) KC173654 Gunppunie BC16 Dickinson, Lee, and Talent 2008-26 |KC173655, KC173656, KC173657, KC173658, (TRT00002452) KC173659, KC173660, KC173661, KC173662 KC173643, KC173644, KC173645, KC173646, KC173647, KC173648, KC173649 ON45 Purich and Talent MP84 KC174184, KC174185, KC174186, KC174187, (TRT 00002249) KC174188, KC174189 ON45 Purich MP98 (TRT00029476) C. xninae-celottiae Crataegus xninae-celottiae and C. xcogswellii (Rosaceae, Maleae)... 9 Taxa Voucher GenBank accession number ON45 Purich and Talent MP85 KC174190, KC174191, KC174192, KC174193, (TRT00002250) KC174194, KC174195 ON45 Purich and Talent MP86 KC173688, KC173689, KC173690, KC173691, (TRT00002251) KC173692, KC173693 KC174266, KC174267, KC174268, KC174269, 21 Dicki BB4 (TRT ON ickinson and Nguyen (TRT) KC174270, KC174271 C. punctata ON31 Purich s.n (TRT) KC174272, KC174273, KC174274, KC174275 NTON23 Purich, Talent, Nguyen, and Lo —|KC173694, KC173695, KC173696, KC173697, MP73 (TRT00002239) KC173698, KC173699, KC173700, KC173701 ods for extracting total genomic DNA, marker amplification, cloning, DNA sequenc- ing, and collapsing original sequences to unique sequences (ribotypes) are described elsewhere (Zarrei et al. http://2012.botanyconference.org/engine/search/index. php?func=detail&aid=536 and unpubl. data). Here we report on analyses of a total of 160 ribotypes (Table 3). A recombination test was performed using RDP4 Beta 4.14 (Martin et al. 2010). The Neighbor-Net analysis (Bryant and Moulton 2004) was un- dertaken using SplitsTree v.4.12.3 (Huson and Bryant 2006) to visualize incompatible splits in the network from uncorrected p-distances calculated with MEGA5 (Tamura et al. 2011). Bootstrap support (BS) was estimated using 1,000 bootstrap pseudorepli- cates (Felsenstein 1985) implemented in SplitsTree. Flow cytometry. Flow-cytometric methods for quantifying nuclear DNA in em- bryo and endosperm followed Talent and Dickinson (2007a). Embryo DNA amounts of 1.48—1.70 pg were taken to indicate diploids, and an endosperm to embryo ratio of approximately 1.5 was taken to indicate sexual reproduction with meiosis. Results and discussion Morphology. Despite differences in sample size, the Pacific Northwest hybrid, Cratae- gus xcogswellii, appears more variable than either of its putative parents, C. monogyna or C. suksdorfii (Fig. 1). The hybrid is clearly intermediate with respect to both leaf lobing (the inverse Dissection Index; Fig. 1) and style number (STYLE; Fig. 1). Prin- cipal components analyses of leaf outlines from Pacific Northwest C. monogyna, C. suksdorfii, and their putative hybrid, demonstrate variation in leaf shape both within and between these three entities (Fig. 2A, B). The first principal component reflects the contrast between the unlobed leaves of C. suksdorfii and the markedly lobed ones of C. monogyna, as well as the intermediacy of the hybrid (Fig. 2A, B), much as illustrated earlier by Love and Feigen (1978; their Fig. 3), and by Wells and Phipps (1989) for the Ontario hybrid and its parents (their Fig. 4). The second principal component reflects variation in the relative overall lengths and widths of the leaf outlines (Fig. 2A). DNA barcode loci. Analyses of both the directly sequenced and the cloned ITS2 ribotypes demonstrate the parentage of both putative hybrids (Fig. 3; Table 10 Knud Ib Christensen et al. / PhytoKeys 36: 1-26 (2014) af) ron) ~~ 3S >) oO a N Oo oo oO o af) a -0.5 0.0 0.5 PC1 (53%) Figure |. Principal components analysis biplot for five morphometric descriptors averaged for each of 41 Crataegus herbarium specimens from the Cogswell-Foster Preserve and other locations in the Pacific Northwest (C. suksdorfii (s), C. monogyna (m), and the putative hybrid, C. xcogswellii (h)): relX, leaf length above the widest point, scaled by the width; re/Z, leaf length below the widest point, scaled by the width; invDI, inverse dissection index = 2(An)'"/P, where A is the leaf area and P is the leaf perimeter; STAM, number of stamens per flower; STYL, number of styles per flower. Both axes shown account for significant portions of the total variance according to the broken-stick criterion (Frontier 1976). 3); no signs of recombination were detected in the cloned ITS2 dataset. ITS2 se- quences from the hybrids resemble either C. monogyna or one of the native North American species. The way in which both parental ribotypes are maintained in each of the hybrids examined here is probably due to how recently the hybrids have been formed: less than 200 years ago in the case of the Ontario hybrids (Douglas 1914; Kirk 1819; Provancher 1863), and less than 100 years ago in the case of the Oregon ones (the earliest specimen of C. monogyna was collected in 1914 in Douglas Co. Oregon; Phipps 1998). These time periods are evidently too short for genome ho- mogenization (concerted evolution) to have taken place, even in diploids reproduc- ing sexually. Our small sample of seed from the hybrids (Table 4) parallels earlier Crataegus xninae-celottiae and C. xcogswellii (Rosaceae, Maleae)... 11 0.1 0.0 PC2 (18%) PC2 (13%) -0.1 -0.2 -0.3 PC1 (57%) PC1 (72%) Figure 2. A Principal components analysis of 39 Fourier amplitudes for 86 subterminal short shoot leaves from 20 Crataegus individuals at the Cogswell-Foster Preserve in Linn Co., Oregon (one C. suks- dorfii (s), seven C. monogyna (m), and 12 putative hybrids (h), C. xcogswellii). Leaf outlines illustrate the shape contrasts responsible for the ordination: in grey, six subterminal leaves from short shoots of a single individual (OR1-8) B Principal components analysis of 39 Fourier amplitudes averaged for leaves sam- pled regardless of position on short shoots of 64 herbarium specimens from the Cogswell-Foster Preserve and (circled points) other locations in the Pacific Northwest (Table 1). In both A and B the two PCA axes shown are significant according to the broken-stick criterion (Frontier 1976). In B arrowed point 1 repre- sents the single individual of C. suksdorfii for which individual leaves are represented in A, while arrowed point 2 represents the averaged data for the six leaves of C. monogyna shown in grey in A. results (Talent and Dickinson 2007a) showing diploidy and sexual reproduction in both parental taxa. Only two of the three chloroplast genome barcode loci showed sufficient variation for individuals from Crataegus section Crataegus to be distinguished from ones belong- ing to either C. section Coccineae or C. section Douglasia (Table 2). Sequence data from both rbcL-a and the trnH-psbA spacer region showed the same two clusters, C. sections Coccineae and Douglasia (Cluster 1), and C. sect. Crataegus (Cluster 2; Table 2). The way in which the hybrids fell into one of these clusters or the other demonstrates that, with one exception, C. monogyna is the female parent of the Ontario hybrids with C. punctata studied here, while C. suksdorfii is the female parent of the Pacific Northwest hybrids. These results corroborate earlier observations based on seed-set in artificial crosses between the parent species (Love and Feigen 1978; Wells and Phipps 1989). In recip- rocal pollinations seed set was greatest (32-34%) when C. monogyna stigmas received pollen from C. punctata (Wells and Phipps 1989). Fruit set was most successful when C. monogyna pollen was applied to the stigmas of C. suksdorfii flowers (mean 42%, range 25-73%, compared to a 29% mean fruit set by C. suksdorfii with open pollina- tion; Love and Feigen 1978). However, all reciprocal crosses between C. monogyna, C. suksdorfii, and their hybrid yielded seeds (R. M. Love, personal communication). 12 Knud Ib Christensen et al. / PhytoKeys 36: 1-26 (2014) TRT129 Crataegus suksdorfi 2006-19 KC861885 TRT140 Crataegus suksdorfii 2006-16 KC861886 RT141 Crataegus suksdorfii 2006-18 KC861887 TRT203 Crataegus xninae-celottiae MP73 KC861842 TRT104 Crataegus punctata MP81 KC861864 TRT105 Crataegus punctata MP56 KC861861 TRT101 Crataegus punctata MP35 KC861865 TRT103 Crataegus punctata MP71 KC861863 TRTO96 Crataegus punctata MP61 KC861862 TRT210 Crataegus punctata 2008-72A KC861867" TRT207 Crataegus xcogswellii EL85 KC861846 : TRT006 Crataegus monogyna EL83 KC861837 1 TRT106 Crataegus Xninae-celottiae MP24 KC861866 ' TRT190 Crataegus monogyna MP83 KC861836 1 TRT208 Crataegus xcogswellii EL79 KC861847 : RT109 Crataegus monogyna 2003-79 KC861839 1 RT211 Crataegus monogyna MP98 KC861841 ; section TRT209 Crataegus monogyna 2008-26 KC861840 , Crataegus ; | | | | section Douglasia | LT | ' . 1 section " Coccineae i | i RT108 Crataegus monogyna MP82 KC861838 RT275 Crataegus monogyna JCO003 (only 204 bp) TRT201 Crataegus xninae-celottiae MP85 KC861843 RT274 Crataegus monogyna JCO01 KC861835 TRT204 Crataegus xninae-celottiae MP84 KC861845! RT202 Crataegus xninae-celottiae MP86 KC8618441 B a b Crataegus monogyna Crataegus suksdorfii Crataegus xcogswellii _ Crataegus xcogswellii Crataegus xninae-celottiae C Crataegus punctata Crataegus *ninae-celottiae 0.01 Figure 3. A Neighbor-joining tree calculated by BOLD for ITS2 DNA barcode sequences amplified directly from genomic DNA (labels include corresponding collector and GenBank number; see dx.doi. org/10.5883/DS-CRATMONO and Table 1 for details). Dashed lines indicate the sectional affinity of the sequences B The corresponding Neighbor-Net network for the cloned ITS2 sequences has three branches representing: (a) ribotypes from individuals of C: monogyna, and from its hybrids with both C. suksdorfii and C: punctata; (b) ribotypes from individuals of C. suksdorfii and C. xcogswellii; and (c) ribotypes from individuals of C: punctata and C x ninae-celottiae (Table 3). The numbers shown are the % bootstrap sup- port for each of the three branches. Crataegus xninae-celottiae and C. xcogswellii (Rosaceae, Maleae)... 13 i> | od ae heap % Crataegus monogyna (TRT, QFA, MT MTMG,UBC) ,, Hi Crataegus xninae-celottiae Holotype a X C. xninae-celottiae (TRT) % C. xninae-celottiae (Wells & Phipps 1989) y\\ ° | 04 : at re mere (0-25 50-75 km Figure 4. Geographic distribution of Crataegus xninae-celottiae K.I. Chr. & T.A. Dickinson nothosp. nov. and C. monogyna in Ontario. Filled square, holotype of Crataegus xninae-celottiae,; Crosses, TRT specimens of C. xninae-celottiae; asterisks, C. xninae-celottiae specimens cited by Wells and Phipps (1989); stars, specimens of C. monogyna in MT, MTMG, QFA, TRT, and UBC. Crataegus punctata occurs throughout the region depicted (Phipps and Muniyamma 1980; this paper also maps additional records for C. monogyna). Table 4. Flow-cytometric results from seeds of the two described Crataegus nothospecies. The ratios shown for endosperm and embryo nuclear DNA contents are well within the ranges observed for sexually reproducing C. monogyna (Talent unpubl. data) and diploid C. suksdorfii (Lo et al. 2013). 54 ee : Total number | Mean embryo | Mean endosperm:embryo Taxon/TRT accession/site/collection Aa aie Ganer et aed) Crataegus xninae-celottiae ON45 2002-13 (TRT00000406) 2 1.56 (2) ON31 EH52 (TRT00002256) 1 1.53 (1) Crataegus xcogswellii OR1 EL-79 (TRT00002657) 3 2.08 pg 1.58 (1) OR1 2009-36 (TRT00002568) 1 1.87 pg 1.60 (1) Our use of data from DNA barcoding is not a test of the value of DNA barcoding in Crataegus, as this is discussed elsewhere (Dickinson et al. http://2011.botanyconfer- ence.org/engine/search/720.html; Zarrei et al. unpubl. data). Rather, we have taken advantage of our barcode sequence data from individuals unequivocally identifiable as C. monogyna, C. punctata, C. suksdorfii and their hybrids in order to use sequence similarity to inform us about the hybridization process. Hybridization. Since its introduction to North America during the late 18" and the 19* centuries (Kirk 1819; Provancher 1863; Douglas 1914), first on the east coast and 14 Knud Ib Christensen et al. / PhytoKeys 36: 1-26 (2014) -120 : L paling Y \ f io oe Ww A wd \ vx Crataegus monogyna (TRT, OSC, UBC, WTU) cca y~ \ X Crataegus xcogswellii f HEC. xcogswellii Holotype 80%). Pollen stainability in the hybrid was found to be variable (27-97%, mostly in the range 60-80%; Purich 2005). Specimens examined, paratypes (in bold, specimens in Tables 2-4). CANADA, Ontario: Peel Co., City of Mississauga, Don Gould Park and E side of Erin Mills Park- way (ON22), 1989-06-02, Dickinson D1480 (TRT00000408!); 1989-06-02, Dick- inson D1482 (TRT00000407!); 1989-05-31, Dickinson D1485 (TRT00000409)); 2000-05-19, Talent NT-03 (TRT00002306!); 2011-05-28, Christensen & Dick- inson s.n. (TRT00024869!). Middlesex Co., Denfield Twp., SE corner Denfield Side Road and Ilderton Road (ON31), 2001-05-17, Harris & Dickinson EH-52 (TRT00002256!); 2001-05-17, Harris & Dickinson EH-54 (TRT00002257!); 2002-07-30, Talent & Dickinson EH52 (TRT00000405!). Durham R.M., Bowman- ville, floodplain of Bowmanville Creek (ON45), 2002-09-30, Dickinson & Nguyen 2002-13 (TRT00000406!), 2004-06-03, Purich 85 (TRT00002250!), 2004-06-03, Purich 86 (TRT00002251!). Crataegus nothosect. Crataeglasia K.I. Chr. & T.A. Dickinson nothosect. nov. (Crataegus sect. Crataegus x sect. Douglasia) Crataegus nothoser. Crataeglasianae K.1. Chr. & T.A. Dickinson nothoser. nov. (C7a- taegus ser. Crataegus x ser. Douglasianae) Crataegus xcogswellii K.I. Chr. & T.A. Dickinson nothosp. nov. (Fig. 7.). — Type: U.S.A., Oregon, Linn Co., Cogswell-Foster Preserve, 44°19.985'N, 123°7.353'W, 3 Sep 2009, Dickinson & Dickinson 2009-40 (holotype TRT00002574!; isotype TRT). (Q Crataegus suksdorfii x SC. monogyna) Ramunculi glabri vel rarius sparsim villoso-lanati. Folia distalia ramorum fertilium quinque-novem-partita, rarius integra, 25-70 mm longa, 15—50 mm lata; stipulae cadu- cae, 4-8 mm longae, plusminusve denticulatae. Inflorescentiae 4-25-florae, laxae, glabrae vel rarius villoso-lanatae; bracteae caducae, plusminusve denticulatae. Sepala integra vel rarius sparsim glandulosa, post anthesin reflexa. Fructus 9-12 mm longus, 12-14 mm latus, lampro-atro-purpureus vel anthracinus; pulpa lutea, mitis et succida; pyrenae 2—5, ventraliter sulcatae vel foveatae. Remarks. Shrub or tree up to ca. 12 m tall. Twigs of the current year glabrous, rarely sparsely villous-lanate; twigs of the previous year dark reddish-brown or pale- or dark- grey; aphyllous thorns 0.5—2 cm long, stout, straight or slightly recurved; spine-tipped, leaf- and dwarf-shoot-bearing branchlets lacking, rarely present. Leaf blades broadly or narrowly obovate, ovate, rhombic-ovate or elliptical, acute at apex, attenuate, cuneate or rounded at base, deeply or shallowly and regularly lobed, rarely some leaves unlobed, lobes with an acute or obtuse apex, basal pair of veins divergent or straight, intercalary 20 Knud Ib Christensen et al. / PhytoKeys 36: 1-26 (2014) veins running to the sinuses usually present; upper surface dull, dark green, sparsely hairy especially along the veins, hairs appressed or semi-patent; lower surface dull, pale green, villous in the vein axils and occasionally along the major veins; margin regularly and + coarsely or finely crenate-serrate or serrate, teeth eglandular or minutely glandular, glands less than 0.1 mm; petiole eglandular or rarely sparsely glandular, narrowly winged in up- per part. Subterminal leaf blade of flowering shoots 25—70 mm long, 15—50 mm wide, deeply or shallowly and regularly lobed, rarely unlobed, lobes (0O—)2—4 pairs, basal pair extending 0.2—0.8 times the width of lamina to midrib, each lobe with 5-18 teeth, ba- sal pair of sinuses in apical 1/3 to basal 1/3 of lamina; petiole 5—15 mm long; stipules persistent or caducous, herbaceous, 5-12 mm long, irregularly or regularly glandular denticulate-serrate or serrate, with 4—30 teeth. Leaf blades of elongate shoots 40-90 mm long, 30-50 mm wide, deeply or shallowly and regularly lobed, lobes 1-4 pairs, basal pair extending 0.4—0.9 times the width of lamina to midrib, each lobe with 7—20 teeth, basal pair of sinuses in basal 1/2—1/5 of lamina; petiole 10—20 mm; stipules persistent or cadu- cous, herbaceous, 6-14 mm long, regularly glandular denticulate-serrate or serrate, with 15-30 teeth. Inflorescence 2.5—5 cm long, lax, corymbose, 4—25-flowered, glabrous, rare- ly sparsely villous-lanate; pedicels 4-11 mm, glabrous, rarely sparsely villous-lanate; bracts caducous or very rarely persistent, membranous or herbaceous, 3-10 mm long, 0.2—2.5 mm wide, linear-lanceolate, 4—10 times as long as wide, regularly glandular-serrate or + ir- regularly glandular-denticulate, with 4—22 teeth. Hypanthium 2—3 mm long, glabrous or rarely sparsely villous-lanate; sepals 1-2.5 mm long, 1.5—2 mm wide, triangular, 0.5—1.7 times as long as wide, entire or very rarely irregularly and minutely glandular-serrate, teeth 0-2, apex acute or obtuse; petals 4-6 mm long and wide; stamens 18-20, occasionally vestigial, anthers 0.6—1 mm long, purple; styles 2-5; hypostyle pilose. Fruit 6-9 mm long, 6-8 mm in diameter, 1—1.2 times as long as wide, globose-subglobose or broadly ellipsoi- dal, epruinose, + lustrous, blackish purple or black, glabrous-subglabrous, crowned by the persistent, reflexed sepals; calyx tube indistinct, 0.4—1 mm long, 3.5—-4.5 mm wide; flesh yellowish, soft and juicy; pyrenes 2—5, irregularly ventro-laterally pitted; hypostyle pilose. Phenology. Flowering in April-May. Fruiting in September. Some individuals strongly parthenocarpic. Reproductive biology. Sexual. 27 = 2x [= 34] (Talent & Dickinson 2005); dip- loid embryos and triploid endosperm. Chromosome number: 27 = 2x = 34, estimated from flow cytometry data (Table 4); chromosome counts have not been made. Distribution. Northwestern U.S.A.; western Oregon (Figure 5); potentially pre- sent in adjacent northwestern California and southwestern Washington where the par- ent species are sympatric. Etymology. Crataegus xcogswellii honours the Cogswell family, and Mr. and Mrs. Lee Foster, of Halsey, Oregon. In 1872 John Cogswell, Mrs. Foster’s grandfa- ther, purchased the land that the Fosters gave to the Oregon Nature Conservancy as the Cogswell-Foster Preserve (Lopez 1971), and at which C. xcogswellii has been most intensively studied (Love and Feigen 1978). Similar taxa. Crataegus xcogswellii differs from C. monogyna in: leaf- and dwarf- shoot-bearing branchlets usually lacking; stipules of leaves of flowering shoots irregu- Crataegus xninae-celottiae and C. xcogswellii (Rosaceae, Maleae)... 21 09 OS Ob Of 4 of 02 OF AA 06 08 OZ Rosaceae Fam.126 Crataegus monogyna Jacg. « C. suksdorfii (Sarg.) Kruschke USA, Oregon, Linn Co, Lat: 44.333082 °N Long: 123.122547 °W Location: Cogswell-Foster Reserve Coll.: Dickinson, T. 2009-40, with Dickinson, J.S., 2009/09/03 Det.: Dickinson, T., 2009/09/03 Repl.: 2 Notes: Single stemmed, ca.7m tall he | Green Plant Herbarium (TRT) - Royal Ontario Museum The ROM Green Plant Herbarium (TRT) ROM Green Plant Herbarium: NTH rect em Crean Figure 7. Holotype of Crataegus xcogswellii K.1. Chr. & T.A. Dickinson nothosp. nov. (2 Crataegus suksdorfii x AG monogyna): TRT00002574, U.S.A., Oregon, Linn Co., Cogswell-Foster Preserve, 44,333082°N 123.122547°W, 3 Sep 2009, Dickinson & Dickinson 2009-40. 22 Knud Ib Christensen et al. / PhytoKeys 36: 1-26 (2014) larly or regularly glandular denticulate-serrate or serrate (not + entire); styles and pyr- enes 2—5 (not 1—(2)); fruit blackish purple or black (not bright or dark red). Crataegus xcogswellii differs from C. suksdorfii in: twigs of the current year occa- sionally sparsely villous-lanate; leaf- and dwarf-shoot-bearing branchlets occasionally present; leaf blades usually deeply or shallowly and regularly lobed, intercalary veins running to the sinuses usually present; inflorescence, pedicels and hypanthia occasion- ally sparsely villous-lanate; hypostyle pilose (not glabrous or sparsely pilose). Specimens examined, paratypes (in bold, specimens in Tables 2—4). U.S.A., Ore- gon: Columbia Co., Sauvie Island (OR11), 2003-06-14, Zika 18482 (TRT00002651!); 2005-08-31, Lo & Dickinson 103.2 (TRT00001918!), Lo 105.2 (TRT00001917!); Lane Co. Eugene, 1993-05-07, Love 9304 (TRT00002644!), 2003-05-13, Love C2003-12 (TRT00002646!), C2003-13 (TRT00002647!); 2003-06-01, Zika 19571 (TRT00001890!); Linn Co., Cogswell-Foster Preserve (OR1), 1987-04-7, 1987-04- 27, 1987-09-20, Love 8707 (TRT00001895!, TRT00001907!, TRT00001912!), 8714 (TRT00001897!, TRT00001899!, TRT00002643!), 8715 (TRT00001901!, TRT00001902!, TRT00001910!), 8716 (TRT00001894!, TRT00001913!), 8717 (TRT00001900!, TRT00001909!), 8718 (TRT00002645!), 8719 (TRT00001893!, TRT00001905!, TRT00001906!), 8720 (TRT00001904!), 1993-05-18, Barbour, Ev- ans & Love 93064 (TRT00001896!), 1997-07-27, Love 9726 (TRT00002196!); 2004- 06-10, Lo, Dickinson & Nguyen 71 (TRT00002650!), 73 (TRT00002660!), 76 (TRT00002658!), 77 (TRT00002659!), 79 (TRT00002657!), 81 (TRT00002655!), 82 (TRT00002656!), 84 (TRT00002653!), 85 (TRT00002654!); 2009-09-03, Dick- inson & Dickinson 2009-22 (TRT00002555!), 2009-23 (TRT00002556!), 2009-24 (TRT00002557!), 2009-28 (TRT00002560!), 2009-33 (TRT00002565!), 2009-34 (TRT00002566!), 2009-36 (TRT00002568!), 2009-38 (TRT00002570!), 2009-39 (TRT00002571!), 2009-41 (TRT00002573!), 2009-42 (TRT00002572!), 2009-43 (TRT00002575!); Marion Co., Salem, 2003-05-01, Zika 18296 (TRT00001889!). Washington: Clark Co., 2003-06-01, Zika 18431 (TRT00001891!). Acknowledgements Dr Peter Wagner, Copenhagen, kindly checked the Latin diagnoses. Diane Celotti gave us biographical information about her daughter, Nina. Rhoda M. Love introduced TAD to Crataegus suksdorfii and its hybrids at the Cogswell-Foster Preserve, and pro- vided unpublished information from her research on these plants; she also arranged with The Nature Conservancy for our access to the Cogswell-Foster Preserve. Ed Alver- son provided bibliographic information for the Lopez article. We obtained the Billings reference thanks to Mike Palmer and the adventive species website (FloraS of North America, http://botany.okstate.edu/floras/). Dale Leadbeater told TAD about the Bowmanville site. Sasa Stefanovi¢ is our collaborator on the Crataegus ITS2 project. Jenn Coughlan, John Dickinson, Rebecca Dotterer, Eric Harris, Eugenia Lo, Rhoda Love, Sophie Nguyen, Melissa Purich, Peter Zika assisted our fieldwork or provided Crataegus xninae-celottiae and C. xcogswellii (Rosaceae, Maleae)... 23 their specimens. Jen Byun and Kathleen Buck helped us collect data from herbarium material. Tara Winterhalt adjusted the contrast in Fig. 6 and Fig. 7. We are grateful to the herbaria mentioned in the figures for lending specimens or making specimen data available to us. Financial support to KIC from the Carlsberg Foundation (Grant 2008_01_0155) and to TAD from the Natural Sciences and Engineering Research Council of Canada (Discovery Grant A3430), the Royal Ontario Museum, the De- partment of Ecology & Evolutionary Biology (formerly the Botany Department) of the University of Toronto, and the Canada Foundation for Innovation and Ontario Research Fund (funding through Canadensys for equipment and personnel for speci- men documentation) are gratefully acknowledged. DNA barcoding was funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (2008-OGI-ICI-03). References Albarouki E, Peterson A (2007) Molecular and morphological characterization of Crataegus L. species (Rosaceae) in southern Syria. Botanical Journal of the Linnean Society 153: 255-263. doi: 10.1111/j.1095-8339.2007.00607.x Billings BJ (1862) List of plants observed growing principally within four miles of Prescott, C. W., and for the most part in 1860. 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