Crataegus ×ninae-celottiae and C. ×cogswellii (Rosaceae, Maleae), two spontaneously formed intersectional nothospecies

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 ×ninae-celottiae K.I. Chr. & T.A. Dickinson (C. monogyna × C. punctata Jacq.). New data are provided to further document nothosp. nov. Crataegus ×cogswellii K.I. Chr. & T.A. Dickinson (C. monogyna × 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.


Introduction
. 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 collection number; principal collector is T. A. Dickinson (D)  der to draw more unbiased samples, with the inevitable consequence that these samples refl ect the greater frequency of the introduced species and its hybrids. To mitigate this, we have included individuals of mostly diploid C. suksdorfi i from other sites in order to refl ect the variation found in this taxon. Note that we distinguish the taxon referred to here as C. suksdorfi i from the other western North American black-fruited hawthorn with 20 stamens per fl ower, C. gaylussacia A. Heller. Th is is because these two taxa are allopatric (Coughlan 2012 and unpubl. data), and diff er in morphology and cytotype. Crataegus gaylussacia has shorter petioles and thorns that are thicker at their base than is the case with diploid C. suksdorfi i (Dickinson unpubl. data). Molecular data are consistent with C. gaylussacia being an autotriploid derivative of diploid C. suksdorfi i (Zarrei et al. http://2012.botanyconference.org/engine/search/index.php?func=detail&aid=536 and unpubl. data; see also Lo et al. 2009). In contrast, the C. suksdorfi i complex has been shown to comprise, in addition to diploids, allotriploids and allotetraploids (Zarrei et al. http://2012.botanyconference.org/engine/search/index.php?func=detail&aid=536 and unpubl. data).
In order to increase our sample for molecular studies we have supplemented fi eld collections of leaf tissue and herbarium vouchers with tissue removed from existing specimens in the ROM Green Plant Herbarium. Historical records of the distribution of C. monogyna were collected from fi ve 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 fi ve northwestern states: Idaho, Montana, Oregon, Washington, Wyoming), 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. Th is approach can be eff ective 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 undefi ned 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 coeffi cients obtained from digitized leaf outlines 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. suksdorfi i , seven C. monogyna , and 12 putative hybrids), and (2) leaves on herbarium specimens from the Cogswell-Foster Preserve and other locations in the Pacifi c Northwest. In the latter the attempt was made to sample the leaf shape variation seen in C. suksdorfi i as widely as possible. In both cases, variation 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 calculate the inverse of the dissection index described by Kincaid and Schneider (1978), i.e. inv(D.I.) = 2( A π) ½ / 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 fl owering 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 summarized 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). Signifi cance of individual principal component axes was evaluated using the broken-stick criterion (Frontier 1976) with the help of R function evplot (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). Th e plastid origin of the markers was used to establish the maternal parentage of the hybrids. DNA was extracted and amplifi ed from leaf tissue of individuals 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 2011a, b). Th is sample overlapped partially with the cloned ITS2 one (below), and provided an additional two C. suksdorfi i , 10 C. monogyna , and fi ve 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.botanyconference.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. suksdorfi i , four C. monogyna , three C. punctata and two each of the two hybrids (Table 3). Meth- Table 2. Results of Neighbor-joining clustering of sequence data for chloroplast DNA barcode loci. GenBank accession numbers indicate cluster affi liation (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.
Flow cytometry. Flow-cytometric methods for quantifying nuclear DNA in embryo 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 diff erences in sample size, the Pacifi c Northwest hybrid, Crataegus × cogswellii , appears more variable than either of its putative parents, C. monogyna or C. suksdorfi i (Fig. 1). Th e hybrid is clearly intermediate with respect to both leaf lobing (the inverse Dissection Index; Fig. 1) and style number (STYLE; Fig. 1). Principal components analyses of leaf outlines from Pacifi c Northwest C. monogyna , C. suksdorfi i , and their putative hybrid, demonstrate variation in leaf shape both within and between these three entities ( Fig. 2A, B). Th e fi rst principal component refl ects the contrast between the unlobed leaves of C. suksdorfi i 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). Th e second principal component refl ects 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   Taxa Voucher GenBank accession number ON45 Purich and Talent MP85  (TRT00002250)   KC174190, KC174191, KC174192, KC174193,  KC174194, KC174195  ON45 Purich and Talent MP86  (TRT00002251) 3); no signs of recombination were detected in the cloned ITS2 dataset. ITS2 sequences from the hybrids resemble either C. monogyna or one of the native North American species. Th e 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). Th ese time periods are evidently too short for genome homogenization (concerted evolution) to have taken place, even in diploids reproducing sexually. Our small sample of seed from the hybrids (Table 4) parallels earlier invDI , inverse dissection index = 2( A π) 1/2 / P , where A is the leaf area and P is the leaf perimeter; STAM , number of stamens per fl ower; STYL , number of styles per fl ower. Both axes shown account for signifi cant portions of the total variance according to the broken-stick criterion (Frontier 1976). results (Talent and Dickinson 2007a) showing diploidy and sexual reproduction in both parental taxa.
Only two of the three chloroplast genome barcode loci showed suffi cient variation for individuals from Crataegus section Crataegus to be distinguished from ones belonging 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). Th e 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. suksdorfi i is the female parent of the Pacifi c Northwest hybrids.
Th ese results corroborate earlier observations based on seed-set in artifi cial crosses between the parent species (Love and Feigen 1978;Wells and Phipps 1989). In reciprocal 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. suksdorfi i fl owers (mean 42%, range 25-73%, compared to a 29% mean fruit set by C. suksdorfi i with open pollination; Love and Feigen 1978). However, all reciprocal crosses between C. monogyna , C. suksdorfi i , and their hybrid yielded seeds (R. M. Love, personal communication).  (Table 1). In both A and B the two PCA axes shown are signifi cant according to the broken-stick criterion (Frontier 1976). In B arrowed point 1 represents the single individual of C. suksdorfi i 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 .  Table 1 for details). Dashed lines indicate the sectional affi nity of the sequences B Th e 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. suksdorfi i and C. punctata ; ( b ) ribotypes from individuals of C. suksdorfi i and C. × cogswellii ; and ( c ) ribotypes from individuals of C. punctata and C × ninae-celottiae (Table 3). Th e numbers shown are the % bootstrap support for each of the three branches.
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.botanyconference.org/engine/search/720.html; Zarrei et al. unpubl. data). Rather, we have taken advantage of our barcode sequence data from individuals unequivocally identifi able as C. monogyna , C. punctata , C. suksdorfi i 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 th and the 19 th centuries (Kirk 1819;Provancher 1863;Douglas 1914), fi rst on the east coast and nov. and C. monogyna in Ontario. Filled square, holotype of Crataegus × ninae-celottiae ; Crosses, TRT specimens of C. × ninae-celottiae ; asterisks, C. × ninae-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. Th e 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. suksdorfi i (Lo et al. 2013).  (Phipps and Muniyamma 1980;Phipps 1998;Lin 2009). Nevertheless, except for isolated occurrences in northern Delaware and adjacent Pennsylvania, as well as in Kentucky, Utah, and the San Francisco Bay area in California, C. monogyna in North America is not found south of 40°N latitude. In Ontario, C. punctata appears to be the only native diploid with a similarly late fl owering period that is also frequently sympatric with C. monogyna ( Fig. 1 in Campbell et al. 1991;Fig. 4). Crataegus suksdorfi i is the only native hawthorn in the Pacifi c Northwest known to include diploid individuals, and these are restricted to Oregon and adjacent California and Washington, west of the Cascades (Fig. 5; Lo et al. 2013). Where they co-occur, diploid C. suksdorfi i and C. monogyna fl ower at the same time, the latter species much more abundantly than the former (Love and Feigen 1978). Crataegus monogyna may never have been commonly planted in boundary hedges in Canada as it was in Europe. Fences and hedges appear to have been only rarely constructed in 17th Century Canada by European settlers to confi ne ruminant animals (Greer 2012); the animals were instead fed indoors, but allowed to roam the arable land for a short season after harvest, confi ned by the wall of surrounding forest. To this day, the hawthorn commonly growing along Ontario fence lines consists of native species, perhaps naturally occurring there. In Ontario forests we often encounter remnants of zig-zag post-and-rail fences, and these had the advantage over a hedge that they could be rapidly constructed as needed to mark property boundaries or to keep animals out of particular areas. In the United States hedging had its advocates in the early nineteenth century, but one of these described the superiority of native species like C. crus-galli ("cockspur" or "Newcastle thorn") and C. marshallii ("parsley-leaved" or "Virginia thorn") over introduced C. monogyna (Kirk 1819; "to sow or plant without fencing, would (in this country) be a useless labour").

Taxon/TRT accession/site/collection
Flow cytometry of seeds from both hybrids was consistent with diploid embryos and triploid endosperm, except that the embryos from C. × cogswellii show slightly higher than diploid measurements, higher than the 1.39-1.66 pg measurements previously obtained from leaf data (Table 4; Talent and Dickinson 2005). Whether the seeds involved would have germinated is unknown, but in contrast to the large healthy looking seeds from C. × ninae-celottiae , those from C. × cogswellii had smaller embryos and were variously misshapen. We noted that some individual trees of C. × cogswellii have a high degree of parthenocarpy-completely seedless fruitand the seeds we collected may therefore have been supernumerary to any strongly viable seeds. We can only state that C. × cogswellii apparently carries out both meiosis and fertilization, as expected of other diploid Crataegus (Table 4; Talent and Dickinson 2007b).
In her examination of hybridization between C. punctata and C. monogyna in Ontario, Purich (2005) found that the styles of C. punctata are signifi cantly longer than those of C. monogyna (mean mono = 4.1 mm; mean punc = 7.3 mm; sample sizes 5/52 and 7/116, individuals/styles). Diff erences between the two species in pollen grain diameter, hence volume, are not signifi cant (Purich 2005). No such diff erence in style length is present when comparing C. monogyna and C. suksdorfi i . Th ese results suggest that in Ontario, at least, the longer styles of C. punctata could act as a barrier to the successful penetration of C. punctata ovules by pollen tubes from C. monogyna pollen grains (Table 2). With style lengths and pollen grain diameters in C. monogyna and C. suksdorfi i similar (Dickinson unpublished data), it may be that the more abundant fl ower production of C. monogyna (Love and Feigen 1978) contributes to its role as the predominant pollen parent of C. × cogswellii . Th e exception to the summary above (TRT203 in Table 2; C. punctata as the maternal parent) refl ects the way in which diff erences in style length likely act to infl uence the direction of hybridization in a probabilistic rather than an absolute way.  (Fig. 6) Remarks. Shrub or tree up to ca. 6 m tall. Twigs of the current year densely to sparsely hairy or glabrous, hairs appressed to patent, straight or slightly curly; twigs of the previous year pale grey or ash-grey; aphyllous thorns 0.5-2 cm long, stout, straight; spinetipped, leaf-and dwarf-shoot-bearing branchlets lacking. Leaf blades ovate, obovate or elliptical, acute at apex, attenuate, cuneate or rounded at base, shallowly or deeply and regularly lobed, lobes with an acute apex, basal pair of veins convergent, straight or slightly divergent, intercalary veins running to the sinuses partly present, upper surface with ± deeply impressed veins at maturity, dull or lustrous bright or dark green, sparsely hairy and often becoming glabrous except along the veins, hairs appressed or semi-patent; lower surface dull, pale green, sparsely hairy throughout or only along the major veins and in the vein axils, hairs appressed or semi-patent; margin regularly crenate-serrate or serrate, teeth minutely glandular, glands less than 0.1 mm; petiole eglandular, narrowly winged in upper part. Subterminal leaf blade of fl owering shoots 30-55 mm long, 16-38 mm wide, shallowly and regularly lobed, lobes 2-5 pairs, basal pair extending 0.2-0.4 times the width of lamina to midrib, each lobe with 6-11 teeth, basal pair of sinuses in apical 1/4 to basal 1/3 of lamina; petiole 6-20 mm long; stipules caducous, membranous or herbaceous, 4-8 mm long, irregularly or regularly glandular-denticulate, with 20-30 teeth. Leaf blades of elongate shoots 35-45 mm long, 25-35 mm wide, shallowly or deeply and regularly lobed, lobes 3-5 pairs, basal pair extending 0.2-0.6 times the width of lamina to midrib, each lobe with 4-11 teeth, basal pair of sinuses in basal 1/2-1/3 of lamina; petiole 8-12 mm; stipules caducous, herbaceous, ca. 6 mm long, regularly glandular denticulate-serrate, with ca. 15 teeth. Infl orescence 3-4 cm long, lax, corymbose, 5-17-fl owered, densely to sparsely hairy, hairs appressed, semi-patent or patent, straight or slightly curly; pedicels 3-18 mm, densely to sparsely hairy, hairs appressed, semi-patent or patent, straight or slightly curly; bracts caducous, membranous or herbaceous, 3-4 mm long, 0.2-0.4 mm wide, linear-lanceolate, 10-15 times as long as wide, irregularly glandular-denticulate, with 5-7 teeth. Hypanthium 3-4 mm long, densely to sparsely hairy, hairs appressed, semipatent or patent, straight or slightly curly; sepals 2-4 mm long, 1.5-2 mm wide, triangular-lanceolate or triangular, 1-2.7 times as long as wide, entire or rarely irregularly and minutely glandular-serrate, teeth 0-2, apex acute or obtuse; petals 6-7 mm long and wide; stamens 18-20, anthers 1-1.2 mm long, pink or purple; styles 2-3; hypostyle pilose. Fruit 9-12 mm long, 8-12 mm in diameter, 1.0-1.1 times as long as wide, globose, broadly ellipsoidal or obovoid, ± lustrous, red or orange, punctate with small, pale brown lenticels, up to ca. 0.2 mm in diameter, sparsely hairy, crowned by the persistent, refl exed sepals; calyx tube indistinct, ca. 0.5 mm long, 3-4 mm wide; fl esh yellowish, hard and mealy; pyrenes 2-3, ventro-laterally smooth; hypostyle pilose. Phenology. Flowering in May-June. Fruiting in August-September. Reproductive biology. Sexual. 2 n = 2 x (2 n = 34? Muniyamma and Phipps 1979;Talent and Dickinson 2005); diploid embryos and triploid endosperm.

Etymology. Crataegus × ninae-celottiae honors Nina Celotti
Crataegus × ninae-celottiae diff ers from C. punctata in: aphyllous thorns shorter, 0.5-2 cm long (not 2-5 cm long); leaf blades regularly lobed almost to the base (not unlobed or shallowly lobed towards apex), intercalary veins running to the sinuses sometimes present; subterminal leaf blade of fl owering shoots usually smaller, up to ca. 55 mm long, and veins 2-5 pairs (not up to ca. 85 mm and veins 6-10 pairs); stipules often herbaceous and irregularly glandular-denticulate; sepals shorter, 2-4 mm long, and wider, 1-2.7 times as long as wide (not 3-7 mm long and 2-4.7 times as long as wide); styles and pyrenes 2-3 (not 3-5); fruit usually smaller, up to ca. 12 mm long and in diameter (not up to ca. 15 mm long and in diameter) and less distinctly punctate with smaller lenticels up to ca. 0.2 mm in diameter (not up to ca. 0.4 mm in diameter).
Phenology. Flowering in April-May. Fruiting in September. Some individuals strongly parthenocarpic.
Distribution. Northwestern U.S.A.; western Oregon ( Figure 5); potentially present in adjacent northwestern California and southwestern Washington where the parent species are sympatric.
Etymology. Crataegus ×cogswellii honours the Cogswell family, and Mr. and Mrs. Lee Foster, of Halsey, Oregon. In 1872 John Cogswell, Mrs. Foster's grandfather, purchased the land that the Fosters gave to the Oregon Nature Conservancy as the Cogswell-Foster Preserve (Lopez 1971), and at which C. × cogswellii has been most intensively studied (Love and Feigen 1978).
Similar taxa. Crataegus × cogswellii diff ers from C. monogyna in: leaf-and dwarfshoot-bearing branchlets usually lacking; stipules of leaves of fl owering shoots irregu- 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 fi gures 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 Department 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 specimen 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).