Watsonia 27: 99–108 (2008) VARIATION IN SORBUS HIBERNICA 99 Genetic variation in Irish Whitebeam, Sorbus hibernica E. F. Warb. (Rosaceae) and its relationship to a Sorbus from the Menai Strait, North Wales R. S. COWAN, R. J. SMITH, M. F. FAY Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB and T. C. G. RICH* Department of Biodiversity and Systematic Biology, National Museum of Wales, Cardiff CF10 3NP ABSTRACT Parnell & Needham (1998) found that S. hibernica was morphologically variable, and Genetic variation within the Irish endemic Sorbus noted that further work was needed to confirm hibernica, Irish Whitebeam, and its relationships to its apomictic nature and also to look more S. aria, S. eminens, S. porrigentiformis and a taxon closely at its relationships to other taxa. In this from the Menai Strait have been assessed using paper, we present a DNA analysis using AFLPs and morphology. Sorbus hibernica is genetically distinct from S. aria, S. eminens and S. amplified fragment length polymorphisms porrigentiformis but is close to the Menai Strait (AFLP; as previously used in Sorbus by Fay et taxon. Sorbus hibernica cannot be separated from the al. 2002) with some comparative morphology Menai Strait taxon using leaf or fruit characters, but to follow up these suggestions. they differ in ploidy. Morphologically S. hibernica looks similar to S. porrigentiformis E. F. Warb., but it is KEYWORDS: AFLP, Ireland, Sorbus aria, Sorbus distinct from it, and differs in having leaves eminens, Sorbus porrigentiformis. with finer, uniserrate toothing and elliptic to obovate leaves (Fig. 1). Studies of plastid DNA INTRODUCTION by Chester et al. (2007) showed that S. porrigentiformis has plastid types C, D, H1, R Sorbus hibernica E. F. Warb., Irish and T, differing from S. eminens, S. hibernica, Whitebeam, is one of the few vascular plants and the Menai Strait Sorbus which all had the endemic to Ireland. It was first collected in plastid type A, which is also common in S. aria 1885 but remained unrecognised until 1933 (L.) Crantz. We have therefore only included a (Pugsley 1934), and was first described by few S. porrigentiformis DNA samples in the Warburg (1952). Rich et al. (2005) reviewed its comparisons, and one sample of the related S. history, distribution and ecology, and reported ‘Bristol porrigentiformis’ sensu Proctor & that it had been recorded from at least 150 Groenhof (1992). Morphologically, S. localities in 76 hectads in 29 vice-counties in hibernica is also easily separated from both Ireland. It is triploid (Bailey et al. 2008), and northern and southern forms of S. eminens (cf. like the other polyploid members of the genus Proctor & Groenhof 1992) which have large, in Britain and Ireland is usually regarded as nearly orbicular leaves; again we have apomictic, although this has not been therefore only included a few DNA samples in investigated experimentally. the comparison. As S. hibernica is very similar Proctor & Groenhof (1992), whilst investi- in leaf shape and toothing to the Menai Strait gating Sorbus populations using isozymes, Sorbus, we have both included DNA samples found similarities between S. hibernica and the and carried out a more detailed morphological English endemic S. eminens E. F. Warb., and analysis. Some DNA samples of the diploid S. with an unnamed taxon previously ascribed to aria from France and England, where it is S. porrigentiformis E. F. Warb. from the Menai native, and from Ireland, where it is introduced Strait, North Wales, and suggested that this (Praeger 1934), were also included for relationship should be investigated further. comparison. *e-mail: [email protected] 100 R. S. COWAN, R. J. SMITH, M. F. FAY AND T. C. G. RICH FIGURE 1. Leaves of Sorbus species from the short, sterile shoots. Sorbus hibernica: 1, Killowen. 2, Kilbeggan. 3, Clifden. 4, Cong. 5, Lough Derg. 6, Kilkenny. 7, Clonbur. 8, Cappagh. Menai Strait Sorbus: 9–12, Bangor. Sorbus eminens: 13, southern form, Shirehampton. 14, northern form, Coldwell. Sorbus porrigentiformis: 15, Stokeleigh. Sorbus ‘Bristol porrigentiformis’: 16, Leigh Woods. Not to scale. VARIATION IN SORBUS HIBERNICA 101 METHODS S. hibernica, 50 leaves of S. aria from Ireland and 56 leaves of the Menai Strait Sorbus on herbarium material in the Welsh National DNA ANALYSIS Herbarium (NMW), Trinity College Dublin DNA samples were collected from 21 S. (TCD) and Belfast (BEL). As many as hibernica trees across Ireland, and compared possible of the trees sampled during the DNA with samples of S. aria (seven samples), S. analysis were included, but some lacked the eminens (both forms, seven samples), S. short shoots or were poor specimens. The porrigentiformis (3 samples), S. ‘Bristol samples used additional to the DNA analysis porrigentiformis’ (one sample) and the Sorbus samples are given in Appendix 1. from the Menai Strait (3 samples). Two leaves The leaf length (excluding petiole), width, from each tree were torn into narrow strips and distance along the mid-vein of the widest point, put into desiccant silica gel in which they were the angle of veins to the midrib at the middle of kept until processed (Chase & Hills 1991). the leaf, and the total number of veins were Voucher specimens were collected at the same measured. From these, the leaf length:width time for NMW, though in some cases leaves ratio and the position of the widest point along from the typical broad, short shoots were not the leaf (expressed as a percentage) were available due to hedge cutting or were out of derived. The seven characters were then reach. The samples included in the DNA analysed using Principal Components Analysis analysis are listed in Table 1 with their origin, (PCA) with a correlation matrix in PAST cross-referenced with the NMW accession version 1.72 (Hammer et al. 2001). numbers and the Kew extraction numbers. Duplicate aliquots of the DNA samples have been deposited in the Irish DNA Bank at RESULTS Trinity College, Dublin. DNA was extracted from approximately 0·2 g of dried material using a modified 2× DNA ANALYSIS CTAB (cetyltrimethyl-ammonium bromide) A total of 187 size fragments were scored of procedure (Doyle & Doyle 1987), purified and which 95% were polymorphic (10 invariable). quantified using a spectrophotometer. In the UPGMA analysis (Fig. 2), the Sorbus AFLPs were performed according to the hibernica samples, Group A, all cluster AFLP Plant Mapping Protocol of PE Applied together and separately from all other taxa. Biosystems Inc. (ABI). For each specimen, 0·5 This is a relatively genetically uniform group µg of DNA were digested using the restriction which represents the main S. hibernica taxon enzymes MseI and EcoRI. Standard adaptors with elliptic-obovate leaves and fine, uniserrate (PE Applied Biosystems) were ligated to the toothing. The relatively tight grouping suggests genomic digests. Twenty four unique primer it is probably (predominantly at least) combinations were screened for their ability to apomictic but that cannot be confirmed from amplify scoreable bands, from which two were these data. selected for this study. Amplification fragments The three Menai Strait samples, Group B, were separated on an ABI 3100 DNA analyser. form a group distinct from, but obviously close Genescan 3.7 and Genotyper 2.0 were used to to, S. hibernica. Group C contains the group of analyse the fragments which were scored as apomictic polyploid taxa from south-west either present (1) or absent (0) for all England, the two forms of S. eminens and the individuals. The resulting binary matrix was form of S. porrigentiformis from Bristol; these analysed using the UPGMA (Unweighted Pair- are clearly closely related to each other and are Group Method using Arithmetic Averages) approximately genetically equidistant from the algorithm in the software package PAUP S. hibernica group and the Menai Strait version 4.0d64 for Macintosh (Swofford 1998) samples. The three British S. porrigentiformis and by Principal Co-ordinates Analysis samples, Group E, form a distinct group that is (PCOA) in the R Package for Multivariate genetically more distant from these groups than Analysis version 4.0 (Casgrain & Legendre S. aria from France and Ireland. 1999) using Jaccard’s coefficient. The samples of the genetically diverse sexual diploid S. aria group by geographic origin. One MORPHOLOGY Group D S. aria specimen was collected at Broad leaves from the short vegetative shoots, Ballynahinch, Galway (vc. H16), the type excluding the oldest and youngest leaf (Aas et locality of S. hibernica (Warburg 1957), where al. 1994), were measured on 88 leaves of we failed to refind S. hibernica. 102 R. S. COWAN, R. J. SMITH, M. F. FAY AND T. C. G. RICH TABLE 1. SORBUS SAMPLES ANALYSED DURING DNA ANALYSIS. SOME S. HIBERNICA, S. ARIA AND S. ‘MENAI STRAIT’ SAMPLES WERE ALSO USED FOR THE LEAF MORPHOLOGICAL ANALYSIS Taxon Locality Vice-county/Region Country Voucher Kew DNA bank no. S. ‘Bristol Leigh Woods 6 North Somerset England 2001.25.273 36.80 porrigentiformis’ S. ‘Menai Strait’ Menai Strait 49 Caernarfonshire Wales 2002.17.68 42.12 S. ‘Menai Strait’ Menai Strait 49 Caernarfonshire Wales 2002.17.66 42.13 S. ‘Menai Strait’ Menai Strait 49 Caernarfonshire Wales 2002.17.67 42.15 S. aria Above Senanque Provence France Civeyrel et al. 46.30 492, K S. aria Ballynahinch H16 West Galway Ireland 2003.20.26 51.01 S. aria Burnham Beeches 24 Buckinghamshire England 2001.48.10 24.10 S. aria Burnham Beeches 24 Buckinghamshire England 2001.49.11 27.20 S. aria Lecarrow H25 Roscommon Ireland 2003.4.1042 49.03 S. aria Roundstone W16 West Galway Ireland 2003.20.24 50.05 S. aria Tallyho H23 Westmeath Ireland 2003.20.30 49.06 S. eminens, southern form Cheddar Gorge 6 North Somerset England 2001.25.141 36.05 S.