Europ.J.Hort.Sci., 74 (3). S. 130–136, 2009, ISSN 1611-4426. © Verlag Eugen Ulmer KG, Stuttgart Historical Cultivars of Buxus sempervirens L. Revealed in a Preserved 17th Century Garden by Biometry and Amplified Fragment Length Polymorphism (AFLP) P. H. Salvesen1,3), B. Kanz2,3) and D. Moe3) (1)Arboretum and Botanical Garden, University of Bergen, Norway, 2)Senckenberg Research Institute, Frankfurt am Main, Germany and 3)Natural History Collections, University of Bergen, Norway) Summary Buxus sempervirens L. extant in a preserved renais- the AFLP data, the three morphs in neighbour-joining sance parterre dating from ca. 1680 at Milde (Bergen, bootstrap analyses formed well supported clades Norway) has been provisionally classified as the (100 % BP) and in a principal coordinate analysis morphs ‘Pendula’, ‘Suffruticosa’, and ”Yellow”. These formed distinctly separated and close clusters, indicat- were thought a priori to represent distinct taxa, viz. ing that the morphs are genetically distinct and the in- cultivars. Shoot length, leaf hairiness, leaf width, leaf dividuals of each morph are closely related. Biometric curvature, and lamina width-to-length ratio measured analysis and finger printing techniques (AFLP) consist- on 30 leaves in each individual allowed separation of ently show that the three morphs are both morpholog- the morphs in discriminant analysis. In AFLP analyses ically and genetically distinct. The three morphs are 7 primer combinations yielded informative data, pro- assumed to represent genetically distinct old cultivars ducing 168 (57 %) polymorphic fragments. Based on introduced during the 17th or 18th century. Key words. boxwood parterre – Buxus sempervirens cultivars – conservation – garden history – Norway – renais- sance garden Introduction pography (FØRLAND 1993). The garden is surrounded by large stone walls and probably dates from before 1530 Buxus sempervirens L. (Common Boxwood) today is native (SALVESEN and MOE 2005; MOE et al. 2006). In written in Southern and Western Europe northwards to Central sources it is, however, only known since the early 18th France and Southern England (TUTIN et al. 1968), and oc- century, and then “flower quarters” and “pyramid trees” curs as a naturalised species further north (DECOCQ et al. are mentioned (SCHÜBELER 1888). The maintenance of the 2004). It has been cultivated for edging and topiary at garden apparently has been sporadic since ca. 1815, and least since Roman Times (CIARALLO 2004), and was well little seems to have been added to its contents by the own- known in medieval gardens of Europe (HARVEY 1981, ers since then (SCHNITLER 1915; MOE et al. 2006). The orig- 1987; DICKSON 1994). The introduction of the species to the inal vegetation is therefore partly still in existence, includ- Nordic countries probably only followed the breakthrough ing some 40 species and cultivars of extant garden plants, of the renaissance garden style that originated in Italy in the and a rectangular parterre (sized 25 m by 15 m) over- 15th century and reached the north during the 16th and grown with a 5–7 m high entangled shrubbery of Buxus 17th centuries (LORENTZON 1998; CHRISTENSEN 1999; DIETZE sempervirens preserved in front of the manor house. The 2000; MOE et al. 2006). Old specimens of Buxus thought Buxus material has been classified into three morphs, to date from these early introductions are still found ex- provisionally named ‘Pendula’, ‘Suffruticosa’, and ”Yel- tant in a few historic gardens and parks in Scandinavia low” (SALVESEN and MOE 2005; MOE et al. 2006). The (MOE 1991; LORENTZON 1998; MOE et al. 2006). former two morphs compare well with known cultivars, whereas the latter does not. It has a notable yellowish The Milde Estate Garden green leaf colour, and was therefore tentatively named ”Yellow”, the double quotes signalling its uncertain affin- The Milde Estate Garden is situated in Bergen, Norway, ity. Six ‘Pendula’ specimens, presumably remnants of the close to the North Atlantic coast (60 ° 15 ’ N. lat., 5 ° 16 ’ E. “pyramid trees” mentioned by SCHÜBELER (1888), are still long., altitude 35 m a.s.l.) with a climate characterized by standing as one-stemmed, bowed trees. The largest, fea- mild winters (mean January temperature range: +4 to turing a stem diameter of 32.6 cm, has been dated by an- 0 °C) and moderate summer temperatures (mean July nual ring counts at 320 ± 47 years of age. The ”Yellow” temperature range: 12 to 16 °C) (AUNE 1993), and an an- morph comprises strongly growing shrubs apparently nual precipitation of 1000 to 2500 mm depending on to- originally trimmed into spherical shapes, each with sev- Europ.J.Hort.Sci. 3/2009 Salvesen et al.: Historical Cultivars of Buxus sempervirens L. 131 eral stems protruding from the base. The age of the larg- five young (last year’s) shoots were picked where possi- est stem (diam. 19.7 cm) is estimated at ca. 200 years. In ble. In each shoot, two well developed leaves near the the smaller ‘Suffruticosa’, making branching shrubs part- middle of the shoot were measured. Thus, in general a to- ly subdued by shade, an age of ca. 205 years has been es- tal of 30 leaves were measured from each of 26 speci- timated for the largest stem (diameter 10.5 cm) (MOE mens. In a few instances additional leaves were measured 1991; SALVESEN and MOE 2005). The age of ‘Suffruticosa’ where variation between measurements was judged to be and ”Yellow” may reflect the time when the maintenance exceedingly large, and occasionally some cases were dis- and renewal of the trimmed hedges of the parterre halt- carded due to missing data (total number of measure- ed. The ages of the ‘Pendula’ morphs presumably give an ments, n=817). The characters scored from each leaf age estimate for the parterre itself, as pollen analysis were: total leaf length, lamina length, and lamina width from the site indicate the presence of Buxus sempervirens (Fig. 1), lamina curvature (arbitrary scale; –2 = strongly since it was established (MOE et al. 2006). concave, –1 = concave, 0 = flat, 1 = convex, 2 = strongly The aim of the present study is to examine if the pro- convex), and hairiness of the petiole and leaf nerve (arbi- visional classification of Buxus morphs is supported by a trary scale; 0 = no hairs, 1 = scattered hairs, 2 = hirsute, 3 more detailed study using biometrical and finger printing = densely hirsute; the mean hairiness of petiole and nerve techniques. And if so, to substantiate if these morphs rep- was entered in the statistical analysis). In addition the resent ancient cultivars, and therefore to establish a basis length of the selected shoot was measured. for the further study of Buxus found in old gardens. AFLP-analysis Materials and Methods Fresh leaf material from each of 26 specimens was sam- pled (P1–6, G1–10, S1–10; Table 1). To test the reproduc- Plant material ibility of our results, nine additional samples were taken Leaves were collected from Buxus sempervirens in the Mil- de Estate Garden for biometric and AFLP analyses (26 samples), from all the 6 specimens of ‘Pendula’ preserved (P1–6), and from ten specimens each of the ‘Suffruticosa’ (S1–10) and ”Yellow” (G1–10) morphs. The samples for biometric and AFLP analyses were all taken from the same stand, but may not include exactly the same speci- mens in the two data sets. The effect this would have on the resulting comparison is expected to be low, as the to- tal number of Buxus specimens in the garden hardly ex- ceeds 30. Biometry Fig. 1. Position where leaf characters total leaf length (mm), From each of the 26 specimens (P1–6, G1–10, S1–10), lamina length (mm), and lamina width (mm) were measured three branchlets were collected, and from each branchlet, in Buxus sempervirens. Table 1. Buxus sempervirens material utilized in the AFLP study. Taxon Specismens Reference / collector Origin (herbarium voucher) ‘Pendula’ P1–10 Salvesen PHS06.01 (BG) Norway, Milde Estate, Bergen P6 1–9 Salvesen PHS06.15 (BG) Norway, Milde Estate, Bergen P10 1–9 Salvesen PHS06.15 (BG) Norway, Milde Estate, Bergen ”Yellow” G1–6 Salvesen PHS06.01 (BG) Norway, Milde Estate, Bergen G1 1–9 Salvesen PHS06.15 (BG) Norway, Milde Estate, Bergen G6 1–9 Salvesen PHS06.15 (BG) Norway, Milde Estate, Bergen ‘Suffruticosa’ S1–10 Salvesen PHS06.01 (BG) Norway, Milde Estate, Bergen S2 1–9 Salvesen PHS06.15 (BG) Norway, Milde Estate, Bergen S3 1–9 Salvesen PHS06.15 (BG) Norway, Milde Estate, Bergen suf1 Kanz (BG) Germany, Atrops tree nursery, Rheurdt not specified Bar Norway, Rosendal Barony, Kvinnherad BG: The Bergen herbarium Europ.J.Hort.Sci. 3/2009 132 Salvesen et al.: Historical Cultivars of Buxus sempervirens L. from two individuals per morph (P1 1–9, P6 1–9, G6 1–9, from 70 individuals using three primer pairs. Matrices G10 1–9, S2 1–9, S3 1–9). In addition to the 26 samples (1) and (2) were converted to distance matrices based on collected at Milde Estate Garden, one sample (Bar) sup- the NEI and LI (1979) (= Dice) index of similarity. plied from a shrub of uncertain morph affiliation in the garden at the Rosendal Barony (Kvinnherad, Hordaland, Data analysis Norway), and one sample (suf1) of the ‘Suffrutico- sa’-morph obtained from the Atrops tree nursery in Ger- Initially the morphological characters separating best be- many were included. Samples were stored in silica gel im- tween the three morphs were selected by evaluating the mediately after collection. Voucher specimens are depos- MORPH effect in separate one-way ANOVAs according to ited in the Bergen herbarium (BG). the model: Morph$ + Specimen$ + Branchl_no + Shoot_no + Constant = 0. Data for these characters were DNA extraction then included in three separate discriminant analyses us- ing the GLM routine in the SYSTAT package v.