A strategy for identifying introduced provenances and translocations* C. FERRIS1, A. J. DAVY2 AND G. M. HEWITT2 Downloaded from https://academic.oup.com/forestry/article/70/3/211/543878 by guest on 29 September 2021 1 Department of Botany, University of Leicester, University Road, Leicester, LEI 7RH, England 2 School of Biological Sciences, University of East Anglia, Norwich, Norfolk, NR4 7TL, England Summary Native species of oak in Britain have been of great importance in our history for their many uses, and have thus been subject to management and planting by man for centuries. Following events such as the Enclosure Acts of the late eighteenth century and the Napoleonic wars, many oaks would have been planted. Translocations and introductions of foreign genotypes were greatly encouraged by early landscape gardeners such as Capability Brown. Britain must, there- fore be a mosaic of native and non-native oaks. A major problem arises when we try to identify non-native trees. Due to their long life-cycle, oaks are of necessity both phenotypically plastic and genetically very variable and it has been virtually impossible to discriminate between native and non-native forms using traditional methods. The advent of new molecular genetic techniques however, now allows us to identify DNA markers that can distinguish between such forms. The geographic patterns for two chloroplast DNA markers will be presented. One clearly differentiates between oaks from eastern Europe versus western Europe and can be used to identify translocations of eastern European oaks into Britain and western Europe. The second identifies genotypes native to East Anglia and can be used to recognize translocations into and out of East Anglia. Introduction County Council, for example, has a biodiversity Biodiversity and its conservation have become initiatlve focusin8 on *c impact of native ver- very important issues in recent years and many *us, no"-nat've trees ™* shrubs in ancient h ed ow A ma)o roblem ln S ch studleS S non-foresters have become more aware of and L 6" ^ . / P " ' . j • r> • • I r ji the identification of native trees. Native can be interested in British forests and the provenance . - . , . c r u • • L- L n it • denned in many ways and here it is synonymous of some of the species within them. Possibilities . ' , , ' •„ c i • r ii- with the term autochthonous. A genotype wil of adaptation or native trees to local environ- . ...... 6 , , , i i- • • ji- • • i- thus be classed native if it is descended from mental conditions, in addition to nationalist . r , . , , , ,, . • • i i i u c that which first co onized the area following the tendencies, have led some groups to call for , . ,, , , 6 c • i • u- last ice-age, i.e. not affected by man. conservation of our native trees. Leicestershire 6 ' * This paper was first presented at the Institute of Chartered Foresters Discussion Meeting, Native and Non-native in British Forestry, held at the University of Warwick in 1995 and has been published in the discussion meeting proceedings. «; Innimtc: (>f Chartered Ft.rotcn, 1997 Forestry, Vol. 70. No. J, 1997 212 FORESTRY At the height of the last ice-age, about 18 000 ation for native plants over a much larger area. years ago, most of Britain was covered with ice That is, we must determine a baseline for trees (Denton and Hughes, 1981), and forests were of known provenance against which to compare restricted to refugial areas in southern Europe trees of unknown or uncertain origin. The sam- (Huntley and Birks, 1983). As the climate pling strategies are, therefore, very important. warmed and the ice melted, the trees were able We must first obtain a sample of native geno- to migrate northwards from these refugia to types and then carefully choose the type of DNA recolonize central and northern Europe. Such in which to look for variation. A map can then migratory responses of plants to long term be drawn of the native genetic variation. Any Downloaded from https://academic.oup.com/forestry/article/70/3/211/543878 by guest on 29 September 2021 changes in climate may be compared to bird trees under investigation that do not conform to migration as a response to seasonal climate this pattern may be attributed to possible intro- changes (Huntley and Webb, 1989). Populations duction or translocation. in different refugia may have been isolated for To reduce time and expense, the strategy for the duration of the ice-age, some 100 000 years, finding DNA markers was initially to sequence a period in which divergence and evolution will the DNA and identify polymorphism directly; have taken place. Any differences that existed and second, using the known sequence differ- between refugial populations for a species, will ences, to determine which restriction enzymes to lead to geographic variation dependent upon the use for a fast restriction analysis of the samples. routes of migration taken (Ferris et al., 1993; The initial sequencing overcomes the lengthy 1995). Further genetic variation may be afforded problems of screening with random enzymes to by mutations fixed during migration and those look for variation, while the subsequent restric- accumulated since recolonization. tion analysis is much cheaper and faster than Genetic variation can be assessed using mor- sequencing all the samples. phological, biochemical or molecular tech- niques. However, many of our tree species are Organisms best suited to molecular studies. Oaks, for example, are phenorypically very plastic and an For the present investigation the native British individual grown under one set of conditions oak species, Quercus robur L. and Q. petraea may appear very different if grown under other (Matt.) Liebl, were chosen. These trees have conditions. Oaks are also genetically very vari- been very important to European history, and able and allozyme studies have revealed very consequently many oaks have been planted. It is high levels of diversity within populations likely, that for the most part, planting will have (Kremer et al., 1991). By contrast, chloroplast been from seed of local origin until about 250 DNA is much less variable and thus easier to years ago. Introductions of oaks for landscaping analyse. occurred during the times of Capability Brown In this paper molecular data is presented on in the second half of the eighteenth century. At two European oaks, Quercus robur L. and Q. the end of the eighteenth century the Enclosure petraea (Matt.) Liebl., and these are used as Acts resulted in the large scale planting of oak, model species in which to present a strategy for as did the depletion of timber for the obtaining molecular markers that can be used to Napoleonic wars in the early nineteenth cen- distinguish between native and non-native geno- tury. The extent to which local seed sources types. were used is not known. We therefore decided to sample trees older than 250 to 300 years, which will pre-date these times of possible intro- ductions. In order to ensure such old age, we Materials and methods sampled trees of exceedingly large girth, and also famous historical trees such as Kett's Oak Strategy in Norfolk, the Queen's Oak in Suffolk, and the In order to isolate markers that can distinguish Mitre Oak in Hereford and Worcester. In addi- between native and non-native plants for a given tion, where such trees were not available, we area, we must first establish the patterns of vari- used samples from ancient forests or wildwood INTRODUCED PROVENANCES AND TRANSLOCATIONS 213 where planting is deemed unlikely. Samples PCR amplification were also available from several sites of dubious PCR was performed using the universal chloro- status; either representing planted forest, trees plast primers C, D and F of Taberlet et al. of young age or where exact location was (1991); C+D amplify the tRNA^"1 intron and unknown, gardens of old halls and estates, and C+F amplify the intron plus an adjacent non- an arboretum in the south of France. In total the coding region. Reaction conditions are as given samples included 125 individuals from 56 sites in Ferris et al (1993). representing 15 countries from mainland Europe (Figures la, lb and 2; Tables 1 and 2); and 103 Downloaded from https://academic.oup.com/forestry/article/70/3/211/543878 by guest on 29 September 2021 individuals from 66 sites representing England, DNA sequencing Ireland and Wales (Figure 3, Table 3). In addi- tion, 50 trees with a girth at breast height Two sequencing methods were used and both greater than 15 feet, were sampled from a 12 X gave equally good results. Double-stranded PCR 12 km area south of Norwich. products were purified using Centricon 30 (Ami- Leaves were stored at -80°C and total con), or double-stranded PCR products were genomic DNA was extracted using the CTAB amplified using primer C biotinylated and sepa- (cetrimide) method (Doyle and Doyle, 1990) rated into single-stranded template using mag- with minor modifications by Howland (1992). netic beads (Dynal). The template was then sequenced using the dideoxy-chain termination method with a fluorescent primer and a T7 Chloroplast DNA Sequencing Kit (Pharmacia, LKB) on an Auto- It was decided to use chloroplast DNA mated Laser Fluorescence sequencing machine, (cpDNA) for two main reasons. First, the ALF (Pharmacia, LKB). extreme variability shown in oaks using Alternatively, double-stranded PCR product allozyme studies, (Kremer et al, 1991), suggests was purified using QIAquick Spin Columns that nuclear DNA may be too variable. The (Qiagen) and sequenced using PCR cycle cpDNA has a slow rate of evolution (Zurawski sequencing with dye-labelled terminators on an et al., 1984), but has been used as a source of automatic sequencing machine (ABI).