A Strategy for Identifying Introduced Provenances and Translocations*

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, therefore 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 techniques. 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 introductions. 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 addition, 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). intraspecific variation in a wide variety of species (reviewed in Soltis et al., 1992). cpDNA Restriction analysis genotypes have been found to be geographically structured in several population studies (Soltis et For the single base mutation T to C at position al., 1989, 1991; Doyle et al, 1990; Lavin et al., 153 of the intron, a single digest with the restric- 1992; Ferris et al, 1993, 1995). tion enzyme C/bl (Gibco BRL) was used. PCR Second, cpDNA is maternally inherited and product (10 u,l) was digested for 1 h at 37°C in no evidence for paternal leakage has been found a total volume of 15 u,l. Products were run on a (Kremer et al, 1991; Whittemore and Schaal, 1 per cent agarose gel and stained with ethidium 1991). This means that where leaf samples are bromide (0.5 u.g ml~') and viewed with a 302- not available, acorns can be used with the nm ultra violet source. For trees possessing the knowledge that there is no contribution from wildtype T, the PCR product is uncut as the the unknown male parent to the DNA being C/bl does not recognize GTGC. However, the used. For genera other than oaks, which may be sequence GCGC is recognized by the C/bl, and less genetically variable, it may be advisable to for trees possessing the mutant C, the PCR use the more variable maternally inherited mito- product is cut. chondrial DNA. The region of the chloroplast For the 13 base pair duplication a double used in this study is the tRNALcul intron found digest with the restriction enzymes Mbol and in the Large Single Copy region, for which Poly- Rsal was used. PCR product (10 u.1) was merase Chain Reaction (PCR) primers were digested overnight at 37°C with both enzymes, readily available (Taberlet et al, 1991). using 'one-phor-all' buffer (Pharmacia), in a total volume of 15 \L\. Products were run on a polyacrylamide gel (12 per cent) in 1 X TBE 214 FORESTRY

Table 1: European samples of Quercus species given in Figures la and lb. The numbers of individuals sequenced for the intron and those digested with C/oI are given, r = Q. robur, p - Q. petraea. Total sample size = 208

No. Site District Country Species Number Number Total sequenced digested

1 Cork Co. Cork Ireland P 0 3 3 2 Elan Valley Powys Wales P 0 2 2 3 Llandcilo Dyfed Wales r 0 1 1 Downloaded from https://academic.oup.com/forestry/article/70/3/211/543878 by guest on 29 September 2021 4 Forest of Dean Gloucester England P 0 2 2 5 Powick Hams Worcester England r&p 0/0 1/2 3 6 Mitre Oak Worcester England r 1 1 1 7 Tamworth West Midlands England r 0 1 1 8 Various East Anglia England r 10 70 70 9 Bcllheme Sarthe France P 1 2 2 10 Le Mans Sarthe France r 0 2 2 11 Blois Loir et Cher France P 0 3 3 12 Fontainbleu Seine ct Marne France r 0 1 1 13 Verdun Meuse France P 0 1 1 14 Bitchc Moselle France r 1 2 2 15 Merz Moselle France P 1 3 3 16 Troncais Allier France P 0 3 3 17 Feurs Loire France r 0 2 2 18 Pilat Loire France r 0 1 1 19 Poitiers Vienne France r 0 2 2 20 Chize Deux Sevres France r 0 2 2 21 Pons Charente Maritime France r 0 2 2 22 Agenais Lot et Garonne France r 0 2 2 23 Montech d'Agre Tarn et Garonne France r 0 4 4 24 Montrejeau Haute Garonne France r 0 1 1 25 Arudy Pyrenees Atlantiques France r 0 1 1 26 Pau Pyrenees Atlantiques France r 0 2 2 27 Cambo Pays Basque France r 0 1 1 28 Elizondo Basque Spain r 0 1 1 29 Ultzama Basque Spain r 0 1 1 30 Montalegre Vilareal Portugal r 0 1 1 31 Tatti Florence Italy P 1 5 5 32 Unknown Zagreb Croatia r 1 3 3 33 Unknown Zagreb Croatia P 1 4 4 34 Stejar Brasov Romania r 2 3 3 35 Gorun Brasov Romania P 2 2 2 36 Lorerto Burgenland Austria P 1 3 3 37 Brunnsee Styria Austria r 1 2 2 38 Stanser Tyrol Austria r 1 2 2 39 Uetliberg Zurich Switzerland r 0 2 2 40 Galm Fribourg Switzerland P 0 2 2 41 Lausanne Vaud Switzerland r 0 1 1 42 Nidderau Hessen Germany P 1 3 3 43 Bad Neuheim Hessen Germany r 2 2 2 44 Hasbruch Niedersachsen Germany r 1 2 2 45 Gohrde Wendland Germany P 0 2 2 46 Ltibeck Holstein Germany r 2 2 2 47 Brzesko Krakow Poland r 1 4 4 48 Lipiny Bialystok Poland P 0 1 1 49 Bialowieza Bialystok Poland r 0 1 1 INTRODUCED PROVENANCES AND TRANSLOCATIONS 215

No. Site District Country Species Number Number Total sequenced digested

50 Skjoldenaesholm Sjaelland Denmark r 1 2 2 51 Scania Veberod Malmohus Sweden r 2 6 6 52 Saarc Saaremaa Estonia r 0 2 2 53 Unknown Unknown Estonia r 0 5 5 54 Gostilizy St. Petersburg Russia r 0 2 2

55 Toksovo Karelian ASSR Russia r 0 3 3 Downloaded from https://academic.oup.com/forestry/article/70/3/211/543878 by guest on 29 September 2021 56 Orehovo Karelian ASSR Russia r 0 2 2 57 Otrodnoe Karelian ASSR Russia r 0 3 3 58 SannSs Uusimaa Finland r 0 2 2 59 Espoo Uusimaa Finland r 0 1 1 60 Bromarv Uusimaa Finland r 0 2 2 61 Lemu Turku Pori Finland r 1 1 1 62 Katariinanlaakso Turku Pori Finland r 1 1 1 63 Taivassalo Turku Pori Finland r 1 1 1 64 Lohja Uusimaa Finland r 0 6 6

Table 2: European samples of Quercus robur given in Figure 2. The numbers of individuals sequenced for the intron and those digested with Mbo\ and Rsal are given. Total sample size = 44

No. Site District Country Number Number Total sequenced digested

1 Sherwood Forest Nottinghamshire England 0 2 2 2 Llandeilo Dyfed Wales 0 1 3 Stoneleigh Warwickshire England 0 1 4 Castor Cambridgeshire England 0 1 5 Downham Market Norfolk England 0 1 6 Knapwell Wood Cambridgeshire England 1 1 7 Hillborough Norfolk England 0 1 1 8 Rackheath Norfolk England 0 1 1 9 Hornnger Suffolk England 0 1 1 10 Staverton Park Suffolk England 1 1 1 11 Epping Forest Essex England 0 1 1 12 West Knoyle Wiltshire England 0 1 1 13 Windsor Great Park Berkshire England 0 4 4 14 Gatwick West Sussex England 0 1 1 15 Le Mans Sarthe France 0 1 1 16 Fontainbleau Seine et Marne France 0 1 1 17 Bitchc Moselle France 1 2 2 18 Chize Deux Sevres France 0 1 1 19 Pau Pyrenees Atlantiques France 0 2 2 20 Feurs Loire France 0 2 2 21 Pilat Loire France 0 1 1 22 Lausanne Vaud Switzerland 0 1 1 23 Uetliberg Zurich Switzerland 0 2 2 24 Stamscr Tyrol Austria 1 1 1 25 Bad Neuheim Hessen Germany 1 2 2 26 Hasbruch Niedersachsen Germany 1 1 1 27 Lubeck Holstein Germany 2 2 2 28 Skjoldenaesholm Sjaelland Denmark 1 1 1 29 Scania Veberod Malmohus Sweden 2 6 6 216 FORESTRY

Table 3: British samples of Quercus species given in Figure 3. Numbers of individuals sequenced for the intron and digested with Mbol and Rsal are given, r = Q. robur, p = Q. petraea. Total sample size = 103

No. Site District Species Number Number Total sequenced digested

1 Sherwood Forest Nottinghamshire P 0 2 2 2 Major Oak Nottinghamshire r/hybrid 0 1 1 3 Wyre Forest Hereford & Worcester r/p 1/0 3/1 4 4 Mitre Oak Hereford & Worcester r 1 1 1 Downloaded from https://academic.oup.com/forestry/article/70/3/211/543878 by guest on 29 September 2021 5 Rushwick Hereford & Worcester r 0 1 1 6 Birmingham West Midlands r 0 3 3 7 Tamworth Staffordshire r 0 1 1 8 Baginton Oak Warwickshire r 0 1 1 9 Stoneleigh Warwickshire r 0 4 4 10 Fosseway Warwickshire r 0 1 1 11 Daventry Northamptonshire r 0 1 1 12 Denton Northamptonshire r 0 1 1 13 Thrapston Northamptonshire r 0 1 1 14 Leicester Leicestershire r 0 1 1 15 Tixover Leicestershire r 0 2 2 16 Castor Cambridgeshire r 0 2 2 17 Knapwell Wood Cambridgeshire r 1 1 1 18 Croxton Cambridgeshire r 0 1 1 19 Hayley Wood Cambridgeshire r 0 2 2 20 Hildersham Cambridgeshire r 0 2 2 21 Bedford Bedfordshire r 0 1 1 1 22 Waltham Abbey Essex r 0 1 2 2 23 Epping Forest Essex r 0 1 1 24 Rettendon Essex r 0 4 4 25 Windsor Great Park Berkshire r 0 5 5 26 Ascot Berkshire r 0 1 1 27 Gatwick West Sussex r 0 1 1 28 Somerset 0 Penselwood r 1 1 29 West Knoyle Wiltshire r 0 1 1 30 Dunkirk Kent P 0 2 2 31 Thorpe Morieux Suffolk r 1 2 2 32 Felshamhall Wood Suffolk r 1 1 1 33 Horringer Suffolk r 0 2 2 34 Staverton Park Suffolk r 1 1 35 Little Glemham Suffolk r 0 1 36 Bramfield Suffolk r 0 1 37 Queen's Oak Suffolk r 1 1 38 Winfarthing Norfolk r 0 3 39 Larling Norfolk r 0 1 40 Attleborough Norfolk r 0 41 Ken's Oak Norfolk r 1 42 Alpington Norfolk r 0 43 Rackheath Norfolk r 0 44 Wroxham Norfolk r 0 45 Blickling Norfolk r 0 46 Bodham Norfolk r 0 47 A148 Norfolk r 0 48 Fakenham Norfolk r 0 49 Reepham Norfolk r 0 50 Sparham Norfolk r 0 INTRODUCED PROVENANCES AND TRANSLOCATIONS 217

No. Site District Species Number Number Total sequenced digested

51 Guesrwick Norfolk r 0 3 3 52 Dereham Norfolk r 0 1 1 53 Seaming Norfolk r 0 1 1 54 Necton Norfolk r 0 1 J 55 Hillborough Norfolk r 0 • 1 1 56 Downham Market Norfolk r 0 1 1 57 King's Lynn Norfolk r 0 1 1 Downloaded from https://academic.oup.com/forestry/article/70/3/211/543878 by guest on 29 September 2021 58 Bradgate Park Leicestershire r 0 1 1 59 Gunthorpe Leicestershire r 0 1 1 60 Harthill South Yorkshire r 0 1 1 61 Castle Howard North Yorkshire r 0 4 4 62 Filey North Yorkshire r 0 3 3 63 Bishop Auckland Durham r 0 5 5

buffer at 5 V cm"1 for 3 h. Gels were stained as distribution confined to Britain except for a above. The PCR product is cut into four frag- single site in the south of Sweden (Figure 2). ments. When the duplication is absent a clear Within Britain the duplication has a mainly East four-banded pattern is produced, but when pres- Anglian distribution (Figure 3). The sites repre- ent the fragment, now 13 bp longer, comigrates sent presumed native genotypes except for sites with another fragment giving a clear three- 3, 26, 27, 30, 40, 47, 60, 62 and 63, which are banded pattern. trees of girth at breast height of 10 feet (3 m) or less, site 44, in part of an old Wroxham estate, and site 61 in the grounds of Castle Howard, York. Results Even when these sites are ignored, a baseline The single base mutation from T to C at posi- pattern is unclear as we still have eight sites tion 153 of the oak chloroplast tRNA1-0"1 intron, within East Anglia which lack the duplication, as identified by restriction analysis, shows an and Windsor Great park in which four very old east-west division across Europe for native sam- trees contain the duplication. ples (Figure la). A polymorphic population Of the 50 old trees sampled south of Norwich occurs at Lubeck in northern Germany (site 46) only one lacked the duplication, a tree in the and may represent the point of contact of the grounds of Stanfield Hall. two cytotypes in this region. This pattern can Again some of the restriction analysis results thus be used as a baseline for native genotypes. were tested by DNA sequencing, 10 with and 13 In total, 13 eastern and 23 western individuals without the duplication, and all sequences con- were sequenced and all results confirmed the firmed the restriction results. restriction analysis results. The pattern for 12 sites across Europe where native status is uncertain, is given in Figure lb. The results for six of the sites do not conform Discussion to the native pattern. The eastern type is found East-west Europe at sites 4, 13-15 and 23, which are within the western region, while the western type is found The tRNA1-*"1 intron is a self-splicing group I at site 39, within the eastern region. intron (Cech, 1988) which has been implicated The 13 base pair duplication at position 335 in the evolution of chloroplasts from bacteria of the oak chloroplast tRNALcul intron, has a (Kuhsel etal., 1990; Xu et al., 1990). The single 218 FORESTRY

(a) Downloaded from https://academic.oup.com/forestry/article/70/3/211/543878 by guest on 29 September 2021

(b)

Figure 1. Polymorphism across Europe for a chloroplast DNA marker in Quercus robur and Q. petraea. (a) Samples from very old trees and from ancient wildwood; (b) Trees of dubious provenance. The arrowed line indicates the presumed line of contact of the two cytotypes following postglacial migration. Sample details are given in Table 1. Filled symbols represent the wildtype condition, a T at position 153 of the tRNA1-™1 intron, while open symbols represent a C. Circles = Q. robur, squares = Q. petraea. INTRODUCED PROVENANCES AND TRANSLOCATIONS 219 Downloaded from https://academic.oup.com/forestry/article/70/3/211/543878 by guest on 29 September 2021

Figure 2. Polymorphism within western Europe for a 13 bp duplication in the chloroplast tRNA1-*™1 intron of Quercus robur. Filled symbols indicate presence of the duplication. Sample details are given in Table 2.

base mutation T to C in the intron occurs in a trees in Florence attests to the western type highly conserved region, the R sequence, and migrating northwards from an Italian refuge. has to date only been found in oaks. It is pres- However, ice would have persisted for a consid- ent in Quercus robur and Q. petraea but was not erable time along mountain ranges and thus found in 13 outgroup Quercus species (Ferris et formed barriers to migration. Fossil pollen data al., 1993). indicates this to be true for the line of the Alps The mutation has a clear eastern distribution around northern Italy (Huntley and Birks, which is best explained by the postglacial his- 1983), and thus it seems possible that for oaks, tory of oak (Ferris et al., 1993). The mutation Italy has to a great extent been isolated from the probably occurred in a glacial refugium in the rest of Europe. The presence of the mutant cyto- Balkan area during the last ice-age and became type across Austria and Switzerland agrees with fixed while the population size was small. When this hypothesis (Figure la). We thus had migra- the oaks migrated north as temperature tion of oaks from Iberia, northward along either increased, the west was colonized from refugia side of the Pyrenees and into western Europe, in southern Iberia and Italy (Huntley and Birks, while the mutant type colonized eastern Europe. 1983), while the mutant cytotype filled eastern The two waves of migration met in the south, parts of Europe. somewhere between Switzerland and France, The results in Figure la show the most com- and formed a line of contact which moves fur- plete data for the native distribution of the ther east as we go north (Figure la), due per- mutation. While the number of samples in Italy haps to a more rapid migration along the east is poor, the absence of the mutation from five coasts of Europe, favoured by the warmer 220 FORESTRY

Those sites that do not conform to the natural pattern must be explained by gene flow or introduction. The Swiss site at Uetliberg in the Zurich region is of unknown status. It is however, fairly close to the likely line of contact and thus could be attributed to natural gene flow. Sites 13—15 represent areas of planted forests, well maintained and with little or no understorey species. For example, at Bitche two plants Downloaded from https://academic.oup.com/forestry/article/70/3/211/543878 by guest on 29 September 2021 were tested. One tree in a stand of pure Q. robur proved to be of eastern type, while a tree from a nearby wooded slope of mixed species, proved to be western type. Similarly, at Metz on the edge of the ForSt de Bezange Grande, a tree in the plantation was of eastern type while a seedling on the other side of the road was of western type. Clearly seed of east European origin has been used for some French oak plantation forests. The trees from site 23 are from the Montech Arboretum near the Forfit d'Agre in the south of France. Again these are obviously good candi- dates for planting. Two eastern type trees were also found at Broadwell in the Forest of Dean. Both are sessile oaks and are part of a stand of uniform age, again with little understorey—presumably a plantation of east European provenance. This chloroplast DNA marker seems very good for identifying oaks of east European provenance. That it can be used for both species of oaks is of great interest and use. First, the Figure 3. Polymorphism within Britain for a 13 bp mutation is likely only to have occurred once duplication in the chloroplast tRNA1-™1 intron of and thus spread from one species to the other Quercus robur. Filled symbols indicate presence of presumably by hybridization and introgression. the duplication. Sample details are given in Table 3. That these species hybridize is well documented (Gardiner, 1970), and fixation by pollen swamp- ing as proposed for Eucalyptus (Potts and Reid, oceanic conditions. There appears to be a clear 1988), is certainly possible. Second, we can use division in the south of Finland. The contact one marker for both species, thus saving time zone given in Figure la, therefore, represents the and effort. It will also be possible to assess the most likely natural /native geographic pattern of relative frequency with which the two oaks have variation in oaks. been used for plantations. The results for trees of dubious provenance that we tested, do not conform to this pattern (Figure lb). Of the 12 sites shown only six pos- East Anglia sess the natural pattern. The Estonian and Croa- The 13 base pair duplication occurs in one of tian trees (sites 53, 32, 33), despite being of the most variable regions of the intron (Ferris et unknown size and origin, do appear to be of al., 1995), and has so far only been found in Q. native eastern stock. Likewise, sites 9, 19 and 51, robur. It has a predominantly East Anglian dis- would appear to contain native western trees. tribution which can be explained by the post- INTRODUCED PROVENANCES AND TRANSLOCATIONS 221 glacial history of the species (Ferris etal., 1995). Of the 50 trees in the 12 X 12 km area south Oaks first migrated into Britain some 10 000 west of Norwich, of girth 15 feet (4.6 m) and years ago, occurring first in the south-west over, only one lacked the duplication. This is a (Godwin, 1975; Huntley and Birks, 1983). Entry tree in the grounds of Stanfield Hall and is a via the eastern landbridge was a little later, very likely candidate for being planted. It is pos- some 8500-9000 years ago, partly due to the sible to look at young trees in this area and test dominance of pine in the east of Britain (God- them for the marker. Preliminary results from win and Deacon, 1974). East Anglia was colo- several small trees in this area show that recent

nized by oak when the conditions became planting of trees from outside East Anglia is Downloaded from https://academic.oup.com/forestry/article/70/3/211/543878 by guest on 29 September 2021 favourable some 8000 years ago. The source quite common. population for this spread was from the south One further site to be explained is in Sweden of England. The absence of the duplication from (Figure 2) where six trees at Scania Veberod all the mainland neighbouring countries (Figure 2), contain the duplication. It would seem that suggests that the mutation occurred within translocations out of East Anglia, even as far as Britain while the oaks were in southern Eng- Sweden, are possible! land. Seemingly oaks have been translocated into When we look at the distribution of the dupli- and out of East Anglia for at least three hundred cation within Britain we see that the pattern is years, this being the approximate age of some of not very clear (Figure 3). We have many old the trees which do not fit the native pattern. The trees in East Anglia which do not contain the maximum age of trees showing translocation duplication, and a few outside of East Anglia across Europe is much less, the non-native plan- which do possess it. First those trees sampled at tation and arboretum trees identified being Ascot and Windsor (sites 25, 26) represent some about a hundred years old at most. of the oldest trees in the country. The largest The two DNA markers presented here give an girth measurement at Windsor was 27 feet (8.2 insight into the patterns of natural genetic vari- m) at breast height. Clearly this tree is many ation formed following the last glaciation. To centuries old. It could be that the trees here were some extent these patterns are still very promi- gifts from Norfolk! Alternatively, the nent despite much planting of foreign genotypes Ascot/Windsor region could be in the area of over the last few centuries. For conservation the source population from which East Anglia purposes the scale of the investigation is very was colonized. Clearly more samples are needed important. First, if we examine the site in south- from this area. Of the trees in the Wyre Forest, ern Sweden using the east-west marker, we find one small Q. robur, contains the duplication that the trees are of native western type as while the others, all Q. petraea, are wild type. expected. This marker fails to show that the site The marker reveals this to be a translocation is likely to represent translocation from East from East Anglia. Similarly, four trees from Anglia. Second, if we examine the trees in the Castle Howard, York, and one from Bishop Forest of Dean using the East Anglian marker, Auckland, all Q. robur, contain the East Anglian we find that the trees lack the marker as duplication. Within East Anglia (sites 20, 31 expected. We fail to show that these trees are of through 57), the samples are all large trees or east European origin. Thus each marker gives its wildwood specimens. Nine sites have trees that own discreet information, and a complementary lack the duplication. How can these be suite of markers is useful. explained? Many of these trees are situated The techniques have proved useful for isolat- alongside major roads into Norfolk, the All, ing chloroplast DNA markers in oaks. For other A148 and A47. Some old roadside trees in East genera, which may be less genetically variable, Anglia, therefore, are not native, despite being mitochondrial DNA would be a good alterna- of old age. Some planting of roadside trees must tive to chloroplast DNA. The strategy for using have taken place centuries ago. Wroxham (44), these markers to identify native versus non- is in the grounds of part of a former old estate. native in oaks is successful. It must be stressed For sites 31, 49 and 51, we have no obvious that only known or presumed native samples explanation. were used for the baseline. In oaks the sampling 222 FORESTRY strategy was to use trees over about three hun- Huntley, B. and Webb, T. Ill 1989 Migration: dred years old, or to use ancient wildwood trees. Species' response to climatic variations caused by This may not be possible for other genera, espe- changes in the earth's orbit. /. Biogeogr. 16, 5-19. cially short-lived species, where the sampling Kremer, A., Petit, R., Zanetto, A., Fougere, V., strategy would need to be modified. Ducousso, A., Wagner, D. and Chauvin, C. 1991 Nuclear and organelle gene diversity in Quercus robur and Q. petraea. In Genetic Variation of For- Acknowledgements est Tree Populations in Europe. M. Ziehe and G. Muller-Starck (eds). Sauerlander's Verlag, Frank-

Part of this work was completed under a grant from furt am Main, 141-166. Downloaded from https://academic.oup.com/forestry/article/70/3/211/543878 by guest on 29 September 2021 the National Environment Research Council Kuhsel, M.G., Strickland, R. and Palmer, J.D. 1990 (GR3/8101). We would like to thank the many people An ancient group I intron shared by eubacteria and who helped to provide us with samples, and Andy chloroplasts. Science 250, 1570-1573. King for technical help. Lavin, M., Mathews, S. and Hughes, C. 1992 Chloro- plast DNA variation in Gliricidia sepium (Legumi- nosae): intraspecific phylogeny and tokogeny. Am. References J. Bot. 78, 1576-1585. Potts, M.M. and Reid, J.B. 1988 Hybridization as a Cech, T.R. 1988 Conserved sequences and structures dispersal mechanism. Evolution 42, 1245-1255. of group I introns—Building an active site for RNA Soltis, D.E., Soltis, P.S., Ranker, T.A. and Ness, B.D. catalysis—A review. Gene 73, 259-271. 1989 Chloroplast DNA variation in a wild plant, Denton, G.H. and Hughes, T.J. 1981 The Last Great Tolmiea menziesii. Genetics 121, 819-826. Ice Sheets. J. Wiley, New York. Soltis, D.E., Mayer, M.S., Soltis, P.S. and Edgerton, Doyle, J.J. and Doyle, J.L. 1990 Isolation of plant M. 1991 Chloroplast DNA variation in Tellima DNA from fresh tissue. Focus 12, 13-15. grandiflora (Saxifragaceae). Am. J. Bot. 78, Doyle, J.J., Doyle, J.L., Grace, J. and Brown, A.H.D. 1379-1390. 1990 Reproductively isolated polyploid races of Soltis, D.E., Soltis, P.S. and Milligan, B.G. 1992 Glyctne tabacina (Leguminosae) had different Intraspecific cpDNA variation: Systematic and chloroplast genome donors. Syst. Bot. 15, 173-181. phylogenetic implications. In Molecular Systemat- Ferris, C, Olivers, R.P., Davy, A.J. and Hewitt, G.M. ics of Plants. D.E. Soltis, P.S. Soltis and J.J. Doyle 1993 Native oak chloroplasts reveal an ancient (eds). Chapman & Hall, New York, 117-150. divide across Europe. Mol. Ecol. 2, 337-344. Taberlet, P., Gielly, L., Pautou, G. and Bouvet, J. Ferris, C, Oliver, R.P., Davy, A.J. and Hewitt, G.M. 1991 Universal primers for amplification of three 1995 Using chloroplast DNA to trace postglacial non-coding regions of chloroplast DNA. Plant migration routes of oaks into Britain. Mol. Ecol. 4, Mol. Biol. 17, 1105-1109. 731-738. Whittemore, A.T. and Schaal, B.A. 1991 Interspecific Gardiner, A.S. 1970 Pedunculate and sessile oak gene flow in sympatric oaks. Proc. Nat. Acad. Sci. {Quercus robur L. and Quercus petraea (Mat- USA 88, 2540-2544. tuschka) Liebl.). A review of the hybrid contro- Xu, M-Q., Kathe, S.D., Goodrich-Blair, H., versy. Forestry 43, 151-160. Nierzwicki-Bauer, S.A. and Shub, D.A. 1990 Bacte- Godwin, H. 1975 The History of the British Flora, 2nd rial origin of a chloroplast intron—Conserved self- edn. Cambridge University Press, Cambridge. splicing group I introns in cycanobacteria. Science Godwin, H. and Deacon, J. 1974 Flandrian history of 250, 1566-1570. oak in the British Isles. In The British Oak. M.G. Zurawski, G., Clegg, M.T. and Brown, A.H.D. 1984 Morris and F.H. Perring (eds), Pendragon Press, The nature of nucleotide sequence divergence Cambridge, 51—61. between barley and maize chloroplast DNA. Howland, D.E. 1992 Molecular variation in populations Genetics 106, 735-749. of Betula L. PhD Thesis. University of East Anglia. Huntley, B. and Birks, H.J.B. 1983 An Atlas of Past Received 20 August 1996 and Present Pollen Maps for Europe: 0-13000 years ago. Cambridge University Press, Cambridge.