BOT.GAZ. 138(4): 512-521. 1977. (h) 1977 by The Universityof Chicago.All rightsreserved. ISOENZYME IDENTIFICATION OF PICEA GLAUCA, P. SITCHENSIS, AND P. LUTZII POPULATIONS1 DONALD L. COPES AND ROY C. BECKWITH USDA Forest Service,Pacific Northwest Forest and Range ExperimentStation ForestrySciences Laboratory, Corvalli.s, Oregon 97331 Electrophoretictechniques were used to identify stands of pure Sitka sprucePicea sitchensis (Bong.) Carr.and purewhite spruceP. glauca (Moench)Voss and sprucestands in whichintrogressive hybridization betweenthe white and Sitka sprucehad occurred.Thirteen heteromorphic isoenzymes of LAP, GDH, and TO were the criteriafor stand identification.Estimates of likenessor similaritybetween seed-source areas were made from 1) determinationsIntrogressed hybrid stands had isoenzymefrequencies that were in- termediatebetween the two purespecies, but the seedlingswere somewhat more like white sprucethan like Sitkaspruce. Much of the west side of the KenaiPeninsula appeared to be a hybridswarm area, with stands containingboth Sitka and white sprucegenes. The presenceof white sprucegenes in Sitka sprucepopula- tions was most easily detectedby the presenceof TO activity at Rm .52. White spruceshowed activity at that positionin 79% of its germinants;only 1% of the pure sitka sprucegerminants had similaractivity. Isoenzymevariation between stands of pure Sitka sprucewas less variablethan that betweeninterior white spruce stands (mean distinctionvalues were 0.11 for Sitka and 0.32 for white spruce). Clusteranalysis showedall six pure Sitka sprucepopulations to be similarat .93, whereaspure white sprucepopulations werenot similaruntil .69. Introduction mining quantitatively the genetic composition of Hybrids between Picea glauca (Moench) Voss and stands suspected of being of hybrid origin would be Sitka spruce Picea sitchensis (Bong.) Carr. were of great use to foresters and researchers. In this first reported by LITTLE(1953) and named P. X case, seed from known hybrid trees is needed for test lutzii. Natural hybridization between white spruce plantations where insect resistance and growth po- and Sitka spruce was found along river drainages in tential for timber production can be evaluated. British Columbia where the two species grew con- In British Columbia, sxmpatric white and Sitka tlguously (DAUBENMIRE1968; ROCHE1 969) . Many spruce populations are not common; but along trees in British Columbia had morphologicalcharac- several river drainages such as the Nass, the Skeena, teristics indicating that introgressive hybridization and the Bulkley Rivers the montane white spruce had occurred. The general form of many trees was and the coastal Sitka spruce populations grow close often similar to that of white spruce, but cone size enough together to interbreed (ROCHE1969). Con- and cone-scale structure were more like Sitka spruce. tinuous gradations in cone-scale morphology were Other trees were intermediate between the two found in the transition stands along the Skeena River species. A similar hybrid situation may occur on the area (DAUBENMIRE1968; ROCHE1969). The inter- Kenai Peninsula in Alaska, Mhere a smooth-bark mediate populations became more like Sitka spruce variety of white spruce, P. glauca var. porsildii at the lower elevations near the Pacific Ocean and Raup., is also found. We questioned the genetic more like white spruce at the higher elevations near composition or species makeup during researchnsork the pure white spruce populations. on the spruce beetle, Dendroctowtus rufipennis(Kby.). The separation of white spruce and Sitka spruce Trees in some areas appeared to have intermediate populations on the Kenai Peninsula of Alaska is morphologicalcharacteristics. somewhat more complex. The Sitka spruce is at the A knowledge of the genetic composition of a tree northern limit of the range of the species (VIERECK is essential when considering growth, form, site and LITTLE1972). On the west side of the peninsula adaptability, and resistance to pests. Interest in and in contrast to British Columbia, a major methods for positive identification of lutsii hybrids> difference in elevation does not separate white has also occurredin Oregon,Washington, and British spruce from Sitka spruce. The factor limiting Columbia because of suspected resistance of the survival has been suggested to be the low tempera- hybrids to the white pine weevil (= Sitka spruce tures during the growing season which cause seedling weevil), Pisso(lesstrobi (Peck) (MITCHELL,JOHNSO N, mortality (FOWELLS1965). and WRIGHT1974). A simple technique for deter- Picea X lutsii areas are diflicult to identify when traditional morphologicaltraits are used to evaluate 1Abbreviations: GDH = glutamatedehydrogenase, LAP = stands in which low levels of introgressionhave oc- leucineaminopeptidase, Rm = relativemobility value, TO = curred.Sitka and white spruce areof recentmonophy- tetrazoliumoxidase, D = mean degreeof diiFference. letic origin (WRIGHT1955); thus most traits are very Manuscript receivedJanuary 1977; revised manuscript received similar. One possible way around this limitation is June 1977. the use of biochemical techniques similar to those 512 20.21.23.12.13.18.14.15.17.19.16.9.8.4.7.2.3.5.1. Skeena AfognakGerstleDryTananaBonanzaCharleyMendenhall,FortPhalarope,HookKodiak,Ninilchik,MooseHaines,Juneau,Soldotna,Seward,Homer,Kenai,MeansRiver, Gulch, St.Lake, Pass, Island,River, B.C River,Creek, AlaskaAlaska AlaskaJames,................. Alaska ........................ AlaskaAlaskaAlaska AlaskaAlaska Alaska Alaska Alaska ................................................B.C........................ 40 ........................ ........................................................................ ........................ ........................ ........................................................................ ............................................50 ... 3 372755 77507240 21687607003020405450 84 8050 0 900 172 3002 0 3 0 0 04 1363 17600 4 2346010486354 11804354100 1121 1009710053 3 379739139928' 05100644 1278 100 37100 3886509535070868432 79 12 740 8015 5337 16100504060 0429743 3 0 153053 037 060 100 100 906591984047357032937083 COPES& BECKWITH IDENTIFICATIONOF ISOENZYMES 513 proposedby HANOVERand WILKINSON(1970). The) stands of pure white spruce were sampled from the used paper chromatographymethods to evaluate interior areas where crossbreedingwith Sitka spruce phenolsfound in needlesof Sitka and white spruce was thought impossible (areas 1-6), and six stands and suspectedhybrids from the SkeenaRiver and of pure Sitka spruce were sampled along the coast found that the three tree tSpes could be separated where no white spruce grew nearby (areas 19-24) by threecompounds. A numericalestimate of degree (table 1). Twelve stands were selected in areas where of introgressionwas not determined. introgressionwas thought possible: 10 of the 12 were This paper reportsanother biochemical method located on the Kenai Peninsula (areas 7-10 and fordetecting genetic variation. Electrophoresis meth- ods wereused to determinethe isoenzymesfound in 12-17); one was across Cook Inlet from the Kenai newlygerminated seedlings for a numberof enzyme Peninsula near Tyonek (area 11); and one was near systems.The seedlingswere grownfrom seeds col- Salvus, British Columbia (area 18), along the Skeena lected in pure Sitka and pure white sprucestands River (table 1). Collectionswere made independently and sprucestands of uncertaingenetic composition. by a number of individuals, and the clean seed was sent to Corvallis, Oregon, for study. The exact Materialand methods number of seed trees sampled from each stand was Seedsfor electrophoretic study were collected from not always known, but for the Kenai stands it was trees growingin 24 differentstands (figs. 1, 2). Six always greater than five. In several mainland iBLE 1 ISOENZYMEBAND FREQUENCIES(WO) FOR LAP, GDH, AND TO IN 24 WHITE,SITKA, AND PUTATIVEHYBRID SPRUCE STANDS Rm LAP TO GDH SEEDLINGSNo SEED SOURCE: SAMPLED .52 .5 4 .5 6 .58 .60 .62 .52 .22 .25 .27 .30 .32 .35 NTO . AN D LOCATION Interior white spruce: 0 76 2 34 22 0 5 56 0 43 12 0 0 38 9 27 49 6 0 45 0 28 37 10 4 59 0 32 32 2 6. Haines Junction, Yukon. 70 0 46 2 54 20 0 57 0 50 0 53 33 0 Means . ........................ ... 1 50 15 49 30 15 79 2 54 2 36 31 3 Kenai Peninsula putative white spruce: 1 39 0 37 61 0 0 0 0 22 87 0 2 12 0 24 71 0 10. Barabara Lake, Alaska ........................50 34 48 0 42 22 0 100 0 27 0 73 0 0 Means ........................ ... 17 26 59 20 32 9 94 1 19 0 39 55 0 Alaskan putative hybrids: 11. Tyonek, .9^laska........................ 40 30 55 35 10 10 0 77 0 38 58 45 8 0 0 30 10 30 50 0 0 88 63 10 0 0 0 46 43 43 22 10 0 65 25 45 0 O 0 63 40 30 0 0 0 58 75 38 3 0 54 2 57 0 55 45 34 12 0 Canadian putative hybrid: 0 12 20 78 O O O 0 86 0 12 14 0 Sitka spruce: 7 4 83 40 11 o o o 3 81 0 14 1 0 o 7 92 8 7 2 0 22. KodiakIsland o 0 98 0 2 O O o O 100 4 0 O O 24. Sooke,B.C 5 0 .................0 ... 0 4 100 86 0 o 4 96 0 0 O O Means ..................... ... O 1 3 99 93 0 1 3 92 9 6 0 0 A L A S K A , I . 2 * \ * \ oFairb;nks \ C A N A D A 6 s7- 10 12- 17 / st22 5 18 *o Ft. St. James z>;,23 FIG. 1.-Location of 24 sampledstands; see table 1 for namesof areas COPES & BECKWITH-IDENTIFICATION OF ISOENZYMES 515 ll 109 12 16 14 a r d Se w ALASKA KENAI PENINSULA, Hl FIG. 2.-Enlarged view of seed-sourcelocations on the Kenai Peninsula,Alaska; see table 1 for namesof areas locations,seed was obtainedfrom commercialcol- liquid and placed at regularintervals on a 3 X lectionsof severalhundred trees. 30-cm strip of thin plastic wrap. The strip was The seedsfrom each stand weregerminated prior rolled so that no wick was ever in direct contact to preparingthem for electrophoreticstudy. Newly with any other wick. Each roll contained10 wicks, germinatedseedlings were used rather than un- which is one-half the number needed to fill all germinatedseeds in order to eliminate variant availablespaces in one gel. Two rolls of wicks were isoenzymesthat wouldhave occurredfrom electro- then placed in 8-ml vials, capped, and stored at phoresisof filled but nonviableseed.