Isoenzyme Identification of Picea Glauca, P. Sitchensis, and P. Lutzii Populations1

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 sprucepopulations 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 Afognak GerstleDryTananaBonanzaCharleyMendenhall,Phalarope,HookFort Kodiak,Ninilchik,MooseHaines,Juneau,Soldotna, Seward,Homer, Kenai,MeansRiver, Gulch, St. Lake, Pass, Island,River, B.C River, Creek, Alaska Alaska AlaskaJames, ...... Alaska ...... Alaska Alaska Alaska Alaska Alaska Alaska Alaska Alaska ...... B.C ...... 40 ...... 50 ... 3 372755 7772 5040216 876070 030 20405450 84 8050 0 900 172 300 20 3 0 00413 631760 0 4 2346010 48 635411 80 4354100 1 1 21 10097100 53 3 379739139928 '05100 644 12 78 100 37100 388650 9535 0708684 3279 12 74 0 80 1553 3716 100 50 40600 4297 43 3 015 3053 037 0 6 0 100 100 9065919840473570 32937083

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 methods 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

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

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

109

a r d Se w ALASKA KENAI PENINSULA,

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. The seedlings -80 C until they were insertedin gels for electro- containedgenes from many of the surroundingtrees phoretic separation.Wicks from each stand were that werethe pollenparents of the seedlingsas well randomizedso that only 10 wicksof one sourcewere as genesfrom the seed trees. placedin any one gel, whilethe other10 wicksin the The possibilitythat foreignor nonlocalgenes are gel werefrom another stand picked at random.Time in the seedlingsis not very likely.The proportionof in-80 C storagevaried from 2 days to 3 mo. No effective pollen producedby surroundingtrees is aberrationsin isoenzymesresulted from freezingor always far greaterthan that shed by trees from a lengthof storage. great distance.Thus with adequatesample size the Electrophoreticapparatus used was similar to seedlingsshould accurately indicate the true genetic that describedby CONKLE(1972). Starch gels (12%o) compositionof the adjacentstand. were preparedabout 1 h before electrophoresis. Seedlingswere grownuntil they were 1.0-2.0 cm Thick gels (16-18 mm) were made so that six long. The seed coats and endospermswere removed, usable2-mm-thick slices could be slicedhorizontally and the seedlingswere maceratedin 1 drop of the from each gel. Each gel contained467 ml of gel gel buffersolution described below. The numberof buffer solution (417 ml of tris-citrate [0.07 M, germinantssampled from each area is listed in pH 8.3], 50 ml of lithiumborate [0.25 M, pH 7.4]), table 1. and 56 g of hydrolyzedstarch. The electrodebuffer Maceratedfluids from each seedling were absorbed waslithium borate (0.25 Mn pH 7.4). Electrophoresis on one 5 X 13-mm paper wick (Whatmanno. 1 was performedat 8 C with a constant currentof chromatographypaper) blotted to remove excess 100 mA (7.6 mA/cm) until the voltage reached BOTANICALGAZETTE [DECEMBER 516 400 V; then constant current was changed to 400 V which white spruce had introgressed into Sitka (30.4 V/cm). Electrophoresis was continued until spruce was TO. This isoenzyme was almost species the bromophenolblue markerdye had traveled 8 cm specific. Of the germinants from pure interior white from the origin slit. Time for electrophoresisvaried spruce stands, 79SO showed activity at Rm .52, from 3.0 to 3.5 h. whereas only 1<7oof the germinants from pure Sitka Peroxidase, esterase, acid phosphatase, GDH, spruce showed activity at the same band position LAP, and TO were the enzymes examined in the (table 1). Germinants grown in suspected hybrid spruce seedlings. Peroxidase, acid phosphatase, es- areas on the Kenai Peninsula had intermediate TO terase, and LAP were visualized with the procedures values averaging 57%. The only stand that could described by SC.\NDALIOS(1969), GDH bs the be suspected of hybridization which showed no TO method of Stt.&wand PRAS.\D(1970), and TO by the activity at Rm .52 was area 18, sampled near the method of BAURand SCHORR(1969). Diagrams of Skeena River in British Columbia. isoenznme patterns were recorded for every gel Isoenzymes LAP and GDH were useful for identi- slice-enzyme combination. Isoenzvme positions were fying the two pure spruce species; their bands had expressed as Rm: the distance the isoenzyme had average frequency values that differed widely be- migrated from the origin toward the anode divided tween species. For example, Sitka spruce seedlings bn the distance the bromophenol hlue marker dye had low average frequencies for LAP at Rm .52 had moved from the origin slit toward the anode. (OSo) .54 (lSo) and .56 (3%o)and high values at Analysis for degree of difference between stands Rm .58 (99%o)and .60 (93%o)(table 1). Interior was calculated for 13 isoenzymes according to the white spruce seedlings had higher average frequen- method of MUHS (1974), whereby the frequency cies than Sitka spruce at Rm .54 (50Co) and .56 values (p) were transformed into radians with the (15%6)and lower values than Sitka spruce at Rm following equation: 0 = sin-1 (1-2p). The D be- .z8 (49So) .60 (30So) and .62 (75%o)(table 1). The tween stands was calculated as most reliable LAP isoenzyme indicators were the Rm .54 and .56 bands. Values above 20<7oor 30%o at one or both Rm positions indicated frequencies kE[(ali-02j)2_( 1-+-1-)] i=l mlj m2j that were common in pure white spruce or in putative hybrid stands where introgression with white Sample size of the two stands being compared was spruce had occurred. At Rm .58 and .60, values of m1 and m2. The number of characters (isoenzymes) 80So-100<70generally represented stands of pure included in the study was k, and in this study Sitka spruce. Only one or two isoenzymes should k = 13. Some isoenzymes were probably controlled not be used for stand identification because some by alleles at the same genetic locus. This dependent overlap of frequency values was noted between condition may have introduced a small amount of several populations of one species and those of the irregularity into some D values, but it was not other species. For example, the LAP Rm .58 band thought to alter the results significantly. for area 4 white spruce was present in 86%oOf the A phenogram was developed after cluster analysis seedlings. That was much higher than that normally by group averages using the Bray-Curtis dissimilar- encountered in white spruce and nearly as high as ity matrix (MCRLIB Oregon State University that found in pure Sitka spruce (97%6-lOOfWo) Computer Center). (table 1). The GDH isoenzyme differencesbetween seedlings Results of Sitka and white spruce followed a pattern similar Sufficientisoenzyme variation between Sitka spruce to that of LAP. High frequency values at Rm .25 and white spruce seedlings was found to allow (92%) (table 1) were common in Sitka seedlings, identification of both species and intermediate whereas lower values were normal in seedlings from populations in which introgressive hybridization white spruce stands (54(JO). More characteristic had apparently occurred. Enzymes useful for this isoenzvmes for separating species were the Rm .30 purpose were LAP, GDH, and TO. The isoenzymes and .32 bands. Isoenzyme frequency of these bands were as follows: LAP = Rm .52, .54, .56, .58, .60, averaged 36So and 31%oin white spruce and only .62; GDH= Rm .22, .25, .27, .30, .32, .35; TO- 6'Xoand 0SO in pure Sitka spruce seedlings. The Rm .52. Other isoenzymes of these enzymes occurred suspected Alaskan hybrids had a mean frequency of but were of no use for identification because no 34%Oat Rm .30 and 12So at Rm .32. This Rm .30 between-species variation was observed. Data on value indicated a closer similarity to white spruce isoenzymes of acid phosphatase, esterase, and peroxi- than to Sitka, but the Rm .32 showed an interme- dase were not included in the report because either diate condition. their isoenzymes were inconsistent in stain develop- Isoenzyme variation within the six pure Sitka ment, or the bands were not distinct enough for spruce stands along the coast of Alaska and Canada accurate measurements. was much less than that noted in the six pure white The most useful enzyme for identifying stands in spruce stands from the interior. Isoenzyme data 1977] COPES& BECKWITH IDENTIFICATIONOF ISOENZYMES 517 were transformed and expressed as D. Small D The area most similar to areas 7 or 8 (D = 0.89 and values denote likeness or similarity, while large D 0.97, respectively) was stand 11. values signify large difTerencesor lack of likeness. Graph presentation of the difference or degree of The D between Sitka stands was only 0.11 (table 2), divergence between each stand from that of the whereas pure white spruce stands from the interior average interior white spruce and average Sitka of Alaska and Canada difTeredfrom each other by spruce stands indicated that the six stands on the D= 0.33. The D within the four Kenai Peninsula Kenai Peninsula, which were suspected of having white spruce stands was 0.95; this abnormally large undergone introgressive hybridization, did indeed divergence was due primarily to atypical data for occupy intermediatepositions between interior white areas 7 and 8. Considerable difTerence(19= 0.52) and Sitka spruce (fig. 3). Their relative position to was detected in area 11 and the six Kenai Penin- the pure species indicated an enzyme similarity sula stands (areas 12-17), where introgression was closer to interior white spruce than to Sitka spruce. suspected. The same general trend is shown from cluster analy- Individual comparisons of each stand with the sis (fig. 4). The morphology of the seed trees also other 23 stands are listed in table 2. Hybrid spruce resembled white spruce more than Sitka spruce. stands (areas 12-17) from the Kenai Peninsula were Areas 11, 12, and 16 showed the closest similarity to greatly difTerentfrom pure Sitka spruce in areas interior white spruce (D= 0.44, 0.49, and 0.42, 19-24 (D= 1.04). The trees in those stands were respectively). At the other extreme, germinants from more alike when compared with each other (D= areas 18, 14, and 16 were the most Sitka-like hybrids 0.52). They were much like interior white spruce in encountered in this study (D = 0.78, 0.71, and 0.75, areas 1-6 (D = 0.59) but very different from Kenai respectively). Note that area 16 was found in both

. . . . Peninsula white spruce in areas 7-10 (D = 1.21). slml. .arlty comparlsons. Lack of similarity of Kenai white spruce areas 7 The phenogram developed from cluster analysis and 8 to interior white spruce areas 1-6 did not showed a slightly difTerentgrouping of populations result from increased similarity to Sitka spruce. (fig. 4). The homogeneous nature of pure Sitka Instead, the difTerenceoccurred because areas 7 and spruce populations was shown by the clustering of 8 had higher frequency values for isoenzymes char- all six populations at .93. The six pure white spruce acteristic of white spruce than interior white spruce populations were not clustered until .69. In this areas or Kenai Peninsula white spruce areas 9 and analysis, stand 17 clustered more closely (.64) with 10. This may have resulted from an insufEcient pure Sitka spruce stands than was shown by D's sampling of seed trees in areas 7 and 8. A large (fig. 3). The uniqueness of stands 7 and 8 was also mean distinctiveness value for within-Kenai white indicated in the phenogram; they did not cluster spruce comparisons (D = 0.95) was noted (table 2). with any other stand until .54. In general, stands

a) 0.2

Q- a) n

. _ o n

a) a) a) cn n

a) o

osl

o n n a)c c a)

. _ n a)

. _

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a) oso oso 0.1 0.2 0.3

Mean distinctiveness from average Sitka spruce (Sooke, B.C., seed source) FIG. 3.-Mean distinctiveness of seedlings from 22 whiten Sitkan and putative hybrid stands in Alaska. The average Sitka or white spruce source with which the other sources were compared is defined as the stand with the least average variation from the other Sve sources of the same species. See tab]e 1 for names of areas. MeansStand ...... means .32 .37 (.33) .38 .41 .27 .24

TABLE 2 MEANDEGREE OF DIFFERENCE (D) BETWEEN24 SPRUCESTANDS IN ALASKAAND CANADA

KENAIPENINSULA CANADIAN INTERIORWHITE SP1DUCE PUTATIVEWHITE SPRUCE ALASEANPUTATIVE HYBRIDS PUTATIVE SITKA SPRUCE 1IYBRID Seed source no. Seed source no. Seed source no. seed Seed source no. SEEDSOURCE: sourceno. NO. ANDLOCATION 1 2 3 4 5 6 Mean 7 8 9 10 Mean 11 12 13 14 15 16 17 Mean 18 19 20 21 22 23 24 Mean

Interiochwhlite sRrvuecre., ...... 30 46 49 07 26 32 74 87 31 .56 .62 .31 70 .69 .61 21 .34 .88 .53 .73 1.10 1.16 1 05 1.54 1.60 1.35 1.30 2 Tanana River. . 51 52 30 23 37 1 60 1 61 .39 .38 1.00 .67 48 .63 1.04 .59 .47 .96 .69 1.08 1.39 1.31 1.23 1.71 1.82 1.57 1.50 3 Bonanza Creek. .29 .39 .25 .38 .95 1.11 .46 .47 .75 .35 .76 .74 .79 .68 .49 .53 .62 .82 1.42 1.35 1.31 1.87 1.94 1.57 1.58 4. Gerstle River ...... 44 .,32 . 41 1.50 1.51 .31 .66 .99 .50 .75 .87 .94 .66 .60 .81 .73 .89 1.39 1.22 1.21 1.65 1.76 1.44 1.45 5 Fort St. James .13 27 95 88 .28 .57 67 40 29 .66 .49 .33 .31 .87 .48 .69 .90 .88 .82 1.26 1.35 1.10 1.05 6 HainesJunction 24 1 29 1 27 .41 40 84 41 42 .78 .68 .46 .28 .55 .51 .49 1.06 .84 .82 1.30 1.42 1.13 1.09

1.17 1.21 .36 .51 .44 .57 .73 .76 .49 .42 .77 . . .78 1.21 1.13 1.07 1 56 1.65 1.36 ... (.81) ( . 59) Kenai Peninsula putative white spruce: 7. Phalarope...... 34 .86 t.36 .85 .89 1.83 2.05 1.47 1.09 1.63 2.09 1.58 1.84 2.56 2.64 2.66 3.28 3.39 2.89 2.90 8. Hook Lake...... 62 1.71 .89 .97 1.44 2.24 1.46 1.27 1.70 2.27 1.62 2.02 2.58 2.70 2.74 3.37 3.53 3.03 2.99 9. Dry Gulch...... 79 .76 .64 .52 1.15 .80 .62 .78 1.26 .82 1.17 1.52 1.49 1.48 2.00 2.13 1.78 1.73 10. BarabaraLake ..... 1.29 .67 .82 .79 1.07 .76 .61 1.00 .82 1.38 1.68 1.59 1.63 2.09 2.25 1.95 1.87 Means ...... 85 .89 .76 1.29 ... .79 1.15 1.56 1.20 .93 1.18 1.66 ... 1.60 2.08 2.11 2.13 2.68 2.83 2.41 ... Stand means..... (-95) (1.21) (1.60) (2.37) Alaskan putative hybrids: 11. Tyonek...... 86 .61 .59 .22 .20 .34 .47 .82 1.11 1.38 1.13 1.24 1.94 1.62 1.40 12. Soldotna...... 59 .46 .74 .44 1.21 .72 1.40 .81 .99 .91 1.20 1.28 1.28 1.08 13. Homer...... 74 .65 .29 .76 .48 1.51 .79 1.28 1.05 1.39 1.31 1.34 1.19 14. Seward...... 35 .48 .81 .57 1.04 .34 .65 .59 .89 .92 .87 .71 15. Ninilchik...... 17 .57 .45 .57 1.36 .77 .70 1.13 1.21 .96 1.02 16. Moose Pass ...... 22 .30 .56 .49 .67 .53 .98 .99 .82 .75 17. Kenai ...... 65 .53 .92 .99 .83 1.47 1.45 1.17 1.14 Means ...... 47 .72 .48 .57 .45 .30 .65 ... .92 .83 .96 .82 1.19 1.30 1.15 ... Stand means..... (.52) (.92) (1.04) Canadianputative hybrid: ... .99 .56 .53 .98 1.02 .63 .78 18. Skeena River ......

Sitka spruce: ... .20 .13 .23 .20 .25 .21 19 Haines...... 03 .08 .13 .04 .10 20. Mendenhall...... 11 .10 .05 .08 21. Juneau...... 22. Kodiak Island, -.03 .04 .09 Afognak Island...... 04 .09 23. Kodiak...... 08 24. Sooke...... 2 1 . 10 . 08 . 09 . 09 . 08 . . . Means ...... ( . 1 1) Stand means.....

NOTE.-All 13 isoenzymes for LAP, GDH, and TO are included in each value. Small values denote likeness, large values denote major differences. | l l l l l l

COPES& BECKWITH-IDENTIFICATIONOF ISOENZYMES 519 which were geographicallyclose, such as 7 and 8, of introgression.Stand 13 in the typical Sitka spruce 22 and 23, and 20 and 21, weremore similar to each belt had isoenzyme frequencies that showed definite otherthan to moregeographically distant stands. evidence of past introgression with white spruce. Much of the west side of the Kenai Peninsula north Discussion from area 13 appeared to be a massive hybrid swarm Correctidentification of sprucestands in which composed of stands with varied degrees of introgres- various levels of introgressivehybridization have sion. The extent of the southern flow of white spruce occurredbetween white and Sitka populationscan genes cannot be determined, because area 13 was often be done with starchgel electrophoretictech- the most southerly Kenai population sampled. Iso- niques.Sufficient enzyme variation existed in three enzyme D figures for LAP, GL)H, and TO indicated enzymes to detect significantmean distinctiveness a closer relationship of the Kenai Peninsula hybrid differences.These estimates were based on 13 spruce to the interior white spruce than to the pure heteromorphicisoenzymes. This method shouldbe Sitka found along the coast near Juneau at areas useful to researchersand foresters interested in 19-21 or on Kodiak or Afognak Islands in areas 23 learningthe genetic compositionof stands of un- and 22. Cluster analysis puts areas 17 and 18 into a known origin. For example,the presenceof white closer fit with Sitka spruce than with white spruce. spruce genes in Sitka populationscan usually be Introgression in the Kenai Peninsula populations detectedby the Rm .52 TO isoenzyme.But whena occurredover a much greater area than was previous- measurementof the amount of introgressionis ly thought. Additional study of more stands sampled desired,the additional12 isoenzymesof LAP and along appropriate east-west and north-south tran- GDII shouldalso be analyzed. sects should reveal more exact information on the KenaiPeninsula spruce stands at areas12-17 and past history of spruce in this area. at area 18 in BritishColumbia all showedevidence Area 18 is from the same latitude and longitude

-22 Agofnak and Kodiak Islonds, AL -23 Kodiak, AL *24 Sooke, B.C. 20 Mendenhall, AL - 21 juneou, AL 19 Haines, AL 17 Kenai, AL 18 Skeena River, B.C. 7 Phalorope, AL 8 Hook Lake, AL 14 Seward, AL 15 Ninilchik, AL 10 Barobara Lake, AL 11 Tyonek, AL 1 Charley River, AL 5 Fort St. James, Yukon 6 Haines Junction, B.C. 16 Moose Pass, AL 2 Tanana River, AL 12 Soldotna, AL 13 Homer, AL 3 Bononza Creek, AL 4 Gerstle River, AL 9 Dry Gulch, AL

.40 .45 .50 .55 .60 .65 .70 .75 .80 .85 .90 .95 1.00 CLUSTERING LEVEL FIG. 4.-Phenogram of the 24 sprucestands. Analysiswas based upon groupaverages (MCRLIB cluster analysis) BOTANICALGAZETTE [DECEMBER 520 as the "Lower Skeena" sampled by HANOVERand that stand yielded progeny more closely resembling WILKINSON(1970). The introgression demonstrated both white and Sitka spruce than did progeny-from by isoenzymes supports their results of hybridity by other introgressed stands. But since the desired or chromatographymethods and the results DAUBEN- requireddegree of introgressionfor weevil resistance MIRE(1968) obtained on the same population by is not known, it is possible that some of the stands morphologicalstudy. similar to Sitka spruce but not as similar to white The Kenai Peninsula collections of white spruce- spruce as were area 11 seedlings may be more like stands at areas 7 and 8 had isoenzyme frequen- weevil resistant than those of Moose Pass seedlings. cies drastically difTerentfrom those of the other two Area 14 is an example of this condition. Its similarity Kenai white spruce stands or any interior white to Sitka spruce was the closest of the introgressed spruce or Sitka spruce. Areas 9 and 10 were very stands (D = 0.71), but it showed the second smallest similar to interior white spruce. It is not certain amount of introgression of any of the six putative whether the unusual results for areas 7 and 9 were hybrid Alaskan areas (D = O.76) . obtained because of chance sampling variation or The histochemical techniques used to make iso- whether part of the northwest Kenai Peninsula area enzymes visible were not always totally satisfactory. contains some distinctly different white spruce Acid phosphatase, esterase, and peroxidase isoen- populations. Further study of stands in that area is zymes were examined but, because of poor band needed before any conclusion can be reached. resolutions, were not included as data in this report. Isoenzymes in seedlings from area 11 closely Heteromorphic variation in acid phosphatase and resembled interior white spruce. Area 11 is isolated esterase was detected, and if better histochemical from other Sitka spruce areas and is in closer proxim- techniques are devised in the future, isoenzymes of ity to pure interior white spruce stands than the these two enzymes may also be useful in further other putative hybrid stands. The area can probably refining introgression estimates or species verifica- be classified as a white spruce population which has tion. Young spruce seedlings had very low levels of just a slight amount of Sitka introgression. peroxidase activity, and it is doubtful whether Isoenzyme methods for determining the genetic peroxidase isoenzymes will be useful unless tissues composition of stands have the advantage of being from older, more mature seedlings are sampled. less subject to the masking effects of local environ- The TO staining techniques used throughout the ment on phenotypic expression. Stands with low first half of this study did not always yield good levels of introgression are difficult to identify ac- results. Light staining of nonenzyme areas of the curately when morphological traits are used as the gels made the negatively stained (white or unstained sole taxonomiccriteria for classification.The accuracy bands) enzyme areas difficult to see. A new method of the electrophoresis method to separate stands for visualizing the TO isoenzrmes was found halfway correctly is doubtful when the difference between through the study, and since it was much better stands is extremely small. Use of young seedlings than the first, it was used during the remaining half grown under uniform conditions rather than buds or of the study. Only the best TO stain recipe is reported needles from the actual trees in each stand removes in the Methods section. If it had been used through- most of the local environmental effect. Laboratory out the entire study, much of the TO variability evaluations based on newly germinated seedlings are reported in table 1 between interior white spruce easy to standardize. Results obtained from uniform stands would have been eliminated. tests of many stands widely separated in the field Electrophoretictechniques can be a useful tool for should be more accurate than measurements made identifying species and populations in which intro- in the field on traits subject to modification by the gressive hybridization has occurred, but initial de- environment. The ease of storage, transfer, and tection of stands suspected of containing hybrids is increased enzyme resolution combine to make seed more easily done with traditional taxonomical or germinated seedling studies preferable to studies methods. Electrophoresis will be useful to verify on more mature tissues gathered in the field from morphologicalobservations and provide a numerical older trees. similarity value for determining the closeness or Selection of putative hybrid stands for Sitka degree of relationship of the stands to either or both spruce weevil resistance can potentially be done by pure species. Electrophoretic methods are accurate electrophoresis. Hybrid stands containing a good and fairly inexpensive, and many stands can be proportion of Sitka spruce genes for growth and sampled under uniform, repeatable test conditions. form and white spruce genes for insect and frost Eflective separations can be made for most, but not resistance would probably be most desirable. In the all, stands. seven putative hybrid areas of the present study, area 16 is especially interesting-its seedlings were Acknowledgments unique because they were most like interior white We are grateful to the following for supplying spruce (D= 0.42) and second most like pure seed used in the study: J. C. ZASADA,Institute of Sitka spruce (19= 0.75). It appeared that trees in Northern Forestry, Fairbanks,Alaska; A. S. HARRIS, 1977] COPES& BECKWITH IDENTIFICATIONOF ISOENZYMES 521

Forestry Sciences Laboratory, Juneau, Alaska; J. Service, Victoria, British Columbia;and ALANLONG, KONISHI,British Columbia Forest Service, Victoria, WeyerhaeuserCompany, Forestry Research Center, British Columbia; R. F. PIESCH,Canadian Forestry Centralia, Washington.

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