GENE DIVERSITY AND GENETIC STRUCTURE IN A NARROW ENDEMIC, TORREY PINE (PINUS TORREYANA PARRY EX CARR.)

F. THOMASLEDIG AND M. THOMPSONCONKLE Institute of Forest Genetics, Pacific Southwest Forest and Range Experiiizent Station, VSDAForest Sewice, P.O. Box 245, Berkeley, Cal$oronzia 94701

Received December 16, 1981. Revised April 8, 1982

Recent reviews have suggested that tree tion also contributes to an understanding species are the most variable of organisms, of the biogeography of the California as measured by proportion of polymorphic Channel Islands. loci or average heterozygosity (Hamrick, Torrey pine has the smallest population 1979; Hamrick et al., 1979). Average het- of any known pine. The 1973 count in the erozygosity is greater than 0.30 for several 445 ha (1,100 acre) Torrey Pines State Re- conifers. By contrast, annual herbaceous serve on the Pacific Coast at San Diego, species have a mean heterozygosity of 0.13 California was 3,401 mature trees (Calif. (Hamrick et al., 1979). Dept. Parks Rec., 1975). Including seed- Conifers have seveial mechanisms that lings, the Reserve's naturalist estimated ca. promote outcrossing, and are expected to 7,000 individuals in 1979 (H. Nicol, pers. maintain high levels of genetic variation. comm.). The population includes only two Despite these mechanisms, it is uncertain other small stands, contiguous with the whether the breeding system is capable of Reserve. Another population occurs on maintaining variability in small popula- Santa Rosa Island, one of the Northern tions and in the absence of migration. Channel Islands off the California coast While the genetic consequences of reduced near Santa Barbara. There may be 2,000 population size have long been understood individuals on the northeast coast of the in theory, empirical evidence is scarce in island. Climatic, edaphic, and floristic plants, particularly in tree species. The characteristics of the two sites were sum- potential to maintain high levels of genetic marized by Haller (1967). The San Diego variation in reduced and scattered popu- and Santa Rosa Island populations are lations is important because of its impli- separated by 280 km and the island is 40 cations for genetic resource conservation, km from the mainland. It is highly un- and in fact, for the ability of species to likely that there has been any significant respond to environmental change and opportunity for gene exchange within re- avoid extinction. Because most conifers are cent centuries. Nor is Torrey pine likely commercially valued and exploited for to exchange genes with its closest rela- lumber and paper products, much of the tives, digger pine (Pinus sabiniana Dougl.) original forest in North America has been and Coulter pine (Pinus coulteri D. Don), destroyed, and the tendency under man- both of which are allopatric. Controlled agement will be to reduce native popula- crosses with Digger pine succeed only with tions to scattered relicts. difficulty and there have been no hybrids One way of forecasting the fate of species with Coulter pine despite several attempts reduced in numbers is to make use of nat- (Critchfield, 1966). ural experiments, by examining the ge- netic structure of species that occur in dis- junct populations (Shaffer, 1981). We Old cones, some in excess of 5 years old, chose to investigate how much variation were collected from stands in the Torrey might be preserved in conifers by observ- Pines Reserve and on Santa Rosa Island ing a narrow endemic, Torrey pine (Pinus in spring 1980. Cones had opened on the torreyana Parry ex Carr.). The investiga- trees and most seed was shed; but in Tor- 80 F. T. LEDIG AND )I. T. CONICLE rey pine, cone scales reflex upon opening The gels were stained to produce iso- to such a degree that some seed is trapped zyme bands in 25 enzyme systems (Table in the basal scales several years after the 1). We used experience gained from our bulk of the seed cohort has dispersed. studies of the inheritance of allozymes in We obtained viable seed from four areas other conifer species (Conkle, 1981) to es- within the Reserve: from 37 contiguous timate the number of loci needed to ac- trees on the windward side of North Grove count for the isozymes within each enzyme overlooking the ocean; 25 trees on High system. We estimated that a total of 59 Point (113 m above sea level) where some loci were required to account for the pat- of the oldest Torrey pine (about 125 years terns in these 25 enzyme systems. old) occur; and 40 trees on two adjacent spur ridges along with three from an in- tervening valley in the Extension Area. Every individual from which seed was Seed was also collected from the largest analyzed at the Torrey Pines State Re- native Torrey pine (ca. 100 cm dbh), lo- serve was identically homozygous to all cated in the Extension Area. The groves others at each of the 59 gene loci. This in the Exteilsion Area were ca. 1.75 km includes trees growing on the coast and NNE of the North Grove, and the North those inland, as well as some of the oldest Grove is ca. 450 m NW of High Point. On and the largest native Torrey pines. Sim- Santa Rosa Island we collected sound seed ilarly, every individual sampled on Santa from 25 trees on the side of one canyon Rosa Island was identically homozygous. facing Beecher's Bay and 26 more from However, the trees on Santa Rosa Is- another canyon ca. 400 m east. land differed from those on the mainland We used starch gel electrophoresis at two, or 3.4%, of the 59 loci. One was (Conkle et al., 1982) to process seed from a malic dehydrogenase locus and the oth- 106 trees in the Reserve and 51 from the er, shikimate dehydrogenase. Because all island. Seeds were stratified under cold- allelic variation is not detectable by elec- moist conditions for 30 days and then ger- trophoresis, the two populations undoubt- minated to the stage where the emerging edly differ at more than 3.4% of their loci. radicals extended 2-4 mm bevond the seed Based on the proportion of amino acid coat. The first analyses used one seed of substitutions producing differences in each parent tree. Samples from the Re- electrostatic charge, only 27.56% of all al- serve were co-electrophoresed with sam- leles should be detectably different (Shaw, ples from the island to maximize the po- 1965), but it has become generally accept- tential for identifying differences in ed that electrophoretic techniques can de- isozyme mobility. Later we examined up tect amino acid changes which produce no to five additional seed per tree for 32 trees change in net charge. Based on empirical (18 from the Reserve and 14 from the is- studies of known forms of hemoglobin, land). 40% of all amino acid substitutions may The female gametophytes and embryos be detectable by electrophoresis under of each seed were analyzed separately at standard conditions (Ramshaw et al., adjacent positions on the starch gel. Iso- 1979). Using Ramshaw et al.'s estimates, zyme bands from the gametophytes gave we expect the mainland and island pop- genetic information on the seed parent. ulations to differ at 0.3410.4 = 0.085 or Pollen parent genotypes were inferred by 8.5% of their gene loci. comparing the diploid pattern from an The observed pattern, all variation be- embryo with the pattern from the corre- tween populations and little or none with- sponding female gametophyte. When tak- in, is unknown for conifers. For all pre- en together, the female gametophyte and viously reported species, genic variation pollen analyses served to evaluate 246 within stands has ranged from 88% to 97% haploid genomes from the Reserve and 132 of the total variation within the species haploid genomes from the island. (O'Malley et al., 1979; Yeh and Layton, GENETIC VARIATION IN PINES 8 1

TABLE1. Enzynze systenzs and estinzates of the lzunzber of loci analyzed in Torrey pine and the number of anzino acid residues as calculated from nzoleczllar weights in Table 6 of Harris and Hopkinson (1976). '

Number Molecular Number of amino acid Enzyme system of loci weight x 10 residues per locus Acid phosphatase Aconitase Alcohol dehydrogenase Aldolase Catalase Diaphorase Esterase Colorimetric Fluorescent Glutamate dehydrogenase Glutamate oxalacetic transaminase Beta-glucosidase Glycerate dehydrogenase Glucose-6-phosphate dehydrogenase Hexoseaminidase Isocitriate dehydrogenase Leucine aminopeptidase Maliate dehydrogenase Menadione reductase Peptidase Peroxidase Phosphoglucomutase 6-phosphogluconate dehydrogenase Phosphoglucoseisomerase Shikimate dehydrogenase Superoxide dismutase

' IvIolecular weight divided by 10i.i, the average weight per amino ac~dresldue in Table 6.2 of Harris and Hopkinson (19i6)

1979; Yeh and O'Malley, 1980; Guries and /3-phellandrene concentrations, although Ledig, 1982). Neither is any other conifer percentages varied less than between pop- so invariable. Even the relatively uniform ulations of many other pines (Zavarin et species, red pine (Pinus resinosa Ait.) and al., 1967). Thus, population structure as western redcedar (Thuja plicata Donn ex revealed by morphological, biochemical, D. Don), have expected heterozygosities of and growth traits supports the contention 0.002 and 0.04, respectively (Guries and based on genetic analysis that there is little O'Malley, pers. comm.; Yeh, pers. genic diversity within populations but mi- comm.). nor differences between them. Allozyme results agree with other infor- The most likely explanation for the lack mation. Measured in the field, the mean of variants is genetic drift. Apparently, difference between populations for a com- both populations were reduced in size in posite score based on cone size, cone and the recent past to even smaller numbers cone scale shape, and foliage color was (much less than 50) than now occur. There twice as large as the spread within popu- have been several climatic fluctations lations, and in a uniform garden trial there which would have influenced the distri- were significant differences between the bution and abundance of Torrey pine. In populations for height growth, tree form, particular, the Xerothermic period 8,500 and needle length (Haller, 1967). Oleores- to 3,000 years B.P. may have reduced in content was "remarkably similar be- Torrey pine at San Diego to a few indi- tween individuals" within populations, but viduals and eliminated intermediate pop- populations were distinct for limonene and ulations. The presence of a disjunct So- 82 F. T LEDIG AND &I.T CONICLE noran desert association at the Torrey cause the longshore current along the Cal- Pines State Reserve indicates a more arid ifornia coast runs north to south, so Tor- climate in the Holocene (Axelrod, 1967). rey pine on Santa Rosa Island must have As few as 20 to 30 generations may have originated on either the Santa Barbara elapsed since the end of the Xerothermic, coast or further north. leaving little opportunity for accumulation Even a single founder had to originate of genetic variants by mutation. With a from a relatively invariable parental pop- mutation rate of per gene per gener- ulation or have suffered subsequent ge- ation only 591106 new variants per indi- netic erosion to explain the lack of vari- vidual would be expected in the most re- ability in Santa Rosa Island Torrey pine. cent generation, and even in 50 generations A naturalized population of pitcher plant (perhaps 5,000 years in Torrey pine), it (Sarmcenia purpurea L.), although known would not be unusual to find no new vari- to be established from only a single found- ants in a sample the size of ours. er, nevertheless had an average hetero- Lack of variation in the population on zygosity of 0.042 compared to a mean of Santa Rosa Island could also be attributed 0.095 for ten other populations (Schwae- to chance acting through the founder ef- gerle and Schaal, 1979). Guries and Ledig fect. To accomodate the presence of fossil (1982) found that heterozygosity in a mar- mammoth remains on Santa Rosa, it was ginal isolate of pitch pine (Pinus rigidu assumed that a land bridge connected the Mill.), probably a relict from the Xero- Northern Channel Islands to the mainland thermic 3,000 years B.P., was only slight- (Valentine and Lipps, 1967). Seismic re- ly less than that in central populations flection profiles and bathymetric maps now (0.120 vs. 0.156). indicate a land bridge was unlikely (Jun- Another possibility is that Torrey pine ger and Johnson, 1980; Vedder and How- on Santa Rosa Island and the mainland at ell, 1980), but about 18,000 years B.P. San Diego were part of the same popula- when sea level was ca. 117 m lower than tion 20 million years B.P. and were sep- now (Nardin et al., 1981), all the Northern arated by tectonic movement. Paleomag- Channel Islands were one large island, netic data indicate that the Northern Santarosae, separated from the mainland Channel Islands were rotated about an axis by only 6 to 7 km (Vedder and Howell, near the eastern end of the Santa Monica 1980; Wenner and Johnson, 1980). If there Range and translated northward during was no land bridge, Torrey pine must have the Miocene (Kamerling and Luyendyk, reached the island as seed stored in a float- 1979; Luyendyk et al., 1980). Santa Rosa ing cone or in the beak of a seed-caching Island was apparently part of the coast bird. Clark's nutcrackers are reported to near San Diego in the mid-Miocene (B. P. cache seed of pinyon pine (Pinus edulis Luyendyk, pers. comm.). Eocene gravels

Engelm.) more than 22 km from the source on Santa Cruz Island., * art of the North- (Vander Wall and Balda, 1977). While ern Channel Islands chain, have correlat- there is no evidence that birds now cache ed with the Poway Conglomerate near San Torrey pine seed, its seed is nearly wing- Diego (Abbott and Smith, 1978). But es- less, not adapted to wind dispersal, and timates of time since genetic divergence suggestive of bird dispersal (Lanner and are an order of magnitude too small to Vander Wall, 1980). Transport by rafting accommodate this possibility. or by a bird might establish Torrey pine Using observed protein differences and on the island. The depauperate and un- length of time since divergence in the fossil balanced Channel Islands fauna support record, Prager et al., (1976) estimated that the view of "sweepstakes" dispersal over it would take 7.5 to 11 million years to a barrier rather than migration over a accumulate a 1% difference in amino acid bridge (Wenner and Johnson, 1980). It is sequencing between plant populations. virtually impossible for seed to have been They used 9 million years in their calcu- rafted from the vicinity of San Diego be- lations. We estimate 24,689 amino acid GENETIC VARIATION IN PINES 83 residues in the 59 loci we examined (Table zuma pine (Pinus rnontezurnae Lamb.) and 1). With two detectable isozyme variants its allies. The paucity of fossils is consis- and a detection rate of 40%, the popula- tent with the picture of a species that may tions differ at five, or 0.02 %, of the amino always have been relatively restricted in acid residues. If divergence between in- range to a progressively shrinking belt of sular and mainland populations of Torrey temperate climate along the coastal strip pine proceeded at the rate of 1% per 9 (Axelrod, 198 1). million years, then these populations have been separated 180 thousand years. Another method of calculating time since Early investigations suggested that ge- divergence uses Nei's (1975 p. 177) stan- netic variability might be associated with dard genetic distance, D. For Torrey pine, longevity, and that tree species were the D = 0.0862 155. Time (t) since divergence most heterozygous of organisms. How- (Nei, 1975 p. 192) is t = D/2a, in which ever, these ideas may be modified as a is the mutation rate. The best estimate species representing a wider range of pop- of mutation rates in higher organisms is ulation structure are investigated. Torrey to (Nei, 1975 p. 28), so time since pine, a California endemic, occurs in only isolation has been between 4.3 to 430 two populations, one on the coast at San thousand years. Diego and one 280 km northwest on Santa We invoke two rare events to explain Rosa Island. Populations number only ca. population structure in Torrey pine: the 7,000 and 2,000 individuals, respectively. reduction of a local San Diego population Each population is composed of identical to such low numbers that all genetic vari- homozygous genotypes at 59 isozyme loci. ation was eliminated and the dispersal of The island population is fixed for alternate a founder to Santarosae across the Santa alleles to those in the coastal population Barbara Channel. Present populations at two of the 59 loci. These results agree must have been derived from a somewhat with other lines of evidence that suggest polymorphic ancestral population, be- Torrey pine is characterized by minor dif- cause they are fixed for different alleles. ferences between relatively uniform pop- There has not been time since the bottle- ulations. neck of the Xerothermic or since the col- Drift is the most likely explanation for onization of Santarosae Island to fix two lack of variability in Torrey pine. It prob- novel mutations. If there had been time ably- ex~erienced- one or more reductions for the fixation of mutation, there would in population size, most recently during also have been time for the accumulation the Xerothermic period 8,500 to 3,000 B.P. of other polymorphic loci. The founder effect may have operated For Torrey pine, growing at the edge of during colonization of Santa Rosa Island aridity, restricted to coastal pockets where ca. 18,000 B.P. Apparently, the Northern fog and high humidity are crucial to its Channel Islands to which Santa Rosa be- survival and reproduction, xerothermic longs were not connected to the adjacent intervals (Axelrod, 1967, 1981) may have mainland in recent geologic history, and fractured the species and created subpop- colonization would require transport across ulations that fluctuated within the demo- at least a 6- to 7-km channel. graphic limits in which drift and fixation Red pine and western redcedar, conifers become the predominant driving force of with greater ranges than Torrey pine, are evolution. There is no reliable fossil record only slightly more variable. Thus, despite of Torrey pine. Needle fascicles from an the presence of mechanisms to maintain Oligocene formation in Oregon attributed high levels of outbreeding, it appears the to Torrey pine (Mason, 1927) are really conifer breeding system can survive in- indistinguishable from those of five-needle, breeding with its consequence of genetic hard pines from Mexico such as Monte- depauperization. 84 I?. T. LEDIG AND NI. T. CONKLE

ACKNOWLEDGMENTS Johnson, and P. Raven (eds.), Topics in Plant Population Biology. Columbia Univ. Press, N.Y. This research was supported in part by HAMRICK,J. L., Y. B. LINHART,AND J. B. MIT- the U.S. Department of the Navy. We are TON. 1979. Relationships between life history grateful to Mr. A1 Vail and the Vail-Vick- characteristics and electrophoretically-detectable ers Ranch for their hospitality and per- genetic variation in plants. Ann. Rev. Ecol. Syst. mission to collect from Torrey pine on 10:173-200. HARRIS,H., AND D. A. HOPKINSON.1976. Hand- Santa Rosa Island. J. Agozino, G. Mc- book of Enzyme Electrophoresis in Human Ge- Masters, D. W. Nicol, and A. M. Wenner netics. North-Holland Publ. Co., Amsterdam. gave valuable advice and helped us to un- JUNGER,A,, AND D. L. JOHNSON. 1980. Was there derstand Torrey pine. W. B. Critchfield's a Quaternary land bridge to the Northern Chan- nel Islands?, p. 33-39. In D. M. Power (ed.),The assistance and stimulating discussion were California Islands: Proceedings of a Multidisci- invaluable. Thanks are due several other plinary Symposium. Santa Barbara Mus. Nat. scientists who graciously contributed un- Hist., Santa Barbara. published information as cited in the text. KAMERLING,M. J., AND B. P. LUYENDYK.1979. Tectonic rotations of the Santa Monica Moun- tains region, western Transverse Ranges, Cali- fornia, suggested by paleomagnetic vectors. Geol. ABBOTT,P. L., AND T. E. SMITH. 1978. Trace Soc. Amer. Bull. Part I, 90:331-337. element comparison of clasts in Eocene conglom- LANNER,R. M., AND S. B. VANDERWALL. 1980. erates, southwestern California and Northwest- Dispersal of limber pine seed by Clark's nut- ern Mexico. J. Geol. 86:753-762. cracker. J. For. 78:637-639. AXELROD,D. I. 1981. Holocene climatic changes LUYENDYK,B. P., M. J. KAMERLING,AND R. in relation to vegetation disjunction and specia- TERRES. 1980. Geometric model for Neogene tion. Amer. Natur. 117:847-870. crustal rotations in southern California. Geol. Soc. . 1967. Geological history of the California Amer. Bull. Part I 91:211-217. insular flora, p. 267-315. In R. N. Philbrick (ed.), MASON,H. L. 1927. Fossil records of some West Proceedings of the Symposium on the Biology of American conifers. Contributions to palaeontol- the California Islands. Santa Barbara Bot. Gar- ogy: additions to the palaeontology of the Pacific den, Santa Barbara. Coast and Great Basin regions of North America. CALIFORNIADEPARTMENT OF PARKSAND RECREA- Carnegie Instit. Publ. 346:139-158. TION. 1975. Torrey Pine State Reserve and State NARDIN,T. R., R. H. OSBORNE,D. J. BOTTJER, Beach. Sacramento, California. AND R. C. ~CHEIDEMANN.1981. Holocene sea- CONKLE,M. T. 1981. Isozyme variation and link- level curves for Santa Monica shelf, California age in six conifer species, p. 11-17. In >I, T. continental borderland. Science 2 13:331-333. Conkle (Tech. Co-ord.), Proceedings of the Sym- NEI, M. 1975. Molecular population genetics and posium on Isozymes of North American Forest evolution. North-Holland Publ. Co., Amster- Trees and Forest Insects, July 27, 1979, Berkeley, dam. California. U.S. For. Serv. Gen. Tech. Rep. PSW- O'MALLEY,D. M., F. W. ALLENDORF,AND G. M. 48. BLAKE. 1979. Inheritance of isozyme variation CONKLE,M. T., P. D. HODGSKISS,L. B. NUNNAL- and heterozygosity in Pinus ponderosa. Biochem. LY, AND S. C. HUNTER. 1982. Starch gel elec- Genet. 17:233-250. trophoresis of pine seed: a laboratory manual. PRAGER,E. M., D. P. FOWLER,AND A. C. WILSON. U.S. For. Serv. Gen. Tech. Rep. In press. 1976. Rates of evolution in conifers (Pinaceae). CRITCHFIELD,W. B. 1966. Crossability and re- Evolution 30:637-649. lationships of the California big-cone pines, p. RAMSHAW,J. A. NI., J. A. COYNE,AND R. C. LE- 36-44. In Joint Proc. Second Genet. Workshop WONTIN. 1979. The sensitivity of gel electro- Soc. Amer. For. Seventh Lake States For. Tree phoresis as a detector of genetic variation. Ge- Improv. Conf., Denver, Colorado. netics 93: 1019-1037. GURIES,R. P., AND F. T. LEDIG. 1982. Genetic SCHWAEGERLE,K. E., AND B. A. SCHAAL. 1979. diversity and population structure in pitch pine Genetic variability and founder effect in the (Pinus rigida Mill.). Evolution 36:387-402. pitcher plant Sariacenia pzlrpurea L. Evolution HALLER,J. R. 1967. A comparison of the main- 33:1210-1218. land and island populations of Torrey pine, p. SHAFFER,M. L. 198 1. Minimum population sizes 79-88. In R. N. Philbrick (ed.), Proceedings of for species conservation. BioScience 31:131-134. the Symposium on the Biology of the California SHAW, C. R. 1965. Electrophoretic variation in Islands. Santa Barbara Bot. Garden, Santa Bar- enzymes. Science 149:936-943. bara. VALENTINE,J. W., AND J. H. LIPPS. 1967. Late HAMRICK,J. L. 1979. Genetic variation and lon- Cenozoic history of the southern California Is- gevity, p. 84-133. In 0. Solbrig, S. Jain, G. lands, p. 21-35. In R. N. Philbrick (ed.), Pro- GENETIC VARIATION IN PINES 8 5

ceedings of the Symposium on the Biology of the YEH, F. C., AND C. LAYTON. 1979. The organi- California Islands. Santa Barbara Bot. Garden, zation of genetic variability in central and mar- Santa Barbara. ginal populations of lodgepole pine (Pinus con- VANDERWALL, S. B., AND R. P. BALDA. 1977. torta ssp. latifolia). Can. J. Genet. Cytol. 2 1:487- Coadaptations of the Clark's nutcracker and the 503. pinon pine for efficient seed harvest and dispers- YEH, F. C., AND D. M. O'MALLEY. 1980. Enzyme al. Ecol. Monogr. 47:89-111. variations in natural populations of Douglas-fir, VEDDER,J. G., AND D. G. HOWELL. 1980. TO- (Pseudotsuga menziesii (Mirb.) Franco) from pographic evolution of the southern California British Columbia. 1. Genetic variation patterns borderland during late Cenozoic time, p. 7-31. in coastal populations. Silvae Genet. 29:83-92. In D. M. Power ied.), The California Islands: ZAVARIN,E., W. HATHAWAY,T. REICHERT,AND Proceedings of a Multidisciplinary Symposium. Y. B. LINHART. 1967. Chemotaxonomic study Santa Barbara Mus. Nat. Hist., Santa Barbara. of Pinus torveyana Parry turpentine. Phytochem- WENNER,A. M., AND D. L. JOHNSON.1980. Land istry 6:1019-1023. vertebrates on the California Channel Islands: sweepstakes or bridges?, p. 497-530. In D. M. Corresponding Editor: S. Jain Power (ed.), The California Islands: Proceedings of a Multidisciplinary Symposium. Santa Bar- bara Mus. Nat. Hist., Santa Barbara.

The Department of Philosophy and the Institute for Molecular Biology at California State University, Fullerton announce the Thirteenth Annual Philosophy Symposium "BIOLOGY AND PHILOSOPHY." Invited papers include those of: Dr. Montgomery Furth, UCLA; Dr. Francisco Ayala, UC Davis; Dr. Larry Wright, UC Riverside; Dr. Gunther Stent, UC Berkeley; Dr. John Campbell, UCLA; Dr. J. William Schopf, UCLA; Dr. Ernst Mayr, Harvard University; Dr. Richard Burian, Drexel University; Dr. Marjorie Grene, UC Davis (Emeritus); Dr. Morton Beckner, Pomona College. The Symposium will be held March 8-11. 1983.