Clonality and sexual reproductive failure in remnant populations ofSantalum lanceolatum (Santalaceae)

Cindy L. Warburton a, Elizabeth A. James b,c, Yvonne J. Fripp c, Stephen J. Trueman d,Helen M. Wallace e,*

aSchool of Environmental Management and Ecology, La Trobe University, Parkers Road, Wodonga, VA 3690, AustraliabRoyal Botanic Gardens Melbourne, Birdwood Avenue, South Yarra, VA 3141, Australia

cDepartment of Genetics, La Trobe University, Bundoora, VA 3083, AustraliadDepartment of Botany, University of Queensland, QN 4072, Australia

eUniversity of the Sunshine Coast, Maroochydore DC, QN 4558, Australia

Received 18 December 1999; received in revised form 1 March 2000; accepted 17 March 2000

Abstract

Habitat fragmentation can have important conservation consequences for clonal plant species that possess self-incompatibilitymechanisms, as lack of genetic variability within remnant populations may result in sexual reproductive failure. Allozymes andRAPDs were used in this study to determine the extent of clonality in remnant Victorian populations of the northern sandalwood,

Santalum lanceolatum (Santalaceae), a species that has been heavily wild-harvested. S. lanceolatum can reproduce asexually by rootsuckers, and each population was identi®ed as a unique single clone composed of numerous ramets of a single genet. Examinationof pollination and fruit set indicated that little or no sexual reproduction was occurring in the remnants, due to pollen sterility in

one population and self-incompatibility or pistil dysfunction in others. Clonality, genetic isolation and sexual reproductive failureindicate that preservation of each population, and possibly the establishment of new ones, should be objectives of the conservationstrategy for the S. lanceolatum remnants. # 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Genetic diversity; Conservation; Pollination; Fruit set; Sandalwood

1. Introduction

The number and size of areas in Australia with naturalor semi-natural vegetation has decreased dramaticallyover the last 200 years, particularly as a result of clearing,burning and over-grazing. Depletion and fragmentationof populations may also result from wild-harvesting ofspecies of economic importance, a process which hasbeen described for many species valued for their timber(Cropper, 1993; Freese, 1997). Habitat loss and com-mercial exploitation may lead to the fragmentation ofpopulations into small, isolated remnants potentiallysusceptible to reduction in habitat quality. For plantpopulations in fragmented landscapes, gene ¯ow bypollen dissemination and seed dispersal may also bedisrupted, resulting in strong genetic di�erentiation

between populations, aided by genetic drift (Brown,1992; Oostermeijer et al., 1996).Many species of sandalwood (Santalum spp.) are

economically important in Southern Asia, Indonesia,Australia and the South Paci®c for timber and fragrantoil production. The northern sandalwood or plumbush,S. lanceolatum, is widespread but not abundant through-out arid and semi-arid Australia, and was harvestedvery heavily in Victoria and New South Wales followingthe gold rushes of the mid- to late-1800s. Clearing, ®reand grazing have also contributed to the species' declineand, at its southern limit in Victoria, it is now restrictedto only seven widely separated populations with a totalof approximately 258 trees (Johnson, 1996). The specieshas been listed as a threatened taxon under Schedule IIof the Victorian Flora and Fauna Guarantee Act 1988.The restriction and isolation of the remnant S. lan-

ceolatum populations has led to the development of aconservation strategy for the species in Victoria (Johnson,1996). In addition to the protection of the remaining

0006-3207/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.

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Biological Conservation 96 (2000) 45±54

www.elsevier.com/locate/biocon

* Corresponding author. Tel.: +61-7-5430-1228.

E-mail address: hwallace@usc.edu.au (H.M. Wallace).

populations, particularly from grazing by rabbits andsheep, the establishment of new populations has beenproposed as a conservation objective. Recruitment hasoccurred in the populations since the construction ofprotective enclosures to exclude grazers, but asexualreproduction by root suckering alone has been pre-sumed responsible for the increase in population sizes.Flowering occurs annually in all populations but fruitproduction has rarely been observed (Johnson, 1996).The present study provided information on the

genetic structure and levels of clonality within remnantpopulations of S. lanceolatum. Genetic diversity wasdetermined for each of the populations using allozymeanalyses. As S. lanceolatum has the ability to reproducevegetatively via root suckers, variation within populationsmay be found on a much smaller scale than allozymeanalysis detects. The RAPD technique is a sensitive toolfor delineating clones in a population at a very smallspatial scale (Steinger et al., 1996) and was included as acomparison with the results of allozyme analysis.Studies of other Santalum spp. have indicated self-

incompatibility within the genus (Bhaskar, 1992; Veerendraand Padmanabha, 1996; Rugkhla et al., 1997), althoughself-compatibility has been reported for S. acuminatum(Sedgley, 1982). If little genetic diversity exists withinpopulations, and if gene ¯ow does not occur betweenpopulations, then the existence of self-incompatibilitymechanisms may result in little or no fruit production.Pollination requirements and natural levels of fruitproduction were therefore assessed in the remnant S.lanceolatum populations. Hand-pollination experimentscompared the e�ects of within-population and across-population pollen transfer to assess whether low levelsof genetic diversity within populations may have con-sequences for sexual reproduction. The combined resultsof the genetic and reproductive biology investigationswere used to determine priorities for the conservation,both in situ and ex situ, of the species in Victoria.

2. Materials and methods

2.1. Study sites

Five of the seven Victorian populations of S. lanceo-latum are found in the north-east region of the state,and these represent the ®ve southernmost populationsof the species (Fig. 1). The populations occur on rockynorthern slopes with well-drained granitic soils, and theassociated vegetation consists of low open woodlandcontaining Eucalyptus blakelyi, E. macrorrhyncha, Aca-cia implexa and A. doratoxylon (Johnson, 1996). Threeof the populations occur on Mount Meg with the closestpopulations approximately 1.5 km apart and the furth-est approximately 2.0 km apart. These populations weredesignated `Mount Meg 1' (MM1), `Mount Meg 2'

(MM2) and `Canaan College' (CAC). Another popula-tion occurs 13 km away at the Warby Range (WBR),and the ®fth population is a further 30 km distant atSpringhurst (SPR). The sites occur in the vicinity of36� S 146� E but, for the protection of the populations,exact locations are not provided. All populations coveronly small areas, ranging from 0.1 to 1.5 ha. Two of thepopulations exist on private land in small, protectivelyfenced enclosures.

2.2. Population structure

Counts were made in 1996 and 1997 to determinetotal population sizes. Visibly separate stems wereregarded as individual trees, whereas stems separated byless than 10 cm were regarded as part of the same indi-vidual but still measured separately. At each site, theheight of each stem was recorded as either less than 2 mor greater than 2 m, and stem maturity was determinedby the presence of ¯owering. Any fruit set was also noted.

2.3. Plant material

Young leaf material (when possible) was collectedfrommost individual trees from all ®ve populations (SPR:44, WBR: 46, CAC: 13, MM1: 80, MM2: 3). Sampleswere wrapped in foil and stored at ÿ70�C until requiredforDNA extraction. The remaining tissue was stored on icefor 1±3 days prior to electrophoretic assays for isozymes.

2.4. Allozymes

Small pieces of young leaf material (�0.5 cm2) wereground in the following grinding bu�er, a modi®cation

Fig. 1. Natural distribution of Santalum lanceolatum (modi®ed from

George, 1984). The location of the ®ve populations in north-east Vic-

toria, Australia (i.e. the study populations) is marked by a `+'.

46 C.L. Warburton et al. / Biological Conservation 96 (2000) 45±54

of the bu�er of Cheliak and Pitel (1984): 10 ml distilledH20, 0.7 g polyvinylpyrrolidone MW 40,000, 0.1 g poly-vinylpyrrolidone MW 360,000, 5 mg ascorbic acid, 45mg diethyldithiocarbamate, 38 mg sodium metabisulphite,17 mg Na2EDTA, 10 mg Bovine Serum Albumin, 1 gsorbitol, 5 mg nicotinamide adenine dinucleotide (NAD), 3mg nicotinamide adenine dinucleotide phosphate (NADP),10 mg dithiothreitol (DTT).Protein electrophoresis was carried out using the

Titan III cellulose acetate system (Helena Laboratories),as described in Coates (1988). Fourteen di�erentenzymes often found to be polymorphic in plants wereexamined using two continuous running bu�ers: (1) TEM,0.08 M Tris, 3 mM disodium ethylenediaminetetraaceticacid, 9.0 mMmaleic acid, pH 8.2 (modi®ed from Coates,1988); and (2) CM, 1:6 dilution of a stock solution of0.04 M citric acid brought to pH 6.1 with morpholine(Clayton and Tretiak, 1972; Weeks et al., 1995).Five enzymes gave reliable banding patterns: glucose±

phosphate isomerase (GPI, EC 5.3.1.9), glycerate dehy-drogenase (GLY, EC 1.1.1.29), malate dehydrogenase(MDH, EC 1.1.1.37), menadione reductase (MDR, EC1.6.99.) and shikimate dehydrogenase (SDH, EC1.1.1.25). The TEM electrophoresis bu�er was used forall enzymes except SDH for which the CM bu�er wasused. All gels were run at 220 V for 20 min.The gels were stained as an agar overlay. For this, 6

ml of 1% agar at 45�C was added to the following stainsolutions, modi®ed from the references cited: GPI Ð 0.6ml 1 M Tris HCl pH 8, 1 mg fructose-6-phosphaste, 0.2ml NADP (10 mg/ml), 0.2 ml methyl thiazole blue(MTT, 2 mg/ml), 0.2 ml phenazine methosulphate(PMS, 1 mg/ml), 5 units glucose-6-phosphate dehy-drogenase (Shaw and Prasad, 1970); GLY Ð 0.6 ml 1 MTris HCl pH 9.5, 6 mg dl-glyceric acid (hemi-calciumsalt), 0.2 ml NAD (10 mg/ml), 0.2 ml MTT, 0.2 ml PMS(Moran et al., 1989); MDH Ð 0.6 ml 1 M Tris HCl pH8, 0.4 ml malic substrate (1 M malic acid, 1 M Na2CO3,pH 7), 0.2 ml NAD, 0.2 ml MTT, 0.2 ml PMS (Shawand Prasad, 1970; Weeks et al., 1995); MDR Ð 0.6 ml 1M Tris HCl pH 8, 5 mg menadione, 1.5 mg nicotina-mide adenine dinucleotide phosphate (reduced form,NADH), 0.2 ml nitro blue tetrazolium (NBT, 15 mg/ml)(Moran and Hopper, 1983); SDH-±15 mg shikimic acid,0.2 ml NADP, 0.2 ml MTT, 0.2 ml NBT, 0.2 ml PMS(Moran and Hopper, 1983).The loci and alleles at each locus were designated

using numbers, with locus 1 and allele 1 representing thelocus and allele with the greatest electrophoretic mobi-lity. All enzymes migrated anodally.

2.5. DNA extraction and ampli®cation

DNA was extracted from 200±400 mg of leaf tissuefrom a subset of samples, using a modi®ed CTABextraction method (Rogers and Bendich, 1985). Sixty

random 10-mer primers (Operon Technologies, Kits A,B, F) were assessed for production of clear RAPD pro-®les in one DNA sample from each population. Fourprimers that consistently ampli®ed polymorphic RAPDproducts were chosen for analysis of multiple samplesfrom each population.Ampli®cation of DNAwas performed in 25 ml reactions

containing 1�bu�er (Perkin±Elmer), 3 mM MgCl2, 0.2mM each dATP, dCTP, dGTP, dTTP, 0.25 mm primer,10 ng genomic DNA and 1 U Taq polymerase Sto�elfragment (Perkin-Elmer). Thermal cycling was per-formed in a DNA thermal cycler (Corbett ResearchFTS-960) with the following pro®le: 5 min at 94�C (1cycle); followed by 40 cycles of 1 min at 94�C, 1 min at36�C, 2 min at 72�C and a ®nal extension step of 3 minat 72�C. Ampli®cation products were separated byelectrophoresis of 1.5% agarose gel at 70 V for 1.5 h inTAE bu�er, stained with ethidium bromide and visua-lised under UV light. DNA fragments were scored forpresence or absence using images from a MitsubishiVideo Image Copy Processor.

2.6. Pollination and fruit set

Pollen viability was assessed in all ®ve populations.Five ¯owers that had reached anthesis were randomlychosen per sample tree at each population. Four treeswere sampled at Springhurst and Warby Range, three atCanaan College and Mt Meg 1, and one at Mt Meg 2.Two anthers from each ¯ower were teased apart on aslide before a drop of ¯uorescein diacetate was added(Heslop-Harrison and Heslop-Harrison, 1970). Using a¯uorescence microscope, 100 pollen grains per samplewere examined to determine the percentage of viablegrains.Pollen tube growth was assessed in two of the popu-

lations, Springhurst andWarby Range, following naturalor hand-pollinations. The pollination treatments were(1) open pollination (i.e. natural pollination), (2) crossedwithin the population, and (3) crossed between popula-tions. Flowers of treatments (2) and (3) were bagged toexclude insects. For treatment (2), ¯owers were hand-pollinated by rubbing a freshly dehisced anther fromanother tree in the same population across the top of thestigma. For treatment (3), ¯owers were hand-pollinatedusing freshly dehisced anthers of ¯owers collected fromthe other population. Four trees were used in eachpopulation, with each tree containing three replicatein¯orescences per treatment and each replicate utilising10±15 ¯owers. All ¯owers were collected 5±6 days afterpollination and ®xed in a 3:1 ethanol:glacial acetic acid(v/v) solution.Pollen tubes were stained with decolorized aniline

blue using the method of Kho and Baer (1968). Pistilswere placed on a slide, dissected longitudinally with ascalpel, squashed gently under a coverslip, and viewed

C.L. Warburton et al. / Biological Conservation 96 (2000) 45±54 47

using a ¯uorescence microscope. The number of pollengrains on the stigma was counted, and the number ofpollen tubes within the transmitting tissue was deter-mined in the stigma, the top of the style, the bottom ofthe style, and in the ovary.The natural levels of fruit set were determined in four

of the populations, using six tagged in¯orescences pertree at each population: Springhurst (®ve trees), WarbyRange (®ve trees), Mt Meg 1 (three trees) and CanaanCollege (two trees). For each in¯orescence, 10±20 open¯owers were left intact while the remaining buds wereremoved. In¯orescences were assessed for fruit set after4 weeks.

2.7. Data analysis

For the pollination experiments, analyses of variancewere conducted regarding trees as blocks. Where a sig-ni®cant di�erence was detected, means were comparedusing Duncan's Multiple Range test (SPSS Version 6.1).For RAPD data, a pairwise similarity matrix between

populations was constructed using a simple matchingcoe�cient (Gordon, 1981). This coe�cient gives equalweighting to a match resulting from a band occurring inboth samples (1,1) or absent from both samples (0,0).Band di�erences between populations were calculatedusing all loci.The treatment of genetic diversity in species capable

of vegetative reproduction is problematic because anindividual genet can be sampled several times, with eachsample being treated in a statistical analysis as a separateindividual. Where numerous individuals of the samegenotype occur clustered together in a population, thequestion arises as to whether these individuals are theasexual descendants of a single ancestral zygote orwhether, by chance, this same genotype was producedmany times independently via sexual reproduction. Thisproblem has been discussed by Parks and Werth (1993),Wide n et al. (1994) and Sydes and Peakall (1998).One approach is to calculate the probability, P [Eq.

(1)], once a particular genotype is found, of obtainingthat same genotype, assuming sexual reproduction, in(nÿ1) subsequently sampled individuals (Parks andWerth, 1993; Sydes and Peakall, 1998), where n is thetotal number of individuals with the same multilocusgenotype. [Note that the probability that one or more ofthe subsequently sampled (nÿ1) individuals are notidentical to the ®rst individual is 1ÿP.] If P is small, itcan be concluded that the most likely explanation forthe observed cluster of individuals of the same genotypeis asexual reproduction.

P � �Pgen�nÿ1 �1�

where Pgen = probability of obtaining the observedmultilocus genotype via sexual reproduction [see Eq.(2)].

For multilocus genotypes at loci where the alleles arecodominant:

Pgen � 2h��xlix2i� �2�h = number of loci at which the genotype is hetero-

zygousx1i = allele frequency of the ®rst allele in the genotype

at locus i. For genotype 11 this would be thefrequency of allele 1, for genotype 23 it would bethe frequency of allele 2.

x2i = allele frequency of the second allele in the geno-type at locus i. For genotype 11 this would be thefrequency of allele 1, for genotype 23 it would bethe frequency of allele 3.

This equation, used for allozyme loci, has been givenpreviously in di�erent algebraic forms by Parks andWerth(1993), Wide n et al. (1994) and Sydes and Peakall (1998).For a RAPD locus, only two phenotypes are possible;

these are band present and band absent. For such loci,Pgen is �xi where xi is the frequency of whichever pheno-type (band presence or absence) was observed at locus i inthe individual being considered (Sydes and Peakall, 1998).For the allozyme and RAPD data collected in the

present study, estimates of Pgen and P were obtained asabove. The problem of estimating allele frequencies forthe calculation of P and Pgen has been discussed byParks and Werth (1993) and Sydes and Peakall (1998).It is especially di�cult in remnant populations, such asSantalum lanceolatum in Victoria, where many rametsof only one or a very few genets remain. In the presentstudy the procedure of Sydes and Peakall (1998) wasfollowed and allele frequencies were calculated using allindividuals assayed.Nei's diversity statistics (HT, HS, GST, DM) were also

calculated for the allozyme loci (Nei, 1973, 1975;Coates, 1988). For these, allele frequencies were esti-mated separately for each population.

3. Results

3.1. Population structure

The number of trees and stems per population rangedfrom three to 88 and from ®ve to 116, respectively(Table 1). Between 71 and 90% of stems were greaterthan 2 m tall in four of the populations but at Springhurst79% of stems were less than 2 m tall. The Warby Rangepopulation contained the highest percentage of ¯oweringstems (31%) and Springhurst the lowest (16%).

3.2. Allozymes

The MDH, MDR and SDH enzymes were poly-morphic in this species whilst GPI and GLY were

48 C.L. Warburton et al. / Biological Conservation 96 (2000) 45±54

monomorphic. The variable loci were designated Mdh-2(the slower of two MDH loci), Mdr and Sdh. Withineach population, all individuals were of the same geno-type. Allele frequencies for each polymorphic enzymeare given in Table 2. Multilocus genotypes and theirprobability estimates are given in Table 3.Of particular importance to the present study, was the

®nding that all individuals assayed from theWarby Rangepopulation were the same heterozygous genotype (12 13)for theMDH and SDH loci. NoMdr or Sdh homozygoteswere observed in 44 and 46 individuals, respectively.

Using these three polymorphic allozyme loci, four dif-ferent multi-locus genotypes were found for these ®vepopulations. The Canaan College and Mt Meg 1 popula-tions were indistinguishable genetically using allozymes.The probability of repeatedly sampling the same gen-

otype within a population simply by chance is extremelylow (Table 3) indicating that the hypothesis that thegenotypes have arisen independently must be rejected.Estimates of the distribution of gene diversity within

and among the populations are given in Table 4. Thelow value for HS (0.06) and the high values for GST

(0.76) and DM (0.24) indicate that the genetic diversityof S. lanceolatum resides among populations rather thanwithin populations.

3.3. RAPDs

A total of 30 reproducible RAPD bands was scoredfrom six primers (OPA-01, OPA-05, OPA-07, OPA-09,OPA-18, OPB-11). Each primer generated between twoand eight RAPDbands and polymorphisms were found in63.3% of bands scored. No variation within populationswas detected by any of the six primers but di�erenceswere detected between populations, consistent withresults from the analysis of variation in allozymes.Importantly, RAPD enabled the establishment of MM1and CAC as di�erent genotypes whereas they wereindistinguishable using allozymes.Similarity between populations ranged from 60.0 to

83.3% and populations were separated by a minimumof ®ve RAPD bands (Table 5). Primer OPA-09 was ableto distinguish between samples from the Warby Range,Mt Meg 2 and Canaan College but could not separatesamples from Mt Meg 1 and Springhurst. Primer OPA-07 generated single unique DNA fragments for samplesfrom Mt Meg 1, Mt Meg 2 and Canaan College but didnot amplify any DNA fragments from the WarbyRange or Springhurst.The probability of individual RAPD phenotypes

being present in all samples from within each popula-tion is too low to consider that they have arisen inde-pendently (Table 6).

Table 1

Structure of the ®ve Santalum lanceolatum populationsa

SPR WBR CAC MM1 MM2

Total no. trees per population 52 50 13 88 3

Total no. stems per population 99 58 28 116 5

Percentage of stems > 2 m tall 21 90 71 78 80

Percentage of stems ¯owering 16 31 25 21 20

a Trees=only individuals more than 10cm from each other; stems

include individuals within 10 cm of each other (trees may comprise

more than one stem).

Table 2

Allele frequencies for the three polymorphic loci for the ®ve Santalum

lanceolatum populations (number of samples assayed)

Allele frequencies Population

Locus Allele SPR WBR CAC MM1 MM2

Mdh2 1 1.0 1.0 1.0 1.0 0.5

2 ± ± ± ± 0.5

(41) (47) (13) (80) (3)

Mdr 1 ± 0.5 1.0 1.0 ±

2 1.0 0.5 ± ± 1.0

(44) (44) (13) (80) (3)

Sdh 1 ± 0.5 ± ± ±

2 ± ± 1.0 1.0

3 1.0 0.5 ± ± 1.0

(45) (46) (13) (80) (3)

Table 3

The genotypes assigned to the four genetic individuals identi®ed with

the three polymorphic allozyme loci, the number of stems sampled for

each genotype and the probabilities of the genotypes arising indepen-

dently

Site Multilocus

genotype

(Mdh2, Mdr, Sdh)

na Prob. genotype

occurring

(Pgen)

Prob. genotype

being present n

times [(Pgen)nÿ1]

SPR 11, 22, 33 41±45 0.022 4.98�10ÿ67WBR 11, 12, 13 44±46 0.047 2.25�10ÿ58CAC 11, 11, 22 13 0.087 1.80�10ÿ13MM1 11, 11, 22 72±80 0.087 3.98�10ÿ76MM2 12, 22, 33 3 3.52�10ÿ4 1.24�10ÿ7

a n, no. stems sampled.

Table 4

Total gene diversity and the distribution of diversity within and among

populations (using all ®ve loci) of Santalum lanceolatuma

Locus HT HS DST GST DM

Mdh2 0.18 0.10 0.08 0.44 (44%) 0.10

Mdr 0.50 0.10 0.40 0.08 (80%) 0.50

Sdh 0.58 0.10 0.48 0.83 (83%) 0.60

Over all ®ve loci 0.25 0.06 0.19 0.76 (76%) 0.24

a HT, total gene diversity;HS, mean gene diversity within populations;

GST=mean proportion of diversity among populations; DST, mean

genetic diversity among populations; DM, absolute measure of gene dif-

ferentiation (measure of net codon di�erences between populations).

C.L. Warburton et al. / Biological Conservation 96 (2000) 45±54 49

3.4. Pollination and fruit set

Viable pollen was observed in four of the populations(SPR: 52.7�4.6%; MM1: 66.6�5.1%; CAC: 68.7�4.5%; WBR: 76.6�4.2%; n=3±4 trees), but no viablepollen was observed in samples collected from theMount Meg 2 population.Pollen deposition on the stigma was detected at the

Springhurst population following both natural and hand-pollinations, but no pollen tube penetration was observed(Table 7). Most ¯owers at Springhurst abscised within 5±6 days after pollination, and so sample sizes were small.Pollen tube penetration was observed following all treat-ments in the Warby Range population. Pollen tube arrestoccurred at all locations in the pistil and irregular tube

growth was common, with many pollen tubes terminat-ing in a callose plug. Pollen tube penetration to theovary was greatest following deposition of pollen col-lected from the other population, signi®cantly greaterthan that following deposition of pollen from within thesame population (P<0.05).Fruit production was not observed on either tagged

or untagged in¯orescences at Canaan College, MountMeg 1 or Springhurst. However, at the Warby Rangepopulation, 1.18�0.39% (n=5 trees) of ¯owers on taggedin¯orescences produced fruit, and mature fruit wereobserved on other trees in the population.

4. Discussion

This study has indicated that each of the ®ve remnantS. lanceolatum populations exists as a single uniqueclone, recruiting individuals only by vegetative repro-duction. This may re¯ect the history of disturbance andfragmentation of the populations due to harvesting,clearing, grazing and ®res, coupled with disrupted gene¯ow and possibly genetic drift. Little or no fruit pro-duction is occurring in the remnant populations, withpollen sterility, pollen±pistil incompatibility or pistildysfunction resulting in little or no fruit initiation.

Table 5

Similarity matrix based on RAPD characters (below diagonal) and

band di�erences between populations (above diagonal)

Site SPR WBR CAC MM1 MM2

SPR ± 5 7 11 8

WBR 83.3 ± 8 10 7

CAC 76.7 73.3 ± 12 9

MM1 63.3 66.7 60.0 ± 7

MM2 73.3 76.7 70.0 73.3 ±

Table 6

Multi-locus RAPD genotypes, the number of stems sampled with these genotypes, the distribution of polymorphic bands, and the probability that

the genotypes have arisen independently

Site Multi-locus

genotype

number

No. stems

sampled

(% of total)

No. polymorphic

bands

(% of total)

Prob.

genotype

(Pdgen)

Prob. being

present n

times [(Pdgen)nÿ1]

SPR A 4 (4.0) 7 (39) 9.84�10ÿ5 9.53�10ÿ13WBR B 5 (8.6) 8 (42) 2.48�10ÿ4 3.78�10ÿ15CAC C 5 (17.9) 5 (29) 3.3�10ÿ6 1.19�10ÿ22MM1 D 5 (4.3) 10 (48) 5.1�10ÿ6 6.76�10ÿ22MM2 E 3 (60.0) 9 (45) 5.88�10ÿ5 3.46�10ÿ9

Table 7

Mean (�S.E.) number of pollen grains on the stigma and pollen tubes within the pistil following pollination of Santalum lanceolatum at Springhurst

and the Warby Rangea

Treatment nb Pollen grains

on stigma

Pollen tubes

in stigma

Pollen tubes

top style

Pollen tubes

bottom style

Pollen tubes

in ovary

Springhurst

Open- pollination 5 3.20�2.06 0 0 0 0

Cross withinPopulation 2 1.00�1.00 0 0 0 0

Cross between populations 9 3.00�1.81 0 0 0 0

Warby Range:

Open- pollination 38 12.97�3.18 3.95�0.65 2.92�0.55 0.55�0.14 0.18�0.08abCross withinPopulation 30 13.90�3.52 5.13�0.96 3.30�0.69 0.47�0.14 0.06�0.11a

Cross between populations 44 12.23�1.85 5.30�0.83 3.64�0.64 0.64�0.17 0.41�0.23ba Signi®cant di�erences between means at a site are indicated by di�erent letters (random block ANOVA and Duncan's Multiple Range test,

P<0.05).

50 C.L. Warburton et al. / Biological Conservation 96 (2000) 45±54

4.1. Population structure

Sandalwoods are typically slow-growing (Rao andBapat, 1995), and plantings of S. spicatum take 7±10years to reach a height of 2 m (Brand et al., 1999). Themajority of stems (71±90%) in four of the ®ve S. lanceola-tum study populations were more than 2 m tall, indicatingthat recent recruitment is occurring in those populationsto only a limited extent. Many of the remnant populationsmay have once consisted of a single stem, with popula-tion expansion occurring by root suckering.Garnet (1967) described the discovery of the Warby

Range stand in the mid-1950s, stating that only two orthree adult trees remained at that stage. The death ofone or two of the trees resulted in a single adult treeremaining. At that stage, S. lanceolatum was thought tobe represented in Victoria by only a single tree at theWarby Range and by two trees at one of the two morenorthern populations not investigated in this study(Johnson, 1996). It was therefore considered one of therarest trees in Victoria, despite having once been com-mon throughout the north central and northeast regionsof the state, including many of the slopes of the WarbyRange.Removal of sheep from the Warby Range site in 1976

and feral goats in 1982, followed by rabbit control pro-grams between 1985 and 1988 have allowed recruitmentin the population. The three populations at Mt Megwere discovered and ®rst surveyed in 1994 and 1995,and previous records of these stands are therefore notavailable.The Springhurst population displays the greatest

amount of recent recruitment. Most of the stems in thepopulation (79%) were less than 2 m tall and only 16%of the stems produced ¯owers. This stand was dis-covered in 1989, and protected by sheep- and rabbit-proof fencing in 1990. The population at that stageconsisted of nine mature trees 3.5±6 m tall (Johnson,1996). Exclusion of grazing animals has allowed extensivevegetative recruitment at this site.

4.2. Genetic diversity

Both allozymes and RAPDs detected no variationwithin populations, suggesting that each populationconsists of a single genet. The results are consistent withthe hypothesis that additional `individuals' in the remnantpopulations have been derived from asexual reproduc-tion via root suckers (Johnson, 1996). Whether this isdue to the Victorian populations being at the southernlimit of the species distribution (see Peck et al., 1998) oris common for the species remains unknown, althoughgrowth consistent with vegetative reproduction has beenobserved in other populations (John Fox, pers. comm.).Clonal patches up to 20 m across within populations ofS. acuminatum in Western Australia have also been

found using ampli®ed fragment length polymorphism(AFLP) analysis (Sieggy Krauss, pers. comm.)For populations such as SPR, CAC and MM1, where

all individuals are the same homozygous genotype, it isnot possible to determine whether recruitment hasoccurred by sexual or asexual reproduction. Where,however, a single heterozygous genotype is present andthe sample size is not small, such as for WBR, asexualreproduction is indicated. If new stems were recruitedvia sexual reproduction (whether cross- or self-pollina-tion), homozygous genotypes would be expected to bepresent. For WBR, the estimated probability of obtain-ing the observed high number of stems with the sameheterozygous multilocus genotype is minute (2.25�10ÿ58, Table 3). Even for the smallest population,MM2, where only three stems (`individuals') comprisethe population, the probability of obtaining identicalgenotypes was 1.24�10ÿ7. The results for S. album (Foxet al., 1995) are consistent with sexual reproduction anda low level of inbreeding in contrast to the clonalreproduction found in the ®ve remnant Victorianpopulations of S. lanceolatum examined in this study.The value of GST, a measure of the degree of di�er-

entiation between populations (Nei, 1973), was high forS. lanceolatum, with 76% of the observed diversity pre-sent between populations. In comparison, the values ofGST for S. album in Timor and India were 0.172 and0.286, respectively (Fox et al., 1995). The di�erence islargely due to the marked di�erence in the mean HS

values. The total gene diversity (HT) value for S. lanceo-latum in Victoria (0.25) was similar to the value for S.album in Timor (0.31) whereas for S. spicatum sampledover a distance of 1000 km in Western Australia, HT

was 0.53 (Brand, 1993). Whether the HT value found inthis study for S. lanceolatum is typical for the species isnot known.RAPD analysis is generally a more sensitive technique

than allozyme analysis for detecting genetic variationbecause it detects variation across the genome ratherthan being restricted to a particular type of gene pro-duct. Importantly in this study, RAPD analysis enabledthe di�erentiation of populations Mount Meg 1 andCanaan College (Fig. 2) that could not be separated onallozyme results because all loci were monomorphic.RAPD analysis has been used successfully in vascularplants for delineating clones of Iliamna corei (Stewartand Porter, 1995) and to demonstrate mixtures of geno-types in populations of Astelia australiana (James andAshburner, 1997). Whilst RAPD analysis cannot provethat there is no variation within the S. lanceolatumpopulations, delineation of clones has been possible inother species using one or a few primers (Stewart andPorter, 1995). The ®ndings of Sydes and Peakall (1998)for Haloragodendron lucasii are unusual because twoclones within a population were indistinguishable withRAPDs but were di�erentiated using allozymes.

C.L. Warburton et al. / Biological Conservation 96 (2000) 45±54 51

4.3. Pollination and fruit set

Flowering occurred in all ®ve study populations butmature fruits were only observed at one location, theWarby Range, where the ®nal fruit set on taggedin¯orescences was 1.18%. This ®nal set is somewhat lessthan values reported for S. album (5.2 and 5.3%, Veer-endra and Padmanabha, 1996; and 10%, Rugkhla et al.,1997) but similar to the ®nal set reported for S. spicatum(1.3%, Rugkhla et al., 1997). Complete lack of viablepollen could not account for poor fruit set in four of thepopulations, where the pollen viability ranged from 52.7to 76.6%. No viable pollen was obtained in the ®fthpopulation, Mt Meg 2, and no fruits were noted in thatpopulation.Limited or failed sexual reproduction resulted from

pollen±pistil incompatibility or pistil dysfunction forfour of the populations. Self-incompatibility partlyexplains the low fruit set in theWarby Range population.Pollen deposition was considerable and many pollentubes entered the stigma following both natural orhand-pollination at the site, but most tubes were arrestedwithin the style or ovary. Pollination using Springhurstpollen (i.e. from a di�erent genotype) did, however,result in greater tube penetration to the ovary than pol-lination using Warby Range pollen (i.e. from the samegenotype). The apparent isolation of the populationensures that all natural pollination is, e�ectively, self-pollination involving pollen ¯ow only within the singlegenet. RAPD analysis of genetic variation in Lyono-thamnus ¯oribundus also revealed that most populationscomprised a single genet, and it was suggested that sexualreproductive failure resulted from self-incompatibilityor inbreeding depression and the con®nement of polli-nators to single genets (Bushakra et al., 1999).No pollen tube penetration was observed at Spring-

hurst despite a small amount of pollen deposition on the

stigmas, even following deposition of pollen fromanother population (the Warby Range). Both theSpringhurst and Warby Range populations producedviable pollen, and Springhurst pollen penetrated to theovary in Warby Range ¯owers. The Springhurst popu-lation may exhibit pistil dysfunction regardless of pollengenotype, a phenomenon previously observed in onegenotype of S. album (Rugkhla et al., 1997). Pollinationsutilising a wider range of genotypes would be requiredto resolve this issue. In any case, the isolation and singlegenet composition of the Springhurst population againensures that only self-pollination is occurring.Pollination in Santalum requires pollen transfer by

insects (Sedgley, 1982; Veerendra and Padmanabha,1996), although the pollinators of S. lanceolatum havenot been determined. Fragmented populations canexhibit disturbed pollination as a result of restrictedpollinator movement, inadequate ¯owering for polli-nator attraction, and insu�cient quantity of pollen orincompatible pollen (Byers, 1995; Oostermeijer et al.,1996). The Springhurst population of S. lanceolatumoccurs on a small rocky outcrop completely surroundedby grazing pastures, and the natural stigma pollen loadswere particularly low in this population. The WarbyRange population, where pollen loads were considerablyhigher, occurs in a much larger tract of natural vegetation.Pollen tube penetration and fruit set can be related to

stigma pollen load even for a species, such as S. lanceo-latum, that produces single-seeded fruits (Hormaza andHerrero, 1994; Trueman and Wallace, 1999). Poor fruitset in the remnant study populations may result frompoor pollen deposition due to limited pollinator visita-tion. However stigma pollen loads were also low fol-lowing hand-pollinations, despite the usually broadperiod of stigma receptivity in Santalum (Sedgley, 1982;Rugkhla et al., 1997), indicating that pistil dysfunctionwas the cause of the poor fruit set at Springhurst.

Fig. 2. RAPD pro®les of all samples from each site generated by the operon primer OPA-09. Within sites, RAPD pro®les do not vary but di�er-

ences are apparent between populations/sites. Samples from the Warby Range (WBR) have a DNA fragment approximately 500 bp in size

(arrowed) which is not present in other populations. Samples from Canaan College (CAC) lack the 640 bp fragment (arrowed) found in the other

populations.

52 C.L. Warburton et al. / Biological Conservation 96 (2000) 45±54

4.4. Conservation

Large numbers of populations may need preservationto adequately represent the genetic diversity of clonalspecies (Peakall and Sydes, 1996). As each of the ®vestudy populations appeared to exist as a unique singleclone, the conservation of S. lanceolatum in Victoriamay require protection of all remnant populations.Similar action has been advised for rare taxa exhibitingclonality such as Astelia australiana (James and Ashbur-ner, 1997), Haloragodendron lucasii (Sydes and Peakall,1998) and Eucalyptus phylacis (Rosetto et al., 1999).The maintenance of several small populations can

have disadvantages in that it promotes the decay ofgenetic variability (Varvio et al., 1986) but this does notappear to be a consideration for populations whichcomprise single genets, such as in S. lanceolatum.Recruitment by root suckering is occurring to somedegree in all populations and there is strong evidencethat exclusion of grazing animals is an important factorfor allowing recruitment. Continuing control measuresto exclude grazing animals appear warranted for theprotection of each remnant.The fragmentation of the Victorian populations into

isolated stands of single genets, exhibiting little or nosexual reproduction, suggests an added need for a broadconservation strategy. It has been suggested thatgenetically depauperate species are unlikely to regaingenetic variation without human intervention (Godt etal., 1995). Whilst the species as a whole is not endan-gered, the retention of the species in Victoria willrequire manipulation of the extant clones if breedingpopulations are the desirable outcome of the conserva-tion strategy. The Warby Range population does pro-duce a small number of fruits but, even there, allrecruitment appears to be occurring asexually.The lack of seedling recruitment in remnant popula-

tions of S. lanceolatum is partly explained by pollen-pistil incompatibility and dysfunction, but could also bethe result of past management practices and/or dis-turbance triggering asexual reproduction. Ellstrand andRoose (1987) and Eriksson (1989) have suggested thatfor species that are clonal, but also exhibit some sexualreproduction, sexual propagules are most likely to havefounded populations even if seedling recruitment is nowrare in established populations. This appears to be thecase with S. lanceolatum. The hemiparasitic nature of S.lanceolatum should not be an impediment to seedlingrecruitment and the active asexual recruitment in mostpopulations suggests that plants have found suitable hosts.Ex-situ cultivation of the remnant genets, as proposed

by Johnson (1996), remains an option for the conserva-tion strategy in Victoria. Sexual reproductive failure inremnant self-incompatible populations poses a threat topersistence (Holsinger and Vitt, 1997) and small popu-lations may be particularly susceptible to catastrophic

events or losses of habitat quality (Cropper, 1993; Oos-termeijer et al., 1996). Construction of new S. lanceolatumstands comprising a range of genotypes may provideself-sustaining populations, capable of sexual reproduc-tion and valuable in the long-term as a source of com-mercial germplasm. Construction of new stands may bepreferable to the introduction of di�erent genotypesinto the existing remnants, as there is some risk of out-breeding depression in the resultant seedlings if parentplants are strongly adapted to di�erent local habitats(Oostermeijer et al., 1996; Peck et al., 1998).This study concentrated on the genetic diversity and

reproductive biology of the remnant southernmostpopulations of S. lanceolatum. Peripheral populationsare often genetically divergent from central populations,and may be important sites for future speciation events(Lesica and Allendorf, 1995). A comparison betweenthe remnant S. lanceolatum populations and those fromother states in Australia would provide a clearer pictureof the natural levels of genetic diversity and recruitmentin more intact populations. With limited resources forconservation, such information is useful when decidingwhether intervention is required to ensure the con-servation of the Victorian populations or whether suchintervention is warranted given that the species occursin other Australian states.

Acknowledgements

We thank Glen Johnson (Victorian Northeast RegionalFlora and Fauna O�cer) for suggesting the project,Karen Clutterbuck and Paul Ratajczyk for help in the®eld, and the property owners for allowing access to thepopulations. Our thanks also to John Fox (Curtin Uni-versity) for providing information on the genus.

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