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Introduction

Delineation of genomic provenanceboundaries of two well-distributed arid

tropical native grass species from thenorth of Western Australia was undertakento establish propagule harvest zones andprotocols to ensure replacement of locallydiverse populations after mining. Main-tenance of the local genetic provenanceprovides the greatest opportunity for suc-cessful establishment of plants, becauselocal genotypes are presumed to be moresuited to prevailing conditions and havebeen shown to out-compete introducedgenotypes. For example, in translocationstudies of Nassella pulchra, local geno-types grew at twice the rate with greatersurvival than non-local genotypes (Knapp

& Dyer 1998). Restoration of local geno-types also facilitates the integration of therestored area with the surrounding matrixof vegetation (Fletcher et al. 1994) andminimizes perturbations to the indigenousbreeding system (Haig et al.1990;Rossettoet al. 1995).

Restorat ion of genet ica l lytyped mater ia l (genet icrestorat ion)

Restoration of common species shouldfollow similar protocols to those used inconservation programmes for rare species(Coates & van Leeuwen 1996), which aimto retain the ebb and flow of local genomedynamics (Mistretta 1994). This necessi-tates delineation of natural genomic

boundaries to allow selection of propag-ules, which maintain the integrity of thelocal provenance (Rossetto et al. 1995)and remove the potential for out-breedingdepression and weed hybridization (Montalvo & Ellstrand 2001).The data alsodetermine the number of individuals fromwhich propagules must be harvested tomaintain natural diversity (von Pergeret al. 1994; Rossetto et al. 1995).

Molecular genetic differentiation ingrass taxa has identified that individualclones can encompass a broad range ofspatial scales. Single clones dominateentire climatic regions (Rice & Mack1991), while changes between clones canoccur within a few metres (Hamrick &Allard 1972). Phenotypic variation mayalso occur within a genotype grown under

A molecular approach to provenance delineationfor the restoration of hummock grasslands(Triodia spp.) in arid-tropical AustraliaBy Grant Wells, Grace Zawko, Maurizio Rossetto and Kingsley Dixon

Grant Wells, Grace Zawko and Kingsley

Dixon are Restoration/Conservation Scientists

with the Botanic Gardens and Parks Authority

(Kings Park and Botanic Gardens West Perth,

Western Australia 6005, Australia. Grant Wells is

the corresponding author. Tel: +61 8 9480 3623;

email: grantw@kpbg.wa.gov.au). Maurizio Ros-

setto is the Conservation Ecologist with Plant Sci-

ences, Royal Botanic Gardens Sydney (Mrs

Macquaries Road, Sydney, NSW 2000, Australia.

Tel: +61 2 9231 8337; email: Maurizio.

Rossetto@rbgsyd.nsw.gov.au). The project was

funded by Argyle Diamonds Pty Ltd as part of a

study to restore endemic hummock grasses to

post-mine sites and supported the authors

research into the conservation genetics of Aus-

tralian flora.

Summary Studies were undertaken to develop genetic principles for the restoration ofspinifex grasses (Triodia spp.) at the Argyle Diamond Mines in northern Australia. Thestudy used random amplified polymorphic DNA (RAPD) DNA markers to provide a molec-ular delineation of provenance boundaries for two species Curly Spinifex (Triodia bitextura)and Triodia bynoei and to provide measures which ensure replacement of local geneticdiversity. Within-population diversity measures (0.44 ± 0.03 T. bitextura and 0.32 ± 0.04T. bynoei) and the proportion of diversity attributed to between-population differences(≈ 12%) by analysis of molecular variance (AMOVA) indicate extensive out-crossing in theseseeder species. As such, both species should be restored using seed-based methods tomaintain natural diversity. Significant groups of populations (P < 0.05) identified by AMOVA

correspond directly to clustering of populations in a dendogram drawn using UPGMA, indi-cating significant provenances in both species. For T. bitextura, a significant genotypic vari-ation identified at 60 km from mine populations was not evident at 30 km. Seed forrestoration at the mine should, therefore, be harvested from within 30 km of the area to berestored to ensure maintenance of the local provenance. Two T. bynoei populations located2 km apart were found to represent a single genotype, all other populations were identifiedas separate genotypes. The majority of the isolated T. bynoei populations, therefore, rep-resent distinct provenances. For both species, in excess of 80% of the measured speciesdiversity occurred in just 20 individuals. As restoration of these grasses will necessitateharvesting seed from thousands of plants the species capacity for evolutionary changeshould be maintained. With continuing improvements in the effectiveness of moleculargenetics technology, the assessment of provenance boundaries through the use of geneticmarkers is becoming a realistic option for the development of more genetically represen-tative sourcing of genotypes in plant restoration programmes.

Key words provenance, random amplified polymorphic DNA, restoration, spinifex, Triodia.

a variety of conditions (ecotypes). Thus,the opportunity to use principles fromother studies of grass genetics are limited.

Signi f icance of spin i fexgrasses

Spinifex grasslands are endemic to Aus-tralia (Lazarides 1997) and cover an esti-mated 22% of the mainland (Jacobs 1992)providing the characteristic hummockgrassland appearance of much of the aridand semiarid regions (Bogusiak et al.1990). The grasses often dominate regionsthey inhabit with cover values of up to95% (Winkworth 1967; Griffin 1984) andprovide a key food resource for nativefauna (Perry 1967; Jacobs 1992) anddomestic stock (Gardner 1952;Perry 1967;Jacobs 1992).

Spinifex deserts have traditional Aborig-inal ownership (89.6%/1.8 million km2),support substantial pastoral and miningactivities (1991–1992: A$10 000 million)and a growing tourist trade (Noble et al.1996). Current and past pastoral practiceshave led to the degradation of large areas.In an analysis by Noble et al. (1996), forexample, 40% of pastoral areas were foundto be in poor condition with a further 2%severely degraded.

Study species

Triodia bitextura

Triodia bitextura has a range extendingacross northern Australia encompassing amosaic of habitats. At the study site, singlestands of this species can cover squarekilometres with projected foliage cover of75%.The species provides refuge for faunaand can produce in excess of 11 000seeds/m2 of habitat providing a valuablefood resource. Plants are typically leftungrazed by domestic stock, but it is animportant drought fodder. The speciesexhibits numerous morphological forms,several of which may occur within metresof each other or a large area may be domi-nated by a single form.Triodia bitextura isa facultative seeder-resprouter with depen-dence on resprouting or seedling recruit-ment varying between populations andpotentially influenced by age (Wells 1999).The species exhibits a capacity for rapid

clonal recruitment producing ramets thatremain integrated by long-lived stolonifer-ous connections, allowing single clones toextend over several metres. The potentialfor high levels of clonality in populationsraised the issue of the diversity of geno-types within and between populations.

Triodia bynoei

Triodia bynoei is found throughout theKimberley region of Western Australia andinto the Northern Territory. At the studysite the plant occurs in a series of isolatedpopulations inhabiting steep rocky slopesand the associated intermittent streamsthat drain them. Soil depauperate ridgesinhabited by the T. bynoei are analogous to many of the landforms created by themining process, indicating that the speciesmay be suited to revegetating these areasin the process of restoration. Triodiabynoei has been observed to recruit solelyfrom seed (Wells 1999) and with seem-ingly more reliable annual yields (occur-ring later in the wet season, with up to60 000 seeds/m2) than neighbouringhummock grasses may provide an impor-tant food source for local fauna.

Isolated populations may be expectedto be closed systems exhibiting a highlevel of localized variation with little geneflow between populations (Ellstrand &Elam 1993).The species has little morpho-logical differentiation of plants betweengeographically isolated populations, suchthat potential genetic differences arephenotypically cryptic. The potential forgenetic differentiation between T. bynoeipopulations necessitated examination ofrelatedness.

Site descr ipt ion

Argyle Diamond Mines operates withinthe Lissadel pastoral lease (16°43′S;128°23′E) in the Kimberley region ofWestern Australia.The region is dominatedby large plains interrupted by hilly tracts,steep-sided tablelands and rock outcrops.Soils are lightly acidic to moderately alka-line; show little structural developmentwith low chemical fertility and littleorganic content (Beard 1979).

Vegetation is characterized by short

grass savannah on calcareous plains andtree steppe on mountainous and rockycountry. There are five recognized grass-land communities of which Triodia spp.are a conspicuous component. The devel-opment of these pyrophilous grasslandsmay date to the Middle Tertiary whenclimate conditions became more con-ducive to fires (Beard 1979). Climate isarid-tropical, with monsoonal rainfall(400–700 mm p.a.) occurring in summer(November–April) when daily maximaexceeding 40°C are commonplace (Beard1979). Rainfall in the remaining months is sporadic and several consecutive drymonths are not uncommon with anaverage of only 60 rain-days per year.

Aims

Interest in restoration of Australian nativespecies has increased in recent times, butlittle is known about the spatial scales overwhich seed may be transferred and suc-cessfully grown while not causing genetic‘pollution’ by introducing out-of-site geno-types (Knapp & Rice 1996). The presentstudy represents the first attempt todevelop a method for evaluation of geneticdiversity in Triodia species. The studyaimed to investigate provenance delin-eation and diversity in T. bitextura andT. bynoei using RAPD. Two main issueswere investigated:

1 Quantification of genetic variabilitybetween individuals within populationsto provide measures of diversity innatural (analogue) communities tomatch to restored sites.

2 Comparison of genetic variabilitybetween populations to identifyprovencal diversity and distinguishprovenance boundaries to allow selec-tion local genotypes for restoration.

Methods

Random amplified polymorphic DNAanalysis was chosen for this study, as themethod has previously been successfullyused to discern genetic provenance andspecies diversity (Williams et al. 1990;Rossetto et al. 1999) including clonal (Castiglione et al. 1993; Bachmann 1994;Steward & Porter 1994) and Western

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Australian taxa (Kennington et al. 1996;Rossetto et al. 1999).

Studies of the provenance of WesternAustralian native species using RAPDanalysis (Rossetto et al. 1995; Rossettoet al. 1997; Zawko et al. 2001; Mattneret al.2002) have used sample sizes rangingfrom one to 12 plants per population. Inthe present study, samples were collectedfrom 10 plants at each of six T. bitexturapopulations (Fig. 1). Forty T. bynoei plantswere sampled, five plants at 4FW and 5FW(smaller populations) and 10 at each of theremaining populations (Fig. 1). Youngleaves were harvested, transported in iceand then stored at –82°C prior toprocessing.

DNA was extracted using the method ofPotter (1991) incorporating an additionalchloroform purification step (Rossettoet al. 1995). Primers (Table 1) were the

same as those used by Rossetto et al. (1995)and the ability to reproduce results was val-idated with duplicate reactions (Williams etal.1990;Keil & Griffin 1994).Of 12 primerstested, six 9mer and two 10mer primers,which generated the highest number ofrepeatable bands, were selected for theanalysis.

Reactions were performed in a totalvolume of 12.5 µL that contained 1.25 µLbuffer (25 mmol/L KCl; 5 mmol/L Tris-HCl(pH 9), 0.05% Triton X100); 1 mmol/LMgCl2, 0.1 mmol/L of each dNTP, 0.5 unitsTaq DNA polymerase, 5 ng primer, 13 ngDNA and the remaining volume consistingof DNA-free deionized water. Reactionconditions were controlled by a HybaidOmnigene thermocycler (Hybaid LtdTeddington, Middlesex, UK) using heatedlids. Polymerase chain reaction (PCR)cycles: 94°C for 5 min; 94°C for 30 s, 34°C

for 1 min and 75°C for 2 min wererepeated 40 times followed by a final stageat 75°C for 10 min.

Amplified DNA fragments were sepa-rated by electrophoresis in 1.5% agarose gel (type 1-B:Low EEO) in 0.5 × TBE solute(Iris-Boric acid and EDTA buffer) at 70 Vfor a period of 4.5 h. Eco RI/Hind III cutλDNA was run on each gel as a fragmentsize marker. Fragments were visualizedunder UV following staining with10 µL/100 mL ethidium bromide. A digi-tized image was saved directly to com-puter through a Sony SSC-M370 SE blackand white video camera, using CREAM®software (Kem-En-tec A/S Copenhagen,Denmark).

Data analys is

Both the presence (1) and absence (0) ofbands was scored to construct a matrix

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200km18016014012010080604020

4FW5FWCH GGN

ADFPpAPpME BW

02040

Figure 1. Schematic diagram of the position and distance between study populations in relation to each other (left to right represents a southto north progression). Grey circles are Triodia bynoei populations, black are Triodia bitextura. The diagram does not depict proximity betweenpopulations of different species. Populations 4FW and AD (T. bynoei and T. bitextura, respectively) represent the local provenance and weresubsequently placed at 0 km.

Table 1. Primer composition and fragment production for Triodia bitextura and Triodia bynoei

Primer Primer Total bands Average bands/Plant (range)code composition Triodia bitextura Triodia bynoei Triodia bitextura Triodia bynoeia TGA GCG GAC A 14 15 2.7 (1–6) 4.2 (1–6)d CAG GCG CAG A 16 9 2.9 (1–7) 2.0 (1–4)R1 CCC ACC AAC 16 11 4.4 (1–11) 4.0 (1–7)R2 CCC TCC TTC 15 12 4.5 (2–8) 2.4 (1–5)R3 GGG TTG TGG 12 9 4.1 (2–7) 2.4 (1–5)R4 GGG AGG AAG 12 14 2.9 (1–5) 3.1 (1–7)R5 GGG TGG TTG 11 9 3.1 (1–5) 3.6 (1–7)R6 GGG AAG GAG 13 16 2.6 (1–6) 3.6 (1–6)

Column statistic 109 95 2.9 (1–7) 3.2 (1–7)

Numbers in bold are the total bands for the species from all primers; italicized bold numbers are the mean value for the species for all primers andnumbers in parentheses the range for a single primer.

depicting the distribution of bands throughall individuals of the species being exam-ined.This distribution of bands is describedboth within and between populations.

Nei and Li’s similarity coefficient (Nei & Li 1979) was chosen to quantify thegenetic distance between study individu-als as it has been shown to be less influ-enced by artefactual bands that may ariseas a result of the RAPD procedures(Lamboy 1994). An estimate of geneticsimilarity (F) was thereby attained:

F = 2nxy/(nx + ny)

where nxy is the number of bands occur-ring in both x and y, nx is the number ofbands recorded for x, and ny is the numberrecorded for y. Genetic distance (D) wasthen calculated:

D = 1 – F

Mean values for the genetic distancebetween all combinations of pairs of indi-viduals both within and between popula-tions were derived. Values for the totalgenetic diversity (HT) of the samples mea-sured (species diversity) and the averagemean distance within populations (HS)were calculated and an estimate of thegenetic diversity attributable to between-population differences determined (Nei &Li 1979):

Diversity between populations= (HT – HS)/(HT) × 100 (%)

A visual representation of the relatednessbetween individuals was drawn from thedata matrix by the unweighted pair-groupmethod using arithmetic averages (UPGMA)clustering from a squared euclidean dis-tance matrix (distance (x,y) = Σ(xI – yI)

2)produced using the RAPDist program.Analysis of molecular variance (AMOVA)(Excoffier et al. 1992; Weir 1996) wasapplied to the data matrix using the Arle-quin program (Schneider et al. 1997) toexamine for significantly different groupswithin the species.

Results

Repeatable fingerprints were obtained for46 (of 60) T. bitextura and 36 (of 40)T. bynoei individuals sampled. Each primer

produced from 11 to 16 bands from allT.bitextura individuals (Table 1) for a totalof 109 unique species bands (SB) from allprimers. Results for T. bynoei were similarwith each primer producing from nine to16 bands for a total of 95 SB. On average,in excess of three bands were producedper individual by each primer for bothspecies with totals of 16–48 produced forindividual plants from all primers.

Distr ibut ion of markersbetween a l l indiv iduals

Triodia bitextura and T. bynoei exhibiteda similar distribution of bands (Fig. 2). Alow (2.1%) proportion of the T. bynoeiSB occurred in all individuals (mono-morphic), over 85% occurred in half of the individuals (Fig. 2) and 9.5% wereexclusive to an individual. Similarly, onemonomorphic band (0.9%) was identifiedfor T.bitextura,over 85% of SB occurred inhalf of the individuals and 10.1% wereexclusive to an individual.

Marker d istr ibut ionbetween populat ions ofspecies

Triodia bitextura

The number of bands varied between pop-ulations (Table 2) indicating differences in

the genotypic composition of plants.Almost half of the SB were common topopulations AD and BW (Table 3), whichare located the shortest distance apart(Fig. 1). The proportion of SB sharedbetween populations often exceeded one-third with the few lower values arisingform the pairing of geographically distantpopulations (Fig. 1).

Triodia bynoei

The number of bands varied betweenpopulations (Table 3) indicating differ-ences in the genotypic composition ofplants. The highest proportion of speciesbands common to two populations andthe highest proportion shared exclusivelyby a pair of populations was evidentbetween the two separated by the shortestgeographical distance, 4FW and 5FW(Fig. 1). Typically, less than one-third of SBwere common to any pair of populations.

Quant i f icat ion of genet icdivers i ty (Nei and L is imi lar i ty coef f ic ient )

Triodia bitextura

The shortest genetic distance betweenpopulations T. bitextura (AD and BW)(Table 4) corresponds with the shortestgeographical distance (Fig. 1). The mean

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0

5

10

15

20

25

30

35

40

45

% plants in population

Triodia bitextura

Triodia bynoei

% b

ands

1–1

0

11

–2

0

21

–3

0

31

–4

0

41

–5

0

51

–6

0

61

–7

0

71

–8

0

81

–9

0

91

–1

00

Figure 2. Distribution frequency of the DNA fragments generated by the RAPD procedure.Bands occur in clusters of frequency throughout all individuals examined, for example, 36.8% ofall bands scored for Triodia bynoei were present in 1–10% of the individuals sampled.

genetic distance between individuals from both populations was lower thanthat recorded within population BW In allother cases,genetic distance between indi-viduals in pairs of populations exceededthe mean distance between individualswithin either population. Total geneticdiversity (HT) of T. bitextura was deter-mined to be 0.54 with almost one-fifth oftotal genetic diversity (18.5%) attributedto between-population differences.

Triodia bynoei

The shortest genetic distance wasrecorded between populations 4FW and5FW (Table 4) separated by the shortestgeographical distance (Fig. 1). Meangenetic distance between individualsfrom both populations was of the samemagnitude as that recorded between indi-viduals within population 5FW. In allother cases, genetic distances betweenindividuals from pairs of populations wassubstantially greater than those recordedbetween individuals within a single popu-lation. Total genetic diversity of T. bynoeiwas calculated to be 0.47 with almostone-third (31.9%) of total genetic diver-sity attributed to between-populationdifferences.

Provenance v isual izat ion(U P G M A)

Both species were generally separated intodistinct clusters for each population byUPGMA (Fig. 3).

Triodia bitextura

For T. bitextura mixing of individualsbetween populations was evident on twooccasions, between populations AD andBW and again with Pp and PpA, depictingcloser relatedness of some individualsbetween the populations than within.AD and BW occurred only 2 km apart, Pp and PpA were separated by 20 km (Fig. 1).A trend of increasing divergence withincreasing geographical distance is alsoevident. AD the ‘local provenance’ (Fig. 1),forms the mixed cluster with populationBW (the nearest neighbour) and thesepopulations form a larger cluster with ME,the next closest population.The remaining

more distant populations form a separatecluster within which Pp and PpA are sepa-rated from F.

Triodia bynoei

An indication of closer relatednessbetween than within populations inT. bynoei was evident only for individualsfrom 4FW and 5FW. These populations

were then linked to CH and GN, the nextclosest populations to form a largercluster, which then links to the mostdistant population G.

Signi f icant provenancegroups (A M O VA)

An AMOVA of the data matrix from theT. bitextura populations identified

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Table 2. Distribution of species bands between populations of Triodia bynoei

4FW 5FW CH GN G

4FW 7.4 0 0 2.15FW 38.9 0 1.1 1.1CH 24.2 27.7 2.1 2.1GN 29.5 23.2 25.3 4.2G 31.6 21.1 24.2 29.5No. bands 48 41 33 47 62

Values in lower corner of table represent the proportion (%) of species bands shared between thepaired populations and values in the top corner are the proportion (%) of species bands sharedexclusively by the paired populations. The number of bands scored for the population is provided inthe bottom row of the column.

Table 3. Distribution of species bands between the populations of the Triodia bitextura

AD BW ME Pp PpA F

AD 3.7 0 3.7 0.9 0.9BW 45.9 0.9 0 0.9 0.9ME 35.8 39.4 0.9 0 0Pp 29.4 29.4 27.5 0.9 0.9PpA 33.0 33.0 36.7 37.6 3.7F 35.8 36.7 33.9 32.1 22.0No. bands 62 70 60 50 62 62

Values in lower corner of table represent the proportion (%) of species bands shared between thepaired populations and values in the top corner are the proportion (%) of species bands sharedexclusively by the paired populations. The number of bands scored for the population is provided inthe bottom row of the column.

Table 4. Matrix of genetic distance (Nei and Li) within (italicised) and between populations ofTriodia bitextura (top) and Triodia bynoei

AD BW Pp PpA ME F

AD 0.34BW 0.46 0.49Pp 0.61 0.64 0.45PpA 0.57 0.61 0.52 0.44ME 0.50 0.55 0.56 0.55 0.39F 0.56 0.62 0.59 0.57 0.55 0.50

4FW 5FW CH GN G

4FW 0.355FW 0.30 0.29CH 0.52 0.55 0.24GN 0.57 0.58 0.49 0.27G 0.69 0.74 0.66 0.68 0.47

significant differences (P < 0.0002)attributing 12% of the measured speciesvariability to between-population differ-

ences, with the remaining 88% attrib-uted to within-population differences.Similar figures (11.7% and 88.3%, respec-

tively, P < 0.0002) were produced forT. bynoei indicating for both species thatthe majority of variation occurs at thelevel of the individual, but also identify-ing significant disparities between popu-lations.

Triodia bitextura

An AMOVA with each population as a sepa-rate group generated high negative φ andVA values (Table 5), indicating that indi-viduals between at least two of the popu-lations were more closely related thanindividuals within populations (Excoffieret al. 1992). AD as the adopted localprovenance (Fig. 1) was then combinedwith its nearest neighbour (BW). Compar-ing each remaining population to thisgroup suggested that all other popula-tions, with the exception of ME, were sig-nificantly genetically different. This isindicated by the significant positive φ andVA values (Excoffier et al. 1992) when ADand BW were grouped together and thenegative and/ or statistically non-significant results obtained when sepa-rated. The zero value obtained for φ andvariance component in comparisons withthe ME population indicates no significantdifference to the AD and BW group;however, the positive φ and VA indicatethat individuals within ME are moreclosely related to one another than to indi-viduals in either AD or BW.

A second series of analyses supportedthis finding, because all other populationsremained significantly different to thegroup following the inclusion of ME(Table 5). A further series of analyses com-pared this group to combinations of theremaining populations with a significantanalysis identifying three distinct groups(Table 5), which replicates the clusteringof populations by UPGMA.

Triodia bynoei

As with T. bitextura, negative φ and VAvalues when populations were analysedindividually indicated some instances ofcloser relatedness between than withinpopulations (Table 6). In the same processadopted for T. bitextura the adopted localgenome (4FW) was grouped with thenearest neighbour 5FW (Fig. 1) and com-

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F1F5F10F8F7F4F3F2PpA5PpA8PpA6PpA4PpA2Pp10Pp8PpA1Pp6Pp5PpA3Pp2BW6BW2BW10BW9BW1BW7BW5BW4AD2AD3AD7AD10AD5AD6AD9AD8AD1ME5ME10ME8ME9ME7ME2ME4ME3ME1G10G9G6G8G7G4G5G3G1FW5-5FW5-2FW5-1FW4-5FW5-4FW5-3FW4-3FW4-2FW4-1CH2CH5CH10CH9CH8CH4CH3CH1GN5GN10GN9GN6GN7GN3GN4GN2GN1

Figure 3. Dendogram derived from upgma indicating relatedness between individuals andpopulations of Triodia bitextura (italicized) and Triodia bynoei.

pared to all remaining populations, whichwere found to be significantly differentfrom this group. The remaining popula-tions were then compared in all possiblecombinations to the 4FW and 5FW groupto determine whether any other grouprepresented a different provenance, andthe significant difference between fourgroups was determined (Table 6).

Discussion

DNA markers were generated for nativeAustralian spinifex grasses as an aid forensuring genetic representativeness ofspecies in ecosystem restoration pro-grammes in the northern Kimberleyregion of Western Australia. The number

of bands produced from the number ofsamples assessed for each species com-pares favourably with previous studies(Barker et al. 1994; Rossetto et al. 1995)indicating that an adequate number ofbands were generated to allow meaningfulanalyses to be conducted.

Tr iodia b i textura

Triodia bitextura was observed to prolif-erate vegetatively in the wild, particularlyin mature communities, where seedlingrecruitment is seldom observed. Clonalorganisms exhibit high band sharing andlow diversity (Brookfield 1992). Con-versely, less than 1% of bands generated forT.bitextura were monomorphic (commonto all individuals); and diversity within

populations (0.34–0.5) exceeded thatidentified within populations of an oblig-ate seeder species Grevillea scapigera(0.20–0.32) (Rossetto et al. 1995) usingthe same RAPD procedure.The proportionof diversity attributed to among-popula-tion differences in T. bitextura (21% byAMOVA and 18% by Nei and Li) is similar to that found for out-crossing seederspecies (Mattner et al. 2002). Populustremuloides (Mitton & Grant 1996) andTetraria capillaris (von Perger et al.1994) are species that reproduce bothfrom seed and asexually and exhibitsimilar diversity to T. bitextura. Taxa thatrecruit vegetatively in mature communi-ties may maintain genetic variability fromperiodic seeding events even if seedlingrecruitment is rare in extant populations(Ellstrand & Roose 1987). The geneticcomposition of T. bitextura populations isdominated by sexual recruitment and, assuch, it is the view of the authors that seedshould be used for restorative purposes tomaintain natural genetic diversity.

Provenance boundaries and propaguleselection

The calculations of Nei and Li (1979)attribute a substantial proportion of themeasured species diversity to between-population differences, which concurswith the results of the AMOVA that identi-fied significant differences between popu-lations. Significant differences betweengroups identified by AMOVA correspondwith the clustering of populations in thedendogram drawn from UPGMA, suggestingthat the differences depicted are signifi-cant. Although the proportion of vari-ability attributed to between-groupdifferences identified by AMOVA was low,the significance of the values suggests thatthe groups identified be treated as differ-ent provenances in the interests of ‘bestpractice’ in restoration. Based on theseobservations, it is suggested that seed forrestoration of T. bitextura be collectedfrom within a 30-km radius of the site to berestored (e.g. distance from AD to ME).

Maintenance of natural diversity

There is little consensus on the speed andflexibility of inheritable adaptive change,

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Table 5. Summary of molecular variance (AMOVA) analyses with Triodia bitextura populations

Population groupings P φCT Variance % Totalcomponent(VA)

AD vs BW vs ME vs Pp vs Ppa vs F < 0.002 – 0.119 – 0.735 11.94AD/BW vs ME < 0.002 0.000 0.000 0.00AD/ME vs BW < 0.002 0.000 0.000 0.00BW/ME vs AD < 0.002 0.000 0.000 0.00AD/BW vs Pp < 0.002 0.033 0.210 3.29BW/Pp vs AD 0.198 – 0.006 – 0.057 0.62AD/Pp vs BW 0.413 – 0.006 – 0.057 0.62AD/BW vs Ppa < 0.002 0.009 0.060 0.91BW/Ppa vs AD 0.371 – 0.004 – 0.040 0.44AD/Ppa vs BW 0.279 – 0.004 – 0.040 0.44AD/BW vs F < 0.002 0.001 0.006 0.10BW/F vs AD < 0.002 – 0.001 – 0.001 0.10AD/F vs BW < 0.002 – 0.001 – 0.001 0.10AD/BW/ME vs Pp < 0.002 0.032 0.203 3.20AD/BW/ME vs Ppa < 0.002 0.009 0.057 0.90AD/BW/ME vs F < 0.002 0.001 0.006 0.10AD/BW/ME vs Pp/Ppa vs F 0.040 0.018 0.111 1.79

Table 6. Summary of molecular variance (AMOVA) descriptives (refer to text) from analyses withTriodia bynoei populations

Population groupings P φCT Variance % Totalcomponent(VA)

4FW vs 5FW vs CH vs GN vs G < 0.002 – 0.117 – 0.702 11.654FW/5FW vs CH < 0.002 0.101 0.571 10.114FW/5FW vs GN < 0.002 0.134 0.796 13.384FW/5FW vs G < 0.002 0.134 0.796 13.384FW vs 5FW/CH 0.495 – 0.040 – 0.220 4.054FW vs 5FW/GN 0.274 – 0.05 – 0.284 5.024FW vs 5FW/G 0.407 – 0.05 – 0.284 5.024FW/5FW vs CH vs GN vs G < 0.002 0.112 0.676 11.17

for example, how the genetic variabilityfor further adaptation can be maintainedwithout compromising the present adap-tiveness of a species (Bachmann 1994).Past studies of Western Australian species(Von Perger et al. 1994; Rossetto et al.1995) suggest that at least 80% of thecalculated diversity of the species shouldbe maintained in restored areas.The selec-tion of 20 individuals at random across thepopulations sampled in the present studywould conserve up to 80% of the mea-sured diversity of T. bitextura (Fig. 3). Therestoration of these grasses currently usesseed harvested from hundreds of plantsand, therefore, it is likely that natural diver-sity will be retained.

Tr iodia bynoei

Triodia bynoei is a highly fecund obligateseeder that does not recruit vegetativelyfrom ramets (G.Wells, unpubl. data, 1999).As for T. bitextura, within-populationdiversity (0.24–0.47) and the proportionof diversity attributed to among-popula-tion differences (12% AMOVA) are similar tothose reported for out-crossing seederspecies. Restoration of this species shoulduse seed-based methods to retain naturaldiversity levels.

Provenance boundaries and propaguleselection

The high proportion of diversity attributedto between-population differences by themethod of Nei and Li (1979) (and substan-tially higher genetic distances betweenindividuals from different populationscompared to within-population distances)indicate substantial differences betweenpopulations. The mean genetic distancebetween individuals when the most iso-lated population was paired with anyother population (0.66–0.74) is indicativeof distantly related individuals (Keil &Griffin 1994). In similar fashion to T. bitex-tura, significant differences betweengroups identified by AMOVA concurred withsplits between groups in the dendogramdrawn from UPGMA, suggesting that thedifferences thus depicted are significant.With the exception of two populationsseparated by a distance of just 2 km (4FWand 5FW) all remaining populations repre-

sent unique genetic provenances.4FW and5FW are located on the banks of intermit-tent streams that drain a common ridge.Deposition of seed from a common parentpopulation or exchange of seed from onepopulation to another during periods ofstream flow may account for this closerelatedness between populations in anotherwise variable species.Each remainingpopulation represents a unique prove-nance requiring precise seed collectionprogrammes to ensure the maintenance oflocal genetic integrity when restoring dis-turbed populations.

Triodia bynoei inhabits harsh, oftensoil-depleted areas; attributes suggestive ofpotential to withstand similar conditionsin severely degraded areas, such as waste-rock dumps following mining. Thus, theenvisaged use of this species could involvetranslocation plantings (introduction to anarea within the documented range of thespecies, but at sites from which it is notpreviously known). As the isolated popula-tions of the species represent uniqueprovenances, harvest of propagules fromplants growing in habitats similar to theareas to be restored (habitat-match post-mine sites with analogous natural commu-nities) may provide the best opportunityof restoring the species.

Maintenance of natura ld ivers i ty

Similar to T. bitextura, maintenance of anexcess of 80% of the measured diversity ofT. bynoei in the present study would beretained in clones taken at random from20 of the individuals used in the presentstudy. As a highly fecund obligate seeder,restoration efforts using seed harvestedfrom hundreds of plants should retainnatural diversity levels.

ConclusionThe methods applied in the present studyto extract DNA and generate ‘fingerprints’have been used previously on Western Aus-tralian native flora (Rossetto et al. 1996).This indicates that the method has poten-tial for use in delineating provenanceboundaries for other native species to beused in restoration programmes. Since thepresent study was undertaken, the field of

restoration genetics has evolved rapidlyand there are now numerous moleculartechniques with improved efficiency andcost than those applied at the time of thepresent study. The ability to accuratelyidentify ‘genetically conserved’ harvestzones for native species restoration is nowa practicality, which, in our view, deservesto be included in the procedures of ‘best-practice’ in restoration programmes, par-ticularly in the mining industry.

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