Habitat characteristics of the rare underground orchidRhizanthella gardneri

Jeremy BougoureA,C, Mark BrundrettA, Andrew BrownB and Pauline F. GriersonA

AEcosystems Research Group, School of Plant Biology M090, University of Western Australia,Crawley, WA 6009, Australia.

BDepartment of Environment and Conservation, Kensington, WA 6151, Australia.CCorresponding author. Email: bougoj01@student.uwa.edu.au

Abstract. Rhizanthella gardneri R.S.Rogers is an entirely subterranean mycoheterotrophic orchid known only from twoisolated populationswithin south-westernWesternAustralia (WA).This rare species appears restricted to habitats dominatedby species of theMelaleuca uncinata complex.R. gardneri purportedly forms a tripartite relationship withMelaleuca1, via aconnectingmycorrhizal fungus, for the purpose of carbohydrate and nutrient acquisition. Here, we quantify key climate, soiland vegetation characteristics of known R. gardneri habitats to provide baseline data for monitoring of known R. gardneripopulations, to better understand how R. gardneri interacts with its habitat and to identify possible new sites for R. gardneriintroduction. We found that the habitats of the two known R. gardneri populations show considerable differences in soilchemistry, Melaleuca structure and Melaleuca productivity. Multivariate analyses showed that both multidimensionalscaling (MDS) and principal components analysis (PCA) ordinations of soil chemical characteristics were very similar.Individual sites within populations were relatively similar in all attributes measured, whereas overall northern and southernhabitats were distinct from each other. These results suggest that R. gardneri can tolerate a range of conditions and may bemore widespread than previously thought, given that there are extensive areas of Melaleuca thickets with similar habitatcharacteristics across south-western WA. Variability within the habitats of known R. gardneri populations suggeststranslocation of this species into sites with similar vegetation may be a viable option for the survival of this species.

Introduction

Many orchid species are on the brink of extinction and as such,intervention via conservation of existing populations,identification of habitat suitable for their reintroduction andrestoration of degraded habitat are paramount to theircontinued survival (Hágsater and Dumont 1996). Restorationand conservation of individual species, particularly threatenedspecies, is generally complex and requires a thoroughunderstanding of the biological systems and processesassociated with both their continued existence and reasons fordecline (Montalvo et al. 1997).Although conservation of existingpopulations and habitat is of primary importance, there is also anincreasing need for ecological restoration and/or translocation ofindividuals produced ex situ to ensure survival and proliferationof endangered species and systems (Young 2000). However,habitat characteristics for many rare or threatened plants are oftenpoorly characterised, particularly in terms of fundamentalecosystem properties such as productivity, soil chemistry andnutrient cycling.

Rhizanthella gardneri R.S.Rogers (western undergroundorchid) is a critically endangered orchid that is entirelysubterranean and mycoheterotrophic, meaning that it has no

ability to photosynthesise and limited capacity toindependently access soil nutrient pools (Dixon and Pate1984). R. gardneri and the only other member of the genus,R. slateri (Rupp) M.A.Clem. & P.J.Cribb (eastern undergroundorchid), are unusual plants in that they remain subterraneanduring flowering (Fig. 1a)—a unique trait even amongmycoheterotrophic species (Leake 1994). It is thought thatR. gardneri is linked, via a mycorrhizal fungus(Thanatephorus gardneri Warcup), to an autotrophic shrub(Melaleuca), to form a tripartite relationship of nutrient andcarbohydrate exchange (Warcup 1985, 1991; Dixon et al.1990). If R. gardneri is indirectly dependent on thesurrounding shrubs for carbon and nutrients then we need anincreased understanding of the range in productivity and nutrientavailability of known habitat in order to restore, create or identifynew habitat.

Habitat considerations are paramount to understanding how tobest conserve and restore populations of rare plant species(Fiedler 2007), including mycoheterotrophic species (Leake1994). Habitat is the range of environments or communitiesover which a species occurs and comprises an abstractformulation of this range in terms of environmental variables

1‘Melaleuca’ in this paper refers to the various species of the Melaleuca uncinata complex that Rhizanthella gardneri is known to associate with.

CSIRO PUBLISHING

www.publish.csiro.au/journals/ajb Australian Journal of Botany, 2008, 56, 501–511

� CSIRO 2008 10.1071/BT08031 0067-1924/08/060501

and the species’ limits in relation to them (Whittaker et al. 1973).Understanding a species’ limits of tolerance and responses tothese environmental variables is paramount tounderstandinghowspecies function and in predicting how they may behave in achanging environment. In the case of R. gardneri, habitatdependence and interactions within their habitat are mostlyunknown, but like most plant species, dependent variables arediverse, with nutrition, climate and biotic interactions probablymost important. Although there are comprehensive climaterecords for all known R. gardneri sites, the nutritional statusand productivity of R. gardneri habitats, i.e. the capacity of the

habitat to maintain nutrient and carbon supply under fieldconditions, remains unknown.

The need for restoration of R. gardneri populations andsurrounding habitat has become increasingly apparent.Monitoring over the past 26 years has identified a suite ofintensifying threats to the survival and proliferation ofR. gardneri, including small population sizes, changes inrainfall distribution (Australian Bureau of Meteorology 2008),limited capability for recruitment because of habitatfragmentation, isolated habitats in excessively clearedlandscapes, possible encroachment by salinisation, increased

Perth Northern population

Southern population

250 kmc

Soil surface

Highest concentration of mycorrhizaformation

Mature infloresence

Fig. 1. (a) Partially exposedfloweringRhizanthellagardneridemonstrating its entirely subterraneannature. (b)Melaleuca scalena individual indicatingwhere aR. gardneriplantwasobserved (Kunjin,WesternAustralia). (c)Distributionof currentlyknownR.gardneripopulations in south-westernWesternAustralia.Eachpopulation consists of three separate sites.

502 Australian Journal of Botany J. Bougoure et al.

weed species competition, unsuitable fire management andherbicide aerial spray drift (Brown et al. 1998). Populations ofR. gardneri have purportedly been in decline since monitoringbegan in 1979,with anecdotal evidence suggesting that decreasedsightings of R. gardneri are correlated with a decline in health ofthe associated Melaleuca habitat. Declining Melaleuca ‘health’has been associated with casual observations of a reduced litterlayer, diminishing ground cover of other species, decreaseddensity of thickets, limited recruitment and the yellowing offoliage. However, there have been no quantitative orqualitative measurements of either the structure or nutritionalstatus of knownR. gardneri habitats, or towhat extent habitat thatis deemed healthy differs from that in decline. Consequently, weassessed vegetation structure, productivity and soil nutrients ofknown R. gardneri habitats. We sought to determine keysimilarities and differences among habitats to help defineprerequisites and limitations to the survival of R. gardneri andto identify new R. gardneri populations and potential sites forR. gardneri introduction.

Materials and methodsStudy sitesField sites analysed in the present study represent habitat of allknown live R. gardneri individuals during 2007. R. gardneri hasbeen found in only two areas of south-westernWestern Australia(WA); three sites within 30 km of Corrigin in the northernwheatbelt (northern population) and three sites ~80 km east ofRavensthorpe in the south-eastern wheatbelt (southernpopulation) (Fig. 1c). The northern and southern populationsare 300 km apart, with many areas of apparently similar habitatbetween these sites; however, there are no records of R. gardnerifrom these intervening areas. The six sites where R. gardneri isknown to occur are between one and eight hectares in area and aregenerally within vegetation remnants directly adjacent toagricultural land. Two sites are located on private property(Dallinup Creek north and south); two sites are in protectednature reserves (Babakin and Sorenson’s Reserve) and twosites are located on unallocated crown land (Oldfield Riverand Kunjin). None of the sites has been directly affected byfire in the past 30 years. However, similar surrounding vegetationthat has been burnt in the past 30 years has rapidly andsuccessfully regenerated.

Both the northern and southern sites are subject to aMediterranean type climate (wet, cool winters with hot, drysummers) as experienced by most of south-western WA. Thenorthern sites receive ~320–350mm annual rainfall comparedwith ~400–450mm for the southern sites. However, annualrainfall is quite variable and it is not uncommon for any of thesites to receive >150mm of rain during typically drysummer months (Australian Bureau of Meteorology 2008).Seasonal temperature fluctuations at northern sites range from5 to 15�C in winter and 15–32�C in summer. Climate conditionsfor the southern sites are slightly milder with average wintertemperatures as low as 7–16�Cduringwinter and 14–28�Cduringsummer (Australian Bureau of Meteorology 2008).

The northern populations of R. gardneri occur in denseMelaleuca scalena Craven & Lepschi thickets (Fig. 2b),whereas the southern populations occur in thickets of

M. hamata Fielding & Gardner, M. uncinata R.Br. and/or athird, apparently undescribed species of the M. uncinatacomplex (Craven et al. 2004). All of the above Melaleucaspecies are taxonomically similar and belong to theM. uncinata complex, a group of species widely distributedthroughout southern Australia (Broadhurst et al. 2004; Cravenet al. 2004). The six sites, encompassing both northern andsouthern populations, although primarily thickets ofMelaleuca, also include more open areas dominated by smallerDryandra spp., large exposed rocks, areas of grasses and sedges,Allocasuarina campestris (Diels) L.A.S.Johnson thickets oroccasional larger Banksia media R.Br. and/or malleeeucalypts. However, it appears that the occurrence ofR. gardneri plants is specific to the Melaleuca-dominatedpatches within each site.

Soils at all six sites are generallywhite–grey sandy loamabovea shallow heavy orange–grey clay layer. The clay layer at theDallinup Creek north and south sites (southern sites) is within afew centimetres of the surface and is hard-setting, so that wateroften pools on the soil surface for a substantial time after heavyrainfall. However, these sites are on slight slopes, which allowsfor some degree of drainage. Soils at northern sites areconsiderably sandier than the loamy soils at southern sites,although they often form hard surface crusts during prolonged

0

200

600

1000

1400

18000–50 cm

50–100 cm

100–150 cm

Sampling distance(a)

Northern population

Litte

r dr

y w

eigh

t (g

m2 )

0

2

6

10

14

18 10 cm

25 cm

50 cm

100 cm

Sampling distance(b)

Litte

r de

pth

(mm

)

Southern population

Northern population Southern population

Fig. 2. (a) Total standing leaf litter of northern and southern populations atthree distances frombases of randomly selectedMelaleuca individuals (mean,s.e., n= 9) collected November 2004. (b) Litter depth (mm) at northern andsouthern populations at four distances from bases of randomly selectedMelaleuca individuals (mean, s.e., n= 18) collected May 2006.

Habitat characteristics of Rhizanthella gardneri Australian Journal of Botany 503

dry spells, especially during summer. A summary of the generalsite characteristics of the northern and southern populations ofR. gardneri, including historical information, is given in Table 1.

Aboveground structure and productivity of MelaleucathicketsThree replicate plots of 10m� 10m were established withinMelaleuca thickets at each of the six sites. Shrub density,canopy cover, stem number and height were measured for 10Melaleuca shrubs within each 10m� 10m plot. The biomass ofMelaleuca at each site was estimated by destructively samplingfive individuals, representative of the range of canopy areas, andmeasuring their aboveground biomass. Individual plants wereseparated into foliage and stem components, weighed and asubsample of foliage measured for total leaf area with aLi-3100 Area Meter (Li-Cor Inc., Nebraska). The subsampleof leaves was then oven-dried at 70�C for 48 h, and specific leafarea (m2 kg–1) was calculated for each shrub. Leaf area per shrubwas calculated as specific leaf area� total dry leaf mass.

Standing leaf litter wasmeasured by collecting all litter within50 cm� 50 cm squares at 0-, 50- and 100-cm distance fromrandomly selected Melaleuca shrubs from within each of thethree plots at each site. Litter was sorted into principal(Melaleuca) components (leaves, fruits and twigs) and those ofother species (leaves and fruits) before being oven-dried at 60�Cfor 48 h and then weighed.

Melaleuca root and living fungal biomass in MelaleucathicketsRoot biomass (<2mm diameter) was estimated from corescollected in June 2005 to coincide with R. gardneri floweringand when soil moisture was consistently greatest. Duplicate soilcores (7.5 cm diameter) were sampled 10, 25, 50 and 100 cm in asouth-westerly direction from three randomly selectedMelaleucashrubs from each of the three plots at each of the six sites. Coreswere bulked by position within each plot after separating into

0–5 cm and 5–15 cm depths for measurement of root biomass.Leaf litter depth (mm) was also recorded at all sampling points.Rootswere separated fromsoil by sieving samples (<1mm)underrunning water and hand-sorting roots. Root samples were thenoven-dried at 60�C for 48 h and all roots <2mm in diameter wereweighed. The gravimetric moisture content of sampled soils wascalculated after drying for 24 h at 110�C.

Living fungal biomass fromasubsample of eachof the0–5-cmsoil cores mentioned earlier in the text was estimated fromergosterol concentrations (Seitz et al. 1977). Duplicatesamples of 5–6 g of field-moist soils were weighed out. Oneduplicate of soil was amended with 100mg ergosterol in 1mLn-hexane-propan-2-ol (98 : 2 v/v) to determine extraction andrecovery efficiency. Ergosterol was then extracted andquantified by reverse phase HPLC using the method describedby Ruzicka et al. (1995) as modified by Grierson and Adams(2000). Ergosterol recovery of ‘spiked’ samples (used to assessergosterol extraction efficiency) was always greater than 75%.

Soil chemistry analysisThree samples from each of the three plots at each site werecollected with 7.5- cm-diameter metal corers, separated into 0–2,2–5, 5–10 and 10–20 cm depths and bulked by plot and depthbefore sieving (<2mm). Soils were then assayed for a range ofchemical attributes. The pH of 5 g fresh soil was measured aftervigorously mixing samples with deionised water (1 : 1) (Thomas1996). Labile phosphoruswasmeasured by two techniques. First,labile inorganic P (Bray Pi) was measured according to themethod of Bray and Kurtz (1945). Briefly, air-dry soil wasmixed with an extraction solution (0.03N NH4F and 0.1NHCl), shaken for 45 s and filtered immediately. Second, labileorganic and inorganic hydroxide-extractable P fractions weremeasured by the method described by Grierson and Adams(2000). Briefly, 10 g of soil in 50mL of 0.1M NaOH solutionswere shaken for 16 h afterwhich extractionswere centrifuged andthe supernatant removed via filtration. One aliquot (OH-Pi) of the

Table 1. General characteristics of known Rhizanthella gardneri sites

Parameter Northern population Southern populationKunjin Babakin Sorenson’s Oldfield Dallinup Dallinup

reserve River Creek CreekNorth South

Land allocation Unallocatedcrown land

Naturereserve

Naturereserve

Unallocatedcrown land

Privateproperty

Privateproperty

Area of Melaleucathicket (ha)

3 5 2 4 8 8

PredominantMelaleuca species(uncinata complex)

M. scalena M. scalena M. scalena M. hamata,unidentified sp.

M. uncinata M. uncinata

Other commonspecies (shrubsor trees)

Allocasuarinacampestris

Allocasuarinacampestris

Allocasuarinacampestris

Banksia media Eucalyptus spp.mallee

Eucalyptusspp. mallee

Time since lastfire (years)

30+ 30+ 30+ 30+ 30+ 30+

Flowers observed(2000–2007)

15 40 40 25 25 3 (2004–2007)

Mean annual rainfall(mm) (1889–2006)A

358 (22%) 325 (24%) 325 (24%) 409 (20%) 442 (19%) 442 (19%)

ACoefficient of variation (CV).

504 Australian Journal of Botany J. Bougoure et al.

filtered supernatant was acidifiedwith HCl, to precipitate organicmatter, and re-filtered. A second aliquot (OH-Pt) of the filteredsupernatant was digested (H2SO4/H2O2). Phosphorus in allextracts was measured by a modified ascorbic acid method ofKuo (1996).Organic P (OH-Po)was estimated as the difference inP between OH-Pt and OH-Pi.

Labile inorganic nitrogen was extracted according to themethod described by Rayment and Higginson (1992). Briefly,5.0 g fresh soil in50mLof1MKClwas shaken for 1 handfilteredbefore supernatant NH4

+-N and NO3–-N concentrations were

determined by automated colourimetric techniques performed ona Technicon Auto Analyser II (Technicon 1977).

Soil and litter samples were analysed for total N and C content(%), and d15N and d13C isotope signatures, with an AutomatedNitrogen Carbon Analyser-Mass Spectrometer consisting of aRoboprep connected with a Tracermass isotope ratiospectrometer (Europa Scientific Ltd, Crewe, UK) in the WestAustralian Biogeochemistry Centre at the University of WesternAustralia. All samples were standardised against a secondaryreference of radish collegate (3.167% N, d15N 5.71; 41.51% C,d13C-28.61) that was in turn standardised against primaryanalytical standards (IAEA, Vienna). Accuracy was measuredat 0.07%,whereas precisionwasmeasured at 0.03%, according tothe stipulations for reporting analytical error in stable isotopeanalysis outlined by Jardine and Cunjak (2005).

Data analysisThe best models for predicting component biomass ofMelaleucaon the basis of canopy area as the independent variable weredetermined after testing several different allometric models. Theallometric models that best fitted the data were chosen byexamining residual distributions and maximum adjusted r2.The behaviour of the models for small diameter and ‘out ofsampled range’ trees were also carefully examined. The modelstested were y= a + bx and y= a + bx + cx2; where x is total canopyarea (m2), y is the mass of different components of sample shrubsand a, b and c are constants.

Analysis of variance (ANOVA) was used to suggest sitedifferences among Melaleuca individual and thicket structure,biomass (Melaleuca aboveground, Melaleuca fine roots(<2mm), leaf litter and fungus) and soil nutrient measuresamong sites. In all cases, data were first checked for normalityand square root or log-transformed where necessary to improvehomogeneity of variances. For analyses where samples weredivided into depths (soil analyses) or distance from Melaleucastems (standing litter biomass, root biomass, ergosterolconcentrations), two-way ANOVA was used to determinewhether there was significant interaction between the twoindependent variables. Significance level for all analyses wasP�0.05 andStatview5.0 software (SAS Institute 1996)wasusedfor all statistical analyses.

Multivariate analyses were used to investigate overallsimilarities and differences among sites. Similarity of the soilcharacteristics of northern and southern sites was calculated byusing principal components analysis and non-metricmultidimensional scaling ordinations (Primer software version6.2, Primer-E Ltd; Clarke and Gorley 2006). Analysis ofsimilarity (ANOSIM) was performed to determine whether

samples within groups were more similar than betweengroups. Similarity percentages (SIMPER) analysis was used todetermine contribution of individual variables to dissimilarity ofgroups. Data were normalised before analysis and Euclideandistance was used to generate the resemblance matrix for data(Clarke and Gorley 2006).

Results

Aboveground structure and productivity of Melaleucathickets

Melaleuca shrub densities at all sites were >3360 individuals perhectare, with little or no evidence of recent Melaleucarecruitment. Heights of Melaleuca were generally uniformwithin sites (~2.5m), even though were in general ~0.25mgreater at the northern than at the southern sites (P = 0.0006;Table 2). Projected canopy areas of individualMelaleuca shrubsranged from0.25 to 6.8m2.Total canopyarea of sites ranged from3875m2 ha–1 at Sorenson’sReserve to 10 011m2 ha–1 atDallinupCreek south. However, these values are likely to be somewhatgreater than total cover at a plot scale as individual Melaleucacanopies overlapped in many instances (Table 2).

Canopy area of individual shrubs was a strong predictorof total shrub biomass, as well as component biomass (woodv. leaves), regardless of site. Allometric equations for predictingMelaleuca biomass did not differ either within or betweennorthern and southern sites, meaning that patterns of totalaboveground biomass allocation were consistent across allsites even though shrub size differed (Table 3). Abovegroundbiomass per hectare of Melaleuca shrubs was estimated byapplying the plot census data. Aboveground biomass of theMelaleuca shrubs was significantly greater at southern(19 070 kg ha–1) than at northern (18 300 kg ha–1) sites. Shrubsalso had more leaves in southern sites, where foliage contributed17.3%of the aboveground biomass comparedwith only 12.9% atnorthern sites (Table 3).

Mean leaf area was similar for Melaleuca from northern(62.7m2 kg–1) and southern (58.8m2 kg–1) sites but wasconsiderably more variable within southern sites. When all sixsites were compared, leaf area per hectare was least at Sorenson’sReserve (8380m2 ha–1) and greatest at Kunjin (16 534m2 ha–1),with no clear distinction between northern and southern sites(Table 2). The variability in leaf area in the southern sites mayreflect different Melaleuca species of the M. uncinata complex(Table 1), which are distinguished in part by leaf size and shape(Craven et al. 2004). We attempted to quantify carbonassimilation as well as transpiration rates of Melaleucaindividuals but were unsuccessful as rates of gas exchangefrom the fine leaves were below levels of detection of a rangeof infrared gas analysers. However, leaf area is also an index ofphotosynthetic potential; whereas melaleucas of northern andsouthern sites were overall similar in their potential to supplyphotosynthate to a mycoheterotrophic orchid, the variabilitywithin the southern sites suggests that R. gardneri can surviveacross a broad range and theKunjin site, for example,may be ableto supply greater carbon from the host plant to any associatedR. gardneri, given the much greater leaf area at that site.

Standing leaf litter at southern sites (~0.6–1.5 kgm2) wasabout twice that of northern sites (P = 0.0019), reflecting the

Habitat characteristics of Rhizanthella gardneri Australian Journal of Botany 505

greater allocation of biomass to foliage and larger total leaf area.Leaf litter accumulated directly at the base of individualMelaleuca plants (Fig. 3). Melaleuca leaves made up themajority of total standing litter (60% at northern and 70% atsouthern sites) andwas strongly correlatedwith total standing leaflitter (r2 = 0.862). When litter depth (rather than litter biomass)wasmeasured, litter depth decreasedwith increasingdistance (10,25, 50 and 100 cm) from the base of individualMelaleuca plants;litter depth was also ~30% greater at southern (6–15mm) than atnorthern (3–12mm) sites (Fig. 2).

Melaleuca root and living fungal biomass in Melaleucathickets

Root (<2mmdiameter) biomass in the top15 cmof the soil profilewas similar at all four sampling distances (10, 25, 50 and 100 cm)from the base of Melaleuca shrubs (Fig. 3). However, root(<2mm diameter) biomass in the top 15 cm of soil at southernsites (3.5 g cm3)wasmore than twice that at northern sites (Fig. 3),consistent with aboveground data and again demonstrating the

higher productivity of these sites. Similar trends were evidentacross soil depths (0–5 cm and 5–15 cm), where root (<2mmdiameter) biomass at southern sites was 3.6 g cm3 (0–5 cm depth)and 3.3 g cm3 (5–15 cm depth), twice that of northern sites.

Ergosterol concentration of soil samples was used as anindicator of living fungal biomass (Davis and Lamar 1992)and were consistently greatest closest to Melaleuca individualscompared with samples further away (Fig. 4). Northern sites hadslightly higher concentrations of ergosterol (3.7mg g–1 soil) thandid southern sites (2.9mg g–1 soil). However, these data are basedona single collectionmade in June2005andare likely tobehighlyvariable according to soil moisture (Grierson and Adams 2000).

Soil chemistry

Soils were significantly more acidic, albeit slightly, at northern(pH ~5.3) than at southern (pH ~5.9) sites (Table 4). Soils wereammonifying with very little nitrate. Soil ammoniumconcentrations (NH4

+-N, 0–20 cm) at northern sites(~12mg g–1) were twice those measured at southern sites

Table 2. Characteristics of Melaleuca thickets at sites where Rhizanthella gardneri is known to exist

Parameter Northern population Southern populationKunjin Babakin Sorenson’s Oldfield Dallinup Dallinup

reserve River Creek north Creek south

Melaleuca density ha–1 380.00 350.00 337.00 603.00 443.00 470.00Canopy area (m2 ha–1) 809.40 591.50 387.50 862.30 562.60 1001.10

Individual Melaleuca shrubs (n= 20)Mean height (m) 2.79 2.64 2.27 2.11 2.26 2.57Mean canopy area (m2) 2.13 1.69 1.15 1.43 1.27 2.13Mean number of stems 5.18 3.85 4.40 6.95 9.90 11.45

Aboveground biomass of Melaleuca individuals (n= 5)Foliage biomass per m2 canopy area (kg dry weight) 0.34 0.38 0.35 0.29 0.56 0.24Leaf area (m2) per kg foliage biomass (dry weight) 62.75 63.21 61.97 53.30 52.19 69.62Foliage—total N (g kg–1) 15.14 15.46 15.01 12.63 10.14 11.21Foliage—total C (g kg–1) 482.50 486.58 481.36 482.14 486.76 484.35Branch/twig/wood biomass per m2 canopy area (kg dry weight) 2.45 2.75 3.23 1.71 2.40 2.09Total aboveground biomass per m2 canopy area (kg dry weight) 3.90 5.83 5.41 3.51 3.90 6.01

Aboveground biomass (kg ha–1)Foliage 274.30 227.20 135.20 249.50 318.30 237.50Branch/twig/wood 1981.20 1621.20 1250.30 1471.20 1351.40 2095.20Total biomassLeaf surface area (m2 ha–1) 172 030 14 358 8380 13 297 16 609 16 534.00

Table 3. Allometric equations used to calculate Melaleuca spp. (uncinata complex) aboveground biomass for all known Rhizanthella gardneri sitesEquations are either in linear (y= ax+ b) or polynomial (y= ax2 + bx+ c) formwith y relating to dryweights (kg) of; total abovegroundbiomass, foliage biomass or

wood/branch biomass and x= canopy area (m2)

Site y Model n a s.e. b s.e. c s.e. r2 P-value

Northern population Total aboveground biomass Polynomial 15 0.15 0.25 2.11 1.09 0.62 0.85 0.90 <0.0001Foliage biomass Linear 15 3.04 0.34 0.41 0.76 0.86 <0.0001Wood/branches biomass Polynomial 15 1.51 2.27 17.98 9.71 5.87 7.59 0.90 <0.0001

Southern population Total aboveground biomass Polynomial 15 0.37 0.10 1.03 0.51 0.48 0.48 0.96 <0.0001Foliage biomass Polynomial 15 –0.33 0.48 4.24 2.38 –0.35 2.26 0.53 0.0100Wood/branches biomass Polynomial 15 4.07 1.06 6.10 5.25 5.14 4.99 0.96 <0.0001

All sites Total aboveground biomass Polynomial 30 1.51 0.52 0.60 0.47 0.28 0.11 0.92 <0.0001Foliage biomass Linear 30 0.28 0.04 0.06 0.09 0.63 <0.0001Wood/branches biomass Polynomial 30 1.10 0.50 0.62 0.45 0.31 0.11 0.92 <0.0001

506 Australian Journal of Botany J. Bougoure et al.

(~6mg g–1), withmost ammonium concentrated in the top 2 cmofsoil (~22mg g–1) (Table 4). However, total N concentrations atboth northern and southern sites were similar(~0.3–1mg g–1 soil), indicating likely variation in organic Nfractions, where more N is likely associated with greaterorganic matter at the southern sites. Bray-Pi was extremelylow in all soils (0.5–1.5mg g–1) (Table 4). However, OH-Piwas slightly greater at northern sites (1.5–3mg g–1 soil;P = 0.0061). C : N ratios of soils were significantly (P = 0.001)lower at southern (22–25) than at northern (23–29) sites. All soilswere enriched in 15N (1–6‰) and isotopic signatures (both d15Nand d13C) did not differ between northern and southern sites(Table 4), reflecting their similarity in organic matter inputs andnutrient cycling processes.

When all soil data were analysed by both MDS (Fig. 5) andPCA (data not shown) analysis, individual quadrats (three at eachsite) separated into two distinct clusters closely representingnorthern and southern sites. Both MDS and PCA ordinationswerevery similar and showedall individual sites tended to cluster.ANOSIM analysis suggested a significant (P = 0.004) global r

value of 0.17, suggesting that northern sites were more similar toeach other than they were to southern sites, and vice versa.SIMPER analysis showed that no soil chemistry variablemeasured had greater than 3.2% contribution to separatingnorthern and southern sites (data not shown).

Discussion

Rhizanthella gardneri is only known from two geographicallydistinct areas in southern WA, which are subject to slightlydifferent climates and also have different soil properties.Although there are many similarities among the habitats ofknown populations, the present study has demonstrated thatthere is a range of conditions under which R. gardneri growsand, presumably, which it is able to tolerate. In addition, thestanding biomass and productivity of the putative autotrophichost, Melaleuca, varies considerably among northern andsouthern sites, suggesting that a wide range of sites could besuitable as habitat for ex situmaterial. These results also suggestthe possibility of additional undiscovered R. gardneripopulations within the broader distribution of the M. uncinatas.l. in southern WA. M. uncinata s.l. is common, with adistribution spanning most of southern Australia. Recentreviews of M. uncinata s.l. taxonomy resulted in therecognition of 11 species of Melaleuca within the group(Broadhurst et al. 2004; Craven et al. 2004), with at least three(and possibly a fourth undescribed species) associating withR. gardneri individuals. However, the concurrent presence ofthe mycorrhizal fungus, T. gardneri, within potential sitesshould also be assessed. Extensive, carefully controlledmicrocosm experiments or discovery of R. gardneri plantsaround other species within M. uncinata s.l. are required toelucidate whether other species can support R. gardneri and itsmycorrhizal fungus.On the basis of a combination of germinationtrials and observations that R. gardneri individuals presentwere always directly at the base of these Melaleuca spp.,Warcup (1985, 1991) suggested that R. gardneri individualsare nutritionally dependent (indirectly via a connectingmycorrhizal fungus) on the presence of a few specificautotrophic hosts (M. uncinata s.l.). However, the only otherRhizanthella species, R. slateri, co-occurs with a range of treeand shrub species, but does not occur with M. uncinata s.l.(Clements and Cribb 1984). Although it is likely that R. slateriassociates with the same mycorrhizal fungus as R. gardneri(J. J. Bougoure, unpubl. data), there is no clear evidencethat R. slateri forms tripartite relationships with surroundingtrees or shrubs, nor has it been determined whether itsmycorrhizal fungus acquires adequate carbon from eitherorganic matter or an autotrophic host to support the orchid.Clearly, further research is required in order to determinewhether the presence of T. gardneri is a key characteristic forRhizanthella habitat.

The density of Melaleuca thickets at both southern andnorthern sites is high (>3500 ha–1) and may be an importantfactor in determining presence or absence of R. gardneri.M. uncinata s.l. often occurs as scattered individuals within avariety of habitat types, whereas R. gardneri plants have neverbeen found in lowdensity thickets or at themargins of knownsiteswhere sites becomemore open. The density ofMelaleuca thickets

0

1

2

3

4

5

10 cm

25 cm

50 cm

100 cm

Sampling distance

Northern population

Dry

wei

ght (

g cm

3 )

Southern population

Fig. 3. Fine root (<2mm) dry weight biomass of top 15 cm of soil profile atfour distances from bases of randomly selectedMelaleuca individuals (mean,s.e., n= 18) collected May 2006.

0

1

2

3

4

5

6

7

10 cm

25 cm

50 cm

100 cm

Sampling distance

Northern population

Erg

oste

rol(µ

g m

–1 s

oil)

Southern population

Fig. 4. Ergosterol concentration (indicator of living fungal biomass) of top5 cm of soil profile at four distances from bases of randomly selectedMelaleuca individuals (mean, s.e., n= 18) collected May 2006.

Habitat characteristics of Rhizanthella gardneri Australian Journal of Botany 507

Tab

le4.

Soilchem

istrycharacteristicsof

Rhizan

thella

gardneripo

pulation

habitats

Soilsamples

aredividedinto

depthandvalues

representmeans

with

standard

errorin

parentheses

Site

Sam

pling

pHTotalC

TotalN

C:N

(soil)

NH4

NO3

BKPi

OHPi

OHPo

d13C

d15N

depth

(mgg–

1(m

gg–

1(m

gkg

–1

(mgkg

–1

(mgg–

1(mgg–

1(mgg–

1

(cm)

soil)

soil)

soil)

soil)

soil)

soil)

soil)

Northernpopulatio

n(n=18

)0–2

5.24

(0.11)

20.21(3.5)

0.87

(0.17)

23.35(1.46)

22.55(5.20)

1.05

(0.18)

1.68

(0.31)

3.26

(0.66)

18.48(1.86)

–25

.85(0.16)

1.13

(0.43)

2–5

5.33

(0.09)

15.29(4.1)

0.57

(0.17)

27.17(1.22)

12.03(2.84)

0.94

(0.34)

1.17

(0.17)

3.16

(0.31)

12.95(5.46)

–25

.92(0.14)

3.78

(0.45)

5–10

5.44

(0.12)

7.60

(0.9)

0.25

(0.02)

29.33(1.60)

7.89

(1.18)

0.54

(0.16)

1.09

(0.11)

2.32

(0.25)

5.49

(0.20)

–26

.12(0.11)

4.88

(0.50)

10–20

5.47

(0.17)

8.20

(2.6)

0.26

(0.06)

28.80(1.76)

5.44

(0.82)

0.26

(0.06)

0.54

(0.08)

1.52

(0.11)

4.19

(0.32)

–26

.05(0.26)

5.54

(0.56)

Southernpo

pulatio

n(n=18

)0–2

5.57

(0.13)

21.89(2.9)

0.99

(0.13)

22.42(0.62)

7.70

(1.19)

2.84

(1.97)

1.2(0.09)

2.32

(0.91)

19.53(2.79)

–25

.90(0.07)

1.54

(0.19)

2–5

6.03

(0.11)

12.05(2.5)

0.52

(0.10)

23.67(1.26)

5.48

(0.34)

0.32

(0.19)

0.86

(0.07)

2.24

(0.34)

7.21

(0.97)

–25

.95(0.17)

3.13

(0.37)

5–10

5.99

(0.15)

10.90(2.5)

0.47

(0.11)

24.74(1.61)

5.52

(0.34)

0.01

(0.04)

0.67

(0.12)

1.94

(0.23)

5.77

(0.78)

–25

.84(0.20)

4.47

(0.37)

10–20

6.03

(0.18)

6.77

(1.3)

0.31

(0.05)

22.92(2.22)

5.60

(0.46)

0.01

(0.04)

0.55

(0.08)

1.33

(0.16)

4.38

(0.59)

–25

.89(0.18)

5.89

(0.28)

Allsites(n=36

)0–2

5.43

(0.09)

21.05(2.2)

0.93

(0.10)

22.89(0.78)

15.13(3.15)

1.94

(0.99)

1.44

(0.17)

2.79

(0.56)

19.01(1.63)

–25

.88(0.09)

1.34

(0.26)

2–5

5.81

(0.11)

13.67(2.3)

0.55

(0.10)

25.42(0.95)

8.76

(1.60)

0.63

(0.20)

1.02

(0.09)

2.70

(0.25)

10.08(2.78)

–25

.94(0.11)

3.46

(0.29)

5–10

5.80

(0.12)

9.25

(1.4)

0.36

(0.06)

27.04(1.23)

6.70

(0.66)

0.27

(0.13)

0.88

(0.09)

2.13

(0.17)

5.63

(0.39)

–25

.98(0.11)

4.68

(0.30)

10–20

5.83

(0.15)

7.49

(1.4)

0.28

(0.04)

25.86(1.55)

5.52

(0.46)

0.13

(0.08)

0.54

(0.06)

1.42

(0.10)

4.29

(0.33)

–25

.97(0.16)

5.72

(0.31)

508 Australian Journal of Botany J. Bougoure et al.

measured here were only ~20% of estimates by Dixon and Pate(1984) for the northern sites and ~80% of that suggested for theOldfield River site (southern site—no previous data for DallinupCreeknorthern and southern sites).Consequently, theremayhavebeen a decline in the density ofMelaleuca, especially within thenorthern sites,which is consistentwith the decrease in the numberof R. gardneri plants observed flowering (not necessarilycorrelating with absolute number of R. gardneri plants) overthe same 24-year period. Unfortunately, no measures ofMelaleuca biomass or canopy cover were made by Dixon andPate (1984) so we are unable to elaborate on any possiblesecondary effects of habitat changes and how these could ordid affect R. gardneri populations. However, we might assumethat with lower total cover there is less biomass, and thus lessproductivity and reduced ability to support any symbionts. Inaddition, microclimate conditions are also likely to be differentwith warmer and possibly drier soils in more open vegetation.

Melaleuca foliage biomass (~0.6–1.6 tonnes C ha–1) was notsignificantly different between northern and southern sites oramong Melaleuca species. However, foliar nitrogen contentswere significantly greater at northern (~15 g kg–1) than atsouthern (~11 g kg–1) sites. As foliar N content correlatesstrongly with rates of CO2 assimilation for a wide range of C3

species (Evans 1989), these results suggest that plants fromnorthern sites were more likely to be actively sequesteringcarbon via photosynthesis and presumably more able tosupport mycorrhizal symbionts. However, the extent of thedependency of R. gardneri and its fungus on carbohydratesfrom its autotrophic host, relative to utilisation of carbon (andnitrogen) from other sources such as organic matter, remainsunknown. Although it is thought that R. gardneri obtainscarbohydrates and nutrients directly from its fungal partner(rather than its autotrophic host), like many ectomycorrhizalfungi, the fungal symbiont associated with R. gardneri(T. gardneri) is likely to be accessing a variety of sources ofcarbon and nutrients (Smith and Read 1997). Therefore,decomposing organic matter, soil and especially leaf litter

(predominantly Melaleuca leaves) might be importantelements of R. gardneri’s nutrition. Accumulated leaf litterwas twice as great at southern sites compared with northernsites, thus representing a significantly greater carbon pool forfungi to access than that at northern sites. This may helpcompensate for the predicted lower photosynthetic capabilityand subsequently reduced carbonpool ofMelaleucaplantswithinsouthern sites. Isotope tracer studies may shed some light on theutilisation of different C and nutrient sources within thisecosystem by Melaleuca, T. gardneri and R. gardneri(McKendrick et al. 2000).

Fine root (<2mmdiameter) biomass in the top fewcentimetresof the soil profile was greatest (almost double) in southern sites.This would presumably be attributed to the greater moisture andnutrient availability at these sites (Cairns et al. 1997). Otherstudies on fine-root formation have found that resource-availability effects are species specific (Lee et al. 2007), sitespecific (Swaty et al. 1998) and further confusedwhen specialisedroots such as ectomycorrhizas are considered (Anderson andCairney 2007). We know that theMelaleuca species associatingwith R. gardneri readily form ectomycorrhizas and arbuscularmycorrhizas (Warcup 1985, 1991); however, it is difficult toelucidate whether mycorrhiza formation affects fine-rootbiomass, is a product of fine-root biomass production or acombination of both. A microcosm study by Bogeat-Triboulotet al. (2004) on Pinus pinaster Ait. showed that percentageectomycorrhizal root tips remained constant regardless ofdecreasing moisture availability, even though fine-rootbiomass increased. If these phenomena are consistent forMelaleuca species, greater fine-root biomass results inT. gardneri having greater access to the autotrophiccarbohydrate pool which might result in a greater carbontransfer to R. gardneri.

Melaleuca biomass, density and associated properties areimportant habitat attributes as these determine overall carbonand nutrient cyclingwithin an ecosystem and affect a range of soilchemical and physical attributes as well as influencing

K1

K2

K3

B1

B2

B3

S1

S2S3

O1

O2

O3

DN1

DN2

DN3

DS1

DS2

DS3

2D Stress: 0.13

Northern population

Southern population

Fig. 5. Non-metricmultidimensional scaling ordinationof soil chemistry profiles of northern and southern sites (n= 9).Datawere normalised and a resemblancematrix based on Euclidean distance was created. Letters represent site (K=Kunjin, B=Babakin, S = Sorenson’s Reserve, O =Oldfield River, DN=DallinupCreek north, DS=Dallinup Creek south) and numbers represent replicate number.

Habitat characteristics of Rhizanthella gardneri Australian Journal of Botany 509

microclimate. However, R. gardneri is directly linked to itsmycorrhizal fungus and thus, dependent on the fungus’tolerances and ability to interact in these specified habitats.Given the nutritional dependence of R. gardneri onmycorrhization and the purported specificity of this association(Warcup 1985, 1991), R. gardneri can survive only when itsfungal partner is active.We found that northern and southern siteshad similar living fungal biomass. Ergosterol concentrations ofsoils sampled in the present studywere remarkably high given thegrowth and climatic conditions, and similar to concentrationsfound in German grassland and forest soils (Djajakirana et al.1996) and about half those of soils of highly productive jarrah(Eucalyptus marginata D.Don ex Sm.) forests of southern WA(Grierson and Adams 2000). However, it is not known whichspecies comprised most of the fungal biomass at our sites.Different fungal species vary in their tolerance to droughtconditions or moisture availability and it is common toobserve changes in ectomycorrhizal community composition(independent of total fungal biomass measures) at varyingmoisture availabilities (Slankis 1974; Shi et al. 2002).However, given the low annual precipitation at field sites inthe present study, T. gardneri probably has some degree oftolerance to reduced moisture availability.

Although we have characterised the known habitat ofR. gardneri, past environmental changes may have influencedcurrent attributes; e.g. it has been more than 30 years since any ofthe R. gardneri sites have been burnt by wildfire. However, it isunknown what the natural fire frequency is in the regions studiedand, consequently, whether fire is necessary for the recruitmentand survival of both Melaleuca and R. gardneri. Extensive landclearing has resulted in very few and small fragmented sites thathave been subsequently protected from fire because of proximityto agriculturally active land.Nevertheless,M.uncinata s.l.has theability to rapidly resprout after low-intensity burns(J. J. Bougoure, pers. obs). In addition, the first R. gardneriplant discovered intact (i.e. not during ploughing and landclearing) was found growing under a ~70 cm high Melaleucaplant resprouting after having its aboveground biomass felled(George 1980). Consequently, disturbance may have positive (orat least not necessarily negative) effects on R. gardneri. A broadrange of terrestrial orchids exhibit greater recruitment andflowering after fire (Coates et al. 2006). For example, thenumber of recruits of the New Zealand terrestrial orchidCorybas carsei (cheeseman hatch) tripled 1 year after fire andincreased by a further 300% in the following year (Norton andDeLang 2003). Similarly, Coates et al. (2006) demonstrated that theAustralian terrestrial orchid Prasophyllum correctum D.L Jonesalso recruited andfloweredmore prolifically in the fewyears afterfire. Although the positive response of some terrestrial orchids todisturbance is not fully understood, it is likely that increasednutrient availability (Bond and van Wilgen 1996) and smoke-induced triggers (Flematti et al. 2004), combined with reducedcompetition from surrounding vegetation, all play a role.

The present research provides information useful for adaptivemanagement for the conservation of R. gardneri. We did notidentify any single habitat characteristic that explained thepresence or absence of R. gardneri. However, given thepurported dependence of R. gardneri on its mycorrhizal fungalpartner, future research must consider how this fungus associates

with its habitat—what conditions are promoting and/or limitingits growth? What is clear from our study is that there are manyareas ofMelaleuca thickets outside those described here that havesimilar habitat characteristics. Consequently, the potential for thisspecies to exist or have existed inmanyparts of southernAustraliais a real possibility and continued exploration of potential habitatshould be encouraged.

Acknowledgements

Financial support for this study was provided by an ARC Linkage grant(LP0454276) in collaboration with the Western Australian Department ofEnvironment and Conservation (DEC).

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Manuscript received 20 February 2008, accepted 12 August 2008

Habitat characteristics of Rhizanthella gardneri Australian Journal of Botany 511

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