hudsonia ericoides and hudsonia tomentosa anatomy of mycorrhizas

NOTE / NOTE

Hudsonia ericoides and Hudsonia tomentosa:Anatomy of mycorrhizas of two members in theCistaceae from Eastern Canada

Hugues B. Massicotte, R. Larry Peterson, Lewis H. Melville, andLinda E. Tackaberry

Abstract: Most species in the family Cistaceae are found in the Mediterranean basin. Several hosts are of special interest,owing to their associations with truffle species, while many are important as pioneer plants in disturbed areas and in soilstabilization. For these reasons, understanding their root systems and their associated fungal symbionts is important. Moststudies of the structure of mycorrhizas in this family involve two genera, Cistus and Helianthemum. The present study ex-amines structural features of mycorrhizas in two North American species, Hudsonia ericoides L. and Hudsonia tomentosaNutt. Root systems of both species are highly branched with most fine roots colonized by mycorrhizal fungi. Based onmorphological features, several mycorrhizal fungi were identified; structural details also provided evidence of more thanone fungal symbiont for each host species. All mycorrhizas had a multi-layered fungal mantle and Hartig net hyphae con-fined to radially elongated epidermal cells; no intracellular hyphae were observed. Although the Hartig net was confinedto the epidermis, the outer row of cortical cell walls lacked suberin, a known barrier to fungal penetration. Mycorrhizas inH. ericoides and H. tomentosa differed from those of Cistus and Helianthemum species that have a Hartig net that extendsinto the root cortex, as well as frequently present intracellular hyphae.

Key words: Cistaceae, Hudsonia, mycorrhiza, anatomy, Hartig net, mantle.

Resume : La plupart des especes de la famille des Cistaceae se retrouvent dans le bassin de la Mediterranee. Plusieurs ho-tes presentent un interet particulier, compte tenu de leurs associations avec des especes de truffes, alors que d’autresconstituent des plantes pionnieres importantes dans les endroits perturbes et pour la stabilisation des sols. Pour ces raisons,il importe de comprendre la biologie de leurs systemes racinaires et de leurs champignons symbiotiques associes. La plu-part des etudes publiees sur la structure des mycorhizes de cette famille portent sur les deux genres Cistus et Helianthe-mum. On examine ici les caracteristiques structurales des mycorhizes de deux especes Nord-americaines, l’Hudsoniaericoides L. et l’Hudsonia tomentosa Nutt. Chez les deux especes, l’on observe des systemes racinaires fortement ramifies,la plupart des racines fines etant colonisees par des champignons mycorhiziens. Sur la base des caracteristiques morpholo-giques, les auteurs ont pu identifier plusieurs especes de champignons mycorhiziens; les details des structures fournissentegalement des preuves de l’existence de plus d’un symbiote fongique chez chaque espece hote. Toutes les mycorhizesmontrent un manteau fongique a plusieurs couches et des hyphes du reseau de Hartig confinees aux cellules epidermiquesradialement allongees; on observe aucune penetration intracellulaire. Bien que le reseau de Hartig soit confine a l’epi-derme, les parois cellulaires de la couche externe de cellules corticales ne possedent pas de suberine, une barriere reconnuepour la penetration fongique. Les mycorhizes chez les H. ericoides et H. tomentosa different de celles des especes de Cis-tus et d’Helianthemum, lesquelles possedent un reseau de Hartig s’etendant dans le cortex racinaire et montrent la presenced’hyphes intracellulaires souvent presentes.

Mots-cles : Cistaceae, Hudsonia, mycorhize, anatomie, reseau de Hartig, manteau.

[Traduit par la Redaction]

Received 27 November 2009. Accepted 21 April 2010. Published on the NRC Research Press Web site at botany.nrc.ca on 27 May 2010.

H.B. Massicotte1 and L.E. Tackaberry. Ecosystem Science and Management Program, University of Northern British Columbia, 3333University Way, Prince George, BC V2N 4Z9 Canada.R.L. Peterson and L.H. Melville. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1 Canada.

1Corresponding author (e-mail: [email protected]).

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Botany 88: 607–616 (2010) doi:10.1139/B10-035 Published by NRC Research Press

IntroductionThe family Cistaceae (the rock rose family) is composed

of eight genera and approximately 180 species (Guzmanand Vargas 2009) centered in temperate Europe and theMediterranean basin where they are often part of importantearly successional stages in disturbed habitats (Guzman andVargas 2005). The genera, based on plastid rbcL and trnL–trnF sequences (Guzman and Vargas 2009), include Cistus,Halimium, Helianthemum, Hudsonia, Lechea, Tuberaria,Fumana, and Crocanthemum. Several genera occur in NorthAmerica. Members of this family have been known to formassociations with mycorrhizal fungi (see Malloch and Thorn1985), and recently, Comandini et al. (2006) published anextensive list of fungal species associated with the genusCistus. Several species of Hebeloma are highly specific toCistus species (Eberhardt et al. 2009). With respect to my-corrhizal associations of Cistaceae species in North Amer-ica, Malloch and Thorn (1985) examined threeHelianthemum species and two species each of Hudsoniaand Lechea (misspelled Lechia in their paper). Based onsporocarps found in the vicinity, the authors listed the prob-able fungal symbionts of these hosts as well as other fungiassociating with Cistus species.

In spite of the large number of species in the Cistaceae,relatively few have been examined for their mycorrhizal sta-tus, and fewer still for the structural details between rootsand associated symbiotic fungi. The majority of structuralstudies have involved Cistus species and various fungalsymbionts: C. incanus L. – Tuber melanosporum Vitt. (Fus-coni 1983; Giovannetti and Fontana 1982; Wenkart et al.2001); Cistus ladanifer L. – Laccaria laccata (Scop.: Fr.)Berk. & Br. (Torres et al. 1995); C. ladanifer L. – Boletusedulis Bull. ex Fr. (Agueda et al. 2006, 2008); and, C. cf.ladanifer L. – Boletus rhodoxanthus Kallenb. (Hahn 2001).All are characterized by having a mantle and a Hartig netthat involves both epidermal and cortical cell layers, featuresthat are shared with conifer ectomycorrhizas. Similar resultswere obtained for four other Cistus species in combinationwith various Tuber species (Giovannetti and Fontana 1982),and for a Cistus sp. associated with both Lactarius tesquo-rum Malencon (Nuytinck et al. 2004) and with Lactariuscistophilus Bon & Trimbach (Comandini and Rinaldi 2008).However, mycorrhizas formed with in-vitro transformed rootclones of C. incanus and fungal isolates of Terfezia boudieriChatin either developed ectomycorrhizal features similar toother Cistus species or had intracellular penetration by hy-phae, depending on the clone and culture conditions (Zar-etsky et al. 2006).

Cleared roots of Helianthemum chamaecistus Mill. – Cen-ococcum graniforme (Sow.) Ferd mycorrhizas showed corti-

cal Hartig net formation as well as intracellular hyphae,although no mention was made concerning mantle develop-ment (Kianmehr 1978). Dexheimer et al. (1985) describedultrastructural features of Hartig net hyphae, as well as intra-cellular hyphae of Helianthemum salicifolium (L.) Mill.colonized by two Terfezia species. Fortas and Chevalier(1992) examined the effect of culture conditions on the col-onization of Helianthemum guttatum (L.) Mill. by three truf-fle species; all species formed ectomycorrhizas with acortical Hartig net but without a mantle in high phosphorusconditions, whereas they developed ectendomycorrhizas(cortical Hartig net and intracellular hyphae) in phosphorus-deficient cultures. Gutierrez et al. (2003) showed that, undergreenhouse conditions, Helianthemum almeriense Pau –T. claveryi mycorrhizas developed a cortical Hartig net withlimited mantle, whereas in-vitro conditions produced a cort-ical Hartig net and a thick, multi-layered mantle. The samehost colonized by Picoa lefebvrei (Pat.) Maire under green-house conditions had limited mantle, cortical Hartig net, andintracellular hyphae. Similarly, cortical Hartig net and intra-cellular fungal hyphae formed in in-vitro Helianthemumovatum (Viv.) Dun. – Terfezia terfezioides (Matt.) Trappemycorrhizas (Kovacs et al. 2003); however, the authors cau-tioned that the root–fungus interaction in vitro might not re-flect the true mycorrhizal association. Malloch and Thorn’s(1985) examination of whole mounts of field-collected Le-chea intermedia roots showed an epidermal and corticalHartig net, but there was no report of intracellular hyphae.The above reports demonstrate how highly variable mycor-rhizal structure is in Cistaceae, perhaps not surprising in afamily that is also taxonomically so diverse. No informationis available for species in the genera Crocanthemum and Tu-beraria.

The two species examined in the present study, Hudsoniaericoides L. and Hudsonia tomentosa Nutt., are native toNorth America. Hudsonia ericoides (pine barren golden-heather; false heather) is an evergreen subshrub that growsin nutrient poor, sandy soil in isolated areas restricted tonortheastern Canada and USA. It is specifically adapted todrought, soil erosion, and extreme soil conditions. Mallochand Thorn (1985) published images of root tips with Ceno-coccum-like mantles, from a collection made in Nova Sco-tia. The second host, H. tomentosa (sand heather; woollybeach heather) is found in sandy pine woods and clearingsfrom Nova Scotia to Alberta, Canada, and in eastern USA.It is often found growing on sand dunes (Smreciu et al.1997). Whole mounts of roots previously collected in NewBrunswick and Ontario showed a Hartig net that appearedto be restricted to the epidermis (Malloch and Thorn 1985).Allen and Allen (1992) noted that H. tomentosa (described

Figs. 1–8. Figs. 1–5. Hudsonia ericoides. Fig. 1. Plant (arrow) growing in sand along an abandoned railroad west of Kingston, Nova Scotia.Fig. 2. Close-up of plant in similar sandy habitat. Trowel scale is in millimetres. Fig. 3. Portion of the root system showing diverse size ofmycorrhizal roots and several morphotypes (arrows). Fig. 4. White rhizomorphic mycorrhiza showing beaded appearance due to growthincrements (arrowheads) along the length of the root, and large attached rhizomorphs (arrow). Scale as in Fig. 5. Fig. 5. Brown Tomentella-like morphotype showing swollen root tips (arrows) and abundant extraradical hyphae. Figs. 6–8. Hudsonia tomentosa. Fig. 6. Excavatedseedling showing the highly branched root system. Scale is in millimetres. Fig. 7. Portion of the same root system showing two morpho-types: Cenococcum mycorrhizas (arrowhead) and white rhizomorphic mycorrhizas (double arrowhead) with large rhizomorphs (arrows).Fig. 8. Mycorrhizal root showing recolonization by Cenococcum (at the apex) and the white rhizomorphic mycorrhiza (basal). Large Ceno-coccum extraradical hyphae (arrowheads) are present.

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by them as an ericaceous shrub) had arbutoid-like mycorrhi-zas; however, this observation was not documented byimages.

The objective of the present study was to use a variety ofmicroscopical methods to more fully describe the anatomicalfeatures of the mycorrhizal associations of H. ericoides andH. tomentosa from field-collected specimens. Of interestwas to compare the results with what is known for thesespecies and for species in the other genera in this large fam-ily.

Materials and methods

Plant materialSamples of H. ericoides were collected on 22 May 2009,

west of Kingston, Nova Scotia (approximately44.9888N, 64.9498W), in sandy habitats along an abandonedrailway bed. Ectomycorrhizal hosts in the vicinity includedseveral Pinus species (e.g., Pinus banksiana Lamb., Pinusresinosa Aiton, Pinus strobus L.), as well as Populus tremu-loides (Michx.) A & D Love, Populus grandidentataMichx., and Betula populifolia Marsh. Collections of H. to-mentosa were made on 9 October 2009, from a sandy beachsite in Petawawa, Ontario (approximately 46.08N, 77.38W);mature P. strobus, P. resinosa, and smaller Populus balsa-mifera L., P. tremuloides, and Betula papyrifera Marsh.trees were in the vicinity. Entire plants with attached rootsand soil were excavated and transported to the University ofGuelph where they were stored briefly at 4 8C; subse-quently, roots were washed in preparation for examinationwith a stereo binocular microscope for morphotype charac-terization and for fixation for further microscopy.

Morphological assessmentStandard light microscopy techniques were used to deter-

mine the different Hudsonia morphotypes (Agerer 1987–2008; Ingleby et al. 1990; Goodman et al. 1996). Mycorrhi-zas were characterized according to colour, texture, lustre,dimensions, tip shape, branching pattern, and presence orabsence of rhizomorphs; root squash mounts were used todescribe mantle features, emanating hyphae, rhizomorphs,and other distinguishing features. Preliminary identificationsto fungal families or genera were designated when possible;otherwise, a descriptive name was assigned.

Resin embedding and light microscopyRoot samples from several plants for each host species

were fixed in 2.5% glutaraldehyde in 0.10 mol/L 4-2-hy-droxyethyl-1-piperazine ethane sulfonic acid (HEPES) buf-

fer at pH 6.8, dehydrated in an ascending series of ethanol,and embedded in LR White resin (London Resin CompanyLtd., Reading, Berkshire, England) using conventional pro-tocols (Ruzin 1999). Thick sections (1–1.5 mm) were cut ona Porter-Blum Ultra-Microtome MT-1 (DuPont Co., New-ton, Connecticut, USA) using glass knives, stained for lightmicroscopy using aqueous 0.05% toluidine blue O (Sigma-Aldrich, St. Louis, Missouri, USA) in 1% sodium borate,and viewed on a Leitz Orthoplan (Leica, Mississauga, On-tario, Canada) microscope. Images were captured using aNikon Coolpix 4500 digital camera (Nikon, Canada, Missis-sauga, Ont.). At least five samples of roots from both siteswere examined for each mycorrhiza type.

Fluorescence microscopyFree-hand sections of roots of both species that had been

stored in 50% ethanol were stained for 1 h with 0.1% (w/v)berberine hemi-sulphate (Sigma, C.I. No. 75160) in dH2O,rinsed several times with dH2O, and mounted in 50% glyc-erol. Sections were viewed under UV and blue light (350–460 excitation wavelength) on a Leitz SM-LUX epifluores-cence microscope (Leica, Mississauga, Ont.). Images werecaptured using the same camera system described above.

Scanning electron microscopySamples fixed in 2.5% glutaraldehyde were dehydrated

through an ascending series of ethanol to 100%, critical-point dried, and mounted on aluminum stubs with two-sidedsticky tape. After coating with gold–palladium, they wereexamined with a Hitachi S-570 (Nissei-Sangyo, Tokyo, Ja-pan) scanning electron microscope. Images were taken withan attached digital camera.

Transmission electron microscopyThin sections (*0.1 mm) of LR-White-embedded material

were sectioned with glass knives on a Reichert ultra-microtome (Model OM-U3; Reichert-Jung (Leica), Wetzlar,Germany), collected on formvar-coated copper grids,stained for 10 min in 2% ethanolic uranyl acetate, andcounter-stained for 5 min with 1.0% lead citrate. Sectionswere examined with a Philips CM10 transmission electronmicroscope (Philips Electron Optics, Eindhoven, Germany)at 80 kV and images were collected with a digital camera.

Results

Morphology of root systemsBoth H. ericoides and H. tomentosa occurred as small

patches in open areas of sandy soil or in semi-open areas

Figs. 9–16. Figs. 9–12. Scanning electron micrographs of Hudsonia ericoides mycorrhizas. Fig. 9. Cenococcum mycorrhiza showing largeextraradical hyphae (arrowheads) extending from the swollen root tip. Fig. 10. Unknown basidiomycete mycorrhiza (likely Tomentella)showing the fungus mantle over the entire root tip. A few extraradical hyphae (arrowhead) are present. Fig. 11. Enlargement of a portion ofthe mycorrhiza in Fig. 10 showing the branched, compact nature of the mantle. Fig. 12. Clamp connections (arrows) on outer mantle hyphaeof a basidiomycete mycorrhiza. Figs. 13–16. Scanning electron micrographs of Hudsonia tomentosa mycorrhizas. Fig. 13. Portion of a rhi-zomorph from a white rhizomorphic mycorrhiza similar to that shown in Fig. 7. Fig. 14. Cenococcum mycorrhiza showing extensive ema-nating hyphae (arrowheads). Fig. 15. Basidiomycete mycorrhiza with few emanating hyphae. Fig. 16. Mycorrhizal root showingrecolonization by Cenococcum (at the apex) and the white rhizomorphic mycorrhiza with loose emanating hyphae (basal). A few largeCenococcum hyphae (arrows) are present.

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near the tree(shrub)–sand zones (Figs. 1 and 2). Branchedshoots consisted of scale-like leaves (Fig. 2). The two spe-cies shared similarities in growth form, rooting patterns, andfungal colonization. Both hosts had a mostly fibrous rootsystem from which grew different order laterals. Most rootswere extremely small (mycorrhizal tips were up to 200 mmwide and approximately 0.5–2.0 mm long) and almost allroot tips that were examined were mycorrhizal (Fig. 3); rootsystems for each host species were colonized by several dif-ferent symbiotic fungi.

Hudsonia ericoides morphotypes included a white rhizo-morphic mycorrhiza that had a compact mantle of felt tonet synenchyma, emanating hyphae (EH) 2–4 mm in diame-ter with clamp connections, and numerous rhizomorphs(Fig. 4) with large vessel-like hyphae. This host also pro-duced a brown Tomentella-like morphotype with a thick netto angular synenchyma mantle, EH 4–5 mm in diameter withclamp connections, and no obvious rhizomorphs (Fig. 5).Minor components of H. ericoides mycorrhizas were formedby dark septate ascomycetes; these consisted of MRA (My-celium radicis atrovirens Melin)-like fungi (Ingleby et al.1990) and Cenococcum geophilum Fr. with isodiametricmantle cells forming a regular synenchyma (see Ingleby etal. 1990) and abundant, large EH. Hudsonia tomentosa roots(Fig. 6) were dominated by two morphotypes: a white rhizo-morphic mycorrhiza similar to that seen for H. ericoides;and Cenococcum geophilum mycorrhizas (Fig. 7). White rhi-zomorphic mycorrhizas had few emanating hyphae; thesewere often short and had a determinant length with enlarged(swollen) hyphal tips, or took the form of enlarged surfacemantle cells. No other associated fungi were identified forH. tomentosa. However, many roots showed frequent suc-cessional colonization patterns with numerous tips being re-colonized by the other fungal morphotype (i.e., eitherCenococcum was at the tip and the white morphotype basal(Fig. 8), or the white mycorrhiza was at the tip and Ceno-coccum basal). The position of the two fungi was variablein recolonized roots.

Scanning electron microscopyRoots of H. ericoides – Cenococcum mycorrhizas, when

viewed by SEM, often had enlarged root apices with largeprominent EH (Fig. 9); other morphotypes formed compactmantles (Fig. 10) with branched mantle hyphae (Fig. 11)

and few to abundant EH with clamp connections (Fig. 12).White mycorrhizas that associated with both host species(as seen in Fig. 7) had abundant, large, branching rhizo-morphs (Fig. 13). Figure 14 shows a H. tomentosa – Ceno-coccum mycorrhiza with numerous, large EH. SomeH. tomentosa mycorrhizas had a compact mantle structurewith fewer EH (Fig. 15), or showed successional recoloniza-tion by two fungal species (Fig. 16).

Light microscopySectioned roots from both plant hosts showed that colon-

ized root tips had a simple anatomy: an epidermis of radiallyelongated cells, only 2–3 rows of cortical cells, and a vascu-lar cylinder consisting of a few xylem tracheary elementsand phloem. Longitudinal sections of LR-White-embeddedmycorrhizal root tips of H. ericoides (Figs. 17–20) showedthat mycorrhizas had a compact, multi-layered mantle(Figs. 17 and 18) and a Hartig net confined to the radiallyelongated epidermal cells; intracellular hyphae were notpresent (Figs. 17 and 18). Other H. ericoides morphotypesalso had compact, fungal mantles of varying thickness thatextended the length of the root and covered the root apex(Fig. 19). The Hartig net was again limited to the radiallyelongated epidermal cells and intracellular hyphae were notpresent (Figs. 19 and 20). Longitudinal sections ofH. tomentosa mycorrhizas (the section illustrated is aCenococcum mycorrhiza) (Figs. 21 and 22) showed similarstructural features to those of H. ericoides; the mantle wasmulti-layered with the outer mantle hyphae showing themelanized walls of Cenococcum and dense inter-hyphaldeposits (Fig. 22). Hartig net hyphae were associated onlywith epidermal cells (as in H. ericoides) and intracellularhyphae were not present (Fig. 22).

Fluorescence microscopyTransverse sections of both H. ericoides and H. tomentosa

mycorrhizal roots, when stained with berberine hemi-sulphate, showed that lignified walls of tracheary elementsand suberized walls of endodermal cells fluoresced whenviewed with UV–blue light (Figs. 23 and 24). Walls ofthe outer layer of cortical cells (hypodermis) and epidermalcells did not fluoresce although these cells and mantle hy-phae showed some background fluorescence (Fig. 23).

Figs. 17–25. Figs. 17–20. Light microscopy of resin-embedded longitudinal sections of Hudsonia ericoides mycorrhizas. Fig. 17. Cenococ-cum mycorrhiza showing a well-developed mantle (arrows) that covers the root tip and Hartig net hyphae (arrowheads) confined to theradially elongated epidermal cells. Fig. 18. Enlargement of a portion of the root shown in Fig. 17 showing the epidermal Hartig net (arrow-head) and thick mantle (arrow). No intracellular colonization was present. Fig. 19. Basidiomycete mycorrhiza showing a thick mantle(arrow) that covers the root tip and epidermal Hartig net (arrowheads). Fig. 20. Enlargement of a portion of the root shown in Fig. 19showing compact mantle (arrow) and epidermal Hartig net (arrowhead). Again, intracellular hyphae are absent. Figs. 21 and 22. Light mi-croscopy of resin-embedded longitudinal sections of Hudsonia tomentosa mycorrhizas. Fig. 21. Cenococcum mycorrhiza showing a well-developed mantle and Hartig net hyphae confined to the radially elongated epidermal cells. The apical meristem (asterisk, *) is obvious.Fig. 22. Enlargement of a portion of the root shown in Fig. 21 showing the epidermal Hartig net, thick mantle, and melanized outer mantlehyphae. No intracellular colonization was present. Figs. 23 and 24. Fluorescence microscopy of free-hand transverse sections of Hudsoniatomentosa mycorrhizas stained with berberine hemi-sulphate. Fig. 23. Section showing xylem elements (arrowhead), suberized endodermalcells (white asterisks), and compact mantle (arrow). Fluorescence is absent in cortical cell walls. Fig. 24. Enlargement of central portion ofroot shown in Fig. 23 showing xylem elements (arrowhead) and suberized endodermal cell walls (white asterisks). Fig. 25. Transmissionelectron micrograph of Hudsonia ericoides outer cortical cells (asterisks) showing that cell walls lack suberin. The thicker dark regions arecollapsed epidermal cells.

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Transmission electron microscopyTransmission electron microscopy confirmed that neither

the walls of the hypodermal cells (Fig. 25) nor the epidermalcells (not shown) contained suberin lamellae.

DiscussionRoot systems of both H. ericoides and H. tomentosa con-

sisted of mostly fibrous roots with numerous laterals, someof a diameter similar to that of ‘‘hair roots’’ of Ericaceaespecies (Peterson et al. 2004). Owing to their minute size,microscopic examination of the fine lateral roots for entireroot systems was essential to determine that most root tipsfor both species were heavily colonized. Despite this, onlyfour distinct fungal morphotypes were characterized for thetwo Hudsonia hosts. Based on observations of other systems(Smith and Read 2008), the number of fungal symbionts in-volved in colonization of both species would possibly in-crease with the use of molecular methods. However, thiswas not the intent of this study; rather, emphasis was on de-termining the structure of the root–fungus associations, tocompare with results of other genera in the Cistaceae.

The presence of several morphotypes, some with an abun-dance of EH, may contribute to the success of these hosts innutrient-poor soils by increasing the absorbing surface of theplant. Also, the well-developed mantles of most morpho-types and the prevalence of rhizomorphs on some may ena-ble these species to exist in drought prone habitats (Smreciuet al. 1997). Morte et al. (2000) showed experimentally thatHelianthemum almeriense was able to withstand imposeddrought conditions when colonized by the desert truffle,T. claveryi. Elsewhere, and in the two habitats sampled,Hudsonia spp. and their mycorrhizal fungal associates mayplay a significant role in soil structure, stabilization and re-storation in sandy soils based on the prevalence of mycorrhi-zal root tips and the abundance of EH (Perry et al. 1990).

The structural features observed for both H. ericoides andH. tomentosa mycorrhizas (a multi-layered mantle, Hartignet restricted to radially elongated epidermal cells, and lackof intracellular hyphae) are shared by the majority of woodyangiosperm ectomycorrhizas (Massicotte et al. 1990; Peter-son et al. 2004; Smith and Read 2008), as well as by Poly-gonum viviparum L. (Polygonaceae) and Kobresia bellardii(All.) Degl. (Cyperaceae), two herbaceous angiosperms(Massicotte et al. 1998). However, in contrast to some ecto-mycorrhizas that have a Hartig net confined to the epider-mis, but which also have a suberized hypodermis believedto restrict hyphal penetration into deeper cortical layers(e.g., Massicotte et al. 1986), the two Hudsonia specieslacked suberized walls in the outer cortical cell layer (hypo-dermis). The lack of suberization suggests that other proper-ties of hypodermal cell walls may exist that impede fungalpenetration; however, absence of suberin in outer corticalcell walls might also allow, under different environmentalconditions or with different fungal associates, the possibilityof intracellular colonization. This has not been explored. Re-ports that H. tomentosa has arbutoid mycorrhizas (Allen andAllen 1992) were not supported by our findings. Although amantle and Hartig net developed, intracellular hyphae werenot present.

Results confirmed true ECM formation for H. ericoides

and H. tomentosa. Jakucs et al. (1999) also reported that thespecies Fumana procumbens (Dun.) Gr. Godr. is an ECMhost (with mantle and Hartig net restricted to the epidermis)for the fungus Hebeloma ammophilum Bohus. These mycor-rhizas differ structurally from some of the other members inthe Cistaceae. For example, mycorrhizas of all Cistus spe-cies that associate with a range of fungal symbionts have amantle and Hartig net involving both epidermal and corticalcells (e.g., Agueda et al. 2006; Fusconi 1983; Giovannettiand Fontana 1982; Nuytinck et al. 2004; Torres et al. 1995;Wenkart et al. 2001). These features are typical of coniferectomycorrhizas (in contrast to the woody angiosperm ecto-mycorrhizas) (Peterson et al. 2004). Structurally, if sectionedmaterial is not median longitudinal (i.e., is submedian ortangential), neighbouring epidermal cells may be cut ob-liquely or at an angle that suggests two cell rows, and thiscould falsely lead to the conclusion that the Hartig net is in-volving cortical cells. In addition, it has been reported thatsome mycorrhizas formed with in-vitro transformed rootclones of C. incanus colonized by T. boudieri had intracellu-lar penetration by hyphae as well as epidermal and corticalHartig net formation, depending on culture conditions (Zar-etsky et al. 2006). This outcome may reflect more the artifi-cial situation under which the roots were colonized.

The mycorrhizal condition reported for species of Helian-themum is highly variable although most host–fungus sym-bioses show both epidermal and cortical Hartig netformation as well as the existence of intracellular hyphae(Dexheimer et al. 1985; Kianmehr 1978; Kovacs et al.2003), features characteristic of ectendomycorrhizas (Pe-terson et al. 2004; Yu et al. 2001). Kovacs and Jakucs(2001) reported the Hartig net in H. ovatum (Viv.) was re-stricted to the outer row of cortical cells and absent fromthe epidermis. Structural differences may partly depend ongrowth and soil conditions, as well as the fungal symbiontinvolved (Gutierrez et al. 2003). The two Hudsonia speciesexamined in our study both came from sandy, mineral soilhabitats and, despite seasonal and geographical samplingdifferences, showed remarkably similar ECM associationsand structure. A larger study might further explore the limitsof location and sampling times for Hudsonia populations,and correlate mycorrhizal status with these and other varia-bles such as soil properties. Fortas and Chevalier (1992) re-ported that phosphorus concentration was important indetermining the type of mycorrhiza formed between threetruffle species and H. guttatum under in-vitro culture condi-tions; whether this might be important in natural systems isnot known. Although a few studies have explored plant–fun-gus signaling in the Cistaceae, these have only involved Cis-tus species and mechanisms underlying these interactionsare still being investigated (see Coughlan and Piche 2005).

For many species in the Cistaceae, the mycorrhizal statusremains undetermined and, as a result, our ability to assessthe range of mycorrhizal categories present in this diversefamily is constrained. Evidence that some species can formarbuscular mycorrhizal associations (Harley and Smith1983) was not seen in the present study but is of interestfor further investigation. The host–fungus species involvedin each association, host age, and (or) the habitat (includinggrowth conditions) are most likely all important contributingfactors.

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AcknowledgementsWe thank the Natural Sciences and Engineering Research

Council of Canada for providing funds through DiscoveryGrants to H.B.M and R.L.P. We also thank Dr. RodgerEvans for taking us to the field site in Nova Scotia and Car-ole Ann Lacroix for confirming the identity of the Hudsoniaspecies. We appreciate the comments on the manuscript of-fered by two anonymous reviewers.

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