Hudsonia ericoides and Hudsonia tomentosa - Canadian Science ...

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Hudsonia ericoides and Hudsonia tomentosa: Anatomy of mycorrhizas of two members in the Cistaceae from Eastern Canada Hugues B. Massicotte, R. Larry Peterson, Lewis H. Melville, and Linda 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 soil stabilization. For these reasons, understanding their root systems and their associated fungal symbionts is important. Most studies of the structure of mycorrhizas in this family involve two genera, Cistus and Helianthemum. The present study examines structural features of mycorrhizas in two North American species, Hudsonia ericoides L. and Hudsonia tomentosa Nutt. Root systems of both species are highly branched with most fine roots colonized by mycorrhizal fungi. Based on morphological features, several mycorrhizal fungi were identified; structural details also provided evidence of more than one fungal symbiont for each host species. All mycorrhizas had a multi-layered fungal mantle and Hartig net hyphae confined to radially elongated epidermal cells; no intracellular hyphae were observed. Although the Hartig net was confined to the epidermis, the outer row of cortical cell walls lacked suberin, a known barrier to fungal penetration. Mycorrhizas in H. ericoides and H. tomentosa differed from those of Cistus and Helianthemum species that have a Hartig net that extends into the root cortex, as well as frequently present intracellular hyphae. Key words: Cistaceae, Hudsonia, mycorrhiza, anatomy, Hartig net, mantle. Re´sume´ : La plupart des espe`ces de la famille des Cistaceae se retrouvent dans le bassin de la Me´diterraneé. Plusieurs hoˆtes pre´sentent un inte´reˆt particulier, compte tenu de leurs associations avec des espe`ces de truffes, alors que d’autres constituent des plantes pionnie`res importantes dans les endroits perturbe´s et pour la stabilisation des sols. Pour ces raisons, il importe de comprendre la biologie de leurs syste`mes racinaires et de leurs champignons symbiotiques associe´s. La plupart des e´tudes publieés sur la structure des mycorhizes de cette famille portent sur les deux genres Cistus et Helianthemum. On examine ici les caracte´ristiques structurales des mycorhizes de deux espe`ces Nord-ame´ricaines, l’Hudsonia ericoides L. et l’Hudsonia tomentosa Nutt. Chez les deux espe`ces, l’on observe des syste`mes racinaires fortement ramifie´s, la plupart des racines fines e´tant coloniseés par des champignons mycorhiziens. Sur la base des caracte´ristiques morphologiques, les auteurs ont pu identifier plusieurs espe`ces de champignons mycorhiziens; les de´tails des structures fournissent e´galement des preuves de l’existence de plus d’un symbiote fongique chez chaque espe`ce hoˆte. Toutes les mycorhizes montrent un manteau fongique a` plusieurs couches et des hyphes du re´seau de Hartig confineés aux cellules e´pidermiques radialement allongeés; on observe aucune peńe´tration intracellulaire. Bien que le re´seau de Hartig soit confine´ a` l’e´piderme, les parois cellulaires de la couche externe de cellules corticales ne posse`dent pas de sube´rine, une barrie`re reconnue pour la peńe´tration fongique. Les mycorhizes chez les H. ericoides et H. tomentosa diffe`rent de celles des espe`ces de Cistus et d’Helianthemum, lesquelles posse`dent un re´seau de Hartig s’e´tendant dans le cortex racinaire et montrent la pre´sence d’hyphes intracellulaires souvent pre´sentes. Mots-cle´s : Cistaceae, Hudsonia, mycorhize, anatomie, re´seau de Hartig, manteau. [Traduit par la Re´daction]

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, 3333 University 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]).

Botany 88: 607–616 (2010)

doi:10.1139/B10-035

Published by NRC Research Press

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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 of mycorrhizal roots and several morphotypes (arrows). Fig. 4. White rhizomorphic mycorrhiza showing beaded appearance due to growth increments (arrowheads) along the length of the root, and large attached rhizomorphs (arrow). Scale as in Fig. 5. Fig. 5. Brown Tomentellalike morphotype showing swollen root tips (arrows) and abundant extraradical hyphae. Figs. 6–8. Hudsonia tomentosa. Fig. 6. Excavated seedling showing the highly branched root system. Scale is in millimetres. Fig. 7. Portion of the same root system showing two morphotypes: 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 Cenococcum extraradical hyphae (arrowheads) are present.

Introduction The family Cistaceae (the rock rose family) is composed of eight genera and approximately 180 species (Guzmań and Vargas 2009) centered in temperate Europe and the Mediterranean basin where they are often part of important early successional stages in disturbed habitats (Guzmań and Vargas 2005). The genera, based on plastid rbcL and trnL– trnF sequences (Guzmań and Vargas 2009), include Cistus, Halimium, Helianthemum, Hudsonia, Lechea, Tuberaria, Fumana, and Crocanthemum. Several genera occur in North America. Members of this family have been known to form associations with mycorrhizal fungi (see Malloch and Thorn 1985), and recently, Comandini et al. (2006) published an extensive list of fungal species associated with the genus Cistus. Several species of Hebeloma are highly specific to Cistus species (Eberhardt et al. 2009). With respect to mycorrhizal associations of Cistaceae species in North America, Malloch and Thorn (1985) examined three Helianthemum species and two species each of Hudsonia and Lechea (misspelled Lechia in their paper). Based on sporocarps found in the vicinity, the authors listed the probable fungal symbionts of these hosts as well as other fungi associating with Cistus species. In spite of the large number of species in the Cistaceae, relatively few have been examined for their mycorrhizal status, and fewer still for the structural details between roots and associated symbiotic fungi. The majority of structural studies have involved Cistus species and various fungal symbionts: C. incanus L. – Tuber melanosporum Vitt. (Fusconi 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. – Boletus ´ gueda et al. 2006, 2008); and, C. cf. edulis Bull. ex Fr. (A ladanifer L. – Boletus rhodoxanthus Kallenb. (Hahn 2001). All are characterized by having a mantle and a Hartig net that involves both epidermal and cortical cell layers, features that are shared with conifer ectomycorrhizas. Similar results were obtained for four other Cistus species in combination with various Tuber species (Giovannetti and Fontana 1982), and for a Cistus sp. associated with both Lactarius tesquorum Malençon (Nuytinck et al. 2004) and with Lactarius cistophilus Bon & Trimbach (Comandini and Rinaldi 2008). However, mycorrhizas formed with in-vitro transformed root clones of C. incanus and fungal isolates of Terfezia boudieri Chatin either developed ectomycorrhizal features similar to other Cistus species or had intracellular penetration by hyphae, depending on the clone and culture conditions (Zaretsky et al. 2006). Cleared roots of Helianthemum chamaecistus Mill. – Cenococcum graniforme (Sow.) Ferd mycorrhizas showed corti-

cal Hartig net formation as well as intracellular hyphae, although no mention was made concerning mantle development (Kianmehr 1978). Dexheimer et al. (1985) described ultrastructural features of Hartig net hyphae, as well as intracellular hyphae of Helianthemum salicifolium (L.) Mill. colonized by two Terfezia species. Fortas and Chevalier (1992) examined the effect of culture conditions on the colonization of Helianthemum guttatum (L.) Mill. by three truffle species; all species formed ectomycorrhizas with a cortical Hartig net but without a mantle in high phosphorus conditions, whereas they developed ectendomycorrhizas (cortical Hartig net and intracellular hyphae) in phosphorusdeficient cultures. Gutie´rrez et al. (2003) showed that, under greenhouse conditions, Helianthemum almeriense Pau – T. claveryi mycorrhizas developed a cortical Hartig net with limited mantle, whereas in-vitro conditions produced a cortical Hartig net and a thick, multi-layered mantle. The same host colonized by Picoa lefebvrei (Pat.) Maire under greenhouse conditions had limited mantle, cortical Hartig net, and intracellular hyphae. Similarly, cortical Hartig net and intracellular fungal hyphae formed in in-vitro Helianthemum ovatum (Viv.) Dun. – Terfezia terfezioides (Matt.) Trappe mycorrhizas (Kovaćs et al. 2003); however, the authors cautioned that the root–fungus interaction in vitro might not reflect the true mycorrhizal association. Malloch and Thorn’s (1985) examination of whole mounts of field-collected Lechea intermedia roots showed an epidermal and cortical Hartig net, but there was no report of intracellular hyphae. The above reports demonstrate how highly variable mycorrhizal structure is in Cistaceae, perhaps not surprising in a family that is also taxonomically so diverse. No information is available for species in the genera Crocanthemum and Tuberaria. The two species examined in the present study, Hudsonia ericoides L. and Hudsonia tomentosa Nutt., are native to North America. Hudsonia ericoides (pine barren goldenheather; false heather) is an evergreen subshrub that grows in nutrient poor, sandy soil in isolated areas restricted to northeastern Canada and USA. It is specifically adapted to drought, soil erosion, and extreme soil conditions. Malloch and Thorn (1985) published images of root tips with Cenococcum-like mantles, from a collection made in Nova Scotia. The second host, H. tomentosa (sand heather; woolly beach heather) is found in sandy pine woods and clearings from 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 New Brunswick and Ontario showed a Hartig net that appeared to be restricted to the epidermis (Malloch and Thorn 1985). Allen and Allen (1992) noted that H. tomentosa (described Published by NRC Research Press

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Figs. 9–16. Figs. 9–12. Scanning electron micrographs of Hudsonia ericoides mycorrhizas. Fig. 9. Cenococcum mycorrhiza showing large extraradical 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 of the mycorrhiza in Fig. 10 showing the branched, compact nature of the mantle. Fig. 12. Clamp connections (arrows) on outer mantle hyphae of a basidiomycete mycorrhiza. Figs. 13–16. Scanning electron micrographs of Hudsonia tomentosa mycorrhizas. Fig. 13. Portion of a rhizomorph from a white rhizomorphic mycorrhiza similar to that shown in Fig. 7. Fig. 14. Cenococcum mycorrhiza showing extensive emanating hyphae (arrowheads). Fig. 15. Basidiomycete mycorrhiza with few emanating hyphae. Fig. 16. Mycorrhizal root showing recolonization by Cenococcum (at the apex) and the white rhizomorphic mycorrhiza with loose emanating hyphae (basal). A few large Cenococcum hyphae (arrows) are present.

by them as an ericaceous shrub) had arbutoid-like mycorrhizas; however, this observation was not documented by images. The objective of the present study was to use a variety of microscopical methods to more fully describe the anatomical features of the mycorrhizal associations of H. ericoides and H. tomentosa from field-collected specimens. Of interest was to compare the results with what is known for these species and for species in the other genera in this large family.

Materials and methods Plant material Samples of H. ericoides were collected on 22 May 2009, west of Kingston, Nova Scotia (approximately 44.9888N, 64.9498W), in sandy habitats along an abandoned railway bed. Ectomycorrhizal hosts in the vicinity included several Pinus species (e.g., Pinus banksiana Lamb., Pinus resinosa Aiton, Pinus strobus L.), as well as Populus tremuloides (Michx.) A & D Lo¨ve, Populus grandidentata Michx., and Betula populifolia Marsh. Collections of H. tomentosa were made on 9 October 2009, from a sandy beach site in Petawawa, Ontario (approximately 46.08N, 77.38W); mature P. strobus, P. resinosa, and smaller Populus balsamifera L., P. tremuloides, and Betula papyrifera Marsh. trees were in the vicinity. Entire plants with attached roots and soil were excavated and transported to the University of Guelph where they were stored briefly at 4 8C; subsequently, roots were washed in preparation for examination with a stereo binocular microscope for morphotype characterization and for fixation for further microscopy. Morphological assessment Standard light microscopy techniques were used to determine the different Hudsonia morphotypes (Agerer 1987– 2008; Ingleby et al. 1990; Goodman et al. 1996). Mycorrhizas were characterized according to colour, texture, lustre, dimensions, tip shape, branching pattern, and presence or absence of rhizomorphs; root squash mounts were used to describe mantle features, emanating hyphae, rhizomorphs, and other distinguishing features. Preliminary identifications to fungal families or genera were designated when possible; otherwise, a descriptive name was assigned. Resin embedding and light microscopy Root samples from several plants for each host species were fixed in 2.5% glutaraldehyde in 0.10 mol/L 4-2-hydroxyethyl-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 Company Ltd., Reading, Berkshire, England) using conventional protocols (Ruzin 1999). Thick sections (1–1.5 mm) were cut on a Porter-Blum Ultra-Microtome MT-1 (DuPont Co., Newton, Connecticut, USA) using glass knives, stained for light microscopy using aqueous 0.05% toluidine blue O (SigmaAldrich, St. Louis, Missouri, USA) in 1% sodium borate, and viewed on a Leitz Orthoplan (Leica, Mississauga, Ontario, Canada) microscope. Images were captured using a Nikon Coolpix 4500 digital camera (Nikon, Canada, Mississauga, Ont.). At least five samples of roots from both sites were examined for each mycorrhiza type. Fluorescence microscopy Free-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% glycerol. Sections were viewed under UV and blue light (350– 460 excitation wavelength) on a Leitz SM-LUX epifluorescence microscope (Leica, Mississauga, Ont.). Images were captured using the same camera system described above. Scanning electron microscopy Samples fixed in 2.5% glutaraldehyde were dehydrated through an ascending series of ethanol to 100%, criticalpoint dried, and mounted on aluminum stubs with two-sided sticky tape. After coating with gold–palladium, they were examined with a Hitachi S-570 (Nissei-Sangyo, Tokyo, Japan) scanning electron microscope. Images were taken with an attached digital camera. Transmission electron microscopy Thin sections (*0.1 mm) of LR-White-embedded material were sectioned with glass knives on a Reichert ultramicrotome (Model OM-U3; Reichert-Jung (Leica), Wetzlar, Germany), collected on formvar-coated copper grids, stained for 10 min in 2% ethanolic uranyl acetate, and counter-stained for 5 min with 1.0% lead citrate. Sections were examined with a Philips CM10 transmission electron microscope (Philips Electron Optics, Eindhoven, Germany) at 80 kV and images were collected with a digital camera.

Results Morphology of root systems Both H. ericoides and H. tomentosa occurred as small patches in open areas of sandy soil or in semi-open areas Published by NRC Research Press

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

near the tree(shrub)–sand zones (Figs. 1 and 2). Branched shoots consisted of scale-like leaves (Fig. 2). The two species shared similarities in growth form, rooting patterns, and fungal colonization. Both hosts had a mostly fibrous root system from which grew different order laterals. Most roots were extremely small (mycorrhizal tips were up to 200 mm wide and approximately 0.5–2.0 mm long) and almost all root tips that were examined were mycorrhizal (Fig. 3); root systems for each host species were colonized by several different symbiotic fungi. Hudsonia ericoides morphotypes included a white rhizomorphic mycorrhiza that had a compact mantle of felt to net synenchyma, emanating hyphae (EH) 2–4 mm in diameter with clamp connections, and numerous rhizomorphs (Fig. 4) with large vessel-like hyphae. This host also produced a brown Tomentella-like morphotype with a thick net to angular synenchyma mantle, EH 4–5 mm in diameter with clamp connections, and no obvious rhizomorphs (Fig. 5). Minor components of H. ericoides mycorrhizas were formed by dark septate ascomycetes; these consisted of MRA (Mycelium radicis atrovirens Melin)-like fungi (Ingleby et al. 1990) and Cenococcum geophilum Fr. with isodiametric mantle cells forming a regular synenchyma (see Ingleby et al. 1990) and abundant, large EH. Hudsonia tomentosa roots (Fig. 6) were dominated by two morphotypes: a white rhizomorphic mycorrhiza similar to that seen for H. ericoides; and Cenococcum geophilum mycorrhizas (Fig. 7). White rhizomorphic mycorrhizas had few emanating hyphae; these were often short and had a determinant length with enlarged (swollen) hyphal tips, or took the form of enlarged surface mantle cells. No other associated fungi were identified for H. tomentosa. However, many roots showed frequent successional colonization patterns with numerous tips being recolonized by the other fungal morphotype (i.e., either Cenococcum was at the tip and the white morphotype basal (Fig. 8), or the white mycorrhiza was at the tip and Cenococcum basal). The position of the two fungi was variable in recolonized roots. Scanning electron microscopy Roots of H. ericoides – Cenococcum mycorrhizas, when viewed by SEM, often had enlarged root apices with large prominent EH (Fig. 9); other morphotypes formed compact mantles (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 rhizomorphs (Fig. 13). Figure 14 shows a H. tomentosa – Cenococcum mycorrhiza with numerous, large EH. Some H. tomentosa mycorrhizas had a compact mantle structure with fewer EH (Fig. 15), or showed successional recolonization by two fungal species (Fig. 16). Light microscopy Sectioned roots from both plant hosts showed that colonized root tips had a simple anatomy: an epidermis of radially elongated cells, only 2–3 rows of cortical cells, and a vascular cylinder consisting of a few xylem tracheary elements and phloem. Longitudinal sections of LR-White-embedded mycorrhizal root tips of H. ericoides (Figs. 17–20) showed that mycorrhizas had a compact, multi-layered mantle (Figs. 17 and 18) and a Hartig net confined to the radially elongated epidermal cells; intracellular hyphae were not present (Figs. 17 and 18). Other H. ericoides morphotypes also had compact, fungal mantles of varying thickness that extended the length of the root and covered the root apex (Fig. 19). The Hartig net was again limited to the radially elongated epidermal cells and intracellular hyphae were not present (Figs. 19 and 20). Longitudinal sections of H. tomentosa mycorrhizas (the section illustrated is a Cenococcum mycorrhiza) (Figs. 21 and 22) showed similar structural features to those of H. ericoides; the mantle was multi-layered with the outer mantle hyphae showing the melanized walls of Cenococcum and dense inter-hyphal deposits (Fig. 22). Hartig net hyphae were associated only with epidermal cells (as in H. ericoides) and intracellular hyphae were not present (Fig. 22). Fluorescence microscopy Transverse sections of both H. ericoides and H. tomentosa mycorrhizal roots, when stained with berberine hemisulphate, showed that lignified walls of tracheary elements and suberized walls of endodermal cells fluoresced when viewed with UV–blue light (Figs. 23 and 24). Walls of the outer layer of cortical cells (hypodermis) and epidermal cells did not fluoresce although these cells and mantle hyphae showed some background fluorescence (Fig. 23). Published by NRC Research Press

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Transmission electron microscopy Transmission electron microscopy confirmed that neither the walls of the hypodermal cells (Fig. 25) nor the epidermal cells (not shown) contained suberin lamellae.

Discussion Root systems of both H. ericoides and H. tomentosa consisted of mostly fibrous roots with numerous laterals, some of a diameter similar to that of ‘‘hair roots’’ of Ericaceae species (Peterson et al. 2004). Owing to their minute size, microscopic examination of the fine lateral roots for entire root systems was essential to determine that most root tips for both species were heavily colonized. Despite this, only four distinct fungal morphotypes were characterized for the two Hudsonia hosts. Based on observations of other systems (Smith and Read 2008), the number of fungal symbionts involved in colonization of both species would possibly increase with the use of molecular methods. However, this was not the intent of this study; rather, emphasis was on determining the structure of the root–fungus associations, to compare with results of other genera in the Cistaceae. The presence of several morphotypes, some with an abundance of EH, may contribute to the success of these hosts in nutrient-poor soils by increasing the absorbing surface of the plant. Also, the well-developed mantles of most morphotypes and the prevalence of rhizomorphs on some may enable these species to exist in drought prone habitats (Smreciu et al. 1997). Morte et al. (2000) showed experimentally that Helianthemum almeriense was able to withstand imposed drought conditions when colonized by the desert truffle, T. claveryi. Elsewhere, and in the two habitats sampled, Hudsonia spp. and their mycorrhizal fungal associates may play a significant role in soil structure, stabilization and restoration in sandy soils based on the prevalence of mycorrhizal root tips and the abundance of EH (Perry et al. 1990). The structural features observed for both H. ericoides and H. tomentosa mycorrhizas (a multi-layered mantle, Hartig net restricted to radially elongated epidermal cells, and lack of intracellular hyphae) are shared by the majority of woody angiosperm ectomycorrhizas (Massicotte et al. 1990; Peterson et al. 2004; Smith and Read 2008), as well as by Polygonum viviparum L. (Polygonaceae) and Kobresia bellardii (All.) Degl. (Cyperaceae), two herbaceous angiosperms (Massicotte et al. 1998). However, in contrast to some ectomycorrhizas that have a Hartig net confined to the epidermis, but which also have a suberized hypodermis believed to restrict hyphal penetration into deeper cortical layers (e.g., Massicotte et al. 1986), the two Hudsonia species lacked suberized walls in the outer cortical cell layer (hypodermis). The lack of suberization suggests that other properties of hypodermal cell walls may exist that impede fungal penetration; however, absence of suberin in outer cortical cell walls might also allow, under different environmental conditions or with different fungal associates, the possibility of intracellular colonization. This has not been explored. Reports that H. tomentosa has arbutoid mycorrhizas (Allen and Allen 1992) were not supported by our findings. Although a mantle and Hartig net developed, intracellular hyphae were not present. Results confirmed true ECM formation for H. ericoides


and H. tomentosa. Jakucs et al. (1999) also reported that the species Fumana procumbens (Dun.) Gr. Godr. is an ECM host (with mantle and Hartig net restricted to the epidermis) for the fungus Hebeloma ammophilum Bohus. These mycorrhizas differ structurally from some of the other members in the Cistaceae. For example, mycorrhizas of all Cistus species that associate with a range of fungal symbionts have a mantle and Hartig net involving both epidermal and cortical ´ gueda et al. 2006; Fusconi 1983; Giovannetti cells (e.g., A and Fontana 1982; Nuytinck et al. 2004; Torres et al. 1995; Wenkart et al. 2001). These features are typical of conifer ectomycorrhizas (in contrast to the woody angiosperm ectomycorrhizas) (Peterson et al. 2004). Structurally, if sectioned material is not median longitudinal (i.e., is submedian or tangential), neighbouring epidermal cells may be cut obliquely or at an angle that suggests two cell rows, and this could falsely lead to the conclusion that the Hartig net is involving cortical cells. In addition, it has been reported that some mycorrhizas formed with in-vitro transformed root clones of C. incanus colonized by T. boudieri had intracellular penetration by hyphae as well as epidermal and cortical Hartig net formation, depending on culture conditions (Zaretsky et al. 2006). This outcome may reflect more the artificial situation under which the roots were colonized. The mycorrhizal condition reported for species of Helianthemum is highly variable although most host–fungus symbioses show both epidermal and cortical Hartig net formation as well as the existence of intracellular hyphae (Dexheimer et al. 1985; Kianmehr 1978; Kovaćs et al. 2003), features characteristic of ectendomycorrhizas (Peterson et al. 2004; Yu et al. 2001). Kovaćs and Jakucs (2001) reported the Hartig net in H. ovatum (Viv.) was restricted to the outer row of cortical cells and absent from the epidermis. Structural differences may partly depend on growth and soil conditions, as well as the fungal symbiont involved (Gutie´rrez et al. 2003). The two Hudsonia species examined in our study both came from sandy, mineral soil habitats and, despite seasonal and geographical sampling differences, showed remarkably similar ECM associations and structure. A larger study might further explore the limits of location and sampling times for Hudsonia populations, and correlate mycorrhizal status with these and other variables such as soil properties. Fortas and Chevalier (1992) reported that phosphorus concentration was important in determining the type of mycorrhiza formed between three truffle species and H. guttatum under in-vitro culture conditions; whether this might be important in natural systems is not known. Although a few studies have explored plant–fungus signaling in the Cistaceae, these have only involved Cistus species and mechanisms underlying these interactions are still being investigated (see Coughlan and Piche´ 2005). For many species in the Cistaceae, the mycorrhizal status remains undetermined and, as a result, our ability to assess the range of mycorrhizal categories present in this diverse family is constrained. Evidence that some species can form arbuscular mycorrhizal associations (Harley and Smith 1983) was not seen in the present study but is of interest for further investigation. The host–fungus species involved in each association, host age, and (or) the habitat (including growth conditions) are most likely all important contributing factors. Published by NRC Research Press

Massicotte et al.

Acknowledgements We thank the Natural Sciences and Engineering Research Council of Canada for providing funds through Discovery Grants to H.B.M and R.L.P. We also thank Dr. Rodger Evans for taking us to the field site in Nova Scotia and Carole Ann Lacroix for confirming the identity of the Hudsonia species. We appreciate the comments on the manuscript offered by two anonymous reviewers.

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