NOTE / NOTE

The effect of mechanical site preparation onectomycorrhizae of planted white spruce seedlingsin conifer-dominated boreal mixedwood forest

Lance W. Lazaruk, S. Ellen Macdonald, and Gavin Kernaghan

Abstract: We characterized the ectomycorrhizae (ECM) of planted white spruce (Picea glauca (Moench) Voss) seedlingsas affected by mechanical site preparation (MSP) of clear-cut conifer-dominated boreal mixedwood forest. Relative abun-dance, richness, and composition of the ECM community were compared among untreated control, mixed, mounded, andscalped site preparation treatments. On >11 000 root tips, we observed 16 ECM morphotypes. Those common to the nurs-ery in which the seedlings were raised were most abundant (Thelephora americana, Wilcoxina-like (E-strain), Amphinemabyssoides, Phialocephala-like (MRA)). Seedlings in the untreated controls had lower abundances of these, but higher abun-dances of other ECM, which were not present in the nursery of origin but were indigenous to these forest stands. In termsof ECM composition, the ‘‘mixed’’ treatment was most similar to the untreated control, while the ‘‘scalped’’ and ‘‘mound’’treatments showed significantly different ECM communities than the controls. Our results suggest that MSP may facilitatecontinued dominance by ECM that establish on seedlings in the nursery while slowing the natural succession towards thenatural forest ECM. MSP treatments that leave some surface organic matter relatively intact may impact ECM less thanthose that remove or bury the organic layer.

Resume : Nous avons caracterise les ectomycorhizes (ECM) de plants d’epinette blanche (Picea glauca (Moench) Voss)affectes par la preparation mecanisee de terrain (PMT) a la suite d’une coupe a blanc dans une foret mixte dominee pardes resineux. L’abondance relative, la richesse et la composition de la communaute d’ECM a ete comparee entre un te-moin non traite et des traitements de preparation de terrain mixtes, d’amenagement de buttes et de scalpage. Nous avonsobserve 16 morphotypes d’ECM sur plus de 11 000 apex racinaires. Ceux qui etaient communs dans la pepiniere ou lessemis avaient ete produits etaient les plus abondants (Thelephora americana, le type Wilcoxina (souche E), Amphinema by-ssoides, le type Phialocephala (MRA)). L’abondance de ces ECM etait plus faible chez les semis des temoins non traitesqui comportaient, cependant, une plus forte abondance d’autres ECM qui n’etaient pas presentes dans la pepiniered’origine, mais qui etaient indigenes a ces peuplements forestiers. En termes de composition en ECM, le traitement mixteetait le plus semblable au temoin non traite alors que le scalpage et l’amenagement de buttes etaient associes a des com-munautes d’ECM significativement differentes de celle du temoin. Nos resultats indiquent que la PMT peut faciliter lemaintien de la dominance des ECM etablies sur les plants en pepiniere tout en ralentissant la succession naturelle vers lesECM de la foret naturelle. Les traitements de PMT qui laissent des superficies relativement intactes de matiere organiquepeuvent avoir moins d’impact sur les ECM que ceux qui enlevent ou enfouissent la couche de matiere organique.

[Traduit par la Redaction]

Introduction

In the North American boreal forest, the regeneration ofwhite spruce (Picea glauca (Moench) Voss) following clear-cutting has often proved difficult (Navratil et al. 1991).White spruce seedlings have slow growth rates (Neinstadtand Zasada 1990); consequently, they are often overtoppedby faster growing broadleaf trees, grasses, and a variety ofearly successional shrubs (Drew 1988; Lieffers et al. 1993).

In addition to competition for light, white spruce seedlingsmust also compete with these species for other essential re-sources such as water and mineral nutrients.

Mechanical site preparation (MSP) is commonly used toimprove the early performance of white spruce seedlings,and ultimately ensure the successful regeneration of harvestedsites. Common treatments include scalping (removing theoverlying organic layer and exposing the mineral soil), mix-ing (incorporating the underlying mineral soil with the over-

Received 12 September 2007. Accepted 6 March 2008. Published on the NRC Research Press Web site at cjfr.nrc.ca on 25 June 2008.

L.W. Lazaruk and S.E. Macdonald.1 Department of Renewable Resources, University of Alberta, Edmonton, AB T6G 2E9, Canada.G. Kernaghan. Biology Department, Mount Saint Vincent University, 166 Bedford Highway, Halifax, NS B3M 2J6, Canada.

1Corresponding author (e-mail: ellen.macdonald@ualberta.ca).

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lying organic layer), and mounding (inverting the soil hori-zons such that the organic layer is sandwiched between theorganic layer and a mineral ‘‘cap’’) (McMinn and Hedin1998). The objectives of MSP include increasing the avail-ability of nutrients, increasing the soil temperature, reducingcompeting vegetation, and, in the case of mounding, elevatingthe seedling above competing vegetation (Orlander et al.1990).

Ectomycorrhizae (ECM) are key components of healthyforest ecosystems, as they facilitate a wide range of inter-actions. They protect host plants from plant pathogens(Kropp and Langois 1990), facilitate uptake of water andmineral nutrients (Smith and Read 1997), and mediate inter-specific competition by transferring carbon and mineralnutrients between interconnected host plants (Simard et al.1997). These interactions are especially important forregenerating seedlings, which are particularly susceptible tocompetitive pressures. Changes to soil conditions and micro-climate that arise from MSP are likely to influence ECMcommunities, yet we have essentially no information on thistopic. Effects of MSP on soil nutrient availability may beparticularly important in terms of effects on ECM commun-ities. In this paper we present the results of a study in whichwe assessed the impact of MSP on the ectomycorrhizal sta-tus of white spruce seedlings planted following forestharvesting in the boreal mixedwood.

Materials and methodsThis study was conducted at the Ecosystem Management

by Emulating Natural Disturbance (EMEND) research site(see Work et al. 2004; Macdonald and Fenniak 2007; www.emend.rr.ualberta.ca), which is within the Lower Boreal-Cordilleran Ecoregion (Strong and Leggat 1992) in north-western Alberta (56847’N, 118821’W). In spring 1999, three0.5 ha plots were established, each in a separate forest standthat had been clear-cut the previous winter. Prior to harvest-ing, the sites were dominated by 113- to 130-year-old whitespruce. Each plot was divided into 50 m � 25 m quadrants,and each quadrant was randomly assigned to one of fourMSP treatments (mound, scalp, mix, or untreated control).The mineral cap of each mound was *80 cm � 100 cm inarea and 10–15 cm deep at the center. The ‘‘scalp’’ treat-ment had the mineral soil exposed in an area of *100 cm2.For the ‘‘mixed’’ treatment, the surface organic layer wasthoroughly mixed with mineral soil in a 140 cm � 100 cmarea. The MSP treatments were created using a 200 seriesexcavator equipped with a mounding bucket (mound andscalp treatments) and a Meri-Crusher high-speed horizontaldrum mulcher (mix treatment). In the 2 years immediatelyfollowing harvesting, the control (un-site-prepared) clear-cutsites supported an understory plant community with gener-ally low cover and richness of herbaceous and shrub species,but in which early successional species, such as Epilobiumangustifolium L. and Calamagrostis canadensis (Michx.)Beauv., had relatively higher abundances (Macdonald andFenniak 2007).

In July 1999, white spruce seedlings (stock type 415B1+0) were obtained from a local nursery (Woodmere Nur-sery, Fairview, Alberta). In the same year, the ECM com-munity of the Woodmere nursery was characterized by

sequencing the DNA of root-associated fungi (Kernaghan etal. 2003). Ten seedlings were planted in each quadrant ofeach plot. The seedlings were collected in May 2001 (twogrowing seasons after planting) by carefully excavating theentire seedling, clipping the seedlings at the root collar, stor-ing the root system in a plastic ‘‘zip-lock’’ bag, and trans-porting them to the University of Alberta where they werestored for up to 3 months at 4 8C until processing. Theisolation of root tips was accomplished by gently rinsingthe root system over a soil sieve (32 mesh/in.; 1 in. =25.4 mm), to remove adhering soil and debris, and cuttingthe roots into 2–3 cm long sections. Sections of the rootsystem were randomly selected, and then 200 fine roottips per seedling were randomly selected for examinationof mycorrhizae. Only roots extending from the soil plugwere sampled. In some instances, fewer than 200 suitableroot tips were available for a given seedling, in whichcase we used as many as met our criteria for inclusion.Root tips were examined using both stereo (Zeiss Stemi2000-C, 7–40�) and compound (Leitz Labrolux K, 500–1000�) microscopes and classified as mycorrhizal or not;mycorrhizal root tips were categorized as to morphotypebased on morphological and anatomical characteristics in-cluding: shape, color, and texture of the ECM system andemanating elements (hyphae and mycelial strands); mantlepatterning; the size, color, ornamentation, and contents ofhyphal cells; the type and frequency of septa; and reactionsto specific chemical compounds (e.g., KOH, sulphovanillin)(following the protocol outlined by Goodman et al. 1996).Root tips with an immature or poorly developed mantle, orthat lacked distinguishable mantle features, were designated‘‘undetermined’’ (Kranabetter and Wylie 1998) and werenot included in analyses of richness or composition.

The relative abundance (% of root tips colonized) and fre-quency of occurrence (% of seedlings on which it wasfound) were calculated for each morphotype. We then sub-divided the ECM morphotypes into those that were com-monly found in the Woodmere nursery (‘‘source nursery’’ECM) (see Kernaghan et al. 2003) and those that were notfound in the nursery, but all of which had been previouslyfound in the conifer-dominated forests at the EMEND site(including unharvested and recently harvested stands;‘‘forest site’’ ECM) (see Lazaruk et al. 2005). We then cal-culated the percentage of root tips colonized and ECM rich-ness (number of morphotypes per seedling) for the twoECM groups separately.

We tested for the effects of MSP on percentage of roottips colonized and on ECM richness per seedling, for‘‘source nursery’’ and ‘‘forest site’’ ECM separately, using amixed-model analysis of variance, in which the MSP treat-ment was treated as a fixed effect and the three plots wereconsidered blocks (random factor); individual seedlings(within plot � treatment) were treated as subsamples. Varia-bles were first tested to ensure they met the assumption ofhomoscedasticity of residuals and that they did not showsubstantial deviations from normality. When the main effectof treatment was significant, we conducted post hoc compar-isons of least-square means. We used PROC MIXED inSAS version 9.1 for these analyses (SAS Institute Inc.,Cary, North Carolina).

To examine the composition of ECM morphotypes on

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seedlings in the different treatments, we conducted a non-metric multidimensional scaling (NMDS) ordination on rela-tive abundance of morphotypes on each seedling (in PC-Ordv.4; (McCune and Mefford 1997)). We used Sorenson’s(Bray–Curtis) distance measure on log-transformed relativeabundances and a stability criterion of 0.000 01; preliminaryruns were conducted to determine the optimal dimension ofthe solution for the final run. To test for significant differ-ences in ECM composition among the MSP treatments, weconducted a multivariate analysis of variance by means ofdistance-based redundancy analysis (dbRDA; Legendre andAnderson 1999; see also Macdonald and Fenniak 2007). Forthis, we first conducted a principle coordinates analysis(using the PrCoord program in CANOCO version 4.5; terBraak and Smilauer (2002)) on the matrix of log-transformedrelative abundances (by morphotype per seedling), selectingthe Bray–Curtis distance measure (as for the NMDS) andcorrection of negative eigenvalues. The resulting matrixwas used as the species data into a redundancy analysis(CANOCO version 4.5), in which the environmental matrixcontained orthogonal dummy variables coding for the MSPtreatments. Significance testing was by Monte-Carlo per-mutation testing restricted to account for the effect of theblocks. Contrasts were then used to compare the differentMSP treatments in pairwise fashion. For both the NMDSand the dbRDA, the four ECM with the lowest fre-quency (Lactarius spp., ‘‘basidiomycete 3,’’ Dermocybespp., ‘‘basidiomycete 2’’) were removed prior to analysis.

Results and discussionOn the more than 11 000 root tips examined, we observed

16 different ECM morphotypes (Table 1; Appendix 1). Themost common were those that were common to the nurseryin which the seedlings were raised (T. americana, Wilcoxina-like (E-strain), A. byssoides, Phialocephala-like (MRA))(Table 1). White spruce seedlings in the untreated controlssupported a lower abundance of these ‘‘source nursery’’fungi, but higher abundance of ‘‘forest site’’ ECM (fungiindigenous to unharvested and recently harvested foreststands in the study area) (Lazaruk et al. 2005), as com-pared with seedlings in the three different types of MSP,which did not differ from one another (Table 1). Therewere no differences among the treatments in terms of thepercentage of roots colonized by ‘‘undetermined’’ fungi,nor in the percentage of nonmycorrhizal root tips. Seed-lings in the control treatment had higher richness of both‘‘source nursery’’ and ‘‘forest site’’ ECM fungi than wasobserved for the other MSP treatments (Table 1).

Our results agree with some previous studies. Jones et al.(1996) found that ECM richness and diversity of lodgepolepine seedlings were higher in control areas than mech-anically site-prepared (scalped) areas. Pennanen et al.(2005), however, found that mounding resulted in higherlevels of ECM colonization for planted Norway spruce seed-lings because mounding was associated with improved rootgrowth than in untreated areas. Adverse effects of soil dis-turbance and compaction on ECM communities were alsoobserved in a related study at the EMEND research site(Lazaruk et al. 2005) and by Page-Dumroese et al. (1998),who reported lower abundance and richness of ECM on

Douglas-fir seedlings as a result of a stump removal treat-ment.

Several studies have shown that ECM are most abundantin the organic layer of forest soils (Harvey et al. 1976;Goodman and Trofymow 1998; Byrd et al. 1999). In site-prepared areas, the highest densities of ECM root tips onwestern white pine and Douglas-fir seedlings were found inthe organic matter (Harvey et al. 1997). We observed thatthe majority of egressed roots on seedlings in the moundtreatment came from the bottom of the root plug, while themajority of egressed roots on the control seedlings origi-nated from the upper half of the root plug. Seedlings col-lected from the mix treatment had uniform and extensiveroot development. The removal or disruption of the forestfloor during MSP likely directly affected the ECM commun-ity through disruption, removal, or burial of the surfaceorganic or litter layer of forest soils.

The removal of competing vegetation, which is also anobjective of MSP, may have had unintended negative im-pacts on ECM, because a number of woody herbs andshrubs found in harvest blocks may be alternate hosts. Thishas been demonstrated for Amelanchier alnifolia Nutt., Arc-tostaphylos sp., Chimaphila umbellata (L.) Bart., Ledumglandulosum Nutt., Salix sp., Shepherdia canadensis (L.)Nutt., Vaccinium sp., and regenerating broadleaf trees (e.g.,alder, aspen, birch) (Largent et al. 1980; Horton et al. 1998;Kernaghan and Currah 1998; Kranabetter 1999; Hagermanet al. 2001). Most of these species or genera are found inconifer-dominated forests at the EMEND site (Macdonaldand Fenniak 2007). Therefore, the exclusion of competingvegetation on the site-prepared microsites may have alsocontributed to the observed reduction in ECM richness andabundance of ‘‘forest site’’ ECM.

The relative abundance of individual morphotypes variedaccording to MSP treatment, reflecting the legacy of ECMfrom the nursery as well as the influence of the MSP treat-ments (Table 1). The majority of ECM were formed by‘‘early stage’’ (e.g., Wilcoxina-like) and ‘‘multistage’’ ecto-mycorrhizal fungi (e.g., A. byssoides, T. americana) (as perDeacon and Fleming 1992; Visser 1995), which are knownto dominate the nursery in which the seedlings were grown(Kernaghan et al. 2003). These were observed on seedlingscollected from all of the treatments, but Wilcoxina-like hadthe highest relative abundance for the control and mix treat-ments, while T. americana was the most abundant for themound and scalp treatments. Amphinema byssoides hadquite low abundance on seedlings in the scalp treatment.Piloderma sp. was only observed on seedlings from site-prepared areas, but had quite low abundance overall. In con-trast, the five most abundant ‘‘forest site’’ fungi (‘‘cf.Laccaria’’, Tomentella spp., Cortinarius spp., Tuber spp.,and Cenococcum geophilum) (Lazaruk et al. 2005) had lowabundance overall on planted seedlings but were markedlymore abundant on seedlings in the control treatment. ‘‘Latestage’’ ECM, such as Piloderma spp., Lactarius spp., andRussula spp. (as per Deacon and Fleming 1992; Visser1995), had quite low abundance in all treatments. It shouldbe noted that, while we are sure that the ‘‘forest site’’ ECMfungi originated from the forest site, and we know whichspecies likely came from the nursery, we can’t be certainthat all of the inoculum for the mycorrhizae classified as

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‘‘source nursery’’ ECM actually came from the nursery, asthose species also existed in recently harvested stands atthese sites (Lazaruk et al. 2005). To verify the nursery ori-gin of these fungi, population level genetic markers (e.g.,simple sequence repeat markers) would have to be employedto follow individual genotypes from the nursery to the field(Gagne et al. 2006).

For the composition of ECM fungi, the control treatmentshowed a significant difference compared with the moundand scalp treatments; all the other paired comparisons werenonsignificant (Table 2). Thus, overall, the mix treatmentseemed to have much less effect on ECM of white spruceseedlings than did the mound or scalp treatment. TheNMDS ordination further illustrated these differences in rel-ative abundance of fungal morphotypes among different sitepreparation treatments. In particular, many seedlings fromthe scalp treatment separated from those in the control andmix treatments, which were more strongly grouped together

(Fig. 1). Our results suggest that the mound and scalp treat-ments were facilitating continued dominance by the ECMthat had established on seedlings in the nursery while reduc-ing colonization by the natural forest mycorrhizae (Table 1).Both Jones et al. (2002) and Pennanen et al. (2005) notedthe dominance of nursery mycorrhizae on seedlings plantedin mounded areas. It has been suggested that disturbed soils,as found in site-prepared areas, may favor continuing domi-nance by nursery ECM over colonization by natural foresttypes (Jones et al. 2002) both through effects on innoculumand on microenvironmental conditions (Jones et al. 2003).

In conclusion, MSP significantly altered the ECM statusof planted white spruce seedlings. Given the variation inecological function among ECM, this has potentially impor-tant implications for survival and growth of planted seed-lings (Jones et al. 2002; Pennanen et al. 2005). MSPtreatments that leave surface organic layers relatively moreintact may result in planted seedlings being colonized by an

Table 1. Total number of root tips sampled, frequency (% of seedlings), and relative abundance (% of root tips colonized) ofectomycorrhizae (ECM) on white spruce seedlings (n = 120) for four mechanical site preparation (MSP) treatments: control,mix, mound, scalp.

Roots colonized (%)

ECM No. of tips Frequency (%) Control Mix Mound Scalp

‘‘Source nursery’’ ECMThelephora americana 3239 46.67 11.86 19.32 43.33 38.92Wilcoxina-like (E-strain) 2294 40.00 23.63 27.14 13.74 14.03Amphinema byssoides 1857 45.00 18.21 20.32 19.67 3.05Phialocephala-like (MRA) 1512 42.50 12.42 20.38 7.79 17.01‘‘Forest site’’ ECM‘‘cf. Laccaria’’ 249 5.00 2.65 2.83 — 3.08Tomentella spp. 227 8.33 5.35 — 1.58 —Cortinarius spp. 213 5.83 1.78 — 3.01 2.17Tuber spp. 190 3.33 6.50 — — —Cenococcum geophilum 141 5.00 4.18 1.00 0.06 —Lactarius spp. 100 0.83 — — — 3.33Piloderma spp. 96 5.83 — 0.62 2.33 0.39‘‘Basidiomycete 3’’ 65 0.83 — — — 2.23Dermocybe spp. 60 0.83 — — 2.00 —Russula spp. 24 1.67 — — 0.78 —Hebeloma spp. 19 2.50 — — — 3.25‘‘Basidiomycete 2’’ 5 0.83 — 0.17 — —‘‘Undetermined’’ 717 25.83 — — — —Nonmycorrhizal 44 5.00 — — — —Total 11052 — — — — —

‘‘Source nursery’’ ECMMean % colonization — — 66.11a 87.17b 84.54b 73.01b95% C.I. — — 11.96 8.57 9.61 13.72Mean richness per seedling — — 2.13a 1.73ab 1.67b 1.43b95% C.I. — — 0.35 0.30 0.25 0.33

‘‘Forest site’’ ECMMean % colonization — — 20.45a 4.62b 9.75ab 14.45ab95% C.I. — — 10.17 5.82 7.24 9.96Mean richness per seedling — — 0.60a 0.20b 0.43ab 0.40ab95% C.I. — — 0.24 0.17 0.22 0.20

Note: Also given are the mean percentages of roots colonized and mean richness (no. of ECM) per seedling (and 95% confidence interval(C.I.)) for each of the MSP treatments for ECM categorized as ‘‘source nursery’’ or ‘‘forest site’’ based on Kernaghan et al. (2003) and La-zaruk et al. (2005). For these, means within a row that have different letters were significantly different (by least-square means at � = 0.008).See Appendix 1 for descriptions of morphotypes.

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ECM assemblage more similar to that found in unpreparedforest sites.

AcknowledgementsWe are grateful for research funding provided by the

Sustainable Forest Management Network and the AlbertaConservation Association (through the Challenge Grants inBiodiversity, University of Alberta). We deeply appreciatethe support of Daishowa-Marubeni International Ltd., Can-adian Forest Products (CanFor, Grande Cache), AlbertaSustainable Resource Development, and Natural ResourcesCanada for their support of the EMEND project. L.W.L.gratefully acknowledges financial support from CanFor, theNatural Sciences and Engineering Research Council

(Canada), and the Faculty of Graduate Studies and Research(University of Alberta). Thanks go to Derek Sidders (Can-adian Forest Service) for use of his silviculture plots forthis study. Charlene Hahn, Kirsten Gregorwich, ChristineLazaruk, and John Dale assisted with the field work andprocessing of samples.

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Table 2. Results of multivariate analysis of variance by means of distance-based redundancy analysis comparingectomycorrhizae (ECM) composition among the four mechanical site preparation (MSP) treatments (control,mix, mound, scalp).

Source of variationNo. of (dummy)variables

� alleigenvalues

� canonicaleigenvalues (‘‘trace’’) F P

MSP treatments 3 0.977 0.032 1.233 0.006*

ContrastsControl vs. mix 1 0.969 0.017 0.999 0.374Control vs. mound 1 0.962 0.031 1.804 0.016*Control vs. scalp 1 0.972 0.036 1.946 0.002*Mix vs. mound 1 0.941 0.023 1.404 0.054Mix vs. scalp 1 0.959 0.024 1.316 0.092Mount vs. scalp 1 0.932 0.018 0.986 0.426

Note: Pairwise comparisons were also conducted. See Legendre and Anderson (1999) and Macdonald and Fenniak (2007)for further explanation of the analytical approach. *, significant at P < 0.05.

Fig. 1. Results of an unconstrained ordination (nonmetric multidi-mensional scaling; NMDS) of relative abundance of ectomycorrhi-zae on white spruce seedlings planted in four different mechanicalsite preparation treatments (MSP: control, mix, mound, scalp).Points are individual seedlings coded as to MSP treatment. A three-dimensional solution was determined to be optimal based on preli-minary runs. Final stress was 11.55, and instability was 0.000 45.

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Appendix ATable A1 appears on the following pages.

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Table A1. Morphological and anatomical features associated with ectomycorrhizae (ECM) morphotypes.

Morphology (dissection microscope) Anatomy (compound microscope)

Ectomycorrhizal system: Emanating elements Outer mantle

Amphinema byssoides Unbranched systems, straight tips;cream to yellow, finely grainy,woolly

Mycelial strands common,smooth or hairy; hyphaecommon, cottony, whiteto yellow

A felt prosenchyma of hyaline cells100 mm � 3–4 mm, no matrix, smoothto finely verrucose, commonly septate,large ‘‘keyhole’’ clamps, H-shapedanastomoses

‘‘Basidiomycete 2’’ Not branched, tips straight to beaded;cream to yellow

Mycelial strands not observed;hyphae common, white tohyaline

A felt prosenchyma of hyaline cells, smooth,clamps common

‘‘Basidiomycete 3’’ Monopodial pinnate, tips straight;dark brown to black, swollen,hyaline to cream apex

Mycelial strands not observed;hyphae common, tortuous,white to hyaline

A felt prosenchyma of hyaline cells(localized areas staining pink in KOH)

Cenococcum geophilum Unbranched system, straight tips;black, coarsely grainy, reflectiveand shiny

Mycelial strands not observed;hyphae common, straight,thick and wiry

A net synenchyma of thick-walled cells20 mm � 4–5 mm wide, blackish red,forming a stellate pattern

Cortinarius spp. Monopodial pinnate systems, straighttips 600 mm wide; bright white topinkish, reflective, cottony tostringy

Mycelial strands common,white, resembling dentalfloss; hyphae white, cottony

A felt prosenchyma of hyaline cells 3–5 mmwide, cylindrical, smooth, clamped

Dermocybe spp. Unbranchedsystem, tips straight orslightly bent; white, woolly,reflective

Mycelial strands common,white, smooth; hyphaecommon, white

A felt or net prosenchyma of hyaline cells4–5 mm wide, smooth, rarely clamped

Hebeloma spp. Unbranched system, tips straight;cottony, white

Mycelial strands not observed;hyphae common, straight,white

A felt prosenchyma of hyaline cells 3–4 mmwide, verrucose ornamentation, clamped

‘‘cf. Laccaria’’ Unbranched to monopodial pinnatesystems, tips straight; creamy whiteto brownish, smooth to felty

Mycelial strands not observed;hyphae rare to common,white to hyaline

A net prosenchyma of smooth, hyaline cells2.5–4 mm wide, clamps rare to common

Lactarius spp. Monopodial pinnate to pyramidal,tips straight; creamy white tobrownish, smooth

Mycelial strands rare; hyphaerare

Regular or interlocking ‘‘jigsaw’’synenchyma, smooth

Phialocephala-like (MRA) Unbranched system, straight tips;black, coarsely grainy, reflectiveand shiny

Mycelial strands not observed;hyphae common, straight,thick and wiry

A net prosenchyma of thick-walled cells2 mm wide (restricted at septum), simpleseptate

Piloderma spp. Irregular systems, tips curved; whitewith some yellow patches, cottony

Mycelial strands common,white; hyphae common, white

A thick felt prosenchyma, cells 3–4 mmwide, verrucose and crystalline ornamen-tation, simple septate

Russula spp. Monopodial pinnate to pyramidalsystems, tips straight; creamy whiteto brownish, smooth (sometimesfuzzy because of cystidia)

Mycelial strands not observed;hyphae not observed

A net prosynchyma of non-interlocking,irregular, synenchyma (some formingrosettes that turn blue in sulphovanillin),smooth, hyaline

Thelephora americana Systems not branched; creamy whiteto brownish, shiny, smooth

Mycelial strands not observed;hyphae not observed

From a thin net synenchyma to anon-interlocking irregular synenchyma ofhyaline cells 4 mm wide, smooth, simpleseptate

Tomentella spp. Systems not branched, tips straight;dark brown to black, coarselygrainy, matte to reflective

Mycelial strands not observed;hyphae rare to abundant,brown, tortuous

From a regular to a non-interlocking irregularsynenchyma of brown to black hyphae4–5 mm wide, smooth

Tuber spp. Monopodial pyramidal systems, tipsstraight to bent; cream to yellow,smooth to felty

Mycelial strands not observed;hyphae not observed

From an irregular to an interlocking or non-interlocking synenchyma of thick-walledhyphae

Wilcoxina like (E-strain) Unbranched system, tips are straight;brown to reddish brown, smoothand glossy

Mycelial strands not observed;hyphae not observed

Thin; from a net synenchyma to a non-inter-locking synenchyma; hyphal cells 6–9 mmwide (restricted at septum), smooth, simpleseptate

Note: The procedure of Goodman et al. (1996a) was used to characterize the morphotypes. For a glossary of terms used in this section refer to Goodman

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Inner mantle Mycelial strands Emanating hyphae Other features

A net prosenchyma of hyaline cells6–15 mm � 3–7 mm, smooth, simpleseptate

Common, loose undifferentiated,smooth to finely verrucose, large‘‘keyhole’’ clamps, H-shapedanastomoses, bright yellow in KOH

Common, 3–4 mm wide, smooth,commonly septate, large ‘‘keyhole’’clamps, H-shaped anastomoses,bright yellow in KOH

None observed

None observed None observed Common, pale yellow to hyaline,smooth, clamps common

None observed

A net synenchyma of hyaline cells None observed Common, thin, tortuous, clamped,abundant globular ornamentation

None observed

Not observed None observed Common, 4–5 mm wide, straight- andthick-walled, rarely septate (simple)

Sclerotia: dark brown toblack, hard, spherical, upto 3 mm in diameter

A net synenchyma of hyaline cells 3–4 mm wide, cylindrical, smooth,rarely clamped

Common, smooth–undifferentiated,hyphal cells cylindrical, smooth,clamped

Common, 2–4 mm wide, smooth,cylindrical, commonly septate,clamped

None observed

A net synenchyma of hyaline cells 6–10 mm wide, smooth, simple septate

Common, loose undifferentiated,ornamentation crystalline, globular,or lacking

Common, 4 5 mm wide, ornamentationcrystalline, globular or lacking;simple septate

None observed

A net synenchyma of hyaline cells3 mm wide, smooth, no clamps

None observed Common, hyaline cells 3–4 mm wide,verrucose, clamped

None observed

A net synenchyma of smooth hyalinecells, simple septate

None observed Common, tortuous, hyaline, 2.5–4 mmwide, irregularly formed clampscommon

None observed

A net synenchyma, smooth, lacticifersabundant (turning blue in sulphova-nillin)

Rare, undifferentiated Rare, hyaline, 2–3.5 mm wide, simpleseptate

None observed

Not observed None observed Common, dark green to black,2–3 mm wide, thick-walled,curved, rarely septate (simple),finely verrucose

None observed

A net synenchyma, cells 3 mm wide,smooth, simple septate

Common, loose, undifferentiated Common, 3–4 mm wide, hyaline, ver-rucose and crystalline ornamenta-tion, simple septate, H-shapedanastomosis common

None observed

A net synenchyma (some with partiallyinterlocking epidermoidal cells),smooth, simple septate, hyaline

None observed None observed Cystidia rare to abundant,depending on species;ampule-shaped, 3.5–6.5 mm wide � 13–30 mm long

A net synenchyma of hyaline cells4 mm wide, smooth, simple septate

None observed None observed Cystidia; rare to common,awl-shaped with basalclamp, 4 mm wide �125 mm long

A net synenchyma 4 mm wide, smooth None observed Rare to abundant, 4 mm wide, brown,clamped, smooth, curly

Cystidia: rare to common;awl-shaped, 50–70 mmlong

An irregular non-interlocking synench-yma

None observed None observed Cystidia: abundant; long(often curved), hyaline,simple septate, bristle toawl-shaped

Not observed None observed Rare; 6–10 mm wide, verrucoseornamentation, simple septate

None observed

et al. (1996b).

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