Iactarius Ectomycorrhizae on Abies Alb Az Morphological Description, Molecular Characteization, and Taxonomic Remarks

Mycologia, 92(5), 2000, pp. 860-873. O 2000 by The Mycotogical Society ofAmerica, f,awrence, KS 6604+8897

Iactarius ectomycorrhizae on Abies alb az morphological description, molecular characteization, and taxonomic remarks

Ursula Eberhardt Kq Words: ectomycorrhizal fungi, ITS scquences, Franz Oberwinkler RT'LP Speziellc Botanik, Mykolngte, [Jniaersität Tübingen, Auf der Mmgenstelle I, D 72076 Tübingen, Germany

Annemieke Verbeken INTR.ODUCTION Labmatorium Plantkunde, Vahgroep Biobgie, Uniaersiteit Gent, Ledeganrhstraat 35, 8-9000 Cent, Though silver fir (Abies alba Miller) is an ecologically Belgium valuable and indigenous tree species in many Euro- pean Andrea C. Rinaldi mountain forests, verv little is known about its ectomycorrhizae (see Comandini et al i998, and ref- Dipartimento di Scienze Med,iche Internistiche, Uniuersitd, di Cagliari, I-09042 Monserrato Cagtiari, erences therein). In the present study, we describe holl the ectomycorrhizae of three Lactarius species on Ä. alba from central Italy, by standard Giovanni Pacioni morphological and anatomical parameters (Agerer Ornella Comandinir 1991): Lactarius subsericatus (Kühner & Romagn.) ex Bon, Lac. inter- Dip ar timento di Scienze Ambient ali, Llnia ersitd. rnedius (Ikombh") Berk. dell'Aquila, Via Vetoio Loc. Coppito, I-67100 & Broome , and Lac. salmom- L'Aquila, Italy icoLor R. Heim & Leclair. Mycorrhizae of the latter species were compared with an existing description (Pillukat 1996). Identification of the fungal symbi- .ll'as Abstract: To date, the ectomycorrhizae formed by onts achieved by means of sequence comparison silver fir (Abies alba), an ecologically valuable and in- between mycorrhizae and sporocarps of the expected digenous tree species in many European mountain lungal partner species, in addition to morphoana- forests, have been poorly investigated. We character- tomical analysis of mycorrhizae. ized the mycorrhizae formed by three Lactarius spe- Lactarius subsericatus (syn. Lac. ichoratus ss. Rom- cies {Lac. subsericatu,s, Lac. intermedius, Lac. salmon- agnesi) is a representative of the subgenus Russularic icolor) on silver fir, on the basis of material originat- (Fr.) Ihuffman sect. Russularia Fr. The species is ing from central Italy. The identification of the fun- closely related to tr ac. fuluissimus Romagn. from gal symbiont was achieved by means of rvhich it differs try the spore ornamentation com- morphoanatomical observations of mycorrhizae, and posed of mostll, isolated warts (more reticulate in by comparison of ITS sequences obtained fiom my- Lac. fuluissimtts).ln the field, Lac. subsericalzs is rec- corrhizae and sporocarps of putative fungal partners. ognized from the latter species by its more greasy Sequences also were obtained from specimens of the pileus surfäce, the milk which stains yeilow on white same species but from different geographic origin or iissues, the darker lamellae and an odor resembling from closely related Lactarius species. A maximum that of Lac. subdulcis (Pers.:Fr.) Gray (rubberJike, likelihood analysis of the data was performed. On the in Lac. fulaissimus much iike the odor of Lepiota cris- whole, the resultant tree is in good agreement with tata (Nb. & Schw. : Fr.) Kummer). For further differ- sporocarp and mycorrhiza morphology. RFLP pat- ences befween these hvo taxa see Schwötrel (1g79)" terns were calculated from sequence data. A discus- The insufficiently known species Lac. {trittanieusReid sion on the main morphoanatomical characters dis- also seems very closely related, if not consp ecrfic. Lac. tinguishing tlne Lactariu.r ectomvcorrhizae reported subsericatus is associated with a broad range of both in this study from those aiready described beionging conif'erous anci deciduous trees on calcareous soil in to related species, is also included. The accuracy of western Europe, but collections of broadleaved hosts diflerent methods to identify mycorrhizae formed by are

860 Egl.ngenor ET AL: Ll.cr,l,tltts EcI'()NIyc()RRHIZ.\F- ()N A. ,u,n.l 861

TenLL, I. Material used for DNA sequencine

Species Host Origin GenBank

I-ac. subsericatus 5"7 Abies alba Italy AF140254 Lac. subscticaf.z.r ECMr' A. alba Ita11, AFI 40255 I-a,c. intermedius S A. alha Ita11, AF1 40256 Lac. intermedlzs ECM A. alba Italy Arl 40257 Lac. salmonicolor Sl A. alba Ital1' 4F140258 Lac. salmonicolorLCM A. alba Iral,v 4F140259 Lac. suh,sericatus 32 Picea abies Germany AFl 40260 Lac. subsnirulus 33 Fagus slhatica Germanl, 4F140261 I-ac. cf. fulaissimus S broadleaves France Ar204678 Lac. Jülaissimus S broadlear.es Belgium AF204679 Lac. scrobiculatus 51. P ahies Germany AFl 40262 Lac. scrobiculatus 32 P abies Germany AFI.10263 Lac. .salmonicolor 32 A. albr.t Germany AFl ,10264 Lac. salmonicolor S3 P abies, E syluatica, Germany 4F140265 Pinus sllaestris (4. alba) Lac. deterrimus Sl P abies Germany AFt,+0266 Lac. deterrimus 32 P a,bie.s G'ermanv AFt 40267 I-ac. semisanguifluus S P sllttestris (11 abies) Germanv AFt 40268 Lac. quieticolor S P sll.uestis Germany 4F140269

" S : Sporocarp. " ECM : Ectomycorrhizae. ed to Lac. scrobiculatus from which it differs by the phoanatomical features, by ITS sequences, and by paler, azonate pileus, the scarcely pubescent margin ITS PCR-RFLP, dealing rvith these closely related spe- soon becoming smooth in mature specimens, the cies. larger spores which are ornamented with finer and less connected warts and ridges, and its exclusive as- \IATERIA]-S,{\D }IETIIODS sociation with Abies,whlle Lac. scrobiculatus is knorr,n Picea (Heilmann-Clausen 1998). to grow with et al S rLmp lin g an d c h ar a c I eriz a ti o n.- I -a ct ariu s e ctomy corrhizae Lactarius intnmedius is only known from calcareous and sporocarps were collected during the growing seasons soils within the natural distribution of its host rree in 1995-1998 from two silver fir stands in the Gran Sassolaga Europe. A detailed description is given by Marchand National Park, central Ital1,: "Fonte Gelata" (artificial stand, ( 1e80). planted in early 1950s; 1000 m asl) and "Colle Pelato" (nat- Lactarius salmonicolor [syn. Z. laeticolor (S. Imai) ural stand; 1100 m asl) (T,rnr-n I). Details of the nto study Imazekil belongs to the subgenus Piperites sect. Dap- areas, including mvcocoenological, phvtosociological and pedological aspects, are reported in Comandini (1997). etes Fr. It is distinguished from the other European Soil cores rvere taken fiom beneath the sporocarps. Sub- members of this group by its exclusive association sequentl)', the samples rvere carefull,v washed in water, and with Abies species and the uniform salmon to orange ectomvcorrhizal roots were separated under a dissecting mi- color without greenish or reddish tinges. For a de- croscope (Leica Wild N,I 10) for macroscopical characteriza- tailed account on this group, we refer to Heilmann- tion, as described by Agerer (1991). Particular care was tak- Clausen et al (1998). Lactarius salmonicolor seems en to isolate mycorrhizal roots of A. alba from those of rvidely distributed in the northern hemisphere and is other host trees in the sampling sites (mainl1,.Fa-grzs syluatica reported from western and central Europe (Heil- L.). Methods to characterize ectomycorrhizae have been mann-Clausen et al 1998), Turkey (Sesli and Baydar comprehensivelv explained b,v Agerer (1986, 1987-1998, 1995), Japan (e.g., Hongo 1960 as Lac. laeticolor), 1991), and a glossary of all terms used has been published Mexico (Heim 1953); and from Abies-plantations out- by the same author (Agerer 1987-1998). Munsell (1975) has been used as reference fcrr the side the natural distributional area. description of colors of ectomycorrhizae. As the three Lactanzs species considered in this Mantle preparations of fresh ectomycorrhizae rvere fixed study all possess closely related species that may occur on slides with polwinvl lactophenol for microscopic inves- in the same area, associated with the same or differ- tigations. For light microscop,v, observations were made with ent tree species, we lvanted to find out about the a Leitz Laborlux S microscope and photographs were taken levels of resolution of different methods for identi- with Ilford Panf Plus 50 films. Cross and lonsitudinal sec- $ring mycorrhizae, i.e., identification by their mor- tions (3-5 pm thick) were cut on a cryotome (Frigocut 862 MrcoLoclq

2800, Reichert-Jrrg). \bucher specimens

Ftc:s. 1, 2. LactaritLs sttbsericatus ectomvcorrhizae. 1. Pseudoparenchvmatous outer mantle laver with poly,gonal hyphal cells. 2. Plectenchl.rnatous inner mantle layernith branched lactifers r.isible through the middle layer. Bars : 10 pm. u.ith white-pale yellow latex. The inner manrle layer unramified ends are usually smooth, yellowish-brown sometimes lacks a distinctive hyphal patrern, but in (10 \R 5/8), slightly grainy in young specimens. Old- other places has a net-like arrangement of hyphal er mycorrhizae become dark yellowish brown. The bundles (Ftc;. 2). Hyphae of the inner layer are mantle secretes a yellonish milk when injured. Ema- branched, with numerous septa and lacking clamp nating hyphae \{ere not found. Rhizomorphs are connections. Anastomoses were not observed. In the quite rare, and generally connected lvith the mantle very tip, the outer part of the mantle is pseudopar- in restricted points; rhizomorphs mostly straight, enchymatous, with the same structure as in the sub- rarely ramified, and with smooth margins. The color apical region. Hiphal cells are densely packed and of rhizomorphs is as in the rest of the mycorrhiza. difficult to measure because of srnall dirnensions. The outer surface of the mantle is completely cov- The inner mantle layer of rhe very tip is plectenchy- ered by a uniform layer of pale yellor,v/brown crys- matous. Lactifers are scarce in the inner layer, rather talloid particles, generally of small dimensions. These abundant in the middle layer. resemble little scales in shape, but bigger ones, main- From longitudinal sections, a mantle 30-60 pm ly rhomboidal or elliptic in shape and measuring 3- thick, with 2-3 distinguishable layers can be ob- 4 pm long, can also be observed (Frc;. 3). Such crys- served. The outermost layer is pseudoparenchyma- talloid particles were present on fresh as well as on tous and formed by polygonal cells, lactifers always glutaraldehyde-preserved mycorrhizae. Because their absent. The rest of the mantle is plectenchymatous. presence prevented microscopical obsertations, we Most of the lactifers are present in the middle layer, attempted to dissolve them by soaking mycorrhizae whereas scarcely observed in the inner one. The very in various solvents. Of these, H,SO., 50% in water (v/ tips show the same structure as the sr.rbapical region, v) for 30 s or more, proved to be most efficient. After- but hyphal cells are more densely packed. The Hartig wards the mycorrhizae were rinsed thoroughly with net surrounds 2-3 rows of cortical cells, forming pal- water. metti-like lobes of 2-7 p"m diam in plan view, and In treated mycorrhizae, observations were as fol- fbrming 1 (2) rows of irregular cells, 2-5 pm thick, lows. The outer mantle has a pseudoparenchymatous in sections. structure with quadrangular or more elongated hy- Lactarius intermedius mycorrhizae are simple or phal cells, oriented in a parallel way in some parts monopodial-pinnate. Mycorrhizal systems range from (Ftc. 4). Hyphal cells are not densely packed, cell 4.4 to 5.8 mm long and from 0.6 to 0.7 mm diam. walls inconspicuous. Lactifers are absent. The middle Unramified ends are senerally bent, more infre- layer is plectenchymatous, hyphae are mixed lvith a quently straight or torruous, from 1.5 to 2.5-3 mm coarse net of straight or branched lactifers, with a Iong and from 0.4 to 0.5 mm diam. The surfaces of yellowish content and inconspicuous cell walls (Frc;. 864 Mr"r]OLOC;IA

Lactarius salmonicolor ectomycorrhizae have already been fully described by Pillukat (1996) on the basis of samples originating from German,v. The outer mantle is plectenchymatous, hyphal cells are rather straight and form characteristic short, often bent protrusions. The inner layer of the sheath is plecten- chpnatous. The interested reader is directed to Pil- lukat's paper for more details (Pillukat 1996). Com- paring the published features with those of the samples we have collected and studied in Italy, it generally appears that no significant differences exist either in the morphological characters of the ectomycorrhizae or in the anatomical structures of the different mantle layers. Only minor details were ob- Ftc;. 3. SEN{ photomicrograph of crystal-like particles on served differentiating our material from the German the surface of untreated Lac. intermedlzs ectomvcorrhizae. samples. In our samples, lactifers, located in the mid- Bar : 5 pm. dle or inner layers, were of more homogeneous di- ameter ranging from 5 to 9 pm fversus 4.5-7 (13) 5). The inner mantle layer is plectenchymatous with pm], and lactifers' cell lvalls were rather thin and not parallel or irregularly arranged hyphae. Anastomoses up to 1.8 pm thick as in the German samples. More- are sometimes present. Hyphae are frequently sep- over, the interhyphal matrix material reported in the tate with inconspicuous cell walls, clamp connections outer and in the inner layers of the mantle for Cer- never observed (Frc. 5). At the very tip, the outer man samples has never been obserr,ed in Italian ec- surface of the mantle is pseudoparenchvmator.rs u'ith tomycorrhizae. The mantle thickness differs conspic- the same structure as in the subapical region. H,vphal uously between Italian and German material, ranging cells are densely packed and difficult to measure as from 30 to 40 pm in the Italian ectomycorrhizae in the inner part of the mantle. Lactifers are verv (Frc;. 7), versus 40-70 p"m for the German samples abundant, especially in the middle layer, shorving the in the subapical region, and from 45 to 50 pm versus same features and dimensions as in the subapical re- 80 pm at the extreme tip. gion. Rhizomorphs are undifferentiated with rather smooth margins, from 30 to 80 pm diam, and densely Dl,{A ana$sls.-For all of the three species, ITS se- covered by crystalloid particles. Within the rhizo- quences of a sporocarp and of mycorrhizae from un- morphs, h,vphae are thin walled, uniform in diam (3- derneath the sporocarp could be obtained (T'Ler.r. I). 4 pm), mostly compactly arranged but in some plac- The length of the sequences from sporocarps and es only loosely woven (Ftc;. 6). Hyphae are often their putative mycorrhizae that were compared'w'ere filled with homogeneous light yellorv content (like 738 bp for Lac. subsericatus, T3l bp for I'ac. salmon- lactifers). In the distal part of the rhizomorphs, abun- icolor, and 675 bp for Lac. intermedius. The less con- dantly connected and agglutinated hyphae can some- served spacer regions ITS1 and ITS2 are completely times be observed (Ftt;. 6). Clamp connections are included in the compared sequences, apart from the absent. ITS2 of Lac. intermedius, dne last 5 bp of which are From longitudinal sections, a mantle 30-50 pm missing from the sequence of the mycorrhizae. No thick can be observed, its surface overlaid by a 5 pm sequence differences could be detected within any of thick dense layer of crystalloid particles. The outer the species, thus confirming that the mycorrhizae de- mantle layer is composed of tangentially elongated scribed above were formed by the respective funsal hlphal cells. More elongated hyphal cells, mixed with species. lactifers, form the middle layer of the mantle. In the Sequence simiktrities. ITS sequences of two sporo- inner part of the mantle more elongated and subgl- carps of Lac. subsericatus and Lac. salmonicolor from obose hyphal cells both are present. The very tip has Germany were obtained and compared to the se- the same structure as the subapical region, but hy' quences of the respective species from Italy and show phal cells are more densely packed. Lactifers are little or no differences (Taems I, II). mostly present in the middle part of the mantle. Gen- Lacta'rius fulaissimus belongs to the same, yet un- erally three rows of cortical cells are surrounded by resolved, species complex as Lac. subsericatus. Two the Hartig net, which extends to a depth of 80-100 sporocarps, morphologically unambiguously assigned pm, forming palmetti-like lobes measuring 3-4 pm to Lac. .fuluissimus, were considered (T,lnt.u I). The in width. ITS sequence of one isolate (named Lac. cf . fulaissi- EspRH,{RDr ET At.: Llc:l',tp.tus ECToMycoRRHIZ.\E oN A. s.rnl, 865

Ftcs. 4-6. Treated Lactarius intermedius ectomycorrhizae. 4. Pseudoparenchymatous outer mantle layer with quadrangular or more elongated hlphal cells. 5. Plectenchymatous inner mantle layer with parallel or irregularly arranged hyphae and rvith branched lactifers visible through the middle layer. 6. Undifferentiated rhizomorphs with isodiametric hyphae, compactly arransed or rather loosely woven, often filled with homogeneous content. Abundantly connected and agelutinated hyphae in the distal part of the rhizomorphs. Bars : 10 pm.

mus in Tenlr,s I, II) was identical to that of the Lac. pine, is available from GenBank as U80999. The ITS subsericatus isolates, the other sequence differed in sequences of two sporocarps of Lac. deterrimus, from ten single base mutations (T,rer.n II). This is more different locations in Germany (ca 300 km apart) dif- than the variation found among Lactarius isolates un- fered only slightly (Teer.r II). Lactarius salmonicolot; ambiguously assigned to one species. The interspe- Lac. deterrimzs and Lac. semisanguifluus, each asso- cific variation among the isolates of other species test- ciated rvith a different host tree, are the most similar ed is higher. Thus, the results do not provide clear in their ITS sequences. The differences among the indications as to the taxonomic status of the two spe- pine-associated species lac. semisanguifluus, Lac. cies. quieticolor and Lac. deliciosus are more pronounced. The ITS of three other species of the section Dap- Multiple inserrions or deletions (up to 20 bp) occur etes were sequenced, Lac. deterrimut associated with in several positions of the alignment (T,rar.r II). spruce, as well as Lac. quieticolor and Lac. semisan- Lactarius scrobiculatus is a close relative of Lac. in- guiJluus associated with pine (Teer-n i). In addition, termedius, and is associated with spruce (Heilmann- the ITS sequence of Lac. deliciosus, associated with Clausen et al 1998). Two sporocarps of Lac. scrobi- 866 Mvcor.oc;Ie

Frcs. 7, B. Anatomical characters of Lactarius ectomycorrhizae. 7. Cross section of Lac. salmonicolorectomycorrhizawith a rather thin mantle. X400. 8. Pseudoparenchymatous outer mantle laver of Lac. subsericttus ectomycorrhiza. Noteworthy, the surface of the mantle lacks an hyphal reticulum. X1000.

culatus, collected ca 40 km apart in southwestern Variation occurs almost exclusively in the spacer Germany (Tenr.t I), showed again little difference. regions, coding regions are mostly identical for all The difference between Lac. intermedius and Lac. sequences. Namely the 5.8S rRNA sequence does not scrobicukr.tus sequences amounted to about 2% (TA- vary betlveen tlne Lactanzs species considered. BLE II). Taking into account the host specificity of Maximum likel)hood analysis. The aim of this anal- these species and the intraspecific homogeneity en- ysis is to show horv well morphologically based taxa countered to date, tlne 2% interspecific variation are confirmed by molecular data. No outgroup spe- should be sufficient (a) to add corroborative evi- cies rvas included, because the data set is too limited dence that Lac. scrobiculatus and Lac. intermedius ate to address phylogenetic questions. The alignment is two separate species and (b) to make an identifica- available from TTeeBASE (5479, M702). Maximum tion of the mycorrhizae of each species possible. likelihood analysis resulted in one optimal tree,

Tetlr-n II. Differences between isolates of the same or related species

Isolates compared Diff'erences Differences (7o)

Ln.c. .urhsericatus S1_S2 0 bp of 719 bp 0.0 S1-S3 2 bp' of 745 bp 0.3 Lac. subsericatus Sl-Lac. cf . fuhissimus 0 bp of 686 bp 0.0 7.1 La.c. Jülaissimus- Lac cf . fulaissimus 10 bp of 705 bp Lac. salmonicolor S1_S2 0 bp of 747 bp 0.0 S1_S2 0 bp of 722 hp 0.0 Lttc. detnrimus S1-S2 4 bp of 694 bp 0.6 section D1.ltetes alignment 668 bp Lac. deliciosu.s-Lac. d,eterrimus SZ 41 bp 6.1 Lac. rlekciosus-Lac. quielicolor 52 bp 7.8 Lac. deliciosus- Lac. salmonicolor 40 bp 6.0 La,c. deliciosus- Lac. semisanguiJlutts 45 bp 6.7 Lac. deterrimus S2-Lac. quieticolor 5l bp 7.6 Lac. detnrimus SZ-Lal. salmonicolor 25 bp Lac. deterrimus 32- Lac. semisanguifluus 25 bp .1. I I.ac. quieticolor- Lac. salmonicolor 53 bp 7.9 Lac. quieticolor- Lac. semisanguiJluus 60 bp 9.0 Lac. salmonicolor- Lac. semisanguiJluus 24 bp 3.6

Lac. s crobicu,larus S1-S2 1 bp of 736 bp 0.1

Lac. intnmedius- Lac. s crobiculatus 32 16 bp of 764bp 2.7

' Ambiguous sequence readings EgnRILARDT ET Ar: Ltc:r,trul ECTo\{ycoRRHIZ\E oN A. tt,r.t 867

Lac. dd. site (T,l.nr,r III). In spite of the small sequence vari- Lac- qui. ation between the Lac. deterrimus tsolates, differences are sufficient to produce a different banding pattern Lac. det. 32 Lac. sal. Sl Lac. sal. ECM with C/oI. The differences in RFLP patterns of eight tac. dd. S1 Lrc. sal. 52 enzymes with 22 cleavage sites bet$,een one of the Lac. sern. Lac. sal. S3 Lac. deterrimzs isolates and Lac. semisanguiJluus are slight (T,ret-l III). Two fragments in the RFLP of Lac. int. S1 tac. int. ECM IIinfl differ in about 20 bp from the respective fragments of the other species. The restriction patterns Lrc. scr. 51 of Lac. intermedius and Lac. scrobiculatus vary in rwo Lac. scr. 52 restriction sites of MöoI (T,utlr III), one of which can be expected to produce a distinctive banding pattern. Among the fungal partners of pine, Lac. deliciosus and Lac. semisanguiJluus can only be distin- Lac. sub. Sl guished by three restriction sites of two different en- Lac. sub. ECM zymes, six enzymes producing similar patterns. Zac- Lac. sub. 52- tarius quieticolor is set apart by its ITS Lac. cf. ful. J the length of Lac. sub. 53 Lac. fut. 0.01 substitutions/site PCR product. In addition, indels change the position of restriction sites such that the resulting fragments FIc. 9. Maximum likelihood- analysis with bootstrap vary notably in size. Lactarius intermedius and Lac. analysis (100 replicates). Lac. Lactarius; del. deliciosus; det. salmonicolor; associates of fir, can be expected to pro- deterrimus; It;J.. int. intermedlz.s; qui. quieticolo4 .fulaissimus; duce distinctly restriction patterns three sal. salmonicoloq scr. scrobicukttus; sem. semisanguiJluus, S different for sporocarp (for numbering see T,tet-t I); ECM ectomycor- of the enzymes. More enzymes distinguish the part- rhizae. Isolates not specified are from sporocarps. ners of spruce, Lac. detnrimus and Lac. scrobiculatus (Tltlr-r III). shown in Ftc. 9. Branch lengths within subgenera are DISC]USSION shorter than the branch separating isolates of the subgenera Pipnites and Russularia. Moreover, this The mycorrhizae of Lac. subsericalzs prer.iously have branch is confirmed by a bootstrap value of 100%. not been described, though descriptions of a number The section Dapetes is backed by a bootstrap value of of different ectomycorrhizae of Lactarlus species sect. 70%, Lac. intermedius and Lac. scrobiculatus of the Russularia are available. Their features are discussed section Zonarii form a cluster with 100% bootstrap by Agerer (1995) and Palfner and Agerer (1996). A1- support. Branches housing conspecific isolates are al- though the host trees associated with these different ways backed by bootstrap values of at least 95% and mycorrhizal types belong to several genera both of mostly of close to zero branch lengths. This implies conifers and broadleaves, several comrnon features that isolates from mycorrhizae always cluster with the within this group can be pointed out, such as the isolates of sporocarps of the same species. As expect- orange-brown color of the ectomycorrhizae, the lack ed from sequence similariry the sequence of Lac. cf. of macroscopically visible lactifers on the surface, and fulaissimus clusters with the isolates of Lac. subseri- a pseudoparenchymatous structure of the outer man- catus. tle layer which is generally covered by thin hyphae Restriction fragment pa,tterns. Not all species can be arranged in a netlike to ramified star-like array. The expected to be separable based on the length of their pseudoparenchymatous structure of the outer mantle ITS PCR products (Taelu III). Putative restriction layer is generally formed by angular cells. Rhizo- fragment lengths of eight endonucleases are given in morphs are quite rare, with differentiated or undif- Tesr.s IIL Closely related species have many restric- ferentiated structures (Palfner and Agerer 1996). tion sites in common, so differentiation will be de- Cystidia have been observed only in few cases, e.g., pendent on the realization of the few predicted dis- in Lac. mitissimus (Fr.:Fr.) Fr. (Weiss 1991) and in tinguishing restriction sites. Lac. rubrocinctusFr. (Brand 1991). In Lac. subdulcis Lactarius subsericatus and Lac. fulruissimus are least and Lac. rubrocinctus ectomycorrhizae, intracellular closely related to the other species, producing restric- infections caused by a second fungus, Leucosqpha tion patterns distinct from those of the species of the leucotricha (4. & S. ex Fr.) Bound (Pezizales) have subgenus Piperites. The divergent isolate of Lac. ful- been observed (Brand 1991, Brand and Agerer uissimus differs from the other Lac. cf. fulruissimus 1986). Most of the common features of the ectomy- isolate and from Lac. subsericatus bv one restriction corrhizae of Lactarius sect. Russularia also can be ob- 868 Mvcor.ocr,r

Tanln III. Presumed restriction fragment lengths of the ITSI,zITS4 PCR products calculated from sequence data

Species' Alul Cfol .EcoRI Haelll llinfl Mbol Mspl Thql

Lac. subsericatus 487 368 406 458 389 263 378 278 qq, 4F140254 161 322 245 263 206 263 271 80 733 25 68 131 87 74 8 106 59 ,9 31 12 Lac. fuluissimus 487 368 406 .158 389 263 611 278 Ar204679 161 cr91 322 245 263 206 87 274 80 133 25 68 131 74 8 106 59 22 31 12 Lac. detctrimus 505 Jta 403 47r 306 269 639 197 AF140266 232 267 334 266 173 197 98 168 98 170 143 119 80 97 117 tt 22 72 I 59 5 Lac. detnrimus 503 267 403 469 306 269 639 197 AFt40267 232 215 332 266 173 797 96 166 155 i68 147 119 98 80 97 177 8 22 72 9 59 5 Lac. semisanguifluus 500 J05 403 466 288 269 615 197

4F 1 40268 lJZ 273 329 266 777 197 87 159 94 I6l 734 127 98 101 119 I 22 72 I 59 5 La.c. delic)osus 501 358 396 475 347 261 635 261 U80999 233 270 338 259 298 198 99 169 106 81 144 125 8 100 120 99 59 I Lac. quieticolor 486 358 396 459 331 262 621 278 4F140269 232 206 322 259 213 9?7 91 262 154 86 197 /.) 80 99 59 8 46 Lac. salmonicolor 504 365 403 470 386 269 642 289 AF140258 232 270 .).).) 266 312 197 94 797 5l 8 139 191 50 100 59

9 Lac. intnmedi,us 506 3t5 410 173 393 267 572 293 AFl 40256 24t 372 3.) I 274 346 206 t75 195 8 149 777 103 B3 22 59 Esl:rulmo-r ET r[: IÄa]zl.lruLs EcroN,ryuoRRHrzAE oN Ä. ,lr.R..r 869

T,qsr-s IIL Continued

Species" Alul Cfol EcoRI HacllI Hinfr Mhol Mspl ToqI I-ac. scrobiculatus 505 374 409 472 392 267 571 292 4F740262 240 371 J.ro ZIJ 345 146 774 r95 tt 139 116 103 83 59 59 99 9 " The sequences employed are identilied by their GenBank accession numbers.

served in Lac. subsericatus mycorrhizae on A. alba, to the endodermis while in Lac. intermedius/fir it with the exception of the hyphal reticulum over the generally surrounds only 3 rows of cortical cells). The pseudoparenchymatous outer mantle layer, which outer mantle layer of Lac. intermedius mycorrhizae, has never been found in our Lac. subsericatus sam- though essentially similar in structure (apart from ples (ftc. 8). Rhizomorphs were never observed in some differences concerning dimensions of hyphal our samples. cells), does not possess fusiform cystidia as described The mycorrhizae of Lac. intnmedius are described byAmiet and Egli (1991) for Lac. scrobiculatus.The here for the first time. Lactarius scrobiculatus ecto- intracellular infection of an unidentified fungus re- mycorrhizae, associated with P abies and Tsuga het- ported byAmiet and Egli (1991) in Lac. scrobiculatus nophylla (Raf.) Sarg., have been comprehensively de- ectomycorrhizae, has never been observed in our scribed byAmiet and Egli (1991) and Kernaghan and samples, nor the lacunae present in the outer mantle Berch (1997), respectively. From a comparison of the layers of Lac. scrobiculatus as described by Kernaghan ectomycorrhizal q,pes formed by Lac. scrobiculatus and Berch (1997). and Lac. intermedius, only few differences can be not- A comprehensive discussion of the main diagnostic ed. The presence of a dense layer of crystalloid par- characters of the described Lactarius ectomycorrhi- ticles on the surface of Lac. intermedius mycorrhizae zae belonging to sect. Dapetes, was carried out by pil- is particularly intriguing. It is a consistent feature of lukat (1996). Subsequenrly, the description of the all mycorrhizae so far examined. This layer was never mycorrhizae formed by Lac. rubrilacteus Hesler & A. observed on Lac. scrobiculatus mycorrhizae (Amiet H. Sm. on Pseudotsuga menziesll (Mirb.) Franco ap- and Egli 1991, Kernaghan and Berch 1997, Egli, Ker- peared (Eberhart and Luoma 1997), as well as a short naghan pers comm). A closer inspection of these par- account of Lac. d,eliciosus var. aerolatus Smith mycor- ticles through a SEM microscopy appararus equipped rhizae on Abies lasiocarpa (Hook.) Nutt. (Kernaghan with an EDS X-ray microanalysis instrument, revealed et al 1997). These mycorrhizae also are rypical for a general appearance of densely packed scales and the section Dapetes in their plectenchymatous mantle an elemental composition rich in Si and O (data not structures and the presence of lactifers concolorous shou,n). Thus, it is possible that these crystalloid par- with the milk in sheath and rhizomorphs. Unlike oth- ticles are indeed soil elements that stick tenaciously er descriptions of mycorrhizae of this section, neither to the surface of the mantle, since they are not re- agglutination of mantle hyphae nor the presence of moved by rinsing the mycorrhiza with water nor by matrix material was observed in our samples. Since brushing. Further work is under way ro shed light on matrix material was described from the mantles of this point. Interestingly, crystalloid particles have re- Lac. salmonicolor mycorrhizae by Pillukat (1996) but cently been observed on the ectomycorrhiza formed was not observed in our specimens, the presence of by another Lactarius species, namely Lac. controuersus matrix material, or at least its presence in distinguish- Pers., on Populus (Jakucs albaL. et al 2000) . able quantities, may not be a constant feature even Most of the other features distinguishin g Lac. in- within the same species. termed,ius and Lac. scrobiculatus mycorrhizae are A comparison of our Italian samples witin Lac. sal- largely due to the differenr host species (Pillukat and monicolor mycorrhizae described from Germany (pil- Agerer 1992), such as the shape of mycorrhizal sys- lukat 1996) revealed that mantle thickness is another tems (monopodial irregularly pinnate to coralloid in distinguishing anatomical feature. Indeed, as report- Lac. scrobiculatus/ spruce mycorrhizae, generally sim- ed above, Italian samples possess mantles only about ple or monopodial-pinnate in Lac. intermed,ius/fir) half as thick as those measured by Pillukat (1996), and the depth of Hartig net (in Lac. scrobiculatus/ both in the subapical region and in the very tip. Of spruce mycorrhizae it surrounds all cortical cells up the numerous samples of Lac. salmonicolormycorrhi- 870 Mvcot.oc;r.c. zae collected in a period spanning four years and in spective taxa, regardless of the taxonomic level, spe- various localities in Italy, all showed a comparable cies, section, or subgenus. Moreover, species known thickness of the mantle. Considerable variation in the or presumed to form morphologically similar mycor- mantle thickness within a single mycorrhizal type rhizae appear in the same clusters. The only excep- (i.e., same mycobiont species on the same host tree tion to the agreement of sporocarp morphology and species), has already been reported in the literature, sequence similarity is Lac. cf. fulaissimus. and differences in mantle thickness have also been Owing to the morphological differences between observed when comparing natural with synthesized Lac. fulaissimus and Lac. subserica,tus, we prefer to mycorrhizae (Agerer and Weiss 1989, Egli et al 1993, treat them as separate species. Others, i.e. Heilmann- Miller et al 1991, Treu 1990, Weiss 1991). However, Clausen et al (1998), do not consider them distinct available data do not allow drawing definitive conclu- species. The molecular verdict is ambiguous. The se- sions as to the influence of specific parameters or quence identity of Lac. cf. ful.uissimzs (morphologi- conditions (environmental factors, growth medium cally unambiguously belonging to Lac. fuluissimus) composition, developmental stage of the mycorrhi- and the Lctc. subsericalzs isolates argues for merging zae, etc.) on the change of mantle thickness. Given both species, following the general opinion that ITS the important functional role played by the fungal sequences are species specific (Baura et al 1992, mantle in the mycorrhiza nutrient input and storage Chen et al 1992, Gardes and Bruns 1993, Gardes et processes, it is conceivable that a marked alteration al 1991, Lee and Taylor 1992, etc.). Nevertheless, lev- in the mantle thickness under different growth con- els of variation lvithin species and among closely re- ditions and habitats might prove to have an adaptive lated species differ considerably besveen different significance. taxa. Compared to the intraspecific variation of ITS The ITS of the nuclear ribosomal genes has be- sequences detected in other ectomycorrhizal fungi come one of the most widely used genomic regions (Anderson et al 1998, Baura et al 1992, Eberhardt et (e.g. for the identification of ectomycorrhizae Erland al 1999, Mankel et al 1998, Shinohara et al 1999), 1996-1998, al et al 1994, Goodman et al .|onsson et the intraspecific variability of the ITS in tl:.e Lactarius 1999, I(är6n et al 1997, Kraigher et al 1995, Pritsch species studied here, is rather low, as is the interspe- most these studies, identification et al 1997). In of cific variation bet\\reen most species of the same sub- by restriction patterns (RFLP) of the was obtained genus. Even an intraspecific variation of 1.47o, the ITS PCR product with a given set of endonucleases. level of variation betrveen La.c. and Lac. For Lac. subsericatus, Lac. intermedius, and Lac. sal- fuhissimus cf. or Lac. subsnicatus, can be considered monicolor we showed that the entire ITS sequences fulaissimus low. On the other hand, Lac. scrobiculatus and Lac. r,vere identical for conspecific mycorrhizae and spo- intermedius are only separated by a few additional sin- rocarps. \A4rile sequence identity between isolates of gle base mlrtations. So levels of inter- and intraspe- the same origin might be expected, we could also cific variability of the ITS may not alrvays be separable show that isolates of Lac. subsericatus and Lac. sal- in Lactarius. As a consequence, species-specificity of monicolor from Germany and ltaly, or Lac. subserica- ITS sequences cannot be taken for granted in this tus and Lac. deterrimzs from distinct locations in Ger- genus. The molecular evidence is not considered suf- many, revealed the same or almost identical sequenc- ficient to der''iate from the view that Lac. subsericatus es. Moreover, even association with different tree spe- ard Lac. are fivo morphologically distinct cies, such as fir, spruce and beech, did not lead to ful.aissimus species. observed sequence variation between divergent ITS sequences for Lac. subsericatus. The Lac. isolates may reflect a high Molecular phylogenetic studies (Bruns et al 1998, tl:e fuluissimuslike Moncalvo et al 2000) result in a common terminal level of intraspecific variability within this species, cluster of Russula and l-actarizs species, thus con- possibly due to onll recent speciation. firming the common origin of the agaricoid Rttssu- Given the widespread application of ITS PCR-RFLP laceae. In recent molecular studies on the phylogeny to the identification of ectomycorrhizae in commu- of the Russulales (Miller et al unpubl, Henkel et al nity studies, fragment lengths were calculated from unpubl), the rnonophylly of the genus Lactarius (in- sequence data. Our putative RFLP results show that cluding species of the same sections or subgenera for identi§ring Lactarius ectomycorrhrzae, the reso- treated in this study) is questioned. Irrespective of lution of ITS PCR-RFLP is not great enough to en- the supposed paraphyly of Lactarius, the result of the sure identification of species of the same section or maximum likelihood analysis presented in this study even subgenus when applying a standard set of three confirms morphological data. Similarity of sporocarp to five endonucleases. Until the presence or absence morphology is reflected by terminal clusters, boot- of certain restriction sites will be known to be of di- strap support, and branch lengths separating the re- agnostic value for the identification of certain spe- Esuxn.tRol nr lr-: I-,tcz,u?/tÄ F.croN{\c()RRHrzAF. ()N Ä. n/.8.4 871

cies, additional means of identification of mycorrhi- morphological analysis. The nrork of U. Eberhardt was sup- zae should not be disregarded. ported by a DFG PhD-scholarship of the Graduiertenkolleg The enzymes arbitrarily selected have been previ- Interaktionen in \Alaldökoq,stemen, Universität Tübingen. ously applied to the identification of ectomycorrhizae by Horton and Bruns (1998), Kär6n et al (1997), and Pritsch et al (1997). In praxi, occasionally additional I-ITER{I'U RI] C]ITI',D (faint) bands may occur in RFLP patterns resulting Agerer R. 1986. Studies on Ectomycorrhizas II. Introducine from incomplete restriction or single base mutations remarks on characterization and identifrcation. Myco- in a minority of the PCR products. The fragment size taxon 26:473-492. of these bands can again be explained by sequence ed. 1987-1998. Colour atlas of ectomvcorrhizas. data, omitting single restriction sites. On the whole, 1 st-l 1 th del. Schu,äbisch Gmünd: Einhorn-\'er1ae. restriction patterns and fragment lengths calculated 1991. Characterization of ectomvcorrhiza. In: Nor- sequence agree (Eberhardt from data rvell unpubl, ris JR, Read D.|, Varma AK, eds. Techniques for the Gandeboeufet al 1997). study of mycorrhiza. Meth Microbbl 23:25-73. Mycorrhizal morphology and anatomy can be em- 1995. Anatomical characteristics of identified ecto- ployed to distinguish mycorrhizae formed by Lactar- mycorrhizas: an attempt towards a natural classifica- lz.r species of the different sections from one anoth- tion. In: Varma A, Hock B, eds. Mycorrhiza: stmcture, er. Within sections, morphological differences tend function, m<;lecular biology and biotechnologl'. Berlin to be slight, though judging from the above compar- Heidelberg: Springer-Verlag. p 685-734. isons, the resolution provided by morphological char- \,!'eiss M. 1989. Studies on ectomycorrhizae. XX. acters is sometimes greater than that of RFLP analy- Mycorrhizae fcrrmed by Thelephora terrestris on Nonvay sis. The presence of rhizomorphs and cystidia in my- spruce. Mycologia 81 :444-453. corrhizae of Lactarius species is variable, and there- Amiet R, Egli S. 1991. Die Ektomykorrhiza des Grubigen fore the mere absence of such structures is not Milchlings (Lactarius scrobiculatus (Scop.: Fr.) Fr.) an sufficient for differentiating between ml,corrhizal Fichte (Picea abies Karst). Schweiz Z Forstwes 112:.53- 60. rypes. Additional characters must be taken into ac- Anderson IC, Chambers SM, Cairney-f\4''G. 1998. Molecular count. Considering the host specificity of Lac. deter- determination of genetic variation in Pisolithus isolates rimus and Lac. semisanguifluus, root morphology from a defined region in New South \{ales, Australia. might help to separate mycorrhizae, while mycorrhi- New Ph,vtol 138:151-162. zal anatomy can be expected to be fairly similar. The Baura G, Szaro TM, Bruns TD. 1992. Gastrosuilhts laricinus mycorrhizae of Lac. semisanguifluus and Lac. quieti- is a recent derivative of Suillus gratillei: molecular eti- color have not been described so far, but nith the dence. Mvcologia 84:592-597. exception of Lac. paradoxus Beardslee (Dan- & Burl. Bon NI. 1980. C16 monographique du genre Lacta,rius (Pers. ielson 1984) mycorrhizal anatomy is fairly, uniform ex Fr.) S.F. Gray. Doc Mycol 10:1-85. within the section (see Pillukat 1996). For distin- Brand F. 1991. Ektom,vkorrhizen an l-agus syluatica. Char- guishing the mycorrhizae of Lac. semisanguifluus, akterisierung und Identifizierung, ökologische Kenn- Lac. quieticolor and Lac. delicioszs, all associated with zeichnuns und unsterile Kultir.ierung. Libri Bot 2:1- pine, environmental conditions may give important 228. hints towards mycorrhizal identiqr. While Lac. delicio- Agerer R. 1986. Studien an Ektomykorrhizen WII. sus t)?ically occurs on neutral, calcareous, often Die Mykorrhizen von Laclarius subdulcis, Lactarius ael- sandy soils with P slfuestris and Juniperus communis kreustnd Laccrtrict, ameth\stina an Buche. Z Mykol 52: L., Lac. semisanguiflzzs is found in grass rich forests 287-320. on calcareous soils, and Lac. quieticolor prefers acid, Bruns TD, Szaro TM, Gardes M, Cullings K\4', Pan.lf, Taylor often sandy soils (Marchand 1980, Michael et al 1983, DL, Horton TR, Iketzer A, Garbelotto M, Li Y 1998. Heilmann-Clausen et al 1998). Site-by-site compari- A sequence database for the identification of ectom1,. sons of sequences of species of the subgenus Piperites corrhizal basidiomycetes by phylogenetic anal,vsis. Mol lead to the impression that adaptation to different Ecol 7:257-272. hosts in Lac. scrobiculatus and Lac. intermedius and Chen \Ar, HoyJW, Schneider RW. 1992. 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