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Home , Austroboletus, Boletaceae, Boletus, Boletus aereus, Boletus reticulatus, Chalciporus

Feddes Repertorium 113 (2002) 1–2, 30–40 Berlin, April 2002

Universität Regensburg, Fakultät für Biologie und Vorklinische Medizin, Botanisches Institut, Regensburg Clark University, Department of Biology, Sackler Science Building, Worcester, Massachusetts

M. BINDER & A. BRESINSKY

Retiboletus, a new for a -complex in the producing retipolides1

With 3 Figures and one Table

Summary Zusammenfassung ornatipes and B. retipes are two closely , eine neue Gattung für einen Arten- related species in , which cannot be Komplex mit Retipoliden innerhalb der Bole- differentiated by morphological and anatomical taceae characters. The 25S rDNA was partly sequenced from several isolates of B. ornatipes, B. retipes, and Boletus ornatipes und B. retipes sind zwei nahe allied species in the Boletaceae to aid identification. verwandte Arten in Nordamerika, die durch - The phylogenetic analysis using maximum likeli- logische und anatomische Merkmale nicht unter- hood suggests that both species are differently dis- schieden werden können. Ein Teil der 25S rDNA tributed. In addition, our results are supported by a mehrerer Isolate von B. ornatipes, B. retipes und previous study of HELLWIG (1999), separating three verwandten Arten wurde sequenziert, um die Identifi- groups in the B. retipes/ornatipes-complex based on zierung zu unterstützen. Die phylogenetische Analyse chemotaxonomical characters. Retipolides the unter Verwendung von Maximum Likelihood weist major pigments in presently analyzed B. ornatipes auf eine unterschiedliche geographische Verbreitung and B. retipes collections and are also detected in beider Arten hin. Zusätzlich werden unsere Ergebnis- B. flavoniger. This group of secondary metabolites se durch eine vorhergehende Studie von HELLWIG is unique in the (for distribution of (1999) unterstützt, in der chemotaxonomische Merk- pigments in Boletales see GILL & STEGLICH 1987) male zur Abgrenzung dreier Gruppen im B. retipes/ and is characteristic of the new genus Retiboletus, ornatipes-Komplex verwendet werden. Retipolide in which B. ornatipes (), B. retipes, and B. fla- bilden die Hauptpigmente in bisher untersuchten voniger are placed. A positive proof of retipolides is Aufsammlungen von B. ornatipes und B. retipes und still missing in B. griseus and nigerrimus. können darüber hinaus in B. flavoniger nachgewie- Both species are transferred to Retiboletus according sen werden. Diese Gruppe von Sekundärmetaboliten to morphological characters and sequence data. ist einzigartig innerhalb der Boletales (zur Verbrei- tung von Pigmenten in Boletales siehe GILL & STEGLICH 1987) und führt zur Etablierung der neuen Gattung Retiboletus, in die B. ornatipes (Typus), B. retipes und B. flavoniger gestellt werden. Ein positiver Nachweis auf Retipolide ist bisher noch ausstehend für B. griseus und Tylopilus nigerrimus. Beide Arten werden aufgrund morphologischer Merkmale und sequenzanalytischer Daten zu Retibo- letus gestellt. Die Gattung Retiboletus wird in der vorliegen- den Studie neu beschrieben. Boletus ornatipes, B. retipes, B. flavoniger, B. griseus und Tylopilus nigerrimus werden in diese neu etablierte Gattung 1 Dedicated to Professor Dr. Hanns Kreisel, Greifs- Retiboletus gestellt und die hierbei notwendigen wald on the occasion of his 70th birthday Neukombinationen durchgeführt.

© WILEY-VCH Verlag Berlin GmbH, 13086 Berlin, 2002 0014-8962/02/1-204-0030 $ 17.50+.50/0 M. BINDER & A. BRESINSKY: Retiboletus, a new genus for a species-complex 31

Introduction already known. According to the presence or absence of particular chemical components, The discovery of retipolides in Boletus ornati- the B. retipes/ornatipes collections could be pes and B. retipes (HERMANN 1980) indicated tentatively subdivided in three groups (Gruppe the occurrence of a novel substance group in 1–3). Thus, HELLWIG’s (1999) findings sug- the Boletaceae. At that time, the Boletaceae gest that B. retipes/ornatipes essentially is a were considered to be absolutely homogenous species-complex including two or more spe- in the production of pigments and other me- cies. tabolites, which basically consist of pulvinic The goal of our study was to evaluate acids and derivatives. Members of the genera the results drawn from chemistry with rDNA Boletus FR., MURR., sequences, using the same set of collections MURRILL, and QUÉL. normally and extending it with other representatives produce derivatives like varie- of the Boletaceae. A main objective was to gatic acid and xerocomic acid (BESL & BRE- verify possible correlations between geo- SINSKY 1978; GILL & STEGLICH 1987). Excep- graphical distribution and taxonomical impli- tions are the genera (CORNER) cations of the B. retipes/ornatipes-complex in WOLFE (not adequately studied), Gyroporus North America. To infer phylogenetic relation- QUÉL., Tylopilus KARST., and ships we generated a data set of nuc-lsu SINGER in which positive proof for pulvinic sequences (25S or 28S rDNA gene), a DNA acid derivatives is still lacking. Instead, an region, which has been successfully used occurrence of cyclopentenones has been de- in previous studies (BRESINSKY et al. 1999; monstrated in case of Gyroporus (BRESINSKY BINDER & BESL 2000; BINDER & BRESINSKY & BESL 1978). 2002). Since the description of B. ornatipes PECK and B. retipes BERK. & CURTIS, it remained uncertain if these are distinct species or Material and methods not (SINGER 1947, 1986; SMITH & THIERS 1971; BOTH 1993). B. ornatipes has a con- Fungal material. – Sources, collection information, spicuous but variable habitus (shown in and GenBank accession numbers (AF456837– BESSETTE et al. 2000) and is usually easily AF456825) are listed in Table 1. Voucher specimens recognized in the field. B. retipes is very are deposited in the Institut für Organische Chemie, similar and differs from B. ornatipes only in a Ludwig-Maximilians-Universität München (IOC), yellowish pulverulent . Apparently, this the Institut für Botanik, Universität Regensburg character is not constant and therefore unreli- (REG) and in The New York Botanical Garden able to distinguish between the two species Herbarium (NY). 32 new nuc-lsu sequences were (BOTH 1993). generated for this study. We also used six sequences from our previous studies and four other sequences In a recent study, HELLWIG (1999) assumed downloaded from GenBank. that previous screenings for chemical com- DNA isolation and polymerase chain reaction. – pounds in B. ornatipes and B. retipes (HER- 20 mg samples from dried herbarium specimens MANN 1980; JUSTUS 1993) were based on were ground in liquid . Cell lysis proceeded mixed collections, because both species have for one hour at 65 °C using 800 PL extraction buffer been regarded as identical. HELLWIG (1999) (50 mM EDTA, 50 mM Tris-HCl, 3% SDS, pH accessed material obtained in an extensive 8.0). The crude preparation (LEE & TAYLOR 1990) collection trip to North America in 1997 un- was followed by a phenol : chloroform : isoamyl dertaken by his colleagues, who kept their alcohol (25:24:1, Amresco) extraction and an collections separately and labelled them as additional step with chloroform. Total DNA was precipitated with 10 PL sodium acetate (3M) and B. retipes/ornatipes. In addition, material from isopropanol (0.54 Vol.%) at –20 °C. DNA pellets Japan and Costa Rica was used for com- were washed three times in 70% ethanol, air dried parison. As a result, HELLWIG (1999) detected and resuspended in 100 PL TE buffer. traces of xerocomic acid in some samples and PCR reactions contained 33 PL DNA solution a wide distribution of an unidentified com- (adjusted to approximately 5 ng), 10 PL PCR reac- pound (Substanz 102) next to the retipolides tion buffer, 2 PL dNTP mix (0.2 mM), 50 pmol each 32 Feddes Repert., Berlin 113 (2002) 1–2 AF456809 AF456805 AF456815 AF456819 AF456817 AF456830 AF456814 AF456825 AF456811 AF456831 AF456810 AF456812 AF456837 AF456836 AF456833 AF456827 AF456834 AF456821 AF456829 AF456828 AF456807 AF456806 AF456824 AF456822 AF456808 AF456823 AF456835 ARUMOTO ARUMOTO EISENHERZ EISENHERZ TEGLICH ALLING ALLING ALLING RNOLD RNOLD RNOLD HIBATA RESINSKY CHREINER CHREINER */* */* */* */* */* */* */* */* */* */* */* N. A */* W. S N. A R. H R. H H. S R. M R. M R. H A. B J. S N. A M. B M. B J. S . 08. 1995 Germany 30. 08. 199608. 1997U.S.A. NC, MA, U.S.A. 27. 08. 1995 Germany 08. 1995 Germany ted nuc-lsu sequencested nuc-lsu stst 10. 07. 1997 17. 08. 1997 Japan Japan Corylus , n numbers of genera of n numbers Quercus Quercus Quercus 219/97forest mixed 01. 09. 1997 MA, U.S.A. 215/97forest mixed 31. 08. 1997 MA, U.S.A. 201/97forest mixed 30. 08. 1997 MA, U.S.A. 186/97forest mixed 30. 08. 1997 MA, U.S.A. 161/97forest mixed 26. 08. 1997U.S.A. NC, 96/97forest mixed 23. 08. 1997U.S.A. NC, 94/97forest mixed 23. 08. 1997U.S.A. NC, 93/97forest mixed 23. 08. 1997U.S.A. NC, 57/97forest mixed 21. 08. 1997U.S.A. NC, 22/97forest mixed 20. 08. 1997U.S.A NC, 11/97 trees decidious 14. 08. 1997 MA, U.S.A. 116/96 5/97 7/94202/97 mixed forestRH7189forest mixed RH7247forest mixed 10. 1994Japan1 30. 08. 1997forest mixed Japan2 24. 11. 1993 mixed fore Japan3 mixed fore MA, U.S.A 08. 06. 1994 U.S.A. RH7567 Costa Rica forest mixed forest mixed Costa Rica n/a 28. 05. 1996 Costa Rica Japan Cam1 mixed forest n/a Germany Bap1 mixed forest 10 .Ba1 , , , . . Bae1 mixed forest . Bf1forest mixed 11. 09. 1994 Germany R ICKS ÓMEZ ÓMEZ ÓMEZV apud . . .: F ALLING ALLING ALLING CHAEFF D ECK ELEN . Bc1forest mixed 07. 09. 1994 Germany S H H H R P ROST ERK ERK ULL V F F ornatipes ornatipes ornatipes ornatipes ornatipes ornatipes ornatipes ornatipes ornatipes ornatipes ornatipes ornatipes ornatipes / / / / / / / / / / / B B / / B . . & L.D.G . & L.D.G . & L.D.G R OSER .: F UELL UELL UELL .) M URTIS URTIS NELL UÉL CHAEFF ECK amarellus Chalciporus (Q Boletus retipes Boletus Boletus retipes Boletus Boletus retipes Boletus Boletus retipes Boletus Boletus retipes Boletus Boletus retipes Boletus Boletus retipes Boletus Boletus retipes Boletus Boletus retipes Boletus Boletus retipes Boletus Boletus retipes Boletus Boletus Boletus calopus coniferarum Boletus griseus Boletus fechtneri Boletus flavoniger Boletus flavoniger Boletus ornatipes Boletus flavoniger Boletus retipes Boletus retipes Boletus retipes Boletus retipes Boletus G.M.M G.M.M G.M.M P & S Boletus appendiculatus Boletus S & C & C Species Coll. no. Date Locality Leg./det. GenBank no. Table 1 Table Species, collection data, and GenBank accessio M. BINDER & A. BRESINSKY: Retiboletus, a new genus for a species-complex 33 )

& of primers LR0R and LR7 (VILGALYS & HESTER

ECK 1990), and 1 U TaqDNA polymerase (Eurogentec). Chal- P MITH

(P The final volume was adjusted to 100 L with ster- ile H2O. The amplifications were run in 37 cycles on a TM3 thermocycler (Biometra) using the fol- q AF456826 AF456818 AF456820 AF456832 AF456813 lowing parameters: denaturation 95 C (1 min), .) A.H. S .) A.H. – U11914; annealing 47 qC (45 sec), extension 72 qC (1.5 min).

PAT PCR products were purified with the QIAquick PCR cleaning Kit (Qiagen). (O Cycle sequencing and sequence analysis. – Se- , Xerocomus quencing reactions were set up with primers LR0R, LR3 and LR5 (primer sequences used in this study Boletus retipes retipes Boletus ELFER RONFELDNER ARUMOTO RNOLD RNOLD RESINSKY were obtained from http://www.botany.duke.edu/ fungi/mycolab/primers.htm) using the ABI Prism N. A W. H A. B R. M N. A M. K

Suillus cavipes BigDye Terminator Cycle Sequencing Ready Reac- tion Kit (Applied Biosystems, California). Each

. – AF139710; . – AF139710; reaction mix included 2 PL BigDye (with AmpliTaq P

. – AF336241; . – AF336241; polymerase), 8 pmol primer, and 3.5 L LR0R-LR7 ARST R product. The PCR program was: 96 °C denaturation

.: F (2 min), 47 °C annealing (15 sec), and 60 °C exten- . – AF098377; .) P.K sion (4 min) in 35 cycles. Cycle sequencing products ERS R

P were run on an ABI 377 automated DNA sequencer ARST .: F (Applied Biosystems) with a 5.25% polyacrylamide gel (PAGE PLUS, 7M Urea, Amresco) at the Uni- ULL

.) P.K versitätsklinikum Regensburg. (B Phylogenetic analyses. – A preliminary align- ment with ClustalX (THOMPSON et al. 1997) was manually adjusted in the editor of PAUP* 4.0b4a Boletus radicans Boletus CHUMACH (SWOFFORD 1998) and submitted to TreeBASE. All 04. 1995 New Zealand (S trees were rooted with Coniophora puteana

30. 08. 1997 U.S.A (SCHUMACH.: FR.) KARST. (Coniophoraceae),

Tylopilus felleus Tylopilus luteus (L.) S.F.GRAY, and S. cavipes (KLOTZSCH in FR.) SMITH & THIERS ().

. – AF336240; Analyses were performed with maximum likeli- R hood (ML) using the complete data set. The ML .: F . – AF139716 RNOLD analysis under the HKY’85 model was performed R

ULL with 100 heuristic search replicates, transition/ – AF042622; – AF042622; Coniophora puteana /N. A B transversion ratio = 2, assumed nucleotide frequen- (L.) F (L.)

RAY cies set to empirical frequencies, number of substi-

ERPIN tution types = 2, rate heterogeneity following the

, T discrete gamma approximation, with four categories and D = 0.5. In addition, one hundred bootstrap replicates (FELSENSTEIN 1985) were run with maxi- Boletus (L.) S.F.G . – AF336244; . – AF336244;

TEGLICH mum likelihood. Heuristic searches were performed AT in PAUP* with the following general settings: 238 mixed forestTyni1 26. n/a 15. 07. 1996 Japan 98/98 trees decidious 26. 08. 1998 U.S.A Pp1 mixedforest 09. 09. 1995 Germany .) B

, W. S MAXTREES set to autoincrease, TBR, random taxa R .) addition sequence, MULTREES on, zero length ) EIM ÉV .: F branches collapsed, gaps treated as “missing”, and ELFER ECK (L ULL R.H steepest descent option not in effect. A constrained

(P analysis was performed using the same data set and (B , W. H settings, forcing the hypothetical of all

INGER retipolide producing including Boletus RNOLD ) S griseus ROST ECK Tylopilus T. nigerri-

sp. 204/97 mixed forest F in P , sp., and mus R.HEIM. Constrained and unconstrained trees – AF139714; – AF071535; – AF071535; . ABB N. A . were compared using the Templeton non-parametric N C

URR test (WSR-test) implemented in PAUP* (TEMPLE- UÉL HIERS INGER pelletieri Phylloporus nigerrimus Tylopilus Tylopilus Q M viscida (M S T ciporus piperatus n/a = not available n/a = not */* = Carolina = North NC MA = Massachusetts; Sequencesobtained from GenBank: TON 1983). 34 Feddes Repert., Berlin 113 (2002) 1–2

Results ship between B. flavoniger and groups 1–3 is indicated. The nuc-lsu rDNA data set of 42 sequences The phylogenetic analysis using maximum included 925 aligned positions. 689 characters parsimony (MP) recovered 216 trees of 560 were constant, 85 variable characters were steps (CI = 0.555, RI = 0.712) in seven islands parsimony-uninformative, and 151 characters (MADDISON 1991). There was no conflict com- were parsimony-informative. The genera paring MP and ML topologies, however, the Boletus and Tylopilus are polyphyletic in both including all retipolides producing maximum likelihood and maximum parsimony boletes collapses in the strict consensus tree analyses, whereas the Boletaceae is confirmed (not shown). In addition, the position of as monophyletic clade. The shortest tree B. griseus and Tylopilus sp., and B. flavoniger (Fig. 1) found using maximum likelihood (-ln remained unresolved. The constrained analysis likelihood = 4357.71191) resulted in three designed to test support for the monophyly of major for the North American groups 1–3 including B. griseus, Tylopilus sp., B. retipes/ornatipes collections, corresponding T. nigerrimus, and B. flavoniger recovered 100 to chemotaxonomical groups 1–3 detected by trees of 562 steps (two steps longer than the HELLWIG (1999). unconstrained trees). None of the trees (p = Group 1 includes collections from Massa- 0,6698–0,8378) was statistically rejected by chusetts and North Carolina and receives the WSR-test. strong support by a bootstrap value of 100%. The nine sequences in group 1 only differ in seven base positions, which suggests that Discussion the isolates are conspecific. Collection B. reti- pes/ornatipes 93/97 shows an additional Nuc-lsu sequence data indicate that the five base pairs deletion at position 510–514. B. retipes/ornatipes-complex is distinct from Nested between groups 1 and 2 are Boletus other groups in the Boletaceae, specifically the griseus, Tylopilus nigerrimus, and Tylopilus genus Boletus. In addition, the results of this sp., which have not been screened for chem- study confirm the findings of HELLWIG (1999), ical compounds in HELLWIG’S study. Except who showed that different patterns of for different colors, B. griseus and T. nigerri- retipolides and other chemical components mus strongly resemble the habitus of B. orna- occur in B. ornatipes and B. retipes collections. tipes and B. retipes and a close relationship Both sequence data and chemotaxonomical is not surprising. Though appearing to be data allow to differentiate between groups in close to B. griseus based on morphology, this puzzling species-complex for the first time. Tylopilus sp. was not unambiguously identi- Still, there are no morphological or ana- fied. tomical characters available to distinguish Group 2, also supported by 100%, contains B. ornatipes from B. retipes. The number of two subclades. The Japanese collections, iden- collections investigated, however, provides tified as B. retipes and B. ornatipes, are puta- evidence of a vicariant geographical distribu- tively conspecific and differ in two base pair tion. B. retipes was described from North positions. They form the to two identical collections from North Carolina and a B. retipes collection from Costa Rica. Together group 2 and group 3, which exclusively in- Fig. 1 ᭤ cludes collections from North Carolina, are Phylogenetic relationships of Retiboletus inferred supported by a bootstrap value of 83%. An- from nuc-lsu (LR0R-LR5) sequences using maxi- other group consists of two B. flavoniger iso- mum likelihood (tree score: –logL = 4357.71191). Numbers above branches are bootstrap frequencies, lates from Costa Rica, which produce the values below 50% are not shown. Groups 1–3 are retipolides found in groups 1–3, and addition- the groups proposed by HELLWIG (1999), based on ally larger amounts of unidentified metabolites the distribution of retipolides. The common node, of (HELLWIG 1999). Supported by morphology which retipolides producing boletes arise, is indi- and chemotaxonomy, a phylogenetic relation- cated by an arrow M. BINDER & A. BRESINSKY: Retiboletus, a new genus for a species-complex 35

B. retipes/ornatipes 5/97, MA B. retipes/ornatipes 94/97, NC B. retipes/ornatipes 11/97, MA B. retipes/ornatipes 93/97, NC B. retipes/ornatipes 215/97, MA 100 B. retipes/ornatipes 201/97, MA group 1 B. retipes/ornatipes 161/97, NC B. retipes/ornatipes 186/97, MA B. retipes/ornatipes 219/97, MA Tylopilus nigerrimus, Japan

100 Tylopilus sp. 204/97, MA Boletus griseus 202/97, MA B. ornatipes, Japan2 100 B. ornatipes, Japan3 B. retipes, Japan1 100 group 2 B. retipes/ornatipes 96/97, NC occurrance 100 B. retipes/ornatipes 22/97, NC of retipolides → Boletus retipes Costa Rica 83 B. retipes/ornatipes 57/97, NC B. retipes/ornatipes 116/96, NC group 3 Boletus retipes U11914

100 Boletus flavoniger RH7247, Costa Rica Boletus flavoniger RH7189, Costa Rica Xerocomus illudens 100 Xerocomus subtomentosus

100 Tylopilus indecisus Fistulinella viscida Boletus reticulatus 100 Boletus aereus Boletus edulis Boletus coniferarum 99 Boletus calopus 91 Boletus radicans → Boletus fechtneri Boletaceae 83 Boletus appendiculatus

100 Chalciporus amarellus

100 Suillus luteus Coniophora puteana 0.01 substitutions/site 36 Feddes Repert., Berlin 113 (2002) 1–2

Carolina by BERKELEY & CURTIS in 1872. point at the presence of retipolides (Arnold, PECK (1878) described B. ornatipes six years pers. comm.) or pulvinic acid derivatives later in Michigan, based on the absence of a (BRESINSKY & BESL 1978) in B. griseus. The pulverulent pileus. Unfortunately, PECK did not occurrence of so-called “chemical albinos” is designate a type, but an authentic collection is not exceptional in the Boletales (e.g. Tylopilus) available (BOTH 1993). Our own observations and explains the different colours in hymeno- from Massachusetts and several other observa- phore and of B. ornatipes and B. griseus, tions from Michigan and New York State which are used for differentiation between the (SMITH & THIERS 1971; BOTH 1993) suggest two species. T. nigerrimus (shown in IMAZEKI that B. retipes is not occurring in the northern et al. 1988) is very similar to B. griseus and is part of Eastern North America. All collections distributed in East . This species is not yet made in Massachusetts (MA) are in group 1, scanned for chemical compounds and has black lack a pulverulent pileus and stipe, and can be tones in pileus and stipe. B. flavoniger is assigned to B. ornatipes. In addition, our re- another species similar to B. ornatipes and sults suggest that B. ornatipes occurs in North B. retipes. It has recently been described from Carolina (NC) as well. For example, sequences Costa Rica and differs from the previously of collections 5/97 (MA) and 94/97 (NC), mentioned species in an orange brown staining respectively, 201/97 (MA) and 161/97 (NC) hymenophore and context and extremely bitter are identical. taste (HALLING & MUELLER 1999). The two B. retipes (groups 2, 3) is apparently more collections of B. flavoniger RH7247 (Holo- widely distributed than B. ornatipes. North type) and RH7189 show differing chemical Carolina is most likely the main distribution components (HELLWIG 1999). Both collections area of B. retipes in North America, where it lack xerocomic acid, Substanz 102 is present. overlaps with the distribution area of B. orna- Retipolide B and retipolide D are only present tipes. SINGER (1947) also reports B. retipes in RH7247, which questions the taxonomical from South Carolina. For this reason, we con- use of both metabolites on the species level clude that B. retipes is limited to the southern again. The nuc-lsu sequences of RH7247 and part of Eastern North America. In addition, RH7189 only differ in one position and the B. retipes occurs in the Neotropics and East collections are putatively conspecific. Asia based on the collections from Costa Rica The results of our analyses compared with and Japan used in this study. A further separa- the results of HELLWIG (1999) suggest that all tion of B. retipes into species originating from retipolide producing fungi, including different continents is difficult with the data at T. nigerrimus, B. griseus, and B. flavoniger, hand. According to HELLWIG (1999), neither form a natural and unique group within the Substanz 102 nor xerocomic acid is detected in Boletaceae. This relationship is partly resolved groups 2 and 3. In contrast, both components in the maximum likelihood analysis and is not were constantly traced in group 1 (B. orna- rejected by the WSR-test using maximum tipes). Group 3 is different from group 2 in parsimony. We create the new genus Reti- producing retipolides B and D (HELLWIG boletus to separate the species mentioned 1999). In regard of missing morphological and above from Boletus and Tylopilus, respectively, anatomical characters, which could be used to and five new combinations are made. separate group 2 and group 3, we cannot con- firm a correlation between chemical and mor- phological evidence in this case. Retiboletus BINDER & BRESINSKY gen. nov. Boletus griseus and Tylopilus nigerrimus are nested between B. ornatipes and B. retipes Retipolida inventa aut partim adsunt, acidi in our study. In some essential characters pulvinici derivata rarius desunt. Carposoma B. griseus resembles B. ornatipes, which led stipitatum pileatum, hymenophoro tubuliformi. SMITH & THIERS (1971) to create a new sub- Pileus siccus aut subtiliter tomentosus, niger, for both species, subsect. Reticulati fusco-griseus vel olivaceus. Color hymeno- SMTIH & THIERS in the genus Boletus. Prelimi- phori pallidus, griseolus vel flavum, brunnes- nary results of chemical screenings do not cens, aurantio-brunnescens, partim immutabi- M. BINDER & A. BRESINSKY: Retiboletus, a new genus for a species-complex 37 lis. Cystidia praesentibus. Stipes centralis, Retiboletus flavoniger (HALLING, G.M.MUELL. reticulatus, basim versus paulatim obscuriore. & L.D.GÓMEZ) BINDER & HALLING comb. Contextus pallidus, flavidus vel luteus, ple- nova rumque in aurantio-brunneum mutatur, aut Basionym: Boletus flavoniger HALLING, partim vix immutabilis. Fibulae nullae. Spo- G.M.MUELL. & L.D.GÓMEZ, Mycologia 91(5): 893– rarum pulvis olivaceo-fuscus aut luteo-fuscus. 899. 1999. Sporae leves, ellipsoideae vel subfusoidae, inamyloideae, vix dextrinoideae. Typus: Boletus ornatipes PECK, Ann. Annotations to (PECK) Rept. N. Y. State Mus. 29: 67. 1878. BINDER & BRESINSKY and R. griseus (FROST Retipolides are frequently occurring or in PECK) BINDER & BRESINSKY exceptionally missing, in addition, pulvinic acid derivatives sporadically occur. Basidio- Retiboletus ornatipes, the type species of the carps are stipitate-pileate with a tubular hy- genus, is one of the most showy representatives menophore. Pilei are convex to plane, dry to within the boletes, quite spectacular in its deep subtomentose, black, dark gray, mustard yel- yellow colours shown in the flesh as well as in low to olive brown. The color of the hymeno- the other parts of the fruit bodies, painting the phores ranges from pallid, grayish to yellow, fingers yellow if touched. Since pulvinic acid changing to brown or orange brown when derivates are missing or at least only present in bruished, sometimes not changing. Cystidia traces, no part of the fruiting body will stain are present. A central stipe shows reticulate blue after bruising. Instead, the colours will ornamentation, the base is usually darker turn to grayish black or partially even black than the apical region. The context is pallid, when the is dried. The taste of the flesh yellow or vivid yellow, changing to orange is somewhat bitter. brown or slightly changing. Clamp connec- As already stated by SMITH & THIERS tions are absent. deposit is olive brown (1971), the cystidia of R. ornatipes (as well as to yellow brown. The smooth are ellip- those of R. griseus) exhibit some special fea- soid to subfusoid, inamyloid and partly dextri- tures not to be found in other boletes. In their noid. monograph it is indicated that the colour of the cystidia in KOH and in Melzer’s reagent is a valuable aid to identify herbarium specimens New combinations as members of the subsection Reticulati in- cluding the two species (R. ornatipes, Retiboletus ornatipes (PECK) BINDER & BRE- R. griseus) mentioned here. The cystidia are SINSKY comb. nova characterized as being “hyaline in KOH when fresh, however, when dried material is revived Basionym: Boletus ornatipes PECK, Ann. Rept. N. Y. State Mus. 29: 67. 1878. in KOH it shows strongly pigmented, ochra- ceous to bister, amorphous masses occupying (FROST in PECK) BINDER & most of the ventricose part of the cystidia. The BRESINSKY comb. nova same colored substances turn to dark red brown Basionym: Boletus griseus FROST in PECK, Ann. in Melzer’s reagent” (the cystidia therefore are Rept. N. Y. State Mus. 29: 45. 1878. said to be dextrinoid). Additional material of R. ornatipes, Retiboletus nigerrimus (R.HEIM) BINDER & checked by us (No. 86-399, USA, North Caro- BRESINSKY comb. nova lina, Macon County, Ball Creek area, Coweeta Basionym: Boletus nigerrimus R.HEIM, Rev. Hydrologic. Lab, Highway 441 near Franklin, Mycol. Paris 28: 281. 1963. 04.09.1986, leg. W. STEGLICH & A. BRESINSKY, specimens deposited in the Botanische Staats- Retiboletus retipes (BERKELY & CURTIS) BIN- sammlung München (M), exhibited the micro- DER RESINSKY & B comb. nova scopical characters (Fig. 2) described by SMITH Basionym: Boletus retipes BERKELEY & CURTIS, & THIERS (1971). The rather slender, bottle Grevillea 1: 36. 1872. like to fusoid cystidia were filled with yellow 38 Feddes Repert., Berlin 113 (2002) 1–2

gitudinal hyphae also balloon-like elements have been found showing the same colour change in Melzer’s. These elements may be interpreted as the remnants of a reduced sterile on the surface of the stipe, however, the basidia of the hymenophore are slender and not globose. The spores are not clearly dextri- noid. In Melzer’s the wall of the spores appears to be coloured a little bit darker, turning to brownish as compared to spores mounted in KOH. According to SMITH & THIERS (1971), the spores are dingy pallid yellow in KOH or a few ochraceous, in Melzer’s pale orange tan. Retiboletus griseus lacks the vivid and deep yellow colors of R. ornatipes. As an exception, a more or less faint yellow pigmentation may be observed. According to SMITH & THIERS (1971), there is some risk in such rare cases and B. griseus may be confused with R. orna- tipes. However, the distinction between the two species is usually possible without any prob- lem. The pallid context of R. griseus does not turn to blue; only a colour change to dingy vinaceous tones may be observed if exposed to the air. Microscopical characters have been reinvestigated in a collection from U.S.A. (No. Fig. 2 86-301, Tennessee, Smoky Mountains National Retiboletus ornatipes (M, No. 86-399, leg. Park, Cades Cove, 28.08.1986, leg. A. BRESINSKY, W. STEGLICH & A. BRESINSKY) specimens deposited in M). The bottle like A — cross section through carpophore; B — net on the shaped cystidia (Fig. 3) exhibited yellowish brown surface of upper part of the stipe; C — cystidia from or brown contents when observed in KOH and the hymenium; D — spores; E — hyphae of ; vivid yellow to brownish yellow masses in Mel- F —hyphae and balloon like cells from cortical layer of zer’ s reagent. The same colours in KOH, and in stipe Hatching applies to colour in Melzer’s (C, E, F), dotted Melzer’ s respectively, have been seen in the parts show colour reaction in KOH hyphae composing the cortical layer of the pi- Scale bar: A, B = 1 cm; C–F = 10 Pm leus and of the stipe as well. FLORES ARZÙ & SIMONINI (2000) made similar observations on a coloured contents distributed partially in the R. griseus collection from Guatemala; as a dif- slender neck, partially also, as stated by SMITH ference to our material, the pleurocystidia & THIERS (1971), in the ventricose part of the showed tobacco brown contents in KOH and cystidia. Pieces of the hymenophore mounted blackish brown contents in Melzer’s reagent. in Melzer’s solution turned their dirty brownish Similar to R. ornatipes, the stipe surface of colour to a very vivid red brown. In this condi- R. griseus is composed of rather broad clavate tion the contents of the cystidia, rather big to balloon shaped elements appearing in nest globular masses and smaller grains, are stained like clusters among longitudinal hyphae which deeply orange brown. A similar (more or less partially are very slender. Most of all these dextrinoid) colour change in Melzer’s could be elements show yellow colours in Melzer’s, observed in case of the hyphae composing the sometimes in form of globular masses (so cortical layers of the cap and of the stipe as especially within the balloon cells; Fig. 3). The well. Here the contents of the hyphae turned in majority of spores is nearly hyalin in Melzer’s; Melzer’s to a vivid yellow colour. In the cortex a few spores, however, appear to be completely of the stipe, besides sometimes branched lon- dextrinoid. M. BINDER & A. BRESINSKY: Retiboletus, a new genus for a species-complex 39

References

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