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 genus for a species-complex in the Boletaceae producing retipolides1 With 3 Figures and one Table Summary Zusammenfassung Boletus ornatipes and B. retipes are two closely Retiboletus, eine neue Gattung für einen Arten- related species in North America, 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 morpho- 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 form 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 Boletales (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 (type), 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 Tylopilus 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., Boletellus MURR., Pulveroboletus sequences, using the same set of collections MURRILL, and Xerocomus QUÉL. normally and extending it with other representatives produce pulvinic acid 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 Austroboletus (CORNER) cations of the B. retipes/ornatipes-complex in WOLFE (not adequately studied), Gyroporus North America. To infer phylogenetic relation- QUÉL., Tylopilus KARST., and Xanthoconium 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 pileus. 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 nitrogen. 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 Table 1 Species, collection data, and GenBank accession numbers of generated nuc-lsu sequences Species Coll. no. Host Date Locality Leg./det. GenBank no. Boletus appendiculatus Bap1 mixed forest 10. 08. 1995 Germany J. SCHREINER AF456837 SCHAEFF.: FR. Boletus aereus BULL.: FR. Bae1 mixed forest 08. 08. 1995 Germany J. SCHREINER AF456836 Boletus calopus FR. Bc1 mixed forest 07. 09. 1994 Germany N. ARNOLD AF456833 Boletus coniferarum DICKS. 7/94 mixed forest 10. 1994 U.S.A. W. STEGLICH AF456827 & SNELL Boletus griseus FROST apud 202/97 mixed forest 30. 08. 1997 MA, U.S.A N. ARNOLD AF456834 PECK Boletus fechtneri VELEN. Bf1 mixed forest 11. 09. 1994 Germany M. BEISENHERZ AF456821 Boletus flavoniger HALLING, RH7189 mixed forest 24. 11. 1993 Costa Rica R. HALLING AF456829 G.M.MUELL. & L.D.GÓMEZ Boletus flavoniger HALLING, RH7247 mixed forest 08. 06. 1994 Costa Rica R. HALLING AF456828 G.M.MUELL. & L.D.GÓMEZ Boletus ornatipes PECK Japan1 mixed forest 10. 07. 1997 Japan H. SHIBATA AF456807 Boletus flavoniger HALLING, Japan2 mixed forest 17. 08. 1997 Japan R. MARUMOTO AF456806 G.M.MUELL. & L.D.GÓMEZV Boletus reticulatus SCHAEFF.Ba1 Quercus, Corylus 27. 08. 1995 Germany M. BEISENHERZ AF456824 Boletus retipes BERK. Japan3 mixed forest n/a Japan R. MARUMOTO AF456822 & CURTIS Boletus retipes BERK. RH7567 mixed forest 28. 05. 1996 Costa Rica R. HALLING AF456808 & CURTIS Boletus retipes/ornatipes 116/96 Quercus 30. 08. 1996 NC, U.S.A. N. ARNOLD AF456823 Berlin Repert., Feddes Boletus retipes/ornatipes 5/97 Quercus 08. 1997 MA, U.S.A. */* AF456812 Boletus retipes/ornatipes 11/97 decidious trees 14. 08. 1997 MA, U.S.A. */* AF456810 Boletus retipes/ornatipes 22/97 mixed forest 20. 08. 1997 NC, U.S.A */* AF456831 Boletus retipes/ornatipes 57/97 mixed forest 21. 08. 1997 NC, U.S.A. */* AF456811 Boletus retipes/ornatipes 93/97 mixed forest 23. 08. 1997 NC, U.S.A. */* AF456825 Boletus retipes/ornatipes 94/97 mixed forest 23. 08. 1997 NC, U.S.A. */* AF456814 Boletus retipes/ornatipes 96/97 mixed forest 23. 08. 1997 NC, U.S.A. */* AF456830 Boletus retipes/ornatipes 161/97 mixed forest 26. 08. 1997 NC, U.S.A. */* AF456817 Boletus retipes/ornatipes 186/97 mixed forest 30. 08. 1997 MA, U.S.A. */* AF456819 113 Boletus retipes/ornatipes 201/97 mixed forest 30. 08. 1997 MA, U.S.A. */* AF456815 (2002) 1–2 Boletus retipes/ornatipes 215/97 mixed forest 31.