299 Mycological Progress 1(3): 299-313, August 2002 Inonotus s. I. in Argentina - morphology, cultural characters and molecular analyses Alexandra M. GOTTLIEB1, Jorge E. WRIGHT 1 * and Jean-Marc MONCALVO 2 A survey of the hymenochaetaceous Inonotus in Argentina was conducted. At least seven Inonotus sensu stricto species were recorded for the region. These are /. ochroporus, I. patouillardii, I. pertenuis, I. quercustris, I. rickii, I. serranus and /. texanus. A detailed description of each species is given, and a key for their identification is provided. Each morphospecies could also be distinguished from both RFLPs and sequence data obtained from PCR products of the internal transcribed spacer region of the nuclear ribosomal RNA gene (ITS). A high level of nucleotide sequence variation was found among Inonotus. Molecular data also indicate that one morphospecies, /. patouillardii, may in fact represent two distinct species. Both cultural and molecular data support the view that Ptychogaster cubensis represents an anamorphic state of /. rickii. Two new combinations are proposed, namely Phellinus crustosus and Inocutis jamaicensis. I. pertenuis is reported for the first time from Argentina. Taxonomical novelties: Inocutis jamaicensis (Murrill) Gottlieb, Wright & Moncalvo Phellinus crustosus (Speg.) Gottlieb, Wright & Moncalvo I the lectotype, while MURRILL (1904), DONK (1933), IMAZEKI generative hyphae lacking clamp connections, the frequent presence of setae, a similar shape and colour of spores, an anoderm pileus surface, etc. According to CORNER (1991) the colour and texture of the flesh is so variable that neither character can really have generic significance. As in other polypores, the context undergoes a colour change in contact with alkali known as xanthochroic reaction. It has been suggested that the precursor of the pigment causative of this chemical reaction is a phenolic polymer called 'hispidin' [4-hydroxy6-(3,4-dihydroxystyryl)-2-pyrone] and that ionisation of phenolic groups produces the extended chromophores res- (1943), CUNNINGHAM (1948), TEDCEIRA (1989) and RYVARDEN ponsible for the darkening (KIRK, LORENZ & LARSEN 1975). (1991) followed the "first species rule" and considered P. cuticularis Bull.: Fr. (=Inonotus cuticularis (Bull.: Fr.) P. Karst.) to be the type. All in all, there is much confusion regarding taxonomic delimitation in Inonotus, and natural relationships of the species in a worldwide scale have still not been clearly established. Some authors like DONK (1974) and FIASSON & NIEMELÁ (1984) considered the genus to be an heterogeneous group that would eventually be broken up into smaller genera. Species of Inonotus s. I. exhibit, as a general rule, a rusty brown coloured fibrous context, the darkening of the flesh on contact with alkali, a monomitic hyphal system composed of However, PARMASTO & PARMASTO (1979) have shown that the xanthochroic reaction is common in fungi, and stated that the same colour may be produced by very different chemical compounds. For example, the flesh of some lignicolous members of the Strophariaceae (Agaricales) also darkens in KOH resembling the xanthochroic reaction (KÜHNER 1980). A monomitic clampless hyphal system is accepted for the genus nonotus was described by KARSTEN in 1879 ("1880" according to DONK 1960) to accommodate pileate poroid fungi with coloured spores. The genus was later emended by DONK (1933) to include other taxa with coloured spores and a rusty brown fibrous context. Many taxonomical problems are associated with Inonotus systematics. For instance, the identity of the type species of the genus is still controversial. PEGLER (1964b), WRIGHT & DESCHAMPS (1975) and CORNER (1991) followed DONK (1960) who considered Polyporus hispidus Bull.: Fr. (=Inonotus hispidus (Bull.: Fr.) P. Karst.) as 1 Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Ciudad Universitaria (C1428EAH). Buenos Aires, Argentina. 2 Department of Biology, Duke University, Durham, NC 27708 USA. * corresponding author: fax: 54-11-4787-2706, e-mail: wright@bg.fcen.uba.ar Inonotus as reflected in GILBERTSON & RYVARDEN (1986) and RYVARDEN (1991), tough a dimitic system, and even a trimitic system for some species have been described (DOMANSKI, ORLOS & SKIRGIELLO 1973, JAHN 1981, CORNER 1991). The use of setal elements as diagnostic characters in distinguishing Inonotus species has been questioned since PEGLER (1967). In addition, structures resembling setae are also known in the agaric genera Marasmius Fr. and Hohenbuehelia Schulzer, but they are thought to be of different origin (DONK 1971). Spore colour typically ranges from hyaline to light yellowish to rusty brown. As stated by FISCHER, AINSWORTH & WAGNER ©DGfM 2002 300 (2001) this character is probably not useful for inter-species systematics in Inonotus because it might have independently evolved in several lineages. At the family level, many modern mycologists followed PATOUILIARD'S (1900) concept of an hymenochaetaceous entity, which unites certain polyporoid, hydnoid and stereoid genera possessing setae, and classify Inonotus in the Hymenochaetaceae Donk, along with genera such as Aurificaría D. A. Reid, Coltricia Gray, Phellinus Quel., Phylloporia Mur- Inonotus s.l. in Argentina also supported and can be distinguished by nucleotide sequence variation in the internal transcribed spacer region of the nuclear ribosomal DNA gene (ITS). The usefulness of ITS data for fungal systematics at lower taxonomic levels (species, sections and genera) has already been shown in many studies, for example, MONCALVO, W A N G & HSEU 1995a,b, Ko, HONG & JUNG 1997, AANEN et al. 2000, GOTTLIEB, FERRER & WRIGHT 2000. rill, etc. (KOTLABA & POUZAR 1957, DONK 1964, RYVARDEN 1978, PARMASTO & PARMASTO 1979, RYVARDEN & RYVAR- Material and methods DEN 1986, RYVARDEN & RYVARDEN 1993). According to RYVARDEN (1991) this family is one of the most homogeneous among the basidiomycetes and a prime example of strong macromorphological variation over a common set of microscopical characters. The monophyletic origin of the hymenochaetaceous fungi is suggested by the widespread presence of clampless hyphae, dolipore apparatuses with imperforate parenthesome, the xanthochroic reaction and the production of white rot. However, some authors separated the genera Inonotus and Phellinus in the family Mucronoporaceae Imazeki & Toki (IMAZEKI 1954, WRIGHT & DESCHAMPS 1975), while some others considered that Inonotus is best classified in the Polyporaceae s. 1. (MURRILL 1908, OVERHOLTS 1953, PEGLER 1964b). The family Coltriciaceae was created by JÜLICH (1981) to unite Inonotus, Aurificaría, Coltricia, Coltriciella Murrill, Cyclomyces Kunze, Onnia P. Karst, and Phylloporia. More recently, the family Inonotaceae was proposed by FIASSON & NIEMELÁ (1984) to accommodate Inonotus, Phylloporia and Inocutis Fiasson & Niemelá. However, this concept was not confirmed by the phylogenetic analyses of WAGNER & FISCHER (2001). OBERWINKLER (1977) raised the family Hymenochaetaceae to the ordinal level. The recognition of a hymenochaetoid clade that includes poroid, toothed and corticioid forms was supported by molecular data, and it also appears to be united by the possession of an imperforate parenthesome (HIBBETT & THORN 2000). Another molecular phylogenetic analysis has shown European species of Inonotus s. I. and Phellinus s. L to be polyphyletic (WAGNER & FISCHER 2001). The amount of molecular data is too fragmentary to reliably delimit generic and family segregation, and thus the evolutionary relationships of Inonotus s. 1. still remain unclear. The main objective of this study was to survey the genus Inonotus in Argentina. Despite the fact that several taxonomical studies on Inonotus have been published for this region, most of them were limited in scale and discussed only one or two species (SPEGAZZINI 1887,1926, WRIGHT & IACONIS 1955, WRIGHT & DESCHAMPS 1975, IBAÑEZ 1995, RAJCHENBERG & WRIGHT 1998, URCELAY & RAJCHENBERG 1999). In addition, none of the previous studies used molecular techniques and cladistic methods to test morphological hypotheses of classification. Here we examine morphological and cultural characters from a regional sampling of Inonotus collections from Argentina, and infer if morphologically defined species are ©DGfM 2002 Morphology. Voucher specimens and culture isolates labelled as Inonotus deposited in the BAFC Herbarium and Culture Collection of the Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires were examined. Full details of the materials studied, including type specimens, are given in the Results section below each species description. Herbarium abbreviations are those of HOLMGREN & HOLMGREN (1992). Colours of macroscopic features are according to MUNSELL (1954). Microscopic characters were examined and measured using a light microscope, in thin sections mounted in 5 % KOH solution or water and phloxine. Species were identified using keys and descriptions of PEGLER (1964b) and Ryvarden (unpublished). Cultures. Mycelia were grown on 9 cm Petri dishes containing 2 % malt extract agar (MEA: malt extract 12 g/L, agar 2 % w/v) and incubated in darkness at 25 °C for 2 wk. Growth characteristics recorded on MEA were mat aspect, colony diameter, colour change in the agar reverse, production of setae and chlamydospores, and presence of any other structure of interest. Isolates were examined under the light microscope by removing mycelial fragments and mounting in phloxine and water. Culture isolates were also grown in liquid medium (sucrose 20 g/L, dextrose 30 g/L, M g S 0 4 . 7 H 2 0 0.5 g/L, K 2 H P 0 4 1 g/L, K H 2 P 0 4 0.46 g/L, yeast extract 10 g/L, peptone 4 g/L) to yield material for DNA isolation. DNA extraction. After 1-2 wk of incubation in darkness and room temperature, mycelia were harvested by vacuum filtration. DNA was extracted following CENIS' (1992) protocol, but 2 vol. 100 % ethanol were used instead of isopropanol. Mycelia were crushed with a sterile glass pestle in 300 uL of extraction buffer (100 mM Tris-HCl pH 8.0, 20 mM EDTA, 0.5 M NaCl and 1 % SDS). Cell debris was pelleted with a 10 min centrifugation at 12000 x g and the aqueous phase was transferred to a fresh tube. DNA was precipitated with 2 volumes of 100 % ethanol (-20 °C) and pelleted with a 5 min centrifugation at 12000 x g. After a 70 % ethanol (ice-cold) wash, genomic DNA was redissolved in 400-500 uL of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0). The DNA extracts were treated with RNAse ONE (Promega Corp., Madison, Wisconsin) for 30 min at 37 °C and then extracted with chloroform, precipitated with 100 % ethanol, and resuspended in 100-200 uL of TE buffer. Mycological Progress 1(3) / 2002 PCR amplification. ITS amplification was carried out by using primers ITS-5 and ITS-4 (WHITE et al. 1990). Amplifi­ cations were performed in 50 uL using 0.225 mM of each dNTP (Promega Corp., Madison, Wisconsin), 2.5 10-7 mM of each primer, 10 % of 10 X buffer (Promega Corp., Madison, Wisconsin), 1.5 mM MgCl2, 1.25 units of Taq DNA poly­ merase (Promega Corp., Madison, Wisconsin), 1 µL of geno­ mic DNA template, and sterile double-distilled water. Condi­ tions for PCR amplification were those of VILGALYS & HESTER (1990). Amplifications were carried out in a Techne Progene Thermal Cycler (Techne Ltd., Cambridge, UK). Control sam­ ples without DNA template were included in each PCR run. PCR products were checked by electrophoresing 1 uL aliquots in 1 % agarose gels (J. T. Baker, Mallinckrodt Baker, Inc., NJ) in 1 X TAE buffer (0.04 M Tris, 0.114 % glacial acetic acid, 1 mM EDTA pH 8.0). Quantification of PCR products was estimated by comparison with known amounts of a DNA molecular size marker (100 bp, Promega Corp., Madison, Wisconsin) which was included in duplicate in all gels. Gels were stained with ethidium bromide for 15-20 min and pho­ tographed under UV light. PCR-RFLPs. Single endonuclease digestions of PCR products were performed directly on an aliquot of the PCR re­ action. Digestions were done using 0.5 units of each endo­ nuclease per µg of amplified DNA following manufacturer's protocol. PCR products were digested with AM, Haelll, Hhal, HinfI, MboI and RsaI (Promega Corp., Madison, Wisconsin). Care was taken to avoid enzymes with overlapping recogni­ tion sites. Incubation was held in a water bath at 37 °C for 3-4 hr. Restriction products were electrophoresed in 2 % aga­ rose gels in 1 X TAE buffer. Size of restriction products was estimated by comparison with the molecular size marker. Sequencing. PCR products were purified by microcentrifugation using ULTRAFREE -MC spin columns (Millipore, Bedford, MA). Cleaned PCR products were sequenced in both directions using primers ITS-1 (WHITE et al. 1990) and ITS-4 and dye-termination chemistries (BigDye terminate cycle se­ quencing kits), following the manufacturer's protocol. Se­ quencing reactions were run on a Perkin-Elmer ABI PRISM model 377 DNA sequencer apparatus according to manufac­ turer's instructions. Sequence chromatograms were assembled and edited with the use of the software package Sequencher 3.1 (Gene Codes Corp., Ann Arbor, MI). Sequences have been deposited in the GenBank database (accession numbers AY072024 to AY072033). Analyses of molecular data. Presence or absence of re­ striction digest banding patterns of given sizes were scored 1 or 0, respectively. A similarity matrix was calculated using the Simple Matching Coefficient in the NTS YS-PC program (ROHILF 1993). To visualise relationships between isolates, a dendrogram was constructed by SAHN clustering method using UPGMA (unweight pair-group method using arithme­ tic averages) in NTSYS-PC. Alignment of nucleotide sequences was conducted in CLUSTALX (THOMPSON et al. 1997) using default settings 301 for GOP and GEP during three consecutive passes (realign­ ments). The resulting alignment was optimised manually to maximise positional homology using BioEdit Sequence Alignment Editor version 5.0.7 (HALL 1999). Six putative Inonotus sequences from the Northern Hemisphere were down­ loaded from GenBank and included in our analyses: /. cuticularis (GenBank accession number AF237730); /. glomeru­ lus (Peck) Murrill (AF247968); /. radiatus (Fr.) P. Karst. (AF237732); /. rheades (Pers.) Bond. & Sing. (AF237731); /. tomentosus (Fr.) Teng (AF237729); and /. circinatus (Fr.) Teng (AF237728). The latter sequence, however, has been renamed /. leporinus (Fr.) Gilb. & Ryv. as recommended by its author (La Flamme, pers. comm.). Two Phellinus igniarius (L.: Fr.) Quél, sequences were also downloaded for comparison purposes (accession number AF56192 and strain KCTC6227 accession number AF110991). A sequence from Phylloporia ribis f. ulicis [Phellinus ribis f. ulicis as appears in GenBank] (AF200237) was used as outgroup. This choice was suggested by traditional taxonomic studies (FIASSON & NIEMELÁ 1984, CORNER 1991) and the phylogenetic analysis of KIM & JUNG (2000). Phylloporia ribis is a monomitic taxon with partly perennial basidiocarps, intermediate between Inonotus s. I and Phellinus s. I. (RYVARDEN 1978, JÜLICH 1981, RYVARDEN & GILBERTSON 1993, WAGNER & FISCHER 2001). Nucleotide sequences were analysed using maximum-parsimony in PAUP* (SWOFFORD 1998). Pairwise nucleotide differences of aligned sequences were calculated using distance matrix option. An heuristic search was conducted via treebisection-reconnection (TBR) branch-swapping algorithm, starting trees were obtained via stepwise addition (random sequence addition of 100 replicates). All characters had equal weight and were unordered, gaps in the alignment were treated as fifth base, regions ambiguously aligned were excluded from the analyses, MaxTrees was set on auto-increase by 100, Multrees was in effect and zero length branches were set to collapse to yield polytomies. A data subset was analysed via a branch-and-bound search and furthest addition sequence. Branch robustness was evaluated from 1000 bootstrap (FELSENSTEIN 1985) replicates with the same settings as in the maximum-parsimony analyses. Sequence alignments have been deposited in TreeBASE (study number S748, matrices accession numbers Ml 182, Ml 183, Ml 184). Results Cultural characters. Cultural characters of the isolates examined are summarised in Tab. 1. After 2 weeks of incubation, the majority of the isolates covered the Petri dishes, or at least 70 % of the surface, with the exception of isolates 0744 (Inonotus patouillardii) and 0994 (Ptychogaster cubensis) which covered 59 % and 47 % of the agar surface, respectively. The production of a soluble pigment that coloured the reverse of the Petri dishes was a common feature among all isolates, with the exception of cultures 2203 (I. rickii) and 1500 and 1508 ©DGfM 2002 302 Inonotus s.l. in Argentina Tab. 1: Cultural data of Argentine Inonotus isolates. CD, colony diameter in cm. AR, coloration on the agar reverse. Chi, chlamydospores. T, tramal setae. F, fiber-like hyphae. P, plechtenchymatous cells. +, present. -, not detected. Isolates in bold type were selected for sequencing. (I. jamaicensis). Mycelial development of isolates ranged from submerged to floccose. Chlamydospores were produced only by I. rickii and Pty. cubensis isolates and by strain 0624 (la­ belled as Inonotus sp. from Buenos Aires) for which no vou­ cher specimen was available. Those cultures also exhibited tramal setae production in vitro, with the exception of isolate 2211, labelled Pty. cubensis. Isolates 0193 (7. quercustris) and 0744 (I. patouillardii) failed to produce tramal setae in cul­ ture although their corresponding basidiomes had long, straight, lanceolate, brown tramal setae (see below). No ventricose setae were observed in any culture. Fibrous hyphae were only observed in isolates 1500 and 1508 (I. jamaicensis), and were rarely branched, with thickened, refringent walls, 1-4 µm ©DGfM 2002 diam. Two I. rickii isolates, 0001 and 2214, together with iso­ late 2211 developed plectenchymatous crust lines formed by irregular cells resembling a jig-saw puzzle. PCR- RFLPs. The size of amplified ITS region with primers ITS-5 and ITS-4 was 700-750 bp. No evident length variation was observed with exception of isolates 1400 and 1499 (I. crustosus), for which a product of about 100 bp longer was obtained. Restriction of ITS region using endonucleases AM, HaeIII, HhaI, HinfI, MboI and Rsal produced 3,5,4,3,4 and 5 distinct banding patterns, respectively. In most cases, there was a good agreement between size estimates for the undiges­ ted PCR products and the sum of fragment sizes, but some Mycological Progress 1 (3) / 2002 anomalies were observed: for instance, an extra band of 600 bp was obtained with Haelll in some I. rickii isolates (numbers 0013 and 0239) and Pty. cubensis isolates (numbers 2201, 2204,2213,2214,2205 and 0994). ITS-RFLPs patterns could not distinguish between the teleomorph, I. rickii, and the anamorphic state Pty. cubensis. Isolates 2339 and 0744, both labelled as I.patouillardii, have different banding patterns, and thus clustered separately from each other in the dendrogram (not shown). On the basis of ITS-RFLPs patterns, all the presumed morphospecies described below could be clearly distinguished. Analyses of nucleotide sequences. Ten isolates (identified in bold type in Tab. 1) were selected for ITS sequencing. Sequence data indicate that the G+C content in the ITS region from Argentine Inonotus s. I. ranged from 41.82 % in isolate 0193, to 48.86 % in isolate 1508. Sequence distance between Inonotus s. I. ranged from 6.4 % to 49.2 % in ITS 1, and from 1 % to 45.5 % in ITS 2. After eliminating the 18S 3'end and the 28S 5'end, the ITS region, including the 5.8S gene, ranges from 685 bp (isolate 1508) to 725 bp (isolate 2213). The I.patouillardii isolates, 0744 and 2339, shared 77 % similarity in their ITS region, whereas the latter and I. quercustris 303 isolate 0193, shared 93 % similarity in the same region based on direct comparison of sequence data. An insert of ca. 70 bp in the ITS 1 of isolate 1499 (I. crustosus) was unique in our data set and was excluded from the analysis. Complete data from the ITS 1 region were not obtained for isolates 0001 (I. rickii), 1400 (I. crustosus) and 1500 (I. jamaicensis) and thus these taxa were removed from some analyses. When the Argentine Inonotus s. 1. sequences, plus Phellinus igniarius and Phylloporia ribis were analysed, the data matrix contained thirteen 5.8 S-ITS 2 sequences (ITS 1 was not included due to the presence of many ambiguous residues). This matrix consisted of 442 characters of which 66 were excluded from the analysis due to ambiguous alignment and 95 were parsimony-informative. The search found 13 most parsimonious trees of length 196 (CI = 0.8673; RI = 0.9004), the strict consensus gene tree is depicted in Fig. 1, along with bootstrap statistical support for branches. At least 3 lineages (numbered 1-3) could be distinguished in the gene tree (Fig. 1). Lineage 1 has 100 % bootstrap support and is composed of three taxa, I. rickii-Pty. cubensis, I. patouillardii and I. quercustris. In vivo tramal setae production was a common feature among members of this lineage, with the exception of culture isolate 2213 which was obtained from a conidiome lacking ©DGfM 2002 304 Inonotus s.l. in Argentina Fig. 2: Gene phylogeny from ITS nucleotide sequences of Southern and Northern Hemispheres hymenochaetoid taxa. The tree is the single most parsimonious gene tree derived from complete ITS region. Values above branches are confidence levels after 1000 bootstrap replications. * branch that collapsed in the bootstrap analysis. In bold type are presented the taxonomical schemes discussed in the text. setae. Nonetheless, culture isolate 2213 formed tramal setae in vitro (Tab. 1). Internal branches within this clade had lower support. For instance, sequences from three chlamydospore forming culture isolates 0001,0013 (I/. rickii) and 2213 (Pty. cubensis) and a non-chlamydogenic isolate 0193 (7. quercustris) formed a polytomy (bootstrap = 58 % ) . I. patouillardii isolates 2339 and 0744, appear to be not monophyletic. Being moderately well supported (bootstrap = 80 % ) , lineage 2 is composed of two I. crustosus and two Phellinus igniarius sequences, each pair showed 100 % bootstrap support. Monophyly between lineages 1 and 2 was moderately well supported (bootstrap = 79 %). On the other hand, lineage 3 which comprises only I. jamaicensis isolates, was poorly supported as sister group of lineages 1 and 2 (bootstrap = less than 50 %). Lineage 1 was re-analysed by adding sequence data from the ITS 1 region. The data subset was composed of 6 taxa and 761 characters. Isolate 0001 was excluded from this analysis due to sequence ambiguities. As a result, 66 parsimony-infor- mative characters yielded, after an exhaustive search, a single gene tree (Fig. 1, right side) of length 331 (CI = 0.9486, RI = 0.7671). The monophyletic origin of isolates 0013 (I. rickii) and 2213 (Pty. cubensis) was fully supported (bootstrap = 100 % ) . The placement of isolate 2339 (I. patouillardii) as a putative sister taxon to the former clade was poorly supported (bootstrap = 63 % ) . Notwithstanding, in this analysis the location of/, quercustris 0193 as a sister taxon was fully supported (bootstrap = 100 %). Nucleotide sequences from complete ITS regions (ITS 15.8 S-ITS 2) belonging to European Inonotus s. I. isolates (i.e.: I. cuticularis, I. glomeratus, I. leporinus, I. radiatus, I. rheades and I. tomentosus) were retrieved from the GenBank and included into the analysis. A "hybrid" ITS complete sequence was constructed for I. crustosus with partially overlapping sequences from isolates 1400 and 1499. Thus, the final data matrix comprised 800 characters, of which 212 were ambiguous to align and were excluded from the analysis. From the re- Mycological Progress 1(3) / 2002 maining variable characters, 205 were parsimony-informative. The search yielded a single gene tree of length 642 (CI = 0.7259; RI = 0.7493) depicted in Fig. 2, along with bootstrap support for branches. The taxonomical scheme proposed by WAGNER & FISCHER (2001) is presented on a side of the gene tree. Three distinct lineages were obtained (named A-C): lineage A showed a similar composition to that of lineage 1 in Fig. 1, but including I. cuticularis with 100 % statistical support. I. glomeratus appeared as a sister taxon with good support (bootstrap = 86 %). Following WAGNER & FISCHER (2001) this lineage would represent the Inonotus s. str. clade. Lineage B, composed by I. jamaicensis and I. rheades (Inocutis according to FIASSON & NIEMALÁ 1984 and WAGNER & FISCHER 2001), showed 95 % bootstrap support, but was poorly related to the sensu stricto clade (bootstrap = 59 %). Within lineage C, sequences of dimitic Phellinus igniarius formed a clade fully supported (bootstrap = 100 %, Phellinus s. str. clade). The "hybrid" sequence of I. crustosus appeared as a sister group to Phellinus s. str. as also shown in Fig. 1, but with higher confidence level (bootstrap = 99 %). The two isolates identified as I. tomentosus and I. leporinus (Onnia according to JAHN 1978 and WAGNER & FISCHER 2001) sampled from conifers, clustered together (100 % bootstrap support). With a lower statistical support (bootstrap = 84 %) I. radiatus (Mensularia, according to WAGNER & FISCHER 2001) was placed as a sister taxon of the above mentioned taxa. Morphological taxonomy Inonotus P. Karst., Medd. Soc. Fauna Fl. Fenn. 5: 39, 1879 [1880]. = Inoderma P. Karst., op. cit. 39 pr. p. = Inodermus Quél., Ench. Fung. p. 173, 1886 pr. p. = Phaeoporus Schroet., Cohn Krypt.-Fl. Schles. Pilze p. 489, 1888 pr. p. = Mucronoporus Ell. & Everh., J. Mycol. 5: 28, 1889 pr. p. = Polystictoides Lázaro, Rev. Real Ac. Cien. Madrid 14: 754. 1916 pr. p. min. = Mensularia Lázaro, op. cit: 736,1916 pr.p. min. =Polyporus Fr., Syst. Mycol. 1: 341,1821 pr. p. =Xantochrous Pat., Cat. rais. PL cellul. Tunisie p. 51, 1897 pr. p. = Xanthoporia Murrill, Mycologia 8: 56, 1916. Basidiocarp annual, resupinate, effused-reflexed, dimidiate, sessile or stipitate, usually light weighted, variable in size. Pileus simple or imbricate, tomentose or glabrous. Hymenophore poroid. Context rusty brown to cinnamon brown [10R 4/4-6-8, 3/6], fibrous to spongy or corky, darkening in KOH. Tubes usually concolorous, brittle when dry. Pores round to angular, often lacerate. Pilear surface usually anoderm. Hyphal system monomitic; generative hyphae branched and septate, without clamp connections, thin- to thick-walled. Tramal setae when present, straight, lanceolate, brown. Hymenial setae when present, ventricose or subulate, straight or curved, brown. Basidia with 4 sterigmata. Spores ovoid, ellipsoid, globose, smooth, typically light to dark brown. Lignicolous, causing either a uniform, mottled or a pocket white rot. Distribution: worldwide. 305 Type species. Inonotus hispidus (Bull.: Fr.) P. Karst., Krit. Finl. Basidsv. p. 330, 1889. Anamorph. Some species have an anamorph identified as Ptychogaster Pat. Inonotus ochroporus (Van der Bijl) Pegler, Trans. Brit. Mycol. Society 47: 183, 1964. Polyporus ochroporus Van der Bijl, in S. Afr. J. Sci. 18: 269,1922. = Daedalea fuscopora Lloyd in Van der Bijl, S. Afr. J. Sci. 21: 308, 1924. Basidiocarp dimidiate, flabelliform, up to 7-7.5 x 12 x 1-4.5 cm. Pileus applanate, broadly attached, single or imbricate, tomentose, yellowish brown to brown [7.5YR 5/6-8 to 3/2]. Hymenial surface yellowish turning ochraceous with age [2.5Y 7/4 to 10YR 5/4]. Context fibrous to somewhat soft, duplex in young specimens, light to dark golden brown [7.5 YR 6/8 to 4/4], 10-24 mm thick. Tubes concolorous, 1-7 mm long. Pores irregular to angular, 2-5 per mm. Margin blunt. Pilear surface anoderm. Generative hyphae thin-walled, hyaline to yellowish, 4-6 urn diam. Tramal setae in dissepiments and in the upper part of the context, and sometimes in the pileus surface, straight, lanceolate, 50-200 x 11-25 µ m. Hymenial setae few, ventricose, mostly 13-20 x 5-8 µ rn, but also 40-50 x 10-15 µ m. Basidia clávate, 10-15 x 5-8 urn. Spores ellipsoid, yellowish, slightly thick-walled, one straight side, 7-8(9) x 5-6.5 µm. Specimens examined. ARGENTINA: Chaco, San Bernardo, V-95, on Casuarina cunninghamiana, BAFC 33722. Corrientes, Capital, Riachuelo, 27-VI-74, BAFC 50291. San Luis, Merlo, XI-98, on Lithraea ternifolia, CORD. Remarks. This species, originally described from South Africa, closely resembles I. patouillardii, from which it is distinguished by having larger spores, the cutis structure and by the duplex consistency of the context. The last feature mentioned, can be difficult to observe particularly in old specimens (RYVARDEN & JOHANSEN 1980). Similarly, the presence of hymenial setae can be easily overlooked. For instance the CORD specimen was previously published under I. quercustris ( U R CELAY & RAJCHENBERG 1999) which exhibits tramal setae only. I. ochroporus was previously recorded for Argentina (Province of Corrientes) by POPOFF (2000). Inonotus patouillardii (Rick) Imazeki, Bull. Tokyo Sci. M u s . 6: 105, 1943. Polystictuspatouillardii Rick, Broteria 6: 89, 1907 (PACA!). Polyporus patouillardii (Rick) Rick, Broteria 4 [31]: 93, 1935. Basidiocarp dimidiate, flabelliform, sessile to pseudostipitate, 12-5 x 9-6 x 1 cm. Pileus convex ungulate, single or imbricate, yellowish brown to brown [7.5YR 5/6-8 to 3/2]. Hymenal surface cream coloured to dark yellowish brown to ferruginous [10YR 8/3-4 to 10YR 3/4 to 2.5YR 5/6-8, 4/6-8]. Context fibrous, with a satin lustre, dark brown [7.5YR 3/2 to 5YR 3/2-3] with lighter streaks, concentrically zonate, 2-20 mm thick; obtuse. Tubes dark reddish brown [5YR 3/2-3], up 306 to 13 mm long. Pores circular, becoming lacerate, 3-6(7) per mm. Margin sterile. Pilear surface formed by a single layer of hyphal terminations, arranged in a parallel disposition resembling a hymeniderm, but composed mainly of septate elements. Generative hyphae of two types: thin and thick- walled, dark brown. Tramal setae abundant in the dissepiments, lanceolate, dark brown, 80-250 x 7-12 µ m. Hymenial setae present or absent, when present few, ventricose, 11-25 x 6-9 µ m. Spores ellipsoid, pale yellow, thick-walled, 5.5-7 x 4-5 µ m. Specimens examined. ARGENTINA: Buenos Aires: Llavallol, Santa Catalina, 5-VI-73, BAFC 23983 (isolate 2339, GenBank AY072024); ibid, V-64, BAFC 23984. Chaco, San Bernardo, V-95, on Casuarina cunninghamiana, BAFC 33722. Corrientes, San Miguel, Estancia Curuzú Laurel, 14-VI-74, BAFC 50615; Corrientes City, Riachuelo, 27-VI-74, BAFC 50291. Entre Ríos, Concordia, Salto Grande, 13-III-73, on Ocotea acutifolia, BAFC 22764. Misiones: Puerto Libertad, Alto Paraná, 11-III-80, BAFC 28522; Cataratas, Iguazú Nat'l Park, 3-III-80, BAFC 26607 (isolate 0744, GenBank AY072028); ibid, 5-III-80, BAFC 26611; ibid, III-80, BAFC 26602. Tucumán: Tafí Viejo, 27-I-65, BAFC 50566; Tafí, Aconquija Park, Sierra San Javier, l-V-52, on Phoebe porphyra, BAFC 50619; Tafí, El Naranjal, 24-V-45, BAFC 50620. Paraguay: Alto Paraná, Reserva Biológica de Itaboá, XI-90, humid underbush, BAFC 34582. Brazil: Rio Grande do Sul, Sao Leopoldo, 1906, Polystictus patouillardii, holotype (PACA!). Remarks. There is some disagreement in Rick's descriptions of I. patouillardii (cfr. RICK 1907,1935,1960) concerning the spore dimensions and the presence of setae. The latter were only mentioned in the 1960 work. There is also disagreement regarding the presence or absence of hymenial setae according to different authors; for example, the materials reported by GILBERTSON (1976) from Arizona did not exhibit hymenial setae. In our case, three collections had hymenial setae, i. e.: BAFC 28522,50615 and 22764. The latter also failed to exhibit an hymenodermis-like type of pilear crust. Inonotus s.l. in Argentina VIII-73, BAFC 24350. Cuba, El Yunque, Underwood & Earle 1071, III-1903, on much decayed wood, holotype (NYBG!). Remarks. According to Ryvarden (pers. comm.) this species also occurs in Costa Rica and Panama. This is the first record of the species for Argentina. It is distinguished by the presence of hymenial setae and small spores. Inonotus quercustris Blackwell & Gilbertson, Mycotaxon 23: 286, 1985. Basidiocarp annual, dimidiate, sessile, 20 x 16 x 7-10 cm. Pileus circular to ungulate, single, glabrous, azonate, rusty brown to dark brown [10R 3/2-4 to 7.5 YR 3/2 to 5YR 3/2-3]. Hymenial surface cream coloured to yellowish brown [10YR 8/34 to 7.5YR 5/6-8]. Context brown to chocolate brown [5YR 2/2, 3/2-4], up to 40 mm thick. Tubes golden brown [7.5YR 6/8 to 10YR 6/6], often up to 10 mm, but sometimes up to 45 mm long. Pores conspicuous, 1-2 per mm. Margin rounded to blunt. Pilear surface anoderm. Generative hyphae hyaline, 5-7um diam. Tramal setae brown, straight, lanceolate, mostly parallel to the tubes, also present in the pileus surface, 100 x 5-11 µm, thick-walled (2 µ m). Hymenial setae absent. Spores pale yellow, slightly thick-walled, 7-10 x 5-7 µ m. Specimens examined. ARGENTINA: Córdoba, Cruz Chica, 24-II-71, on Schinus sp., BAFC 50289 (isolate 0193, GenBank AY072026). Entre Ríos, Gualeguay, Estancia El Retiro, 13-I-51, on Acacia caven, LPS 31336. Remarks. This species, originally described from Louisiana, USA (BLACKWELL & GILBERTSON 1985), is characterised by its base-tapered setal hyphae, the absence of hymenial setae and the large coloured spores. It closely resembles /. patouillardii and I. ochroporus but differs in spore dimensions and in the presence of tramal setae only. Our collections had slightly smaller spores, but otherwise fit well with the diagnosis. It was previously recorded from La Rioja in Argentina Inonotus pertenuis Murrill, North American Flora 9: 87, 1908. by URCELAY & RAJCHENBERG (1999). Polyporuspertenuis (Murrill) Sacc. & Trott., Syll. Fung. 21: 273, 1912. Inonotus rickii (Pat.) D. A. Reid., K e w Bull. 12: 141, 1957 Basidiocarp resupinate, dimidiate to imbricate, 3 x 5 x 2 cm. Pileus sometimes striate, hispid-squamulose, yellowish brown [7.5YR 5/6-8]. Hymenial surface ferruginous to dark yellowish brown [2.5YR 5/6-8,4/6-8 to 5YR 4/6-8]. Context light brown to yellowish brown [7.5YR 5/4 to 5/6-8], corky, 1-15 mm thick. Tubes ferruginous [2.5YR 5/6-8, 4/6-8], fragile, 1-5 mm long. Pores circular, entire to lacerate, 5-7(10) per mm. Margin very thin, lobed. Pilear surface anoderm. Generative hyphae light brown, 3-7 µ m diam. Setal hyphae in the trama dark brown, thick-walled, somewhat branched, 4-7.2 µm diam. Tramal setae absent. Hymenial setae ventricose, straight, 18-45 x 5-16.5 µrn. Spores smooth, hyaline to pale ferruginous, ovoid to ellipsoid, 4.3-6.5 x 3-4.5 µm. Specimens examined. ARGENTINA: Buenos Aires: Delta, INTA, 31-VIII-73, on dead Quercus or Fraxinus, BAFC 50616; Buenos Aires City; Ciudad Universitaria, 30-I-99, on Pinus sp., BAFC 50164. Corrientes, Empedrado, Empedrado River and Hway 12, 31- Xanthochrous rickii Pat., Bull. Soc. Mycol. Fr. 24: 6, 1908. Polyporus rickii (Pat.) Sacc. & Trott, Saccardo 21: 270,1912. = P. (Inonotus) rickii (Pat.) Sacc. & Trott. f. sp. negundinis J. E. Wright & Iaconis, Rev. Invest. Agr. 9: 100, 1955. = P. hispidus Speg. (non Fr.), Bol. Acad. Nac. Cs. Córdoba 11: 163, 1887. = P. fuscobadius Bres. sec. Speg., Bol. Acad. Nac. Cs. Córdoba 25: 22,1921 (NYBG!). = P. calcuttensis Bose, Ann. Mycol. 23: 179, 1925. = P. corruscans Fr. sec. Speg., Bol. Acad. Nac. Cs. Córdoba 28: 372, 1926. Basidiocarp sessile, dimidiate to imbricate, usually 1 5 x 7 cm. Pileus applanate or ungulate, slightly glabrescent, hispid, yellowish to brownish to dark brown [10YR 6/6-8, 5/4-8 to 7.5YR 5/6-8, 3/2]. Hymenial surface cream coloured to ferruginous [10YR 8/3-4 to 2.5YR 5/6-8]. Context often dark brown [7.5YR 3/2 to 5YR 3/2-3], lighter towards the margin, brittle, radiate, zonate, fibrous, 10-75 mm thick. Tubes concolorous, fragile when dry, 1-20 mm long. Pores angular, entire to lacerate, 2-5 per mm. Margin obtuse, glabrous, yel- Mycological Progress 1(3) / 2002 lowish-grey [5Y 7/2-3]. Pilear surface anoderm. Generative hyphae hyaline to yellowish, 3-4 µm diam. Tramal setae present or absent, when present straight, lanceolate, 65-500 x 8-12 µm. Hymenial setae abundant, ventricose, reddish brown, 20-30 x 6-12 µ m. Spores abundant, coloured when fully mature, yellowish to ochraceous, ovoid to broadly ellipsoid, smooth, 6-8 x 4-7 µ m. Chlamydospores immersed in the trama and over the pileus surface, pyriform, globose or irregular, forming small chains, 9-15 µ m, thick-walled (2-3 µ m thick). F o r m a anamorphosis Ptychogastercubensis Pat., Bull. Soc. Mycol. Fr. 12:133,1896 = Ceriomyces stuckertii Speg., An. Soc. Cient. Arg. 47: 265, 1899 Hemispheric to pulvinate, ferruginous brown [2.5YR 5/6-8, 4/6-8 to 10R 3/2-4] when dry, formed by a velvety cortical layer and by concentric layers of tramal hyphae and chlamydospores. Hymenial setae absent. Specimens examined. ARGENTINA: Buenos Aires City: Belgrano, 9-V-94, on Platanus sp., BAFC 34121; Thames 975, 18-IV94, on Platanus sp., BAFC 34113 (isolate 2205); Facultad de Agronomía, IV-87, on Casuarina cunninghaniana, BAFC 31046 (isolate 0013, GenBank AY072025); Carranza 2158, IV-94, on Acer sp., BAFC 34116 (isolate 2209); Llerena 2896, 10-V-94, on Platanus sp., BAFC 34111; Lezama Park, 5-V-94, on Celtis australis, BAFC 34124 (isolate 2203); Echeverría 3155,29-IV-1950, on Acer sp., LPS 31206; Echeverría 3159,25- III-1951, on Acer negundo, LPS 31204. Buenos Aires: Acassuso, Estrada 1039, 20-VI-69, on A. negundo, BAFC 23979; Llavallol, Santa Catalina: 6-V-93, BAFC 33145; ibid., 22-V-72, on C. spinosa, BAFC 23980; ibid., 6-II-76, on C. spinosa, BAFC 24104; Lomas de Zamora, 25-V-69, on Lippia citriodora, BAFC 23977; Camino Gral. Belgrano-Pila, 10-IV-71, BAFC 23978 (isolate 0239). Córdoba, La Población near Yacanto, 25-II-79, on C. tala, BAFC 50288. Corrientes, Santo Tomé, Estancia Europa, near River Uruguay, VI-65, on rotted stump, BAFC 24332 (isolate 0001, GenBank AY072032). Entre Ríos: Gualeguay, 23-II-73, BAFC 24333; Rosario del Tala, Caseros, Palacio San José, 14-II-73, on A. negundo, BAFC 22778. Tucumán, VIII-1918, sub P. corruscans Fr., LPS 18614. Chile, leg. C. Spegazzini, det. Bresadola (sub Polyporus), 1919, BAFC 50290. Anamorph: Buenos Aires City: Saavedra, l-V-79, on Acer sp., BAFC 50293; Armenia 1450, IV-94, BAFC 34123 (isolate 2201); Virrey del Pino 3500,4-V-94, on dead Platanus sp., BAFC 34125 (isolate 2214); Vidal 2050, 20-IV-48, on A. negundo, LPS 31235; Echeverría 3024,29-IV-1950, on A. negundo, LPS 31238; Mendoza 2900, on Acer sp., LPS 31203; Azcuénaga 1721, 31-X-1951, on A. negundo, LPS 31240; Piyol and Méndez de Andes, on A. negundo, LPS 31242; Avellaneda Park, 12-V-94, on Acacia melanoxylon, BAFC 34115 (isolate 2213, GenBank AY072027); Lezama Park, 15-V-94, on C. australis, BAFC 34114 (isolate 2204). Buenos Aires: La Plata, Parque Facultad de Agronomía, 18-IV-72, on Acer sp., BAFC 23981; Martínez, 29-IV-63, BAFC 23982. Philippines: Mindanao, Worcester, VIII-1912, Polyporus fuscobadius, cotype (NYBG!). Isolates with no voucher specimens available, determined by WRIGHT & IACONIS (1955) as /. rickii: Buenos Aires, on A. negundo, isolate 0782; as Ptychogaster cubensis: isolate 0462; isolate 0467 on Platanus sp. and isolate 0994 from La Plata, on A. negundo. From Buenos Aires City, Guatemala 5016, IV-94, on Acer sp., labelled as Pty. cubensis, isolate 2211. Remarks. Chlamydospore production is widespread throughout the Basidiomycetes, especially in culture (KEND- 307 RICK & WATLING 1979). It is a helpful character to distinguish between morphologically similar species, but has little value for inferring natural relationships at higher taxonomic levels (NOBLES 1958, STALPERS 1978, MONCALVO, W A N G & HSEU 1995a,b). The link between Inonotus rickii and the anamorphic fungus, Ptychogaster cubensis, was first suggested by PATOUILLARD (1908), and was later experimentally confirmed on the basis of culture studies (DAVIDSON, CAMPBELL & W E BER 1942, WRIGHT & IACONIS 1955, STALPERS 2000). Thus, Inonotus rickii constitutes an easily recognisable species due to chlamydospore production both in vivo and in vitro. This feature was also detected in Inonotus niduspici Pilát ( G I L BERTSON & RYVARDEN 1986, Ryvarden pers. comm.). Nonetheless, neither PILAT (1953) nor PEGLER (1964b) mentioned the existence of chlamydospores in the latter species. Concerning the anamorph of I. rickii, STALPERS (2000) pointed out (:178) that Ptychogaster fici Pat. is the anamorph of the former, which he synonymized with Pty. cubensis. However, upon describing the latter he stated that Pty.fici deviates from Pty. cubensis precisely in the main features that characterise it. As a consequence, we do not consider Pty.fici as a synonym of Pty. cubensis, until material of the former can be studied. A newformae specialis of Inonotus rickii was published as a physiological adaptation to a particular substrate, Acer negundo, by WRIGHT & IACONIS (1955). In contrast, PEGLER (1964b) regarded this variation within the range of l. rickii. The same consideration is maintained in this study. Later on, PEGLER (1967) synonymized P. calcuttensis with I. rickii and stated that setal hyphae occur on the pileus surface of both tropical American and Indian specimens. Setal elements were found in the pileus surface of some of our materials (e. g. BAFC 31046, 34121, 24332). Inonotus rickii was previously recorded for Chaco Prov. by POPOFF (2000), and for Misiones by IBAÑEZ (1995) but in the latter case, under an erroneous name (I. patouillardii). Inonotus serranus Robledo, Urcelay & Rajchenb. Mycologia (submitted). Basidiocarp effused to effused-reflexed, up to 9 x 7 x 2 cm. Pileus, of the reflexed portions, tomentose to glabrous, separated from the context by a thin, black line; dark brown [7.5YR 3/2 to 5YR 3/2-3]. Hymenial surface dark brown, glancing, becoming yellowish [7.5YR 5/6-8 to 3/2] towards the margin. Context brown [10R 4/4-6-8, 3/6] corky, up to 3 mm thick. Tubes concolorous, up to 6 mm long. Pores angular, with entire dissepiments, 4-6 per mm. Margin velutinate, concolorous with hymenial surface. Generative hyphae hyaline and thin-walled, or chestnut and thick-walled, simple septate, 2.4-6(7) µrn diam. Tramal and hymenial setae absent. Basidia broadly clávate to ovoid, 9.6-12.8 x 6.4-7.2 µm. Spores ellipsoid to ovoid, with one straight side, thick-walled, pale golden brown, 5.5-7.2 x 4-5 µ m. Specimens examined. ARGENTINA: Córdoba, Quebrada del Condorito, Pampa de Achala, Depto. de San Alberto, Robledo # 3, 19-III-00, on dead trunk of Polylepis australis. 308 Remarks. The taxon was described solely from altitudinal grasslands of Córdoba Prov. According to Robledo, Urcelay & Rajchenberg (unpubl.) the presence of a distinct black line separating tomentum and context, in combination with annual to biannual habit, the lack of setae and the host substrate, Polylepis australis, distinguish this taxon from other Inonotus. Additionally it differs from Phellinidium (Kotl.) Fiasson & Niemelä by the absence of hyphoid setae. Inonotus texanus Murrill, Bull. Torr. Bot. CI. 31: 597, 1904 (NYBG!) Basidiocarp sessile, effuse to dimidiate, convex to ungulate, 6-7 x 4-5 x 1.5-4.5 cm. Pileus circular, glabrous, concentrically cracking, dark reddish brown to almost black [10R 2/12]. Hymenial surface cream coloured to dark yellowish brown [10YR 8/3-4 to 4/4]. Context corky, thin, 1-3 mm thick, golden brown [7.5YR 5/6-8], with a rudimentary granular core or lacking it. Tubes fragile, ferruginous to chocolate [10R 4/46-8, 3/6 to 5YR 2/2, 3/2-4], 10-30 mm long. Pores irregular, lacerate, 1-3 per mm. Margin markedly obtuse. Pilear surface anoderm. Generative hyphae thin to thick-walled, yellowish, 4-6 urn diam., branched or not. Tramal and hymenial setae absent. Spores abundant, yellowish, subglobose, smooth, thick-walled (1 urn thick), 7-10 x 6-7(8) µ rn. Specimens examined. ARGENTINA: Catamarca, Dept. Capayán, 22-III-95, BAFC 33667. Chubut, Los Alerces Nat'l Park, Lake Futalaufquen, Cerro Dedal, 9-V-97, on fallen branch oí Diostea juncea, BAFC 34591. La Rioja, Río La Carpintería, I-2001, on Acacia furcatispina, BAFC 50971. San Luis, Carpintería, IV-71, on Prosopis sp., BAFC 50287. Santiago del Estero, Dept. Copo, Reserva El Copo, III-86, on Schinopsis sp., BAFC 30686. U. S. A.: Texas, Austin, leg. Underwood, 24-XI-1891, on mesquite? holotype (NYBG!). Remarks. Ryvarden (pers. comm.) stated that the context of this species exhibits a distinct granular core. However, MURRILL (1904) made no comments on the presence of this feature. Furthermore, PEGLER (1964b) explicitly stated the absence of such a structure, and our specimens agree with that. The type material is in poor condition, only small fragments of the context remain, and thus it was impossible to check for the existence of a granular core. Specimens studied showed homogeneous morphological characters, i. e. a thin context, large tubes and pores, and identical spore dimensions (7-8 x 5-6 urn). Inonotus texanus and I. jamaicensis {Inocutis, see below) have some features in common, that are an homogeneous context without tramal setae, an hymenial layer lacking hymenial setae and in some cases, a granular core more or less developed. On the other hand, I. texanus is distinguished by its larger spores. Related taxa Inocutis Fiasson & Niemelä, Karstenia 24: 24, 1984. Type species. Polyporus rheades Pers., Mycol. Eur. 2: 69, 1825. = Inonotus rheades (Pers.) Bond. & Sing., Ann. Mycol. 39: 56, 1941 (L!). Inonotus s.l. in Argentina Remarks. Although the genus Inocutis is characterised by having a distinct granular core (KOTLABA & POUZAR 1969, 1970), at present, no such structure was observed in the lectotype material. Another characteristic feature is the total absence of setae. Inocutis jamaicensis (Murrill) Gottlieb, Wright & Moncalvo comb. nov. Bas.: Inonotus jamaicensis Murrill, Bull. Torr. Bot. Club 31: 597, 1904 (NYBG!). = Polyporus jamaicensis (Murrill) Sacc. & Trott, Syll. Fung. 21: 274, 1912. = Polyporus rheades Pers. var. cognatus Bres., Ann. Mycol. Berl. 18: 34, 1920 (S!). = P. cognatus (Bres.) Speg., Bol. Acad. Nac. Cs. Córdoba 28: 371-372, 1926. = Inonotus rheades (Pers.) Bond. & Sing. var. cognatus (Bres.) Pegler, Trans. Brit. Mycol. Soc. 47: 188,1964. = Inonotus ludovicianus (Pat.) Murrill, Southern Polyp., p. 41, 1915 (isotype? FH!). = Polyporus hispidusBull: Fr. var. minorRick, Iheringia Bot. 7: 231, 1960 (PACA!). = Inonotus venezuelicus Ryv., Mycotaxon 28: 259, 1987 (NYBG!). Basidiocarp annual, dimidiate to triquetrous to resupinate, sometimes effused-reflexed, sessile, simple or imbricate, attached by a broad base, 3-8 x 2-5 x 1.5-2.5 cm. Pileus subungulate, convex, velutinate, yellowish to brownish [10YR 6/6-8 to 5/48]. Hymenial surface dark brown [7.5 YR 3/2 to 5YR 3/2-3]. Context fibrous, dark reddish brown to chocolate brown [5YR 2/2, 3/2-4], 1-25 mm thick. Tubes concolorous, 1-25 mm long. Pores polygonal to irregular, 3-4(5) per mm. Margin blunt to acute. Pilear surface formed by agglutinated hyphae, anoderm. Generative hyphae thin to thick-walled, yellowish to dark brown, 2.8-6 µ rn diam. Tramal and hymenial setae absent. Basidia and basidioles subglobose. Spores abundant, yellowish to reddish brown, ellipsoid to ovoid, slightly thickwalled, smooth, with one straight side, 1-2 guttulate, 6(7) x 4-5 µrn. Specimens examined. Argentina: Buenos Aires: Alsina, Campo Los Pinos, 31-VIII-78, on dead Eucalyptus viminalis, BAFC 24384; ibid., BAFC 24385; Castelar, INTA, 24-VI-93, on trees, BAFC 33333; Magdalena, Estancia El Destino, 28-VIII-84, BAFC 30218; Otamendi, INTA-Delta, 27-VII-78, on rotten stump in Taxodium distichum woods, BAFC 24386; ibid., 31-VIII-73, on Quercus or Fraxinus, BAFC 23985; Ramallo, Fiplasto, 22-VIII-78, on Eucalyptus stump, BAFC 24383; La Plata, Villa Elisa, 26-V-1921, on Eucalyptus sp., LPS 21859; ibid., V-1921, on E. globulus, LPS 21860. Chubut: Lago Puelo Nat'l Park, near Los Trineos stream, 10V-96, on stem and branches of a dead Diostea júncea in Austrocedrus forest, BAFC 34575 (isolate 1500, GenBank AY072033); Futaleufú, Los Alerces Nat'l Park, Lake Verde, 9-V-96, on fallen trunk of Lomatia hirsuta in Nothofagus forest, BAFC 34592 (isolate 1508, GenBank AY072029). Córdoba: near Yacanto, Quinta Mariska, II79, on ornamental plant, BAFC 27391; Alto San Pedro, Villa Giardino, 17-VII-2000, on rotted stump near conifers, BAFC 50689; Cordoba, 26-IV-1899, T. Stuckertn°6847, LPS 21857; ibid., T. Stuckert n°6889, LPS 21856; leg. C. Spegazzini, Polyporus rheades var. cognatus, holotype (S!). Mendoza: Tupungato, Ancón, 25-IV-48, BAFC 28767. Tucumán: Sierra San Javier, Anta Muerta, 25-I-65, on Bocconia pearcei or Tessaria absinthoides BAFC 50564; ibid, BAFC 50567; Sierra San Javier, 9-XII-50, on stump, BAFC 50621; Anta Muerta, alt 1000 m, 31-X-49, on Allophylus, Cedrela, Phoebe, Durantia, Prunus and Celtis, BAFC 23463. Brazil: Paraná, Gral. Carneiro, Fazenda Sao Pedro, 31-V-1989, on dead dicot tree, BAFC 309 Mycological Progress 1(3) / 2002 Identification key of South American morphospecies of Inonotus and related taxa 1 Setae present in context and tubes 2 Only tramal setae present; spores 7-10 x 5-7 2* Tramal and hymenial setae or only hymenial setae present 3 Tramal and hymenial setae present 4 Chlamydospores lacking µm I. quercustris 5 Spores ellipsoid, 5 . 5 - 7 x 4 - 5 µm; cutis resembling an hymeniderm I. patouillardii 5* Spores ellipsoid, 7-8(9) x 5-6.5 µm; cutis anoderm I. ochroporus 4* Chlamydospores in context and pileus, thick-walled, 9-15 µ m diam.; spores ovoid to broadly ellipsoid, 6-8 x 4-7 µm I. rickii 3* Only short ventricose to subulate hymenial setae present 6 Spores 9.2-10.8 x 6.6-8.2 µm; basidiome resupinate Ph. crustosus 6* Spores 4.3-6.5 x 3-4.5 µm; basidiome dimidiate to imbricate I. pertenuis 1 * Setae lacking 7 Context and tomentum separated by a black line, spores 5 . 5 - 7 . 2 x 4 - 5 7* Context not exhibiting a black line, but sometimes with a rudimentary granular core µm I. serranus 8 Spores ellipsoid to ovoid, 6(7) x 4-5 µm; basidiome triquetrous to effuse-reflexed to resupinate Inocutis jamaicensis 8* Spores 7-10 x 6-7 µm; basidiome dimidiate to ungulate I. texanus 34765; Rio Grande do Sul, Sao Leopoldo, Rick, 1938, on Prunus persica, Polyporus hispidus var. minor, holotype (PACA!). Jamaica, Mabess River, at 3.000 ft, Underwood, 23-IV-1903, on broad-leaf tree, Inonotus jamaicensis, holotype (NYBG!). U. S. A.: Louisiana, St. Martinville; AB Langlois 2741, 14-VII-1898, on the foot of a dead trunk, Polyporus ludovicianus Pat., isotype? (FH!). Uruguay: Canelones, XI-99, on E. globulus, BAFC 50377; Montevideo, C. Spegazzini, V-1914, LPS 21858. Venezuela, Mérida, Parque Nacional Sierra Nevada, on Polylepis sp., Inonotus venezuelicus, holotype (NYBG!). Remarks. I. jamaicensis was originally described as having a dimidiate, convex, sessile and broadly attached basidiome (MURRILL 1904).The species concept, under Inonotus, was later broadened to include effused-reflexed material (PEGLER 1964b). The synonymy with Polyporus hispidus var. minor was established by RAJCHENBERG (1987). More recently, RAJCHENBERG & WRIGHT (1998) recorded the species from Buenos Aires and Chubut provinces in Argentina and undertook a cultural study including oxidase reactions of three strains, of which only two are extant. From the study of the type of Polyporus rheades var. cognatus, we concluded that the Argentine collection, sent by Spegazzini to Bresadola for its identification, fits well with the concept of I. jamaicensis. The latter is very difficult to distinguish from Inonotus tenuicarnis Pegler & Reid, I. porrectus Murrill and I. ludovicianus. All these taxa lack setae and have coloured spores with overlapping dimensions [I. tenuicarnis: 4.6-6 x 3.5-4.3 µm; I. porrectus: 5-7 x 4-5 µm (6 x 4.4 µ m); I. ludovicianus: 5-7 x 3.5—4 µ m]. However, I. porrectus exhibits a peculiar pilear structure formed by somewhat branched and swelled hyphal endings, not seen in our specimens. We were unable to study the holotype of I. tenui- carnis, originally described from India, so the nature of the pileus remains to be checked. PEGLER (1964a) describe I. tenuicarnis having a crust formed of simple, unbranched, strongly agglutinated, erect hyphae, thus fitting in the concept of I. jamaicensis. In PEGLER's identification key (1964b) I. ludovicianus is separated from I. jamaicensis on the basis of their attachment to the substrate, which is a poor character. The isotype material of I. ludovicianus lacks a pilear layer, otherwise it is identical with I. jamaicensis. Phellinus Quel., Ench. Fung. p. 172, 1886. Type species: Phellinus igniarius (L.: Fr.) Quel, Ench. Fung., p. 172, 1886. Remarks. For a comprehensive description of the genus refer to LARSEN & COBB-POULLE (1990). Phellinus crustosus (Speg.) Gottlieb, W r i g h t & M o n calvo c o m b . nov. Bas.: Polyporus (Resupinatus) crustosus Speg., Bol. Acad. Nac. Cs. Córdoba 11: 64,1887. = Poria crustosa (Speg.) Speg. in Herbarium Spegazzini. = Inonotus crustosus (Speg.) J. E. Wright & J. R. Deschamps, Fl. Cript. Tierra del Fuego 11: 22, 1975. Basidiocarp effused, resupinate, coriaceous when fresh, rigid and fragile when dry, 4-10 cm diam. Context 1-2 mm thick, ferruginous clay colour to tobacco red [10R 4/1, 3/3-4]. Tubes concolorous, decurrent, oblique, 2-7 mm long. Pores lacerate, 2-4 per mm. Margin irregularly lobate, not reflexed, acute, strongly adhered to the subiculum,. Generative hyphae of two types: a) thin-walled, 2.6-A.6 µm diam., and b) thick-walled, 3.6-10 µ m diam., some conducting-like hyphae present, 310 4.1-7.7 urn diam. Tramal setae absent. Hymenial setae ventricose, some bent, 24-30 x 8.2-13.4 urn. Spores smooth, yellowish, thick-walled, broadly ellipsoid to ellipsoid or ovoid, 9.2-10.8 x 6.6-8.2 µ rn. Specimens examined. ARGENTINA: Neuquén: Lanín Nat'l Park, Lake Lácar, Quenil-hué stream, 10-IV-92, in Nothofagus dombeyi forest, BAFC 32836 (isolate 1400, GenBank AY072031); Lácar, Lake Queñí, 28-IV-96, on N. dombeyi, BAFC 50703 (isolate 1499, GenBank AY072030); Tierra del Fuego, Ushuaia, Estancia Moat, 26-II-88, BAFC 32865. Remarks. This is apparently a rare, endemic taxon known only from Southern Argentina. LOWE (1966) discarded the idea of this taxon belonging to Poria without making many comments; nowadays Poria is rejected as a nomen dubium (RYVARDEN 1991). Later on, WRIGHT & DESCHAMPS (1975) transferred the species to Inonotus but were unable to study other material than the holotype [Argentina, Tierra del Fuego, Ushuaia, Slogget Bay, on tree bark, C. Spegazzini, VI-1882 (LPS)]. Almost one hundred years after Spegazzini's collection, RAJCHENBERG (1993) recorded this species in the region for a second time. The species is characterised by resupinate basidiocarps, spore shape and dimensions, and by the presence of hymenial setae. Molecular data indicate that our specimens do not share a more recent common ancestor with Inonotus sensu stricto. The fact that the molecular analyses pointed to a close relationship with Phellinus and that two types of hyphae are distinguishable in the trama, which could be interpreted as a dimitic hyphal system, led us to consider its inclusion in Phellinus. Discussion This study reveals the presence of at least seven morphologically distinct taxa of Inonotus s. str. in Argentina, namely: Inonotus ochroporus, I. patouillardii, I. pertenuis, I. quercustris, I. rickii, I. serranus and I. texanus. Two additional taxa, formerly belonging to Inonotus, are presented under new combinations taking into account the evidence from molecular data, namely Phellinus crustosus and Inocutis jamaicensis. Regarding Inonotus texanus, we followed a conservative approach until new evidence would imply a different rapport, e.g. Inocutis. Cladistic analyses of morphological data combined with additional molecular data, might clarify the relationships between I. texanus, I. tenuicarnis and Inocutis jamaicensis. All of the species studied can also be distinguished from each other using PCR-RFLPs or nucleotide sequence data from the ITS region (Figs. 1, 2). In addition, both molecular and cultural data could discriminate between two morphologically similar collections (isolates 2339 and 0744) referred to as I. patouillardii. Therefore, our results suggest that these isolates probably represent two distinct species, although we were unable to differentiate them on the basis of morphological characters. This would point out that similarities in a complex pilear structure resembling an hymeniderm, are not necessarily indicative of a close taxonomic relationship. A Inonotus s.l. in Argentina similar case of convergence of morphological characters was also observed, for example, in Argentine species of Ganoderma (GOTTLIEB, FERRER & WRIGHT 2000) suggesting that it is not uncommon. Though discussion of taxonomical position of taxa not found in Argentina is out of the scope of this study, evidence from molecular data suggests that Inonotus glomeratus, which was originally described from North America, might be an Inonotus s. str. For a complete morphological description refer to PEGLER (1964b). Low inter-taxa sequence divergence was found between I. tomentosus and I. leporinus (Onnia according to JAHN 1978 and WAGNER & FISCHER 2001, yet Ryvarden accepts them in Inonotus, pers. comm.) differing only in 7 % of nucleotide positions. Similarly, a low divergence value was obtained between sequences of I. rickii and Pty. cubensis, which differ only in 3 positions. The latter result supports the connection between the teleomorph I. rickii and the anamorph Pty. cubensis, as previously established from cultural data (DAVIDSON, CAMPBELL & WEBER 1942, WRIGHT & IACONIS 1955, STALPERS 2000). Therefore, when basidiocarps or conidiomes are lacking, molecular data and other cultural characteristics appear to be useful for identification purpose in Inonotus. Consequently, we have assigned the unknown culture isolate 0624 to the I. rickii- Pty. cubensis group, on the basis of both chlamydospore production in culture and RFLP patterns. Incomplete homogenisation of ITS copies might be the cause for the presence of the 600 bp additional band obtained in the Haelll RFLP pattern of several I. rickii- Pty. cubensis isolates. Direct sequencing of the PCR product of I. rickii isolate 2213 produced an ITS sequence that lacks an Haelll target site, contrasting with the PCR-RFLP pattern observed. This suggests that, in the present case, PCR amplification combined with endonuclease digestion detected, at least, more than one type of ITS while only the ITS type lacking a Haelll target site was obtained by direct sequencing. On the other hand, the ca. 70 bp insert detected by direct sequencing of ITS 1 of isolate 1499 (Phellinus crustosus) is in agreement with the retarded migration of the amplified ITS fragment in agarose gels. The high level of ITS variability encountered among Inonotus species is similar, for instance, to that encountered in Trichaptum (Ko, HONG & JUNG 1997) where ITS dissimilarity between species varied from 14.6 - 53.5 % in ITS 1 and 15.6 - 61.2 % in ITS 2. This contrasts with the low level of ITS variation observed in other genera, e. g. in Ganoderma, where the highest distance values between Argentine species of different subgenera were 17.7 % in ITS 1 and 20.1 % in ITS 2 (GOTTLIEB, FERRER & WRIGHT 2000). If ITS divergence bet- ween taxa is indicative of divergence time, then our results would indicate that Inonotus has a similar age than Trichaptum and these two genera are much older than Ganoderma. In this study, we found a good correlation between morphological, cultural, and molecular data in distinguishing between Argentine species of Inonotus, but could not find Mycological Progress 1(3) / 2002 morphological or cultural characters that would support all of the lineages obtained from phylogenetic analyses of molecular data (Figs. 1, 2). For instance, the distribution of the hymenial setae over the gene tree (Fig. 1) does not follow any discernible pattern. In the taxonomic scheme proposed by FIASSON & NIEMELÄ (1984) while studying European Inonotus, the type of host substrate, the presence of a granular core in the basidiocarp, and the production of styrylpyrone (a compound related to hispidin), lack correlation with other characters at ordinal level, although those features were valuable in defining new genera. All of the Argentine specimens examined were collected from deciduous hosts and none exhibited a distinct well-developed granular core, thus these features were of little use. Surprisingly, the xanthocroic reaction tested on every specimen studied showed variation within species. In a number of cases the reaction was transient while in others the colour change was permanent. In addition, context colour changes ranged from deep purplish to almost black. Because of the variation observed, its taxonomic significance was not considered. The identification key presented in this paper constitutes the first practical tool for field identification of South American Inonotus species, at least in Argentina. Additional sampling of taxa and molecular data in a broader geographical scale is still necessary for a better understanding of the taxonomy, distribution, and morphological evolution of Inonotus species. In this perspective, this study indicates that nucleotide sequence variation in the ITS region might be appropriate to address these questions among closely related species, but data from slower-evolving genes would be more appropriate for revisiting the generic and family classification of Inonotus and related hymenochaetoid fungi. 311 BLACKWELL M, GILBERTSON RL (1985) A new species of Inonotus (Aphyllophorales, Hymenochaetaceae) on oak in Louisiana. -Mycotaxon 23: 285-290 CENIS JL (1992) Rapid extraction of fungal DNA for PCR amplification. - Nucleic Acid Research 20: 2380 CORNER EJH (1991) Ad Polyporaceas VII. 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In: Petersen, RH (ed.) Evolution in the higher Basidiomycetes, pp 3-24, Knoxville, USA DONK MA (1974) Check list of European Polypores. North Holland Publ. Co.-Amsterdam, London FELSENSTEIN J (1985) Confidence limits on phylogenies: an approach using bootstrap. - Evolution 39: 783-791 FIASSON JL, NIEMELÄ T (1984) The Hymenochaetales: a revision of the European poroid taxa. - Karstenia 24: 14-28 FISCHER M, AINSWORTH AM, WAGNER T (2001) Phellinus cavicola: a species not assignable to European subgroups of Phellinus s. 1. - The Mycologist 15: 16-18 Acknowledgments AMG and JEW wish to thank L Ryvarden for providing a still unpublished identification key of I. Inonotus species; ME Ranalli and collaborators for the use of the PCR apparatus; thanks are due to BO Saidman, C Besega, A Nudelman and E Canepa for kindly supplying many of the reagents used in this work; to M Rajchenberg for sending cultures BAFC 1400, 1499,1500 & 1508. This is publication number 144 of the PRHIDEB-CONICET. 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