Cystoderma, Cystodermella and Ripartitella in Atlantic Forest, São Paulo State, Brazil

(Cystoderma, Cystodermella and Ripartitella in Atlantic Forest, São Paulo State, Brazil). This paper reports on the genera Cystoderma, Cystodermella and Ripartitella from Atlantic Rainforest, Southeast Brazil. They are represented by Cystoderma chocoanum, Cystodermella contusifolia, C. sipariana and Ripartitella brasiliensis. Cystoderma chocoanum is reported for the fi rst time outside the type locality (Colombia) and its relationship with others species of Cystoderma, based on nLSU rDNA sequences, is discussed.


Introduction
The species from genus Cystoderma Fayod was separated in two distinct genera, Cystoderma s. str.and Cystodermella by Harmaja (2002), considering the amyloidity of basidiospores; previously unused differences or tendencies present in the genus, like 'harpoon' cystidia, arthrospores in fruit body and/or mycelium culture, liability to Squamanita, bryophily; nuclear DNA content, and the results of phylogenetic analysis.The phylogenetic analysis clusters Cystodermella close to Ripartitella Singer and Cystoderma to Floccularia Pouzar [F.albolanaripes (G.F.Atk.) Redhead].
Ripartitella was erected by Singer (Singer 1947) as a monotypic genus close to Cystoderma with the species R. squamosidisca (Murrill) Singer.According to Singer (1946) the two genera differ in three important characteristics: the covering layer structure of pileus and stipe, which is an epithelium in Cystoderma and a trichodermium in Ripartitella; the spore wall, always smooth in Cystoderma and echinulate in Ripartitella, and the eccentric position of the stipe in a large number of basidioma of Ripartitella, whereas Cystoderma is centrally stipitate.Singer (1949) considered only one species in the genus, reducing R. squamosidisca to synonym of R. brasiliensis (Speg.)Singer.The late species was based on Pleurotus brasiliensis Speg.collected in Apiaí, São Paulo State, by Puiggari (Spegazzini 1889).Later, R. sipariana (Dennis) Dennis (Dennis 1970), R. ponderosa (A.H.Sm. & Singer) Franco-Mol.(Franco-Molano 1993) and R. alba Halling & Franco-Mol.(Halling & Franco-Molano 1996) were added to the genus.Of these species, R. sipariana has smooth basidiospores, which exclude it from Ripartitella and is better classifi ed under Cystodermella, since the basidiospores are also inamyloid.Halling & Franco-Molano (1996) demonstrated that Cystoderma ponderosa A.H. Sm. & Singer has ornamented spores and no sphaerocytes at the pileipellis.Singer (1986) classified Cystoderma and Ripartitella in tribus Cystodermateae family Agaricaceae.However other authors as Thoen (1969), Heinemann & Thoen (1973) and Harmaja (1979) considered Cystoderma in Tricholomataceae without mention on Ripartitella while Pegler (1983) considered Cystoderma in Agaricaceae following Singer (1986) and Ripartitella in Tricholomataceae.The fi rst published paper with molecular data (Johnson & Vilgalys 1998) suggested the exclusion of tribus Cystodermateae from the family Agaricaceae, which was followed by Kirk et al. (2001).In further studies (Moncalvo et al. 2002) Ripartitella and Cystoderma (including C. granulosum transferred to Cystodermella) remained outside the clade Agaricaceae and the family Tricholomataceae was split in several clades without the presence of Ripartitella, Cystoderma and Cystodermella.The last edition of the "Dictionary of the Fungi" (Kirk et al. 2008) considers the three genera in Agaricaceae with the observation that studies with more species of these genera are required before they can be addressed to a defi nitive family.
In this paper the specimens belonging to these genera from Herbarium SP were revised, including a recent collection of a Cystoderma species that resembles C. amianthinum.The molecular analysis of the nLSU gene was also made, with the aim of ordering the Brazilian species and its relationship with C. amianthinum, a species essentially from temperate, not tropical region (Heinemann & Thoen 1973).

Material and methods
Sampling -The studied material was collected at Parque Estadual das Fontes do Ipiranga (PEFI), a forest reserve in the south of São Paulo City (23°39'S and 46°37'W), Reserva Biológica de Paranapiacaba (RBP), in Santo André City (23°46'S and 46°37'W), both remainings of Atlantic Rainforest in urban area, and Parque Estadual da Ilha do Cardoso, in the south of São Paulo State (25°10'S and 48°W), a preserved Rainforest.Morphological study -The microscopic analysis was made from dried material rehydrated in 70% ethanol, followed by 5% KOH and Melzer's reagent.The Q m represents the mean length/width quotient of the total spores measured.The colours of fresh material were compared with Küppers (1979) and specimens are deposited at Herbário do Estado Maria Eneyda P. Kauffmann Fidalgo (SP).
Molecular study -The nLSU rDNA sequences were used for phylogenetic analysis, in order to elucidate the relationship of C. chocoanum found in São Paulo, Brazil with sequences deposited in the GenBank (table 1).DNA extraction -Procedures for DNA extraction were according to an adapted protocol of Ferreira & Grattapaglia (1995) using lyophilized basidiomata previously grounded to a fine powder in liquid nitrogen.The sample was resuspended in 50 μL of TE, incubated at 37 °C for 30 min after the addition of RNase A (0.01 mg μL -1 ) and stored at -20 o C. PCR amplifi cation and DNA sequencing -The 5' end of the nLSU rDNA was targeted for amplifi cation.The nLSU region was amplifi ed using the primer set LR16 and LR0R (Moncalvo et al. 2000).PCR reaction, containing 0.5 U of Platinum® Taq DNA Polymerase -Brazil (Invitrogen, São Paulo City, SP, Brazil), 0.2 mM of each dNTP, 1 mM of MgCl 2 , 1% of polyvinylpyrrolidone (Sigma, St. Louis City, MO, USA) and 0.1 μM of each primer of the selected region in 50 μL, was performed in a Progene (Techne, Staffordshire, UK) thermocycler.The program was initiated by a 5-min denaturation step at 92 °C, followed by 40 cycles of 40 sec at 92 °C, 90 sec at 40 °C and 2 min at 72 °C.The polymerization was completed by a 5-min incubation at 72 °C.Amplifi cation products were electrophoresed in a 1.5% agarose gel containing 0.1 μg mL -1 ethidium bromide.PCR products were then purifi ed using PureLink PCR Purifi cation Kit (Invitrogen, São Paulo City, SP, Brazil).
DNA sequencing reactions were performed with the Applied Biosystems (ABI) BigDye Terminator Cycle Sequencing Kit v.3.1.in an ABI Prism 377 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instruction.The sample was sequenced in both directions with the same primers.The sequence was deposited in the GenBank.Data analysis -Initially, a blast search was conducted in the GenBank to compare the sequence of C. chocoanum with the existing sequence data.Phylogenetic analysis was done using nLSU sequence determined in this study with nine sequences available in the GenBank (table 1).
The sequences were analyzed using BioEdit version 7.0.5.3 (Hall 1999) and then automatically aligned in Clustal W (Thompson et al. 1994).The alignment was deposited in the TreeBase.Parsimony analysis was performed with PAUP version 4.0b10 (Swofford 2001).Most parsimonious tree was obtained by heuristic searches with simple sequence addition in 1,000 replicates, employing tree-bisectionreconnection (TBR) branch-swapping algorithm.Characters from the extreme 5` and 3` ends of the sequences were deleted from all taxa to obtain individual datasets that had identical start and end positions, gaps were treated as missing, all characters were unordered and equally weighted, multistate taxa interpreted as uncertainty, starting trees obtained via stepwise addition, one tree held at each step during stepwise addition, steepest descent option not in effect, initial MaxTrees was set to auto-increase, branches of zero length were collapsed (creating politomies), and MulTrees options in effect.
Branch and branch node supports were determined using 1,000 bootstrap replicates.Estimated levels of homoplasy and phylogenetic signal (retention and consistency indexes) were determined.Trees generated were rooted to Lepiota cristata Barla as the outgroup taxa.

Results and Discussion
Molecular analysis -Ten taxa plus the outgroup Lepiota cristata were aligned.The alignment dataset consisted of 1,480 characters, including gaps.Prior to analysis, 859 characters from 5' and 3' ends of the sequences were excluded.Out of the 621 characters included in the analysis, 279 characters were constant, 307 variable characters were parsimony-uninformative and 35 were parsimony informative.
The parsimony tree generated from nLSU sequence data using species of Cystoderma and Ripartitella revealed two major clades (fi gure 1).The upper clade, clustered C. granulosum and R. brasiliensis with 83% of bootstrap support.This was demonstrated before by Moncalvo et al. (2002) and utilized by Harmaja (2002)  In the analysis of Moncalvo et al. (2002), Floccularia albolanaripes (G.F.Atk.) Redhead (GenBank AF261380) appears as a sister group of Cystoderma with a low bootstrap support (< 50%).They presented no explanation for this fact because there are no obvious morphological relations between the two genera.This sequence appeared in the blast search made with the sequence of C. chocoanum obtained for this study, and, when added to the dataset analyzed, F. albolanaripes appears in the Cystoderma clade with a bootstrap support of 58% (data not shown).Floccularia Pouzar (Pouzar 1957) was placed in Amanitaceae mainly because of the bilateral lamellar trama and amyloid basidiospores.It comprises six species distributed in the United States and Europe, and F. albolanaripes is the only species with sequence in the GenBank.This species, according to Mitchel & Smith (1976, as Armillaria albolanaripes G.F. Atk.) does not have divergent hyphae in lamellar trama, has weakly amyloid basidiospores and do not have any kind of sphaerocytes at the pileipellis.
Habitat: Gregarious to caespitose on decaying wood.
Known distribution: Brazil (Bononi et al. 1981, Capelari 1989, Pegler 1997), Colombia (Franco-Molano 1993).Little morphological differences were observed between the Brazilian material and the original description for the Colombian material (Franco-Molano 1993).Macroscopically the pileus surface of the Brazilian collections is not areolate and the lamellae attachment is more decurrent than mentioned by Franco-Molano (1993).The basidiospores have comparable dimensions, but the pear-to-kidney shape mentioned was not observed in the Brazilian collections, all samples were ellipsoid.The others characteristics are in accordance.
Cystoderma chocoanum was previously registered from Brazil as C. amianthinum by Bononi et al. (1981), Capelari (1989) and Pegler (1997).The citations of C. amianthinum made by Singer (1969) for Argentine, Chile and Bolivia, and by Dennis (1961) for Venezuela, probably also represent C. chocoanum or another species, since, at least once, Singer (1969) said that the South American collections are close to the typical C. amianthinum, with differences at pileus surface and basidiospores.
Cystoderma austrofallax Smith & Singer, described from Chile is also close to C. chocoanum, differing in the pileus colour and small basidiospores (3.5-5 × 3 μm), strongly amyloid reaching a violaceous colour and a strong reaction of the pilear surface to intense ferruginous-orange with KOH (Singer 1969).
Molecular analysis with the North and South American species of Cystoderma will be necessary to show the relations between C. fallax, already mentioned by Franco-Molano (1993) as close to C. chocoanum, C. austrofallax and the others South American species, in order to confi rm the presence of C. amianthinum in South America.
Habitat: on soil.
This species was described based on material collected in Martinique.The Brazilian material agrees with the original description, except for pileus colour, that is very pale ochraceous, and the slightly bigger cheilocystidia (18-38 × 4-10 μm) mentioned by Pegler (1983).The identifi cation of the Brazilian material was confi rmed by Dr. Pegler and, as far as we know, it is represented only by the type and this collection.
Material examined: BRAZIL.SÃO PAULO: Cananéia, Parque Estadual da Ilha do Cardoso, Restinga do Pereirinha, 9-I-1990, M. Capelari et al. 3010 (SP).Pegler (1983) presents a description of this species and the only material that could be assigned to it, present at Herbarium SP, is completely contaminated by molds.Few observations were recovered.The pileipellis has sphaerocytes peculiar to Cystodermella, and basidiospores are smooth and inamyloid.Pegler (1997) cited four Brazilian materials under this species.From them, SP193723, also from Parque Estadual da Ilha do Cardoso is Lepiota abruptibulba Murrill (Capelari 1989) as mentioned under this late species (Pegler 1997, p. 38).The two materials from Parque Estadual de Campos do Jordão were not found at Herbarium SP and the last (Capelari et al. 3010) is very moldy.Until now the species has not been found again.
Ripartitella alba Halling & Franco-Mol., described from Costa Rica is very close to R. brasiliensis, differing according to Halling & Franco-Molano (1996) by the less squamulose to glabrous pileus, less and different pigmentation, smaller habit, wider spacing between lamellae, smaller spores, and smaller cystidia of R. alba.It is evident in macroscopic appearance of R. alba, according to published photographs of type material (Halling & Franco-Molano 1996, Halling & Mueller 2005) that it is quite different from R. brasiliensis.Bandala et al. (2005) mentioned R. alba as occurring in Mexico.But, by the macroscopic illustrations they presented, the pileus "initially covered with a more or less compact (interrupted), brownish-orange or dull brownish-orange, tomentosesquamulose layer breaking after pileus expansion…", and the absence of cheilocystidia (as in R. brasiliensis), it probably represents a collection of R. brasiliensis, not R. alba [compare also with the photography published by Ovrebo (1988)].Wartchow et al. (2007) mentioned R. alba for northeast Brazil.
to create the genus Cystodermella segregated from Cystoderma.Meanwhile, all other species of Cystoderma appears in the lower clade separated into two groups with 77% of bootstrap support.Cystoderma chocoanum from Brazil appears in the fi rst group with C. chocoanum from Colombia, in a clade with 100% of bootstrap support.It shows that the Brazilian species belongs to C. chocoanum, also confi rmed by the morphological analysis.C. jasonis and C. amianthinum clustered in the second group.

Table 1 .
Collection data and GenBank accession number of the taxa analyzed.Cladogram generated by parsimony analysis of partial LSU rDNA sequences.Bootstrap values ≥ 50% are shown above branches.GenBank accession numbers are shown after each taxon name.