the genus pluteus , typified by p. cervinus , includes saprobic agaricoid fungi characterized by free lamellae , no universal veil ( a partial veil may be present in a few species ) , pinkish spore - print , inverse hymenophoral trama , and inamyloid , cyanophilic spores ( vellinga & schreurs 1985 , orton 1986 , singer 1986 , vellinga 1990 , heilmann - clausen 2012 ) .
it belongs to the pluteoid clade as circumscribed by several authors on the basis of molecular data ( moncalvo et al .
the infrageneric taxonomy is based primarily on the characteristics of the hymenial cystidia and pileipellis .
historically , three sections based on morphological features are widely accepted ( homola 1975 , horak & heinemann 1978 , orton 1986 , singer 1986 , citrin & eyssartier 1998 , malysheva et al .
pluteus , with thick - walled ( metuloid ) pleurocystidia , usually with apical hooks , and pileipellis as a cutis ; sect .
hispidoderma with non - metuloid pleurocystidia , and a pileipellis consisting of elongated , filamentous elements organized either as a cutis , a hymeniderm or a trichoderm ; and sect .
celluloderma with non - metuloid pleurocystidia and a pileipellis composed of swollen clavate or spheropedunculate elements organized in a hymeniderm , with transitions to an epithelium . singer ( 1956 , 1958 , 1986 ) sometimes also recognized two subsections in section celluloderma : mixtini and eucellulodermini , differing respectively in the presence or absence of elongated cystidium - like elements in the pileipellis .
finally , vellinga & schreurs ( 1985 ) proposed an infrageneric classification for pluteus differing from singer s classificatory scheme only with respect to the placement of species with non - metuloid cystidia and a filamentous pileipellis : they reduced sect .
hispidoderma to a subsection of celluloderma , including only species with a hymenodermal or trichodermal pileipellis , and established sect .
this system has not been widely accepted by later authors ( e.g. banerjee 1992 , banerjee & sundberg 1993 , citrin & eyssartier 1998 , rodrguez & guzmn - dvalos 2001 ) .
justo et al . ( 2011a , b ) showed a general agreement between the morphological subdivision of pluteus and the molecular phylogeny they obtained , even though : ( 1 ) some species with non - metuloid pleurocystidia and a pileipellis which was a differentiated cutis ( formerly sect .
celluloderma , together with the species characterized by a hymenidermal pileipellis , or in sect .
celluloderma into subsections mixtini and eucellulodermini was not supported by the phylogenetic analyses of the molecular data ; species with a mixture of vesiculose - clavate and elongate elements in the pileipellis were not monophyletic and were distributed over sections celluloderma and hispidoderma . here
we fully describe , based on recent italian collections , the rarely reported pluteus variabilicolor , which was originally assigned to subsect .
a phylogenetic analysis of its sequences was performed to : ( 1 ) provide molecular insights on its status and taxonomic placement within pluteus ; and ( 2 ) assess whether this species , with a mixture of elongated and short elements in the pileipellis , was phylogenetically closer to species in sect .
the its sequence of the isotype collection was also obtained to confirm our identifications of the recent collections .
the optical microscope used was a nikon eclipse e400 trinocular with plan - achromatic objectives .
the primary mounting media used were : distilled water for the observation of the pigments , and ammoniacal congo red for the other structures .
spore measurements were based on 90 elements in ammoniacal congo red and randomly selected from three collections .
the following abbreviations are used : l = number of lamellulae between each pair of entire lamellae ; and q = the length divided by width of the spores in side view , qm being the average quotient .
descriptive terms used in the descriptions follow vellinga ( 1988 ) , and collection acronyms follow index herbariorum except that  tl  and  vm  refer to the personal reference collections of tomaso lezzi and vincenzo migliozzi .
genomic dna was isolated from 1 mg per specimen of four dried specimens ( tm20130521 - 01 , vm20111105 - 01 , vm20130504 - 01 and bp - fn 56936 ) using the dneasy plant mini kit ( qiagen , milan , italy ) .
amplification reactions were performed in a pe9700 thermal cycler ( perkin - elmer , applied biosystems ) in a 25 l reaction mixture using the following final concentrations or total amounts : 10 ng dna , 1pcr buffer ( 20 mm tris / hcl ph 8.4 , 50 mm kcl ) , 0.4 m of each primer , 2.5 mm mgcl2 , 0.25 mm of each dntp , 0.5 units of taq polymerase ( promega ) . the pcr program was as follows : 3 min at 95 c for one cycle ; 30 s at 94 c , 45 s at 50 c , 2 min at 72 c for 35 cycles , and 10 min at 72 c for one cycle .
pcr products were resolved on a 1.0 % agarose gel and visualized after staining with ethidium bromide .
the pcr products were purified with the ampure xp kit ( beckman ) and sequenced by macrogen ( seoul , republic of korea ) .
sequence assembly and editing were performed using geneious v. 5.3 ( drummond et al . 2010 ) .
the sequences are deposited in genbank ( www.ncbi.nlm.nih.gov/ ) under the accession numbers given in fig .
the sequences obtained in this study were combined with published pluteus its rdna sequences selected from genbank and unite ( http://unite.ut.ee ) databases on the basis of the greatest similarity based on blastsearch , outcomes of recent phylogenetic studies focused on pluteus ( justo et al .
2011a , b , pradeep et al . 2012 ) , and subsequent phylogenetic analysis ( preliminary trees not shown )
the sequence alignments were then imported into mega v. 5.10 ( tamura et al . 2011 ) for manual adjustment .
pluteus diettrichii ( hm562143 ) and pluteus seticeps ( hm562199 ) were used as outgroup taxa following justo et al .
best - fit models were estimated by both the akaike information criterion ( aic ) and the bayesian information criterion ( bic ) with jmodeltest v. 0.1.1 ( posada 2008 ) to provide a substitution model for the alignment .
phylogenetic analyses were performed using the bayesian inference ( bi ) and maximum likelihood ( ml ) approaches .
the bi was performed with mrbayes v. 3.1.2 ( huelsenbeck & ronquist 2001 ) with four incrementally heated simultaneous monte carlo markov chains ( mcmc ) run over 10 million generations , under the gtr+ evolutionary model .
trees were sampled every 1 000 generations resulting in an overall sampling of 10 001 trees ; the first 2 500 trees were discarded as  burn - in  ( 25 % ) . for the remaining trees ,
a majority rule consensus tree showing all compatible partitions was computed to obtain estimates for bayesian posterior probabilities ( bpp ) .
ml estimation was performed through raxml v. 7.3.2 ( stamatakis 2006 ) with 1 000 bootstrap replicates ( felsenstein 1985 ) using the gtrgamma algorithm to perform a tree inference and search for a good topology .
support values from bootstrapping runs ( mlb ) were mapped on the globally best tree using the  -f a  option of raxml and  -x 12345  as a random seed to invoke the novel rapid bootstrapping algorithm .
only bpp values over 0.70 and mlb over 50 % are reported in the resulting tree ( fig .
( 2012 ) ; pairwise % identity values of its sequences were calculated using mega 5.10 ( tamura et al . 2011 ) .
the optical microscope used was a nikon eclipse e400 trinocular with plan - achromatic objectives .
the primary mounting media used were : distilled water for the observation of the pigments , and ammoniacal congo red for the other structures .
spore measurements were based on 90 elements in ammoniacal congo red and randomly selected from three collections .
the following abbreviations are used : l = number of lamellulae between each pair of entire lamellae ; and q = the length divided by width of the spores in side view , qm being the average quotient .
descriptive terms used in the descriptions follow vellinga ( 1988 ) , and collection acronyms follow index herbariorum except that  tl  and  vm  refer to the personal reference collections of tomaso lezzi and vincenzo migliozzi .
genomic dna was isolated from 1 mg per specimen of four dried specimens ( tm20130521 - 01 , vm20111105 - 01 , vm20130504 - 01 and bp - fn 56936 ) using the dneasy plant mini kit ( qiagen , milan , italy ) .
universal primers its1f / its4 were used for the its region amplification ( white et al .
amplification reactions were performed in a pe9700 thermal cycler ( perkin - elmer , applied biosystems ) in a 25 l reaction mixture using the following final concentrations or total amounts : 10 ng dna , 1pcr buffer ( 20 mm tris / hcl ph 8.4 , 50 mm kcl ) , 0.4 m of each primer , 2.5 mm mgcl2 , 0.25 mm of each dntp , 0.5 units of taq polymerase ( promega ) . the pcr program was as follows : 3 min at 95 c for one cycle ; 30 s at 94 c , 45 s at 50 c , 2 min at 72 c for 35 cycles , and 10 min at 72 c for one cycle .
pcr products were resolved on a 1.0 % agarose gel and visualized after staining with ethidium bromide .
the pcr products were purified with the ampure xp kit ( beckman ) and sequenced by macrogen ( seoul , republic of korea ) .
the sequences are deposited in genbank ( www.ncbi.nlm.nih.gov/ ) under the accession numbers given in fig . 1 .
the sequences obtained in this study were combined with published pluteus its rdna sequences selected from genbank and unite ( http://unite.ut.ee ) databases on the basis of the greatest similarity based on blastsearch , outcomes of recent phylogenetic studies focused on pluteus ( justo et al .
2011a , b , pradeep et al . 2012 ) , and subsequent phylogenetic analysis ( preliminary trees not shown )
the sequence alignments were then imported into mega v. 5.10 ( tamura et al . 2011 ) for manual adjustment .
pluteus diettrichii ( hm562143 ) and pluteus seticeps ( hm562199 ) were used as outgroup taxa following justo et al .
best - fit models were estimated by both the akaike information criterion ( aic ) and the bayesian information criterion ( bic ) with jmodeltest v. 0.1.1 ( posada 2008 ) to provide a substitution model for the alignment .
phylogenetic analyses were performed using the bayesian inference ( bi ) and maximum likelihood ( ml ) approaches .
the bi was performed with mrbayes v. 3.1.2 ( huelsenbeck & ronquist 2001 ) with four incrementally heated simultaneous monte carlo markov chains ( mcmc ) run over 10 million generations , under the gtr+ evolutionary model .
trees were sampled every 1 000 generations resulting in an overall sampling of 10 001 trees ; the first 2 500 trees were discarded as  burn - in  ( 25 % ) . for the remaining trees
, a majority rule consensus tree showing all compatible partitions was computed to obtain estimates for bayesian posterior probabilities ( bpp ) .
ml estimation was performed through raxml v. 7.3.2 ( stamatakis 2006 ) with 1 000 bootstrap replicates ( felsenstein 1985 ) using the gtrgamma algorithm to perform a tree inference and search for a good topology .
support values from bootstrapping runs ( mlb ) were mapped on the globally best tree using the  -f a  option of raxml and  -x 12345  as a random seed to invoke the novel rapid bootstrapping algorithm .
only bpp values over 0.70 and mlb over 50 % are reported in the resulting tree ( fig .
( 2012 ) ; pairwise % identity values of its sequences were calculated using mega 5.10 ( tamura et al . 2011 ) .
both bayesian and maximum likelihood analyses produced the same topology ; therefore , only the bayesian tree with both bpp and mlb values is shown ( fig .
the its data matrix comprised 46 sequences ( including 38 from genbank and 4 from unite ) .
the three italian collections of p. variabilicolor and the isotype collection cluster with the three collections of p. castri ( holotype included ) forming a well - supported clade ( /variabilicolor ; bpp = 1 , mlb = 100 % ) within the pluteus leoninus clade of sect .
the clade is in sister position to /chrysaegis ( bpp = 1 , mlb = 100 % ) .
the clade consisting of /variabilicolor and /chrysaegis is sister ( bpp = 1 , mlb = 100 % ) to /leoninus (= the p. leoninus complex ) .
the p. leoninus complex includes collections identified as p. leoninus , p. roseipes , p. flavofuligineus , and two provisionally undescribed taxa ( pluteus aff .
10 : 470 ( 2011 ) . selected descriptions : babos ( 1978 : 193195 ) ; lohmeyer et al .
( 1994 : 9698 ) ; migliozzi ( 2011 : 47 ) ; justo et al .
( 1998 : 96 , 127 ) ; albert ( 2011 , two unnumbered figs ) ; migliozzi ( 2011 : 4 , 8) ; justo et al .
pileus medium - sized , 3080 mm broad , smooth , lined up to middle , colour distinctly yellow - orange , with central darker umbo , often radially rugulose - veined , especially in mature specimens , hygrophanous , clearly striate or not at margin .
lamellae free , quite crowded , ventricose , first white then pink , with the presence of lamellulae ( l = 02 ) , with concolorous or whitish , flocculose edge .
stipe 3070  412 mm , cylindrical , slightly enlarged at the base , streaked - fibrillose over the entire length , yellow , with reddish tinges at the base in mature specimens .
context white - yellowish , yellowish orange under the pileus surface , without distinctive smell and taste .
basidiospores 5.57.0  4.55.5(6.0 ) m , on average 6.0  4.9 m , q = 1.101.35 ; qm = 1.22 , broadly ellipsoid to subglobose , thin - walled .
cheilocystidia fusiform to lageniform , 5090  2530 m , hyaline , thin - walled , frequently with short , wide appendix at the apex ( mucronate ) .
pileipellis a hymeniderm consisting of clavate , rounded terminal elements and cylindrical , elongated cells 40200
the hymeniderm with short cells is strongly predominant ; in other parts the elongated cells are strongly predominant .
often the two types of elements are mixed independently from whether they are found : in the centre or in the margin of the pileus .
caulocystidia present over the whole length of the stipe , 1370  315 m , cylindrical to claviform , fusiform , sometimes mucronate , usually grouped in clusters .
habitat : on decaying wood of quercus cerris and branches of castanea sativa on the ground . specimens examined ( of p. variabilicolor ) : italy : lazio : manziana ( rm ) , on decaying wood of quercus cerris , 21 may 2013 , t. lezzi ( tl20130521 - 01 ) ; loc .
, 26 july 2013 , l. perrone ( tl20130726 - 01 ) ; lombardia : parco di monza ( mi ) , on decaying wood of castanea sativa , 30 may 2013 , m. biraghi ( tl20130530 - 01 ) ; lazio : manziana ( rm ) on decaying wood of quercus cerris , 5 nov . 2011 , v. migliozzi ( vm20111105 - 01 ) ; loc .
 hungary : near szrliget , on a pile of sawdust , 06 july 1977 , m. babos & a. friesz ( bp - fn 56936  isotype ) ; budakeszi , on a pile of sawdust , 27 july 1980 , l. babos ( bp - fn 65137 ) ; vallis lepence , near visegrd , on a pile of sawdust , 31 july 1981 , m. babos ( bp - fn 72875 ) ; com .
specimens examined ( of p. castri ) : japan : fukuoka prefecture : kyushu , on piled wood chips , 5 may 2007 , m. shintani & s. takehashi ( tnsf 17602  holotype ) ; ibaraki prefecture : honshu , tsukuba , 25 oct .
 russia : central russia , moscow region , prioksko - terrasny state reserve , on decaying wood of populus tremula , 15 aug .
1991 , g. e. levitskaya ( le 216873 ) ; samara region , near pribrezhny , on decaying wood of deciduous tree , 1 july 2007 , e. f. malysheva ( le 212090 ) .
1 ) the four sequenced collections of pluteus variabilicolor ( isotype included ) and the three of p. castri retrieved from genbank ( holotype included ) are conspecific .
pluteus variabilicolor was originally described from hungary and primarily characterized by a pileus which was at first yellowish orange and then becoming chrome - yellow ( hence the epithet  variabilicolor  ) , rugose - veined at the centre , and a pileipellis consisting of dimorphic elements , viz . a mixture of spheropeduncolate - vesiculose and elongated , more or less cystidioid elements , the presence of caulocystidia , and growth on decaying sawdust ( babos 1978 ) .
apart from hungary , where it was found several times after the first report ( e.g. babos 1981 , 1989 , 1991 , rimczi & vetter 1990 , lukcs 2010 , albert 2011 ) , p. variabilicolor is reported only from austria ( lohmeyer et al .
1998 , migliozzi 2011 ) , romania ( bres 2012 ) , slovenia ( jogan et al .
pluteus variabilicolor is mainly reported from sawdust deposits , even though our finds and those of migliozzi ( 2011 ) and musumeci ( pers .
comm . ) are on branches and rotten wood of fagaceae ( mainly quercus species ) . as suggested by babos ( 1978 , 1981 ) and courtecuisse ( 2006 ) , the species may not be native to europe and could have been introduced through timber import . in support of this hypothesis ,
all the nations in which the species was found are geographically related . on the other hand ,
natural  habitats , so it may very well be a native eurasian species that is just not common .
our description , based on italian specimens , fits quite well with the protologue , with the morphologic features reported by later authors ( lohmeyer et al .
1998 , migliozzi 2011 ) , and the hungarian collections we have checked ( isotype included ) .
2011a ) based on collections from japan ( on piled wood chips , holotype ) and central russia ( on decaying wood of deciduous trees ) .
the main difference with respect to the original description of p. variabilicolor and the italian collections found was in the structure of the pileipellis . in p.
castri we did not observe the elongated elements ( to 200 m long ) found in other collections of p. variabilicolor .
however , molecular data indicate that all collections of p. castri should be considered to represent p. variabilicolor . in the phylogenetic analysis ( fig .
1 ) p. variabilicolor , in spite of the presence of both vesiculose - clavate and elongate elements in the pileipellis , clustered into the leoninus clade of sect .
( 2012 ) , where it is sister to p. chrysaegis and very close to the p. leoninus complex .
most species of the leoninus clade are characterized by yellowish tinges on the pileus surface .
africanus ) resembles p. variabilicolor in the general structure of the pileipellis and the yellow colouring of the basidiome , the veined pileus surface , and the presence of caulocystidia ( pradeep & vrinda 1996 , pradeep et al .
however , it differs in the even shorter elements of the pileipellis ( to 40 m long ) , and the non - mucronate , slightly thick - walled cheilocystidia ( horak & heinemann 1978 , pradeep et al .
2012 ) . the species in the p. leoninus complex ( i.e. p. leoninus , p. roseipes , p. flavofuligineus , p. aff .
leoninus ii ) all have a pileipellis made up exclusively of narrowly fusiform elements to 230 m long ( justo et al . 2011a ) and , with the exception of p. aff .
 based on several collections from italy , a detailed description ( including also macrophotographs , microphotographs , and drawings ) of the morphological characters of the poorly known pluteus variabilicolor , originally described from hungary , is provided . 
 the analysis of the its sequences placed this species within the p. leoninus clade of sect . 
 hispidoderma , in spite of the presence of clavate elements in the pileipellis . 
 according to the molecular comparison of the type collections , pluteus castri , a species recently described on the basis of material collected in japan and central russia , is reduced to a synonym of p. variabilicolor . 
INTRODUCTION
MATERIAL AND METHODS
Morphology
DNA extraction, amplification and sequencing
Sequence alignment and phylogenetic analysis
RESULTS 
TAXONOMY
DISCUSSION