Diversity trapped in cages: Revision of Blumenavia Möller (Clathraceae, Basidiomycota) reveals three hidden species

Basidiomata of Phallales have a diversified morphology with adhesive gleba that exudes an odor, usually unpleasant that attracts mainly insects, which disperse the basidiospores. The genus Blumenavia belongs to the family Clathraceae and, based on morphological features, only two species are currently recognized: B. rhacodes and B. angolensis. However, the morphological characters adopted in species delimitations within this genus are inconsistent, and molecular data are scarce. The present study aimed to review and identify informative characters that contribute to the delimitation of Blumenavia species. Exsiccates from America and Africa were analyzed morphologically, and molecularly, using ITS, LSU, ATP6, RPB2 and TEF-1α markers for Maximum Parsimony, Bayesian and Maximum likelihood analyses, and also for coalescent based species delimitations (BP&P), as well as for bPTP, PhyloMap, Topo-phylogenetic and Geophylogenetic reconstructions. According to our studies, seven species can be considered in the genus: B. rhacodes and B. angolensis are maintained, B. usambarensis and B. toribiotalpaensis are reassessed, and three new species are proposed, B. baturitensis Melanda, M.P. Martín & Baseia, sp. nov., B. crucis-hellenicae G. Coelho, Sulzbacher, Grebenc & Cortez, sp. nov., and B. heroica Melanda, Baseia & M.P. Martín, sp. nov. Blumenavia rhacodes is typified by selecting a lectotype and an epitype. Macromorphological characters considered informative to segregate and delimit the species through integrative taxonomy include length of the basidiomata, color, width and presence of grooves on each arm as well as the glebifer position and shape. These must be clearly observed while the basidiomata are still fresh. Since most materials are usually analyzed after dehydration and deposit in collections, field techniques and protocols to describe fugacious characters from fresh specimen are demanded, as well as the use of molecular analysis, in order to better assess recognition and delimitation of species in Blumenavia.


Introduction
The Phallales E. Fisch. are gasteroid fungi that commonly possess zoochoric dispersion of their basidiospores, through insects that are attracted by the unpleasant odor of the mucilaginous gleba. The usual appearance and smell gave rise to the popular denomination of "stinkhorns " or "cage fungi" [1,2]. Blumenavia Möller is a genus belonging to this order and is included in the family Clathraceae Chevall. Möller [3] proposed this genus based on specimens discovered in Blumenau, Santa Catarina (Brazil), with Blumenavia rhacodes Möller as the type species; this author segregated Blumenavia from Laternea Turpin based on the pale-yellow basidiomata color, triangular to irregular wing-like glebifers occupying the external borders of the whole length of the arms, and a wide groove at the external face of the arms present in B. rhacodes.
Until now, based on morphological data, three other species have been recognized in the genus: Blumenavia angolensis (Welw. & Curr.) Dring [4], basionym Laternea angolensis Welw. & Curr. [5]; B. usambarensis Henn. [6]; and B. toribiotalpaensis Vargas-Rodr [7]. In addition, Dring [4] proposed B. usambarensis as a synonym of B. angolensis, since both present white basidiomata, absence of a groove in the outer face of the arms, and glebifers restricted to a quarter or a third of the receptacle; characters that differentiate them from B rhacodes. This taxonomic treatment proposed by Dring [4] has been widely adopted by subsequent studies [8][9][10][11]. However, despite the proposed synonymy, the author did not analyze the type specimen of B. usambarensis, from Tanzania, but only exsiccates from Brazil and Trinidad and Tobago.
The most recent species described, using as diagnostic characters color of basidiomata, size of basidiospores, arrangement of the gleba in the arms of the receptacle, and cross-section of the arm, was B. toribiotalpaensis [7]. However, Calonge et al. [12] synonymized B. toribiotalpaensis with B. rhacodes, considering as similar characters: basidiomata staining, number and anatomy of arms and basidiospore dimensions. This synonymy has been mostly adopted by later studies [10][11][12][13].
The scarcity of morphological characters adopted to segregate the species and the ephemeral basidiomata are limitations in studies on Phallales. The hypothesis of our work is that the use of morphological and molecular data, as well as geographical distribution, are effective to delimit species in the genus Blumenavia. In this context, the aim of the present study was to review collections of the genus Blumenavia, including type specimens and specimens from type localities, using morphological and molecular analyses, to revise the taxonomy and systematics of the genus.

Sampling
For this study 23 exsiccates from Brazil (South and Northeast), Mexico (Jalisco and Xalapa), and Tanzania were analyzed, including type specimens, this collections were borrowed from: under the Centro de Pesquisas e Conservação da Natureza Pró-Mata's authorization. These material were depositad in UFRN-Fungos, SMDB (Herbário SMDB, Jardim Botânico da Universidade Federal de Santa Maria, Centro de Ciências Naturais e Exatas) and ICN, respectively.
All of those materials cited were included in the morphological analyses, and 13 were used in molecular analyses. All herbaria gave permissions for morphological and molecular studies.

Morphological studies
Specific literature was consulted to determine the analyzed characters [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. The colors of the structures follow Küppers [22]. For each specimen the size, color and texture of expanded basidiomata, receptacles, glebifers, volva, as well as rhizomorphs were analyzed at a macroscopic level, therefore are written in the description based on fresh basidiomata. In addition, microscopic observations were obtained from the hyphae forming the rhizomorphs, basal and apical arms, glebifers, basal and apical exoperidia, mesoperidia, and endoperidia. Since these characters were not informative to segregate species, microscopic analyzes of basal and apical arms, glebifers, basal exoperidia, mesoperidia and endoperidia are not included in taxonomical descriptions (see S1 Table). However, this study is the first in which the different peridium layers have been analyzed in Blumenavia (see layers detail in Fig 9A). In this paper, only the informative characters are included in the description of each species. These microstructures were mounted in 5% KOH (potassium hydroxide) and 1% Congo red. The shape and size of basidiospores of each specimen were observed mounted in 5% KOH, Melzer's reagent, cotton blue and 1% Congo red. These dyes can provide essential information through chemical reactions characteristic of certain taxa [1], also, it is the first time that microstructure reactions were analyzed in Blumenavia.
To describe the shape of basidiospores, Q value (height/width quotient) was calculated according to Bas [23]; Qm represents the mean Q value, where "x" is the mean of values previously presented, and "±" is the standard deviation, as observed under the 100× objective. For each microscopic structure thirty hyphae/basidiospores were measured for each basidiomata.
ExoSAP-IT™ PCR Product Cleanup Reagent (USB Corporation, OH, USA) was used for purification of the amplifications, following the fabricant instructions. Sequencing of the PCR products was carried out at Macrogen in Seoul, South Korea. All the sequences are deposited in Genbank1 (http://www.ncbi.nlm.nih.gov/). Phylogenetic analyses. The sequences obtained in this study are listed in Table 1. These sequences were aligned in MEGA X through the Muscle algorithm followed by manual adjustment with the sequence of B. angolensis obtained from NCBI/GenBank database and published in Degreef et al. [8], and two Clathrus species included as outgroup. Then this dataset was submitted to the Partition Homogeneity (Incongruence Length Difference) Test in software PAUP � v.4.0a166 [30].
Phylogenetic analyses were conducted for the concatenated five loci dataset under maximum parsimony (MP) analysis, Bayesian, and maximum likelihood (ML). Alignment gaps were marked with "-" and missing data were indicated with "N". The parameters used in the phylogenetic analyses were modified from Sousa et al. [31]. MP analysis was conducted in software PAUP � , initial trees for 10,000 bootstrap simulations (MPbs) of Heuristic Search under TBR swapping algorithm were obtained by 10x randomized stepwise addition, and the number of trees obtained in each bootstrap replication was not restrained. In the Bayesian analysis, performed using Mr.Bayes v.2.3.6 [32] implemented in CIPRES Science Gateway (Cyberinfrastructure for Phylogenetic Research web portal, http://www.phylo.org), partitions were set to use specific substitution models chosen by the software jModelTest 2 [33] for each marker under the Akaike Informational Criterion (AIC) and corrected Akaike Informational Criterion (AICc). Substitution rates, proportions of invariant sites, gamma shapes and base frequencies were independently estimated among partitions from initial priors obtained by jModelTest. Ten million generations were used, and trees were sampled each 5,000 generations. Convergence and sampling were checked by Potential Scale Reduction Factor (PSRF) and Estimated Sample Size (ESS) as recommended by MrBayes developers. Convergence was also visualized in Tracer v.1.7.1 [34]. Stationarity of MCMC were assessed in Tracer v.1.7.1 software and also manually in MCMC output from MrBayes v.2.3.6 when Average Standard Deviation of Split Frequencies (AvgStdDev) dropped below 0.01. In the Bayesian analysis we tested two partition models: one considering five partitions (ITS1-5.8S-ITS2/LSU/ATP6/ RPB2/TEF-1a), and another considering seven partitions (ITS1/5.8S/ITS2/LSU/ATP6/RPB2/ TEF-1a). ML analyses were performed using RAxML software [35] implemented in CIPRES Science Gateway (Cyberinfrastructure for Phylogenetic Research web portal, http://www. phylo.org) and 1,000 bootstrap replicates (MLbs) under GTRGAMMA model.
For poorly supported terminal clades in which morphological variation among terminals was inconspicuous, additional species delimitation analyses were carried out based on a reduced alignment without outgroups, to increase homology in alignment, and refine results in subtree analyses. The software BP&P v.3.3 (Bayesian Phylogenetic & Phytogeography version 3.3) [36] was used for simultaneous species recognition and delimitation by Bayesian Markov chain Monte Carlo (MCMC) under a multispecies coalescent model (MSC) using algorithms 0 and 1, and considering equal probabilities for the number of species and for the rooted species trees [37][38][39] by 100,000 replicates sampled every 5 generations and burnin of 2,000 sampled generations. Theta and Tau priors where adjusted after previous run of the program for estimation of parameters under the MSC model. MCMC step lengths where set to automatic adjustment during the burnin phase. Additionally, Bayesian analysis was carried out following the same methodology as in the general analysis to generate an input tree for bPTP (Bayesian Poisson Tree Processes) [40], PhyloMap and Topo-phylogenetic [41] reconstructions. Although bPTP was designed for single locus analysis, it is commonly used for multilocus trees [42][43][44]. To illustrate the distribution of Blumenavia spp. in the world, the Geophylogeny was made using the world map with the phylogenetic tree with CorelDraw1 Graphics Suite X8 software.

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.
In addition, new names contained in this work have been submitted to MycoBank, from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix at http://www. mycobank.org/MB. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS and Digital-CSIC.

Molecular phylogeny
For this study, DNA sequences of 13 specimens of Blumenavia were obtained: 12 ITS, 9 LSU, 7 ATP6, 10 RPB2 and 11 TEF-1a (Table 1), except B. usambarensis. Incongruence Length Difference test has a P value = 1.00. The concatenated alignment obtained has 3,803 unambiguously Table 1. Blumenavia specimens analyzed in molecular analysis. Genbank accession numbers for each region of the sequences included in this study. Accession numbers in bold are from newly generated sequences. Absences of sequences are marked with "-". x-access-code= a21d01f584a54a1997c0c42350b6f8f1&format=html. Bayesian analyses considering two partition models retrieved the same topology. The posterior probabilities result was slightly higher in the analysis with seven partition, so we chose to show only the results for this analysis (Fig  1). Bayesian analysis gave a best tree with -lnL = -6,633.82. The supports of the other two analysis as also show in the Fig 1. In the three phylogenetic of all analyses (MP, ML, BI), five clades and a singleton were recovered for the genus Blumenavia (Fig 1), corresponding to the three known species (B. angolensis, B. rhacodes, B. toribiotalpaensis), and three species that we consider new to science: B. baturitensis sp. nov., B. crucis-hellenicae sp. nov., and B. heroica sp. nov. Also, the morphological analysis made it possible to confirm B. usambarensis, as an independent species; it was not possible to obtain molecular data from these old specimens, which were collected in 1900.
MP, ML and BI show B. baturitensis sp. nov., B. heroica sp. nov. and B. rhacodes grouped together in a clade, but in ML B. angolensis is grouped in the middle with B. rhacodes with low support. In MP Blumenavia angolensis forms a singleton in a bigger clade of B. baturitensis, B. rhacodes and B. heroica, and in BI as a singleton together with the clades B. toribiotalpaensis and B. crucis-hellenicae. An explanation is that B. angolensis has only LSU sequence, in contrast with more than one with the others.
Blumenavia toribiotalpaensis and B. crucis-hellenicae are grouped in a clade in BI and ML, in BI this clade shares a common ancestor with B. angolensis, as mentioned before. In MP B. crucis-hellenicae is a sister group of the clade with B. angolensis and the one with B. baturitensis, B. rhacodes and B. heroica, B. toribiotalpaensis in this analysis appears out of all these clades.
An evolutionary relationship for B. angolensis may be unclear because the lack of some markers, but these differences between the trees don't reveal an incongruency in the separation of the species. Defining infrageneric relationship was not intended in this work, so evolutionary placement of B. angolensis can made in future works with better sampling of this species.
As shown in Fig 1, Blumenavia baturitensis sp. nov., B. heroica sp. nov. and B. rhacodes grouped together in a clade, within three independent groups, but with low support values; and, since these species show few unremarkable morphological differences, in order to test species delimitation hypothesis, this clade was chosen for secondary analyses. The BP&P analysis obtained a posterior probability of 0.9746 for three species in the dataset, which is visualized in Fig 2 with bPTP Phylomap program; the same results were obtained (three species recorder) using Topo-Phylogeny.
Additional data that supports the three species delimitation, as well as in the case of the other species in Blumenavia, are regarding the geographical distribution (Fig 3). Blumenavia angolensis identified in the Southern and Northeastern Brazil are the new species B. crucis-hellenicae sp. nov. and B. baturitensis sp. nov., respectively. Both segrageted from B. angolensis from Africa. The type location of Blumenavia rhacodes is Southern Brazil, collection from this region maintains its identity and was shown to be distinct from others identified as B. rhacodes from Mexico; the latter is a novelty for science: B. heroica sp. nov. In addition, in Mexico, B. toribiotalpaensis was considered by other authors [10][11][12][13] as B. rhacodes, but both were shown to be distinct, assuming the invalidation of this synonymization. Given the restrict distribution found for all species in our revision on genus Blumenavia, our results rise the possibility for

Morphological characters
After all analysis the diagnostic characters for Blumenavia genus are macroscopically: length of the basidiomata; color and width of the arms, presence of a groove on the external and internal face of each arm and glebifer position and shape; microscopically: basidiospore size and shape; apical exoperidia hyphal type and wall thickness of rhizomorph hyphae.
Some characters are revealed to be uninformative for species delimitation. Macroscopically are they: number of arms, volva color and shape, rhizomorph and gleba color; microscopically: receptacle, glebifer, basal exoperidia, mesoperidia and endoperidia hyphae length.
In relation to chemical reaction of the microstructures the basidiospores are cyanophilous in all species, and they do not react in 1% Congo red or Melzer's reagent. The hyphae of all parts of the basidiomata are hyaline, and react becoming light red in 1% Congo red, except the hyphae of the apical part of the exoperidia that have a dark brown lumen.    Fig 7).

Taxonomy
Description. Since specimens were not located, this description is based on Degreef et al. [8], and on the color photography included in Desjardin and Perry [11], see Fig 4A and 4B. Expanded basidiomata 80-85 mm length × 35-40 mm wide. Exoperidium white (N00Y00M00) smooth base, cracked into brownish gray (N 60 Y 00 M 00 -N 90 Y 00 M 00 ) scales above. Receptacle 4 arms united above, free in the base, white (N 00 M 00 C 00 ), arms even thickness throughout the length, without grooves. Membranous glebifers, adhered to the anterolateral angles of the arms, triangular, quadrangular or irregular, regularly spaced along the full length of the arms, glebifers covered with glebal mass. Basidiospores ellipsoid, (3. Habit and habitat. Epigeous and solitary, saprotrophic in secondary forest. Known distribution. Afrotropic: Angola, Pungo Andongo [5]; São Tomé and Príncipe, São Tomé [8,11]. Comments. Specimens of this species were not analyzed because recent collections were not located. We contacted Dr. Jerome Degreef (Belgium) to study the specimen ( Fig 4A) described in Degreef et al. [8], since there is an available LSU sequence in GenBank; Dr. Degreef confirmed to us that the specimen was not located, nor the DNA isolate, thus it was not possible to obtain sequences from more loci. The specimen from Desjardin and Perry [11] was not conserved, because the authors did not collect the specimens, they took only pictures of them ( Fig  4B).
Dring [4] based on the image protologue ( Fig 4C) and specimens from Brazil and Trinidad and Tobago made an illustration for B. angolensis (Fig 4D). Degreef et al. [8] based their analysis in one specimen from Sao Tome and Principe (Fig 4A). Dring [4] and Degreef et al. [8] separated B. angolensis from B. rhacodes according to the distribution of the glebifer, and the color of their basidiomata. However, our study confirms the importance of color for distinguishing the basidiomata of these species, but not the glebifer distribution, since both species have their glebifers distributed along the arms.
Desjardin and Perry [11] mentioned Degreef et al. [8] for the description of the photographed basidiomata from Sao Tome and Principe. This photograph (Fig 4B) shows the glebifers regularly spaced along the length of the arms. Although this character differs from the protologue [5] (Fig 4C), and from the description provided by Degreef et al. [8], we adopted this image (Fig 4B) to describe this character and exemplify the species B. angolensis in the phylogenetic tree (Fig 1). Degreef et al. [8] probably analyzed an old basidioma with the arms not joined at the apex. Also, because of zoochoric dispersion, insects may have consumed Etymology. The name of the species refers to the type locality, located in Baturité microregion, Ceará, Brazil.
Description. Expanded basidiomata 91-119 mm length × 35-55 mm wide. Peridium composed of three layers. Exoperidium light yellow to dark yellow (N 10 C 00 Y 30 -N 10 C 00 Y 60 ) smooth base, cracked into brownish gray (N 60 Y 00 M 00 -N 90 Y 00 M 00 ), dark brown (N 90 Y 80 M 40 ) or black (N 99 Y 30 M 00 ) scales above. Single, branched, white rhizomorph (N 00 M 00 C 00 ). Receptacle 4 to 5 arms united above, free in the base, whitish yellow (Y 70 M 10 C 00 ) or white (N 00 Y 00 C 00 ) near the volva becoming yellowish (N 00 Y 50 M 00 ) at the apex, the arms are thinned from the middle to the top of the basidiomata, without grooves (Fig 5A-5C). Membranous glebifers adhered to the anterolateral angles of the arms, formed by wrinkled, tentacled (finger-like) projections, grouped parallel and regularly spaced in the upper half of the basidiomata (Fig 5A-5D, see the arrows), in some points near the apex these projections coming from each side of the arm are joined and form a larger projection leaving a fusiform space between them (Fig 5E), this basidiomata have torn and twisted projections from the middle to the base of the arms (Fig 5F and  5G), glebifers covered with glebal mass.
Habit and habitat. Epigeous and solitary, lignicolous; in 'Brejo de altitude' forest (Northeastern fragment of Atlantic Forest), on decaying wood and on litter. Comments. Specimens of this new species (collection UFRN-Fungos 1943) were identified based on morphological characters alone in Rodrigues and Baseia [9] and Trierveiler-Pereira et al. [10] as B. angolensis and B. rhacodes, respectively. However, B. baturitensis sp. nov. differs from B. angolensis by having larger basidiomata (91-119 mm), yellowish and thick arms, tapering from the middle of the basidiomata to the apex (Fig 5A and 5B), and glebifers as wrinkled tentacular projections (Fig 5D). While B. rhacodes has groove in the outer face of the arms, and the glebifer shape different, as also confirmed by molecular data. The new species shows similar characteristics to B. toribiotalpaensis; however, the glebifer projections are tentacular ( Fig 5D) in B. baturitensis sp. nov. and lacerated in B. toribiotalpaensis (Fig 10F) in the upper part of basidiomata. In our phylogenetic tree (Fig 1)  Comments. Trierveiler-Pereira et al. [10] identified the collections ICN 177268 and ICN 177269 (studied here) as B. angolensis, but B. crucis-hellenicae sp. nov. differs from B. angolensis, and from the other species in the genus, by the elongated (oblong) basidiospores (Fig 6K), with a Qm = 1.93 ± 0.16 unlike the other species that have a range between Qm = 2.22 ± 0.15 to Qm = 2.42 ± 0.20. Moreover, B. crucis-hellenicae sp. nov. has a cross-shaped glebifer ( Fig  6D), and grooves in the inner part of the arms (Fig 6A and 6B). Molecular analyses do not confirm the existence of a cluster with white colored species as mentioned by other authors [4,10] since the white species Blumenavia crucis-hellenicae sp. nov. and B. angolensis shares a common ancestor with B. toribiotalpaensis, one of the yellowish species (Fig 1) Etymology. Named in honor to the type locality, Veracruz, also known as Heroica Veracruz.
Description. Expanded basidiomata 45-80 mm length × 13-30 mm wide. Peridium composed of three layers. Exoperidium all dark grey, almost black (N 99 Y 10 M 00 ) smooth, or white (N 00 Y 00 M 00 ) smooth base, cracked into light to dark brown (N 30 Y 60 M 30 -N 90 Y 80 M 40 ) scales above. Receptacle 3 to 4 arms united above, free in the base, whitish yellow to pale yellow (Y 70 M 10 C 00 -Y 70 M 20 C 10 ), the arms even thickness throughout its length (Fig 7A) or are thinner from the middle to the top of the basidiomata (Fig 7B) with a marked longitudinal groove in the outer face (Fig 7B and 7C, see the arrows). Membranous glebifers adhered to the anterolateral angles of the arms. Glebifers composed by parallel lines curving inward (Fig 7A and 7D, see the arrows) forming triangular, quadrangular or irregular points projections regularly spaced along the length of the arms or parallel lines curving inward not sparse in the upper half of the basidiomata (Fig 7E, see the arrow) with triangular to quadrangular spaced projections to the base (Fig 7F, see the arrows), glebifers covered with glebal mass.
• Laternea rhacodes (Möller) Lloyd, Synopsis of the known phalloids (7) Description. Expanded basidiomata 85-130 mm length × 25-50 mm wide. Peridium composed of three layers (Fig 9A). Exoperidium white to light brown (N 00 Y 00 M 00 -N 40 Y 70 M 30 ) smooth base, cracked into gray to dark brown (N 70 Y 00 M 00 -N 90 Y 80 M 40 ) scales above. Single branched, white rhizomorph (N 00 M 00 C 00 ). Receptacle 3 to 4 arms united above, free in the base, whitish yellow to light orange (Y 70 M 10 C 00 -Y 99 M 40 C 20 ), arms even thickness throughout its length (Fig 9B-9E, arrows in B and C indicating glebifers), outer face with a marked longitudinal groove (Fig 9F, see the arrow). Membranous glebifers adhered to the anterolateral angles of the arms, triangular, quadrangular or irregular, regularly spaced along the full length of the arms (Fig 9G-9I, see the arrows), glebifers covered with glebal mass.
The phylogenetic trees (Fig 1) reveal B. rhacodes as a sister clade to B. baturitensis sp. nov. Blumenavia rhacodes is morphologically similar to B. baturitensis sp. nov, B. heroica sp. nov. and B. toribiotalpaensis by the arm color; but, B. toribiotalpaensis and B. baturitensis sp. nov. have irregularly shaped glebifers along the arms, only filamentous hyphae in the apical exoperidium and the arms are thinned from the middle to the top of the basidiomata. On the other hand, B. heroica sp. nov. has apical exoperidium composed of filamentous and subglobose to ellipsoid hyphae and the basidiomata up to 80mm length, mine while, B. rhacodes has filamentous and elongated hyphae in the apical exoperidium and the basidiomata above to 80mm length. Description. Expanded basidiomata 121-153 mm length × 38-56 mm wide. Peridium composed of three layers. Exoperidium light yellow to dark yellow (N10C00Y30-N 10 C 00 Y 60 ) smooth base, cracked into gray (N 70 C 00 Y 20 ) from the middle until dark gray (N 90 C 00 Y 10 ) in the apical part. Single branched, white rhizomorph (N 00 M 00 C 00 ). Receptacle 3 to 4 arms united above, free in the base, whitish yellow to light orange (Y 70 M 10 C 00 -Y 99 M 40 C 10 ), the arms are thinned from the middle to the top of the basidiomata, outer face with a marked longitudinal groove (Fig 10A-10E). Membranous glebifers adhered to the anterolateral angles of the arms, formed by large lacerated projections, slightly spaced from one another at apex to the upper half (Fig 10F), with small, more spaced, torn and twisted points to the base (Fig 10G and 10H), glebifers covered with glebal mass.

Conclusion
Scarce molecular data, together with the use of a limited set of informationally poor morphological characters, led to misidentifications and synonymization in Blumenavia species. There are a small number of macromorphological characters that are useful for delimiting species, but some of these must be observed while the basidiomata are still fresh, such as color oh the basidiomata as well as the arrangement and morphology of the glebifers, since they become difficult to observe in fungarium collections. Even so, the analysis of morphological characters, integrated with molecular data, as well as geographical distribution allowed the definition of diagnostic characters for Blumenavia species, affirming the hypothesis of this work. The results obtained reaffirm the importance of the integration with different data, and the importance of comparative revision work with materials deposited in collections, especially type species, to delimit and identify the species in Blumenavia, as in another gasteroid with confusing taxonomy.