Diversity of the Ganoderma species in Uruguay

ABSTRACT Ganoderma is a cosmopolitan genus that includes a great diversity of species. Many of them have been historically described based only on morphological characteristics; however, due to their morphological plasticity, there is no complete understanding about their relationship and taxonomic status. Commonly applied names, particularly in the southern Neotropics, come from species of North Hemisphere distribution (e.g. G. lucidum, G. resinaceum and G. applanatum). The objective of the present work was to perform a survey of Ganoderma species thriving in Uruguay. We aimed to identify and characterize them through molecular, morphological and ecological analysis. The results confirm the presence of four reddish laccate species first registered for Uruguay (G. dorsale, G. platense, G. martinicense and G. mexicanum), and one non-laccate species (G. australe s.l.) composed of two clades. The species are morphologically differentiated mainly by its stipe, pilear surface, context, pores, basidiospores and cutis cells. Regarding the ecological data, the species present differences in substrate preferences. In addition, a taxonomic discussion regarding phylogenetic relationships and taxonomic status of Uruguayan Ganoderma species is presented.


Introduction
Ganoderma (P. Karst.) harbors at least 220 species, being the most diverse genus of Ganodermataceae [1][2][3][4]. However, due to their high diversity and phenotypic plasticity, the phylogenetic relationship and taxonomic status of many species remain unclear until now [5,6]. In the last 20 years, the use of molecular tools, mainly through the amplification and sequencing of Internal Transcribed Spacer (ITS), has been incorporated into systematic studies and into circumscription of Ganoderma species around the world. In this sense, through phylogenies based on molecular characters, the tendency in recent years has been to reinterpret the variations in characters of morphologically defined species, reinterpret the ecological relationships (relationship with hosts), determine their distribution and arrive at an understanding of the biogeographic processes that shape it [5,[7][8][9][10][11].
Ganoderma species are morphologically characterized by the formation of sessile to stipitate basidiomata, with a glossy reddish laccate to opaque non-laccate cover, ellipsoid to ovoid double-walled basidiospores with truncated apex and endosporium with columnar ornamentations [12]. This cosmopolitan genus is comprised of parasitic and saprophytic species that decay the wood of plants from temperate and tropical areas around the world [6,13,14]. These fungi are described as white rot decayers that play a critical role in the dynamics of wood decomposition in tropical forests [15]. Moreover, they are the main cause of tree deterioration in public ornamental and commercial plantations [10,[15][16][17][18].
In the last decades, some researchers have tried to elucidate the diversity of Ganoderma genus in the Neotropics, particularly Bazzalo and Wright [19], Gilbertson and Ryvarden [20], Gottlieb and Wright [21,22], Gottlieb et al. [7], Ryvarden [12,23], Torres-Torres and Dávalos [24], Torres-Torres et al. [25,26], and more recently Cabarroi-Hernández et al. [6] and Loyd et al. [11] for the northern limit of Neotropical distribution. Uruguay in particular harbors a great biodiversity due to its transitional condition and ecoregions diversity [27]. Some Ganoderma species have been historically recorded in Uruguay (Table 1), while Gazzano [28,29] and Martinez [30] have made more recent contributions to the diversity of the genus in Uruguay.
Some Uruguayan cited species as G. applanatum (Pers.) Pat. (= G. lipsiense (Batsch) G.F. Atk.), G. sessile Murrill, G. lucidum P. Karst. and G. resinaceum Boud were distributed out of the southern Neotropics [14,35,36], while others as G. lorenzianum (Kalchbr.) Pat., G. nitens (Fr.) Pat. lack of phylogenetic studies and their taxonomic status is unclear. Until now, there is no complete understanding on the systematic of the Ganoderma genus in Uruguay and its species diversity remains unknown.
The objective of the present work was to perform an updated survey of the Ganoderma species thriving in Uruguay and characterize them through molecular, morphological and ecological analysis. We also aimed to discuss the taxonomic status of the species of Uruguay and contrast with previous reports. We hypothesized that the Ganoderma specimens of Uruguay correspond to native species that have not been previously reported for the country. Those species are different from those registered to date (with names of species described from the Northern Hemisphere).

Fungal specimens, basidiomata description and host characterization
Fresh Ganoderma basidiomata were collected from indigenous and urban ecosystems of Uruguay, during 2017 and 2018 field expeditions. Geographic location, host species and substrate condition (living tree, dead trunk, roots and stump) data were taken at the collection site following Urcelay and Robledo [37]. For fresh basidiomata, small pieces were aseptically taken from the context, placed into 2% malt extract agar (MEA) and incubated in darkness at 25°C. Pure cultures and basidiomata were deposited in MVHC. In addition, specimens from national herbaria (MVHC and MVM) were examined. Herbarium acronyms follow Thiers [38] (continuously updated, http://sweetgum.nybg.org/).
For morphological analyses and basidiomata identification, macroscopic and microscopic observations were made on basidiomata following the terminology and methodology according to authors [6,7,19,[22][23][24][25][26]. Macroscopic features of basidiomata were analyzed and measured, particularly: pileus dimension, colour, texture, shape and appearance of surface, margin and stipe. The colour, presence of melanoid deposits and texture of context were also inspected and pores were described and measured (pores/mm). Microscopic features (basidiospores, chlamydospores in context, generative and somatic hyphae and cuticular cells) were analyzed with an optical microscope by mounting small sections of basidiomata with 5% KOH or Melzer's Reagent (to test for dextrinoid or amyloid reaction). In particular, for the analysis of the hyphal system, sections of basidiomata were treated for 24-48 h in 3% NaOH at 50-60°C [39]. Thirty basidiospores were measured from each specimen (length and width) and values were expressed as rank of mean values. Width was measured in the widest part of the spore and the length considered from the base to the truncated apex of the basidiospore. Then, Q ratio was calculated as the relation: length/width. Measures of cuticular cells were made from the middle part of the basidiomes.
Host relationships of each Ganoderma species in Uruguay were characterized by host range and native/exotic status. The preference of substrate condition was analyzed through the relative frequency of each Ganoderma species in each substrate condition (LT = stem of living tree, DT = dead trunks, S = stumps and R = soil, arising from roots of living or dead trees) following Urcelay and Robledo [37]. Then, substrate preference was determined by transforming this relative frequency into a percentage.

Phylogenetic analyses
The sequences obtained were manually edited (visual inspection of sequences and chromatograms, resolution of conflicts and pair the extremes) with Bioedit V.7.0.5.3 [47] and incorporated into alignments with sequences of specimens from other parts of the world obtained from GenBank (Table 2). Multiple alignment was made using ProbCons 1.12 from the CIPRES Science Gateway [48]. Subsequently, the best evolutionary model for each region (ITS1, 5.8S and ITS2) was estimated using the Corrected Akaike Informational Criteria (AICc), implemented by the jModelTest2 v.1.6 software [49]. The phylogenetic analysis was conducted in two independent ways: Bayesian Inference (BI) and Maximum Likelihood (ML), performed with MrBayes 3.2.7 [50] and RAxML 8.2.12 [51], respectively, in CIPRES Science Gateway [52]. Cristataspora coffeata (Murrill) Robledo, Costa-Rezende and de Madrignac Bonzi (FLOR 50933) and Foraminispora rugosa (Berk.) Costa-Rezende, Drechsler-Santos and Robledo (FLOR 52191 and HUEFS DHCR560) were used as outgroup [4,53]. For the BI, two independent runs were performed, starting with random trees, with four independent and simultaneous chains, 10,000,000 MCMC generations, and maintaining 1 tree every 1000 generations. Burn in discarded values was indicated as 0.25. The estimated models for each partition were incorporated, as indicated below (see Results
Morphologically assigned species are presented in Figures 1 and 2. Ganoderma species of Uruguay were primarily differentiated by their pilear surface into two groups: reddish laccate and non-laccate (Table 3 and Figure 1).
The first group is composed of two species with great and robust basidiomes (G. martinicense and G. platense) and two species with smaller basidiomes (G. dorsale and G. mexicanum).
Ganoderma martinicense is characterized by a large, commonly substipitate basidiomata with a tuberculous concentric zonated pilear surface and conspicuous melanoid deposits in the homogeneous context. Microscopically, it is characterized by the presence of a distinct smooth basidiospores ornamentation, formed by free pillars and cutis cells with weak  reaction and spheroid-pedunculated shape. On the other hand, G. platense basidiomata are smaller, semiorbicular zonate, homogeneous colored and always sessile. Its context is also different, presenting extremely inconspicuous melanoid lines and microscopically characterized by particular cutis elements with apical constrictions (Figure 2). Ganoderma mexicanum produces stylized, flabelliform, laterally and horizontally stipitate basidiomata characterized by an almost light-colored context and basidiospores with fine ornamentation, whereas G. dorsale produces stylized, spatuliform, shellshaped and almost laterally and vertically stipitate basidiomata with no homogeneous distinctive context. Its basidiospores present notorious rough ornamentation and almost cylindrical cutis cells with a strong amyloid reaction (Figure 2).
Specimens with non-laccate surfaces are morphologically very similar, discernible however by their pilear surface, which is distinctly tubercular in G. australe M1 and distinctly zonate in Ganoderma australe M2.
Herbaria specimens showed a morphology consistent with the species recorded in this study. In that sense, G. lucidum is a name previously used for specimens corresponding to the G. dorsale, G. martinicense, G. platense and G. mexicanum species. The name G. resinaceum was previously used to name specimens corresponding to G. platense and G. martinicense. The names G. lipsiense, G. applanatum and G. marmoratum were used to refer to specimens of G. australe in the broad sense (from now on sensu lato or s.l.). A morphological key for Ganoderma species found in Uruguay is presented below.
Host relationships of each Ganoderma species in Uruguay and presence in distinct departments are presented in Table 4. Ganoderma martinicense and G. australe were found alternatively in several departments growing on native or exotic trees. On the other hand, G. platense was only recorded in the southeast (Montevideo, Canelones and Maldonado departments), growing preferentially on exotic trees, G. mexicanum was only found on dead stems of native forest, and G. dorsale, almost exclusively on native trees. Regarding their substrate preferences, G. mexicanum and G. australe M2 were found preferentially on dead stem wood (100% and 46%, respectively); G. martinicense and G. dorsale, preferentially on roots (87% and 58%, respectively); and G. australe M1 and G. platense, preferentially on live stems (45% and 85%, respectively).

Phylogenetic analyses
A total of 53 new sequences were generated from Uruguayan specimens. The dataset alignment resulted in 140 DNA sequences comprising 639 bp. The best evolutionary models for each partition were as follows: K80 + G (ITS1), JC (5.8 S), K80 + G (ITS 2). The partition scheme was K80 + G (ITS1) with -lnL = 1373.0169, and equal base frequencies as follows: A = 0.25, C = 0.25, G = 0.25, T = 0.25, JC (5.8 S) with -lnL = 300.8947 with equal base frequencies, and K80 + G (ITS 2) with 1498.7406 and equal base frequencies. The Bayesian Inference consensus tree is presented in Figure 3.
A total of 19 supported clades were recovered through a phylogenetic analyses (Figure 3). Sequences corresponding to Uruguayan specimens

Discussion and integrative taxonomy
The taxonomic status of Ganoderma species in Uruguay was evaluated through ITS-based phylogenetic analyses in combination with morphological and ecological data. The topology recovered in our phylogenetic analyses is congruent with previous works [4][5][6]. Three main clades were recovered. One of them is composed of Ganoderma species with reddish laccate basidiomata, stipe and pilear surface, including traditional G. lucidum and G. resinaceum complexes (0.99/67). The second one is composed of almost-sessile Ganoderma species with non-laccate pilear surface, including G. australe and G. applanatum complexes (1/76). The third one is a small subclade of 2 reddish laccate species including G. zonatum and G. ecuadoriense (0.96/66). Studied Uruguayan Ganoderma specimens are distributed in six clades representing species whose taxonomic resolutions are discussed below.
Ganoderma martinicense is characterized by developing short stipitate basidiomata of large dimensions (up to 30 cm upper view), with the pilear surface concentrically zonate, with melanoid incrustations in the context, pores 5-6/mm, basidiospores measuring 10-12 × 5-7 µm and non-amyloid pear-shaped to shortly cylindrical cutis cells. The species is distributed from SW North America through the Caribbean and the Neotropical Atlantic Rain Forests, [11,56], reaching Uruguay and NE Argentina. Ten of the studied specimens fit very well with that morphological description. Moreover, they are grouped with G. martinicense specimens, including the type in a strongly supported clade (1/88). Other names were used to label sequences of specimens that are conspecific with sequences of this lineage (Figure 3), i.e. G. tuberculosum [7], G. oerstedii (Fr.) Murrill [13] and G. parvulum Murrill [57]. Ganoderma tuberculosum and G. parvulum form two different, distant and unrelated lineages [6,11]. Ganoderma oerstedii has been described as presenting a context lacking resinous lines, longer basidiospores with semi-rough columnar ornamentations: 12-15 × 8-10 µm [12] and 9-14 × 6-9 µm [19]. The pileipellis is formed by irregular, lobed and branched cells with up to seven short, wide protuberances [25]. Sequences of specimens from the type locality (San Juan de Puerto Rico) need to be included to determine the taxonomic status and the phylogenetic relationships of G oerstedii. The closest relative of Ganoderma martinicense is G. multipileum (1/94), an Asian species with small pores: 6-8/mm [9].
Five sequences of Uruguayan specimens are grouped together in a distinct, well-supported clade (1/100), unrelated to any available sequence. Specimens of this clade are characterized by a stylized basidiomata, laterally to centrally slender stipite, shiny reddish orange to violet colored radial and concentric zonate pilear surface, and melanoid bands in the context. Microscopically, the cutis is composed of clavate cells ( Figure 2) and basidiospores with thick, often anastomosed endosporic ornamentations. The macro-morphological features suggest some Neotropical taxa including Ganoderma elegantum Ryvarden [12], G. concinnum [23] and G. dorsale [58]. Ganoderma elegantum is characterized by pores 6-7/mm, branched cutis cells (with up to six branches) and basidiospores with thin endosporic ornamentations [12]. Ganoderma concinnum, recorded in Colombia and Bolivia, is characterized by the formation of basidiomata with slender stipes of up to 20 cm long and it forms a different phylogenetic lineage [12,53]. Ganoderma dorsale (= G. lucidum var. dorsale in accordance with Torres-Torres et al. [25]) was described from Río Grande Do Sul in southern Brazil [59]. The macro and micro morphological characteristics of the specimens of this lineage agree with the description of the type specimen of G. dorsale [22,25] and fit well with the studied specimens: basidiomata shape and size, presence of melanoid lines in the context, basidiospores' shape and size, chemical reactions and shapes of pileipellis cells. Thus, considering morphology and distribution, Ganoderma dorsale is the most suitable name for the new clade of Uruguayan specimens. The closest relative is a phylogenetic clade named "G. lucidum" from NW Argentinean Yungas [7]. More sequences and morphological reassessments are necessary to evaluate the taxonomic status of the "G. lucidum" clade sensu Gottlieb et al. [7].
Ganoderma platense is characterized by developing sessile basidiomata, usually imbricated, with a semiorbicular zonate pilear surface context, usually with inconspicuous and discontinuous melanoid deposits, thin dissepiments and large pores (2-4/mm), pileipellis formed by cylindrical to slightly claviform elements, with apical constrictions and basidiospores measuring 9-13 × 5-8 µm, with thin, endosporic ornamentations [7,22,62]. The morphological characteristics of the studied specimens constituting this clade (Figures 1 and 2) agree with the description of G. platense and this name is hereinafter applied to the clade. Originally described from Buenos Aires (Argentina), it is currently known for growing on Platanus acerifolia trees of urban ecosystems of its type locality and on stumps of gallery forests of the Parana and Uruguay rivers [7,22,62]. Within the G. platense clade, two sequences correspond to specimens labelled as G. sessile and G. zonatum [7]; however, both species were phylogenetically circumscribed to distinct North American clades and are characterized by growing on hardwoods wood and monocots, respectively [11]. Ganoderma platense is grouped in the so called "resinaceum clade", together with G. resinaceum s.l. in Cabarroi-Hernández et al. [6], G. polychromum and G. sessile sensu Loyd et al. [11]. Ganoderma resinaceum encompasses North American, European and Asian populations, including more than one phylogenetic species. Nevertheless G. resinaceum sensu auctores from Eurasian descriptions present cutis cells with laterally diverticulate branches [22,25,63,64]. It contrasts with G. platense, which presents cylindrical to slightly clavate cells with apical constrictions. Ganoderma polychromum (Copel.) Murrill is distributed in North America, growing on hardwoods and characterized by a context without melanoid deposits, smaller pores (4-5 mm) and larger basidiospores measuring 10.8-13.2 × 6-7.5 µm [11].
Ganoderma mexicanum is known for its occurrence in Brazil, Colombia, Costa Rica, Cuba, French Guiana, Mexico, Nicaragua, South-eastern USA (Florida) [6], and now reported in Uruguay.
Morphologically, the studied specimen presents the diagnostic characters of light-colored context with variably abundant dextrinoid, smooth chlamydospores [6].
The sequences of non-laccate Ganoderma specimens collected in Uruguay were distributed in two clades: one of them with Southern Hemisphere specimens and the other one with specimens from South America and Asia [5]. Although the morphological analysis did not allow us to discriminate the specimens of each clade, the pilear surface generally appears tubercular and non-zonate in G. australe M1 and concentrically zonate G. australe M2. In addition, there seems to be ecological differences regarding host preferences: G. australe M1 specimens develop basidiomata on living hosts, whereas G. australe M2 on dead hosts. It was established that species of the G. australe/ applanatum complexes could have a recent origin (not earlier than 30 Ma), with a distribution pattern explained by a large-scale, episodic colonization model and subsequent distance isolation [5]. The recent origin, in addition to the remaining interfertility between specimens from both clades [5], may be the plausible explanation of the crypticity and lack of clear morphological differences for the specimens of the two clades of G. australe. Many names were proposed for sequence of specimens grouped in each clade: G. tornatum (Pers.) Bres., G. lobatum (Schwein.) G.F. Atk., G. annulare (Lloyd) Boedijn and G. lipsiense [7]. Ganoderma annulare and G. tornatum are largely considered synonyms of G. australe, and Ganoderma lipsiense was long considered a synonym of G. applanatum [21,25]. Ganoderma applanatum type locality is in the Northern Hemisphere and previous phylogenetic analyses suggested that it could be represented by a Northern Hemisphere clade [5,8]. Morphologically, G. applanatum lacks melanoid lines in the context [25], whereas all Uruguayan specimens present melanoid elements in the context. From the morphological, ecological and distribution data, M1 and M2 specimens should remain as G. australe s.l.
In this context, the previous records of Ganoderma in Uruguay (Table 1) should be questioned, and due to the absence of herbarium specimens, only speculations can be made in relation to published descriptions. Records of G. lorenzianum [65,66] have characteristics that resemble G. mexicanum due their morphological similarities regarding light colored context (picture in reference [67]), stipe and ovoid, smooth basidiospores (9-10 × 6-7 µm). G. lorenzianum is an earlier name than G. mexicanum but the description and picture offered in bibliography [67] and the low number of specimens of G. mexicanum analyzed in Uruguay is insufficient to assess the epitypification of these species. Focused studies on specimens corresponding to this species are urgently needed. In Uruguay, Ganoderma orbiforme (Fr.) Ryvarden (=G. fornicatum (Fr.) Pat.) was recorded by Felippone [66], but it currently forms a different phylogenetic clade [57] and no specimens or sequences corresponding to this species were recorded in the country. Ganoderma nitens was recorded by Patouillard [65] characterized by warty basidiospores, 10 × 7 µm, the type is currently lost [68], so this record could be attributed to G. dorsale, a single reddish laccate Ganoderma species of Uruguay with warty spores.

Conclusions
An integrated comprehensive approach was carried out using morphological, molecular and ecological evidence to assess the diversity of Ganoderma in Uruguay. A total of five species (forming six supported clades) were found through the analysis of 163 basidiomata and 53 sequences. Among those species, G. martinicense, G. mexicanum, G. platense and G. dorsale were confirmed and identified for the first time in Uruguay. Particularly, G. platense and G. dorsale were first recovered in the phylogenetic analyses. On the other hand, non-laccate specimens were distributed in two clades and so far considered as G. australe s.l.