Discovery or Extinction of New Scleroderma Species in Amazonia?

The Amazon Forest is a hotspot of biodiversity harboring an unknown number of undescribed taxa. Inventory studies are urgent, mainly in the areas most endangered by human activities such as extensive dam construction, where species could be in risk of extinction before being described and named. In 2015, intensive studies performed in a few locations in the Brazilian Amazon rainforest revealed three new species of the genus Scleroderma: S. anomalosporum, S. camassuense and S. duckei. The two first species were located in one of the many areas flooded by construction of hydroelectric dams throughout the Amazon; and the third in the Reserva Florestal Adolpho Ducke, a protected reverse by the INPA. The species were identified through morphology and molecular analyses of barcoding sequences (Internal Transcribed Spacer nrDNA). Scleroderma anomalosporum is characterized mainly by the smooth spores under LM in mature basidiomata (under SEM with small, unevenly distributed granules, a characteristic not observed in other species of the genus), the large size of the basidiomata, up to 120 mm diameter, and the stelliform dehiscence; S. camassuense mainly by the irregular to stellate dehiscence, the subreticulated spores and the bright sulfur-yellow colour, and Scleroderma duckei mainly by the verrucose exoperidium, stelliform dehiscence, and verrucose spores. Description, illustration and affinities with other species of the genus are provided.


Introduction
Amazonia is the largest and most diverse of the world's tropical rainforests, encompassing more than 6 million km2 in nine countries of South America. According to the Rainforest Conservation Fund [1], in the rainforest most of the organisms are undescribed and unknown.
Recent studies indicate at least 427 amphibians, 1294 birds, 3,000 fishes, 378 reptiles, 427 mammals, and 40,000 plant species in Amazonian rainforest [2]. Studies on fungi from the Brazilian Amazon forest have reported about 1000 species of macrofungi [3]. Knowledge of fungal diversity is amplified through advances in laboratory methodologies and computational analysis [4,5]. Molecular studies combined with morphological knowledge has led to a better delimitation of taxonomic groups, determining which morphological characters are informative, or not, so as to detect cryptic species. On the other hand, there seems to be consensus that these rainforests are reservoirs of the greatest amount of biodiversity as yet uncatalogued by science [6,7,8], which makes the destruction of the tropical rainforests the main challenge facing the discovery of fungi that are still unknown.
To ensure energy independence and exploit mineral resources, the governments of Amazonian countries are embarking on a major dam building drive on the basin's rivers, with 191 dams finished and a further 246 planned or under construction. This rush to reap the basin's renewable energy has come without considering the negative environmental consequences to the most speciose freshwater and terrestrial biomes of the world [9].
Brazil has emerged as one of the few countries where deforestation is falling, due to programs aimed at protecting forest areas such as blacklisting on deforestation. Critical districts with high annual forest loss are included in blacklists published regularly by the Brazilian Ministry of the Environment, and farms in those blacklists face new administrative rules to obtain licenses for clearing forests. This practice contributed to reducing the average deforestation in the years 2002 to 2012 [10,11]. Extensive projects on biodiversity studies were implemented and helped to demarcate, justify and maintain biological reserves across the country, for example, the Research Program on Biodiversity (PPBio) and the Integrated Studies Center of the Amazonian Biodiversity (CENBAM). However, the construction of increasing numbers of hydroelectric dams throughout the Amazon has led to destruction and irreversible ecological imbalance in many areas [12,13,14].
The diversity of macrofungi species present in the tropical rainforest is still insufficiently known, and Hawksworth [7] considers this biome the largest reserve of biodiversity on the planet. So far, only around 1000 species of macrofungi have been described for the Amazon forests [3]. For gasteroid fungi, 20 species have been described distributed in the states of Amazônia, Pará and Rondônia [15,16,17,18,19,20,21].
The genus Scleroderma was described in 1801 by Persoon and is currently included in the order Boletales [22]. In accordance with Guzmán et al. [23], Scleroderma is divided into three sections based on the surface structure of the basidiospores and on the presence or absence of a clamp connection: (1) Reticulatae, characterized by reticulated spores, (2) Scleroderma, with echinulate spores, and (3) Sclerangium, presenting subreticulated spores. Molecular studies, based on comparison of Internal Transcribed Spacer (ITS) nrDNA, confirm this classification [24,25].
On March 28, 2015, some of the authors of this article (NKI, IFF, SU and NM) visited Camassú, one of about 50 islands that would be flooded due to construction of Belo Monte Dam; they collected a number of Scleroderma specimens that were not possible to assign to any known species.
This work describes novelties of the genus Scleroderma from the Amazon rainforest with analyses based on morphological and molecular data.

Collections studied
The species were collected from native vegetation of the Brazilian Amazon rainforest, and were deposited in Brazilian and Spanish collections: UFRN (Rio Grande do Norte), INPA (Amazonas) and MA-Fungi (Madrid). Data of collections studied are included in Table 1. All necessary permits were obtained for studies issued by the Curators of the Herbaria (reference document number UFRN-02/2015, INPA-13/2015).

Morphological analysis
The morphological analyzes with dry material followed preliminary studies [23,30,40,41,42,43], and were performed in the fungal biology laboratory at the Universidade Federal do Rio Grande do Norte. Measurements were performed using a ruler attached to the microscope with smallest divisions at 1 mm. For microscopic analysis hand-cut sections of the layers of the peridium and spores, mounted in 5% KOH, Melzer's reagent and Congo Red were examined with the light microscope. The standardization of the colors followed Kornerup and Wanscher [44].

Molecular analyses
Samples for DNA extraction were excised from dry basidiomes. To avoid contamination by other fungi, pseudotissues were taken from the inner part of the basidiome. DNA extraction, amplification, and sequencing of the ITS regions including the 5.8S of the ribosomal RNA gene cluster followed the protocols mentioned by Phosri et al. [24]. The ITS regions were amplified with Ready-To-Go TM PCR Beads (GE healthcare Life Sciences, NJ, USA), using the primers ITS1F [45] and ITS4 [46], and the cycling protocol described in Martín and Winka [47]. Aliquots of the purified products were mixed separately with the direct and reverse primers before sending them to Macrogen (South Korea) for sequencing. Consensus sequences were assembled using Sequencher software (Gene Codes Corporation Inc, Ann Arbor, Michigan, USA). Previous to the alignment, sequences were compared with homologous sequences from the EMBL/GenBank/DDBJ [48] using the BLASTn algorithm [49]. All new sequences have been deposited on the EMBL-EBI database and their accession numbers are presented in Table 1.
The alignment was analyzed using the programms PAUP 4.0a147 [50], MrBAYES 3.2.2 [51] and RAxML [52] using the CIPRES portal (http://www.phylo.org/portal2/) [53]. Pisolithus arhizus FM213365 was used as outgroup, since this species is closely related to Scleroderma [54]. First, a parsimony analysis under a heuristic search was conducted. Gaps were treated as missing data. The tree branch robustness was estimated by bootstrap (MP-BS) analysis [55] employing 10000 replicates, using the fast-step option. The starting branch lengths were obtained using the Roger-Swofford approximation method and the starting trees for branch swapping were obtained by stepwise addition. The tree bisection-reconnection (TBR) branchswapping algorithm was used with the Multitrees options. The data were further analyzed using a Bayesian approach [56,57]. The posterior probabilities (PP) were approximated by sampling trees using the MCMC method. The Bayesian analysis was performed assuming the general time reversible model [58] including estimation of invariant sites and assuming a discrete gamma distribution with six rate categories (GTR+I+G). A run with 2M generation starting with a random tree and employing 12 simultaneous chains was executed. Every 100th tree was saved into a file. The log-likelihood scores of sample points were plotted against the number of generations using TRACER 1.0 (http://evolve.zoo.ox.ac.uk/software.html) to determine that stationarity was achieved when the log-likelihood values of the sample points reached a stable equilibrium value [51]. The initial 1000 trees were discarded as a burn-in before calculating posterior probabilities (PP). Using the "sumt" command of MrBAYES, the majority-rule consensus tree was calculated from 19K trees sampled after reaching likelihood convergence to calculate the posterior probabilities. A third maximum likelihood bootstrapping analysis was performed with RAxML 7.2.8 [52], using the default parameters as implemented on the CIPRES NSF XSEDE resource with bootstrap statistics calculated from 1000 bootstrap replicates (ML-BS) under GTR + I + G model of evolution. The phylogenetic tree was drawn with the program TreeView [59] and edited in Adobe Illustrator CS3; names of clades and subclades are according to Phosri et al. [24], Rusevska et al. [25], and Crous et al. [39]. A combination of MP-BS, ML-BS, and PP was used to assess confidence for a specific node [60,61].

Nomenclature
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Molecular studies
The sequences obtained from Amazonian specimens have been deposited within EMBL (http://www.ebi.ac.uk/embl) with the accession numbers indicated in Table 1. The topologies of the three analyses performed (Maximum parsimony, Maximum likelihood and Bayesian) were similar to each other; the 50% majority-rule consensus tree from the Bayesian analysis is shown in Fig 1  Description. Basidiomata epigeous, sessile, subglobose when closed, up to 90 mm diam × 45 mm high; when mature, stellate dehiscence forming 5-7 irregular branches, the expanded basidiomata up to 115 mm diam × 75 mm high, with rhizomorphs aggregated at the  Table 1. base (Fig 2A). Surface reticulated, brown (5F6, 5F7) to dark brown (6F6) at maturity, with aggregated soil particles (Fig 2B). Peridium 450-600 mm thick, with three layers (Fig 2C): the outer layer made of cylindrical hyphae, yellowish in KOH, 2.5-6.5 μm diam, walls up to 1.0 μm thick, winding ( Fig 2D); the middle layer consists of cylindrical hyphae, with rounded ends at the surface, hyaline in KOH, 4.5-16 μm diam, walls up to 2.5 μm thick; and inner layer pale yellow (3A3), composed of interwoven cylindrical hyphae, hyaline in KOH, 4.0-6.5 μm diam, walls up to 1.0 μm thick, clamp connections rare. Gleba when mature greyish brown (6E3), compact to powdery at maturity, protected by the inner layer of peridium. Basidiospores 3.5-5.3 × 3.8-5.4 μm diam including ornamentation, globose to subglobose, hyaline to yellowish in KOH, smooth under LM (Fig 2E), with small granules on the surface under SEM (Fig 2F).
Remarks. Scleroderma anomalosporum is characterized mainly by the smooth spores under LM in mature basidiomata and the large size of the basidiomata, being capable of achieving up to 120 mm in diameter when expanded, and the stelliform dehiscence. In accordance with Guzmán et al. [22], smooth spores in the genus Scleroderma are found in immature basidiomes, and when mature, the spores vary between reticulated, subreticulated and echinulate. The spores of S. anomalosporum under SEM present small, unevenly distributed granules, a characteristic not observed in other species of the genus. Scleroderma polyrhizum (J.F. Gmel) Pers. and S. texense Berk. present basidiomata that can reach up to 150 mm and 140 mm, respectively, when expanded. However, they have larger spores (7-11 μm in diameter) than S. anomalosporum, and different ornamentation: subreticulated and lightly echinulate spores in S. polyrhizum [23,30], and reticulated in S. texense [23,30]. Isotype. INPA 271114 Diagnosis. Basidiomata epigeous, sessile or pseudostipitate, opening by a dehiscence irregular to stellate, up to 20 mm diam, surface scaly to verruculose. Peridium up to 0.5 mm thick, consisting of three layers. Basidiospores 6.4-8.0 × 5.6-7.5 μm diam, globose to subglobose, subreticulated under LM, irregular reticulum under SEM.
Remarks. Scleroderma camassuense is characterized mainly by the irregular to stellate dehiscence, the subreticulated spores and the yellow sulfur colour. Scleroderma sinnamariense, S. verrucosum (Bull.) Pers., S. citrinum Pers. and S. uruguayense (Guzmán) Guzmán also present a dark yellow peridium, but can be distinguished by the size of the larger basidiomata (up to 45 mm in diameter) and the presence of pilocystidia in the external layer of the peridium in S. sinnamariense [30,42]; by the larger and echinulate spores (9-12 μm in diameter) in S. verrucosum [43]; and by the larger and reticulated spores (11-14 μm in diameter) in S. citrinum and S. uruguayense [30,43].

Discussion
The centers of endemism in Amazonia are well established for animals (vertebrates) and plants [63], and the type locality of the two new Scleroderma species, S. anomalosporum and S. camassuense, are within these areas of high endemism. According to Haffer [64], there are many hypotheses proposed to explain barrier formation separating populations and causing the differentiation of species in Amazonia during the course of geological history based on different factors. Among them there is the river hypotheses, due to the barrier effect of Amazonian rivers. Several anthropogenic activities such as accelerated deforestation and flooded areas from the construction of dams, contribute to the rapid habitat degradation in the Central Amazon. These events, and the disorderly growth of cities in Northern Brazil, in association with climate changes, makes the scientific community recognize the urgency in learning about the biodiversity in this kind of megadiverse area, before the current species become extinct due to human activities [65,66,67,68,69].
The results obtained through the morphological and molecular studies show an interesting species richness of Scleroderma with peculiar morphology, as in the case of S. anomalosporum that presents unusual spores in comparison with other species of this genus. The sequences obtained from Amazonian specimens as indicated in Fig 1, show that the Amazonian specimens are grouped in three different clades and well-supported to be considered independent taxa. Scleroderma anomalosporum and S. camassuense are described from Camassú island (type locality) that is now under the unnatural level of the Xingu River waters; and the third new species, S. duckei, is described from a protected reserve by the INPA. After decades of collecting in rainforests, these three species have not been described before, and they could be endemic to their respective habitats. In a recent study of diversity and distribution of ectomycorrhizal fungi in white-sand forests in Amazonia (along the Cuieiras river) and French Guiana [70], only S. minutisporum [17] is mentioned. In Amazonia, species have restricted distribution [71], being very sensitive to any changes in their habitats [72,73]. Based on complete checklists of published flora data from Brazil (Ducke Reserva), French Guiana (Saül region) and Peru (the Iguits area), Hopkins [74] pointed that it is extremely rare to found a species in any locality; based on this, authors claim that the conservation of Amazonian biodiversity requires actions in all landscapes, not only in protected areas [75].
There are enormous shortfalls in biological knowledge of the Amazon rainforest [76]. Although great efforts to develop international research networks are gathering existing data about species diversity, the number of species that the Amazonia contains it is not yet known [77]. With the exiting data, how can we best protect Amazonia's biodiversity? Authors agree that the more knowledge we have, the better prepared we will be to protect and maintain the Amazonian biodiversity [75]. However, the scientific process of describing new species is slow compared with the high rates of destructions of natural landscapes [75]. At least, two of the What do we already know about fungal extinctions and how much does it matter? When looking through public databases for studies related to fungus (or fungi) and extinction, almost all papers listed concern pathogenic fungi associated, for example, with the risk of extinction and decline of amphibians [78,79], bees [80], bats [81,82,83], or with recent hypotheses about their role in mass extinction of dinosaurs [84], also some related to invasive plants diseases [85]. However, the information related to the extinction of fungi per se is limited [86]. It is well known that fungi play a key role in all biomes as organic matter decomposers and, the great contribution that ectomycorrhizal fungi make to plant nutrition in infertile soils [87], such as Scleroderma species. Many Scleroderma species found in Brazil forms ectomycorrhiza with introduced Pinus spp. and Eucalyptus spp.; however, two species where located in native vegetation of the Amazon rainforest [17,39], as well as, the three species described here. The extinction of any mycorrhizal fungi can be a very important matter to the associate plant.
Our results support the designation of the Amazon Forest as a hotspot [88] with high diversity and several taxa still unknown to Science. Inventory studies are urgent, mainly in the areas most endangered by human activities, where species could be in risk of extinction before being described and named.