REVISÃO DA FAMÍLIA GEASTRACEAE CORDA (, ) COM ÊNFASE EM ESPÉCIES NEOTROPICAIS

Julieth de Oliveira Sousa ______Tese de Doutorado Natal/RN, fevereiro de 2019 JULIETH DE OLIVEIRA SOUSA

REVISÃO DA FAMÍLIA GEASTRACEAE CORDA (GEASTRALES, BASIDIOMYCOTA) COM ÊNFASE EM ESPÉCIES NEOTROPICAIS

ORIENTADOR: IURI GOULART BASEIA (UFRN)

CO-ORIENTADORA: MARÍA PAZ MARTÍN (RJB-Espanha)

Natal - RN 2019

JULIETH DE OLIVEIRA SOUSA

REVISÃO DA FAMÍLIA GEASTRACEAE CORDA (GEASTRALES, BASIDIOMYCOTA) COM ÊNFASE EM ESPÉCIES NEOTROPICAIS

Tese apresentada ao Programa de Pós- graduação em Sistemática e Evolução da Universidade Federal do Rio Grande do Norte, em cumprimento às exigências para obtenção do título de Doutora em Sistemática e Evolução.

Aprovada em: 26 de fevereiro de 2019.

Comissão Examinadora:

______Dr. Iuri Goulart Baseia – UFRN (presidente)

______Dr. Bruno Tomio Goto – UFRN

______Dra. Raquel Cordeiro Theodoro – UFRN

______Dra. Bianca Denise Barbosa da Silva – UFBA

______Dra. Rhudson Henrique Santos Ferreira da Cruz – UFOB

Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI

Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial Prof. Leopoldo Nelson - •Centro de Biociências - CB

Sousa, Julieth de Oliveira.

Revisão da família Geastraceae corda (Geastrales, Basidiomycota) com ênfase em espécies neotropicais / Julieth de Oliveira Sousa. - Natal, 2019.

164 f.: il.

Tese (Doutorado) - Universidade Federal do Rio Grande do Norte. Centro de Biociências. Programa de Pós-Graduação em

1. Fungos gasteroides - Tese. 2. Estrelas-da-terra - Tese. 3. Barcode - Tese. 4. Phallomycetidae - Tese. 5. Taxonomia - Tese.

I. Baseia, Iuri Goulart. II. Martín, María Paz. III. Universidade Federal do Rio Grande do Norte. IV. Título. RN/UF/BSE-CB CDU 582.281.21

Elaborado por KATIA REJANE DA SILVA - CRB-15/351

DEDICATÓRIA

Dedico este trabalho a todos os pesquisadores brasileiros, incluindo os alunos de pós-graduação. AGRADECIMENTOS

Agradeço a Universidade Federal do Rio Grande do Norte, instituição a qual estou vinculada há 10 anos, por proporcionar o suporte necessário para minha formação desde a graduação (2009) até o doutorado (2019). Sou grata aos órgãos de fomento: Coordenação de Aperfeiçoamento de Pessoa de Nível Superior (CAPES) e Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) pelo financiamento deste trabalho e pelas Bolsas de Doutorado (por um ano em 2015/2016) e Doutorado Sanduíche (por quatro meses em 2017). Agradeço também a Pós-Graduação em Sistemática e Evolução. Hoje, como servidora da UFRN, eu devo agradecer ao Departamento de Nutrição, onde estou lotada, por ter me concedido afastamento por quatro meses para realizar Doutorado Sanduíche no Real Jardín Botánico de Madrid- Espanha.

Aos meus orientadores Prof. Iuri Baseia e Profa. María P. Martín sou extremamente grata. O Prof. Iuri, que é meu orientador desde a graduação, eu devo agradecer pela confiança que sempre depositou no meu trabalho e por todo o suporte que me conferiu durante esses anos. A Profa. María eu agradeço imensamente por tudo que vem me ensinando e por tornar este trabalho possível.

Sou grata aos professores Bianca Silva, Bruno Goto, Raquel Theodoro e Rhudson Cruz por aceitarem participar da avaliação deste trabalho.

Agradeço a todos os coautores dos artigos resultado desta tese e os colegas do Laboratório de Biologia de Fungos que colaboraram de alguma forma para a realização deste trabalho.

Os agradecimentos a familiares e amigos serão feitos pessoalmente, mas deixo aqui registrado a gratidão a todos que contribuíram direta ou indiretamente para que eu finalizasse esta etapa.

RESUMO

Cerca de 97% das espécies fúngicas existentes ainda não foram descritas pela ciência, mesmo a identificação sendo embasamento para uma gama de estudos aplicados (e.x. bioprospecção, evolução, ecologia, conservação). A utilização dos códigos de barras moleculares (barcodes) vem auxiliando a delimitação de espécies. Sobretudo para os fungos gasteroides (Basidiomycota), o espaçador transcrito interno do DNA ribossômico nuclear (ITS) vem demostrando eficiência para o descobrimento de uma diversidade escondida. A família Geastraceae é constituída pelos gêneros gasteroides e Myriostoma, sendo os espécimes popularmente conhecidos como estrelas- da-terra (earthstars). Embora seja uma das mais ricas da ordem Geastrales, o conhecimento sobre a diversidade desta família apresenta lacunas, especialmente na região Neotropical, onde há países megadiversos, “hotspots” e ecossistemas tropicais, os quais demonstram elevado potencial para abrigar uma diversidade escondida. Assim, objetiva-se revisar coleções de representantes da família Geastraceae, enfatizando os que apresentam distribuição Neotropical. As análises basearam-se em dados morfológicos e moleculares. Foram investigadas 215 amostras provenientes de 10 diferentes herbários nacionais e internacionais; sendo destas 14 coleções tipo. A metodologia consistiu em uma profunda revisão dos caracteres morfológicos, além de análises filogenéticas moleculares das regiões ITS, LSU, ATP6, RPB2 e TEF1α, sobre os critérios de Máxima Parcimônia, Máxima Verossimilhança e Inferência Bayesiana. Foram geradas 186 sequências novas, as quais foram comparadas com 294 sequências homólogas provenientes do banco de dados GenBank. Foram descritas 12 espécies novas de Geastrum: G. laevisporum J.O. Sousa & Baseia; G. pusillipilosum J.O. Sousa et al.; G. verrucoramulosum T.S. Cabral, J.O. Sousa & Baseia; G. magnosporum J.O. Sousa et al.; G. caatingense J.O. Sousa, M.P. Martín & Baseia; G. parvistellum J.O. Sousa, M.P. Martín & Baseia; G. baculicrystallum J.O. Sousa et al.; G. brunneocapillatum J.O. Sousa et al.; G. courtecuissei P.-A. Moreau & C. Lécuru, G. neoamericanum J.O. Sousa et al.; G. rubellum P.-A. Moreau & C. Lécuru; G. rubropusillum J.O. Sousa et al. O nome Geastrum hirsutum Baseia & Calonge foi revalidado e teve sequência de seu parátipo disponibilizano GenBank. Para o gênero Myriostoma duas espécies novas foram descritas: M. calongei Baseia, J.O. Sousa, & M.P. Martín e M. australianum J.O. Sousa Baseia & M.P. Martín; ademais, foram propostas duas combinações novas: M. areolatum (Calonge & M. Mata) M.P. Martín, J.O. Sousa & Baseia e M. capillisporum (V.J. Stanek) L.M. Suz et al. Foram propostos, também, um lectótipo e um epitipo para a espécie M. coliforme (Dicks.) Corda. Os dados gerados por esta revisão modificaram as interpretações sobre a sistemática de Geastraceae, sendo possível comprovar que os complexos de espécies existentes subestimavam a diversidade da família Geatraceae em região Neotropical.

Palavras chave: barcode; estrelas-da-terra; fungos gasteroides; Phallomycetidae; taxonomia.

ABSTRACT

About 97% of existing fungi have not been described by science, although the identification being the basis for many appliced studies (ex: bioprospecting, evolution, ecology, conservation). Barcodes have been very useful to species delimitation. Mainly for gasteroid fungi (Basidiomycota), the Internal Transcribed Spacer of Nuclear rDNA (ITS) has demonstrated to be very efficacy to discover hidden diversity. The family Geastraceae is constituted by the gasteroid Geastrum and Myriostoma, being the specimens popularly known as earthstars. Although it is one of the richest families in the Geastrales order, the knowledge about the diversity of this family has gaps, especially in the Neotropical region, where there are megadiverse countries, “hotspots” and tropical ecosystems, which have high potential to shelter hidden diversity. Thus, this study aimed to review collections of family Geastraceae, emphasizing those with Neotropical distribution. Two hundred and fifteen samples from 10 distinct international and national fungal collections were investigated, of these, 14 collections. The methodoly consisted in a deep revision of morphological characters, besides the molecular phylogenetic analyses of the DNA regions ITS, LSU, ATP6, RPB2 e TEF1α, following Maximum Parsimony, Maximum Likelihood and Bayesian inference criteria. There were generated 186 new sequences, which were compared with 294 homologue sequences from GenBank data. Twelve news species of Geastrum were described: G. laevisporum J.O. Sousa & Baseia; G. pusillipilosum J.O. Sousa et al.; G. verrucoramulosum T.S. Cabral, J.O. Sousa & Baseia; G. magnosporum J.O. Sousa et al.; G. caatingense J.O. Sousa, M.P. Martín & Baseia; G. parvistellum J.O. Sousa, M.P. Martín & Baseia; G. baculicrystallum J.O. Sousa et al.; G. brunneocapillatum J.O. Sousa et al.; G. courtecuissei P.-A. Moreau & C. Lécuru, G. neoamericanum J.O. Sousa et al.; G. rubellum P.-A. Moreau & C. Lécuru; G. rubropusillum J.O. Sousa et al. For the genus Myriostoma, two new species were described: M. calongei Baseia, J.O. Sousa, & M.P. Martín and M. australianum J.O. Sousa Baseia & M.P. Martín; moreover, two new combinations were proposed: M. areolatum (Calonge & M. Mata) M.P. Martín, J.O. Sousa & Baseia and M. capillisporum (V.J. Stanek) L.M. Suz et al. A lectotype and an epitype for the specie M. coliforme (Dicks.) Corda was elected. The data generated by this revision changed the systematic interpretations about the family Geastraceae, it was possible to prove that there were species complex which underestimated the knowledge about the richness of this family in Neotropical region. Keywords: barcode; earthstar; gasteroid fungi; Phallomycetidae; .

LISTA DE FIGURAS

Figura 1. Estruturas tradicionalmente utilizadas nas descrições taxonômicas de Geastrceae. A-B. Geastrum. C-D. Myriostoma...... 26 Figura 2. Exemplos de géis de eletroforese obtidos neste trabalho. A. Gel de comprovação amplificação de TEF1α (EF1-1018F/EF11620R). B. Gel de comprovação amplificação de RPB2 (RPB2-5F/RPB2-7.1R nested com bRPB2-6F/bRPB2-7R2). C. Gel de comprovação de purificação poços de 1-6 região ITS (ITS5/ITS4), poços 7-12 região LSU (LR0R/LR5). D. Gel de comprovação de purificação poços de 1-5 região LSU (LR0R/LR7), poços de 6-12 região LSU ( LR0R/LR5)...... 33

Capítulo I

Figura 3 (Fig. 1) Site in “Caatinga”, Brazil. A. Map of the “Caatinga” biome in Brazil. B. Paraíba State. C-D. landscape images...... 56 Figura 4 (Fig. 2) Basidiomata in situ (A-B) and ex situ (C-D). A. Basidiomata with arched exoperidium. B. Detailed mycelial layer. C. Basidiomata with strongly hygroscopic rays. D. Detailed peristome...... 57 Figura 5 (Fig. 3) Basidiospores under LM (A-D)...... 58 Figura 6 (Fig. 4) SEM images. A-B. Basidiospores. C. Hyphae of endoperidium. D. Eucapillitium...... 59

Capítulo II

Figura 7 (Fig. 1) Strict consensus tree of the eight most parsimonious trees of concatenated ITS and LSU nrDNA sequences of Geastrum indicated in Table 1...... 74 Figura 8 (Fig. 2) Geastrum caatingense sp. nov. A. Fresh basidiomata in situ (UFRN– Fungos 2843, holotype). B. Fresh basidioma in situ. C (UFRN–Fungos 2960, isotype). Peristome detail. D. Endoperidial body detail. E. Endoperidium surface under SEM. F. Eucapillitialy under SEM. G. Basidiospores under LM. H. basidiospore under SEM...... 75 Figura 9 (Fig. 3) Geastrum parvistellum sp. nov. A. Fresh basidioma in situ (UFRN– Fungos 2841, holotype). B. Fresh basidioma in situ (UFRN–Fungos 2961, isotype). C.

Peristome detail. D. Pedicel detail. E. Endoperidium surface under SEM. F. Eucapillitial hyphae under SEM. G. Basidiospores under LM. H. Basidiospore under SEM...... 76

Capítulo III

Figura 10 (Fig. 1) Bayesian trees of ITS (on the left) and ITS/LSU (on the right) nrDNA sequences of Geastrum species...... 83 Figura 11 (Fig. 2) Type collections of Geastrum hirsutum and Geastrum schweinitzii. (A) Geastrum hirsutum MA-Fungi 67886, paratype. (B) Geastrum hirsutum UFRN- Fungos 245, holotype. (C) Geastrum schweinitzii K (M) 180188, type. (D) Geastrum schweinitzii K (M) 180187 ...... 85 Figura 12 (Fig. 3) Analysis involving species of Geastrum schweinitzii complex. (A) Bayesian analysis conducted in Beast 2 software along 50 million generations. (B) Topo- phylogenetic and (C) phylogenetic network representations...... 87 Figura 13 (Fig. 4) Geastrum schweinitzii and allies. (A). Geastrum neoamericanum sp. nov. (B) Geastrum baculicrystallum sp. nov. (C). Geastrum courtecuissei sp. nov. (D) Geastrum rubropusillum sp. nov...... 88 Figura 14 (Fig. 5) Geastrum hirsutum and allies. (A) Geastrum brunneocapillatum sp. nov. (B) Geastrum hirsutum. (C) Geastrum pusillipilosum. (D) Geastrum rubellum sp. nov...... 90

Capítulo IV

Figura 15 (Fig. 1) The 50% majority-rule consensus tree of ITS/LSU nrDNA sequences of Myriostoma species using a Bayesian approach...... 108 Figura 16 (Fig. 2) Myriostoma areolatum (MA-Fungi 68596, isotype). (a) Dried expanded basidioma ex situ, bar = 10 mm. (b) Stomata, bar = 5 mm. (c) Basidiospores under SEM, bar = 2 μm. (d) Endoperidial surface under SEM, bar = 50 μm...... 110 Figura 17 (Fig. 3) Myriostoma calongei. (a) Fresh expanded and unexpanded basidiomata in situ (UFRN-Fungos 2019, holotype), bar = 20 mm. (b) Endoperidial surface (UFRN-Fungos 386, paratype), bar = 1 mm. (c) (UFRN-Fungos 386, paratype), bar = 2 mm. (d) Basidiospores under LM (UFRN-Fungos 2020, isotype), bar = 10 μm. (e) Capillitium under LM (UFRN-Fungos 2019, holotype), bar = 10 μm. (f) Basidiospores under SEM, bar = 2 μm. (g) Endoperidial surface under SEM (UFRN- Fungos 2019, holotype), bar = 50 μm...... 112

Figura 18 (Fig. 4) Myriostoma capillisporum. (a–b) Dried expanded basidiomata ex situ (KM205483 and K(M)205482, respectively), bar = 20 mm. (c–d) Basidiospores under LM (K(M)205483), bar = 10 μm. (e) Basidiospores under SEM (K(M)205483), bar = 2.5 μm. (f) Endoperidial surface under SEM (K(M)205483), bar = 50 μm...... 113 Figura 19 (Fig. 5) Myriostoma coliforme. (a–b) Dried expanded basidiomata ex situ (K(M)138625, epitype), bar 10 mm. (c) Endoperidial surface (PC0723885), bar = 1 mm. (d) Stoma (MJ8714), bar = 1 mm. (e) Basidiospores under LM (K(M)138625, epitype), bar = 10 μm. (f) Capillitium under LM (MJ8714), bar = 10 μm. (g) Basidiospores under SEM (K(M)138625, epitype), bar = 2.5 μm. (h). Endoperidial surface under SEM (K(M)138625, epitype), bar = 100 μm...... 116 Figura 20 (Fig. 6 )Dickson’s illustration of Myriostoma coliforme (lectotype) published in 1785 as (Tab. III: Fig 4)...... 117 Figura 21 (Fig. 7) Distribution map of the Myriostoma specimens included in the phylogenetic analyses of this study (geometric figures in colour)...... 117

Capítulo V

Figura 22 (Fig. 1) Phylogenetic tree, 50% Bayesian majority rule combined consensus tree of ITS and LSU nrDNA...... 133 Figura 23 (Fig. 2) Myriostoma australianum (holotype MEL 2305388). A: Expanded dried basidiomata B: Stoma in detail. C: Basidiospore (LM). D–E: Basidiospores (SEM)...... 134 Figura 24 (Fig. 3) Myriostoma australianum (holotype MEL 2305388), Exoperidium layers under LM. A. Mycelial layer under LM B. Fibrous layer C. Pseudoparenchimatous layer...... 135 Figura 25 (Fig. 4) Line drawing of basidiospores. A. Myriostoma australianum (holotype MEL 2305388). B. M. capillisporum (K(M)205483)...... 135

Anexo I

Figura 26. Overview Mucoromycotina and Agaricomycotina phylogeny ...... 140 Figura 27. One of the 19 equally most parsimonious trees of ITS nrDNA sequences obtained after a heuristic search using SeaView v. 4.6 (Gouy et al. 2010)...... 142 Figura 28. Colour illustrations. Brazil, Paraíba, Reserva Biológica Guaribas, field track where the type species was collected; a. Basidiomata in situ (UFRN- Fungos 2316,

isotype); b. detail of hairy exoperidium (UFRN-Fungos 2315, holotype); c. basidiospores under the light microscope (UFRN-Fungos 2314); d. verrucose basidiospore with columnar warts (UFRN-Fungos 2314)...... 142

Anexo II

Figura 29 (Fig. 1) Phylogenetic tree obtained by Bayesian analysis derived from concatenated data (atp6 and nuc-LSU), with representatives of section Exareolata. Codes after species names are herbarium vouchers; in bold the new species Geastrum verrucoramulosum. Numbers on nodes indicate support values (posterior probabilities values above, and percentage of bootstrap below)and the scale bar indicates substitution per site...... 155 Figura 30 (Fig. 2) Geastrum verrucoramulosum sp. nov., fresh (A, B) and dried (C–F) basidiomata. A: LABEV 6059, paratype (Photo: Wendeson Castro). B: INPA264956, holotype (Photo: D.L. Komura). C–F: UFRN-Fungos 2782, paratype (Photos: Wendeson Castro). C: Exoperidium with densely verrucose surface. D: Non- delimitated peristome. E: Cross-section expanded basidioma. F: Ramulose stipe…………………………………………………………………………………. .. 156 Figura 31 (Fig. 3) Geastrum verrucoramulosum sp. nov., micro-structures of UFRN- Fungos 2782 (paratype) under scanning electron microscope (Photo: Iuri G. Baseia). A, B: Basidiospores. C: Hyphae of endoperidium surface. D: Hyphae of eucapillitium.. 157 Figura 32 (Fig. 4) Geastrum verrucoramulosum sp. nov., micro-structures of stipe (A, B). A: Under 400 × of light microscope. B: Under 1000 × of light microscope...... 157

Anexo III

Figura 33. Overview Mucoromycotina and Basidiomycota phylogeny...... 162 Figura 34. Colour illustrations. Brazil, Paraíba, Reserva Biológica Guaribas, SEMA II, open area of Atlantic rainforest where the type species was collected; expanded basidiomata in situ (UFRN – Fungos 2312, holotype); expanded basidiomata ex situ (UFRN – Fungos 2312, holotype); basidiospores under LM; basidiospores under SEM; eucapillitium under SEM. Scale bars = 2.5 mm (basidiomata in situ), 2 mm (basidiomata ex situ), 10 μm (basidiospores under LM), 1 μm (basidiospores and eucapillitium under SEM)...... 164

Figura 35. The first of three equally most parsimonious trees of the ITS nrDNA sequence alignment were obtained from a heuristic search...... 164

LISTA DE TABELAS

Tabela 1. Regiões alvos da amplificação ...... 29 Tabela 2. Espécies novas descritas neste trabalho...... 32 Tabela 3. Material utilizado nas análises moleculares...... 34

Capítulo II

Tabela 4 (Table 1) Geastrum species included in the molecular analyses with their country, collection number and GenBank accession numbers of ITS and LSU of nuclear ribosomal DNA. The new sequences in bold...... 72 Tabela 5 (Table 2) Comparative table with morphologic characteristics from species of section Corollina...... 73

Capítulo III

Tabela 6 (Table 1) Specimens and sequences included in this study...... 81

Capítulo IV

Tabela 7 (Table 1) Specimens and sequences included in this study...... 105 Tabela 8 (Table 2) Matrix of pairwise Kimura-2-parameter (K2P) distance between ITS sequences from the four species analysed in this paper...... 109

Capítulo V

Tabela 9 (Tabela 1) Specimens and sequences used to reconstruct the phylogenetic trees. New ITS and LSU sequences are indicated in bold...... 127 Tabela 10 (Tabela 2) Comparative tableo of morphologic characteristics of Myriostoma australianum and M. capillisporum...... 131

Anexo II

Tabela 11 (Table 1) Sequences used in aligment 2. Species names, herbarium vouchers, localities, and Genbank accession numbers...... 154

SUMÁRIO 1. INTRODUÇÃO ...... 18 1.1. Geastraceae...... 20 1.1.1. Geastrum ...... 23 1.1.2. Myriostoma ...... 24 2. OBJETIVOS...... 25 3. MATERIAL E MÉTODOS ...... 25 3.1. Análises Morfológicas...... 25 3.2. Análises Moleculares ...... 27 4. RESULTADOS E DISCUSSÃO ...... 31 4.1. Geastrum ...... 40 4.2. Myriostoma ...... 44 Capítulo I — Geastrum laevisporum: a new earthstar with uncommon smooth ...... 54 Introduction ...... 55 Materials & Methods ...... 56 Results...... 57 References ...... 60 Capítulo II — Contribution to Neotropical data of Geastrum section Corollina (Basidiomycota): Two new earth–stars from Caatinga vegetation, Brazil ...... 62 Introduction ...... 65 Materials and methods ...... 65 Results… ...... 66 Discussion ...... 68 References ...... 69 Capítulo III — Hidden fungal diversity from Neotropics: Geastrum hirsutum, G. schweinitzii (Basidiomycota, Geastrales) and their allies ...... 77 Introduction ...... 78 Materials and methods ...... 79 Results ...... 82 Discussion ...... 96 References ...... 99 Capítulo IV — More than one fungus in the pepper pot: Integrative taxonomy unmasks hidden species within Myriostoma coliforme (Geastraceae, Basidiomycota) ...... 102

Introduction ...... 103 Materials and methods ...... 104 Results… ...... 106 Discussion ...... 118 References ...... 119 Capítulo V — Strengthening Myriostoma (Geastraceae, Basidiomycota) diversity: Myriostoma australianum sp. nov ...... 122 Taxonomy ...... 129 References ...... 131 6. CONCLUSÃO GERAL ...... 136 7. CONSIDERAÇÕES FINAIS ...... 136 Anexo I — Geastrum pusillipilosum J.O. Sousa, Alfredo, R.J. Ferreira, M.P. Martín & Baseia, sp. nov...... 137 Anexo II — A remarkable new species of Geastrum with an elongated branched stipe 143 Anexo III — Geastrum magnosporum J.O. Sousa, B.D.B. Silva, P. Marinho, M.P. Martín & Baseia, sp. nov...... 158

1. INTRODUÇÃO

O conhecimento sobre a diversidade do Reino Fungi (cerca de 135 mil espécies descritas) está a uma distância abissal quando comparado aos outros Reinos eucarióticos, como Plantae (cerca de 400 mil espécies descritas) e Animalia (cerca de 1 milhão de espécies descritas) (Mora et al., 2011; Hibbett et al., 2016; RBQ Kew, 2016). A incipiência sobre o estudo dos fungos fica ainda mais evidente frente às estimativas de diversidade. Na era pré-estudos de biologia molecular, Hawksworth (1991) estimou o número de espécies fúngicas em 1.5 milhões, baseado na relação média de fungos encontrada por espécie de planta (6:1) em Ilhas britânicas. Estudos moleculares mais atuais estimam que exista aproximadamente cinco a seis milhões de espécies de fungos (Blackwell, 2011; Taylor et al., 2014), sendo, portanto quase 97% das espécies existentes ainda nem sequer descritas pela ciência.

Os fungos são organismos heterotróficos extremamente múltiplos em suas formas. Estão “virtualmente onipresentes”, sendo encontrados na forma unicelular ou pluricelular. São fundamentais agentes na decomposição, além de realizarem importantes interações ecológicas mutualísticas, parasitárias ou predatórias com animais e plantas. Quando “domesticados”, os fungos têm alta aplicabilidade em vários setores da economia mundial: como fabricação de pão, cerveja e vinho, proveniente da fermentação realizada pela levedura Saccharomyces cerevisiae Meyen ex E.C. Hansen; produção de fármacos como a penicilina, que foi o primeiro antibiótico descoberto a partir de uma espécie do gênero Penicillium Link; cultivo de cogumelos comestíveis como Agaricus bisporus (J.E.Lange) Imbach, Lentinula edodes (Berk.) Pegler e Tuber melanosporum Vittad. (conhecidos na gastronomia como Champignon, Shitake e Trufa Negra, respectivamente), os quais têm elevado valor comercial (Carlile et al., 2001; Webster & Weber, 2007; Willis, 2018).

O degrau elementar para que se possa utilizar qualquer organismo, independente de sua aplicabilidade, é a identificação. A taxonomia é a ciência responsável por esta tarefa, além de ser a base para estudos evolutivos, ecológicos e de conservação. A taxonomia do Reino Fungi passou por uma revolução nas últimas décadas, devido ao emprego da ferramenta molecular. A utilização de porções da sequência do DNA como caracteres vem auxiliando na delimitação de espécies, uma vez que a utilização de apenas caracteres morfológicos, e os diferentes pesos dados a eles, pode se tornar um

18

obstáculo para classificar os mais variados grupos (Stielow et al., 2011; Haelewaters, 2018).

Regiões do DNA ribossomal (subunidade menor do rDNA ribossômico “SSU”; subunidade maior do rDNA ribossômico “LSU”; espaçador transcrito interno do rDNA ribossômico “ITS”) e mitocondriais (subunidade 6 da ATP sintase-DNA mitocondrial “ATP6”) são preferencialmente empregadas para responder questões da sistemática e evolução de fungos em diferentes níveis taxonômicos. Essas regiões são principalmente utilizadas pela facilidade em amplificação, uma vez que apresentam muitas cópias ao longo da cadeia de DNA; e devido à presença de iniciadores (primers) universais (White et al., 1990; Gardes & Bruns, 1993; Matheny et al., 2002). Em 2012 a região ITS foi designada como região código de barra (barcode) universal do Reino Fungi (Schoch et al., 2012). Entretanto, estudos prévios demonstraram também resultados satisfatórios na sistemática em nível de espécie utilizando-se regiões codificadoras de proteínas como: maior subunidade da RNA polimerase II “RPB1”; segunda subunidade maior da RNA polimerase II “RPB2” e fator de elongação 1 – α da RNA polimerase II “TEF1α” (Matheny et al., 2002, 2007; Matheny, 2005).

O filo Basidiomycota é caracterizado pela estrutura microscópica denominada basídio, a partir de onde são originados os basidiósporos (Webster & Weber, 2007). Este filo, juntamente com Ascomycota, compõe o sub-reino Dicarya (Hibbett et al., 2007). Sobretudo para os fungos gasteroides, agrupamento polifilético do filo Basidiomycota distinguido pelo basidioma angiocárpico e basidiósporos estatismosporos, o emprego da ferramenta molecular utilizando o barcode universal do reino Fungi (região ITS nrDNA) vem demostrando eficiência na taxonomia, possibilitando o descobrimento de uma diversidade escondida (Martín et al., 2013; Phosri et al., 2014; Baseia et al., 2016).

A diversidade da região Neotropical é ainda pouco estudada quando comparada com as regiões Neárticas e Paleárticas (Olson et al., 2001; Brooks et al., 2006). Contudo, esta região abrange países megadiversos, sete “hotspots” (Mesoamérica, Ilhas do Caribe, Andes Tropicais, “Tumbes-Chocó-Magdalena”, Cachoeiras do Chile e Florestas Valvidias, Cerrado e Mata Atlântica) e ecossistemas tropicais, os quais demonstram elevado potencial para abrigar espécies de fungos ainda não conhecidas pela ciência (Myers et al., 2000; Hawksworth, 2001). Além disso, países como o Brasil vêm

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apresentando grande representatividade na descoberta de novos táxons dentro da família Geastraceae Corda, onde da última década até o presente momento foram descobertas uma dezena de espécies (exceto as descritas nesta tese): Geastrum aculeatum B.D.B. Silva & Baseia, G. echinulatum T.S. Cabral, B.D.B. Silva & Baseia (Silva et al., 2013); G. caririense R.J. Ferreira et al. (Crous et al., 2017); G. entomophilum Fazolino (Fazolino et al., 2008); Calonge & Baseia; G. hirsutum Baseia & Calonge (Baseia & Calonge, 2006); G. inpaense T.S. Cabral B.D.B. Silva & Baseia (Cabral et al., 2014a); G. ishikawae Accioly, J.O. Sousa Baseia & M.P. Martín (Crous et al., 2016); G. piquiriunense Accioly, A.A. Lima, J.O. Sousa, M.P. Martín & Baseia (Crous et al., 2018); G. rusticum Baseia, B.D.B. Silva & T.S. Cabral (Cabral et al., 2014b); G. setiferum Baseia (Baseia & Milanez, 2002). Análises filogenéticas, baseadas tanto na morfologia da família Geastraceae quanto em análises moleculares, enfatizando espécies Neotropicais são escassas. Desta forma, almejamos investigar as relações filogenéticas dos representantes da família Geastraceae, utilizando novos caracteres morfológicos e moleculares, dando ênfase em táxons com distribuição neotropical. Pretendemos confirmar a seguinte hipótese: “Existem complexos de espécies que subestimam o conhecimento sobre a diversidade de Geastraceae na região Neotropical”.

1.1. Geastraceae

Os representantes da família Geastraceae Corda (Geastrales, Basidiomycota) são visualmente reconhecidos pelos corpos de frutificação em forma de estrela, devido à deiscência do exoperídio em raios na maturidade, sendo denominados comumente “earthstars”. Baseado em estudos moleculares mais recentes esta família é constituída por dois gêneros: Geastrum Pers. (Persoon, 1801) e Myriostoma Desv. (Desvaux, 1809) os quais apresentam basidiomas gasteroides e basidiósporos liberados passivamente através do mecanismo de fole (Hosaka et al., 2006; Jeppson et al., 2013; Zamora et al., 2014). Apesar da maioria dos representantes de Geastrum apresentar basidioma em forma de estrela, alguns podem ser classificados como falsas-trufas, sendo caracterizados pelo basidioma esférico e hábito geralmente hipógeo. Essas espécies eram anteriormente classificadas no gênero Radiigera Zeller, o qual, atualmente, é um sinônimo de Geastrum (Jeppson et al., 2013). O crescimento hipógeo dos espécimes anteriormente

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classificados em Radiigera demonstra ser um caráter homoplásico dentro do gênero Geastrum, tendo surgido em várias ocasiões dentro da história evolutiva do gênero (Hosaka et al., 2006; Trappe et al., 2009; Zamora et al., 2014).

Sunhede (1989) considera oito gêneros na família: Geastrum, Myriostoma, Geasteropsis Long, Trichaster Czern, Phialastrum Sunhede, Pyrenogaster Malençon e Riousset, Radiigera e Terrostella Long (= Geasteropsis). No entando, alguns desses gêneros, além de Radiigera, como Trichaster e Geasteropsis são considerados por Kirk et al. (2008) sinônimos de Geastrum. Outros como Phialastrum e Pyrenogaster (= Schenella T. Macbr.) necessitam ser mais bem investigados para comprovar sua classificação (Estrada-Torres et al., 2005). Os gêneros que compõe Geastraceae são essencialmente saprofíticos, sendo encontrados principalmente no solo decompondo madeira morta ou liteira. Entretanto, há relato que esses fungos podem realizar associações ectomicorrízicas facultativas, favorecendo o estabelecimento de comunidades vegetais (Carlile et al., 2001; Tedersoo et al., 2010). Existem vários registros etnomicologicos sobre a utilização desses fungos na medicina popular, principalmente para uso hemostático e cicatrização (Shepard et al., 2008). Ensaios bioquímicos demostram potencial anti-inflamatório e antioxidante desses fungos (Dore et al., 2007; Sevindik et al., 2017).

Geastraceae apresenta uma distribuição global, sendo cosmopolita (Kasuya et al., 2012; Jeppson et al., 2013). Geastrum é um dos gêneros gasteroides mais diversos, com cerca de 100-120 espécies de acordo com Zamora et al. (2014). Enquanto Myriostoma foi considerado monoespecífico até 2017, constituído apenas por Myriostoma coliforme (Dicks.). Corda (Sunhede, 1989; Sousa et al., 2017, Capítulo VII desta tese); espécie que, ainda que amplamente distribuída, tem ocorrência rara, sendo classificada na lista vermelha de espécies ameaçadas com “status” de ameaçada de extinção em algumas localidades europeias, como no Reino Unido (Jeppson et al., 2013).

De acordo com Kirk et al. (2008), no total esta família abrange 64 espécies, porém condigno com as recentes publicações de novos táxons para ciências, além de espécies crípticas, este número está subestimado, não refletindo a quantidade real de espécies conhecidas até o momento. Geastraceae é a família tipo da ordem Geastrales Hosaka et Castellano. Ainda assim, não foi definida uma sinapomorfia que caracterize a família.

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Acredita-se que com base na morfologia da rizomorfa seja possível elucidar caracteres que definam este clado (Agerer, 2006; Hosaka et al., 2006).

No século XX alguns estudos sobre a sistemática e taxonomia da família Geastraceae foram realizados (Coker & Couch, 1928; Ponce de Léon, 1968; Sunhede, 1989). Porém, esses estudos levaram em conta exclusivamente caracteres morfológicos, não apresentando uma classificação baseada na filogenia do grupo. Além disso, as análises tiveram foco principalmente em espécimes do Hemisfério Norte, sendo mínima ou ausente a representatividade de táxons da região Neotropical.

Apenas nos últimos anos foram publicados trabalhos sobre Geastraceae utilizando dados de filogenia molecular. Cabral et al. (2014a, b), Caffot et al. (2016) Crous et al. (2015, 2017), Douanla-Meli et al. (2005) e Silva et al. (2013) empregaram a ferramenta molecular para comprovar a descoberta de novos táxons. Enquanto, Kasuya et al. (2012) propuseram a polifilia da espécie Geastrum triplex Jungh. Contudo, esses estudos realizados não tiveram uma amostragem significativa de espécimes neotropicais, além de não utilizarem o tipo da espécie nas análises. No ano seguinte, Jeppson et al. (2013) realizaram estudo de Sistemática das espécies europeias, propondo a sinonimização do gênero Radiigera com Geastrum.

Posteriormente, Zamora et al. (2014) realizaram um amplo estudo morfológico e de filogenia molecular utilizando as sequências das regiões gênicas: ITS, LSU, RPB1 e ATP6, no qual foi proposta divisão do gênero Geastrum em 14 seções e 9 subseções. Neste estudo, além dos caracteres tradicionalmente utilizados na taxonomia de Geastraceae, novos caracteres com proposição de importância taxonômica foram apresentados, incluído morfologia de cristais presente nas rizomorfa dos espécimes. Contudo, das 139 amostras analisadas por Zamora et al. (2014), apenas 23 são provenientes da região Neotropical (13 da Argentina, 6 do Brasil, 2 da Bolívia e 2 do Peru). Desta forma, algumas seções propostas por Zamora et al. (2014) necessitam ser melhor investigadas, principalmente para adição de dados Neotropicais.

As seções Myceliostroma (Henn.) P.Ponce de León e seção Exareolata De Toni são as únicas seções com espécimes capazes de produzir subículo (densa camada esbranquiçada de hifas que se estendem sobre o substrato), característica a qual, de acordo com Ponce de Léon (1968), está presente exclusivamente em espécies com

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distribuição tropical. Enquanto a seção a seção Corollina J.C. Zamora apresenta espécies tipicamente americanas (G. morganii Lloyd, G. saccatum Fr. e G. violacum Rick), porém com alguns representantes ainda não identificados em nível de espécie. Assim, essas três seções serão as principais abordadas neste estudo.

1.1.1. Geastrum

O gênero Geastrum é facilmente reconhecido pelo basidioma estreliforme e um único ostíolo apical. O nome do gênero faz referência à deiscência dos corpos de frutificação em raios; “geo”, em grego, significa terra; enquanto “aster” significa corpo celeste ou estrela. O gênero foi proposto por Persoon em 1974, apresentando como espécie tipo Geastrum coronatum Pers. (Zamora, 2014). Além de Radiigera, outros sinônimos de repercussão de Geastrum são: Coilomyces Berk. & M.A., publicado em 1853 com espécie tipo C. schweinitzii Berk. & M.A. Curtis (= Geastrum schweinitzii Berk. & M.A. Curtis) (Zeller, 1948); e Trichaster Czern., publicado em 1845 com a espécie tipo T. melanocephalus Czern. (= Geastrum melanocephalus Czern.) (Capellano & Riousset, 1968; Kasuya et al., 2012). Geastrum destaca-se dentre os fungos gasteroides por sua riqueza, abundância e ampla distribuição, sendo encontrado em variados tipos de ecossistemas. Assim, é um gênero considerado subcosmopolita, com ocorrência em todos continentes, excluindo-se a Antártida. Ocorre desde florestas tropicais e subtropicais, como Mata Atlântica e os Pampas brasileiros; ou ainda em desertos como Sonora no México, e regiões semiáridas como a Caatinga no Brasil (Silva et al., 2011; Sousa et al., 2015; Trierveiler-Pereira et al. 2017).

Segundo o Index Fungorum (www.indexfungorum.org) são encontradas 337 espécies agrupadas neste gênero. Baseado em Kirk et al. (2008), há 50 espécies válidas agrupadas em Geastrum, enquanto que de acordo com Zamora et al. (2014), 100-120 espécies pertencem a este gênero. Na região Neotropical, em consonância com levantamento bibliográfico realizado neste estudo, existe cerca de 90 espécies, sendo 60 destas (67%), ocorrentes em território brasileiro, Entretanto, estes números são incertos, devido à grande quantidade de sinônimos, escassez de dados neotropicais e espécies novas publicadas recentemente e de maneira recorrente.

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1.1.2. Myriostoma

O gênero Myriostoma é um raro fungo gasteroide com basidioma estreliforme. Apresenta macro-morfologia muito similar a outros gêneros gasteroides com este mesmo formato, tais como Astreus Morgan; e, sobretudo, Geastrum. Contudo, Myriostoma é claramente distinguido pela presença de vários estomas na superfície apical do endoperidio, além de apresentar mais de um pedicelo abaixo do corpo endoperidial e basidiósporos com ornamentação reticulada (Phosri et al., 2014; Sousa et al., 2017). Devido aos múltiplos estomas, por onde a pulverulenta e amarronzada é liberada, o gênero foi denominado. “Myri” em grego significa muitos, enquanto “stoma” significa boca ou orifício. Os espécimes deste gênero são popularmente conhecidos como “pepper pot earthstar” (estrela da terra pote de pimenta) ou “colaender ” (bufa-de-lobo coador) (Sousa et al., 2017).

A primeira citação de um representante do gênero Myriostoma foi realizada por Withering em 1776 como Lycoperdon coliforme With. Este mesmo nome foi utilizado por Dickson (1785) em trabalho com ilustração da espécie. Em 1801, Persoon propõe Geastrum coliforme (Dicks.) Pers. baseando-se no trabalho de Dickson. O primeiro nome válido do gênero foi apenas proposto em 1809, baseado na espécie Myriostoma anglicanum Desv., cujo material tipo, atualmente, encontra-se perdido (Bates, 2004). Gray (1821) propôs o gênero Polystoma (Dicks.) Gray, baseando-se na espécie Lycoperdon coliforme do trabalho de Dickson, contudo, por Polystoma ser sinônimo de Myriostoma, e baseando-se na prioridade da publicação mais antiga, o nome considerado válido para o gênero é Myriostoma (Sunhede, 1989). Em 1842, Corda propôs a combinação Myriostoma coliforme (Dicks.: Pers.) Corda. A ilustração de Dickson (1785) [Fasc. pl. crypt. brit. (London) 1:24] baseada em Lycoperdon colliforme (= Myriostoma coliforme (With.: Pers.) Corda) foi considerada até 2017 seu único material tipo.

Desde o século XIX Myriostoma coliforme vinha sendo reconhecida como a única espécie do gênero, apresentando distribuição global. Em 2017 foi publicada revisão do gênero, resultando em 4 espécies classificada neste gênero. Esta atualização de Myriostoma desmistifica o “status” de monoespecífico do gênero e faz parte dos resultados desta tese.

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2. OBJETIVOS

Geral: Revisar espécies de Geastraceae com distribuição Neotropical, interpretando as relações filogenéticas a fim de ampliar a compreensão da sistemática do grupo.

Específicos: . Caracterizar molecular e morfologicamente representantes da família Geastraceae da região Neotropical; . Elucidar novos caracteres a serem utilizados nos estudos taxonômicos da família; . Revisar os espécimes tipo utilizando base morfológica moderna; . Realizar estudos comparativos com espécimes tipo. . Estudar a relação morfologia-filogenia dos espécimes Neotropicais de Geastraceae.

3. MATERIAL E MÉTODOS

3.1. Análises Morfológicas

O estudo dos espécimes foi realizado no Laboratório de Biologia de Fungos (UFRN, Natal, Brasil) e no Departamento de Micología del Real Jardín Botánico (RJB-CSIC, Madrid, Espanha). Foram analisadas amostras coletadas durante o decorrer desta revisão (expedições de campo realizadas por membros do Lab. De Biologia de Fungos UFRN entre os anos de 2015-2018) e depositadas em herbários, dando ênfase aos espécimes coletados em regiões Neotropicais e amostras tipo. Os principais herbários brasileiros investigados foram: Universidade Federal do Rio Grande do Sul (ICN, Porto Alegre) Universidade Federal do Rio Grande do Norte (UFRN-Fungos, Natal), Universidade Federal de Pernambuco (URM, Recife), Instituto Anchietano de Pesquisa/UNISINOS PACA (Rio Grande do Sul, São Leopoldo), Instituto Nacional de Pesquisas na Amazônia (INPA, Manaus). Bem como, foram consultados os herbários onde se concentram amostras provenientes da região Neotropical, a saber: Herbarium of the Muséum national d'Histoire Naturelle (PC, Paris, França), Naturalis (L, Leiden, Holanda), U.S. National Fungus Collections (BPI, Beltsville-MD, Estados Unidos), Royal Botanic Gardens (K, Londres, Inglaterra) e Real Jardín Botánico (MA-Fungi, Madrid, Espanha). Descrições detalhadas das características macro e micro

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morfológicas (Fig. 1) foram desenvolvidas baseando-se nas metodologias propostas por Sunhede (1989), Calonge (1998) e Zamora et al. (2013).

Figura 1. Estruturas tradicionalmente utilizadas nas descrições taxonômicas de Geastrceae. A-B. Geastrum. C-D. Myriostoma.

Para o estudo das características macroscópicas caracteres como disposição do exoperídio (arqueado, saculiforme, fornicado), morfologia do peristômio (fribriloso, sulcado), presença ou ausência de pedicelo, tamanho e cor dos basidiomas foram analisados. A definição da coloração dos espécimes foi baseada na tabela de cores proposta por Kornerup & Wanscher (1978) ou Küppers (2002). Para análise das micro- estruturas, lâminas foram montadas com auxílio de pinças e o conteúdo foi reidratado em KOH 5% para visualização em microscópio óptico, utilizando principalmente as objetivas de 40 e 100 . Basidiósporos, basídios, capilícios, hifas do exoperídio e da rizomorfa foram analisados. Nas análises dos basídios foi utilizado o corante Vermelho Congo. De cada coleção, foram medidos no mínimo trinta basidiósporos aleatoriamente, todas as medições incluíram a ornamentação. Após medição do diâmetro dos basidiósporos, a ornamentação foi medida separadamente. Medidas estatísticas como média do diâmetro e altura dos basidiósporos, desvio padrão (x ± SD, respectivamente), além do quociente entre largura e altura média (Qm) foram realizadas, como também,

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foram realizadas análises de ultraestrutura com auxílio de microscópio eletrônico de varredura (MEV); a preparação do material analisado em MEV seguiu Silva et al. (2011).

3.2. Análises Moleculares

Os procedimentos de extração, amplificação e purificação de DNA foram realizados no Laboratório de Genética Molecular de Plantas do Centro de Biociências- Universidade Federal do Rio Grande do Norte, coordenado pelo Professor Doutor Paulo Marinho; e no Laboratório de Sistemática Molecular - Real Jadrín Botánico de Madrid, onde os trabalhos foram coordenados pela Dr. María P. Martín. Para extração de DNA fragmentos de aproximadamente 10 mg foram retirados do basidioma herborizado e adicionados a tubos de 1,5 ml, dando preferência à porção fértil (gleba). Os fragmentos de basidioma foram macerados com um micro pistilo ou trituradas com auxílio de micro-esferas de vidro no equipamento TissueLyser (Qiagen). Os kits utilizados para a extração de DNA foram: QIAGEN DNeasy Plant Mini Kit (Qiagen, Valencia, California, U.S.A.) ou The Speedtools Tissue DNA Extraction Kit (Biotools B&M Labs.S.A). Os protocolos de extração foram adaptados com base em Martín & Winka (2000). Uma das modificações aplicadas ao protocolo original dos fabricantes dos Kits de extração foi submeter às amostras + Buffer 1 a temperatura 56-60ºC durante 1-2 dias. Para espécies herborizadas, com data de coleta muita antiga, ou espécies com problemas na extração utilizando a metodologia citada anteriormente, foi utilizado Kits forense de extração (FTA Cards), seguindo a metodologia de extração baseada em Telleria et al. (2014).

A amplificação foi realizada através de PCR (Reação em Cadeia de Polimerase) utilizando “PCR beads” (Illustra™ PuReTaq Ready-To-Go PCR Beads). Nos tubos de PCR beads foi adicionado 1 μl de cada iniciador a uma concentração de 10 M, 1-23 μl do DNA dependendo de sua concentração, e água ultra pura estéril, resultando em um volume final de 25 μl. Os produtos da PCR foram analisados por eletroforese em gel de agarose, utilizando-se marcadores de pares de base padrão (1 kb ladder) para confirmar o tamanho das bandas (Figura 1). As regiões alvo do DNA foram: subunidade maior do rDNA (LSU= 28S); espaçador transcrito interno do rDNA ribossômico (ITS = ITS1 + 5.8S+ ITS2); segunda maior subunidade da RNA polimerase II (RPB2); fator de

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elongação 1 – alpha da RNA polimerase II (TEF1α). Os indicadores para cada região são os seguintes (Tabela 1):

- ITS. Foi utilizado o par de iniciadores ITS1F/ITS4 (Gardens & Bruns, 1993; White et al., 1990). Quando necessário (bandas ausentes ou fracas na eletroforese) foi realizada uma PCR-nested; para isto, uma primeira amplificação com os iniciadores ITS1F/ITS4B (Gardes & Bruns, 1993), nas quais se incluiu 1 μl de DNA, na segunda amplificação se utilizou 1 μl do produtor da primeira amplificação e os iniciadores ITS5/ITS4 (White et al., 1990). Informações do programada utilizado: 1ª fase desnaturação inicial (94ºC por 5 minutos); 2ª fase - 5 ciclos: (desnaturação 94º por 30 segundos) + (anelamento a 54ºC por 30 segundos) + (extenção a 72ºC por 1 minuto); 3ª fase- 33 ciclos: (desnaturação 94º por 30 segundos) + (anelamento a 48ºC por 30 segundos) + (extenção a 72ºC por 1 minuto); 4ª fase (extensão a 72 º por 10 minutos); 5ª fase (armazenamento a 4 ºC ∞).

- LSU. Foram utilizados os iniciadores LR0R/LR7 (Rehner & Samuels, 1994; Vigalys & Hester, 1990). Também, quando necessário foi realizada uma PCR- seminested com os iniciadores LR0R/LR5 (Vigalys & Hester, 1990). Informações do programada utilizado: 1ª fase desnaturação inicial (94ºC por 5 minutos); 2ª fase - 36 ciclos: (desnaturação 94º por 30 segundos) + (anelamento a 52ºC por 30 segundos) + (extenção a 72ºC por 1 minuto); 3ª fase- (extensão a 72 º por 10 minutos); 5ª fase (armazenamento a 4 ºC ∞).

- RPB2. Foi realizada diretamente uma PCR-nested, com iniciadores externos RPB2-5F/RPB2-7.1R (Matheny, 2005) e os internos bRPB2-6F/bRPB2-7R2 (Matheny, 2005), com as mesmas quantidades de DNA que para a região ITS. Informações do programada utilizado: 1ª fase desnaturação inicial (95ºC por 5 minutos); 2ª fase - 35 ciclos: (desnaturação 94º por 1 minuto) + (anelamento a 55ºC por 1 minuto) + (extenção a 72ºC por 1 minuto); 3ª fase- (extensão a 72 º por 10 minutos); 5ª fase (armazenamento a 4 ºC ∞).

- TEF1α. Foram utilizados os iniciadores EF1-1018F/EF11620R (Matheny et al., 2007) para uma PCR direta, utilizando 1 μl de cada iniciador e 23 μl do produto de extração. Informações do programada utilizado: 1ª fase desnaturação inicial (94ºC por 5

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minutos); 2ª fase - 40 ciclos: (desnaturação 94º por 1 minuto) + (anelamento a 48ºC por 1 minuto) + (extenção a 72ºC por 1 minuto); 3ª fase- (extensão a 72 º por 7 minutos); 5ª fase (armazenamento a 4 ºC ∞).

Anteriormente ao sequenciamento, os produtos foram purificados mediantes os protocolos do kit com colunas da marca Speedtools PCR Clean-Up Kit (Biotools B&M Labs. S.A), quando nos géis de eletroforese se observaram mais de uma banda; ou mediante a enzima ExoSAP-IT1(USB Corporation, OH, USA), quando se observou banda única na eletroforese (Fig. 2).

Tabela 1. Regiões alvos da amplificação

Iniciadores Utilizados Sequência do Iniciador

5’ – 3’

ITS ITS1F CTT GGT CAT TTA GAG GAA GTA A

ITS4B CAG GAG ACT TGT ACA CGG TCC AG (ITS1+5.8S+ITS2)

ITS5 GGA AGT AAA AGT CGT AAC AAG G

TCC TCC GCT TAT TGA TAT GC ITS4

LSU LR0R ACC CGC TGA ACT TAA GC

LR7r TAC TAC CAC CAA GAT CT (28S)

LR5r TCC TGA GGG AAA CTT CG

RPB2 RPB2-5F GAYGAYMGWGATCAYTTYGG

RPB2-7.1R CCCATRGCYTGYTTMCCCATDGC

bRPB2-6F TGGGGYATGGTNTGYCCYGC

ACYTGRTTRTGRTCNGGRAANGG bRPB2-7R2

TEF1α EF1-1018F GAYTTCATCAAGAACATGAT

EF11620R GACGTTGAADCCRACRTTGTC

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O sequenciamento bidirecional foi realizado na empresa Sul Coreana Macrogen, utilizando os mesmos iniciadores que geraram as amplificações de PCR. As sequências consenso foram obtidas com auxílio do programa Sequencher 5.2.4 (Gene Codes Corp., USA). Os consensos foram submetidos a uma busca no Genbank/ncbi.nlm.nih.gov utilizando a ferramenta BLAST (Basic Local Alignment Search Tool), este procedimento foi necessário para que sequencias nucleotídicas errôneas não fossem utilizadas na análise. Os alinhamentos das sequências obtidas neste trabalho e sequências retiradas do Genbank foram inicialmente realizados com o algoritmo do MAFFT e posteriormente editados manualmente no software Seaview version 4.7 ou Molecular Evolutionary Genetics Analysis-MEGA (Gouy et al., 2010). As análises filogenéticas foram realizadas obtidas sobre três critérios: Máxima Parcimônia (MP), Máxima Verossimilhança (MV) e Inferência Bayesiana. As analises foram realizadas com as regiões alvo da amplificação por separado e, posteriormente, também concatenadas. As análises de MP foram realizadas no software PAUP* (Phylogenetic Analysis Using Parsimony) v.4.0b10 (Swofford 2003) utilizando a busca heurística e análise de bootstrap (MPbs) com 10 mil replicações mediante a opção rápida. As análises de MV utilizaram RAxML no portal CIPRES portal (CIPRES Science Gateway v.3.3) com o modelo evolutivo GTRGAMMA ou GTR+I+G e bootstrap (MVbs) de mil replicações. As análises de Inferência Bayesiana foram realizadas no programa MrBayes, assumindo o modelo GTR+I+G. As probabilidades posteriores (PP) e os valores de bootstrap foram levados em conta para identificar o grau de confiabilidade dos clados (Lutzoni et al., 2004). As árvores obtidas foram visualizadas no FigTree, sendo posteriormente exportadas para programas de edição de imagem.

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4. RESULTADOS E DISCUSSÃO

Foram analisadas 215 coleções, das quais 14 corresponderam a exemplares tipos: Geastrum aculeatum (UFRN-Fungos 1681, holótipo); G. echinulatum (UFRN-Fungos 1682, parátipo); G. entomophilum, (UFRN-Fungos 504, holótipo); G. hariotii Lloyd (PC0084345, holótipo): G. hirsutum (UFRN-Fungos 245, holótipo; MA-Fungi 67886, parátipo); G. lloydianum Rick (BPI 841471, lectotype); G. pleosporum Douanla-Meli (MA–Fungi 56971, isótipo); G. schweinitzii (Berk. & M.A. Curtis) Zeller (K(M) 180187, isótipo); G. triplex Jungh. (L0053171, holótipo); G. velutinum Morgan (K(M)179863, holótipo); G. welwitschii Mont. (PC073886, holótipo); Myriostoma coliforme var. areolatum Calonge & M. Mata (MA-Fungi 68596, isótipo; MA-Fungi 36165, parátipo). Ao final dessa revisão foram descritas 14 espécies novas (Tabela 1).

Foram realizadas no total 184 extrações de DNA. No total foram realizadas 349 reações de amplificação da região ITS, sendo destas 139 reações de PCR “nested”; 142 de LSU, sendo destas 37 de PCR “semi-nested”; 40 da região RPB2, sendo todas estas de PCR “nested”; além de 35 reações de amplificação da região TEF1α. Como se pode observar na Fig. 1, nem todos os produtos de amplificação tiveram a mesma qualidade (banda única e brilhante), desta forma, foram realizadas 356 reações de purificação de produtos de PCR e enviados para sequenciamento 308 purificaçãos. Quando editados os electroferogramas, foram obtidas 86 sequências de ITS, 77 de LSU, 18 de RPB2 e 16 de TEF1 (Tabela 2), que correspondem aos dois gêneros estudados. Como pode observar na Tabela 1, apenas foi detectado nove sequências de outros gêneros (contaminações).

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Tabela 2. Espécies novas descritas neste trabalho.

ESPÉCIES NOVAS

1- G. laevisporum J.O. Sousa & Baseia Capítulo I, publicado

2- G. pusillipilosum J.O. Sousa et al. Anexo I, publicado

3- G. verrucoramulosum T.S. Cabral, J.O. Sousa & Baseia Anexo II, publicado

4- G. magnosporum J.O. Sousa et al. Anexo III, publicado

5- G. caatingense J.O. Sousa, M.P. Martín & Baseia Capitulo II, aceito

6- G. parvistellum J.O. Sousa, M.P. Martín & Baseia Capitulo II, aceito

7- G. baculicrystallum J.O. Sousa et al. Capítulo III, publicado

8- G. brunneocapillatum J.O. Sousa et al. Capítulo III, publicado

9- G. courtecuissei P.-A. Moreau & C. Lécuru Capítulo III, publicado

10- G. neoamericanum J.O. Sousa et al. Capítulo III, publicado

11- G. rubellum P.-A. Moreau & C. Lécuru Capítulo III, publicado

12- G. rubropusillum J.O. Sousa et al Capítulo III, publicado

13- M. calongei Baseia, J.O. Sousa, & M.P. Martín Capítulo IV, publicado

14- M. australianum J.O. Sousa Baseia & M.P. Martín Capítulo V, publicado

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Figura 2. Exemplos de géis de eletroforese obtidos neste trabalho. A. Gel de comprovação amplificação de TEF1α (EF1-1018F/EF11620R). B. Gel de comprovação amplificação de RPB2 (RPB2-5F/RPB2-7.1R nested com bRPB2-6F/bRPB2-7R2). C. Gel de comprovação de purificação poços de 1-6 região ITS (ITS5/ITS4), poços 7-12 região LSU (LR0R/LR5). D. Gel de comprovação de purificação poços de 1-5 região LSU (LR0R/LR7), poços de 6-12 região LSU ( LR0R/LR5). M= marcador (1Kb Ladder); Negativos: poços 27A e 18B. Seta vermelha= banda indicando amplificação com presença de DNA degradado, sendo necessário purificação com Kit de colunas. Seta verde= banda indicando amplificação com banda única e “limpa”, material a ser purificado com enzima ExoSAP. Seta amarela= banda “fraca” indicando pouca quantidade de produto amplificado. Seta azul= ausência de banca após a purificação, indicação de necessidade de repetir amplificação.

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Tabela 3. Material utilizado nas análises moleculares. Em negrito sequências enviadas GenBank; (Acc.Nº) = sequências a serem enviadas ao GenBank; (elec. duplo) = sequências com eletroferogama duplo; (seq. curtas) = sequências curtas; (cont.) = contaminação; * = extrações realizadas no Real Jardín Botánico de Madrid.

Espécie Código Coleção Local de coleta Código Extração ITS LSU RPB2 TEF1α G. aculeatum UFRN Fungos 1681, holótipo Serra da Confusões-PI E15/13-2 (Acc.Nº) - - - G. albonigrum (FN-40) Brasil E17/34-1* (Acc.Nº) (Acc.Nº) - - G. albonigrum UFRN-Fungos 2279 (JM151) Brasil, Areia-PB E16/6-8 (Acc.Nº) - - - G. baculicrystallum sp. nov. UFRN-Fungos 2835 (JM137) Brasil, Areia-PB E15/13-6 MH634995 MH635028 - - G. brunneocapillatum sp. nov. UFRN-Fungos 2834 (JM117) Brasil, Areia-PB E15/2-5 MH634997 MH635030 - - UFRN-Fungos 2131 (JM45) Brasil, Areia-PB E15/3-8 (Acc.Nº) (Acc.Nº) - - UFRN-Fungos 2132 (JM63) Brasil, Areia-PB E15/3-4 (Acc.Nº) (Acc.Nº) - - UFRN-Fungos 2286 (JM101) Brasil, Guaribas-PB E15/2-7 MH634996 MH635029 - - UFRN-Fungos 2287 (JM106) Brasil, Guaribas-PB E15/2-8 (Acc.Nº) - - - UFRN-Fungos 2851 (Ovrebo Costa Rica E15/15-5 MH634998 - - - 2203) G. caatingense sp. nov. UFRN-Fungos 2843 (PB04) Brasil, Triunfo-PB E16/6-6 MH253884 MH253886 - - G. cf. arenarium UFRN-Fungos 355 Brasil, PE E16/6-11 MG938500 MG938501 - - G. cf. calceum UFRN-Fungos 2842 (PB03) Brasil, Triunfo-PB E15/1-10 (Acc.Nº) (Acc.Nº) - - G. cf. hariotii (JM127) Brasil,Areia-PB E15/17-6 (Acc.Nº) (Acc.Nº) - - G. cf. hirsutum (AMO 705) Brasil, AC E16/6-2 (Acc.Nº) - - - (DT174) Brasil, Serra das - (Acc.Nº) (Acc.Nº) - - Confusões-PI UFRN-Fungos 115 Brasil, Natal-RN E15/3-3 (cont.) - - -

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Espécie Código Coleção Local de coleta Código Regiões Amplificadas Extração ITS LSU RPB2 TEF1α G. cf. hirsutum UFRN-Fungos 1500 Brasil, Natal-RN E15/2-4 (elec. duplo) - - - UFRN-Fungos 1781 Brasil, Baía Formosa- E15/3-1 (elec. duplo) - - - RN UFRN-Fungos 2244 Brasil, Manaus-AM E15/2-11 (elec. duplo) - - - G. cf. javanicum MA-Fungi 82855 Costa Rica E17/34-4* (Acc.Nº) (Acc.Nº) - - MA-Fungi 78294 Brasil E17/34-5* (Acc.Nº) (cont.) - - MA-Fungi 34133 Venezuela E17/34-6* (elec. duplo) - - - UFRN-Fungos 3002 (Ovrebo Panamá E16/6-4 (Acc.Nº) (Acc.Nº) - - 3757) (Ovrebo3704) Panamá E15/17-1 (seq. curta) - - - G. cf. lloydianum BPI 841471, lectótipo Brasil E17/33-4 * (cont.) (Acc.Nº) - - E17/33-5* MA-Fungi 80070 República Dominicana E17/34-7* (Acc.Nº) (Acc.Nº) - - UFRN-Fungos 2840 (JM115) Brasil, Guaribas-PB E15/17-5 (Acc.Nº) (Acc.Nº) - - UFRN-Fungos 2836 (JM135) Brasil, Areia-PB E15/17-7 (Acc.Nº) (Acc.Nº) - - G. cf. pectinatum UFRN-Fungos 1796 Brasil, Baía Formosa- E15/17-11 (Acc.Nº) (Acc.Nº) - - RN UFRN-Fungos 1798 Brasil, Natal-RN E15/17-10 (seq. curta) - - - G. cf. saccatum Ovrebo 4044 Panamá E15/17-3 (Acc.Nº) (Acc.Nº) - - G. cf. schweinitzii Ovrebo 2843 Costa Rica E16/6-3 (Acc.Nº) - - - G. cf. stiptatum MA-Fungi 39161 Camarões E17/45-2* (cont.) - - - G. cf. triplex L 0837171 México E17/34-9* (Acc.Nº) (Acc.Nº) - - L 0837172 Russia E17/34-10* (Acc.Nº) (Acc.Nº) - - L 3961257 Áustria E17/42-9* (Acc.Nº) - - - L 3961340 Indonésia, Java E17/44-4* (Acc.Nº) - - - E17/44-5*

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Espécie Código Coleção Local de coleta Código Regiões Amplificadas Extração ITS LSU RPB2 TEF1α UFRN-Fungos 2306 (JM154) Brasil, Areia-PB E16/6-7 (Acc.Nº) (Acc.Nº) - - G. cf. triplex L 396331 França E17/42-1* (Acc.Nº) (Acc.Nº) - - L 3961324 Suíça E17/42-6* (elec. duplo) - - - L 3961343 EUA, Tennessee E17/34-8* (Acc.Nº) - - - L 3961344 EUA, Oregon E17/42-5* (elec. duplo) (cont.) - - G. cf. velutinum MA-Fungi 73247 India E17/34-3* (Acc.Nº) (Acc.Nº) - - G. echinulatum UFRN-Fungos 1682, parátipo Brasil, Serra da Jibóia- E15/13-13 (Acc.Nº) - - - BA G. entomophilum UFRN-Fungos 504, holótipo Brasil, Natal-RN E15/13-3 (Acc.Nº) (Acc.Nº) - - G. hariotii PC084345, tipo Brasil E17/33-7* (elec. duplo) - - - PC00844350 Nova Caledônia E17/34-2 (elec. duplo) (Acc.Nº) - - G. hirsutum MA-Fungi 67886, paratipo Brasil, Recife-PE E17/33-1* MH538295 (cont.) - - E17/33-2* UFRN-Fungos 245, holótipo Brasil, PE E15/2-13 (cont.) - - - INPA 259950 Braisl, Manaus-AM E15/3-7 MH634993 MH635026 - - G. laevisporum sp. nov. UFRN-Fungos 2587 Brasil,Triunfo-PB E15/1-9 (elec. duplo) - - - UFRN-Fungos 2258 Brasil,Triunfo-PB E17/42-11* (Acc.Nº) (Acc.Nº) - - G. magnosporum sp. nov. UFRN-Fungos 2310 Brasil,Guaribas-PB E15/17-8 MG938498 MG938499 - - UFRN-Fungos 2312 (JM110) Brasil,Guaribas-PB E15/17-9 MG938496 MG938497 - - G. neoamericanum sp. nov. UFRNFungos 2850 (DAS 279) Brasil, MG E16/6-10 MH635000 MH635032 - - UFRN Fungos 2302 (JM90) Brasil,Guaribas-PB E15/13-5 MH635001 MH635040 - - G. parvistellum sp. nov. UFRN-Fungos 2841 (PB02) Brasil, Triunfo-PB E16/6-5 MH253885 MH253887 - - G. pusillipilosum sp. nov. UFRN - Fungos 2759 (JD171) Brasil,Guaribas-PB E15/3-10 KX61177 KX761178 - - UFRN-Fungos 2256 Brasil, Crato-CE E14/15-2 KX761180 - - -

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Espécie Código Coleção Local de coleta Código Regiões Amplificadas Extração ITS LSU RPB2 TEF1α G. pusillipilosum sp. nov. (RJFnº23) Brasil, Crato-CE E14/15-2 (seq. curta) - - - UFRN-Fungos 2316 (JM103) Brasil,Guaribas-PB E14/12-12 KX761179 - - - UFRN-Fungos 1934 Brasil, MG E15/3-5 (seq. curta) - - - UFRN-Fungos 2315 (JM100) Brasil,Guaribas-PB E15/2-9 KX761175 KX761176 - - G. rubellum sp. nov. UFRN-Fungos 2844 (AMO604) Brasil, AC E16/6.1 MH634999 MH635031 - - UFRN-Fungos 1214 Brasil, Baía Formosa- E15/3.2 (Acc.Nº) (Acc.Nº) - - RN G. rubropusillum sp. nov. UFRN2308 (JM36) Brasil, Areia-PB E15/13.11 MH634994 MH635027 - - G. rusticum UFRN-Fungos 2301 (JM105) Brasil,Guaribas-PB E15/17.4 (Acc.Nº) (Acc.Nº) - - (Ovrebo 3620) Panamá E15/17.2 (seq. curta) - - - UFRN-Fungos 1217, holótipo Brasil, Baía Formosa- E15/13.12 (elec. duplo) - - - RN G. schweintizii K(M)180187, isótipo Surinam - MH635016 (Acc.Nº) - - G. setiferum URM 77077, parátipo Brasil, Rebio Serra E15/13.1 (elec. duplo) - - - Negra-PE G. triplex L 053171, tipo Indonesia, Java E17/44-1 (cont.) (cont.) - - E17/44-2 G. velutinum K(M) 179863, holótipo EUA - (Acc.Nº) (Acc.Nº) - - G. welwitschii PC073886, tipo Portugal E16/1-6 (Acc.Nº) - - - G. xerophilum UFRN-Fungos 944 Brasil, Catimbau-PE E15/17-12 (seq. curta) - - - Geastrum sp. JM146 Brasil, Areia-PB E16/6-9 (seq. curta) - - - JM146 Brasil, Areia-PB E15/13-10 (seq. curta) - - - UFRN-Fungos 2125 ( JM49) Brasil, Areia-PB E15/13-9 (Acc.Nº) (Acc.Nº) - - UFRN-Fungos 2837 (JM161) Brasil, Areia-PB E15/6-2 (Acc.Nº) (Acc.Nº) - - UFRN-Fungos 2290 (JM123) Brasil, Guaribas-PB E15/6-3 (Acc.Nº) (Acc.Nº) - -

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Espécie Código Coleção Local de coleta Código Regiões Amplificadas Extração ITS LSU RPB2 TEF1α Geastrum sp. UFRN-Fungos 2291 (JM143) Brasil, Guaribas-PB E15/13-7 (Acc.Nº) (Acc.Nº) - - (JD170) Brasil, Guaribas-PB E15/3-9 (Acc.Nº) - - - (Ovrebo 4065) Panamá E16/6-12 (elec. duplo) - - - E15/15-6 UFRN-Fungos 2839 (JM156) Brasil, Areia-PB E15/13-8 (seq. curta) (Acc.Nº) - - M. areolatum comb. nov MA-Fungi 36165 Costa Rica E15/99.5* KY096673 KY096690 (Acc.Nº) (Acc.Nº) M. australianum sp. nov. MEL 310277 Austrália, Sydney E17/43-5* (Acc.Nº) (Acc.Nº) (elec. (Acc.Nº) duplo) MEL 2310279 Austrália, Sydney E17/43-6* (Acc.Nº) (Acc.Nº) (Acc.Nº) (elec. duplo) MEL 2091620 Austrália, Sydney E17/43-2* MG675903 MG675884 - (Acc.Nº) MEL 2305388 Austrália, Sydney E17/43-4* MG675901 MG675882 (Acc.Nº) (Acc.Nº) MEL 2060796 Austrália, Sydney E17/43-1* MG675902 MG675883 (elec. - duplo) MEL 2095275 Austrália, Sydney E17/43-3* MG675904 MG675885 - - M. calongei sp. nov. L 3961249 Brasil E17/35-4 * MG675905 MG675886 (Acc.Nº) (Acc.Nº) ICN 175617 Brasil, RS E17/27-3 * MG675906 MG675887 (Acc.Nº) (Acc.Nº) ICN 177080 Brasil, RS E17/27-4* MG675907 MG675888 (Acc.Nº) (Acc.Nº) URM 31429 Eua, New mexico E17/27-5* (elec. duplo) (Acc.Nº) - - URM 31433 Eua, New mexico E17/27-2 MG675908 MG675889 - - UFRN-Fungos 2019 Brasil, Baía Formosa- E15/15-2 KY096676 KY096693 - - RN UFRN -Fungos 2020 Brasil, Baía Formosa- E15/15-1 KY096677 KY096694 - - RN UFRN-Fungos 386 Brasil, Catimbau-PE E15/15-4 KY096674 KY096691 - - UFRN-Fungos 990 Brasil, Catimbau-PE E15/15-3 KY096675 KY096692 - -

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Espécie Código Coleção Local de coleta Código Regiões Amplificadas Extração ITS LSU RPB2 TEF1α M. coliforme L 3961237 Bulgária E17/35-9* MG675912 MG675893 (Acc.Nº) (seq. curta) L 3961244 Áustria E17/35-15* MG675910 MG675891 (Acc.Nº) (Acc.Nº) L 3961240 França E17/35-5* MG675914 MG675895 (Acc.Nº) (Acc.Nº) L 3961245 Áustria E17/35-13* - (Acc.Nº) (Acc.Nº) - L 3961239 Ilha de Jersey E17/35-6* MG675913 MG675894 - (seq. curta) L 3961250 USA, Ohio E17/35-1* MG675919 MG675900 (Acc.Nº) (Acc.Nº) L 3961251 Holanda E17/35-7* MG675917 MG675898 (Acc.Nº) (Acc.Nº) L 3961243 Hungry E17/35-2* MG675916 MG675897 - - L 3961241 Austria E17/35-12* MG675909 MG675890 (Acc.Nº) (Acc.Nº) L 3961246 Austria E17/35-11* (elec. duplo) (Acc.Nº) (Acc.Nº) (Acc.Nº) L 3961247 Austria E17/35-14* MG675911 MG675892 - (Acc.Nº) L 3961248 Hungria E17/35-8* (elec. duplo) (Acc.Nº) - - L 3961242 Alemanha E17/35-10* MG675915 MG675896 (Acc.Nº) - L 3961238 Slovakia E17/35-3* MG675918 MG675899 (Acc.Nº) (Acc.Nº) MA-Fungi 31316 Porugal E15/99-4* - - (Acc.Nº) - Ma-Fungi 60898 Espanha E15/99-7* - - (Acc.Nº) - MA-Fungi 40288 Espanha E15/99.-3* - (elec. duplo) - (Acc.Nº) PC 0072885 França E16/1-10 KY096684 - - -

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4.1. Geastrum

A revisão morfológica e análises moleculares permitiram delimitar e identificar 12 espécies novas para o gênero Geastrum: G. laevisporum J.O. Sousa & Baseia (Capítulo I); G. pusillipilosum J.O. Sousa et al. (Anexo I); G. verrucoramulosum T.S. Cabral, J.O. Sousa & Baseia (Anexo II); G. magnosporum J.O. Sousa et al. (Anexo III); Geastrum caatingense J.O. Sousa, M.P. Martín & Baseia e G. parvistellum J.O. Sousa, M.P. Martín & Baseia (Capítulo II); G. baculicrystallum J.O. Sousa et al., G. brunneocapillatum J.O. Sousa et al.; G. courtecuissei P.-A. Moreau & C. Lécuru, G. neoamericanum J.O. Sousa et al.; G. rubellum P.-A. Moreau & C. Lécuru e G. rubropusillum J.O. Sousa et al. (Capítulo III). Além de revalidar o nome Geastrum hirsutum Baseia & Calonge (Capítulo III).

Três seções do gênero Geastrum foram principalmente investigadas: Corollina (duas novas espécies adicionadas: G. caatingense e G. parvistellum), Exareolata (uma nova espécie adicionada: G. verrucoramulosum) e Myceliostroma (seis novas espécies adicionadas: G. baculicrystallum, G. brunneocapillatum, G. courtecuissei, G. neoamericanum, G. rubellum, G. rubropusillum). Essas seções são compostas principalmente por espécies com ocorrência Neotropical, sendo abordadas raramente em estudos anteriores. Duas espécies novas descritas neste estudo, baseado em análises filogenéticas das regiões ITS e LSU, não se agruparam em nenhuma das seções propostas nos estudos anteriores, são elas: G. magnosporum e G. laevisporum.

A morfologia dos basidiósporos (tamanho, forma) e sua ornamentação (tamanho e forma) são caracteres primordiais na distinção a nível específico em todas as seções analisadas. Particularmente para a seção Myceliostroma, a morfologia da camada micelial (tipo de ornamentação, tamanho dos tufos, coloração e outros) foi também essencial. Como o estudo dessas características foi possível elucidar a identificação de espécies novas dentro de complexos que envolviam Geastrum schweinitzii e Geastrum hirsutum (Capítulos III e Anexo I). Adicionalmente, novos caracteres, nunca antes descritos para o gênero Geaastrum, são aqui apresentados: basidiósporos com parede lisa; estipe ramuloso e longo abaixo do basidioma; hifas pseudoparequimatosas na camada micelial.

Geastrum laevisporum é principalmente caracterizado por exoperídio arqueado, camada micelia incrustada com sedimentos, raios higroscópicos, peristômio fibriloso

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delimitado, basidiósporos subglobosos a ovais com 6,3–9 (9,9) × 5–8,2 μm diam. Apresenta característica nunca antes descrita no gênero Geastrum: basidiosporos com superfície lisa. Apesar disso, a macro morfologia desta espécie é muito similar a representantes da seção Geastrum Pers. (camada micelial incrustada, presença de pedicelo, peristômio fibriloso, exoperídio arqueado). O caractere exclusivo dos basidiósporos (que além de lisos são os maiores descritos para o gênero até o momento e apresentam parede espessa) sugere uma possível adaptação ao clima semiárido da Caatinga, domínio onde G. laevisporum foi coletado. Uma vez que o maior tamanho, aliado à parede espessa, proporcionaria mais nutriente e água para que os basidiósporos se tornassem viáveis por mais tempo durante os períodos de seca.

Esta espécie foi incialmente descrita e publicada apenas com base em dados morfológicos, porém análises filogenéticas posteriores das regiões ITS e LSU confirmaram seu status de nova espécie. Além disso, mesmo com as análises filogenéticas dessas regiões, não foi possível observar um agrupamento claro em nenhuma das seções propostas anteriormente. Análises moleculares de outros marcadores (ATP6; RPB1; RPB2; TEF1α) são necessárias para elucidar o posicionamento de G. laevisporum dentro das seções propostas anteriormente, ou para propor novas seções em estudos futuros.

Geastrum pusillipilosum é reconhecido pelo basidioma muito pequeno (com até 17 mm de largura quando expandido), peristômio fibriloso delimitado, exoperídio densamente piloso (pelo com até no máximo 0,3 mm de altura) e basidiósporos globosos com 5.0–6.8 μm diam. Os basidiomas desta espécie foram coletados crescendo sobre madeira ou liteira e foi observada produção de subículo. As características citadas previamente aliadas aos dados de filogenia molecular (ITS e LSU) agrupam esta espécie na seção Myceliostroma subsebção Epigaea. A descrição de G. pusillipilosum embasou a hipótese de que Geastrum apresenta complexo de espécies com exoperídio piloso, tema o qual será abordado no Capítulo III desta revisão.

Outra espécie com características nunca antes descrita para o gênero Geastrum é G. verrucoramulosum, a qual apresenta estipe ramificado abaixo do basidioma com até 41 mm de altura. Esta espécie foi agrupada na seção Exareolata com base em análises filogenéticas das regiões ATP6, ITS e LSU. A presença de estipe abaixo do basidioma demonstra ser um caractere encontrado apenas na seção Exareolata até o momento,

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espécies como G. echinulatum, igualmente agrupada nessa seção de acordo com nossas análises, também apresentam essa característica. Além de estipe ramuloso e grande, esta espécie apresenta como característica distintiva verrugas no exoperídio composta por hifas pseudoparenquimatosas, característica encontrada em gêneros de “” como Morganella Zeller, mas nunca antes descrita para o gênero Geastrum. A presença de estipe longo, comparado a outras espécies de Geastrum, pode sugerir adaptação de G. verrucoramulosum a ambientes inundados ou de solo encharcado, como a Floresta Amazônica, onde foi coletado. Uma vez que, o mecanismo de fole, responsável pela liberação passiva dos basidiósporos em Geastrum, apenas funciona se a gleba pulverulenta estiver seca.

Geastrum magnosporum distingue-se por basdiomas pequenos (com no máximo 19 mm de largura quando expandido), camada micelial incrustada com sedimentos, peristômio fibriloso com delimitação curta ou ausente, pedicelo curto (até 0,6 mm de altura), basidiósporos com 6 – 8,5 μm de diam e verrugas longas (até 1,3 μm de altura). Apresenta morfologia semelhante às espécies da seção Papillata de Toni (peristômio fibriloso indistintamente delimitado, micelial incrustada e raios involutos) e seção Geastrum subseção Arenaria J.C. Zamora (peristômio fibriloso, micelial incrustada e raios involutos). Contudo, diferenças morfológicas baseadas principalmente no tamanho dos basidiósporos e presença de pedicelo em G. magnosporum permitiram distinguir esta espécie e não a considerar integrante das seções citadas anteriormente, inclusive baseadas em dados moleculares de ITS e LSU.

Geastrum caatingense é distinguido por apresentar raios higroscópicos, peristômio fibriloso distintamente delimitado, camada micelial incrustada, camada fibrosa coriácea com raios longitudinais, endopéridio não pruinoso e basidiósporos com 5,7–6.5 × 5,2– 6.2 μm. Enquanto, Geastrum parvistellum apresenta basidioma muito pequeno (6–12 mm de lagura), peristômio fracamente delimitado, endoperidio não pruinoso, camada micelial incrustada e basidiósporos com 4,8–6,5 × 4,8–6,3 μm.

Além de Geastrum laevsiporum, outras duas espécies descritas nesta revisão foram coletadas em região de clima semiárido (vegetação de Caatinga sensu stricto): Geastrum caatingense e G. parvistellum. Ambas apresentam raios higroscópicos ou sub-higroscópicos. A higroscopia é um mecanismo encontrado em gêneros como Geastrum e Morgan (“false earthstar”) que consiste no “movimento” do

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exoperídio de acordo com a umidade ambiental. Quando o ambiente está úmido, os raios ficam expandidos, expondo o endoperídio para que os basidiósporos sejam dispersos. Enquanto o ambiente se torna seco, os raios se recurvam em direção ao endoperídio e o cobrem, impedindo ação do mecanismo de fole. Em ambientes áridos ou semiáridos este mecanismo pode aumentar a viabilidade dos basidiomas, uma vez que esses organismos necessitam de água para se desenvolver.

Geastrum baculicrystallum apresenta basidiomsta pequenos (8–10 mm quando expandido), exoperídio tomentoso a rugoso, peristômio delimitado por linha marrom acinzentada e basidiósporos globosos com 3,6–4,5 μm diam. É uma espécie semi- criptica com Geastrum schweinitzii.

Geastrum brunneocapillatum é principalmente caracterizada pela presença de pelos com marrons escuros com até dois mm de altura, peristômios fribriloso delimitado por linha amarelada e basidoposros com 2,8–4 μm diam. Esta espécie é semi-criptica com Geastrum hirsutum.

Geastrum courtecuissei diferencia-se por exibir basidiomas pequenos (até 20 mm de largura), exoperdidio levemente equinulado quando imaturo, peristômio fibriloso deprimido no endoperidio e delimitado por linha marrom claro, basidiósporos com 3,8– 5 μm diam.

Geastrum neoamericanum caracteriza-se por basidiomas pequenos (8–10 mm de largura), exoperidio branco amarelado e tomentoso, peristômio mamiforme delimitado por linha acinzentada, basidiósporos globosos a subglobosos Qm = 1,03), 3,7–5,1 μm diam. É uma espécie semi-criptica com Geastrum schweinitzii.

Geastrum rubellum distingue-se pela camada micelial avermelhada quando fresco, camada micelial com pelos espaçados e curtos (0,34–0,68 mm de altura), peristômio fibriloso delimitado por linha cinza alaranjada e basidiósporos com 4,4–6,3 μm diam. Esta espécie é semi-criptica com Geastrum hirsutum.

Geastrum rubropusillum é reconhecido por apresentar basidiomas pequenos (7–9 mm de largura), camada pseudoparenquimatosa avermelhada quando fresco, camada micelial tomentosa marrom clara, peristômio levemente deprimido no endoperidio, basidiósporos subglobosos a ovais (Qm = 1.07) com 4,2–5,9 μm diam.

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A ferramenta molecular comprovou eficácia em auxiliar nos casos de complexos de espécies abordados neste estudo, sobretudo com a utilização da região ITS, código de barras molecular para fungos. Para a amplificação desta região, houve maior eficiência quando realizada PCR “nested” com os iniciadores ITS1F/ITS4B, na primeira reação; e ITS5/ITS4, na segunda reação.

A partir desses resultados foi possível contribuir em 13% sobre o conhecimento da riqueza do gênero Geastrum na região Neotropical.

4.2. Myriostoma

Para o gênero Myriostoma foram descritas duas espécies novas, já publicadas: M. calongei Baseia, J.O. Sousa, & M.P. Martín (Capítulo IV) e M. australianum J.O. Sousa Baseia & M.P. Martín (Capítulo V). Além disso, foram realizadas duas combinações novas: M. areolatum (Calonge & M. Mata) M.P. Martín, J.O. Sousa & Baseia e M. capillisporum (V.J. Stanek) L.M. Suz et al. (Capítulo IV).

Foi proposto um epitipo (K(W) 138625) com descrição morfológica moderna e dados moleculares (ITS e LSU) e um lectótipo (ilustração de Dickson 1785) para a espécie Myriostoma coliforme. Sendo geradas e disponibilizadas no GenBank 70 novas sequências dos marcadores ITS e LSU. Os resultados do Capítulo IV demonstraram que a distribuição de M. coliforme, proposta pela Lista Vermelha de Fungos (Fraiture & Otto, 2015), está sobrestimada, aumentando assim a atenção sobre o “status” de conservação desta espécie.

Para a delimitação de espécies em Myriostoma, caracteres como superfície endoperidial (presença ou não de verrugas, forma e altura), tamanho e ornamentação dos basidiósporos além de morfologia dos estomas foram essenciais. As regiões ITS e LSU, nunca antes utilizadas como ferramente taxonômica para este gênero, foram importantes na delimitação das cinco espécies propostas para compor Myriostoma até o momento.

Myriostoma areolatum é facilmente diferenciado por apresentar grande quantidade de ostíolos (até 42) com morfologia tubular (1 mm altura por 1 mm diam.), levemente deprimidos no endoperídio e distintamente por aréola, além de basidiósporos com 5.6– 6.9 μm diam.

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Myriostoma calongei é principalmente caracterizado por apresentar 3-11 ostíolos com boca fibrilosa e levemente delimitados, endoperidio com hifas proeminentes em forma de processos triangulares (0,13–0,28 mm), basidiósporos com 5,6–8,7 μm diam.

Myristoma capillisporum distingue-se, sobretudo, por exibir basidiósporos grandes 7,4–10,9 μm diam e ornamentação longa (2,9–6,6 μm de altura), além de poucos ostíolos na superfície do endoperidio (até 4) e pedicelos com 2,3–2,9 mm de altura.

Myriostoma australinaum caracteriza-se por apresentar 3–5 ostiolos, pedicelos com 2,8–6,1 mm de altura, basiosporos com 6,7 –8,3 μm diam. e ornamentação com 1,32–3 μm de altura.

Análises moleculares, posteriores às publicações dos Capítulos IV e V, utilizando as regiões RBP2 e TEF1α, concatenadas com as regiões ITS e LSU, e com adição de novos espécimes, confirmaram a delimitação filogenéticas das cinco espécies de Myriostoma.

Além disso, essas análises com quatro marcadores moleculares contribuem com a elucidação de hipóteses de bio e filogeogrofia, previamente lançadas nos Capítulos IV e V. As espécies com ocorrência na Oceania, América do Sul e África do Sul, respectivamente M. australianum, M. calongei e M. capillisporum, compõe um único clado, irmão de M. coliforme, espécie a qual tem distribuição restrita ao Hemisfério Norte. A topologia obtida com as análises corroboram com a hipótese de ancestral comum entre M. australianum, M. calongei e M. capillisporum o qual provavelmente habitou o supercontinente Gondwana.

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Capítulo I — Geastrum laevisporum: a new earthstar fungus with uncommon smooth spores

Publicado: Sousa J.O., Baracho G.S., Baseia I.G. 2015. Geastrum laevisporum: a new earthstar fungus with uncommon smooth spores. Mycosphere 6(4), 501–507. Doi 10.5943/mycosphere/6/4/12

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Mycosphere 6 (4): 501–507(2015) ISSN 2077 7019 www.mycosphere.org Article Mycosphere Copyright © 2015 Online Edition Doi 10.5943/mycosphere/6/4/12

Geastrum laevisporum: a new earthstar fungus with uncommon smooth spores

Sousa JO1, Baracho GS2 and Baseia IG1

1Universidade Federal do Rio Grande do Norte, Departamento de Botânica e Zoologia, Programa de Pós-graduação em Sistemática e Evolução, Campus Universitário, 59072-970, Natal, RN, Brazil. 2Universidade de Pernambuco, Instituto de Ciências Biológicas, Laboratório de Biologia Vegetal, Rua Arnóbio Marques, 310, Santo Amaro, CEP 50.100-130, Recife, PE, Brazil.

Sousa JO, Baracho GS, Baseia IG 2015 – Geastrum laevisporum: a new earthstar fungus with uncommon smooth spores. Mycosphere 6(4), 501–507, Doi 10.5943/mycosphere/6/4/12

Abstract Geastrum laevisporum is found occurring in the xerophitic shrubland biome named “Caatinga”, from Paraíba State, Brazil. Growing in groups on soil, several specimens were collected and studied. The fundamental diagnostic characteristic for this new species is the smooth basidiospore surface. Description, discussion and photographs of this new taxon are given.

Key words – Geastraceae – gasteromycetes – taxonomy – biodiversity

Introduction Studies of the genus Geastrum Persoon in the semiarid region of Northeastern Brazil have been intensified in recent years. However, the occurrence of species of this genus predominate in Caatinga enclaves of tropical altitudinal moist forests, regionally known as “Brejos de Altitude”, and few species occur in more xerophytic plains as well as savanna areas and seasonally dry forests (Leite et al. 2007, Drechsler-Santos et al. 2008, Silva et al. 2011, 2013, Trierveiler-Pereira et al. 2011, Sousa et al. 2014a, b, c). The Caatinga is an exclusively Brazilian biome, which covers 10% of the country‟s area (about 800.000 km2). Typical “Caatinga” vegetation is adapted to water deficiency (low and irregular rainfall), and high temperatures and sun incidences. Trees and shrubs lose their leaves in the driest seasons, decreasing evapotranspiration. The landscape then becomes comprised of naked, whitish wood, which explains the name Caatinga, meaning “white forest” in Tupi, a Brazilian indigenous language (Leal et al. 2005, Prado 2003, Machado et al. 2012). In general Geastrum species presents basidiospores ornamented with columnar, conical or cylindrical processes (Zamora et al. 2014a). However, species with smooth spores are unusual in this group. The only record of a single species with a sub-smooth basidiospore is Geastrum pleosporum Douanla-Meli described from Cameroon, which presents variable shape of spores (Douanla-Meli et al. 2005). Thus, this work aimed to describe a new species of Geastrum found from the Caatinga biome, Brazil.

Submitted 08 May 2015, Accepted 29 June 2015, Published online 24 August 2015 Corresponding Author: Iuri G. Baseia – e-mail – [email protected]

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Materials & Methods

Collection site Paraíba state is situated in the eastern portion of the Northeast region of Brazil, with coordinates between 6o and 8o S and between 34o and 38o W, placing it within the tropical zone (Figure 1). It comprises an area of 56,372 km2 and is divided into four mesoregions („Mata Paraibana‟, „Borborema‟, „Agreste Paraibano‟ and „Sertão Paraibano‟) and 23 geographic microregions, and includes a total of 223 municipalities (CEPED/UFSC 2011). The samples were collected from an open semiarid area in the extreme western part of the state and in the south- central portion of the ecoregion of „Depressão Sertaneja Setentrional‟, one of the areas most impacted by the anthropogenic actions of the „Caatinga‟ biome (Velloso et al. 2002). This area is dominated by the „Sertão Paraibano‟ (remote backland), and characterized by its Cretaceous age formation. It is a component of the „Uiraúna-Brejo das Freiras‟ region, a smaller intracratonic basin with 480 km2 of great paleontological and geological diversity and relevance. The climate is semi- humid (Aw‟), warm and dry but with relative humidity of 70%, susceptible to prolonged drought periods, with mean annual precipitation of approximately 770–800 mm. The local flora is characterized by a diversity of many different plant species from the Caatinga to elements of Atlantic Rainforest and Cerrado, including cacti and bromeliads, Solanaceae, Malvaceae, Euphorbiaceae, and many grasses and legumes. The water availability and precipitation seasonality determined by pulses of rainfall separated by intervening dry periods of variable lengths are events that influence and may promote changes in plant performance in arid and semiarid regions (Snyder & Tartowski 2006).

Figura 3 (Fig. 1) Site in “Caatinga”, Brazil. A. Map of the “Caatinga” biome in Brazil. B. Paraíba State. C-D. landscape images.

Morphologic study Morphological analysis was performed on nineteen dry basidiomata in variable stages of development. All samples were deposited in the Herbarium of the Federal University of Rio Grande do Norte (UFRN-Fungos), Brazil. Macro and micro morphological descriptions were based on specific literature: Bates (2004), Calonge (1998), Sunhede (1989) and Zamora et al. (2014b, 2015). 56

Color terms followed Kornerup & Wanscher (1978). A Nikon H600L stereomicroscope coupled with a Nikon DS-Ri camera were used for macro morphological study and image capturing. A Nikon Eclipse Ni light microscope coupled with a Nikon DS-Ri camera were used for performing light microscopy (LM). Blades with portions of gleba (basidiospores and eucapillitium) and exoperidial hyphae were all mounted in 5% KOH (w/v). A Shimadzu SSX-550 was used for scanning electron microscope (SEM) analysis. For this, preparation of the material examined under SEM followed Silva et al. (2011). Basidiospores of the nineteen basidiomata were analyzed; sixteen randomly selected basidiospores were measured using LM at 1000× magnification. Basidiospore abbreviations follow Sousa et al. (2014b): n = number of randomly measured basidiospores; x = mean ± standard deviation of basidiospore diameter and height (including ornamentation); Qm = mean height/width quotient.

Results

Geastrum laevisporum J.O. Sousa & Baseia Fig. 2–4 MYCOBANK: 812507 Etymology – From Lat.: leavi (smooth). Named in reference to the smooth spore surface. Diagnosis – Expanded basidiomata arched, 7–25 mm wide. Exoperidium mostly arched, strongly hygroscopic. Mycelial layer densely intermixed with sediments, not persistent. Stalk very short, 0.5–0.7 mm high. Peristome fibrillose, delimited, slightly depressed on endoperidium. Basidiospores yellowish, polymorphous, subglobose, oval to ellipsoid, 6.3–9 (9.9) × 5–8.2 μm [Qm = 1.11], thick-walled (up to 0.9 μm diam.), smooth to sub-smooth under SEM.

Figura 4 (Fig. 2) Basidiomata in situ (A-B) and ex situ (C-D). A. Basidiomata with arched exoperidium. B. Detailed mycelial layer. C. Basidiomata with strongly hygroscopic rays. D. Detailed peristome. Description – Unexpanded basidioma not observed. Expanded basidiomata arched, 5–15 mm high (including peristome) × 7–25 mm wide. Exoperidium splitting into 8–10 rays, mostly arched, sometimes planar to involute, often completely covering the endoperidial body, strongly hygroscopic. Mycelial layer orange white (5A1), densely intermixed with sediments, felted, peeling away in irregular patches with age exposing the fibrous layer, not persistent. Fibrous layer yellowish white (4A1), coriaceous. Pseudoparenchymatous layer brown (6E5) to dark brown (6F6),

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rimose on tips of rays, peeling away in irregular patches with age, absent on some basidiomata. Endoperidial body depressed, globose to subglobose, 5–9 × 5–8 mm, stalked, glabrous, non- pruinose, grayish brown (6D2) to grayish orange (6B2). Apophysis absent. Stalk very short, 0.5– 0.7 mm high, lighter than endoperidium. Peristome fibrillose, delimited or weakly delimited, delimitation orange white (5A1), slightly depressed on endoperidium, concolorous with to lighter than the endoperidium, mammiform. Columella not observed. Gleba brown (7E4). Basidiospores yellowish, polymorphous, subglobose, oval to ellipsoid, 6.3–9 (9.9) × 5–8.2 μm [x = 7.8 ± 0.7 × 6.9 ± 0.7, Qm = 1.11, n = 60], thick-walled (0.6 – 0.9 μm diam.), ornamentation inconspicuous under LM; smooth to sub-smooth under SEM. Basidia not observed. Eucapillitium yellowish to hyaline, thin walls (0.5–1 μm), 3.9–5.9 μm diam., sinuous, surface slightly verrucose, encrusted, lumen evident. Mycelial layers composed of hyaline, thin-walled hyphae (0.4–0.6 μm), 1.8–3.8 μm diam., encrusted, lumen evident. Fibrous layer composed of hyaline, thin-walled hyphae (0.4–1 μm), 4.4– 7.4 μm diam., sinuous, lumen evident. Pseudoparenchymatous layer composed of thin-walled (0.5– 1 μm) hyphal cells, subglobose to oval, 13.4–32.1 ×11.9–19.3 μm. Endoperidium comprised of interlaced hyphae, with an absence of protuberant hyphae. Habitat – sand soil covered by litter, „Caatinga‟ biome (dry forest). Habit – gregarious. Specimens examined – BRAZIL – Paraíba State, Triunfo municipality, sítio Jenipapeiro, remnant of Caatinga vegetation, 6º36'18" S, 38º32'44" W, 273 m a. s. l., 28/02/2015, leg. G.S. Baracho 12.179, holotype UFRN Fungos-2587, isotype UFRN Fungos-2588.

Figura 5 (Fig. 3) Basidiospores under LM (A-D).

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Figura 6 (Fig. 4) SEM images. A-B. Basidiospores. C. Hyphae of endoperidium. D. Eucapillitium.

Remarks – Geastrum laevisporum has a macro morphology typical of the genus Geastrum: star-shaped basidiomata with expoperidium pluristratified splitting in rays, one apical stoma margin by the peristome, and pulverulent mature gleba with true capillitium and basidiospores (Zamora et al. 2014a). This species is mainly characterized by small-sized basidiomata (up to 25 mm wide); exoperidium densely intermixed with sediments; strongly hygroscopic rays; peristome fibrillose, delimited; and endoperidial body slightly stalked. Geastrum laevisporum is easily differentiated through a microscopic analysis of basidiospores, with size, ornamentation and shape uncommon in the genus Geastrum. The basidiospores are subglobose, oval to ellipsoid (Qm = 1.11), have smooth to sub-smooth surface, and height up to 9.9 μm. These basidiospores patterns were found in the nineteen specimens analyzed in this study. One species whose basidiospores are a closely related partner is Geastrum pleosporus Douanla-Meli. The basidiospores are also of variable sizes and shapes: globose, cylindrical, elliptic, reniform, and club-shaped basidiospores with (3.5) 4–6 (8) × (3) 4–5 (6) μm Although these are smaller and verruculose under SEM. Moreover, G. pleosporus has a macro morphology completely distinct, differing by saccate basidiomata, delimited peristome, sessile endoperidium, presence of subiculum, exoperidium not encrusted, and non-hygroscopic rays (Douanla-Meli et al., 2005). Another species with oval basidiospores is Geastrum ovalisporum Calonge & Moreno- Arroyo, but differs by its distinct delimited peristome; long and small pedicel, up to 2 mm high; and verrucose basidiospores, up to 3.5 μm diam. (Calonge et al. 2000). The largest basidiospores reported before of G. laevisporum are found in Geastrum campestre Morgan and G. platense Spegazzini, both with basidiospores just until 8 diam., they differ from G. laevisporum in some macro morphological traits and mainly in shape and ornamentation of basidiospores. Geastrum campestre has plicate peristome, a largest basidiomata (until 40 mm wide), endoperidium surface covered with warts, and globose, densely verrucose basidiospores. While Geastrum platense has non-delimited peristome, endoperidial body sessile and verrucose basidiospores (Sunhede 1989, Soto & Wright 2000, Bates 2004). 59

Geastrum hungaricum Hollós is a very closely related species in macro morphology. Both G. hungaricum Hollós and G. laevisporum have small-sized basidiomata, exoperidium densely intermixed with sediments, strongly hygroscopic rays, mycelial layer not persistent, fibrous layer whitish, and peristome fibrillose. They are distinguished from each other in that G. hungaricum presents distinctly a delimited peristome and globose, verrucose, smaller basidiospores, up to 6 μm (Sunhede 1989). Other species with similar macro morphology are Geastrum corollinum (Batsch) Hollós and G. floriforme Vittad. Both have strongly hygroscopic rays and peristome fibrillose. However, these two species present verrucose basidiospores up to 5 and 7 μm in diam., respectively. G. corollinum is distinct in macro morphology, as its peristome is delimited and exoperidium not encrusted; G. floriforme has a sessile, furfuraceous endoperidium (Sunhede 1989, Sousa et al. 2014b). Until now, there has been no description of any species in the genus Geastrum with the pattern of basidiospores found in Geastrum laevisporum. The morphological evidence and discussion above are sufficient to propose this species as new.

Acknowledgements The authors are grateful to the Programa de Pesquisa em Biodiversidade do Semiárido (PPBio Semiárido), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and to Universidade de Pernambuco (UPE/PFA, Programa de Fortalecimento Acadêmico) for financial support; and Maria Batista de Freitas (D. Carminha) and family (sítio Jenipapeiro, Triunfo, PB), for the logistical support, kindness, and assistance during fieldwork.

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Capítulo II — Contribution to Neotropical data of Geastrum section Corollina (Basidiomycota): Two new earth–stars from Caatinga vegetation, Brazil

Aceito: Sousa J.O., Baracho G.S., Martín M.P., Baseia I.G. 2019. Contribution to Neotropical data of Geastrum section Corollina (Basidiomycota): Two new earth–stars from Caatinga vegetation, Brazil. Nova Hedwigia.DOI:10.1127/nova_hedwigia/2019/0524

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Contribution to Neotropical data of Geastrum section Corollina (Basidiomycota): Two new earth–stars from Caatinga vegetation, Brazil Julieth de Oliveira Sousa*1, George Sidney Baracho 2, María P. Martín 3, Iuri Goulart Baseia 4

*[email protected] Programa de Pós–Graduação em Sistemática e Evolução Universidade Federal do Rio Grande do Norte Campus Universitário Natal 59072–970, Brazil

2 Universidade de Pernambuco, Instituto de Ciências Biológicas Laboratório de Biologia Vegetal, 50.100–130, Recife, Brazil.

3 Departamento de Micología Real Jardín Botánico–CSIC Plaza de Murillo 2, Madrid, Spain

4 Departamento de Botânica e Zoologia Universidade Federal do Rio Grande do Norte Campus Universitário Natal 59072–970, Brazil

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Abstract

The Caatinga is a unique phytogeographical domain of semi–arid vegetation in northeastern Brazil. Although, it includes rare and endemic birds and mammals, it is poorly represented in the Brazilian Conservation Area Network. In this paper we describe two new species of the genus Geastrum section Corollina: G. caatingense and G. parvistellum, based on morphological data and molecular analyses of ITS/LSU nrDNA. Species description, images, and taxonomic discussion are provided.

Keywords gasteroid fungi · Geastraceae · phylogeny · systematic · taxonomy

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Introduction Caatinga is a unique phytogeographical domain in Brazil, comprising about 10% of the territory of this “continental” country. The vegetation grows under semi–arid conditions, a climate unusual in a tropical region. It is mainly characterized by scarcer and concentrated rains (means of less than 1,000 mm per year), mean temperature of 26–28 ºC and a high level of evapotranspiration (Prado 2003, Leal et al. 2005, Moro et al. 2016). The Caatinga includes rare and endemic taxa, but just 2% of its area is protected in conservation units (Castelletti et al. 2003). Recently, the genus Geastrum has been a focus of systematic and taxonomic studies (Kasuya et al. 2012, Jeppson et al. 2013, Zamora et al. 2014). Historically the genus is considered as of subcosmopolitan distribution, and has been investigated more intensively in temperate areas (Paleartic and Neartic regions) than in tropical areas. However, this scenario has changed in the last decade, during which more than ten new species of the genus were discovered from Brazil (Fazolino et al. 2008, Silva et al. 2013, Cabral et al. 2014a, Cabral et al. 2014b, Sousa et al. 2015, Crous et al. 2016, 2017, 2018, Cabral et al. 2017). One of the 14 sections recently proposed for the genus Geastrum is the section Corollina J.C. Zamora. It groups species with very heterogeneous morphology, mainly recognized by peristome fibrillose to irregularly plicate, basidioma frequently saccate, but rarely arched, exoperidium hygroscopic or not, mycelial layer normally not encrusting debris, rarely encrusted, endoperidial body sessile (Zamora et al. 2014). The sect. Corollina is divided into three subsections: subsect. Lageniformia J.C. Zamora, mainly characterized by strongly delimited, fibrillose peristome, presence of horn–like crystal on rhizomorphs and mycelial layer composed of generative hyphae; Marginata P. Ponce de León, mainly characterized by strongly delimited fibrillose peristome, presence of acicular crystals on rhizomorphs and two sub–layers in the mycelial layer, outer layer with skeletal hyphae and inner layer with generative hyphae; and subsect. Plicostomata V.J. Staněk, mainly characterized by non–delimited or weakly delimited peristome, irregularly plicate, presence of acicular crystals on rhizomorphs and two sub–layers in the mycelial layer, outer layer with skeletal hyphae and inner layer with generative hyphae. The sect. Corollina has some undetermined species (Geastrum sp.) (Zamora et al. 2014). Furthermore, there are few studies with Corollina’ specimens from Neotropical Region, where exists high potential to found undescribed species for science (Hawksworth, 2001). Thus, the aimed of this study is to enhance the knowledge of the sect. Corollina, describing two news species of from Neotropical region, Brazil (Caatinga vegetation), with analyses based on morphological and molecular data.

Materials and methods

Colletion Site and Morphological Analysis The specimens were collected during the rainy season of 2015 in an area of Caatinga sensu stricto vegetation (6º36'18" S, 38º32'44" W, 273 meters above sea level), localized in the ecoregion of “Depressão Sertaneja Setentrional” (Velloso et al. 2002). Morphological studies were performed according to Sousa et al. (2014a, 2014b) with dried basidiomata at variable stages of development. Color definition was based on Kornerup & Wanscher (1978). For light microscopy (LM), slides containing basidiospores, eucapillitial, and exoperidial hyphaemounted on 5% (w/v) KOH were examined under a Nikon Eclipse Ni light microscope coupled with a Nikon DS–Ri camera. Thirty basidiospores were measuremed at 1000 × magnification including ornamentation. Scanning Electron Microscopy (SEM) was used to observe ultrastructure of basidiospores ornamentation, eucapillitium and endoperidial surfaces. Statistical measurements of basidiospores given in the description followed Sousa et al. (2017). All analyzed samples have been deposited in the collection of the Federal University of Rio Grande do Norte, Natal, Brazil (UFRN–Fungos).

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Molecular Analyses Genomic DNA was extracted from approximately 10 mg of gleba from dried basidiomata as described in Sousa et al. (2017). Amplifications were carried out using illustraTM PureTaqTM Ready–To–GoTM PCR Beads (Healthcare, Buckinghamshire, UK) with a final volume of 25 µl. Two regions were amplified by Polymerase Chain Reaction (PCR): Internal Transcribed Spacer (ITS nrDNA), including the 5.8S of the ribosomal RNA, and Large SubUnit (LSU nrDNA), with the primer pairs ITS1F/ITS4B (White et al. 1990, Gardes & Bruns 1993) and LR0R/LR7 (Vilgalys & Herster 1990, Rehner & Samuels 1994), respectively. When amplifications were weak (less than 10 ng/ µl), nested–PCR to ITS and seminested–PCR to LSU was done using 1 µl of amplification product from the first PCR. For the nested–PCR to ITS, the primers ITS5/ITS4 were used (White et al. 1990), and for seminested–PCR to LSU, the primers LR0R/LR5 were used (Vilgalys & Herster 1990, Rehner & Samuels 1994). Before sequencing, 20 μl of the amplification products were purified using Speedtools PCR Clean–up Kit (Biotools, B & M Labs, S.A). Purified PCR products were then sequenced at Macrogen (Seoul, Korea), with the primer pairs used in the amplification. Editing and consensus assembly of DNA sequences were performed using Sequencher v.4.1.4 (Gene Codes, Ann Arbor, Michigan, USA). Sequences were submitted to GenBank under the accession numbers indicated in Table 1. Both ITS and LSU sequences were compared with homologous Geastrum sequences retrieved from GenBank. The multiple alignments were optimized visually in MEGA v. 5.2 (Kumar et al. 2016). Sequences of two specimens of Geastrum hungaricum (MJ9317 and MJ8915) were included as outgroup. The concatenated alignment was analyzed by Maximum Parsimony (MP), using PAUP* v.4.0b10 during phylogenetic analysis. A heuristic search was performed, with branch swapping and using the TBR algorithm, with initial trees obtained by stepwise addition of random additional sequences repeated 10 times, and bootstrap (MPbs) of ten thousand replicates. A Maximum Likelihood Analysis (ML) was also carried out using RAxML in the CIPRES portal (CIPRES Science Gateway v.3.3), with GTRGAMMA as the model of evolution (Stamatakis 2014), with bootstrap (MLbs) of one thousand replicates.

Results

Molecular analyses The concatenated dataset (ITS/LSU) included 38 specimens of Geastrum (36 ingroup sequences of Sect. Corollina, and two of G. hungaricum as outgroup). Seventy–two sequences were retrieved from GenBank and four were newly generated in this study (Table 1, the new sequences are shown in bold.). In MP analysis of 1643 positions, 1262 positions were constant, 140 parsimony–uninformative, and 241 were parsimony– informative. Gaps were treated as "missing data". Parsimony tree scores were identical for the eight most parsimonious trees obtained: Consistency Index (CI) = 0.5457, Retention Index (RI) = 0.7033and Homoplasy Index (HI) = 0.4543. The strict consensus of these trees is shown in Fig. 1. The three ML consensus trees have similar topology (not shown). Bootstrap percentages (MPbs and MLbs) are indicated in Fig.1. Morphological and phylogenetic analyses of the specimens represent two species new to science, i.e. Geastrum caatingense J.O. Sousa, M.P. Martín & Baseia, and Geastrum parvistellum J.O. Sousa, M.P. Martín & Baseia. Both are included in sect. Corollina. Geastrum caatingense grouped in subsect. Marginata, as a sister species of two specimens from Spain (Geastrum sp. 1) in Fig. 1; however this relationship is not well supported (less than 50 bootstrapsupport). Geastrum parvistellum grouped in subsect. Plicostomata, with a sequence of undetermined Geastrum (Geastrum sp. 2) in Fig. 1 from Australia (MEL 2382911) and this relationship is strongly supported by good bootstrap values (MPbs = 98, MLbs = 100).

Taxonomy

Geastrum caatingense J.O. Sousa, M.P. Martín & Baseia (Fig 2) 66

MYCOBANK: MB825153

Diagnosis: This species is mainly recognized by hygroscopic rays, distinctly delimited, fibrillose peristome, ephemeral mycelial layer which expose the coriaceous fibrous layer with longitudinal cracks. Geastrum caatingense is close to Geastrum corollinum, however G. caatingense differs by non–pruinose endoperidium, non–ephemeral mycelial layer with presence of encrustations, and larger basidiospores (5.7– 6.5 × 5.2–6.2 μm). Geastrum saccatum is also similar to G. caantingense, but G. caatingense has hygroscopic rays, as well as an encrusted mycelial layer and a fibrous layer with longitudinal cracks.

Types: BRAZIL. Paraíba State: Triunfo, 28.II.2015, G.S. Baracho 12.180 (holotype UFRN–Fungos 2843, isotype UFRN–Fungos 2960). GenBank accession ITS = MH253884, LSU = MH253886).

Etymology: “caatingense” refers to the vegetation of the type locality. Expanded basidiomata saccate, 7–14 mm high (including peristome) × 11–29 mm wide. Exoperidium splitting into 6–10 rays, revolute when fresh, involute, rolling up above the endoperidial body or planar when dry, hygroscopic; Mycelial layer pallid orange (5A3), papery, strongly encrusted with debris, peeling away in irregular patches with age; Fibrous layer white to orange white (5A2), coriaceous, with longitudinal cracks; Pseudoparenchymatous layer dark brown (6E4; 7F4), persistent when young or peeling away in irregular patches with age. Endoperidial body orange gray (6B2), brownish gray (6C2) to yellowish white (5A2), depressed globose to subglobose, 5–12 × 9–16 mm, subsessile, surface glabrous, without pruinose material. Apophysis absent or reduced, lighter color than endoperidium. Pedicel inconspicuous (< 0.5 mm high), lighter than endoperidium. Peristome finely fibrillose, distinctly delimited by orange white (5A2) line, strongly conic (up to 3.2 mm high, up to 5.2 mm diam.) lighter than endoperidium. Gleba grayish brown (6F3). Mycelial layer composed of hyaline to yellowish hyphae, 2.5–3.2 μm diam., unbranched, thick– walled (0.5–1.0 μm), surface encrusted, lumen evident. Fibrous layer composed of hyaline hyphae, 4.2–7.9 μm diam., thin–walled (0.7–1.2 μm), surface not encrusted, lumen evident. Pseudoparenchymatous layer composed of hyaline subglobose to elongated hyphal cells, 17.0–46.0 × 17.8–34.1 μm, thick–walled (0.8– 1.5 μm). Endoperidium composed of interlaced hyphae. Eucapillitial hyphae yellowish, 3.8–6.7 μm wide, thin walled (0.5–0.8 μm), surface encrusted, densely verrucose, lumen evident. Basidiospores brownish, subglobose, 5.7–6.5 × 5.2–6.2 μm [x = 6.0 ± 0.2 × 5.8 ± 0.2, Qm = 1.04, n = 30], with ornamentation less conspicuous under LM; densely verrucose under SEM, with columnar to triangular warts (0.9–1.7 μm high), slight pointed or rounded apex, with some confluent tips; apiculus inconspicuous.

Habitat and distribution: Found in Caatinga vegetation, growing on sandy soil covered by leaf–litter.

Geastrum parvistellum J.O. Sousa, M.P. Martín & Baseia Fig 3

MYCOBANK: MB825154

Diagnosis: This species is mainly recognized by small basidiomata (6–12 mm wide), sub–hygroscopic rays, encrusted mycelial layer, irregularly plicate and weakly delimited peristome. Geastrum parvistellum is close to G. morganii, however G. parvistellum has smaller basidiomata (6–12 mm wide), involute rays, ephemeral and encrusted mycelial layer. Geastrum arenarium is also a similar species to G. parvistellum, but G. parvistellum has smaller basidiomata (6–12 mm wide), non–pruinose endoperidial body, weakly delimited peristome, and larger basidiospores (4.8–6.5 × 4.8–6.3 μm).

Type: BRAZIL. Paraíba State: Triunfo, 28.II.2015, G.S. Baracho 12.181 (holotype UFRN–Fungos 2841, isotype UFRN–Fungos 2961). GenBank accession ITS= MH253885, LSU = MH253887).

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Etymology: “parvistellum” (derived from Latin: parvo = small; stella = star) refers to the small size of the basidiomata. Expanded basidiomata saccate to arched, 6–8 mm high (including peristome) × 6–12 mm wide. Exoperidium splitting into 5–7 involute rays, rolling up under the endoperidal body, sub–hygroscopic; Mycelial layer grayish orange (5B3), wooly, strongly encrusted with debris, persistent or peeling away in some basidiomata; Fibrous layer white (5A1), coriaceous; Pseudoparenchymatous layer brown (6E5) to dark brown (7F5), persistent, rimose. Endoperidial body orange gray (5B2), brownish orange (5C3) to dark brown (7F4), subglobose, 3–7 × 5–8 mm, sub–sessile, surface furfurcaeous. Apophysis absent. Pedicel absent or inconspicuous (< 1 mm high), lighter than endoperidium. Peristome irregularly plicate, fibrillose with age, weakly delimited, slightly depressed on the endoperidium or mammiform, concolorous or lighter than endoperidium (up to 1 mm high, up to 5.2 mm diam.) Gleba grayish brown (5F3). Mycelial layer composed of hyaline hyphae, 1.2–3.2 μm diam., thin-walled (< 0.5μm) surface encrusted, lumen evident, some clamped hyphae present. Fibrous layer composed of hyaline hyphae, 3.3–4.9 μm diam., thin–walled (0.5–0.9 μm), surface non-encrusted, lumen evident. Pseudoparenchymatous layer composed of subglobose, oval to elongated hyphae, 17.3–36.7 × 14.4–23.9 μm, thick–walled (0.4–1.2 μm). Eucapillitial hyphae brownish, sinuous, umbranched, 3.2–5.3 μm diam., thin-walled (0.5–0.9 μm), surface slight verrucose, encrusted, lumen evident. Basidiospores brownish, globose to subglobose, 4.8–6.5 × 4.8–6.3 μm [x = 5.5 ± 0.5 × 5.4 ± 0.4, Qm = 1.02, n = 30], ornamentation less conspicuous under LM; verrucose under SEM, formed by triangular to cylindrical warts, slightly truncated, with planar to slightly pointed apex, 0.9–1.2 μm high; apiculus inconspicuous.

Habitat and distribution: Found in Caatinga vegetation, growing on sandy soil covered by leaf–litter.

Discussion

Geastrum caatingense is morphologically very similar to species of subsect. Marginata, and our phylogenetic analyses confirm the placement of the species in this subsection. G. caatingense is related to G. corollinum (Batsch) Hollós and G. diosiae J.C. Zamora, both have saccate basidiomata, very well delimited peristome, and hygroscopic rays; however, these two species have smaller basidiospores (4.5–5 μm diam. in G. corollinum and 4–5 μm diam. in G. diosiae) (Sunhede 1989, Crous et al. 2015). Moreover, G. corollinum has a pruinose endoperial body, and ephemeral mycelial layer without encrustations in contrast to G. caatigense; and G. diosiae has sessile and pruinose endoperial body, and absence of longitudinal cracks on the fibrous layer (Sunhede 1989, Crous et al. 2015). Geastrum flexuosum (L.S. Domínguez & Castellano) Jeppson & E. Larss. and Geastrum saccatum Fr. are phylogenetic close to G. caatingense. Nevertheless, the morphology could clearly separate G. flexuosum, because it has hypogeuos sequestrate basidiomata (Jeppson et al. 2013); while G. saccatum is distinct to G. caatingense by non-hygroscopic rays, absence of longitudinal cracks on the fibrous layer and non-encrusted mycelial layer (Sunhede 1989) (Table 2). Another species with morphology similar to G. caatingense is G. hungaricum Hollós. Although these species are phylogenetic distant (G. hungaricum is grouped in Geastrum section), both have involute rays, distinct delimited peristome, mycelial encrusted and palling away with age. However, G. hungaricum has strongly hygroscopic rays, a pseudoparenchymatous layer with thicker walled hyphal cells (> 1.5 μmick vs upto 1.5 μm), and basidiospores with smaller ornamentation (up to 0.7 μm vs up to 1.7 μm) (Sunhede 1989, Zamora et al. 2015). The phylogenetic analyses showed Geastrum parvistellum grouped in subsect. Plicostomata with Geastrum morganii Lloyd and Geastrum violaceum Rick. These three species have irregularly plicate peristome with delimitation weak or absent. On the other hand, G. violaceum is easily distinct to G. parvistellum by its endoperidium and exoperidium color, purple to violet, and smaller basidiospores (2.7– 3.1 μm diam.) (Sousa et al. 2014a); while G. morganii is distinct to G. parvistellum by mycelial layer

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persistent, non-encrusted, revolute rays and larger basidiomata (9−28 mm wide) (Sousa et al. 2014b) (Table 2). In the section Corollina, another species are similar to G. parvistellum, as G. corollinum. But, G. corollinum has a pruinose endoperidium, distinctly delimited peristome, ephemeral mycelial layer without encrustations and smaller basidiospores (4–5 μm) (Sunhede 1989, Kuhar et al. 2012). Geastrum lageniforme and G. saccatum could also recall G. parvistellum, however, these two species differ from G. parvistellum due to absence of encrustations on the mycelial layer and distinctly delimited peristome (Sunhede 1989). Additionaly, Geastrum saccatum var. parvulum Speg. is similar to G. parvistellum, mainly due to the small size of the basidiomata (15–20 mm wide). However, G. saccatum var. parvulum is distinct by having non-hygroscopic rays, sessile endoperidial body and smaller basidiospores (3–5 μm diam.) (Dios et al. 2017). Although the phylogenetic analyses showed that G. parvistellum belongs to subsect. Plicostomata, morphologically it is very similar to Geastrum arenarium Loyd. (sect. Geastrum); both have an encrusted mycelial layer, semi–hygroscopic rays and subsessile endoperidial body. Nevertheless, G. arenarium has larger basidiomata (15–30 mm wide), a pruinose endoperidial body, distinctly delimited peristome and smaller basidiospores (up to 4 μm diam.) (Bates 2004, Kuhar et al. 2012).

Acknowledgements The first author would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazilian agency) for four months of doctorate international scholarship in Madrid–Spain; the second author thanks the Universidade de Pernambuco (Programa de Fortalecimento Acadêmico/PFA), Maria Batista de Freitas (D. Carminha) and family (sítio Jenipapeiro, Triunfo, PB), for the logistical support and assistance during fieldwork. Authors are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq—Brazil, Projeto Pesquisador Visitante Especial PVE/407474/2013–7) for providing the financial support; and also to Prof. Marian Glenn for English revision.

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Crous, P.W., Wingfield, M.J., Le Roux, J.J., Richardson, D.M., Strasberg, D. et al. (2015): Fungal Planet Description Sheets: 371–399. – Persoonia. 35: 264–327. Dios, M.M., Moreno, G., Zamora, J.C. & Altés, A. (2017): Algunos hongos gasteroides epigeos interesantes de Catamarca (Argentina) – Lilloa. 54: 154–169. Fazolino, E.P., Calonge, F.D. & Baseia, I.G. (2008): Geastrum entomophilum, a new earthstar with an unusual spore dispersal strategy. – Mycotaxon. 104: 449–453. Gardes, M. & Bruns, T.D. (1993): ITS primers with enhanced specificity for Basidiomycetes applications to the identification of mycorrhizae and rusts. –Molecular Ecology. 1: 113–118. Hawksworth, D. L. (2001): The magnitude of fungal diversity: the 1.5 million species estimate revised. – Mycological Research 105 (12): 1422–1432. Jeppson, M., Nilsson, H.R. & Larsson, E. (2013): European earthstars in Geastraceae (Geastrales, Phallomycetidae) – a systematic approach using morphology and molecular sequence data. – Systematics and Biodiversity. 11 (4): 437–465. Kornerup, A. & Wanscher, J.H. (1978): Methuen Handbook of Colours. – 3rd edn. Eyre Methuen, London. Kasuya, T., Hosaka, K., Uno, K. & Kakishima, M. (2012): Phylogenetic placement of Geastrum melanocephalum and polyphyly of Geastrum triplex. – Mycoscience. 53(6): 411–426. Kuhar, F., Castiglia, V. & Papinutii, L. (2012): Geastrum species of the Riojia province, Argentina. – Mycotaxton. 122, 145–156. Kumar, S., Stecher, G. & Tamura, K. (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. 2016. – Mol. Biol. Evol 33(7): 1870–1874. Leal, I.R., da Silva, J.M.C., Tabarelli, M. & Lacher, J.R. T.E. (2005): Mudando o curso da conservação da biodiversidade na Caatinga do Nordeste do Brasil. – Megadiversidade. 1: 139–146. Moro, M.F., Lughadha, E.M., Araújo, F.S. & Martins, F.R. (2016):A Phytogeographical Meta analysis of the Semiarid Caatinga Domain in Brazil. – TheBotanical Review. 82: 91–148. Prado, D. (2003): As Caatingas da América do Sul. – In: Leal, I., Tabarelli, M. & Silva, J.M.C. (eds). Ecologia e Conservação da Cattinga. Recife. Ed. Universitária UFPE. Rehner, S.A. & Samuels, G.J. (1994): Taxonomy and phylogeny of Gliocladium analyzed from nuclear large subunit ribosomal DNA sequences. – Mycological Research. 98: 625–634. Silva, B.D.B., Cabral, T.S., Marinho, P., Ishikawa, N.K. & Baseia, I.G. (2013): Two new species of Geastrum (Geastraceae, Basidiomycota) from Brazil. – Nova Hedwigia. 96: 445–456. Silva, B.D.B., Sousa, J.O. & Baseia, I.G. (2011): Discovery of Geastrum xerophilum from the Neotropics. – Mycotaxon. 118: 355–359. Sousa, J.O., Baracho, G.S. & Baseia, I.G. (2015): Geastrum laevisporum: a new earthstar fungus with uncommon smooth spores. – Mycosphere 6: 501–507. Sousa, J.O., Morais, L.A., Nascimento, Y.M. & Baseia, I.G. (201a) – Updates on the geographic distribution of three Geastrum species from Brazilian semi-arid region. – Mycosphere 3: 467– 474. Sousa, J.O., Silva, B.D.B. & Baseia, I.G. (2014b): Geastrum from the Atlantic Forest in northeast Brazil – new records for Brazil. –Mycotaxon 129: 169–179. Sousa, J.O., Suz, L.M., García, M.A., Alfredo, D.S., Conrado L.M. et al. (2017): More than one fungus in the pepper pot: integrative taxonomy unmasks hidden species within Myriostoma coliforme (Geastraceae, Basidiomycota). – PloS ONE 12(6): e0177873. Stamatakis, A. (2014): RAxML Version 8: A tool for phylogenetic analysis and post– analysis of large phylogenies. – Bioinformatics. 30: 1312–1313. Sunhede, S. (1989): Geastraceae (Basidiomycotina): morphology, ecology, and systematics with special emphasis on the North European species. – Synopsis Fungorum 1. Fungiflora, Oslo, Norway. Velloso, A.L., Sampaio, E.V.S.B. & Pareyn, F.G.C. (2002): Ecorregiões propostas para o bioma Caatinga. Associação Plantas do Nordeste, Instituto de Conservação Ambiental. – The Nature Conservancy do

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Brasil, Recife. Available from http://www.mma.gov.br/estruturas/203/_arquivos/ecorregioes_site_203.pdf Vilgalys, R. & Hester, M. (1990): Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. – Journal of Bacterioly. 172: 4238–4246. PMID: 2376561 White, T.J., Bruns, T. & Taylor, J. (1990): Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. – In: Innes, M.A., Gelfand, D.H., Sninsky, J.J. & White, T.J. (eds), PCR protocols: A guide to methods and applications. cademic Press, Inc.; pp. 315−322. California, San Diego. Zamora, J.C., Calonge, F.D., Hosaka, K. & Martín, M.P. (2014): Systematics of the genus Geastrum (Fungi: Basidiomycota) revisited. – Systematics and Phylogeny. Taxon. 63: 447–497. Zamora, J.C., Calonge, F.D. & Martín, M.P. (2015): Integrative taxonomy reveals an unexpected diversity in Geastrum section Geastrum (Geastrales, Basidiomycota). – Persoonia. 34: 130–165.

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Tabela 4 (Table 1) Geastrum species included in the molecular analyses with their country, collection number and GenBank accession numbers of ITS and LSU of nuclear ribosomal DNA. The new sequences in bold.

Species Locality Collection number GenBank accession number ITS LSU Geastrum caatingense sp. nov. Brazil UFRN–Fungos 2843 MH253884 MH253886 Geastrum corollinum Sweden MJ2322 KC581972 KC581972 Spain MA–Fungi 5746 KF988359 KF988481 Sweden Herb. Sunhede 7744 KF988360 KF988482 Geastrum diosiae Argentina MA–Fungi 83788 KF988452 KF988587 Argentina Ma–Fungi 83789 KF988453 KF988588 Geastrum flexuosum Sweden UPS F–119844 KF988371 KF988493 Geastrum lageniforme Spain Herb. Zamora 207 KF988388 KF988513 Spain Herb. Zamora 316 KF988339 KF988514 Slovakia MJ7337 KC581966 KC581966 Geastrum aff. lageniforme Argentina MA–Fungi 83768 KF988389 KF988516 Niger COFC Hama 327 KF988390 KF988517 Argentina MA–Fungi 83770 KF988391 KF988518 Argentina MA–Fungi 83769 KF988392 KF988519 Portugal MA–Fungi 78398 KF988393 KF988520 Spain Herb. Ribes 221210–01 KF988394 KF988521 Geastrum morganii Canada Herb. Lebeuf HRL0177 KF988406 KF988534 France MJ8422 KC581971 KC581971 Geastrum aff. morganii Spain Herb. Zamora 525 KF988408 KF988536 Spain Herb. Zamora 367 KF988407 KF988535 Argentina MA–Fungi 83772 KF988409 KF988537 Argentina MA–Fungi 83773 KF988410 KF988538 Geasrtum parvistellum sp. nov. Brasil UFRN–Fungos 2341 MH253885 MH253887 Geastrum cf. saccatum Argentina MA–Fungi 83775 KF988427 KF988555 Bolivia MA–Fungi 47185–2 KF988426 KF988554 Australia Herb. Sunhede 7749 KF988343 KF988556 Japan UPS F–530056 KF988428 KF988558 Argentina MA–Fungi 83778 KF988433 KF988563 Niger COFC Hama 343 KF988432 KF988562 Spain Herb. Zamora 260 KF988430 KF988560 Geastrum sp.1 Spain MA–Fungi 31143 KF988454 KF988589 Spain MA–Fungi 37546 KF988455 KF988590 Geastrum sp.2 Australia MEL 2382911 KP012780 KP012780 Geastrum sp.3 Australia TNS KH–AUS10–74 JN845177 JN845301 Geastrum violaceum Argentina BAFC 51671 KF988450 KF988585 Argentina MA–Fungi 82487 KF988451 KF988586 Outgroup Geastrum hungaricum Hungary MJ8915 KC581963 KC581963 Slovakia MJ9317 KC581964 KC581964

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Tabela 5 (Table 2) Comparative table with morphologic characteristics from species of section Corollina.

Section Corollina Subsec. Plicostomata Subsec. Marginata Subsec. Lageniformia G. morganii G. parvistellum G. violaceum G. caatingense G. corollinum G. diosiae G. saccatum G. flexuosum G. lageniforme Basidiomata 9−28 6–12 9–26 11–29 11–30 5–14 8–44 20–40 15–44 wide (mm) Exoperidium non- sub- hygroscopic non- hygroscopic strongly strongly non- non- non-hygroscopic hygroscopic hygroscopic hygroscopic hygroscopic hygroscopic hygroscopic Mycelial layer non- encrusted, rarely non-encrusted, encrusted, non-encrusted, encrusted, non-encrusted, encrusted, non-encrusted, encrusted, non-peristent pesistent rarely non- ephemeral normally persistent, peristent persistent, with pesistent peristent non-peristent rarely with longitunidal cracks with longitunidal cracks Endoperidium with furfuraeous glabrous glaborus pruinose glabrous or glabrous reduced or glabrous suface protruding incospicuous lacking hyphae pruinose Pedicel absent inconspicuous absent absent or absent or absent absent absent absent (< 0.5 mm high) inconspicuous inconspicuous (< 1 mm high) Peristome irregularly irregularly plicate irregularly fibrillose fibrillose fibrillose fibrillose absent fibrillose plicate plicate Peristome absent absent or weak absent distinct distinct distinct distinct absent distinct delimitation Basidiopores 4.5–6 4.8–6.5 2.7–3.1 5.2–6.5 4.5–5 4–5 4.5–6 4–5 4.5–5 size (μm diam.)

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Figura 7 (Fig. 1) Strict consensus tree of the eight most parsimonious trees of concatenated ITS and LSU nrDNA sequences of Geastrum indicated in Table 1. Numbers over branches are parsimony bootstrap (MPbs) and maximum likelihood bootstrap (MLbs) values. Holotypes of the new species here described are indicated in bold. Locality, collection numbers, and GenBank codes are indicated in Table 1.

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Figura 8 (Fig. 2) Geastrum caatingense sp. nov. A. Fresh basidiomata in situ (UFRN–Fungos 2843, holotype). B. Fresh basidioma in situ. C (UFRN–Fungos 2960, isotype). Peristome detail. D. Endoperidial body detail. E. Endoperidium surface under SEM. F. Eucapillitialy hypha under SEM. G. Basidiospores under LM. H. basidiospore under SEM. Scale bars: A–B = 5 mm, C–D = 2 mm, E = 20 µm, F = 2 µm, G = 10 µm, H = 1 µm.

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Figura 9 (Fig. 3) Geastrum parvistellum sp. nov. A. Fresh basidioma in situ (UFRN–Fungos 2841, holotype). B. Fresh basidioma in situ (UFRN–Fungos 2961, isotype). C. Peristome detail. D. Pedicel detail. E. Endoperidium surface under SEM. F. Eucapillitial hyphae under SEM. G. Basidiospores under LM. H. Basidiospore under SEM. Scale bars: A–B = 5 mm, C–D = 1 mm, E = 20 µm, F = 2 µm, G = 10 µm, H = 1 µm.

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Capítulo III — Hidden fungal diversity from Neotropics: Geastrum hirsutum, G. schweinitzii (Basidiomycota, Geastrales) and their allies

Publicado: Accioly T*., Sousa J.O*., Moreau P-A., Lécuru C., Silva B.D.B., Roy M., Gardes M., Baseia I.G., Martín M.P. 2018. Hidden fungal diversity from Neotropics: Geastrum hirsutum, G. schweinitzii (Basidiomycota, Geastrales) and their allies. . PLoS ONE 14(2): e0211388. https://doi.org/10.1371/journal.pone.0211388

* Autores com contribuições iguais para este trabalho.

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RESEARCH ARTICLE Hidden fungal diversity from the Neotropics: Geastrum hirsutum, G. schweinitzii

OPEN ACCESS (Basidiomycota, Geastrales) and their allies

Citation: Accioly T, Sousa JO, Moreau P-A, Le´curu Thiago Accioly 1☯, Julieth O. Sousa1☯, Pierre-Arthur Moreau2, Christophe Le´curu3, Bianca C, Silva BDB, Roy M, et al. (2019) Hidden fungal D. B. Silva4, Me´lanie Roy5, Monique Gardes5, Iuri G. Baseia6, María P. Mart´ın 7* diversity from the Neotropics: Geastrum hirsutum, G. schweinitzii (Basidiomycota, Geastrales) and 1 Programa de Po´s-Graduac¸ão em Sistema´tica e Evoluc¸ão, Universidade Federal do Rio Grande do Norte, their allies. PLoS ONE 14(2): e0211388. https://doi. Natal, Rio Grande do Norte, Brazil, 2 EA4483 IMPECS, UFR Pharmacie, Universite´ de Lille, Lille, France, org/10.1371/journal.pone.0211388 3 Herbarium LIP, UFR Pharmacie, Universite´ de Lille, Lille, France, 4 Departamento de Botaˆnica, Instituto de Biologia, Universidade Federal da Bahia, Ondina, Salvador, Bahia, Brazil, 5 Laboratoire UMR5174 Evolution et Editor: Sabrina Sarrocco, Universita degli Studi di Diversite´ Biologique (EDB), Universite´ Toulouse 3 Paul Sabatier, Toulouse, France, 6 Departamento de Pisa, ITALY Botaˆnica e Zoologia, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil, 7 Departamento de Micolog´ıa, Real Jard´ın Bota´nico-CSIC, Madrid, Spain Received: October 16, 2018

Accepted: January 11, 2019 ☯ These authors contributed equally to this work. * [email protected] Published: February 6, 2019

Copyright: © 2019 Accioly et al. This is an open access article distributed under the terms of the Abstract Creative Commons Attribution License, which permits unrestricted use, distribution, and Taxonomy of Geastrum species in the neotropics has been subject to divergent opinions reproduction in any medium, provided the original among specialists. In our study, type collections were reassessed and compared with author and source are credited. recent collections in order to delimit species in Geastrum, sect. Myceliostroma, subsect. Data Availability Statement: All sequences files Epigaea. A thorough review of morphologic features combined with barcode and phyloge- are available from the Genbank database. The complete list of Accession numbers are included in netic analyses (ITS and LSU nrDNA) revealed six new species (G. neoamericanum, G. Table 1 and figures, as well as in the figures rubellum, G. brunneocapillatum, G. baculicrystallum, G. rubropusillum and G. courtecuis- included as Supporting Information. They will be sei). In additon, the presence of hairs on the exoperidium, a commonly used feature to public after the paper is published. diagnose Geastrum species, proved to be ineffective because it is a derived character Funding: This work was supported by: (TA and JS) within subsect. Epigaea. Coordenac¸ão de Aperfeic¸oamento de Pessoal de N´ıvel Superior (CAPES—Brazil) for the PhD scholarships awarded to Thiago Accioly and Julieth Sousa (PDSE-2017); (IGB and MPM) Conselho Introduction The Neotropical biogeographic realm, or Neotropics, comprises Central America, most of South America (except Patagonia), and the southern portion of North America. It is consid- ered the most diverse region for well-studied terrestrial taxa, mainly animals (amphibians, rep- tiles, birds and mammals) and plants (Angiosperms) [1,2,3,4,5]. However, knowledge regarding neotropical fungi is still insufficient. Indeed, the neotropical region represents a pri- ority area for taxonomic studies since it encompasses megadiverse countries (Brazil, Colombia, Costa Rica, Ecuador, Mexico, Peru, Venezuela) with hotspot areas, such as the Atlantic Rain- forest, Cerrado and Caribbean Islands; and tropical ecosystems where many potentially new taxa are threatened by human impacts [6,7,8,9]. Geastrum Pers., is a genus of gasteroid fungi in which the outer layers of the fruitbodies (basidiomata) open in a stellate pattern once the spores are mature, which makes them

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Hidden fungal diversity from Geastrum species in the Neotropics

Nacional de Desenvolvimento Cient´ıfico e popularly known as earthstars. Moreover, Geastrum is one of the most diverse genera of gas- Tecnolo´gico (CNPq—Brazil, Projeto Pesquisador teroid fungi, with 100–120 species around the world [10]. Although intensive inventories of Visitante Especial PVE/407474/2013–7, María P. gasteroid fungi in Brazil began only in the last two decades, already 56 Geastrum species have Mart´ın and Iuri G. Baseia); (PAM, MR and MG) The French Laboratories of Excellence Labex TULIP been reported or described from this country, which accounts for approximately half of the (ANR-10-LABX-41; ANR-11-IDEX-0002-02) and global Geastrum diversity. In recent years, at least one new species per year has been described Labex CEBA (ANR-10-LABX-25-01), the National from Brazil, including Geastrum ishikawae Accioly et al.; G. pusillipilosum J.O. Sousa et al.; G. Forest Office (ONF, France), the program ANR E- verrucoramulosum T.S. Cabral, J.O. Sousa & Baseia; G. caririense R.J. Ferreira et al.; G. magnos- TRICEL (National Research Agency, France) and porum J.O. Sousa et al. [11,12,13,14,15]. the DREAL Martinique (France) provided financial However, a great diversity remains hidden, especially in poorly sampled areas, as well as within support for this study. species that have been considered of wide distribution, such as Geastrum triplex Jungh. [16]. Competing interests: The authors have declared that Based on molecular data (ITS and LSU nrDNA loci; as well as a fragment of the atp-6 gene), no competing interests exist. Kasuya et al. [16] reported different groups according to their geographical origin, con- firming that the epithet G. triplex represents, in fact, a complex of cryptic or semi-cryptic spe- cies [17,18,19,20,21] awaiting description. In another recent study about species complexes in sect. Geastrum, Zamora et al. [22] dis- covered seven new species through integrative taxonomy by analysing not only molecular data, but also chemical and ultra-structural features usually not studied in the group. However, these studies reported mainly efforts on temperate or sub-tropical species, without much atten- tion to tropical and neotropical regions, where greater diversity is expected [7,23,24]. Subiculose Geastrum species are generally found in tropical habitats and are encompassed by section Myceliostroma [10]. The subiculum is a macroscopic, whitish layer, composed of thin, interlaced, sinuous hyphae [25]. This type of mycelium occurs mainly in lignicolous spe- cies, but it was also recorded in species growing on soil [26]. The most widespread neotropical species with this feature are Geastrum schweinitzii (Berk. & M.A. Curtis) Zeller and G. hirsu- tum Baseia & Calonge. Both G. schweinitzii and G. hirsutum are included in subsect. Epigaea Dissing & M. Lange within sect. Myceliostroma (P. Henn.) P. Ponce de Leo´n, and there has been an on-going con- troversy about their identities. In 1989, Ponce de Leo´n synonymized ten epithets under the name G. schweinitzii, including G. mirabile Mont., G subiculosum Cooke & Massee, and G. tri- chiferum Rick; and considered a stipitate : Geastrum schweinitzii var. stipitatum (Solms) P. Ponce de Leo´n [27]. The species concept of Ponce de Leo´n has been widely applied by others since then. When the epithet G. trichiferum (= Geaster trichifer) was revived by Trierveiler-Pereira & Silveira [28], G. hirsutum was synonymized into it, mainly based on its hirsute exoperidium. However, the identity of these hairy species is controversial. [29,30]. This study aimed to enhance the knowledge about neotropical Geastrum species richness and taxonomy by investigating potential morphospecies complexes within sect. Myceliostroma, subsect Epigaea.

Materials and methods Morphological studies Macro and micro morphological analyses were performed with fresh and mainly with dry basi- diomata from Andre´ Maur´ıcio Vieira de Carvalho Herbarium, Cocoa Research Center (CEPEC, Itabuna-BA, Brazil); Fungal Collection of the Federal University of Rio Grande do Norte (UFRN–Fungos, Natal-RN, Brazil); Herbarium Anchieta (PACA, Porto Alegre-RS, Bra- zil); Kew Fungarium of the Royal Botanic Garden (K(M), London, England); National Insti- tute of Amazonian Research (INPA, Manaus-AM, Brazil); U.S. National Fungus Collections (BPI, Beltsville-MD, USA). The descriptions were based on specific literature [10,25,31,32,33].

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Comparative analyses were done including the following type collections: Geastrum hirsutum, BRAZIL–Pernambuco, Recife, Reserva

Ecolo´gica do Gurjau´, 12 Jul. 2003, leg. I.G. Baseia (Ma- Fungi 67886 paratype); Geastrum pleosporum, CAMEROON, Centre Province, Department of Nyong & So’o, in the Mbalmayo forest reserve, 47 km South east of Yaounde, 16 Oct. 2002, leg. C. Douanla–Meli

(MA–Fungi 56971 isotype); Geastrum pusillipilosum, BRAZIL, Para´ıba, Mamanguape, REBIO Guaribas, SEMA II, 26 Jun.2014, leg. J.O. Sousa JM100 (UFRN–Fungos 2315 holotype, ITS sequence GenBank KX761175); Geastrum trichiferum PACA 15970, BPI 706088

(Rick’s original collection), and BPI 706086; and Geastrum schweinitzii, SURINAME, (K (M) 180187 type, K(M) 180187 isotype). Colour descriptions were based on [34,35]. Sample observations and macro morphological image capturing were done using a stereomicroscope Nikon H600L coupled with a Nikon DS–Ri camera. Micro morphological studies were carried out using a Nikon Eclipse Ni light microscope (LM) coupled with a Nikon DS–Ri camera, and scanning electron microscope (SEM) analysis was done under a Shimadzu SSX–550. For light microscopy of basidiospores, eucapillitium, rhizomorphs and exoperidial hyphae, the samples were mounted in 5% KOH (w/v). Also, samples were mounted in Congo Red to observe the basidia; and Melzer’s reagent was used to test the exoperidium, subiculum and rhizomorph hyphae. Preparation of the mate- rial examined under SEM followed Silva et al. [36]. At least thirty randomly selected basidio- spores were measured using LM at 1000× magnification, including surface ornamentation; and the height of the ornamentation was also measured. Basidiospore abbreviations follow [37]: n = number of randomly measured basidiospores; x = mean ± standard deviation of basidiospore diameter and height (including ornamentation); Qm = mean height/width quo- tient. Geographic distributions of delimited taxa followed Biogeographic Realms, Biomes and Ecoregions proposed by Dinerstein et al. [5].

Molecular analyses For UFRN–Fungos, INPA-Fungos and CEPEC samples, the extractions of DNA were per- formed utilizing 10 mg of gleba from dry basidiomata, preferably mature gleba. For the DNA isolation, DNeasyTMPlant Mini Kit (Qiagen, Valencia, CA) was used following manufactur- er’s instructions; except that the incubation in the lysis buffer was done at 55–60 ˚C over- night. For all other samples, fungal DNA was extracted from fragments of dried fruitbodies by using the Wizard Genomic Purification kit (Promega, Charbonnière les Bains, France) according to the manufacturer’s recommendations, and the final pellet resuspended in 40 μl of sterile water. Internal Transcribed Spacer (ITS) region of the nuclear ribosomal gene, including the 5.8S subunit (ITS nrDNA), and Large Subunit region of nuclear ribosomal DNA (LSU nrDNA) were the loci selected for molecular analyses. DNA amplification, purifi- cation and sequencing protocols are deposited in protocols.io (dx.doi.org/10.17504/ protocols.io.wpdfdi6). Sequences obtained in this study were submitted to Genbank under the accession numbers indicated in Table 1. The newly-generated ITS and LSU sequences, and homologous sequences retrieved from EMBL/GenBank/DDBJ databases, mainly from [10,12,16,38], were separately aligned in MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 [39]. Alignment gaps were marked with “–” and unresolved nucleotide positions were indicated with “N”. Geastrum velutinum was included as outgroup since this species is in section Myceliostroma, subsection Velutina J.C. Zamora, sister clade of subsect. Epigaea [10]. The maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference analy- ses are also deposited in protocols.io under the doi indicated above.

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Hidden fungal diversity from Geastrum species in the Neotropics

Tabela 6 (Table 1). Specimens and sequences included in this study. Clades as indicated from the bottom-up in Fig 1. Accession numbers in bold are from newly generated sequences.

Clade—Species Fungarium Number Country Genbank Acc. N˚ Genbank Acc. N˚ ITS LSU Clade I—G. schweinitzii K(M)180188 Type Suriname - - K(M) 180187 Isotype Suriname MH635016 - INPA 143435 Brazil MH635017 - Clade II—G. mirabile TNS 36758 Japan, Bonin Island JN845108 JN845226 TNS 36761 Japan, Bonin Island JN845109 JN845227 Clade IIII—G. courtecuissei sp. nov. LIP PAM/GUAD 11.04 Paratype Guadeloupe Island MH635002 - (France) LIP FH2004090503 Holotype Guadeolupe Island MH635003 MH635033 (France) MA–Fungi 83779 (under G. schweinitzii) Argentina KF988437 KF988567 Paratype Clade IV—G. pleosporum MA–Fungi 56971 Isotype Cameroon KF988416 KF988544 Clade V—G. hirsutum LIP MR/GUY 12.171 French Guiana MH635004 - LIP RC/GUY 12.086 French Guiana MH635005 - INPA 259950 Brazil MH634993 MH635026 MA–Fungi 67886 Paratype Brazil MH538295 - Clade VI—G. rubropusillum sp. nov. LIP RC/MART 03.015 Holotype France, Martinique Island MH635006 - LIP CL/MART 08.112 Paratype France, Martinique Island MH635007 MH635034 MA–Fungi 36141 (under G. schweinitzii) Panama KF988438 KF988568 Paratype UFRN–Fungos 2308 Paratype Brazil MH634994 MH635027 Clade VII—G. pusillipilosum MA–Fungi 83780 (under G. schweinitzii) Argentina KF988439 KF988569 UFRN–Fungos 2315 Holotype Brazil KX761175 KX761176 UFRN–Fungos 2256 Paratype Brazil KX761180 - UFRN–Fungos 2759 Brazil KX761177 KX761178 Clade VIII—G. baculicrystallum sp. LIP RC/GUY 08.035 Paratype French Guiana MH635008 - nov. UFRN–Fungos 2835 Holotype Brazil MH634995 MH635028 UFRN–Fungos 1857 Paratype Brazil MH635018 MH635035 Clade IX—G. brunneocapillatum sp. UFRN–Fungos 2286 Holotype Brazil MH634996 MH635029 nov. UFRN–Fungos 2834 Paratype Brazil MH634997 MH635030 UFRN–Fungos 2851 Paratype Costa Rica MH634998 - Clade X—G. minutisporum CORD-MLHC 14 Holotype Argentina KM260664 - CORD-MLHC 15 Argentina KM260665 - CORD-MLHC 16 Argentina KM260666 - Clade XI—G. rubellum sp. nov. LIP CL/MART 08.067B Paratype France, Martinique Island MH635009 MH635035 LIP PAM/MART 12.100 Holotype France, Martinique Island MH635010 MH635037 UFRN–Fungos 1214 (under G. hirsutum) Brazil KJ127029 JQ683662 Paratype UFRN–Fungos 2844 Paratype Brazil MH634999 MH635031 (Continued )

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Tabela 5 (Table 1). (Continued)

Clade—Species Fungarium Number Country Genbank Acc. N˚ Genbank Acc. N˚ ITS LSU Clade XII—G. neoamericanum sp. nov. LIP CL/GUAD 06.010 Paratype France, Guadeloupe Island MH635011 - LIP CL/GUAD 06.056 Paratype France, Guadeloupe Island MH635012 - LIP CL/MART 04.001 Paratype France, Martinique Island MH635013 - LIP JLC12030103 Paratype French Guiana MH635014 MH635038 LIP MR/GUY 12.004 Paratype French Guiana MH635015 - UFRN–Fungos 348 Paratype Brazil MH635019 - UFRN–Fungos 2850 Paratype Brazil MH635000 MH635032 CEPEC 1391 Paratype Brazil MH635020 - UFRN–Fungos 2149 Paratype Brazil MH635021 MH635039 UFRN–Fungos 2302 Holotype Brazil MH635001 MH635040 INPA 259949 Paratype Brazil MH635025 MH635041 UFRN–Fungos 1741 Paratype Brazil KJ127030 MH635042 UFRN–Fungos 2655 Paratype Brazil MH635022 - UFRN–Fungos 168 Paratype Brazil MH635023 - UFRN–Fungos 1803 Paratype Brazil MH635024 MH635043 Outgroup—G. velutinum MA-Fungi 83785 Argentina KF988446 KF988581 https://doi.org/10.1371/journal.pone.0211388.t001

Results The ITS dataset included 48 sequences of Geastrum specimens, of which 34 were generated in this study (Table 1) and 14 obtained from EMBL/GenBank/DDBJ databases. In addition to the new species, sequences were obtained from type collections of five previously described species (Table 1). The ITS alignment resulted in 657 unambiguously aligned nucleotide positions (317 con- stant, 103 parsimony-uninformative, and 237 parsimony-informative). MP analysis resulted in one most parsimonious tree (S1 Fig) with 862 steps (CI = 0.572, RI = 0.823, RC = 0.471, HI = 0.428). The ITS/LSU concatenated dataset included 28 samples with both ITS and LSU sequences, 1583 unambiguously aligned nucleotide positions: 1242 constant, 72 parsimony uninformative, and 269 parsimony-informative. Parsimony analyses resulted in a most parsi- monious tree (S3 Fig) with 748 steps (CI = 0.591, RI = 0.779, RC = 0.460, HI = 0.409). Maximum likelihood analyses with the default model GTRCAT gave a best ITS tree with— lnL = -2,900.087 (S2 Fig). Concatenated ITS/LSU likelihood analyses gave a best tree with— lnL = -4340.387 (S4 Fig). The evolutionary models chosen by jModel-Test for Bayesian inference were HKY+G for ITS dataset (according to all criteria: AIC, BIC, AICc and DT), and TIM3+I+G for LSU dataset (according to AIC and AICc criteria—BIC and DT suggested TIM3+I, so we decided to use the more thorough algorithm). In the Bayesian analyses, the first 2,000 trees from the non-sta- tionary phase were discarded. Maximum Clade Credibility tree and Posterior Probabilities (PP) were calculated from the 18,002 remaining trees. The summarized MCC tree has lnL = -2962.852. Concatenated ITS/LSU analysis gave us a MCC tree with lnL = -4329.812 (S5 Fig), which has been summarized from 18,002 trees after excluding 2,000 initial samples from the non-stationary phase. Tree topologies generated by maximum parsimony (S1 and S3 Figs), maximum likelihood (S2 and S4 Figs), and Bayesian analyses (Fig 1 and S5 Fig) were similar, showing equivalent clustering patterns at terminal nodes. Except in ML analyses, G. schweinitzii isotype K(M) 180187 (seq. MH635016) and the sample INPA143435 (seq. MH635017) do not cluster together, and appear as singletons. Excluding the outgroup, eleven delimited clades and a singleton (clade IV) are shown in Fig 1, corresponding to 12 species. The singleton is the isotype of G. pleosporum Douanla-Meli from Cameroon (Africa); among these delimited clades, six of them constitute new species:

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Figura 10 (Fig. 1) Bayesian trees of ITS (on the left) and ITS/LSU (on the right) nrDNA sequences of Geastrum species. Geastrum velutinum was used as out-group. Terminal branches are labeled with GenBank accession numbers. For further details, see Table 1. Numbers at the nodes indicate the Maximum Parsimony bootstrap, bootstrap values obtained from Maximum likelihood, and Posterior Probabilities from Bayesian analysis (MPbs /MLbs/ PP). Thick-lined branches are those with higher support (MPbs and MLbs higher than 85%, and PP higher than 0.85). Asterisk denotes fully supported branches in all three analyses. https://doi.org/10.1371/journal.pone.0211388.g001

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Hidden fungal diversity from Geastrum species in the Neotropics

Clade I. Geastrum schweinitzii isotype from Suriname (seq. MH635016), and a collection from Roraima-Brazil (seq. MH635017) grouped together. Specimens were collected in “Guiana Shield” biogeomorphological and pristine area of the Amazon forest, where there is a high level of endemism [40,41,42,43,44,45,46]. Clade II. The two sequences identified as Geastrum aff. mirabile Mont. obtained from Gen- Bank (seqs. JN845108/JN845226 and JN845109/JN845227); [16]) formed a strongly supported group (MPbs = 100, MLbs = 100, PP = 1). According to the authors, specimens were collected in Asia (Bonin Island-Japan). In the protologue, these specimens of G. mirabile, are described as having small basidiomata, sessile endoperidial body, basidiospores 3.5–5.0 μm diam., and lignicolous habit. Leprieur’s revision of the original type collections of G. mirabile from French Guiana held at PC (PC0084351, Leprieur 849, 2 plates), not successfully sequenced, suggests that G. mirabile is a morphological synonym of G. schweinitzii. Since we have not been able to analyze the Japanese collections of Kasuya et al. [16] we cannot ensure whether their morphol- ogy matches that of the PC vouchers of G. mirabile. However, all available photographs of the samples alleged to be G. mirabile at on-line TNS fungarium databases (including source mate- rials of the sequences used in our analysis) are from specimens with small mycelial tufts in a slightly hirsute exoperidium (S6 Fig). Thus, Japanese material from TNS claimed to be G. mirabile needs reassessment because they did not group in any of the clades presenting ‘schweinitzii-like’ morphology, and do not exhibit this morphological pattern. On the other hand, original PC vouchers can be either a synonym of G. schweinitzii or a cryptic species, and only molecular assessment of this material can clarify its real identity. Clade III. The specimens LIP PAM/GUAD 11.04 (seq. MH635002) and LIP FH2004090503 (seqs. MH635003/MH635033) from the Caribbean (Guadeloupe Island, France), and MA-Fungi 83799 (under Geastrum schweinitzii; seqs. KF988437/KF988567) from South Amer- ica (Argentina), grouped with maximum support (MPbs = 100, MLbs = 100, PP = 1). Both morphological, unique barcode sequence and concatenated ITS/LSU phylogenetic analysis lead us to describe a new species, G. courtecuissei sp. nov. Clade IV (singleton). Geastrum pleosporum isotype (MA–Fungi 56971; seqs. KF988416/ KF988544), from Africa (Cameroon), is the sister species of the previous one. Clade V. Two collections from Brazil, including the paratype of G. hirsutum (MA-Fungi 67886; seq. MH538295, Fig 2) from Brazil, grouped with two collections, LIP MR/GUY 12.171 (seq. MH635004) and LIP RC/GUY 12.086 (seq. MH635005), from French Guiana. This clade is highly supported (MPbs = 100, MLbs = 97, PP = 1). Clade VI. Four specimens, LIP RC/MART 03.015 (seq. MH635006), LIP CL/MART 08.112 (seqs. MH635007/MH635034), MA-Fungi 36141 (under G. schweinitzii, seqs. KF988438/ KF988568) and UFRN 2308 (seqs. MH634994/MH635027), from Central America (Panama), the Caribbean (Martinique Island) and South America (Brazil), respectively, grouped together with strong support (MPbs = 100, MLbs = 99, PP = 1). Both morphological and unique bar- code sequence led us to describe a new species, G. rubropusillum sp. nov. Clade VII. The holotype UFRN-Fungos 2315 (seqs. KX761175/KX761176) and paratype UFRN-Fungos 2256 (seq. KX761180) of G. pusillipilosum, a recently published species [12], grouped together (MPbs = 100, MLbs = 30, PP = 1). They were collected in South America (Brazil and Argentina). Clade VIII. A specimen from French Guiana, LIP RC/GUY 08.035 (seq. MH635008), and two from Brazil, UFRN-Fungos 1857 and 2835 (seqs. MH635018/MH635035 and MH634995/ MH635028) joined together in a highly supported group (MPbs = 100, MLbs = 96, PP = 1). Both morphology and a unique barcode sequence led us to describe a new species, G. baculi- crystallinum sp. nov. Clade IX. Sample UFRN-Fungos 2851 (seq. MH634998) from Central America (Costa Rica), and samples UFRN-Fungos 2286 and 2834 (seqs. MH634996/MH635029 and MH634997/MH635030) from South America (Brazil) grouped together in a well-supported

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Figura 11 (Fig. 2) Type collections of Geastrum hirsutum and Geastrum schweinitzii. (A) Geastrum hirsutum MA-Fungi 67886, paratype. (B) Geastrum hirsutum UFRN- Fungos 245, holotype. (C) Geastrum schweinitzii K (M) 180188, type. (D) Geastrum schweinitzii K(M) 180187 (M) 180187, isotype. Photo credits C, D: Donis Alfredo. A2, B2, C2, D2 bar = 5mm. https://doi.org/10.1371/journal.pone.0211388.g002

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Hidden fungal diversity from Geastrum species in the Neotropics clade (MPbs = 91, MLbs = 76, PP = 1). Both morphology and a unique barcode sequence led us to describe a new species, Geastrum brunneocapillatum sp. nov. Clade X. Three sequences of G. minutisporum from Argentina including the holotype (seqs. KM260664, KM260665 and KM260666) grouped together (MPbs = 100, MLbs = 98, PP = 1). Clade XI. Two specimens, LIP CL/MART 08.067B (seqs. MH635009/MH635035) and LIP PAM/MART 12.100 (seq. MH635010/MH635037), from the Caribbean (Martinique Island), and two from South America (Brazil), UFRN-Fungos 1214 and 2844 (seqs. KJ127029/ JQ683662 and MH634999/MH635031), came together in a well-supported group (MPbs = 85, MLbs = 86, PP = 0.9). Both morphology and unique barcode sequence led us to describe a new species, G. rubellum sp. nov. Clade XII. Fifteen specimens from Brazil, French Guiana and Caribbean Islands clustered together in a highly supported group (MPbs = 100, MLbs = 99, PP = 1). Both morphology and unique barcode sequence led us to describe a new species, G. neoamericanum sp. nov. The morphological ambiguity in type collections of G. schweinitzii K(M) 180187 (Fig 2) along with its not-fully-supported node among our molecular analyses lead us to create a new alignment with its clade (K(M) 180187 plus INPA 143435; seqs. MH635016 plus MH635017), and those species showing morphotypes in accordance with descriptions of G. schweinitzii (G. neoamericanum sp. nov., G. cf. mirabile, and G. baculicrystallum). The align- ment resulted in 22 sequences (Table 1), with 441 nucleotide positions, and 326 constant nucleotides. Bayesian analysis of this short alignment, conducted in Beast 2 software, along 50 million generations is shown in Fig 3a, along with Topo-Phylogenetic (Fig 3b) and Network represen- tations (Fig 3c). The Maximum Clade Credibility tree summarized after discarding the non- stationary burn-in has lnL = -1291.281;also, Topo-Phylogeny and Phylogenetic Network clearly delimited the clade formed by INPA 143435 (seq. MH635017) plus K(M) 180187 (G. schweinitzii isotype; seq. MH635016) as a solid taxon.

Taxonomy Geastrum baculicrystallum J.O. Sousa, Accioly, Baseia & M.P. Mart´ın, sp. nov., Fig 4, B1–B5 Mycobank MB 827089. Etymology. Referring to the rod-shaped crystals of the rhizomorphs.

Holotype. BRAZIL, Para´ıba, Areia, Mata do Pau Ferro, 22 Jul. 2014, leg. J.O. Sousa, D.S. Alfredo & E.J. Souza, JM137 (UFRN–Fungos 2835, ITS sequence GenBank MH634995, LSU sequence GenBank MH635028). Diagnosis. Geastrum baculicrystallum is very close to G. neoamericanum sp. nov. in mor- phology; however, G. baculicrystallum is differentiated by the cylindrical (2.3×0.9 mm), smaller warts of basidiospores (0.02–0.08 μm high), inconspicuous in light microscope, and presence of cystidia in subiculum hyphae. Description. Unexpanded basidiomata epigeous, brownish grey (5C2 to 5B2), citriform, 6–8 × 5–8 mm, surface tomentose to rugose when mature, not encrusted with debris. Subicu- lum white (4A1), covering the substrate, with exudate. Expanded basidiomata saccate, 5–7 mm × 8–10 mm wide. Exoperidium splitting into 5–6 revolute, 3–4 mm diam, triangular rays, non–hygroscopic. Mycelial layer yellowish white (4A2), surface papery, rugose, not encrusted with debris, persistent. Fibrous layer white orange (5A2), papery. Pseudoparenchymatous layer light brown (5D4), persistent. Endoperidial body grayish brown (6E3) subglobose to depressed globose, 2–4 × 3–6 mm, sessile, glabrous. Peristome fimbriate, delimited by a line, grayish brown (6D3), lighter than endoperidium, mammiform, depressed on endoperidium. Columella cylindrical, 2.3 × 0.9 mm, central, inconspicuous, white (4A1).

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Hidden fungal diversity from Geastrum species in the Neotropics

Figura 12 (Fig. 3) Analysis involving species of Geastrum schweinitzii complex. (A) Bayesian analysis conducted in Beast 2 software along 50 million generations. (B) Topo- phylogenetic and (C) phylogenetic network representations. https://doi.org/10.1371/journal.pone.0211388.g003

Subiculum composed of hyaline, filamentous, slender, hyphae, 0.6–1.0 μm diam, dextri- noid, sinuous, crystals not seen, presence of cystidia. Rhizomorphs composed of hyaline hyphae, surface covered by crystals with narrow oblique prism shape, 24.2–41.7 × 8.4– 12.3 μm. Mycelial layer formed of hyaline hyphae, thin-walled (0.5–0.9 μm diam), 3.1–7.0 μm diam, dextrinoid, surface encrusted, lumen slightly evident, branched. Fibrous layer formed of hyaline hyphae, 5.4–9.2 μm diam, surface non–encrusted, lumen evident.

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Hidden fungal diversity from Geastrum species in the Neotropics

Figura 13 (Fig. 4) Geastrum schweinitzii and allies. (A). Geastrum neoamericanum sp. nov. (B) Geastrum baculicrystallum sp. nov. (C). Geastrum courtecuissei sp. nov. (D) Geastrum rubropusillum sp. nov. A1, B1, C1, D1 Expanded basidiomata. A2, B2, C2, D2 Unexpanded Basidiomata. A3, C3, D3 Exoperidium detail. B3. Rhizomorphs under SEM. A4, B4, C4, D4 Basidiospores under LM. A5, B5, D5 basidiospores under SEM. A1, B1, D1 bar = 5 mm; C1 bar = 3 mm; A2, B2, C2 bar = 2 mm; A3, D2 bar = 1 mm; A4, B3, C4, D4 bar = 5 μm; A5, B5, D5 bar = 1 μm. https://doi.org/10.1371/journal.pone.0211388.g004

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Hidden fungal diversity from Geastrum species in the Neotropics

Pseudoparenchymatous layer formed by brownish hyphal cells, subglobose to oval, 30.4– 34.9 × 21.1–30.1 μm, thin–walled (0.6–0.9 μm). Eucapillitium brownish, thick-walled (0.4–0.8 μm diam), 2.6–4.7 μm diam, surface slightly encrusted, lumen not evident. Basidia, yellow- ish, thin to thick walls (0.4–1.2 μm), subglobose to oval, 9.4–16.4 × 6.4–10.0 μm. Basidiospores brownish, subglobose, 3.8–4.5 × 3.6–4.4 μm (x = 4.1 ± 0.2 × 3.9 ± 0.2, Qm = 1.04, n = 30), orna- mentation inconspicuous under LM, verrucose under SEM, warts very small (0.02–0.08 μm high). Ecology and distribution. This species has a Neotropical distribution. Found in the biome Tropical & Subtropical Moist Broadleaf Forests of Brazil (Pernambuco coastal forests ecore- gion), and French Guiana (Guianan lowland moist forests) on decaying wood with gregarious or caespitose growth.

Additional material examined. BRAZIL, Para´ıba, Areia, Mata do Pau Ferro, 17 Jul. 2012, leg. D.S. Alfredo (UFRN–Fungos 1857 paratype, ITS sequence GenBank MH635018, LSU sequence Genbank MH635035). FRENCH GUIANA, Sau¨l, Layon des Grands Arbres, 3 May 2008, leg. R. Courtecuisse (LIP RC/GUY 08.035, ITS sequence GenBank MH635008). Remarks. This species is basically characterized by small basidiomata (8–10 mm wide when expanded), exoperidium tomentose to rugose; peristome delimited by a grayish brown line, depressed on the endoperidium; columella cylindrical (2.3 × 0.9 mm); basidiospores globose to subglobose 3.6–4.5 μm diam, presence of cystidia on subiculum hyphae. It is morphologi- cally related to G. neoamericanum. However, there are small differences between them: the basidiospores of G. neoamericanum are slightly larger (3.8– 5.1 × 3.7–4.9 μm), with longer warts; the basidia differ, with clavate, pyriform to lageniform shapes in G. neoamericanum, and the crystals with rod shape in G. baculicrystallum. The bacullar crystals on the rhizo- morphs, and more inconspicuous warts on the basidiospores in G. baculicrystallum can differ- entiate this species from G. schweinitzii; we can consider G. baculicrystallum a semi- cryptic species of G. schweinitzii.

Geastrum brunneocapillatum J.O. Sousa, Accioly, M.P. Mart´ın & Baseia, sp. nov., Fig 5, A1–A5, Mycobank MB 827086. Etymology. Referring to the dark brown hairs on the exoperidium. Holotype. Para´ıba, Mamanguape, Reserva Biolo´gica de Guaribas, 26 Jun. 2014, leg. J.O. Sousa, Y.M. Nascimento & E.J. Souza JM101 (UFRN–Fungos 2286, ITS sequence GenBank MH634996, LSU sequence GenBank MH635029). Diagnosis. This species is morphologically close to G. pusillipilosum; but the sparsely hairy mycelial layer, longer exoperidium hairs (0.5–2.0 mm high) and smaller basidiospores (2.7– 4 μm diam) in G. brunneocapillatum, clearly differentiates these species. Also G. rubellum resembles G. brunneocapillatum, but the latter has longer (0.5–2.0 mm high) hairs and smaller basidiospores 2.8–4.0 μm diam. Description. Unexpanded basidiomata epigeous, brown (5E5) to brownish orange (5C4), subglobose, pyriform, obpyriform to oval, 7–13 × 6–10 mm, surface hairy, not encrusted. Subi- culum orange white (6A2) under some cespitose basidiomata. Expanded basidiomata saccate, 4.1–11 mm high × 8–26 mm wide. Exoperidium splitting into 5–7 revolute, triangular rays, non–hygroscopic. Mycelial layer yellowish (5D4), not encrusted with debris, persistent or peel- ing away in irregular patches. Hairs dark brown, long (0.5–2 mm high), ephemeral when fully mature. Fibrous layer yellowish white (4A2), papery. Pseudoparenchymatous layer yellowish (5D4), persistent, glabrous. Endoperidial body globose, 4–9 × 3–11 mm, sessile, glabrous, grey orange (5B2). Columella cylindrical, 4.9 × 1.7 mm, central, inconspicuous, grey orange (6B2). Peristome finely fibrillose, delimited, delimitation yellowish (5D4), darker than endoperidium. Gleba dark brown (6F4).

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Hidden fungal diversity from Geastrum species in the Neotropics

Figura 14 (Fig. 5) Geastrum hirsutum and allies. (A) Geastrum brunneocapillatum sp. nov. (B) Geastrum hirsutum. (C) Geastrum pusillipilosum. (D) Geastrum rubellum sp. nov. A1, B1, C1, D1 Expanded basidiomata. A2, B2, C2, D2 Unexpanded Basidiomata. A3, B3, C3, D3 Exoperidium detail. A4, B4, C4, D4 Basidiospores under LM. A5, B5, C5, D5 basidiospores under SEM. A1 bar = 2 mm; A2, A3, C2, bar = 1 mm; B1, B2, B3, C1, D2 bar = 2 mm; C3 bar = 0.1 mm; D1 bar = 4 mm; A4, B4, C4, D4 bar = 5 μm; A5, B5, C5, D5 bar = 1 μm. https://doi.org/10.1371/journal.pone.0211388.g005

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Subiculum composed of hyaline, filamentous, slender, hyphae, 0.9–1.0 μm diam, dextri- noid, sinuous. Subiculum and rhizomorphs with surface covered by crystals, with coarser and more irregular oblique prism shape. Mycelial layer composed of hyaline to yellowish, thin– walled hyphae (<1 μm), 1.66–3.04 μm diam, dextrinoid, lumen not evident. Hairs dark brown, thick–walled (0.7–1.0 μm.), skeletal hyphae, 3.2–4.5 μm diam, strongly dextrinoid, sin- uous, lumen not conspicuous. Fibrous layer composed of hyaline, sinuous, thick–walled hyphae (0.7–1.15 μm), 3.8–7.6 μm diam, encrusted, branched, lumen evident. Pseudoparen- chymatous layer composed of light brown, thin–walled (0.75–1.48 μm) hyphal cells, oval, ellip- soid, lageniform to pyriform, 30.5–65.6 × 21.1–55.5 μm. Eucapillitium yellowish brown, thin walls (0.5–1.0 μm), 1.5–3.8 μm diam, encrusted or not, lumen evident. Basidia, yellowish, thin- walled (0.4–0.7 μm), clavate, pyriform to lageniform, 12.2–19.3 × 3.8–6.7 μm. Basidiospores yellowish, globose to subglobose, 2.8–4.0

× 2.7–4.0 μm (x = 3.3 ± 0.4 × 3.2 ± 0.4, Qm = 1.03, n = 90), ornamentation inconspicuous under LM, verrucose under SEM; warts short (up to 0.4 μm high), rounded tips, apiculus reduced. Ecology and distribution. This species has a Neotropical distribution. Found in the biome Tropical & Subtropical Moist Broadleaf Forests of the Brazil (Pernambuco coastal forests and Pernambuco interior forests ecoregions) and Costa Rica (Isthmian-Atlantic moist forests ecor- egion) on soil covered by leaf–litter, decaying wood or termites nest, with gregarious or caespi- tose growth.

Additional material examined. BRAZIL, Para´ıba, Areia, Mata do Pau Ferro, Trilha Boa Vista, 21 Jul. 2014, leg. J.O. Sousa, D.S. Alfredo & E.J. Souza, JM117 (UFRN–Fungos 2834, paratype, ITS sequence GenBank MH634997). COSTA RICA, La Selva Biological Station, C. Ovrebo 2303 (UFRN–Fungos 2851, paratype, ITS sequence GenBank MH634998).

Additional species examined. Geastrum hirsutum, BRAZIL–Pernambuco, Recife, Reserva Ecolo´gica do Gurjau´, 12 Jul. 2003, leg. I.G. Baseia (MA-Fungi 67886, paratype, ITS sequence GenBank MH538295) (Fig 5, B1–B5); Amazonas, Manaus, Reserva Florestal Adolfo Ducke, 02 Jul. 2014, leg. M.D.F. Santana (INPA 259950, ITS sequence GenBank MH634993 and LSU sequence GenBank

MH635026). Geastrum pusillipilosum, BRAZIL. Para´ıba, Mamanguape, REBIO Guaribas, SEMA II, 26 Jun. 2014, leg. J.O. Sousa JM100 (UFRN–Fungos 2315, holo- type, ITS sequence GenBank KX761175 and LSU sequence GenBank KX761176) (Fig 5, C1– C5); Ceara´, Crato, Floresta Nacional do Araripe, 01 Apr. 2014, leg. R.J. Ferreira (UFRN–Fun- gos 2256, paratype, ITS sequence GenBank KX761180); Para´ıba, Mamanguape, Reserva Biolo´- gica Guaribas, 11 Jul. 2015, leg. J.O. Sousa, J.F. Freitas-Neto (UFRN–Fungos 2759, ITS sequence GenBank KX761177 and LSU sequence GenBank KX761178). Remarks. This species is recognized by its yellowish exoperidium; with ephemeral, long (0.5–2.0 mm high), dark brown hairs; peristome delimited by a yellowish line; and basidio- spores globose to subglobose, 2.8–4.0 μm diam. It is morphologically close to other species with hairy exoperidium, such as G. pusillipilosum and G. rubellum; however, these four species are fundamentally distinct by their larger basidiospores (5.0–6.5 μm diam and 4.4–6.3 μm diam., respectively). Additionally, G. pusillipilosum and G. rubellum have smaller hairs in the exoperidium (up to 0.3 mm; and up to 0.68 mm longer, respectively) than G. brunneocapilla- tum. We can consider G. brunneocapillatum a semi-cryptic species of G. hirsutum (Fig 5, B1– B5), since the latter species has slightly larger basidiospores 4.1–4.9 μm. Another species with morphological features similar to G. brunneocapillatum is G. minutisporum described without subiculum and with very tiny basidiospores (2–)2.5–3 mm diam [47].

Geastrum courtecuissei P.-A. Moreau, C. Lécuru, sp. nov., Fig 4, C1–C5 Mycobank MB 827090. Etymology. In honor to Dr. Courtecuisse, research scientist from France.

Holotype. GUADELOUPE, Saint-Claude, Matouba, along the Victor Hugues trail, on litter of Cupressus cf. macrocarpa, leg. F. Hairie, 9 Sep. 2004 (LIP FH2004090503, ITS sequence Gen- Bank MH635003, LSU sequence GenBank MH635033). Diagnosis. This species is similar to G. baculicrystallum, but G. courtecuissei is distinguished by the presence of echinulate exoperidium and basidiospores with longer warts, conspicuous under LM. Another morphologically closel-related species is G. pleosporum, which has poly- morphic basidiospores (4.0–7.0 × 3.2–5.0 μm), while, G. baculicrystallum has globose to sub- globose basidiospores (3.7–4.9 μm diam).

Description. Unexpanded basidiomata epigeous, light brown (N10Y30M10), subglobose to citriform, 2.5–22 × 4.1–20 mm, surface slightly tomentose to rugulose, papery to cottonous, not encrusted. Subiculum whitish (N10Y20M10), covering the substrate. Expanded basidiomata saccate, 2.8–8.1 mm high × 4.8–35 mm wide. Exoperidium splitting into 4–8 revolute rays, non–hygroscopic. Mycelial layer at first echinulate with short, whitish hairs, easily broken, forming collapsed ochre–brown patches and veins on whitish ground, light brown (N10Y40M10), not encrusted. Fibrous layer papery, white (N00Y10M00). Pseudoparenchymatous layer orange brown (N00Y40M10), peeling-off in irregular patches. Endoperidial body surface steel grey when fresh, brown (N30Y30M20) when dry, subglobose, 1.5–2.6 × 3.0–15 mm sessile, covered with a persistent whitish tomentum. Peristome fimbriate, delimited by light brown line (N20Y20M10), lighter or darker than endoperidium, mammiform to papilla-like, < 1 mm high, 2–2.5 mm wide, depressed on endoperidium. Columella not seen. Mature gleba greyish brown (N70Y20M10). Subiculum composed of hyaline, filamentous, slender hyphae, <1–1.8 μm diam, dextrinoid, strongly sinuous, thick–walled, sometimes

91

Hidden fungal diversity from Geastrum species in the Neotropics coiled; in depth, made of more or less parallel slen- der hyphae 1–2.5 μm diam, with sparse broader skeletal hyphae 2.3 μm diam, all smooth and colorless, slightly dextrinoid; with crystals coarser and more irregular oblique prisms. Rhizo- morphs composed of hyaline, slender hyphae, 1.0–3.0 μm diam, lumen not evident, some with clamps and ampulliform septa, surface covered by crystals, with narrow oblique prism shape. Mycelial layer formed of hyaline, thick-walled (0.5–3.5 μm diam) hyphae, 2.9–6.0 μm diam, dextrinoid, surface not encrusted, lumen evident, not branched, some with yellow granular content; terminations rounded to attenuate, smooth, always with yellow content. Fibrous layer formed of hyaline, thin-walled (1.0–1.1 μm diam) hyphae, 3.9–7.5 μm diam, surface encrusted, lumen evident. Pseudoparenchymatous layer formed by hyaline to yellowish hyphal cells, sub- globose, ellipsoid to pyriform, 19.5–37 × 18.0– 36.5 μm, thin–walled (0.8–1.0 μm diam). Euca- pillitium light brown, 2.5–3.9 μm diam, thin–walled (<1 μm diam), surface not encrusted, no lumen evident. Basidia cylindrical, pyriform to elongated 9–20 × 2–6.5 μm, 2–4 sterigmata. Basidiospores brownish, globose to subglobose, 3.8–5.0 × 3.7–4.9 μm (x = 4.1 ± 0.4 × 4.0 ± 0.3, Qm = 1.03, n = 30), ornamentation conspicuous in LM. Ecology and distribution. This species has a Neotropical distribution. Found in the biome Tropical & Subtropical Moist Broadleaf Forests of Argentina (Alto Parana´ Atlantic forest ecor- egion) and Guadeloupe Island (Leeward Islands moist forest ecoregion).

Additional material examined. ARGENTINA, Puerto Iguazu´, no date, leg. E. Grassi (MA– Fungi 83779, paratype, ITS sequence GenBank

KF988437, LSU sequence GenBank KF988567). GUADELOUPE, Saint–Claude, Matouba, along the Victor Hugues trail, on litter of Cupressus cf. macrocarpa, 10 Aug. 2011, leg. P.-A. Moreau, LIP PAM/GUAD 11.04, paratype, ITS sequence GenBank MH635002). Remarks. This species has an exoperidium layer at first echinulate, forming collapsed ochre–brown patches and veins on whitish ground when fully mature; peristome delimited by a light brown line, depressed on endoperidium; basidiospores globose to subglobose with 3.8–5.0 μm diam. It resembles G. pusillipilosum, however, G. pusillipilosum has a densely hairy exoperidium and larger basidiospores (5.0–6.5 μm diam). Geastrum schweinitzii, and G. neoa- mericanum are other species with very closely related morphology, but these species do not have the echinulate exoperidium. Geastrum pleosporum is very close to G. courtecuissei in the phylogenetic tree, but G. pleosporum differs in having a reddish pseudoparenchymatous layer, polymorphic basidiospores (4.0–7.0 × 3.2–5.0 μm) with smooth to nearly smooth ornamentation.

Geastrum neoamericanum J.O. Sousa, Accioly, M.P. Martíın & Baseia, sp. nov., Fig 4, A1–A5, Mycobank MB 827091. Etymology. Referring to the widespread occurrence in Latin America.

Holotype. BRAZIL, Para´ıba, Mamanguape, REBIO Guaribas, SEMA II, 25 Jun. 2014, J.O. Sousa, Y.M. Nascimento & E. Souza JM90 (UFRN–Fungos 2302, ITS sequence GenBank MH635001, LSU sequence GenBank MH635040). Diagnosis. This species is morphologically similar to Geastrum baculicrystallum, but G. neoamericanum is differentiated by ampuliform columella 4.5×0.6 mm, longer warts of basid- iospores (0.43–0.91 μm high), conspicuous in light microscope, and absence of cystidia in sub- iculum hyphae. Description. Unexpanded basidiomata epigeous, yellowish white (4A2), citriform to lacri- miform, 5–9 × 3–8 mm, surface tomentose to rugulose when mature, not encrusted. Subicu- lum white (4A1), covering the substrate, producing exudate in some basidiomata. Expanded basidiomata saccate, 3–8 mm high × 8–10 mm wide. Exoperidium splitting into 5–6 revolute, triangular rays, 2–3 mm diam, non–hygroscopic. Mycelial layer yellowish white (4A2) to orange white (5A2), surface rugose, tomentose to papery when mature, not encrusted, persis- tent. Fibrous layer papery, white (4A1). Pseudoparenchymatous layer brown (6D4) to orange grey (5B4), persistent. Endoperidial body greyish brown (6F3) to orange grey (5C3), subglo- bose to depressed–globose, 3–5 × 4–6 mm sessile, glabrous. Peristome fimbriate, with folds on apex, delimited by a line brownish grey (6F2) to greyish brown (5C3), lighter than endoperi- dium, mammiform, < 1 mm high. Columella ampuliform, 4.5 × 0.6 mm, central, inconspicu- ous, orange white (5A2). Subiculum composed of hyaline, filamentous, slender hyphae, 0.6–1.7 μm diam, dextrinoid, strongly sinuous. Rhizomorphs composed of hyaline, slender hyphae, 1.2–2.5 μm diam, sur- face encrusted, lumen not evident, surface covered by crystals, with narrow oblique prism shape. Mycelial layer formed of hyaline, thick-walled (0.5–1.2 μm diam) hyphae, 5.3–10.1 μm diam, dextrinoid, surface not encrusted, lumen evident, branched apex. Fibrous layer formed of hyaline, thick-walled (0.6–1.3 μm diam) hyphae, 5.1–9.5 μm diam, surface encrusted, lumen non–evident. Pseudoparenchymatous layer formed by brownish hyphal cells, subglobose to pyriform, 32.6– 59.5 × 23.4–40.8 μm, thick–walled (0.9–1.8 μm diam). Eucapillitium dark brown, 2.7–4.0 μm diam, thin–walled (0.5–0.8 μm diam), surface slightly encrusted, no lumen evident. Basidia yellowish, thin-walled (0.4–0.7 μm), clavate, pyriform to lageniform, 9.2– 18.7 ×

7.5–15.5 μm. Basidiospores brownish, globose to subglobose, 3.8–5.1 × 3.7–4.9 μm (x = 4.3 ± 0.3 × 4.2 ± 0.3, Qm = 1.03, n = 90), ornamentation conspicuous in LM, densely ver- rucose under SEM, medium warts (0.43–0.91 μm high) with planar tips. Ecology and distribution. This species has a Neotropical distribution. Found in the biome Tropical & Subtropical Moist Broadleaf Forests of the Brazil (Alto Parana´ Atlantic forests, Bahia coastal forests, Pernambuco coastal forests, Pernambuco interior forests, Uatumã- Trombetas moist forests) and the Guiana shield (Suriname and French Guiana, Guyanan low- land moist forest ecoregion),

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Hidden fungal diversity from Geastrum species in the Neotropics and in the Caribbean (Guadeloupe, Martinique). Cespitose basi- diomata grow on abundant subiculum, on decaying wood.

Additional specimens examined. BRAZIL, Para´ıba, Areia, Mata do Pau Ferro, Trilha Engenho Triunfo, 18 Jul. 2013, J.O. Sousa & D.S Alfredo, JM64 (UFRN–Fungos 2149, paratype, ITS sequence GenBank MH635021, LSU sequence GenBank MH635039); Rio Grande do Norte, Parnamirim, Mata da Base Ae´rea de Natal, 23 Aug. 2007, leg. E.P. Fazolino (UFRN–Fungos 348, paratype, ITS sequence GenBank MH635019); Rondoˆnia, Mo´dulo Abunã, 25 Jun. 2014, leg. M.D.F. Santana (INPA 259949, paratype, ITS sequence GenBank MH635025, LSU sequence GenBank MH635041); Minas Gerais, Santa Rita do Sapuca´ı, Reserva Biolo´gica Municipal Santa Rita M´ıtzi Brandão, 2015, leg. D.S. Alfredo & P. Lavor, DSA279 (UFRN–Fun- gos 2850, paratype, ITS sequence GenBank MH635000, LSU sequence GenBank MH635032); Bahia, Ilhe´us, RPPN Mãe da Mata, 19 Dec. 2006, leg. J.L. Bezerra 858 (CEPEC 1391, paratype, ITS sequence

GenBank MH635020). GUADELOUPE, Petit-Bourg, Carrère, Forêt de´partemen- talo-domaniale de Bois Sergeant, 21 Nov. 2006, leg. C. Le´curu (LIP CL/Guad 06.010, paratype, ITS sequence GenBank MH635011); Petit-Bourg, Route forestière de Jules, domaine de Duclos, 24 Nov.

2006, leg. C. Le´curu (LIP CL/Guad 06.056, paratype, ITS sequence GenBank MH635012). MARTINIQUE, Saint-Esprit, morne David, 23 Aug.

2004, leg. C. Le´curu (LIP CL/ Mart 04.001, paratype, ITS sequence GenBank MH635013). FRENCH GUIANA, 1 Mar. 2012, leg. J.-L. Cheype (LIP JLC12030103, paratype, ITS sequence GenBank MH635014, LSU sequence GenBank MH635038); 2012, leg. M. Roy (LIP MR– GUY–12–004, paratype, ITS sequence GenBank MH635015). Remarks. This species is mainly characterized by small basidiomata (9–10 mm wide when expanded); exoperidium whitish to yellowish and rugose to tomentose; pseudoparenchyma- tous layer brown to orange grey; peristome mammiform, delimited by a brownish grey to grey- ish brown line; columella ampuliform; basidiospores globose to subglobose (Qm = 1.03), 3.7–5.1 μm diam, with medium warts (0.43–0.91 μm high). It is a semi-cryptic species with G. schweinitzii, being distinct only by its slightly larger basidiospores 3.7–5.1 μm diam (against 3.5–4.2 μm in G. schweinitzii), and by longer warts on basidiospores (0.43–0.91 μm high), which are conspicuous under LM (against inconspicuous in G. schweinitzii). It also resembles. G. rubropusillum, but the darker mycelial and pseudoparenchymatous layers (brownish and reddish, respectively), peristome depressed on the endoperidium, and subglobose to oval basidiospores (Qm = 1.07) distinguish G. rubropusillum from G. neoamericanum.

Geastrum rubellum P.-A. Moreau & C. Le´curu. sp. nov., Fig 5, D1–D5, Mycobank MB 827092. Etymology. Named in reference to its reddish-coloured exoperidum.

Holotype. MARTINIQUE, Le Prêcheur, Anse Couleuvre, path towards Anse Le´vrier, 17 Aug. 2012, leg. P.–A. Moreau (LIP PAM/Mart 12.100, ITS sequence GenBank MH635010, LSU sequence GenBank MH635037). Diagnosis. This species is morphologically similar to G. pusillipilosum (Fig 2, C1–C5), but the smooth and sparsely hairy mycelial layer, longer exoperidium hairs (0.34–0.68 mm high), grayish endoperidium, and reddish pseudoparenchymatous layer in G. rubellum, clearly differ- entiate these species. Also G. rubellum resembles G. brunneocapillatum, but G. rubellum has reddish to brownish exoperidium with smaller hairs. Description. Unexpanded basidiomata, globose to slightly attenuate at base, somewhat umbonate (more conspicuously when sectioned) before opening, 5–12 mm wide. Expanded basidiomata saccate, 10 mm high × 8.5–30 mm wide. Exoperidium splitting into 5–7 rays, involute or revolute, sometimes bifid, non–hygroscopic. Subiculum white (4A1) to yellowish white, found only on basidiomata base or widely spread. Mycelial layer pale pinkish white at first then becoming pinkish red, brown (5E6) when dried, surface not encrusted, rugose, slightly hirsute and persistent. Hairs concolorous with mycelial layer, spaced apart, 0.34–0.68 mm high, becoming vinaceous pink–red when collapsing. Fibrous layer pinkish gray when fresh, then yellowish white (4A2), papery. Pseudoparenchymatous layer purplish pink when fresh, brownish grey (6D3) when dried, persistent. Endoperidial body grey (6D3), subglobose, 6 × 3–10 mm, sessile, surface glabrous. Peristome fimbriate, delimited by a line, orange grey

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Hidden fungal diversity from Geastrum species in the Neotropics

(6B2), mammiform, concolorous with endoperidium. Columella distinct, intruding 2/3 into the glebal mass, white, cylindrical. Mature gleba dark brown (6F4). Subiculum composed of hyaline, filamentous, slender hyphae, dextrinoid, 0.9–1.6 μm diam, sinuous, presence of abundant crystals, with coarser and more irregular oblique prism shape, often clustered in rosette–like aggregates, mostly on surfaces but also internal, 3–25 μm long. Mycelial layer composed of hyaline, thin–walled hyphae (0.5–0.7 μm), 2.3–4.5 μm diam, dex- trinoid, sinuous, surface not encrusted, lumen inconspicuous. Hairs composed of thick–walled (0.5–1.2 μm), brownish, skeletal hyphae, 4.7–6.4 μm diam, strongly dextrinoid, surface not encrusted, lumen not evident or slightly evident. Fibrous layer composed of hyaline, sinuous, thick–walled hyphae (0.6–0.9 μm), 4.3–7.2 μm diam, slightly encrusted, lumen evident. Pseudoparenchymatous layer composed of light brown, thin– walled (0.8–1.3 μm) hyphal cells, subglobose to oval, 33.7–57.0 × 41.4–37.1 μm. Eucapillitium brownish, thick walls (0.5–1.1 μm), 2.3– 5.5 μm diam, surface encrusted, lumen evident. Basidia globose before maturity, then vesiculose to lageniform with a subcapitate neck bearing the insconspicuous sterigmata, 6.5–12 x 5–6.8 μm. Basidiospores dark brown, globose to subglobose, (3.5) 4.4–6.3 × 4.4–6.0 μm

(x = 5.5 ± 0.5 × 5.3 ± 0.1, Qm = 1.03, n = 60), ornamentation conspicuous under LM, warts 0.6–1.2 μm high. Ecology and distribution. This species has a Neotropical distribution. Found in the biome Tropical & Subtropical Moist Broadleaf Forests of Brazil (Atlantic Coast restingas and South- west Amazon moist forests ecoregions), and Martinique Island (Windward Islands moist for- ests ecoregion), growing in groups on soil covered by leaf–litter or decaying wood.

Additional material examined. BRAZIL. Rio Grande do Norte, Ba´ıa Formosa, Reserva Partic- ular do Patrimoˆnio Natural Mata Estrela, 09 Jun. 2009, leg. B.D.B Silva, I.G. Baseia, T.S. Cabral (UFRN–Fungos 1214, paratype, ITS sequence GenBank KJ127029, LSU sequence GenBank JQ683662); Acre, Floresta Nacional do Macauã, 29 Jan. 2016, leg. A.M. Ottoni, AMO 604 (UFRN–Fungos 2844, paratype,

ITS sequence GenBank MH634999, LSU sequence GenBank MH635031). MARTINIQUE, Le Prêcheur, Anse Couleuvre, along the road, in secondary meso- phytic forest, 25 Aug. 2008, leg. C. Le´curu (LIP CL/MART 08.067B, paratype, ITS sequence GenBank MH635009, LSU sequence GenBank MH635035). Remarks. This species is recognized by the reddish to brown exoperidium in fully mature basidiomata, and verrucose to hairy mycelial layer, with ephemeral, short (0.34–0.68 mm high) sparse hairs; grey endoperidium; peristome delimited by an orange grey line; and basid- iospores globose to subglobose, 4.4–6.3 μm diam. The morphology of G. rubellum is very simi- lar to G. hirsutum and taxonomic misunderstandings can occur, as happened with the collection UFRN–Fungos 1214 (seq. KJ127029/JQ683662), which was previously determined as G. hirsutum [44]. However, G. hirsutum is distinguished by lighter endoperidium (grey orange 5B2), and by longer (0.6– 1.8 mm high) and darker (dark brown) hairs on the exoperi- dium. The collection UFRN–Fungos 1214 is described here as G. rubellum sp. nov.

Geastrum rubropusillum J.O. Sousa, Accioly, M.P. Martíın & Baseia, sp. nov., Fig 4, D1– D5, Mycobank MB 827094. Etymology. Referring to the reddish pseudoparenchymatous layer and the small size of basidiomata.

Holotype. MARTINIQUE, Le Prêcheur, anse Couleuvre, 1 Sep. 2003, leg. R. Courtecuisse (LIP RC/MART 03.015, ITS sequence GenBank MH635006). Diagnosis. This species is morphologically close to Geastrum pleosporum, but G. rubropusil- lum is distinguished by its mammiform peristome and subglobose to oval basidiospores, (3.8–5.9 × 3.7–5.3 μm) with short warts (0.1–0.5 μm high). Description. Unexpanded basidiomata epigeous, napiform to citriform, light brown (5D4), 4.3–8.0 mm × 4.0–7.4 mm, surface densely tomentose to rugulose when mature, not encrusted with debris. Subiculum white orange (5A2) to yellowish (4A2), covering the substrate,

94

Hidden fungal diversity from Geastrum species in the Neotropics rhizomorphs light brown (5D4) attached to the substrate. Expanded basidiomata saccate, 2.9– 7 mm high × 7.0–9.5 mm wide. Exoperidium splitting into 5–6 revolute, triangular rays, 3–4 mm diam, non–hygroscopic. Mycelial layer light brown (5D4) to orange white (N00Y30M10), surface papery, slightly tomentose to rugose, not encrusted with debris, persistent. Fibrous layer papery, yellowish white (4A2). Pseudoparenchymatous layer pale red (7A3) when fresh and light brown (6D4) to brown (N70Y90M50) when dried, persistent or cracking on the base of rays. Endoperidial body brownish gray (6D2) to greyish brown (N70Y40M20), globose to sub- globose, 3.0–5.5 × 3.0–6.0 mm sessile, glabrous. Peristome fibrillose, delimited by a line, brownish gray (6E2), lighter than endoperidium, mammiform, 2 mm wide, up to 1 mm high, slightly depressed on endoperidium. Columella cylindrical, 4.2 × 0.4 mm, central, conspicu- ous, yellowish white (3A2). Mature gleba brown (6E4). Subiculum composed of hyaline, filamentous, slender hyphae, 0.9–1.2 μm diam, dextrinoid, sinuous. Rhizomorphs composed of hyaline, slender hyphae, lumen not evident, surface cov- ered by crystals, with coarser and more irregular oblique prism shape, 1.1–2.9 × 6.2–8.2 μm. Mycelial layer formed of hyaline to yellowish hyphae, thin walled (1 < μm diam), 2.8–4.8 μm diam, dextrinoid, surface encrusted, lumen evident, unbranched. Fibrous layer formed of hya- line hyphae, 4.6–8.7 μm diam, surface not encrusted, lumen not evident. Pseudoparenchyma- tous layer formed by brownish hyphal cells, subglobose to pyriform, 29.4–53.2 × 22.5–40.5 μm, thin–walled (<1 μm). Eucapillitium 2.2–4.4 μm diam, thick–walled (>1 μm diam), sinuous, branched, surface slightly verrucose, encrusted, lumen evident, dark brown in 5% KOH. Basidia subglobose to clavate, 4.5–21.4 × 3–6.6 μm, 3–5 sterigmata, yellowish brown in 5% KOH.

Basidiospores subglobose to oval, 3.8–5.9 × 3.7–5.3 μm (x = 4.3 ± 0.5 × 4.6 ± 0.4, Qm = 1.05, n = 60), yellowish, ornamentation inconspicuous under LM, verrucose under SEM, warts short (0.1–0.5 μm high), slightly columnar, with planar to rounded tips, apiculus reduced surrounded by columnar processes. Ecology and distribution. This species has a Neotropical distribution. Found in the biomes Tropical & Subtropical Moist Broadleaf Forests of Brazil (Pernambuco coastal forests ecore- gion) and Martinique Island (Windward Islands moist forests), and in the biome Mangroves of Panama´ (Southern Mesoamerican Pacific mangroves ecoregion) on decaying wood, fruiting is gregarious, rarely cespitose.

Additional material examined. BRAZIL, Para´ıba, Areia, Mata do Pau Ferro, Trilha do Cumbe, 15 Jul. 2013, leg. J.O. Sousa, D.S. Alfredo & R.A Lima JM36 (UFRN–Fungos 2308, paratype, ITS sequence GenBank MH634994, LSU sequence GenBank MH635027). PANAMA, Coiba Island, 15 Nov. 1996, leg. F. Pando & M.P. Nu´ñez (MA–Fungi 36141, paratype, ITS sequence GenBank KF988438, LSU sequence

GenBank KF988568). MARTINIQUE, Le Robert, bois Pothau, 30 Aug. 2008, leg. C. Le´curu (LIP CL/MART 08.112, paratype, ITS sequence Gen- Bank MH635007, LSU sequence GenBank MH635034). Remarks. This species has small basidiomata (7–9 mm wide when expanded), exoperidium light brown, tomentose to rugulose when mature; pseudoparenchymatous layer reddish when fresh to light brown when mature; peristome slightly depressed on the endoperidium; basidio- spores subglobose to oval (Qm = 1.07) with 4.2–5.9 μm diam, warts short (0.1–0.5 μm high) with planar to rounded tips. Based on morphology, G. rubropusillum is very similar to G. schweinitzii, and this explains some misidentifications, such as the collection MA–Fungi 36141 (seqs. KF988438/KF988568), which was previously identified as G. schweinitzii [10] but it belongs to the species G. rubropusillum. According to our data, G. schweinitzii differs from G. rubropusillum by having lighter pseudoparenchymatous layers (whitish when fresh), peristome non–depressed on the endoperidium, and globose basidiospores. Another species similar to G. rubropusillum is G. pusillipilosum, which is distinguished by its densely hairy exoperidium and globose basidiospores (Qm = 1.00).

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Hidden fungal diversity from Geastrum species in the Neotropics

Discussion This study uncovered a hidden richness of subiculose neotropical Geastrum species. From our analyses 12 species were recovered, mainly collected in South and Central America, of which six were species unknown to science. Thus, we confirm the underestimated biodiversity of the genus Geastrum in the Neotropical region, illustrated by two cases of species complexes involv- ing G. schweinitzii and G. hirsutum. The barcode sequence (ITS) of the isotype of G. schweinitzii (K (M) 180187) grouped with only one sequence (INPA 143435) in Clade 1. It is interesting to note that these two collections are from the same biogeomorphological region: the Guiana Shield, a pristine Amazonian area with minor modifications during landscape evolution [42] revealing a possible scenario for allopatric or parapatric speciation dynamics in which G. schweinitzii may be endemic, since the Pakaraima Mountains are an ecological barrier for many organisms [44,48,49]. Keeping this in mind, the ten synonyms of Geastrum schweinitzii proposed by Ponce de Leo´n [27] need to be reassessed. Furthermore, some of the putative synonyms are from very distinct ecore- gions [5]: e.g. G. papyraceus Berk. & M.A. Curt. from Bonin Islands (Indomalayan Realm); G. lignicola Berk. from Australia (Australasia Realm); G. rhizophorum Dissing & M. Lange from Congo (Afrotropic Realm); and G. subiculosum from Australia (Australiasia Realm). Fifteen samples with the morphology traditionally associated to the name G. schweinitzii, collected from widespread areas of the neotropical region, appear in clade XII in Fig 1, illus- trating a case of semi-cryptic species [17,18,20,21] and evolutionary convergence in their mor- phology. Another case of semi-cryptic species of G. schweinitzii is G. baculicrystallum, the two species could be distinguished only by details in basidiospore size and ornamentation. Three ITS sequences from GenBank previously identified under the name G. schweinitzii, KF988437, KF988438, and KF988439, are in fact three different species: G. courtecuissei, G. rubropusillum, and G. pusillipilosum, respectively. In these cases, morphological features dis- tinguish each of these species. The presence of hairs on the exoperidium is a recurrent feature of subiculose species. How- ever, presence or absence of hairs as a single decisive feature for taxonomic identification could result in misidentification. Five of the species recognized here included this same fea- ture. These semi-cryptic species are distinguished by molecular data, but discriminatory mor- phological features are unremarkable. Geastrum brunneocapillatum, G. rubellum and G. hirsutum are semi-cryptic species, and are not even sibling/sister species [17,18,20], reinforc- ing the statement that the presence of hairs on the exoperidium alone is not a suitable feature for species delimitation in Geastrum, but, instead, it represents an evolutionary convergence. Recently a synonymization of Geastrum trichiferum to G. hirsutum was proposed [28]. Geastrum trichiferum is a mysterious species involved in taxonomic and nomenclatural prob- lems in recent years [28,29,30]. Trying to better understand the nomenclature and taxonomic status of this species, we analyzed the collections PACA 15970 (packet labeled holotype in PACA), BPI 706088 (Rick’s original collection alleged by Zamora & Parra [30] and BPI 706086 (lectotype designated by Trierveiler-Pereira & Silveira [28] (S7 Fig). It was possible to distinguish BPI and PACA collections from other species studied in this paper. By morphological analysis, we realized that these two exsiccates are notably different from each other, and they should probably be treated as distinct species: PACA 15970 has a basidiome with non-delimited peristome and small basidiospores (2.7–4 μm diam); while, BPI 706086 has delimited- peristome and larger basidiospores (4.4–6.5 μm diam). Thus, besides the nomenclatural problems involving its protologue, G. trichiferum has ambiguous type collec- tions, since no voucher was indicated in the original description.

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When compared to G. hirsutum, the collection BPI 706086 of G. trichiferum shows that specimens have larger basidiospores (4.4–6.5 μm diam) and lighter hairs (light brown), while morphologic analysis of the collection PACA 15970 shows that the single basidioma is distinct from G. hirsutum by the non–delimited peristome and short hairs on the exoperidium. The type collections of G. trichiferum (S7 Fig) were also compared to G. pusillipilosum, a morphologically similar species, which also exhibits a hairy exoperidium. Our morphological analysis demonstrated that they can be differentiated mainly by basidiospore size and orna- mentation. The collection PACA 15970 is distinguished from G. pusillipilosum by its non- delimited peristome and smaller basidiospores (2.7–4 μm diam) with inconspicuous ornamen- tation under LM, composed of small (0.1–0.6 μm high) warts with rounded tips under SEM; while the specimens of the BPI 706086 collection grow on a developed subiculum, the hairs on the exoperidium are longer (0.8–1.3 mm high), basidiospores have similar ornamentation: inconspicuous under LM, small (0.2–0.6 μm high) warts with rounded wart tips under SEM. Unfortunately, DNA extraction was not allowed by the herbarium and definitive conclusions are not possible. We note that it is necessary to be extremely careful with species synonymization and conse- quent under-estimating of biodiversity. The integration of molecular and bioinformatic approaches for taxonomic and systematic studies seems to be essential for species delimitations in Geastrum, especially when dealing with species complexes.

Supporting information S1 Fig. Maximum parsimony tree of ITS nrDNA sequences of Geastrum species. One sequence of Geastrum velutinum was used as out-group. Terminal branches are labeled with appropriate specimen codes. For further specimen details, see Table 1. Numbers at the nodes indicate the maximum parsimony bootstrap. (TIF) S2 Fig. Maximum likelihood tree of ITS nrDNA sequences of Geastrum species. One sequence of Geastrum velutinum was used as out-group. Terminal branches are labeled with appropriate specimen codes. For further specimen details, see Table 1. Numbers at the nodes indicate the maximum likelihood bootstrap. (TIF) S3 Fig. Maximum parsimony tree of concatenated ITS/LSU nrDNA sequences of Geastrum species. One sequence of Geastrum velutinum was used as out-group. Terminal branches are labeled with appropriate specimen codes. For further specimen details, see Table 1. Numbers at the nodes indicate maximum parsimony bootstrap. (TIF) S4 Fig. Maximum likelihood tree of concatenated ITS/LSU nrDNA sequences of Geastrum species. One sequence of Geastrum velutinum was used as out-group. Terminal branches are labeled with appropriate specimen codes. For further specimen details, see Table 1. Numbers at the nodes indicate maximum likelihood bootstrap. (TIF) S5 Fig. Bayesian tree of concatenated ITS/LSU nrDNA sequences of Geastrum species. One sequence of Geastrum velutinum was used as out-group. Terminal branches are labeled with appropriate specimen codes. For further specimen details, see Table 1. Numbers at the nodes indicate the posterior probabilities. (TIF)

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S6 Fig. Geastrum mirabile. (A) Original collection of G. mirabile (PC0084351). (B, C) Japa- nese collection (TNS 36748) identified as G. mirabile by Kasuya et al. [16], adapted from http://db.kahaku.go.jp/webmuseum_en/mediaDetail?cls=col_b2_01&pkey=36748&lCls= med_b2_01&lPkey=B07- 019855&detaillnkIdx=0. A, B, C bar = 10mm. (TIF) S7 Fig. Collections of Geastrum trichiferum. (A) protologue in Lloyd (1907). (B) BPI 706088, material that correspond to Lloyd´s illustration (Fig 147–148), according to Zamora & Parra (2016), adapted from: http://nedoko.sakura.ne.jp/sblo_files/nedoko/image/RIMG2757.JPG (photo by Taiga Kasuya). (C) BPI 706086. (D) PACA 15970. (C1, D1) Herbarium data. (C2, D2) Basidiomata of collection. (C2, D3) Basidiospores under scanning electronic microscope. Bar C2, D2 = 5 mm, C3, D3 = 1 μm. (TIF)

Acknowledgments The analyses of specimens of the genus Geastrum Pers. from distinct neotropical localities (Antilles Islands, Argentina, Brazil, Costa Rica, French Guiana, Suriname) in this work was only possible thanks to the collaboration of taxonomic and systematic researchers from many institutions of the world: Universite´ de Lille and Universite´ de Toulouse (France); Real Jard´ın Bota´nico de Madrid (RJB- CSIC, Spain); Universidade Federal do Rio Grande do Norte and Universidade Federal da Bahia (Brazil). The authors would like to express their thanks to the Coordenac¸ão de Aperfeic¸oamento de Pessoal de N´ıvel Superior (CAPES—Brazil) for the PhD scholarships awarded to Thiago Accioly and Julieth Sousa (PDSE-2017); to the Conselho Nacional de Desenvolvimento Cient´ı- fico e Tecnolo´gico (CNPq—Brazil, Projeto Pesquisador Visitante Especial PVE/407474/2013– 7, María P. Mart´ın and Iuri G. Baseia). The authors are also thank Donis Alfredo for the photos from Kew´s collections, Dr. Clark Ovrebo for the collection UFRN-Fungos 2851 from Costa Rica, Angelia Ottoni for the collection UFRN-Fungos 2844 from Acre state, Brazil, Marian Glenn for English revision, the curators of herbaria K, LIP and PC for having made collections available to them, Jean-Louis Cheype, Re´gis Courtecuisse, Franc¸ois Hairie, Jean- Pierre Fiard and Me´lanie Roy for providing interesting material from French West Indies. We are grateful to Sophie Manzi for help with molecular labwork, and we also thank Jona- than Dubousquet and Morgan Ada who have contributed to this work by conducting some DNA extractions and amplifications at the laboratory EDB. The French Laboratories of Excel- lence Labex TULIP (ANR-10-LABX-41; ANR-11-IDEX-0002-02) and Labex CEBA (ANR-10- LABX-25-01), the National Forest Office (ONF, France), the program ANR E- TRICEL (National Research Agency, France) and the DREAL Martinique (France) provided financial support for this study.

Author Contributions Conceptualization: Thiago Accioly, Julieth O. Sousa, Pierre-Arthur Moreau, Bianca D. B. Silva, Monique Gardes, Iuri G. Baseia, María P. Mart´ın. Data curation: Thiago Accioly, Julieth O. Sousa, Pierre-Arthur Moreau, Christophe Le´curu, Me´lanie Roy, Iuri G. Baseia, María P. Mart´ın. Formal analysis: Thiago Accioly, Julieth O. Sousa, María P. Martín. Funding acquisition: Me´lanie Roy, Monique Gardes, Iuri G. Baseia, María P. Mart´ın.

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Investigation: Thiago Accioly, Julieth O. Sousa, Pierre-Arthur Moreau, Christophe Le´curu, Bianca D. B. Silva, Monique Gardes, Iuri G. Baseia, María P. Mart´ın. Methodology: Thiago Accioly, Julieth O. Sousa, Me´lanie Roy, Monique Gardes, Iuri G. Baseia, María P. Martíın. Project administration: Pierre-Arthur Moreau, Monique Gardes, Iuri G. Baseia, María P. Martín. Resources: Pierre-Arthur Moreau, Monique Gardes, Iuri G. Baseia, María P. Martíın. Software: Thiago Accioly, Julieth O. Sousa, María P. Martrín. Supervision: Iuri G. Baseia, María P. Martín. Validation: Thiago Accioly, Julieth O. Sousa, Pierre-Arthur Moreau, Iuri G. Baseia, María P. Martín. Visualization: Thiago Accioly, Julieth O. Sousa, Pierre-Arthur Moreau, Iuri G. Baseia, María P. Martín. Writing – original draft: Thiago Accioly, Julieth O. Sousa, Pierre-Arthur Moreau, Bianca D. B. Silva, Monique Gardes, Iuri G. Baseia, María P. Martín. Writing – review & editing: Thiago Accioly, Julieth O. Sousa, Pierre-Arthur Moreau, Bianca D. B. Silva, Monique Gardes, Iuri G. Baseia, María P. Martín.

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Capítulo IV — More than one fungus in the pepper pot: Integrative taxonomy unmasks hidden species within Myriostoma coliforme (Geastraceae, Basidiomycota)

Publicado: Sousa J.O., Suz L.M., García M.A., Alfredo D.S., Conrado L.M., Marinho P., Ainsworth A.M., Baseia I.G., Martín M.P. 2017. More than one fungus in the pepper pot: Integrative taxonomy unmasks hidden species within Myriostoma coliforme (Geastraceae, Basidiomycota). Plos One 12: e0177873. https://doi.org/10.1371/journal.pone.0177873

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RESEARCH ARTICLE More than one fungus in the pepper pot: Integrative taxonomy unmasks hidden

OPEN ACCESS species within Myriostoma coliforme Citation: Sousa JO, Suz LM, Garc´ıa MA, Alfredo (Geastraceae, Basidiomycota) DS, Conrado LM, Marinho P, et al. (2017) More than one fungus in the pepper pot: Integrative Julieth O. Sousa1, Laura M. Suz2, Miguel A. García3, Donis S. Alfredo1, Luana taxonomy unmasks hidden species within M. Conrado4, Paulo Marinho5, A. Martyn Ainsworth2, Iuri G. Baseia6, Maríıa P. Martín7* Myriostoma coliforme (Geastraceae, Basidiomycota). PLoS ONE 12(6): e0177873. 1 Programa de Po´s-Graduac¸ão em Sistema´tica e Evoluc¸ão, Universidade Federal do Rio Grande do Norte, https://doi.org/10.1371/journal.pone.0177873 Natal, Rio Grande do Norte, Brazil, 2 Jodrell Laboratory, Royal Botanic Gardens Kew, Richmond, Surrey, England, 3 Department of Biology, University of Toronto, Mississagua, Ontario, Canada, 4 Graduac¸ão em Editor: Craig Eliot Coleman, Brigham Young Ciências Biolo´gicas, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil, University, UNITED STATES 5 Departamento de Biologia Celular e Gene´tica, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil, 6 Departamento de Botaˆnica e Zoologia, Universidade Federal do Rio Grande do Received: February 12, 2017 Norte, Natal, Rio Grande do Norte, Brazil, 7 Departamento de Micolog´ıa, Real Jard´ın Bota´nico-CSIC, Plaza de

Accepted: May 1, 2017 Murillo 2, Madrid, Spain

Published: June 7, 2017 * [email protected]

Copyright: © 2017 Sousa et al. This is an open Abstract access article distributed under the terms of the Creative Commons Attribution License, which Since the nineteenth century, Myriostoma has been regarded as a monotypic genus with a widespread permits unrestricted use, distribution, and distribution in north temperate and subtropical regions. However, on the basis of morphological reproduction in any medium, provided the original characters and phylogenetic evidence of DNA sequences of the internal transcribed spacer (ITS) author and source are credited. regions and the large subunit nuclear ribosomal RNA gene (LSU), four species are now delimited: M. Data Availability Statement: All relevant data are areolatum comb. & stat. nov., M. calongei sp. nov. M. capillisporum comb. & stat. nov., and M. within the paper; the new sequences generated in this study are already located in GenBank coliforme. Myriostoma coliforme is typified by selecting a lectotype (iconotype) and a modern (Accession Numbers are included in Table 1). sequenced collection as an epitype. The four species can be discriminated by a combination of Funding: The authors would like to express their morphological characters, such as stomatal form, endoperidial surface texture, and basidiospore size thanks to the Coordenac¸ão de Aperfeic¸oamento de and ornamentation. Pessoal de N´ıvel Superior (CAPES—Brazil) for the PhD scholarships awarded to Julieth de Oliveira Sousa and Donis S. Alfredo in 2014/2015 and to Introduction the Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnolo´gico (CNPq—Brazil, Projeto Correct species recognition is an essential requirement for the understanding of systematics, Pesquisador Visitante Especial PVE/407474/2013– 7 evolution and ecology. Furthermore, it is a prerequisite for population biological studies, reli- and Projecto de Pesquisa em Biodiversidade do able Red List assessments and effective conservation action. Recent molecular studies suggest Semiarido PPBio/457476/2012–5; Maríıa P. Martíın that the magnitude of fungal taxonomic diversity is seriously underestimated [1–3]. Basidio- and Iuri G. Baseia) for the scholarship of scientific initiation awarded to Luana Conrado. Natural mycete taxonomy has been revolutionized by the use of molecular techniques, which have England (Species Recovery Programme) is been particularly valuable in revealing component cryptic or semi-cryptic taxa within species thanked for funding part of the sequencing work complexes or aggregates [4–6]. The drawbacks associated with the traditional morphology- carried out in England. only approach are succinctly expressed by Stielow et al. [7]: “The difficulties in defining char- Competing interests: The authors have declared that acters and their states, and particularly the fact that distinct taxonomists assigned distinct no competing interests exist.

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More than one fungus within Myriostoma coliforme weights to morphological characters, have probably been the largest obstacles to the establishment of broadly acceptable classifications of numerous difficult groups of fungi”. Historically, the gasteroid genera Astraeus Morgan and Pisolithus Alb. & Schwein. (Bole- tales) have been regarded as monotypic. However, recent molecular analyses, mainly of the universally adopted DNA barcode region for fungi [8], the internal transcribed spacer region of nuclear ribosomal DNA (ITS), have revealed the presence of previously hidden taxa. Several species have now been described within these genera and several more await formal naming and description [9–12]. The genus Myriostoma Desv., typified by Myriostoma anglicum Desv. (an illegitimate name for M. coliforme) is a very distinct and rare gasteroid genus of the family Geastraceae (Basidio- mycota) which, until now, has been regarded as monotypic. Although M. coliforme (With.) Corda has been adopted as the correct name by some authors [13,14], the correct authorship for the name is M. coliforme (Dicks.) Corda. The single species is considered to have a world- wide distribution [13,15–22]. It is popularly known as pepper pot earthstar, and historically as cullender (colander) puffball, and is considered to be easily recognizable by its combination of unique characters, such as multiple endoperidial stomata (pores) and pedicels (stalks) and basidiospores with a wing-like reticulate ornamentation [21,23], characters that are absent in the earthstar genus Geastrum Pers. Two varieties have been described based on morphological characters: M. coliforme var. capillisporum V.J. Staneˇk from Cape Province, South Africa [24], and M. coliforme var. areolatum Calonge & M. Mata from Costa Rica [16]. Although M. coliforme is considered to be rare across the continent and red-listed in 20 European countries, evidence of large-scale population decline is lacking [22]. Moreover, contributors to the IUCN Global Fungal Red List Initiative (http://iucn.ekoo.se/iucn/species/ 122233/) indicated that populations are increasing in some European countries and likely to be currently underestimated globally. The main aim of the current study is to apply a com- bined molecular and morphological (integrative taxonomic) analysis to specimens identified as M. coliforme to investigate whether the name has been applied to a suite of hidden species as was previously shown to be the case in Astraeus and Pisolithus.

Materials and methods Morphological studies The morphological analyses were performed on specimens, including types, deposited in the Fungal Collection of the Federal University of Rio Grande do Norte (UFRN Herbarium), the collection of fungi of the Real Jard´ın Bota´nico of Madrid (MA-Fungi), the cryptogamy collec- tion (PC) at the Herbarium of the Muse´um national d’Histoire naturelle (MNHN—Paris), and the Fungarium of the Royal Botanic Gardens, Kew (K), (Table 1). Macromorphological studies were based on 26 exsiccates using a Nikon H600L stereomicroscope coupled with a Nikon DS-Ri camera for image capture. Colour descriptions followed Kornerup and Wanscher [25]. For micromorphological features, such as basidiospores, eucapillitium and exoperidial hyphae, a Nikon Eclipse Ni light microscope (LM) coupled with a Nikon DS- Ri camera was used. Basidiospore measurements were made at 1000× magnification following Sousa et al. [21] and include ornamentation. Scanning electron microscopy (SEM) was used to observe the patterns of ornamentation on basidiospores, eucapillitium and endoperidial surfaces.

DNA extraction, PCR amplification and sequencing Genomic DNA was extracted from approximately 10 mg of gleba of mature dry basidiomata. The DNeasyTMPlant Mini Kit (Qiagen, Valencia, CA) was used to isolate DNA from UFRN and MA-Fungi specimens, following the manufacturer’s instructions with the following modifications: glebal masses were macerated in 1.5 ml tubes with a micropestle before suspension in lysis buffer and again after overnight incubation at 55–60˚C. Both ITS and the 5’– 1450–base region of the LSU were analysed using the primer pairs ITS1F/ITS4 [26–27] and LR0R combined with LR7 or LR5 [28–29] respectively. DNA amplifications were carried out using illustraTM PureTaqTM Ready-To-GoTM PCR Beads (Healthcare, Buckinghamshire, UK), adding 1 μl [10 μM] of each primer and 23 μl of isolated DNA [1.5–5.0 ng/μl]. Cycling conditions followed Martín and Winka [30]. PCR products were verified on 1% agarose gels (UtraPureTM Invitrogen), purified using ExoSAP-IT1 (USB Corporation, OH, USA) and sequenced bidirectionally in Macrogen Inc. (Seoul, South Korea). DNA from specimens K(M)138625 and K(M)61641 was extracted using an enzymatic digestion-glass fibre filtration protocol in 96-well plate format with a vacuum-manifold as described in Dentinger et al. [31]. PCR amplifications and sequencing were performed follow- ing Dentinger and Suz [32]. DNA from the rest of the specimens from the Kew Fungarium was extracted and ITS and LSU regions amplified using Extract-N-Amp (Sigma, Dorset, UK). The resulting sequences were edited and the consensus sequence was obtained using Sequencher 5.2.4 (Gene Codes Corp., USA). Preliminary identifications were performed through megablast searches comparing the newly-generated sequences with those in GenBank [33]. Sequences were submitted to GenBank under the accession numbers indicated in Table 1.

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Tabela 7 (Table 1). Specimens and sequences included in this study.

Species Country: Locality Collection Fungarium number GenBank accession year number ITS LSU Myriostoma areolatum comb. & stat. Costa Rica: San Jose´ 1991 MA-Fungi 36165, paratype KY096673 KY096690 nov. Costa Rica: Guanacaste 2005 MA-Fungi 68596, isotype - - Myriostoma calongei sp. nov. Argentina: Colo´n 2012 MA-Fungi 83759 (as M. coliforme), KF988467 KF988348 paratype Brazil: Pernambuco 2006 UFRN-Fungos 386, paratype KY096674 KY096691 Brazil: Pernambuco 2007 UFRN-Fungos 990, paratype KY096675 KY096692 Brazil: R´ıo Grande do Norte 2012 UFRN-Fungos 2019, holotype KY096676 KY096693 Brazil: R´ıo Grande do Norte 2006 UFRN-Fungos 2020, isotype KY096677 KY096694 Myriostoma capillisporum comb. & South Africa: Grahamstown 1930s K(M)205482 (as M. coliforme) KY096678 KY096695 stat. nov. South Africa: Groot River 1930s K(M)205483 (as M. coliforme) KY096679 KY096696 South Africa: Cape of Good pre 1885 K(M)205540 (as M. coliforme) KY096680 KY096697 Hope Myriostoma coliforme Channel Islands: Jersey 1996 K(M)37233 EU784376 KY096698

Channel Islands: Jersey 1999 K(M)61641 KY096681 KY096699

UK: England, East Suffolk 2006 K(M)138625, epitype - KY096700

UK: England, East Suffolk 2010 K(M)166470 KY096682 -

UK: England, East Suffolk 2014 K(M)195584 - KY096701

UK: England, West Norfolk 1880 K(M)81165 KY096683 -

France: Re´gion de Nay 1964 PC 0723885 KY096684 - Hungary: Felsolajos 2003 M. Jeppson 8714* KC582020 KC582020 Portugal: Leiria 1993 MA-Fungi 31316 KY096685 KY096702 Portugal: Madeira Island 2007 MA-Fungi 75818 KY096686 - Russia: Rostov Region 2004 K(M)154620 KY096687 KY096703 Spain: Menorca 1998 MA-Fungi 40288 KY096688 - Spain: Jae´n 2004 MA-Fungi 60898 KY096689 KY096704 Spain: Madrid - JC. Zamora 496* KF988337 KF988466 USA: Hawai‘i - TNS: TKG-GE-50801 JN845203 JN845328 outgroup Geastrum saccatum Sweden - TK950910 KC581968 KC581968 Sweden 2000 GH000909 KC581969 KC581969

New sequences in bold. *Personal Fungarium. https://doi.org/10.1371/journal.pone.0177873.t001

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Sequence alignments and phylogenetic analyses Both ITS and LSU sequences were aligned separately using Se-Al v. 2.0a11 Carbon [34]. To infer phylogenetic relationships among Myriostoma specimens, homologous sequences retrieved from the EMBL/GenBank/DDBJ databases were included in the alignment [35]. Since Geastrum is the sister genus of Myriostoma [36], two sequences of Geastrum saccatum Fr. were included as outgroup. Where ambiguities in the alignment occurred, the alignment generating the fewest potentially informative characters was chosen [37]. Alignment gaps were marked “-”, unresolved nucleotides and unknown sequences were indicated with “N”. Three types of analyses were carried out for ITS and LSU individual alignments and the combined ITS/LSU alignment: maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference. The combined ITS/LSU alignment was submitted to the TreeBASE Number. In the MP analyses, minimum length Fitch trees were constructed using heuristic searches with tree-bisection-reconnection branch swapping, collapsing branches if maximum length was zero, with the MulTrees option in PAUP*4.0b10 [38], and a default setting to stop the analyses when reaching 100 trees. Gaps were treated as missing data. Nonparametric bootstrap (MPbs) support [39] for each clade, based on 10,000 replicates using the fast stepwise-addition, was tested [40]. The consistency index, CI [41], retention index, RI [42], and rescaled consis- tency index, RC [42], were obtained. The ML approach was carried out using RAxML [43] in the CIPRES portal (CIPRES Science Gateway v.3.3) assuming a GTR+I+G model as selected by PAUP*4.0b10; MLbs support for each clade, based on 1,000 replicates was tested. The Bayesian analysis [44–45] was performed using MrBayes 3.2 [46], and assuming the general time reversible model [47], including estimation of invariant sites and assuming a discrete gamma distribution with six categories (GTR+I+G), as selected by PAUPm4.0b10. Two in- dependent and simultaneous analyses starting from different random trees were run for 2.000.000 generations with four parallel chains and trees and model scores saved every 100th generation. The default priors in MrBayes were used in the analysis. Every 1.000th generation tree from the two runs was sampled to measure the similarities between them and to deter- mine the level of convergence of the two runs. The potential scale reduction factor (PSRF) was used as a convergence diagnostic and the first 25% of the trees were discarded as burn-in before stationary was reached. The 50% majority-rule consensus tree and the posterior probability (PP) of the nodes were calculated from the remaining trees with MrBayes. A combination of both bootstrap proportion and PP was used to assess the level of confidence for a specific node [2,48]. The phylogenetic trees were visualized using FigTree v. 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/) and edited with Adobe Illustrator CS3 v. 11.0.2 (Adobe Systems). Moreover, Kimura-2-parameter (K2P) pairwise distances between ITS sequences were obtained using PAUPmVersion 4.0b10, to delimit species following a barcoding approach [8].

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 Nomen- clature for algae, fungi, and plants, and hence the new names contained in the electronic publi- cation 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 follow- ing digital repositories: PubMed Central, LOCKSS and Digital-CSIC.

Results This study generated 32 new Myriostoma sequences (Table 1). The ITS dataset included 24 sequences, 17 generated in this study and seven obtained from EMBL/GenBank/DDBJ data- bases. The alignment resulted in 613 unambiguously aligned nucleotide positions (496 constant, 40 parsimony-uninformative, and 77 parsimony-informative). The 100 most parsimonious trees gave a length of 129 steps, CI = 0.9535, HI = 0.0465, and RC = 0.9688. The ML tree and the 50% Bayesian majority rule combined consensus tree (not shown) showed essentially the same topology as the parsimony strict consensus tree (not shown). Myriostoma sequences were resolved as monophyletic in a highly supported clade (MPbs = 100%, MLbs = 100%, PP = 1.0). The specimen from Costa Rica (MA-Fungi 36165) was sister to the other Myriostoma specimens (MPbs = 100%, Mlbs = 100%, PP = 1.0). The rest of the Myriotoma sequences clustered in two main groups: sequences from Europe and USA (Hawai‘i) formed a highly supported clade (MPbs = 99%, MLbs = 100%, PP = 1.0), whereas sequences from Argentina, Brazil and South Africa grouped together in two subgroups, one including sequences of South Africa [K(M) 205482,

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K(M)205483 and K(M)205540] and the other one with those of Argentina and Brazil. The LSU dataset included 21 sequences, 15 generated in this study and six obtained from sequence databases. The alignment resulted in 1391 unambiguously aligned nucleotide positions (1302 constant, 20 parsimony-uninformative, and 69 parsimony-informative). The 100 most parsimonious trees gave a length of 100 steps, CI = 0.9300, HI = 0.0700 and RC = 0.9533. The ML tree and the 50% Bayesian majority rule combined consensus tree (not shown) showed essentially the same topology as the parsimony strict consensus tree (not shown). In the three analyses, the sequences from South Africa [K(M)205482, K(M)205483 and K(M) 205540] were sister to the other Myriostoma sequences, although this relationship was weakly supported (MPbs = 52%, MLbs = <50%, PP = 0.65). In the parsimony strict consensus tree, the sequence from Costa Rica appeared as the sister group to those from Argentina and Brazil, but this relationship had very low support (MPbs = 61%); moreover, in the ML and Bayesian analyses, the sequence from Costa Rica was the sister group to the clade formed by sequences from Europe and Hawai‘i, a relationship with moderate support (MLbs = 54%, PP = 0.91). In the ITS/LSU combined dataset there were 2004 unambiguously aligned nucleotide posi- tions (1797 constant, 61 parsimony- uninformative and 146 parsimony-informative). The 100 most parsimonious trees gave a length of 235 steps, CI = 0.9234, HI = 0,0766, and RC = 0.9474. The ML tree (not shown) and the 50% Bayesian majority rule combined consensus tree (Fig 1) showed essentially the same topology as the parsimony strict consensus tree (not shown). Two main Myriostoma clades were produced: Clade I which comprised all sequences obtained from specimens from the Southern Hemisphere, in which the three sequences from South Africa grouped together (MPbs = 96%, MLbs = 54%, PP = 0.99) as did those from Argentina and Bra- zil (MPbs = 81%, MLbs = <50%, PP = 0.99); and Clade II which comprised all sequences originating from material collected in the Northern Hemisphere, in which all sequences from Europe and the one from Hawai‘i form a highly supported group (MPbs = 100%, MLbs = 85%, PP = 1.0) separated from the Costa Rica sequence. A revision of the morphological characters present in the analysed material, such as basidio- spore size and shape, together with endoperidial surface texture and stomatal morphology, supports the recognition of four distinct species: in Clade I, M. capillisporum comb. & stat. nov. from South Africa, with basidiospores 7.0–10.9 μm diam with long warts (2.9–6.6 μm high), and a wrinkled to slightly verrucose endoperidial surface; and M. calongei sp. nov. from Argentina and Brazil, with basidiospores 5.6–8.7 μm, verrucose endoperidium with prominent triangular processes (warts 0.13–0.28 mm high); and in Clade II, M. coliforme, with basidiospores 6.1–8.0 μm, with a wrinkled to slightly verrucose endoperidial surface (warts < 0.1 mm high), and M. areolatum comb. & stat. nov. from Costa Rica, with basidiospores 5.6–6.9 μm diam, with a similar endoperidial surface to that of M. coliforme, and M. capillisporum. Furthermore, the K2P pairwise distance of Myriostoma ITS sequences included in Table 1 show high genetic variation between the four species considered (Table 2). There are clearly defined barcoding gaps within the ITS sequences of Myriostoma such that interspecific varia- tion exceeds intraspecific variation [8]. Based on these results, a new species is described and two varieties are elevated to specific rank. No type material of M. coliforme is known [13,15] and as Persoon [49] referred to Dickson’s (not Withering’s) name [50], thereby sanctioning it, Dickson’s illustration is selected as lectotype and a recently sequenced collection from the same English region (East Anglia) is designated as epitype (see below).

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Figura 15 (Fig. 1) The 50% majority-rule consensus tree of ITS/LSU nrDNA sequences of Myriostoma species using a Bayesian approach. Two sequences of Geastrum saccatum were used as outgroup. Terminal branches are labelled with appropriate specimen codes and countries of origin. For further specimen details, see Table 1. Numbers at the nodes indicate the percentage bootstrap values obtained from parsimony and maximum likelihood analysis and the posterior probabilities from Bayesian analysis (MPbs/MLbs/PP). https://doi.org/10.1371/journal.pone.0177873.g001

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Tabela 8 (Table 2). Matrix of pairwise Kimura-2-parameter (K2P) distance between ITS sequences from the four species analysed in this paper.

1 2 3 4 1. Myriostoma areolatum -* 2. M. capillisporum 0.06918 0.00000 3. M. calongei 0.05623 0.01278 0.00227 4. M. coliforme 0.07711 0.03446 0.05219 0.00885

Maximum intraspecific distances in bold; the other values are the minimum interspecific distances. * The intraspecific value for M. areolatum is not given in the table, since only one collection was sequenced. https://doi.org/10.1371/journal.pone.0177873.t002

Taxonomic treatment Key to Myriostoma species

1.Basidiomata with areolate and tubular stomata ...... M. areolatum 1.Basidiomata with non-areolate and non-tubular stomata ...... 2 2.Endoperidial surface strongly verrucose with warts > 0.1 mm high with (SEM) blunt triangular shape ...... M. calongei 2.Endoperidial surface wrinkled to slightly verrucose with warts < 0.1 mm high having (SEM) rounded apices ...... 3

3.Basidiospores (7.0)7.4–10.9 μm diam with prominent ornamentation (2.9–6.6 μm high) comprising (SEM) a reticulum of branching perfo- rated ridges, crests and warts forming arcs and broken circles in face view ...... M. capillisporum 3. Basidiospores 6.1–8.0 μm diam with ornamentation (1.2–1.6 μm high) comprising (SEM) a relatively low simpler reticulum with less curvature in face view ...... M. coliforme

Myriostoma areolatum (Calonge & M. Mata) M.P. Martín, J.O. Sousa & Baseia, comb. & stat. nov.–Fig 2, Mycobank MB 818615 Etymology. Referring to the numerous areolate stomata in the endoperidium. Basionym. Myriostoma coliforme var. areolatum Calonge & Mata, Bol. Soc. Micol. Madrid 30:116 (2006), MB 546689.

Holotype. COSTA RICA, Guanacaste, Parque Nacional Barra Honda, La Capilla, Caverna Pequeña, in soil, 1 Aug. 2005, leg. C. Aguilar 130–05 (USJ 82231!, under Myriostoma coliforme var. areolatum Calonge & M. Mata). Diagnosis. Myriostoma areolatum can be distinguished from other known Myriostoma spe- cies by its tubular (up to 1 mm high) and areolate (up to 4 mm diam.) stomata. This species is very close to M. coliforme, but M. areolatum has smaller basidiospores (5.6–6.9 μm diam). Description. Expanded basidiomata arched, 50–80 mm wide. Exoperidium splitting into 7–9 rays, revolute to horizontal, non- hygroscopic. Endoperidial body 35–45 mm wide, shiny, verrucose. Multiple circular stomata (up to 42) of about 4 mm diam, which have an areolate, tubular and fimbriate peristome (1 mm high, 1mm diam). Endoperidial surface ornamenta- tion comprised of small processes with rounded tips (sub SEM). Eucapillitial hyphae brownish, 2.0–5.0 μm diam, surface smooth or with rounded warts, lumen yellowish. Basidiospores glo- bose to subglobose, 5.6–6.9 μm diam, with an ornament of winged ridges 0.8–1.5 μm high; under SEM, the ornamentation is reticulate, comprising warts and branching ridges with pla- nar and curved apices. Known distribution. Central America (Costa Rica).

Additional specimens examined. COSTA RICA, Guanacaste, Parque Nacional Barra Honda, La Capilla, Caverna Pequeña, in soil, 1 Aug. 2005, leg. C. Aguilar 130–05 (MA-Fungi 68596, under M. coliforme var. areolatum Calonge & M. Mata, isotype); San Jose´, Ciudad Colo´n, Finca “El Rodeo”, 13 Jun. 1991, leg. M.P Nu´ñez (MA-Fungi 36165, under M. coliforme var. are- olatum Calonge & M. Mata, paratype).

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Figura 16 (Fig. 2) Myriostoma areolatum (MA-Fungi 68596, isotype). (a) Dried expanded basidioma ex situ, bar = 10 mm. (b) Stomata, bar = 5 mm. (c) Basidiospores under SEM, bar = 2 μm. (d) Endoperidial surface under SEM, bar = 50 μm. https://doi.org/10.1371/journal.pone.0177873.g002

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Myriostoma calongei Baseia, J.O. Sousa, & M.P. Martín, sp. nov., Fig 3, Mycobank MB 818593. Etymology. In honour of Prof. Francisco Diego Calonge, for his great contribution to the study of gasteroid fungi.

Holotype. BRAZIL, Rio Grande do Norte: Ba´ıa Formosa, RPPN Mata Estrela, 6˚22’31.8”S 35˚ 01’22.4”W, 61 msl, fruiting on leaf litter, under Ficus sp., 15 July 2012, leg. B.D.B. Silva & J.O. Sousa (UFRN-Fungos 2019!). Diagnosis. Myriostoma calongei differs from other Myriostoma species mainly by the verru- cose endoperidium, with prominent triangular processes (warts 0.13–0.28 mm high). It is closely related to M. capillisporum, but M. calongei has smaller basidiospores (5.9–8.7 μm diam) with less prominent ornamentation (1.0–2.3 μm high). Description. Unexpanded basidiomata semi-hypogeous, globose to depressed globose, 22– 30 mm × 18–37 mm, surface brown (6E4), papery, with longitudinal cracks, not encrusted to slightly encrusted with debris. Expanded basidiomata arched to saccate, 15–59 × 23– 120 mm. Exoperidium splitting into 4–8 rays, arched to involute, rolling up under the endoperidial body, semi-hygroscopic to non- hygroscopic. Mycelial layer brown (6E4), dark brown (6F4) to greyish brown (6F3), papery, slightly encrusted to not encrusted with debris, peeling off in lon- gitudinal cracks or in irregular patches. Fibrous layer brownish orange (5C3), white (5A1), orange white (5A2), greyish orange (5B3), coriaceous. Pseudoparenchymatous layer dark brown (6F4; 7F4), brown (6E4; 6E5), persistent or peeling off in irregular patches. Endoperi- dial body greyish brown (6D3), light brown (6D4) to orange grey (6B2), brownish grey (6D2), depressed globose to globose, 9–22 × 15–55 mm, surface slightly metallic and shiny, verrucose, warts 0.13–0.28 mm high. Multiple pedicels (5–13), 1.6–3.6 mm high, concolorous with the endoperidium, laterally compressed. Multiple stomata (3–11), fibrillose, scattered across the surface of the endoperidial body, slightly delimited, non-depressed on the endoperidium, lac- erate with age, 2.5–3.8 mm diam. Gleba brown (6E5) to dark brown (6F3; 6F5). Endoperidial surface with prominent triangular warts, 0.13–0.28 mm high under SEM. Mycelial layer composed of hyaline to brownish hyphae 2.5–5.1 μm diam, thin-walled (0.6–1.1 μm), non-incrusted, lumen not seen. Fibrous layer composed of hyaline sinuous hyphae 3.6–6.5 μm diam, thin-walled (0.4–1.0 μm), lumen not seen. Pseudoparenchymatous layer composed of hyaline to yellowish, thin to thick-walled hyphal cells, pyriform, subglobose to elongated, 20.4–41.1 × 10.5–32.8 μm. Eucapillitium of brownish hyphae 1.6–4.7 μm diam, thick-walled (0.3–0.9 μm), sinuous, encrusted, lumen seen. Basidiospores yellowish, subglo- bose, 5.9–8.7 × 5.6–7.6 μm [x = 6.9 ± 0.6 × 6.6 ± 0.5, Qm = 1.06, n = 120], warts prominent (1.0– 2.3 μm high) under LM; under SEM, the ornamentation is reticulate formed by confluent warts and ridges which are planar or curved when seen in face view. Known distribution. South America (Brazil and Argentina).

Additional specimens examined. ARGENTINA, Colo´n, Ubajay, El Palmar, S. Suaza, next to Allophyllus edulis and Ligustrum lucidum, 31

May 2012, leg. J. Maller & J.C. Zamora (MA- Fungi 83759, paratype). BRAZIL, Rio Grande do Norte, Ba´ıa Formosa, RPPN Mata Estrela, 6˚22’32.1”S 35˚01’21.6”W, 12 Jun. 2006, leg. B.D.B. Silva, J.O. Sousa & A.G. Leite (UFRN-Fungos 2020, isotype); Pernambuco, Bu´ıque, Parque Estadual Vale do Catimbau, 8˚30’22”S 37˚ 19’23”W, fruiting on humid ground, 4 Aug. 2006, leg. J.F. Silva (UFRN- Fungos 386, paratype); Morro do Cachorro, 8˚34’01”S 37˚14’19”W, fruiting on ground under Ziziphus sp., 16 Apr. 2007, leg. T. Ottoni, (UFRN-Fungos 990, paratype). Remarks. Specimens of this new species were identified in Sousa et al. [21] as M. coliforme. In Brazil, this species occurs in two vegetation types with quite different characteristics: Atlan- tic Rain Forest and “Caatinga”. The Atlantic Rain Forest is a “hotspot” of biodiversity compris- ing tropical forest formations, which extend along the east coast of the South American continent, while “Caatinga” is a vegetation type endemic to Brazil found in semiarid regions and specialized for life in a dry climate [51,52,53,54]. According to the specimen label, in Argentina this species was found next to endemic (Allophyllus edulis) and introduced (Ligus- trum lucidum) plants.

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Figura 17 (Fig. 3) Myriostoma calongei. (a) Fresh expanded and unexpanded basidiomata in situ (UFRN-Fungos 2019, holotype), bar = 20 mm. (b) Endoperidial surface (UFRN-Fungos 386, paratype), bar = 1 mm. (c) Stoma (UFRN-Fungos 386, paratype), bar = 2 mm. (d) Basidiospores under LM (UFRN-Fungos 2020, isotype), bar = 10 μm. (e) Capillitium under LM (UFRN-Fungos 2019, holotype), bar = 10 μm. (f) Basidiospores under SEM, bar = 2 μm. (g) Endoperidial surface under SEM (UFRN-Fungos 2019, holotype), bar = 50 μm. https://doi.org/10.1371/journal.pone.0177873.g003

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Figura 18 (Fig. 4) Myriostoma capillisporum. (a–b) Dried expanded basidiomata ex situ (KM205483 and K(M)205482, respectively), bar = 20 mm. (c–d) Basidiospores under LM (K(M)205483), bar = 10 μm. (e) Basidiospores under SEM (K(M)205483), bar = 2.5 μm. (f) Endoperidial surface under SEM (K(M)205483), bar = 50 μm. https://doi.org/10.1371/journal.pone.0177873.g004

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Myriostoma capillisporum (V.J. Staneˇk) L.M. Suz, A.M. Ainsw., Baseia & M.P. Martín, comb. & stat. nov., Fig 4, Mycobank MB 818616. Etymology. Derived from Latin capillus, meaning hair and referring to the prominent hair- like spore ornamentation. Basionym. Myriostoma coliforme var. capillisporum V.J. Staneˇk, in Pila´t (ed.) Flora ČSR B1 –Gasteromycetes: 402 (1958), MB 347330.

Type. SOUTH AFRICA, Cape Province, Belvidere, A.V. Duthie No. 31355 (Herb?). Diagnosis. The basidiospore size, (7.0–)7.4–10.9 μm, and ornamentation comprising prom- inent warts under LM (2.9–6.6 μm high), which under SEM are formed by warts and ridges with confluent tips forming arcs and circles in face view, clearly distinguish this species from the other Myriostoma spp. Etymology. Derived from Latin capillus, meaning hair and referring to the prominent hair- like spore ornamentation. Expanded basidiomata arched 31–37 × 33–68 mm. Exoperidium splitting into 6–7 rays, arched to revolute, rolling up under the basidioma, non-hygroscopic. Pseudoparenchymatous layer ephemeral, absent in some basidiomata. Endoperidial body depressed globose, 11–12 × 26–35 mm (excluding pedicels), slightly verrucose, stalked. Multiple pedicels (6–7), 2.3–2.9 mm high, paler than or concolorous with the endoperidium. Multiple stomata (up to 4), fibrillose, scattered across the surface of the endoperidial body, lacerate with age. Endoperidial surface with irregular processes under SEM. Basidiospores light yellowish, subglobose, (7.0–) 7.4–10.9 μm [x = 8.5 ± 0.8 n = 27], warts prominent (2.9–6.6 μm high) under LM; the ornamentation is reticulate under SEM, formed by warts and ridges with confluent tips, forming arcs and circles in face view. Known distribution. South Africa.

Additional material studied. SOUTH AFRICA, Cape of Good Hope, pre 1885, Mac Owan (ex herb. M.C. Cooke), (K(M)205540, Kew Fungarium); Grahamstown, 1930s, leg. N.J.G. Smith, Smith 334, (K(M)205482, Kew Fungarium); Groot River, 1930s, leg. N.J.G. Smith, Smith 320, (K(M)205483, Kew Fungarium).

Myriostoma coliforme (Dicks.) Corda 1842 [55], Fig 5, Mycobank MB 122233 Etymology. The specific epithet coliforme means colander- or strainer-like, because “colum” means strainer in Latin, referring to the multistomatous endoperidium. Lectotype (designated here). Dickson’s illustration [as Lycoperdon colliforme], in Dickson (1785) Fasc. pl. crypt. brit. (London) 1:24 (Tab. III: Fig 4), reproduced as Fig 6, IF 552745.

Epitype (designated here). UNITED KINGDOM: England. East Suffolk (vice county 25), north of Ipswich, Nat. Grid Ref. TM15, fruiting in sandy soil, 19 February 2006, leg. C. Povey (K(M) 138625!), IF 552746. Supported lectotypification. See above. Basionym. Lycoperdon coliforme Dicks., Fasciculus plantarum cryptogamicarum Britanniae 1:24 (1785), MB 228521. Diagnosis. Myriostoma coliforme is closely related to M. areolatum, but clearly distinguished by the presence of flattened stomata, whereas in M. areolatum the stomata are areolate and tubular. Description. Expanded basidioma arched 38–135 mm wide. Exoperidium splitting into 6–11 rays arched, involute to horizontal, non- hygroscopic. Mycelial layer greyish yellow (3D4), papery, encrusted with debris, squamous or slightly peeling in longitudinal cracks in some basidiomata. Fibrous layer pastel yellow (1A4) to pale yellow (2A2), papery. Pseudopar- enchymatous layer greyish yellow (3C4; 4B4) to light brown (5C6), persistent or peeling in some basidiomata. Endoperidial body pale yellow (2A3), blond (4C4) to yellowish brown (5D4), depressed globose to globose, 18–40 × 24–50 mm (excluding pedicels), surface wrinkled to slightly verrucose (warts < 0.1 mm high with rounded tips), stalked. Multiple pedicels (3– 9), 3–10 mm high, concolorous with the endoperidial body. Multiple stomata (6–24), >30 visible in some Swedish specimens illustrated in Sunhede (1989), fibrillose, flattened, non- delimited, lacerate with age, 1.5– 5 mm diam. Gleba light brown (5E5) to brown (6E7). Endoperidial surface comprised of small processes under SEM. Eucapillitial hyphae brown- ish 3.3–4.0 μm diam, thick-walled (0.9–1.5 μm), sinuous, non-encrusted, lumen seen. Basidio- spores yellowish, subglobose 6.1–8.0 μm [x = 7.0±0.4 n = 60], warts 1.2–1.6 μm high under LM; under SEM, the ornamentation is reticulate, formed by warts and ridges with planar or slightly curved tips in surface view. Known distribution. Europe, North America and Oceania (Hawai’i).

Additional material studied: CHANNEL ISLANDS, Jersey, Les Vaux Cuissin, fruiting on sandy soil, 23 May 1999, leg. B.M. Spooner (K(M)61641, Kew Fungarium); St Ouen’s, fruiting on old sand dunes, Feb. 1996, leg. N. Armstrong (K(M)37233, Kew Fungarium). FRANCE,

64 Pyre´- ne´es-Atlantiques, Re´gion de Nay (Basses Pyre´ne´es), 1964, leg. J. Beller (PC 0723885). HUNGARY, Ba´cs-Kiskun, Lada´nybene, Felsolajos, 8 Sep. 2003, leg. L. Nagy & M. Jeppson (personal her- barium MJ8714; ITS and LSU nrDNA Acc. Number KC582020).

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PORTUGAL, Leiria, Mato das Acacias, 2 Apr. 1983, leg. L. Freire & M. Castro (MA-Fungi 31316); Maderia, Jardines de la Universidad, under Cupressus sp., 8 Aug. 2007, leg. M. Sequeira (MA-Fungi 75818). RUSSIA, Rostov region, Sholokhovsky District, Schebunyaevsky village, fruiting on pasture soil, 20

July 2004, leg. Y. Rebriev 1090 (K(M)154620, Kew Fungarium). SPAIN, Jae´n, Andu´jar, Las Viñas, under Eucalyptus sp. 18 Nov. 2004, leg. F. Jime´nez (MA-Fungi 60898); Balearic Islands, Menorca, Mao´, Sant Antoni, under Quercus ilex, 27 Nov. 1998, leg. B. Mateo (MA-Fungi

40288). UNITED KINGDOM: England, East Suffolk, near Harleston, fruiting on sandy soil, 30 July 2010, leg. N. Mahler (K(M)166470, Kew Fungarium); idem, 18 Nov. 2014, leg. N. Mahler (K (M)195584, Kew Fungarium); England, West Norfolk, Hillington, fruiting on soil, Oct. 1880, leg. P. Hebgin (via Lady Ffolkes) (K(M)81165, Kew Fungarium). Sequences retrieved from GenBank (specimens not studied morphologically). USA, Hawai‘i, TKG-GE-50801 (TNS Herbarium; ITS sequence Acc. Number JN845203; LSU sequence Acc. Number JN845328). Remarks. Although we have not analysed DNA from specimens from the USA, other than those from Hawai‘i, this species is widespread in North America. Descriptions of North American material provided in Coker and Couch [56] and Bates [15] accord with the lectoty- pification proposed here. Specimens were recorded under desert hackberry (Celtis pallida), juniper (Juniperus spp.), mesquite (Prosopis spp.) or cactus species [15]. The presence of M. coliforme on Hawai‘i Island was also recorded in Smith and Ponce de Leo´n [57] and Gilbertson et al. [58], under , an endemic Fabaceae; moreover, Hemmes and Desjar- din [18] collected numerous specimens in Manuka Wayside Park (Hawai‘i Island) under sev- eral introduced and endemic plants. Tejera et al. [59] provided descriptions of specimens identified as M. coliforme from the Canary Islands that are also in accordance with the lectotypified concept. Esqueda-Valle et al. [19,60] recorded M. coliforme in Mexico (Sonora Desert) under Prosopis sp.; however, there are no descriptions to confirm that these authors are referring to the lectotypified concept. In South America, there are several records of M. coliforme, mostly from southeast Brazil [61–62] and from areas of “Caatinga” vegetation in northeast Brazil: under Spondias tuberos [63] and under Ficus sp. [21, 64], With the exception of the specimens in Sousa et al. [21], the South American collections were not subjected to DNA sequence analysis, but based on morphologi- cal characters alone they should be assigned to M. calongei.

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Figura 19 (Fig. 5) Myriostoma coliforme. (a–b) Dried expanded basidiomata ex situ (K(M)138625, epitype), bar 10 mm. (c) Endoperidial surface (PC0723885), bar = 1 mm. (d) Stoma (MJ8714), bar = 1 mm. (e) Basidiospores under LM (K(M)138625, epitype), bar = 10 μm. (f) Capillitium under LM (MJ8714), bar = 10 μm. (g) Basidiospores under SEM (K(M)138625, epitype), bar = 2.5 μm. (h). Endoperidial surface under SEM (K(M)138625, epitype), bar = 100 μm. https://doi.org/10.1371/journal.pone.0177873.g005

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Figura 20 (Fig. 6 )Dickson’s illustration of Myriostoma coliforme (lectotype) published in 1785 as (Tab. III: Fig 4). https://doi.org/10.1371/journal.pone.0177873.g006

Figura 21 (Fig. 7) Distribution map of the Myriostoma specimens included in the phylogenetic analyses of this study (geometric figures in colour). This figure was made using the software Quantum Gis 2.8 (QGIS), a free and open source geographic information system (https://www.qgis.org/es/site/about/index.html). The dark grey areas correspond to the distribution of Myriostoma coliforme s.l. according to http://iucn.ekoo.se/iucn/species_view/122233/ (accessed on 12.01.2017) and Fraiture & Otto [22]. https://doi.org/10.1371/journal.pone.0177873.g007

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Discussion Since the nineteenth century, the genus Myriostoma has been regarded as monotypic. Pegler et al. [14] indicated that M. coliforme is widespread in north temperate and subtropical regions. However, our study reveals that the name M. coliforme has been applied to at least four members of a species complex each of which is well characterized by a combination of morphological characters, of which the stomata, endoperidial surface and spore size and orna- mentation are the most important. Consequently, the distribution of M. coliforme in the original sense has been overestimated (IUCN webpage: http://iucn.ekoo.se/iucn/species_view/ 122233/; [22]; Fig 7). Although further worldwide sampling is clearly required, current DNA- based evidence supports a European and North American range for M. coliforme. A lack of knowledge about dispersal mechanisms coupled with insufficient molecular data on Neotropical fungi have resulted in speculative interpretations of their biogeographic distribution, especially for saprotrophic taxa such as Myriostoma [20,65]. Recent studies demonstrate that fungal species with a cosmopolitan distribution are the exception [66,67]. In general, most names applied to species with an apparent worldwide distribution probably represent species complexes rather than good species [68]. Based on the conclusion of Kasuya et al. [20] regarding the earthstar Geastrum triplex Jungh., which has a similar, bellows-like, spore dispersal mechanism, Myriostoma dispersal capacity is not expected to be very effective over long distances. This work opens new perspectives on this striking genus through the application of integra- tive taxonomy using a combined molecular and morphological approach. Indeed, the production of revised distribution maps of Myriostoma species is an essential prerequisite for ecological studies and robust and reliable IUCN-compliant conservation assessments.

Acknowledgments The authors would like to express their thanks to the Coordenac¸ão de Aperfeic¸oamento de Pessoal de Níıvel Superior (CAPES—Brazil) for the PhD scholarships awarded to Julieth de Oliveira Sousa and Donis S. Alfredo in 2014/2015 and to the Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnolo´gico (CNPq—Brazil, Projeto Pesquisador Visitante Especial PVE/ 407474/2013–7 and Projeto de Pesquisa em Biodiversidade do Semiarido PPBio/457476/ 2012–5; María P. Martíın and Iuri G. Baseia) for the scholarship of scientific initiation awarded to Luana Conrado. Natural England (Species Recovery Programme) is thanked for funding part of the sequencing work carried out in England. The first author would like to thank Bianca Silva and Judcleidson Cavalcante for their help with the Brazilian field expedition. Bart Buyck is thanked for granting permission for specimen loans from the Herbarium of the Muse´um national d’Histoire naturelle (MNHN—Paris), and Mikael Jeppson for loan of the sample MJ8714. Thanks to M. Teresa Telleria and Margarita Dueñas from the Real Jardíın Bota´nico-CSIC (Madrid, Spain) for continuously supporting the study of gasteroid fungi and thanks to Paul M. Kirk (RBG, Kew) for nomenclatural advice. Thanks to Sandra Nogal-Prata for Myriostoma areolatum Fig 2A and 2B. The authors would also like to thank Maríıa Conejero (RBG, Kew) and Yolanda Ruiz (RJB, Madrid) for technical support with the SEM images.

Author Contributions Conceptualization: JOS LMS MAG AMA IGB MPM. Data curation: JOS LMS AMA IGB MPM. Formal analysis: MPM JOS. Funding acquisition: IGB MPM JOS DSA LMS AMA. Investigation: JOS LMS DSA LMC AMA IGB MPM. Methodology: JOS LMS MAG AMA IGB MPM. Project administration: IGB AMA MPM.

Resources: IGB PM AMA MPM. Software: JOS LMS MAG MPM. Supervision: MPM IGB. Validation: MPM JOS LMS AMA MAG IGB. Visualization: JOS LMS DSA LMC AMA IGB MPM. Writing – original draft: JOS LMS AMA IGB MPM. Writing – review & editing: MPM JOS LMS MAG DSA AMA IGB.

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Capítulo V — Strengthening Myriostoma (Geastraceae, Basidiomycota) diversity: Myriostoma australianum sp. nov

Publicado: Sousa J.O., Baseia I.G., Martín M.P. 2019. Strengthening Myriostoma (Geastraceae, Basidiomycota) diversity: Myriostoma australianum sp. nov. Mycoscience 60(2019): 35-30. https://doi.org/10.1016/j.myc.2018.07.003

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Strengthening Myriostoma (Geastraceae, Basidiomycota) diversity: Myriostoma australianum sp. nov.

Julieth O. Sousaa, Iuri G. Baseiab, María P. Martínc*

a Programa de Pós-Graduação em Sistemática e Evolução, Universidade Federal do Rio Grande do Norte, Campus Universitário Natal 59072-970, Brazil b Departamento de Botânica e Zoologia, Universidade Federal do Rio Grande do Norte, Campus Universitário Natal 59072-970, Brazil c Departamento de Micología, Real Jardín Botánico-CSIC, Plaza de Murillo 2, Madrid, Spain

*Corresponding author

María P. Martín

Tel: +34 914203017

Fax: +34 914200157

E-mail: [email protected]

Text: 9 pages; tables: 2; figures: 4

ABSTRACT

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A new species in the genus Myriostoma (Geastraceae, Basidiomycota) is described from Australia. Phylogenetic analyses of the internal transcribed spacer (ITS) and large subunit (LSU) of nuclear ribosomal DNA, as well as morphological data are evidence that the new species, Myriostoma australianum, is closely related to M. capillisporum from South Africa. Additional collections under M. coliforme from Brazil and USA (New Mexico) were analyzed and confirmed as belonging to M. calongei.

Keywords

Distribution; Earthstar; Gasteroid fungi; Systematics; Taxonomy

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The genus Myriostoma Desv. is a rarely-occurring, star-shaped gasteroid fungus. It is very similar in macro morphology to other star-shaped gasteroid genera, such as Geastrum Pers. and Astraeus Morgan, but Myriostoma is clearly distinct mainly by the presence of an endoperidium with several stomata and pedicels, and reticulate basidiospores (Phosri et al. 2014; Sousa et al. 2014, 2017).

Until Sousa et al. (2017) the genus was considered monotypic. Based on morphological features, as well as molecular data (ITS and LSU nrDNA), these authors recognized four species. Sousa et al. (2017) proposed the epitype to Myriostoma coliforme (With.: Pers.) Corda, type species of the genus, and the species M. areolatum (Calonge & M. Mata) M.P. Martín, J.O. Sousa and Baseia from Costa Rica, M. calongei Baseia, J.O. Sousa, and M.P. Martín from Argentina and Brazil, and M. capillisporum (V.J. Staněk) L.M. Suz, A.M. Ainsw., Baseia and M.P. Martín from South Africa. Moreover, according to their data, M. coliforme is restricted to the Northern Hemisphere. However, Rees et al. (2005) and Moore and O’Sullivan (2014) cited this species from Australia.

Therefore, following Sousa et al. (2017), this work aimed to examine more specimens of Myriostoma worldwide, and to investigate whether specimens of M. coliforme from Australia belong to this species or, as we hypothesize, correspond to a new species. Phylogenetic analyses of ITS and LSU nrDNA are presented, along with morphological description, illustrations, and discussion of the new species in relation to the other species of the genus. This work raises the known number of Myriostoma species to five.

The new specimens analyzed were located in four fungus collections: Naturalis (L, Netherlands, Leiden); Universidade Federal do Rio Grande do Sul (ICN, Porto Alegre, Rio Grande do Sul, Brazil,); Universidade Federal de Pernambuco (URM, Recife, Pernambuco, Brazil), and National Herbarium of Victoria (MEL, Melbourne, Victoria, Australia) (Table 1). Macro- and micro-morphological studies followed Sousa et al. (2017); basidiospore measurements were made at 1000×, and include ornamentation. Color descriptions were based on Küppers (2002). Analyses of Scanning Electron Microscopy (SEM) of basidiospores and capillitium were performed with a Hitachi S-3000N microscope.

Genomic DNA was extracted from approximately 10 mg of peridium or gleba from dry basidiomata. The Speedtools Tissue DNA Extraction Kit (Biotools B&M Labs.S.A) was used to isolate DNA based on the manufacturer's instructions with the following modifications: fungal material was macerated in 1.5 mL tubes with glass balls utilizing TissueLyser (Qiagen), and incubated 24–48 h at 56 ºC. PCR amplifications, purifications, sequencing, and alignments followed Sousa et al. (2017). Preliminary identifications were performed through megablast searches (Altschul et al. 1997) comparing the newly-generated sequences with those in GenBank. The new ITS and LSU Myriostoma sequences were compared with homologous sequences from GenBank, mainly

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published in Sousa et al.. (2017) (Table 1), but only from collections with both sequences. The two alignments were optimized visually in MEGA v. 5.2. As in Sousa et al. (2017) three types of analyses were carried out for the combined ITS/LSU alignment, including Geastrum saccatum as outgroup: maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference. The combined ITS/LSU alignment was submitted to the TreeBASE Number; the only modification was related to the ML analyses that were performed using PAUP* v.4.0b10 (Swofford 2003). The ML and the Bayesian analyses were performed assuming the general time reversible model (Rodriguez et al. 1990) including estimation of invariant sites and assuming a discrete gamma distribution with six categories (GTR+I+G), as selected by PAUP* v.4.0b10. A combination of both bootstrap proportions and PP was used to assess the level of confidence for a specific node (Lutzoni et al. 2004; Wilson et al. 2011). The phylogenetic trees were visualized using FigTree v. 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/) and edited with Adobe Illustrator CS3 v. 11.0.2 (Adobe Systems).

Thirty-eight new sequences of Myriostoma are provided (Table 1, in bold). In the MP analysis of 1,590 positions, 1,390 positions were constant, 51 parsimony-uninformative and 138 parsimony-informative; gaps are treated as "missing" character. Parsimony tree scores were identical for all the 100 trees retained: length = 220, consistency index (CI) = 0.9136, retention index (RI) = 0.9637 and homoplasy index = 0.1131. The 100 MP consensus tree (not shown) and the three ML consensus trees (not shown) have topology identical to the 50% Bayesian majority rule combined consensus tree (Fig.1); bootstrap percentages (MPbs and MLbs), as well as the posterior probability (PP) values are indicated in Fig. 1.

The collections ICN 175617, ICN 177080, L 3961249 and URM 31433, previously identified as M. coliforme, generated sequences which grouped into the clade of M. calongei (Fig. 1). The morphology of these exsiccates was revised, and the prominent warts on endoperidium surface, as well as the basidiospore size and ornamentation, confirmed them as belonging to M. calongei. Exsiccates from Leiden, except L 3961249, were confirmed as indicated in the table, as belonging to M. coliforme. However, the sequences obtained from collections from Australia (MEL 2060796, MEL 2091620, MEL 2095275, and MEL 2305388), previously identified as M. coliforme, are grouped in their own well-supported clade (MPbs = 93 %, MLbs = 94%, PP= 0.99), forming a sister group of M. capillisporum specimens. Based on molecular analyses and morphological data, we propose the new species Myriostoma australianum described here.

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Tabela 9 (Tabela 1) Specimens and sequences used to reconstruct the phylogenetic trees. New ITS and LSU sequences are indicated in bold.

Species Locality Year Collection number GenBank accession number

ITS LSU Myriostoma areolatum Costa Rica: San 1991 MA-Fungi 36165, paratype KY096673 KY096690 José Myriostoma australianum Australia: Sydney 1979 MEL 2060796 MG675902 MG675883 sp. nov. (as M. coliforme), Australia: Sydney 1978 MEL 2091620 MG675903 MG675884 (as M. coliforme) Australia: Sydney 1978 MEL 2095275 MG675904 MG675885 (as M. coliforme), Australia: Sydney 2007 MEL 2305388 MG675901 MG675882 (as M. coliforme), holotype Myriostoma calongei Argentina: Colón 2012 MA-Fungi 83759 KF988467 KF988348 (as M. coliforme), paratype Brazil - L 3961249 MG675905 MG675886 (as M. coliforme) Brazil: 2006 UFRN-Fungos 386, paratype KY096674 KY096691 Pernambuco Brazil: 2007 UFRN-Fungos 990, paratype KY096675 KY096692 Pernambuco Brazil: Río 2012 UFRN-Fungos 2019, holotype KY096676 KY096693 Grande do Norte Brazil: Río 2006 UFRN-Fungos 2020, isotype KY096677 KY096694 Grande do Norte Brazil: Rio 2011 ICN 175617 MG675906 MG675887 Grande do Sul (as M. coliforme)

Brazil: Rio 2011 ICN 177080 MG675907 MG675888 Grande do Sul (as M. coliforme)

USA: New 1963 URM 31433 MG675908 MG675889 Mexico (as M. coliforme)

Myriostoma capillisporum South Africa: 1930s K(M)205482 KY096678 KY096695 Grahamstown (as M. coliforme)

South Africa: 1930s K(M)205483 KY096679 KY096696 Groot River (as M. coliforme) South Africa: pre K(M)205540 KY096680 KY096697 Cape of Good 1885 (as M. coliforme) Hope Myriostoma coliforme Austria: 1985 L 3961241 MG675909 MG675890 Niederösterreich

Austria: 1985 L 3961244 MG675910 MG675891 Niederösterreich 127

Austria: 1986 L 3961247 MG675911 MG675892 Bulgaria: 1977 L 3961237 MG675912 MG675893 Channel Islands: 1996 K(M)37233 EU784376 KY096698

Jersey Channel Islands: 1999 K(M)61641 KY096681 KY096699 Jersey

United Kingdom: 2003 L 3961239 MG675913 MG675894 Isle of Jersey France: 1983 L 3961240 MG675914 MG675895 Languedoc- Roussillon

Germany: - L 3961242 MG675915 MG675896 Hungary: Bács- - L 3961243 MG675916 MG675897 Kiskun Hungary: 2003 M. Jeppson 8714 KC582020 KC582020 Felsolajos Netherlands: 1980 L 3961251 MG675917 MG675898 Noord-Holland Portugal: Leiria 1993 MA-Fungi 31316 KY096685 KY096702

Russia: Rostov Region2004 K(M)154620 KY096687 KY096703 Slovakia: 1981 L 3961238 MG675918 MG675899 Spain: Jaén 2004 MA-Fungi 60898 KY096689 KY096704 England: East 2006 K(M)138625, epitype KY096682 KY096700

USA: Hawai‘i - TNS: TKG-GE-50801 JN845203 JN845328 USA: Ohio 1911 L 3961250 MG675919 MG675900 outgroup Geastrum saccatum Sweden - TK950910 KC581968 KC581968 Sweden 2000 GH000909 KC581969 KC581969

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Taxonomy

Myriostoma australianum J.O. Sousa, Baseia, & M.P. Martín, sp. nov. Fig 2, 3

MycoBank no.: MB 823778.

Diagnosis: Myriostoma australianum is closely related to M. capillisporum, but M. australianum has smaller basidiospores 6.7–8.3 μm (x = 7.9) and shorter warts (1.3–3 μm high) than M. capillisporum, which has basidiopsores with 7.4–10.9 μm (x = 8.5) diam. and warts with 2.9–6.6 μm high.

Type: AUSTRALIA, New South Wales, Central Coast, National Park Mort Bay, Balmain, 33º51’15” S 151° 11’01” E, 1 May 2007, leg. Wilson, K.L. 10443 (holotype MEL 2305388, ITS and LSU sequence GenBank, MG675901 and MG675882).

Etymology: In reference to the type locality.

Expanded basidiomata arched, 27–55 × 24–51 mm. Exoperidium splitting into 6–9 rays, mostly arched, rarely involute, rolling up under the endoperidial body, non-hygroscopic. Mycelial layer brown (N80Y50M40), slightly encrusted to not encrusted with debris, peeling off in irregular patches. Pseudoparenchymatous layer brown (N70Y60M40) to dark brown (N80Y70M40), peeling off, rimose or absent. Endoperidial body greyish brown (N30Y20M10 to N60Y20M10), depressed globose to globose, 19–54 mm wide, surface slightly metallic and shiny, verrucose. Multiple pedicels (5–8), 2.8–6.1 mm high, concolorous with the endoperidium, laterally compressed. Multiple stomata (3–5), fibrillose, scattered across the surface of the endoperidial body, non- depressed on the endoperidium, slightly conic, lacerate with age, 1.1–3.4 mm diam. Gleba pulverulent, brown (N50Y50M40).

Mycelial layer composed of yellowish to brownish, thick-walled hyphae (0.5–0.9 µm), 2.7–6.8 µm diam., some sinuous, non-incrusted, lumen not seen. Fibrous layer composed of hyaline sinuous, thick-walled hyphae (0.5–1.0 µm), 3.5–6.3 µm diam., lumen conspicuous. Pseudoparenchimatous layer composed of hyaline to brownish, thick-walled hyphal cells, pyriform, subglobose to oval, 19.1–35.1 × 16.5–29.0 µm. Basidiospores yellowish, subglobose, (6.5) 7.1–8 × (6.3) 6.7 –8.3 μm [x = 7.9 – 0.5 × 7.5 – 0.5, Qm = 1.052, n = 30], warts prominent (1.32–3 μm high) under LM; under SEM, the ornamentation is reticulate formed by warts and ridges with confluent tips, forming arcs and circles in face view.

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Distribution: Australia, Sydney.

Habitat: Specimens from the collection MEL 2305388 were found on garden bed amongst leaf litter and woodchip mulch, next to young eucalypts. Specimens from collection MEL 2091620 were found on ground among leaf litter.

Additional specimens/cultures examined: AUSTRALIA. New South Wales, Central Coast, Royal Botanic Gardens, Sydney, 33º52’ S 151°13’ E, 22 May 1979, Coveny, R. GAC X27 (paratype MEL 2060796, ITS and LSU sequence GenBank, MG675902 and MG675883); Royal Botanic Gardens, Sydney, 33º52’S 151°13’ E, 03 Jun 1978, Coveny, R. F172 (MEL 2091620, ITS and LSU sequence GenBank, MG675903 and MG675884); Royal Botanic Gardens, Sydney, 33º52’52” S 151°13’ E, May 1978, Coveny, R. s.n. (MEL 2095275, ITS and LSU sequence GenBank, MG675904 and MG675885).

Recently, based on integrative taxonomy, we demonstrated the hidden high diversity in the genus Myriostoma (Sousa et al. 2017); however, none specimens from Oceania were included. From this continent, there are records only from Australia, and specimens were identified as M. coliforme (Rees et al. 2005; Moore & O’Sullivan 2014). In our study, after molecular and morphological analyses, specimens analyzed from Australia represent a distinct and new species of this genus, Myriostoma australianum.

The new species is very closely related to M. capillisporum, and the current geographic distribution of these two species (Australia and South Africa) could suggest the follow hypothesis: common origin in the Gondwanan age, then by allopatric speciation, new species were established; although, more collections and loci should be studied before confirming the origin and distribution of Myriostoma species. However, these species are distinct, not only by ITS and LSU sequences, but also by the pedicel height, higher in M. australianum (2.8–6.1 mm high) than in M. capillisporum (2.3–2.9 mm high); and mainly by the size of basidiospores, which have different medians: M. australianum 7.9 ± 0.5 μm, and M. capillisporum 8.5 ± 0.8 μm (Fig. 4; Table 2).

Another species close to M. australianum is M. calongei, but the latter species has distinct ornamentation of basidiospores, formed by confluent warts and ridges, which are planar or curved when seen in face view, and slightly delimited stomata. Myriostoma areolatum is easily differentiated from M. australianum by the morphology of the stomata (tubular and delimited by an areola); whereas M. coliforme differs by its slightly verrucose endoperidium surface, and the greater number of stomata (6–24).

This study confirms that the genus Myriostoma is even more diverse than recently published, and that M. coliforme is restricted to the Northern Hemisphere.

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Tabela 10 (Tabela 2) Comparative table of morphologic characteristics of Myriostoma australianum and M. capillisporum.

M. australianum M. capillisporum Pedicel height 2.8–6.1 mm high 2.3–2.9 mm high Spores size 6.7 –8.3 μm diam. 7.4–10.9 µm diam. Spores media 7.9 μm diam. 8.5 µm diam.

Spores warts 1.32–3 μm high 2.9–6.6 μm high

Acknowledgements

We would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazilian agency) for the four-month doctorate international scholarship in Madrid, Spain for the first author; and to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq—Brazil) for providing the financial support of the “Projeto Pesquisador Visitante Especial” (PVE-407474/2013-7). Also, we are grateful to the fungal collection curators Dr. Nicolien Sol (L), Dr. Leonor Costa Maia (URM), Dr. Mara Rejane Ritter (ICN), and Dr. Nimal Karunajeewa (MEL) for the loans. The authors would like to thank Rhudson Cruz for the drawing; Profa. Marian Glenn for checking the English; Dra. Yolanda Ruiz for her technical assistance with SEM analysis; Dra. Margarita Dueñas, MA-Fungi curator, for helping with the request of loans; and Sandra Nogal-Prata and Lucia Vergara for help in the molecular laboratory of RJB-Madrid.

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Sousa JO, Suz LM, García MA, Alfredo DS, Conrado LM, Marinho P, Ainsworth AM, Baseia IG, Martín MP, 2017. More than one fungus in the pepper pot: Integrative taxonomy unmasks hidden species within Myriostoma coliforme (Geastraceae, Basidiomycota). Plos One 12: e0177873. https://doi.org/10.1371/journal.pone.0177873

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Figura 22 (Fig. 1) Phylogenetic tree, 50% Bayesian majority rule combined consensus tree of ITS and LSU nrDNA. Numbers over branches are parsimony bootstrap (MPbs), maximum likelihood bootstrap (MLbs) values, and posterior probabilities (PP). Voucher numbers, locality and GenBank codes are indicated in Table 1.

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Figura 23 (Fig 2) Myriostoma australianum (holotype MEL 2305388). A: Expanded dried basidiomata B: Stoma in detail. C: Basidiospore (LM). D–E: Basidiospores (SEM). Bars: A 20 mm; B 5 mm; C–E 5 μm.

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Figura 24 (Fig. 3) Myriostoma australianum (holotype MEL 2305388), Exoperidium layers under LM. A. Mycelial layer under LM B. Fibrous layer C. Pseudoparenchimatous layer. Bars: A–C 10 μm.

Figura 25 (Fig. 4) Line drawing of basidiospores. A. Myriostoma australianum (holotype MEL 2305388). B. M. capillisporum (K(M)205483). Bars: A, B 10 μm.

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6. CONCLUSÃO GERAL

Os resultados proporcionados por este trabalho demonstraram o quão subestimada vem sendo a riqueza da família Geastraceae, sobretudo na região Neotropical, confirmando, assim, nossa hipótese: Existem complexos de espécies que subestimam a diversidade de Geastraceae na região Neotropical.

7. CONSIDERAÇÕES FINAIS

A leitura conservadora da taxonomia de Myriostoma levou este gênero a ser considerado monoespecífico durante mais de um século, mesmo com ocorrência em quase todos os continentes do globo e diferenças morfológicas sutis encontradas nos espécimes. A mesma linha conservadora, somada à falta de ferramentas adicionais a taxonomia alfa, também proporcionou sinonimizações dentro do gênero Geastrum, contribuindo com a concepção de complexos de espécies. Os trabalhos de revisão taxonômica em fungos se tornam essenciais no presente momento, devido à revolução que a utilização do barcode nas últimas décadas vem causando na sistemática desses organismos. Entretanto, trabalhos que envolvam grandes táxons ou amplas regiões de ocorrências apresentam alguns entraves que limitam a atuação dos taxonomistas, são eles: burocracia exacerbada para empréstimo de coleções; negativa de autorização para extração de DNA; mau acondicionamento dos espécimes; grandes distâncias geográficas entre o taxonomista e as coleções; altos valores necessários para utilização de métodos de análises moleculares, entre outros. Assim, esta revisão só foi possível devido a uma rede de trabalho (network) proporcionada pelas parcerias dos meus orientadores com pesquisadores de diversas áreas de atuação e provenientes de diferentes áreas do globo. As novas exigências da taxonomia integrativa demandam análises que sobrepõem apenas os dados proporcionados pelos caracteres morfológicos. A ferramenta molecular demonstra ser primordial para a elucidação de complexos de espécies. No entanto, a maioria das coleções tipo de espécies descritas nos séculos anteriores não apresenta condições para extração de DNA, pelo longo tempo de coleta, ou por não apresentarem acondicionamento adequando para conservação do DNA. Em alguns casos, também não estão aptas para análises morfológicas detalhadas. Dessa forma, a eleição de coleções epitipo se torna necessária em diversos casos, tanto para proporcionar informações sobre os dados moleculares das coleções tipos, tanto para descrições morfológicas atuais. Apesar dos oito produtos gerados por este trabalho, as perspectivas apontam para uma expansão desses resultados, sobretudo porque os dados gerados poderão ser “substrato” para análises de bio e filogeografia, além de elucidação de outros complexos de espécies.

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Anexo I — Geastrum pusillipilosum J.O. Sousa, Alfredo, R.J. Ferreira, M.P. Martín & Baseia, sp. nov.

Publicado: Crous, P.W., Wingfield, M.J., Burgess, T.I., Hardy, G.E.St.J., Crane, C., Barrett, S., Cano-Lira, J.F., Leroux, J.J., Thangavel, R., Guarro, J., Stchigel, A.M., Martín, M.P., Alfredo, D.S., Barber, P.A., Barreto, R.W., Baseia, I.G., Cano-Canals, J., Cheewangkoon, R., Ferreira, R.J., Gené, J., Lechat, C., Moreno, G., Roets, F., Shivas, R.G., Sousa, J.O., Tan, Y.P., Wiederhold, N.P., Abell, S.E., Accioly, T., Albizu, J.L., Alves, J.L., Antoniolli, Z.I., Aplin, N., Araújo, J., Arzanlou, M., Bezerra, J.D.P., Bouchara, J.-P., Carlavilla, J.R., Castillo, A., Castroagudín, V.L., Ceresini, P.C., Claridge, G.F., Coelho, G., Coimbra, V.R.M., Costa, L.A., da cunha, K.C., da silva, S.S., Daniel, R., de beer, Z.W., Dueñas, M., Edwards, J., Enwistle, P., Fiuza, P.O., Fournier, J., García, D., Gibertoni, T.B., Giraud, S., Guevara-Suarez, M., Gusmão, L.F.P., Haituk, S., Heykoop, M., Hirooka, Y., Hofmann, T.A., Houbraken, J., Hughes, D.P., Kautmanová, I., Koppel, O., Koukol, O., Larsson, E., Latha, K.P.D., Lee, D.H., Lisboa, D.O., Lisboa, W.S., López-Villalba, Á., Maciel, J.L.N., Manimohan, P., Manjón, J.L., Marincowitz, S., Marney, T.S., Meijer, M., Miller, A.N., Olariaga, I., Paiva, L.M., Piepenbring, M., Poveda-Molero, J.C., Raj, K.N.A., Raja, H.A., Rougeron, A., Salcedo, I., Samadi, R., Santos, T.A.B., Scarlett, K., Seifert, K.A., Shuttleworth, L.A., Silva, G.A., Silva, M., Siqueira, J.P.Z., Souza-Motta, C.M., Stephenson, S.L. 2016. Fungal Planet description sheets: 469–557. Persoonia 37: 218–403. https://doi.org/10.3767/003158516X694499

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RESEARCH ARTICLE Persoonia 37, 2016: 218 – 403 www.ingentaconnect.com/content/nhn/pimj http://dx.doi.org/10.3767/003158516X694499 Fungal Planet description sheets: 469– 557 P.W. Crous1,2, M.J. Wingfield3, T.I. Burgess4, G.E.St.J. Hardy4, C. Crane5, S. Barrett6, J.F. Cano-Lira7, J.J. Le Roux8, R. Thangavel9, J. Guarro7, A.M. Stchigel7, M.P. Martín10, D.S. Alfredo11, P.A. Barber12, R.W. Barreto13, I.G. Baseia14, J. Cano-Canals15, R. Cheewangkoon16, R.J. Ferreira17, J. Gené7, C. Lechat18, G. Moreno19, F. Roets20, R.G. Shivas21, J.O. Sousa14, Y.P. Tan21, N.P. Wiederhold22, S.E. Abell23, T. Accioly14, J.L. Albizu24, J.L. Alves13, Z.I. Antoniolli26, N. Aplin25, J. Araújo27, M. Arzanlou28, J.D.P. Bezerra29, J.-P. Bouchara30, J.R. Carlavilla19, A. Castillo19, V.L. Castroagudín31, P.C. Ceresini31, G.F. Claridge32, G. Coelho33, V.R.M. Coimbra34, L.A. Costa35, K.C. da Cunha36, S.S. da Silva35, R. Daniel37, Z.W. de Beer 2, M. Dueñas10, J. Edwards38, P. Enwistle39, P.O. Fiuza35, J. Fournier 40, D. García7, T.B. Gibertoni34, S. Giraud30, M. Guevara-Suarez7, L.F.P. Gusmão35, S. Haituk16, M. Heykoop19, Y. Hirooka41, T.A. Hofmann42, J. Houbraken1, D.P. Hughes27, I. Kautmanová43, O. Koppel44, O. Koukol45, E. Larsson46, K.P.D. Latha47, D.H. Lee48, D.O. Lisboa13, W.S. Lisboa13, Á. López-Villalba19, J.L.N. Maciel49, P. Manimohan 47, J.L. Manjón19, S. Marincowitz 2, T.S. Marney 21, M. Meijer1, A.N. Miller 50, I. Olariaga 51, L.M. Paiva29, M. Piepenbring 52, J.C. Poveda- Molero53, K.N.A. Raj 47, H.A. Raja54, A. Rougeron30, I. Salcedo51, R. Samadi28, T.A.B. Santos35, K. Scarlett55, K.A. Seifert44, L.A. Shuttleworth37, G.A. Silva29, M. Silva13, J.P.Z. Siqueira7, C.M. Souza-Motta29, S.L. Stephenson56, D.A. Sutton22, N. Tamakeaw16, M.T. Telleria10, N. Valenzuela-Lopez7, A. Viljoen57, C.M. Visagie44, A. Vizzini58, Wartchow59, B.D. Wingfield48, E. Yurchenko60, J.C. Zamora61, J.Z. Groenewald1 Key words Abstract Novel species of fungi described in this study include those from various countries as follows: Australia: Apiognomonia lasiopetali on Lasiopetalum sp., Blastacervulus eucalyptorum on Eucalyptus adesmophloia, Bul- ITS nrDNA barcodes lanockia australis (incl. Bullanockia gen. nov.) on Kingia australis, Caliciopsis eucalypti on Eucalyptus marginata, LSU Celerioriella petrophiles on Petrophile teretifolia, Coleophoma xanthosiae on Xanthosia rotundifolia, Coniothyrium novel fungal species hakeae on Hakea sp., Diatrypella banksiae on Banksia formosa, Disculoides corymbiae on Corymbia calophylla, systematics Elsinoë eelemani on Melaleuca alternifolia, Elsinoë eucalyptigena on Eucalyptus kingsmillii, Elsinoë preissianae on Eucalyptus preissiana, Eucasphaeria rustici on Eucalyptus creta, Hyweljonesia queenslandica (incl. Hyweljonesia gen. nov.) on the cocoon of an unidentified microlepidoptera, Mycodiella eucalypti (incl. Mycodiella gen. nov.) on Eucalyptus diversicolor, Myrtapenidiella sporadicae on Eucalyptus sporadica, Neocrinula xanthorrhoeae (incl. Neocrinula gen. nov.) on Xanthorrhoea sp., Ophiocordyceps nooreniae on dead ant, Phaeosphaeriopsis agava- cearum on Agave sp., Phlogicylindrium mokarei on Eucalyptus sp., Phyllosticta acaciigena on Acacia suaveolens, Pleurophoma acaciae on Acacia glaucoptera, Pyrenochaeta hakeae on Hakea sp., Readeriella lehmannii on Eucalyptus lehmannii, Saccharata banksiae on Banksia grandis, Saccharata daviesiae on Daviesia pachyphylla, Saccharata eucalyptorum on Eucalyptus bigalerita, Saccharata hakeae on Hakea baxteri, Saccharata hakeicola on Hakea victoria, Saccharata lambertiae on Lambertia ericifolia, Saccharata petrophiles on Petrophile sp., Sac- charata petrophilicola on Petrophile fastigiata, Sphaerellopsis hakeae on Hakea sp., and Teichospora kingiae on Kingia australis. Brazil: Adautomilanezia caesalpiniae (incl. Adautomilanezia gen. nov.) on Caesalpina echinata, Arthrophiala arthrospora (incl. Arthrophiala gen. nov.) on Sagittaria montevidensis, Diaporthe caatingaensis (en- dophyte from Tacinga inamoena), Geastrum ishikawae on sandy soil, Geastrum pusillipilosum on soil, Gymnopus pygmaeus on dead leaves and sticks, Inonotus hymenonitens on decayed angiosperm trunk, Pyricularia urashimae on Urochloa brizantha, and Synnemellisia aurantia on Passiflora edulis. Chile: Tubulicrinis australis on Lophosoria quadripinnata. France: Cercophora squamulosa from submerged wood, and Scedosporium cereisporum from fluids of a wastewater treatment plant. : Beltraniella acaciae, Dactylaria acaciae, Rhexodenticula acaciae, Rubikia evansii and Torula acaciae (all on Acacia koa). India: Lepidoderma echinosporum on dead semi-woody stems, and Rhodocybe rubrobrunnea from soil. Iran: Talaromyces kabodanensis from hypersaline soil. La Réunion: Neocordana musarum from leaves of Musa sp. Malaysia: Anungitea eucalyptigena on Eucalyptus grandis  pellita, Camptomeriphila leucaenae (incl. Camptomeriphila gen. nov.) on Leucaena leucocephala, Castanediella communis on Eucalyptus pellita, Eucalyptostroma eucalypti (incl. Eucalyptostroma gen. nov.) on Eucalyptus pellita, Melanco- niella syzygii on Syzygium sp., Mycophilomyces periconiae (incl. Mycophilomyces gen. nov.) as hyperparasite on Periconia on leaves of Albizia falcataria, Synnemadiella eucalypti (incl. Synnemadiella gen. nov.) on Eucalyptus pellita, and Teichospora nephelii on Nephelium lappaceum. Mexico: Aspergillus bicephalus from soil. New Zealand: Aplosporella sophorae on Sophora microphylla, Libertasomyces platani on Platanus sp., Neothyronectria sophorae (incl. Neothyronectria gen. nov.) on Sophora microphylla, Parastagonospora phoenicicola on Phoenix canariensis, Phaeoacremonium pseudopanacis on Pseudopanax crassifolius, Phlyctema phoenicis on Phoenix canariensis, and Pseudoascochyta novae-zelandiae on Cordyline australis. Panama: Chalara panamensis from needle litter of

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Abstract (cont.) Pinus cf. caribaea. South Africa: Exophiala eucalypti on leaves of Eucalyptus sp., Fantasmomyces hyalinus (incl. Fantasmomyces gen. nov.) on Acacia exuvialis, Paracladophialophora carceris (incl. Paracladophialophora gen. nov.) on Aloe sp., and Umthunziomyces hagahagensis (incl. Umthunziomyces gen. nov.) on Mimusops caffra. Spain: Clavaria griseobrunnea on bare ground in Pteridium aquilinum field, Cyathus ibericus on small fallen branches of Pinus halepensis, Gyroporus pseudolacteus in humus of Pinus pinaster, and Pseudoascochyta pratensis (incl. Pseudoascochyta gen. nov.) from soil. Thailand: Neoascochyta adenii on Adenium obesum, and Ochroconis capsici on Capsicum annuum. UK: Fusicolla melogrammae from dead stromata of Melogramma campylosporum on bark of Carpinus betulus. Uruguay: Myrmecridium pulvericola from house dust. USA: Neoscolecobasidium agapanthi (incl. Neoscolecobasidium gen. nov.) on Agapanthus sp., Polyscytalum purgamentum on leaf litter, Pseudopithomyces diversisporus from human toenail, Saksenaea trapezispora from knee wound of a soldier, and Sirococcus quercus from Quercus sp. Morphological and culture characteristics along with DNA barcodes are provided.

Article info Received: 1 October 2016; Accepted: 12 November 2016; Published: 21 December 2016.

1 CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, Fed- eral de Santa Maria, Av. Roraima n°1000, Campus, Bairro Camobi, The Netherlands; corresponding author e-mail: [email protected]. CEP 97105-900, Santa Maria, RS, Brasil. 34 2 Department of Microbiology and Plant Pathology, Forestry and Agricultural Departamento de Micologia, Centro de Ciências Biológicas, Universidade Biotechnology Institute (FABI), University of Pretoria, P. Bag X20, Pretoria Federal de Pernambuco, Av. Prof. Nelson Chaves, s/n, 50670-901 Recife, 0028, South Africa. Pernambuco, Brazil. 3 Forestry and Agricultural Biotechnology Institute (FABI), University of 35 Departamento de Ciências Biológicas, Universidade Estadual de Feira Pretoria, Pretoria 0002, South Africa. de Santana, Av. Transnordestina s/n, Novo Horizonte, 44036-900, Feira de 4 Centre for Phytophthora Science and Management, Murdoch University, Santana, BA, Brazil. 90 South Street, Murdoch, WA 6150, Australia. 36 Dermatology Laboratory (SML), University Hospital of Geneva, Rue 5 Department of Parks and Wildlife, Vegetation Health Service, Locked Bag Gabrielle Perret-Gentil 4, 1205 Genève, Geneva, Switzerland. 104, Bentley Delivery Centre, Bentley, WA 6983, Australia. 37 Elizabeth Macarthur Agricultural Institute, Department of Primary Indus- 6 Department of Parks and Wildlife Albany District, 120 Albany Highway, tries, Private Bag 4008, Narellan 2567, Australia. Albany, WA 6330, Australia. 38 AgriBio Centre for AgriBiosciences, Department of Economic Develop- 7 Mycology Unit, Medical School and IISPV, Universitat Rovira i Virgili (URV), ment, Jobs, Transport and Resources, 5 Ring Road, LaTrobe University, Sant Llorenç 21, 43201 Reus, Tarragona, Spain. Bundoora, Victoria 3083 Australia. 8 Centre for Invasion Biology, Department of Botany & Zoology, Stellenbosch 39 North East Agricultural Services, McLeans Ridges 2480, NSW, Australia. University, Matieland 7602, South Africa. 40 Las Muros, 09420 Rimont, France. 9 Plant Health & Environment Laboratory, Ministry for Primary Industries, 41 Biodiversity (Mycology), Agriculture and Agri-Food Canada, Ottawa, ON, Manatū Ahu Matua, 231 Morrin Road, St Johns, Auckland 1072, P.O. Box K1A 0C6, Canada; Department of Clinical Plant Science, Faculty of Bio- 2095, Auckland 1140, New Zealand. science, Hosei University, 3-7-2 Kajino-cho, Koganei, Tokyo, Japan. 10 Departamento de Micología, Real Jardín Botánico-CSIC, Plaza de Murillo 42 Herbarium UCH, Mycological Research Center (CIMi), Autonomous Uni- 2, 28014 Madrid, Spain. versity of Chiriquí (UNACHI), 0427, David, Chiriquí Province, Panama. 11 Pós-graduação em Sistemática e Evolução, Universidade Federal do Rio 43 Slovak National Museum-Natural History Museum, P.O. Box 13, 810 06 Grande do Norte, Natal, Rio Grande do Norte, Brazil. Bratislava, Slovakia. 12 ArborCarbon, 1 City Farm Place, East Perth, Western Australia, 6004 44 Biodiversity (Mycology), Agriculture and Agri-Food Canada, Ottawa, ON, Australia. K1A 0C6, Canada; Department of Biology, University of Ottawa, 30 Marie- 13 Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, Curie, Ottawa, ON K1N 6N5, Canada. 36570-900, MG, Brazil. 45 Department of Botany, Faculty of Science, Charles University, Benátská 14 Departamento de Botânica e Zoologia, Universidade Federal do Rio 2, CZ-12801, Praha 2, Czech Republic. Grande do Norte, Natal, Rio Grande do Norte, Brazil. 46 Department of Biological and Environmental Sciences, University of 15 I.E.S Gabriel Ferrater i Soler, Ctra. de Montblanc, 5-9, 43206 Reus, Tar- Gothenburg, Box 463, 405 30 Göteborg, Sweden. ragona, Spain. 47 Department of Botany, University of Calicut, Kerala, 673 635, India. 16 Department of Entomology and Plant Pathology, Faculty of Agriculture, 48 Department of Genetics, Forestry and Agricultural Biotechnology Institute Chiang Mai University, Chiang Mai 50200, Thailand. (FABI), University of Pretoria, P. Bag X20, Pretoria 0002, South Africa. 17 Pós-graduação em Biologia de Fungos, Universidade Federal de Per- 49 Brazilian Agriculture Research Corporation-Wheat (EMBRAPA-Trigo), nambuco, Recife, Pernambuco, Brazil. Caixa Postal 3081, Rodovia BR-285 Km 294, 99050-970 Passo Fundo, Rio 18 Ascofrance, 64 route de Chizé, 79360 Villiers en Bois, France. Grande do Sul, Brazil. 19 Departamento de Ciencias de la Vida (Unidad Docente de Botánica), 50 University of Illinois Urbana-Champaign, Illinois Natural History Survey, Universidad de Alcalá, E-28805 Alcalá de Henares, Madrid, Spain. 1816 South Oak Street, Champaign, Illinois, 61820, USA. 20 Department of Conservation Ecology and Entomology, Stellenbosch 51 University of the Basque Country (UPV/EHU), Apdo. 644, E-48080 Bilbao, University, South Africa. Spain. 21 Department of Agriculture and Fisheries, GPO Box 267, Brisbane 4001, 52 Department of Mycology, Cluster for Integrative Fungal Research (IPF), Queensland, Australia. Institute for Ecology, Evolution and Diversity, Goethe University, Max-von- 22 Fungus Testing Laboratory, Department of Pathology, University of Texas Laue-Str. 13, DE-60438 Frankfurt am Main, Germany. Health Science Center, 7703 Floyd Curl Dr., San Antonio, Texas 78229- 53 C/Federico García Lorca 52-1, Riba-roja de Túria, Valencia, Spain. 3900, USA. 54 University of North Carolina, Department of Chemistry and Biochemistry, 23 Australian Tropical Herbarium, James Cook University, PO Box 6811, Greensboro, North Carolina, 27402, USA. Cairns 4870, Queensland, Australia. 55 Faculty of Agriculture and Environment, The University of Sydney, Sydney 24 Aranzadi Society of Sciences, Mycology section, Zorroagagaina 11, P.C. 2006, Australia. 200014, Donostia-San Sebastián, Spain. 56 Department of Biological Sciences, University of Arkansas, Fayetteville, 25 21 Shetland Close, Pound Hill, Crawley, West Sussex RH10 7YZ, England, Arkansas 72701, USA. UK. 57 Department of Plant Pathology, University of Stellenbosch, Private Bag 26 Programa de Pós-graduação em Ciência do Solo, CCR, Universidade X1, Stellenbosch 7602, South Africa. Federal de Santa Maria, Av. Roraima n°1000, Campus, Bairro Camobi, 58 Department of Life Sciences and Systems Biology, University of Torino, CEP 97105-900, Santa Maria, RS, Brasil. Viale P.A. Mattioli 25, I-10125 Torino, Italy. 27 Center of Infectious Disease Dynamics, Millennium Science Complex, 59 University Park Campus, Pennsylvania State University, USA. Departamento de Sistemática e Ecologia, Centro de Ciências Exatas e 28 Plant Protection Department, Faculty of Agriculture, University of Tabriz, da Natureza, Universidade Federal da Paraíba, 58051-900 João Pessoa, P.O. Box 5166614766, Tabriz, Iran. Paraíba, Brazil. 60 29 Departamento de Micologia Prof. Chaves Batista, Universidade Federal Department of Biotechnology, Paleski State University, Dnyaprouskai de Pernambuco, Recife, Brazil. flatylii str. 23, BY-225710, Pinsk, Belarus. 61 30 GEIHP - EA 3142, Université d’Angers, Institut de Biologie en Santé PBH- Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad IRIS CHU, 4 Rue Larrey, 49933 Angers Cedex 9, France. Complutense de Madrid, Madrid, Spain. 31 UNESP-University of São Paulo State, Av. Brasil no. 56, 15385-000, Ilha Solteira, São Paulo, Brazil.32 P.O. Box 529, Gatton 4343, Queensland, Australia. 33 Departamento de Fundamentos da Educação, CCR, Universidade 139

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Acknowledgements Paulo C. Ceresini acknowledges permission for scien- and Jaime de Frutos (Mycological Society of Segovia) and Celestino Gelpi tific activities # 39131-3 from the Brazilian Ministry of Environment (MMA) / (Mycological Society of Extremadura), for sending collections of Gyroporus ‘Chico Mendes’ Institute for Conservation of Biodiversity (ICMBIO). Vanina pseudolacteus; to L. Monje and A. Pueblas of the Department of Drawing L. Castroagudín is supported by a Post-Doctoral research fellowship from and Scientific Photography at the University of Alcalá for their help in prepar- São Paulo Research Foundation – FAPESP / Higher Education Personnel ing the digital photographs; to J. Rejos, curator of the AH herbarium for his Improvement Coordination – CAPES, Brazil (PDJ 2014/25904-2, from 2015 – assistance with the specimens examined in the present study. The survey 2016). Paulo C. Ceresini is supported by a research grant from FAPESP which yielded the material considered herein was supported in part by a grant (2015/10453-8) and a fellowship grant from the Brazilian National Council (DEB-0316284) from the National Science Foundation to the University of for Scientific and Technological Development – CNPq (307295/2015-0). Arkansas. The assistance of Lal Singh in carrying out the fieldwork in India Santiago Català and Carlos Rojo helped by providing the DNA sequences is gratefully acknowledged. K.N. Anil Raj acknowledges support from the of Cyathus ibericus used in this study. The research was on-going while J.C. University Grants Commission (UGC), India, in the form of a Rajiv Gandhi Zamora was a recipient of funding from the Ministerio de Economía y Com- National Fellowship (Grant No. F. 14-2(SC)/2009 (SA-III)). K.P. Deepna Latha petitividad (Juan de la Cierva-formación program, FJCI-2014-19801, Spain). acknowledges support from the Kerala State Council for Science, Technology Christian Lechat and Nick Aplin acknowledge Amy Y. Rossman (Oregon State and Environment (KSCSTE) in the form of a PhD fellowship (Grant No. 001/ University, Corvallis, USA) for her advice and scientific assistance. Jacques FSHP/2011/CSTE), and is also grateful to the Principal Chief Conservator of Fournier, Las Muros, 09320 Rimont, France, is thanked for the material he forests, Kerala State, for granting permission (No. WL10-4937/2012, dated collected. Thiago Accioly and co-workers acknowledge Marian Glenn (Seton 03-10-2013) to collect agarics from the forests of Kerala. Margarita Dueñas Hall University, New Jersey) for the English revision of the Geastrum text. and co-workers acknowledge financial support from the Agreement Endesa Gabriel Moreno and co-workers express their gratitude to Antonio Sánchez and San Ignacio de Huinay Foundations and Consejo Superior de Investiga-

Figura 26. Overview Mucoromycotina and Agaricomycotina phylogeny

Consensus phylogram (50 % majority rule) of 2 394 trees resulting from a Bayesian analysis of the LSU sequence alignment (58 taxa including outgroup; 874 aligned positions; 507 unique site patterns) using MrBayes v. 3.2.6 (Ronquist et al. 2012). Bayesian posterior probabilities (PP) are shown at the nodes and thickened lines represent nodes with PP = 1.00. The scale bar represents the expected changes per site. Families, orders and classes are indicated with coloured blocks to the right of the tree. GenBank accession or Fungal Planet numbers are indicated behind the species names. The tree was rooted to Saccharomyces cerevisiae (GenBank Z73326) and the taxonomic novelties described in this study for which LSU sequence data were available are indicated in bold face. The alignment and tree were deposited in TreeBASE (Submission ID S20202).

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Geastrum pusillipilosum

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Fungal Planet 473 – 21 December 2016 Geastrum pusillipilosum J.O. Sousa, Alfredo, R.J. Ferreira, M.P. Martín & Baseia, sp. nov.

Etymology. Named in reference to reduced size of basidiomata and the G. pusillipilosum. However, it is easily distinguished by its larger hirsute exoperidium. basidiomata (25 – 20 mm wide), subicular base, long, dark- Classification — Geastraceae, Geastrales, Agaricomycetes. brown hairs (1.5 – 3 mm in length) and smaller basid- iospores Unexpanded basidiomata epigeous, globose to subglobose, 3 (2.5 – 3 μm diam) (Baseia & Calonge 2006). Other species in the –10 mm wide, surface densely hairy, short hair (< 1 mm high), genus Geastrum that present an exoperidium with hairs are G. presence of subiculum under some basidiomata, yellowish inpaense and G. albonigrun. However, these species are brown (4C8, 5D5, Kornerup & Wanscher 1978). Expanded distinguished by non-delimited peristome, dark brown basidiomata saccate, 4 – endoperidium and larger basidiomata, 25 – 40 mm and 32 – 40 –17 mm wide. Exoperidium splitting into 5 – 9 revolute, tri- mm wide, respectively (Calonge & Mata 2004, Cabral et al. angular rays, non-hygroscopic. Mycelial layer not encrusted 2014). Geastrum schweinitzii is another species similar to with debris, persistent, pale yellow (colour 3A3), densely hairy, G. pusillipilosum, both having small basidiomata, subiculum, and formed of hyaline to greenish hyphae, 2.5 –7 μm diam, lumen lignicolous habit, but G. schweinitzii differs by not having hairs on evident, hairs formed by brownish, interlaced hyphae, 5.5 – 9 the exoperidium and having smaller basidiospores, up to 5 μm μm diam, thick-walled (0.5 –1.5 μm), lumen evident. Fibrous diam (Cortez et al. 2008, Sousa et al. 2014). The ITS nrDNA (see layer papery, yellowish white (colour 4A2), formed of brown- tree figure in G. ishikawae (FP472)) and LSU sequences of G. ish, sinuous hyphae, 3.5 –7.5 μm diam, thin-walled (< 1 μm), pusillipilosum show that it is a well-delimited species. lumen evident. Pseudoparenchymatous layer persistent, gla- brous, brown (colour 5E6), formed by brownish hyphal cells, subglobose, oval to pyriform, 15 – 34 15 – 31.5 μm, thin-walled (< 1 μm). Endoperidial body globose, 1–7 mm wide, sessile, glabrous, brown (colour 5E4) to greyish brown (colour 5D3). Peristome finally fibrillose, delimited, delimitation dark brown (colour 6F4), concolorous or lighter than endoperidium, up to 1 mm high. Columella elliptical, central, inconspicuous, white (colour 4A1). Mature gleba greyish brown (colour 5F3). Basi- diospores globose, 5 – 6.5 μm diam, dark brown in 5 % KOH, ornamentation densely verrucose, warts columnar, slightly truncated, with planar tips, apiculus reduced. Basidia clavate, pyriform to lageniform, 10.5 –16.5 5 –7.5 μm, 3 – 4 sterigmata, yellowish in 5 % KOH. Eucapillitium 2 – 4.5 μm diam, thin-walled (< 1 μm diam), sinuous, unbranched, surface slightly verrucose, encrusted, lumen not evident, brownish in 5 % KOH. Ecology & Distribution — The specimens present a gre- garious habit and colonise two different types of substrates: leaf-litter and decaying wood. Until now, the distribution of G. pusillipilosum is restricted to Brazil and Argentina. In Bra- zil, this species occurs in the North-east (Paraíba and Ceará States) and South-east (Minas Gerais State) regions. Speci- mens were found in three Conversation Units of the Brazilian Atlantic Rainforest domain (Reserva Biológica Guaribas and Reserva Biológica Mitzi Brandão), and Caatinga domain (Flo- Figura 27. One of the 19 equally most parsimonious trees of ITS resta Nacional do Araripe), in different phytophysiognomies: nrDNA sequences obtained after a heuristic search using tropical montane forest low broadleaf; upland, wet forest SeaView v. 4.6 (Gouy et al. 2010). The two new Geastrum enclaves and coastal tableland. The Atlantic Rainforest is a species described in this issue are marked with rectangles: G. ‘hotspot’ of biodiversity and Caatinga is an endemic vegeta- ishikawae and G. pusillipilosum (see Fungal Planet 473 in this tion formation of Brazil; however these areas are extremely manuscript). The accession numbers from EMBL/GenBank degraded, and these Conservation Units represent a few databases are indicated on the tree. Bootstrap support values remaining of this domain (Galindo-Leal & Câmara 2005, Melo greater than 50 % are indicated on the branches, as well as Santos et al. 2007). Typus. Brazil, Paraíba, Mamanguape, Reserva Biológica Guaribas, alt. 150 m, posterior probabilities obtained after Bayesian analyses. S06°44'28.0" W35°08'23.8", on soil covered by leaf-litter or decay- ing wood, 26 Geastrum elegans was included as outgroup. June 2014, J.O. Sousa et al. (holotype UFRN-Fungos 2315, ITS sequence GenBank KX761175, LSU sequence GenBank KX761176, isotype UFRN-Fungos Figura 28. Colour illustrations. Brazil, Paraíba, Reserva 2316, ITS sequence GenBank KX761179, MycoBank MB812875). Notes — Geastrum pusillipilosum is recognised by its small Biológica Guaribas, field track where the type species was basidiomata (up to 17 mm wide), fibrillose, delimited peristome, collected; a. Basidiomata in situ (UFRN- Fungos 2316, isotype); exoperidium totally covered by short hairs (up to 1 mm in length) b. detail of hairy exoperidium (UFRN-Fungos 2315, holotype); c. and basidiospores 5.0 – 6.8 μm diam, with columnar warts. basidiospores under the light microscope (UFRN-Fungos 2314); The presence of hairs on the exoperidium is a rare feature in d. verrucose basidiospore with columnar warts (UFRN-Fungos the genus Geastrum. One species with this characteristic is G. 2314). Scale bars: a = 2.5 mm; b = 0.5 mm; c = 10 µm; d = 1 µm. hirsutum, which has a morphology closely related to Julieth O. Sousa & Donis S. Alfredo, Pós-graduação em Sistemática e Evolução, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil; e-mail: [email protected] & [email protected] Renato J. Ferreira, Pós-graduação em Biologia de Fungos, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil; e-mail: [email protected] María P. Martín, Departamento de Micología, Real Jardín Botánico-CSIC, Plaza de Murillo 2, 28014 Madrid, Spain; e-mail: [email protected] Iuri G. Baseia, Departamento de Botânica e Zoologia, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil; e-mail: [email protected]

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Anexo II — A remarkable new species of Geastrum with an elongated branched stipe

Publicado: Cabral T., Sousa J.O., Silva B.D.B., Martín M.P., Clement, C.R., Baseia I.G. 2017. A remarkable new species of Geastrum with an elongated branched stipe. Mycoscience 58 (5): 344–350. https://doi.org/10.1016/j.myc.2017.03.004

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A remarkable new species of Geastrum with an elongated branched stipe

Tiara S. Cabrala, Julieth O. Sousab, Bianca D.B. Silvac, María P. Martínd,*, Charles R. Clemente, Iuri G. Baseiaf

a Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva, Instituto de Pesquisa da Amazônia, Manaus 69067- 375, Brazil. b Programa de Pós-Graduação em Sistemática e Evolução, Universidade Federal do Rio Grande do Norte, Campus Universitário, Natal 59072-970, Brazil.

c Departamento de Botânica, Universidade Federal da Bahia, Instituto de Biologia, Ondina, Salvador 40170115, Brazil. d Departamento de Micología, Real Jardín Botánico–CSIC, Plaza de Murillo 2, 28014 Madrid, Spain. e Coordenação de Tecnologia e Inovação, Instituto Nacional de Pesquisas da Amazônia, Manaus 69067-375, Brazil.

f Departamento de Botânica e Zoologia, Universidade Federal do Rio Grande do Norte, Campus Universitário, Natal 59072-970, Brazil.

* Corresponding author:

María P. Martín

Tel: +34 914203017 Fax: +34 914200157 E-mail: [email protected]

Text: 8 pages; table: 1; figures: 4.

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Abstract Based on morphological and molecular analysis, we describe the new species Geastrum verrucoramulosum, discriminated from other species in the section Exareolata mainly by an elongated, verrucose, branched stipe. This new species is currently known from two forest locations in central and southwestern Amazonia. Species description, images, and taxonomic discussion of both morphological and molecular data are provided.

Keywords Geastraceae, Neotropics, Phallomycetidae, Phylogeny, Taxonomy

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The order Geastrales K. Hosaka & Castellano (Hosaka et al. 2006) has been the subject of studies to elucidate the phylogenetic relationships among its genera and species (Hosaka and Castellano 2008; Kasuya et al. 2012; Zamora et al. 2013, 2014). However, the diversity of tropical earthstars is still poorly known. New discoveries and the inclusion of DNA sequences of tropical species are needed, and may modify the current phylogeny. In recent years, intensive fieldwork focusing on gasteroid fungi (Basidiomycota) has been carried out in several Brazilian biomes, including the Semi-arid, Atlantic Rainforest, and Amazon Rainforest, especially focusing on Geastrum species (Cabral et al. 2014b; Sousa et al. 2014a, b). These studies have revealed exceptional new and unusual diversity, totaling 54 species described so far for Brazil (Baseia and Milanez 2002; Baseia and Calonge 2006; Fazolino et al. 2008; da Silva et al. 2013; Cabral et al. 2014a; Sousa et al. 2015). In this study, our goal is to provide new information about the genus Geastrum in the Amazon Rainforest by describing a remarkable new species with an elongated and branched stipe. Specimens were collected during the rainy season of 2012 in the Brazilian Amazon rainforest. One specimen (LABEV 6059) was collected during an ecology field course conducted by the Federal University of Acre (UFAC) and the Research Program on Biodiversity (PPBio), and sent to the first author by the collectors. Descriptive terminology and taxonomy are based on Sunhede (1989), Baseia and Milanez (2002), and da Silva et al. (2013). Color codes follow Kornerup and Wanscher (1978). For light microscopy, free-hand sections were mounted in 5 % (w/v) KOH andthen examined with a Nikon Eclipse Ni light microscope (Nikon Corporation, Tokyo) coupled with a Nikon DS-Ri camera (Nikon Corporation, Tokyo), supported by NIS-Elements AR 4.00.03 software (Nikon Corporation, Tokyo). Thirty randomly selected basidiospores were measured using light microscopy, under the 100× immersion oil objective, with 10× oculars. All measurements include basidiospore ornamentation. Basidiospore abbreviations follow Sousa et al. (2014a): n = number of randomly measured basidiospores; x = mean ± standard deviation of basidiospore diameter and height (including ornamentation); Qm= mean height/width quotient Scanning electron microscopy studies were performed at the Universidade Federal do Rio Grande do Norte (UFRN) with a Philips XL 20 (Philips Company, Amsterdam), in accordance with previously described methods (da Silva et al. 2011). Specimens are deposited in the fungal collection of the INPA and UFRN Herbaria (Manaus and Natal, Brazil). DNA extraction was performed from a small piece of the dried basidioma, following da Silva et al. (2013). Two DNA regions were amplified, the nuclear large subunit rDNA (nuc-LSU) and the mitochondrial ATPase subunit 6 coding region (atp6), using primers developed by Vilgalys and Hester (1990) and Kretzer and Bruns (1999). The PCR fragments were purified with ExoSAP-IT (Affymetrix

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Inc., Thermo Fisher Scientific, Waltham), and sequenced with BigDye™ Terminator Cycle Sequencing Ready Reaction Kit version 3.1 (Applied BiosystemsTM, Thermo Fisher Scientific, Waltham). The nuc-LSU and atp6 sequences generated in this study, and those published by Zamora et al. (2014) and Cabral et al. (2014b), were aligned and manually edited in Geneious R6.1 (Biomatters Ltd., New Zealand), treating each DNA region separately. Two datasets were analyzed. The first was used to determine in which section the new species belongs; preliminary analyses were done including sequences of representatives from all sections (alignment 1), according to Zamora et al. (2014), and using Myriostoma coliforme (Dicks.) Corda as outgroup. In order to delimitate the new species, the second dataset was analyzed using only representatives of the section in which the new sequences belong, and using G. schmidelii Vittad. as outgroup (alignment 2) (Table 1). The GTR substitution model was chosen by MrModelTest 2.3 (Nylander 2004) for both atp6 and nuc-LSU. The two blocks of aligned sequences from each DNA region were concatenated to form one single matrix. This matrix was used to perform maximum parsimony and Bayesian phylogenetic analyses. The maximum parsimony analysis was conducted with PAUP* 4.0 (Swofford 2003). The trees were calculated with a heuristic search, with branch swapping, using the TBR algorithm, with initial trees obtained by stepwise addition of random additional sequences repeated 100 times, and bootstraps of 1000 replicates. The Bayesian analysis was performed with MrBayes v.3.1.2. (Huelsenbeck and Ronquist 2001), where trees were calculated using two different runs with four incrementally heated simultaneous MCMC simulations over 10 million generations for the first analysis and 2 million for the second analysis, with trees sampled at every 1000 generations. To estimate posterior probabilities and calculate the consensus tree, part of the trees was discarded as a burn-in stage observing the average standard deviation of split frequency values. The trees were edited with FigTree (http://tree.bio.ed.ac.uk/software/figtree/). All molecular data can be accessed at TreeBase under ID 19668. Alignment 1 consisted of 133 taxa and 1512 characters (862 nuc-LSU and 649 atp6), among which 508 were parsimony-informative characters. The maximum parsimony and Bayesian analyses resulted in similar phylogenetic trees, where the new species clustered with species from section Exareolata De Toni (trees not shown; see TreeBase ID 19668). In alignment 2, only species from section Exareolata were included, with G. schmidelii as outgroup. The concatenated matrix consisted of 16 taxa and 1496 characters (906 corresponding to nuc-LSU and 589 corresponding to atp6), among which 236 were parsimony-informative characters. We obtained one most parsimonious tree with 543 steps and CI = 0.705, RI = 0.788, RC = 0.555. Both maximum parsimony and Bayesian analyses resulted in similar trees (Fig. 1), where the new species is a sister 147

clade of Geastrum cf. stipitatum [determined in Zamora et al. (2014)], also from Brazilian Amazon Rainforest. Due to the unique LSU and atp6 sequences, as well as unique morphological characters, the new species Geastrum verrucoramulosum is here described.

Taxonomy

Geastrum verrucoramulosum T.S. Cabral, J.O. Sousa, & Baseia, sp. nov. Figs. 2–4. MycoBank no.: MB 817844. Diagnosis: Unexpanded basidioma epigeous, caespitose, surface densely verrucose, developed above a prominent and ramulose stipe (17– 41 mm high). Endoperidium comprised of irregularly arranged hyphae. Peristome not truly plicate, becoming fibrillose with age, not delimited. Columella circular. Basidiospores globose, 3.6–4.5 × 3.6–4.4 μm, verrucose, short warts, columnar, with flattened to rounded apex.

Type: BRAZIL, Amazonas, Manaus, Estação Experimental de Manejo Florestal ZF-2, on clay soil, 17 Mar 2012, leg. D.L. Komura 286 (holotype, INPA 264956), Genbank KX670829 (atp6), KX670831 (nuc-LSU).

Etymology: verrucoramulosum (Lat.), referring to the ramulose stipe and the verrucose surface of the exoperidium.

Unexpanded basidiomata epigeous, caespitose, subglobose, 9–17 mm high (not including stipe) × 6–13 mm wide, surface densely verrucose (warts pyramidal, up to 1.5 mm high), rugose with age, light brown (6D4) to brown (6E4), not encrusted with debris, developed above a prominent stipe, absence of a subiculum. Stipe ramulose, trumpet-like, 17–41 mm high × 4–6 mm wide, surface longitudinal-striated, yellowish brown (5D5) to yellowish (5D4). Expanded basidiomata saccate, 8–15 mm high (not including stipe) × 15–19 mm wide. Exoperidium splitting into 6–7 triangular rays; rays triangular, often involute, non-hygroscopic. Mycelial layer dark brown (7F3), surface rugose, not encrusted with debris, persistent. Fibrous layer yellowish white (4A2), papery. Pseudoparenchymatous layer light brown (6D8) when fresh, becoming brownish gray (8F2) when dry, peeling

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away in irregular patches from the base of the rays, persistent. Endoperidium subglobose, 9–10 mm high × 8–10 mm wide, sessile, apophysis absent, surface glabrous, grayish brown (7F3). Peristome not truly plicate, becoming fibrillose with age, non-delimitated, conic (up to 3 mm high), lighter than the endoperidium. Columella circular, central, 1.5 mm wide, yellowish white (4A1) in cross-section. Gleba pulverulent, grayish brown (8F3). Warts from outer part of peridium composed of thin-walled (< 1 μm) sphaerocysts, subglobose, pyriform to oval, 14.4–30.7 × 13–27.4 μm, brownish. Mycelial layer composed of thin sinuous- walled (< 1 μm) hyphae, 3.8 × 5.7 μm diam, surface not encrusted, lumen evident, hyaline to yellowish. Fibrous layer composed of thin-walled hyphae (< 1 μm), 3.7–6.3 μm diam, surface not encrusted, lumen evident, hyaline. Pseudoparenchymatous layer composed of thick-walled (> 1 μm) hyphal cells, subglobose, oval to pyriform hyphae, 24–52.4 × 21–32.9 μm, brownish. Endoperidium comprised of irregularly arranged hyphae, 2.4–2.6 μm diam. Stipe composed of filamentous, thick walled hyphae (0.5–1.0 μm), 4.7–5.9 μm diam., surface encrusted with amorph material, lumen evident, light brown to yellowish. Eucapillitium 3.8–6.2 μm diam, thin walls (< 1 μm), surface rugulose, strongly encrusted, lumen evident, branched, yellowish brown. Basidia 7.3–32 × 2.8–7.5 μm, clavate, guttulate, 2 sterigmata.

Basidiospores globose, 3.6–4.5 × 3.6–4.4 μm [x = 4.0 ± 0.2 × 4.0 ± 0.2, Qm = 1.01, n = 30], strongly brownish, warts inconspicuous under light microscope, verrucose under SEM; warts short, columnar, with flattened to rounded apex; apiculus inconspicuous. Habitat and distribution: terrestrial, found both in open forest composed mainly of shrubs on white-sand soil (called ‘campinarana’) and in upland dense forest on clay soil. So far the new species is restricted to the Amazon Rainforest.

Additional specimens examined (paratype): BRAZIL, Acre, Mâncio Lima, Santa Bárbara community (Módulo do PPBio, LO I parcela P50), on white-sand soil, 30 Nov 2012, leg. J.C.N. Rosario 25 (LABEV 6059, UFRN-Fungos 2782), Genbank KX670830 (atp6), KX670832 (nuc-LSU).

The molecular phylogenetic analyses placed the new specimens from Amazon Rainforest within the genus Geastrum. Based on the phylogenetic tree and morphological analyses, it can be confirmed that the new species belongs to the section Exareolata. Despite the morphological heterogeneity of this section, G. verrucoramulosum shares characteristics with other species assigned to this section, such as: exoperidium with well-developed stipe, non-delimited peristome, and mycelial layer subdivided into two layers (Zamora et al. 2014). This species is grouped in a clade with other species included by Zamora et al. (2014) in the Exareolata section, such as 149

Geastrum aculeatum B.D.B. Silva & Baseia, and G. echinulatum T.S. Cabral, B.D.B. Silva & Baseia. These species present exoperidium with protruding hypha, non-delimited peristome and saccate basidiomata like G. verrucoramulosum, but they are easily distinguished by the absence of a stipe and the morphology of exoperidium tufts, which in G. verrucoramulosum are warts composed of sphaerocysts. Geastrum verrucoramulosum presents a prominent branched stipe below the basidiomata, making it unique. The presence of the long branched stipe can be considered an autapomorphy within the genus, found only in the new species described here. Furthermore, this species has a range of characteristics that together makes it distinct from the rest of the genus: caespitose growth, epigeal development, non-delimited peristome, saccate basidiomata, sessile endoperidium, verrucose exoperidium and spores with short warts. One species that presents a stipe below the basidiomata is Geastrum stiptatum, described by Solms as 'Geaster stipitatus' (Fischer 1893), distributed throughout Java, the Congo, and Brazil (Lloyd 1907; Dissing and Lange 1962). Another species that also presents a stipe is Geastrum congolense Dissing & Lange (Dissing and Lange 1962). Mainly because stipe development started from a subiculum, Ponce de León (1968) proposed the combination of G. stipitatum and Geastrum congolense in Geastrum schweinitzii var. stipitatum (Solms) P. Ponce de León. Geastrum schweinitzii var. stipitatum presents characteristics in common with G. verrucoramulosum, such as saccate basidiomata, caespitose habit and the presence of pyramidal warts in the external portion of the peridium. However, G. schweinitzii var. stipitatum is different from G. verrucoramulosum by having a less conspicuous unbranched stipe (up to 10 mm in height × up to 3 mm in width), the presence of a subiculum, delimited peristome, and smaller spores (up to 3.5 μm in diam) that are sparsely ornamented (Dissing and Lange 1962; Ponce de León 1968; Calonge and Daniëls 1998). Another species described as having a stipitate exoperidium is Geastrum juruense Henn. (as 'juruensis') described from Amazon Rainforest in 1904, and it is different from G. verrucoramulosum by having a smaller unbranched stipe (up to 10 mm in height), and presence of subiculum and smaller basidiospores (up to 2.5 μm in diam) (Hennings 1904). The caespitose form of development of the basidiomata is a habit observed in G. verrucoramulosum and shared by a restricted group of earthstar species that also occur in tropical regions, such as G. schweinitzii (Berk. & M.A. Curtis) Zeller, G. schweinitzii var. stipitatum and G. hirsutum Baseia & Calonge. These species are differentiated from G. verrucoramulosum by the presence of subiculum, delimited fibrillose peristome, mycelial layer with prominent hyphae (hirsute or tomentose), and absence of a prominent unbranched stipe under the basidiomata (Dissing and Lange 1962; Ponce 1968; Calonge and Daniëls 1998; Baseia and Calonge 2006). The basidiospores found in G. verrucoramulosum present short warts with flattened or rounded apex, inconspicuous under light microscope, very similar to the warts found on the basidiospores of G. hirsutum. Nevertheless, these species are 150

easily separated, for G. hirsutum presents a hairy mycelial layer, delimited peristome, presence of a subiculum, and the absence of a branched stipe under the basidiomata (Baseia and Calonge 2006).

Disclosure The authors declare no conflicts of interest. All work undertaken in this study complies with the current laws of the country where they were performed. Acknowledgments The authors thank Dirce L. Komura and Júlio Nauan Caruta do Rosario for providing the specimens presented in this article, and Ricardo Braga Neto for facilitating the contact between Acre’s collectors and the first author. Thanks to Marian Glenn (Seton Hall University, South Orange, New Jersey, USA) for the English revision. The authors also thank the funding agencies onselho Nacional de Desenvolvimento Científico e Tecnológico (MCTIC/PCI 300775/2016-4, Universal 473422/2012-3 and PVE 407474/2013-7), and Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM 3137/2012).

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Tabela 11 (Table 1) Sequences used in aligment 2. Species names, herbarium vouchers, localities, and Genbank accession numbers.

Species Herbarium Locality nuc-LSU atp6 voucher Geastrum albonigrum UFRN-Fungos 1989 Brazil KJ127019 KJ127015 Geastrum albonigrum MA-Fungi 36140-2 Panama KF988468 KF988738 Geastrum aculeatum UFRN-Fungos 1681 Brazil JQ683661 JQ683668 Geastrum argentinum LSP 48446 Argentina KF988472 KF988742 Geastrum argentinum MA-Fungi 82605 Argentina KF988473 KF988743 Geastrum echinulatum INPA 240001 Brazil JQ683659 JQ683665 Geastrum echinulatum INPA 240005 Brazil JQ683660 JQ683666 Geastrum inpaense INPA 239990 Brazil KJ127017 KJ127013 Geastrum inpaense INPA 255834 Brazil KJ127018 KJ127014 Geastrum rufescens Zamora 274 Spain KF988553 KF988820 Geastrum rufescens Zamora 253 Spain KF988552 KF988819 Geastrum schmidelii UPS F-560805 Sweden KF988565 KF988832 Geastrum schmidelii Zamora 279 Spain KF988564 KF988831 Geastrum cf. stipitatum Zamora 528 Brazil KF988576 - Geastrum verrucoramulosum INPA 264956 Brazil KX670831 KX670829 Geastrum verrucoramulosum LABEV 6059 Brazil KX670832 KX670830

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Figura 29 (Fig. 1) Phylogenetic tree obtained by Bayesian analysis derived from concatenated data (atp6 and nuc- LSU), with representatives of section Exareolata. Codes after species names are herbarium vouchers; in bold the new species Geastrum verrucoramulosum. Numbers on nodes indicate support values (posterior probabilities values above, and percentage of bootstrap below) and the scale bar indicates substitution per site.

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Figura 30 (Fig. 2) Geastrum verrucoramulosum sp. nov., fresh (A, B) and dried (C–F) basidiomata. A: LABEV 6059, paratype (Photo: Wendeson Castro). B: INPA264956, holotype (Photo: D.L. Komura). C–F: UFRN- Fungos 2782, paratype (Photos: Wendeson Castro). C: Exoperidium with densely verrucose surface. D: Non- delimitated peristome. E: Cross-section expanded basidioma. F: Ramulose stipe. Bars: A, B 10 mm; C–E 2 mm; F 5 mm.

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Figura 31 (Fig. 3) Geastrum verrucoramulosum sp. nov., micro-structures of UFRN-Fungos 2782 (paratype) under scanning electron microscope (Photo: Iuri G. Baseia). A, B: Basidiospores. C: Hyphae of endoperidium surface. D: Hyphae of eucapillitium. Bars: A, B 1 µm; C 20 µm; D 2 µm.

Figura 32 (Fig. 4) Geastrum verrucoramulosum sp. nov., micro-structures of stipe (A, B). A: Under 400 × of light microscope. B: Under 1000 × of light microscope. Bars: A 20 μm; B 10 μm.

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Anexo III — Geastrum magnosporum J.O. Sousa, B.D.B. Silva, P. Marinho, M.P. Martín & Baseia, sp. nov.

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RESEARCH ARTICLE Persoonia 40, 2018: 240 – 393 ISSN (Online) 1878-9080 www.ingentaconnect.com/content/nhn/pimj https://doi.org/10.3767/persoonia.2018.40.10

Fungal Planet description sheets: 716 – 784 P.W. Crous1,2, M.J. Wingfield3, T.I. Burgess 4, G.E.St.J. Hardy 4, J. Gené5, J. Guarro5, I.G. Baseia6, D. García5, L.F.P. Gusmão7, C.M. Souza-Motta8,13, R. Thangavel9, S. Adamčík10, A. Barili11, C.W. Barnes12, J.D.P. Bezerra13, J.J. Bordallo14, J.F. Cano-Lira5, R.J.V. de Oliveira13, E. Ercole15, V. Hubka16,17, I. Iturrieta-González5, A. Kubátová16, M.P. Martín18, P.-A. Moreau19, A. Morte14, M.E. Ordoñez11, A. Rodríguez14, A.M. Stchigel5, A. Vizzini15, J. Abdollahzadeh20, V.P. Abreu21, K. Adamčíková22, G.M.R. Albuquerque8, A.V. Alexandrova23,24, E. Álvarez Duarte37, C. Armstrong-Cho25, S. Banniza25, R.N. Barbosa8, J.-M. Bellanger26, J.L. Bezerra13, T.S. Cabral27, M. Caboň10, E. Caicedo11, T. Cantillo7, A.J. Carnegie28, L.T. Carmo7, R.F. Castañeda-Ruiz29, C.R. Clement30, A. Čmoková17, L.B. Conceição7, R.H.S.F. Cruz6, U. Damm31, B.D.B. da Silva32, G.A. da Silva13, R.M.F. da Silva13, A.L.C.M. de A. Santiago13, L.F. de Oliveira34, C.A.F. de Souza13, F. Déniel33, B. Dima35, G. Dong36, J. Edwards38, C.R. Félix39, J. Fournier40, T.B. Gibertoni13, K. Hosaka42, T. Iturriaga43, M. Jadan44, J.-L. Jany33, Ž. Jurjević45, M. Kolařík16,17, I. Kušan44, M.F. Landell39, T.R. Leite Cordeiro13, D.X. Lima13, M. Loizides46, S. Luo36, A.R. Machado13, H. Madrid47, O.M.C. Magalhães13, P. Marinho48, N. Matočec44, A. Mešić44, A.N. Miller 43, O.V. Morozova49, R.P. Neves13, K. Nonaka50, A. Nováková17, N.H. Oberlies51, J.R.C. Oliveira-Filho13, T.G.L. Oliveira13, V. Papp52, O.L. Pereira53, G. Perrone54, S.W. Peterson55, T.H.G. Pham24,41, H.A. Raja51, D.B. Raudabaugh43, J. Řehulka56, E. Rodríguez-Andrade5, M. Saba57, A. Schauflerová58, R.G. Shivas59, G. Simonini 60, J.P.Z. Siqueira5, J.O. Sousa61, V. Stajsic62, T. Svetasheva49,63, Y.P. Tan64, Z. Tkalčec 44, S. Ullah65, P. Valente66, N. Valenzuela-Lopez5,67, 68 34 36 13 1 M. Abrinbana , D.A. Viana Marques , P.T.W. Wong , V. Xavier de Lima , J.Z. Groenewald

Key words Abstract Novel species of fungi described in this study include those from various countries as follows: Australia, ITS nrDNA barcodes Chaetopsina eucalypti on Eucalyptus leaf litter, Colletotrichum cobbittiense from Cordyline stricta  C. australis LSU hybrid, Cyanodermella banksiae on Banksia ericifolia subsp. macrantha, Discosia macrozamiae on Macrozamia new taxa systematics miquelii, Elsinoë banksiigena on Banksia marginata, Elsinoë elaeocarpi on Elaeocarpus sp., Elsinoë leucopogonis on Leucopogon sp., Helminthosporium livistonae on Livistona australis, Idriellomyces eucalypti (incl. Idriellomyces gen. nov.) on Eucalyptus obliqua, Lareunionomyces eucalypti on Eucalyptus sp., Myrotheciomyces corymbiae (incl. Myrotheciomyces gen. nov., Myrotheciomycetaceae fam. nov.), Neolauriomyces eucalypti (incl. Neolauriomyces gen. nov., Neolauriomycetaceae fam. nov.) on Eucalyptus sp., Nullicamyces eucalypti (incl. Nullicamyces gen. nov.) on Eucalyptus leaf litter, Oidiodendron eucalypti on Eucalyptus maidenii, Paracladophialophora cyperacearum (incl. Paracladophialophoraceae fam. nov.) and Periconia cyperacearum on leaves of Cyperaceae, Porodiplodia livi• stonae (incl. Porodiplodia gen. nov., Porodiplodiaceae fam. nov.) on Livistona australis, Sporidesmium melaleucae (incl. Sporidesmiales ord. nov.) on Melaleuca sp., Teratosphaeria sieberi on Eucalyptus sieberi, Thecaphora aus• traliensis in capsules of a variant of Oxalis exilis. Brazil, Aspergillus serratalhadensis from soil, Diaporthe pseudo• inconspicua from Poincianella pyramidalis, Fomitiporella pertenuis on dead wood, Geastrum magnosporum on soil, Marquesius aquaticus (incl. Marquesius gen. nov.) from submerged decaying twig and leaves of unidentified plant, Mastigosporella pigmentata from leaves of Qualea parviflorae, Mucor souzae from soil, Mycocalia aquaphila on decaying wood from tidal detritus, Preussia citrullina as endophyte from leaves of Citrullus lanatus, Queiroziella brasiliensis (incl. Queiroziella gen. nov.) as epiphytic yeast on leaves of Portea leptantha, Quixadomyces cearen• sis (incl. Quixadomyces gen. nov.) on decaying bark, Xylophallus clavatus on rotten wood. Canada, Didymella cari on Carum carvi and Coriandrum sativum. Chile, Araucasphaeria foliorum (incl. Araucasphaeria gen. nov.) on Araucaria araucana, Aspergillus tumidus from soil, Lomentospora valparaisensis from soil. Colombia, Corynespora pseudocassiicola on Byrsonima sp., Eucalyptostroma eucalyptorum on Eucalyptus pellita, Neometulocladospori• ella eucalypti (incl. Neometulocladosporiella gen. nov.) on Eucalyptus grandis  urophylla, Tracylla eucalypti (incl. Tracyllaceae fam. nov., Tracyllalales ord. nov.) on Eucalyptus urophylla. Cyprus, Gyromitra anthracobia (incl. Gyromitra subg. Pseudoverpa) on burned soil. Czech Republic, Lecanicillium restrictum from the surface of the wooden barrel, Lecanicillium testudineum from scales of Trachemys scripta elegans. Ecuador, Entoloma yanacolor and Saproamanita quitensis on soil. France, Lentithecium carbonneanum from submerged decorticated Populus branch. Hungary, Pleuromyces hungaricus (incl. Pleuromyces gen. nov.) from a large Fagus sylvatica log. Iran, Zymoseptoria crescenta on Aegilops triuncialis. Malaysia, Ochroconis musicola on Musa sp. Mexico, Cladosporium michoacanense from soil. New Zealand, Acrodontium metrosideri on Metrosideros excelsa, Polynema podocarpi on Podocarpus totara, Pseudoarthrographis phlogis (incl. Pseudoarthrographis gen. nov.) on Phlox subulata. Nigeria, Coprinopsis afrocinerea on soil. Pakistan, Russula mansehraensis on soil under Pinus roxburghii. Russia, Baoran•

© 2018 Naturalis Biodiversity Center & Westerdijk Fungal Biodiversity Institute You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights.

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Abstract (cont.) gia alexandri on soil in deciduous forests with Quercus mongolica. South Africa, Didymocyrtis brachylaenae on Brachylaena discolor. Spain, Alfaria dactylis from fruit of Phoenix dactylifera, Dothiora infuscans from a blackened wall, Exophiala nidicola from the nest of an unidentified bird, Matsushimaea monilioides from soil, Terfezia morenoi on soil. United Arab Emirates, Tirmania honrubiae on soil. USA, Arxotrichum wyomingense (incl. Arxotrichum gen. nov.) from soil, Hongkongmyces snookiorum from submerged detritus from a fresh water fen, Leratiomyces tesquorum from soil, Talaromyces tabacinus on leaves of Nicotiana tabacum. Vietnam, Afroboletus vietnamensis on soil in an evergreen tropical forest, Colletotrichum condaoense from Ipomoea pes•caprae. Morphological and culture characteristics along with DNA barcodes are provided.

Article info Received: 1 March 2018; Accepted: 10 May 2018; Published: 13 July 2018.

1 Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, 26 CEFE UMR5175, CNRS – Université de Montpellier – Université Paul-Va- The Netherlands; léry Montpellier – EPHE – INSERM, 1919, route de Mende, F-34293 corresponding author e-mail: [email protected]. Montpellier Cedex 5, France. 2 Department of Genetics, Biochemistry and Microbiology, Forestry and 27 Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Agricultural Biotechnology Institute (FABI), University of Pretoria, P. Bag Norte, Brazil. X20, Pretoria 0028, South Africa. 28 Forest Health & Biosecurity, NSW Department of Primary Industries, Level 3 Forestry and Agricultural Biotechnology Institute (FABI), University of 12, 10 Valentine Ave, Parramatta NSW 2150, Locked Bag 5123, Par- Pretoria, Pretoria 0002, South Africa. ramatta NSW 2124, Australia. 4 Centre for Phytophthora Science and Management, Murdoch University, 29 Inst. de Investigaciones Fundamentales en Agricultura Tropical ‘Alejandro 90 South Street, Murdoch, WA 6150, Australia. de Humboldt’, Calle 1 Esq. 2, C.P. 17200, Santiago de Las Vegas, C. 5 Mycology Unit, Medical School and IISPV, Universitat Rovira i Virgili, Sant Habana, Cuba. Llorenç 21, 43201 Reus, Spain. 30 Instituto Nacional de Pesquisas da Amazônia, Manaus, Amazonas, Brazil. 6 Departamento de Botânica e Zoologia, Universidade Federal do Rio 31 Senckenberg Museum of Natural History Görlitz, PF 300 154, 02806 Grande do Norte, Natal, Rio Grande do Norte, Brazil. Görlitz, Germany. 7 Departamento de Ciências Biológicas, Universidade Estadual de Feira de 32 Universidade Federal da Bahia, Salvador, Bahia, Brazil. Santana, Av. Transnordestina s/n, NovoHorizonte, 44036-900, Feira de 33 Université de Brest, EA3882 Laboratoire Universitaire de Biodiversité et Santana, BA, Brazil. Ecologie Microbienne, IBSAM, ESIAB, Technopôle Brest-Iroise, 29280, 8 URM Culture Collection, Universidade Federal de Pernambuco, Recife, Plouzané, France. Brazil. 34 Universidade de Pernambuco- Campus Serra Talhada, Serra Talhada, 9 Plant Health and Environment Laboratory, Ministry for Primary Industries, Brazil. P.O. Box 2095, Auckland 1140, New Zealand. 35 Eötvös Loránd University, Department of Plant Anatomy, Budapest, Hun- 10 Department of Plant Pathology and Mycology, Institute of Forest Ecology gary. Slovak Academy of Sciences Zvolen, Akademická 2, SK-949 01 Nitra, 36 University of Sydney, Plant Breeding Institute, 107 Cobbitty Rd, Cobbitty Slovakia. 2570, New South Wales, Australia. 11 Escuela de Ciencias Biológicas, Pontificia Universidad Católica del 37 Mycology Unit, Biomedical Sciences Institute, University of Chile, Santiago, Ecuador, Av. 12 de octubre 1076 y Roca, Quito, Ecuador. Chile. 12 Instituto Nacional de Investigaciones Agropecuarias, Estación Experimen- 38 Agriculture Victoria, School of Applied Systems Biology, La Trobe Univer- tal Santa Catalina, Panamericana Sur Km 1, Sector Cutuglahua, Pichincha, sity, Bundoora 3083, Victoria, Australia. Ecuador. 39 Instituto de Ciências Biológicas e da Saúde – ICBS, Universidade Federal de 13 Departamento de Micologia Prof. Chaves Batista, Universidade Federal Alagoas, Maceió, Brazil. de Pernambuco, Recife, Brazil. 40 Las Muros, 09420 Rimont, France. 14 Departamento de Biología Vegetal (Botánica), Facultad de Biología, 41 Saint Petersburg State Forestry University, 194021, 5U Institutsky Str., Universidad de Murcia, 30100 Murcia, Spain. Saint Petersburg, Russia. 15 Department of Life Sciences and Systems Biology, University of Torino, 42 National Museum of Nature and Science, Tsukuba, Ibaraki, Japan. Viale P.A. Mattioli 25, I-10125 Torino, Italy. 43 University of Illinois Urbana-Champaign, Illinois Natural History Survey, 16 Department of Botany, Faculty of Science, Charles University, Benátská 1816 South Oak Street, Champaign, Illinois, 61820, USA. 2, 128 01 Prague 2, Czech Republic. 44 Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia. 17 Laboratory of Fungal Genetics and Metabolism, Institute of Microbiology 45 EMSL Analytical, Inc., 200 Route 130 North, Cinnaminson, NJ 08077, of the CAS, v.v.i, Vídeňská 1083, 142 20 Prague 4, Czech Republic. USA. 18 Departamento de Micología, Real Jardín Botánico-CSIC, Plaza de Murillo 46 P.O. Box 58499, 3734 Limassol, Cyprus. 2, 28014 Madrid, Spain. 47 Centro de Genómica y Bioinformática, Facultad de Ciencias, Universidad 19 Université de Lille, Faculté de pharmacie de Lille, EA 4483, F-59000 Lille, Mayor de Chile, Camino La Pirámide 5750, Huechuraba, Santiago, Chile. France. 48 Departamento de Biologia Celular e Genética, Universidade Federal do 20 Department of Plant Protection, Faculty of Agriculture, University of Kur- Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil. distan, P.O. Box 416, Sanandaj, Iran. 49 Komarov Botanical Institute of the Russian Academy of Sciences, 197376, 2 21 Departamento de Microbiologia, Universidade Federal de Viçosa, 36570- Prof. Popov Str., Saint Petersburg, Russia. 900, Viçosa, Minas Gerais, Brazil. 50 Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, 22 Branch for Woody Plants Biology, Institute of Forest Ecology, Slovak Minato-ku, Tokyo 108-8641, Japan. Academy of Sciences Zvolen, Akademická 2, SK-949 01 Nitra, Slovakia. 51 Department of Chemistry and Biochemistry, University of North Carolina, 23 Lomonosov Moscow State University (MSU), Faculty of Biology, 119234, Greensboro, USA. 1, 12 Leninskie Gory Str., Moscow, Russia. 52 Szent István University, Department of Botany, Budapest, Hungary. 24 Joint Russian-Vietnamese Tropical Research and Technological Center, 53 Departamento de Fitopatologia, Universidade Federal de Viçosa, 36570- Hanoi, Vietnam. 900, Viçosa, Minas Gerais, Brazil. 25 Crop Development Centre / Dept. of Plant Sciences, University of Sas- 54 Institute of Sciences of Food Production, CNR, Via Amendola 122/O, katchewan, 51 Campus Drive, Saskatoon S7N 5A8, Canada. 70126 Bari, Italy.

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55 Mycotoxin Prevention and Applied Microbiology Research Unit, Agricultural 62 Royal Botanic Gardens Victoria, Birdwood Avenue, Melbourne 3004, Research Service, U.S. Department of Agriculture, 1815 North University Victoria, Australia. Street, Peoria, IL 61604, USA. 63 Biology and Technologies of Living Systems Department, Tula State Lev 56 Department of Zoology, Silesian Museum, Nádražní okruh 31, 746 01 Tolstoy Pedagogical University, 125 Lenin av., 300026 Tula, Russia Opava, Czech Republic. 64 Plant Pathology Herbarium, Department of Agriculture and Fisheries, 57 Department of Botany, University of Gujrat, Hafiz Hayat campus, Gujrat Dutton Park 4102, Queensland, Australia. 65 50700, Pakistan. Department of Botany, Hazara University, Mansehra, Pakistan. 66 58 Veterinary clinic Fénix, Velehradská 19, 13000 Prague 3, Czech Republic. Departamento de Microbiologia, Imunologia e Parasitologia, Instituto de 59 Centre for Crop Health, University of Southern Queensland, Toowoomba Ciências Básicas e da Saúde, Universidade Federal do Rio Grande do Sul, 4350, Queensland, Australia. Porto Alegre, Brazil. 67 Microbiology Unit, Medical Technology Department, Faculty of Health 60 Via Bell’Aria 8, I-42121 Reggio nell’Emilia, Italy. Science, University of Antofagasta, Av. Universidad de Antofagasta s/n, 61 Pós-graduação em Sistemática e Evolução, Universidade Federal do Rio 02800 Antofagasta, Chile. Grande do Norte, Natal, Rio Grande do Norte, Brazil. 68 Department of Plant Protection, Faculty of Agriculture, Urmia University, P.O. Box 165, Urmia, Iran.

Acknowledgements The study of Olga V. Morozova and Tatiana Yu. University in Opava) and Jitka Koubková (Veterinary Laboratory Labvet) for Svetasheva was carried out within the framework of an institutional research providing photo documentation and material for mycological examinations; project of the Komarov Botanical Institute RAS ‘Biodiversity and spatial struc- Czechoslovak Microscopy Society for support (CSMS scholarship 2016). The ture of fungi and myxomycetes communities in natural and anthropogenic research of V. Hubka was supported by Charles University Research Centre ecosystems’ (АААА-А18-118031290108-6) using equipment of its Core program No. 204069 and the grant of the Czech Ministry of Health (AZV Facility Center ‘Cell and Molecular Technologies in Plant Science’. Alina 17-31269A). Alfredo Vizzini and colleagues thank Jan Holec for administer- V. Alexandrova acknowledges financial support from the Russian Science ing of the loan of European material from PRM herbarium (Prague, Czech Foundation (project N 14-50-00029). Republic). Soňa Jančovičová helped with the line drawings. Jozef Šibík and Daniela de A. Viana Marques acknowledges Universidade de Pernambuco David Cooper are acknowledged for the support during the field collections for financial support. Jan Borovička is thanked for providing the Portuguese in Colorado (USA) that was financed by the Slovak American Foundation. collection of Baorangia emileorum and its ITS and LSU sequences, and Ales- The sequencing of samples was funded by the Slovak national project Vega sia Tatti for sending the Sardinian collections of B. emileorum. Taimy Cantillo, 02/0018/18. Željko Jurjević acknowledges Filomena Epifani and Sammy Luis F.P. Gusmão, Luana T. do Carmo, Lucas B. Conceição, Julieth O. Sousa, Sedky for their excellent technical support. Malka Saba acknowledges the Luiz F. de Oliveira, Renan N. Barbosa, Rhudson H.S.F. Cruz, André L.C.M. Higher Education Commission (HEC), Islamabad, Pakistan, for financial de A. Santiago, Carlos A.F. de Souza, Diogo X. Lima, Rafael J.V. de Oliveira assistance during field trips in Pakistan and the Slovak national project and Thalline R.L. Cordeiro, Olinto L. Pereira, Rejane M.F. Silva, Rafael J.V. APVV-15-0210 for sequencing of Russula mansehraensis. The research of Oliveira, José L. Bezerra, Gladstone A. Silva Ciro R. Félix, Melissa F. Landell, Alena Nováková and Miroslav Kolařík was supported by the Ministry of Thays G.L. Oliveira, Jadson D.P. Bezerra, Alexandre R. Machado, Cristina M. Education, Youth and Sports of the Czech Republic (grant number LO1509). Souza-Motta and Oliane M. C. Magalhães, Tatiana B. Gibertoni, Vitor Xavier Asunción Morte, Juan Julián Bordallo and Antonio Rodríguez were supported de Lima and José R. C. Oliveira-Filho acknowledge financial support and/or by projects 19484/PI/14 (FEDER and Fundación Séneca - Agencia de Ciencia y scholarships from the Coordenação de Aperfeiçoamento de Pessoal de Nível Tecnología de la Región de Murcia, Spain) and CGL2016-78946-R (AEI Superior (CAPES), the Conselho Nacional do Desenvolvimento Científico e and FEDER, UE); they also thank Aurelio Garcia Blanco, Andries Gouws, Tecnológico (CNPq) and the Fundação de Amparo à Ciência e Tecnologia de Tom de Wet, Ali Hassan and Faisal Abdullab for their observations and as- Pernambuco (FACEPE); the Fundação de Amparo à Pesquisa do Estado de sistance with field work. Daniel B. Raudabaugh and colleagues thank the Minas Gerais (FAPEMIG), the Instituto Chico Mendes de Conservação da Commonwealth of Pennsylvania, Pennsylvania Department of Conservation Biodiversidade (ICMBio), Parque Memorial Zumbi dos Palmares and Usina and Natural Resources, Pennsylvania Bureau of State Parks, and Black Caeté – Grupo Carlos Lyra and Nordesta AS for suport during field trips. Moshannon State Park for research support, the Mycological Society of Maria E. Ordoñez and colleagues acknowledge the Secretaria de Educación America and University of Illinois Urbana-Champaign School of Integrative Superior, Ciencia, Tecnología e Innovación del Ecuador (SENESCYT), Arca Biology for financial support, and Michael Woodley for field support. Cheryl de Noé Initiative, and the Pontificia Universidad Católica del Ecuador, project Armstrong-Cho and Sabine Banniza acknowledge funding and support by N13415 for financial support. Hugo Madrid was partially funded by Comisión the Saskatchewan Ministry of Agriculture, the Western Grains Research Nacional de Investigación Científica y Tecnológica (CONICYT), Fondo Na- Foundation, the Herb, Spice and Specialty Agriculture Association and the cional de Desarrollo Científico y Tecnológico (FONDECYT), Chile, project Saskatchewan Crop Insurance Corporation. Shuming Luo and colleagues no. 11140562. Vit Hubka and colleagues express their gratitude to Marek thank Mui-keng Tan for helpful advice during this study. Kiecoň, Pavel Malík and Tereza Krasnokutská (National Heritage Institute) for providing information on archaeological research; Hana Rajhelová (Silesian The USDA is an equal opportunity provider and employer.

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Figura 33. Overview Mucoromycotina and Basidiomycota phylogenyConsensus phylogram (50 % majority rule) of 57 752 trees resulting from a Bayesian analysis of the LSU sequence alignment (118 taxa including outgroup; 862 aligned positions; 551 unique site patterns) using MrBayes v. 3.2.6 (Ronquist et al. 2012). Bayesian posterior probabilities (PP) > 0.84 are shown at the nodes and thickened lines represent nodes with PP = 1.00. The scale bar represents the expected changes per site. Families, orders, classes, subdivisions and phyla are indicated with coloured blocks to the right of the tree. GenBank accession and/or Fungal Planet numbers are indicated behind the species names. The tree was rooted to Phytophthora moyootj (GenBank KP004499.1) and the taxonomic novelties described in this study for which LSU sequence data were available are indicated in bold face. The alignment and tree were deposited in TreeBASE (Submission ID S22745).

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Geastrum magnosporum

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Fungal Planet 731 – 13 July 2018 Geastrum magnosporum J.O. Sousa, B.D.B. Silva, P. Marinho, M.P. Martín & Baseia, sp. nov.

Etymology. Referring to the size of basidiospores, being larger than the mean hygroscopic rays, a sessile endoperidium and smaller basidio- spores size in the genus Geastrum. (up to 7 μm diam) (Sunhede 1989, Calonge 1998). An- other similar species is G. arenarium, although, the latter differs in its well-delimited Classification — Geastraceae, Geastrales, Agaricomycetes. peristome, hygroscopic rays and smaller basidiospores (up to 4 μm diam) (Bates 2004). Geastrum hi• eronymi and G. minimum also Unexpanded basidioma hypogeous, orange white (5A2; Kor- nerup & resemble G. magnosporum, but these two species have a longer Wanscher 1978), subglobose, 7  6 mm, surface papery to cottony, pedicel (up to 3 mm long) and smaller basidiospores (up to 5 μm and strongly encrusted with sand. Expanded basi• diomata, arched, rarely 6.5 μm, respectively) (Bates 2004, Kuhar et al. 2012). Other species saccate, 6 –16 mm (including peri- stome)  10 –19 mm. Exoperidium with large basidiospores in the genus are G. laevisporum (up to 10 splitting into 6 – 8 rays, arched, revolute, some involute, rolling up under μm diam), G. campestre (up to 8 μm diam) and G. platense (up to 8 endoperidial body, non-hygroscopic. Mycelial layer yellowish white μm diam). Geastrum laevisporum is distinct due to its smooth (4A2), surface papery to cottony, strongly encrusted with sand and basidiospores and hygroscopic rays; G. campestre in the plicate debris, persistent or peeling away in irregular patches, com- posed of peristome and verrucose endoperidium; and G. platense in the larger yellowish, thin-walled (< 1 μm) hyphae, 2 – 2.5 μm diam, surface not basidiomata (up to 26 mm wide), hygroscopic rays and sessile encrusted, lumen not seen. Fibrous layer orange white (5A2), surface endoperidium (Sunhede 1989, Soto & Wright 2000, Bates 2004, coriaceous, composed of hyaline, thick-walled hyphae (> 1 μm), surface Sousa et al. 2015). encrusted, lumen seen. Pseudoparenquimatous layer, dark brown (7F4, 6F4), rimose, absent in some basidiomata, composed of brownish, thick- walled (> 1 μm) hyphae cells, subglobose, pyriform to ovoid, 30.5 – 63  27– 46.5 μm. Endoperidial body orange grey (6B2), depressed- globose to subglobose, 3 – 5  6 – 9 mm, subsessile, surface furfuraceous. Apophysis absent or inconspicuous. Pedicel absent or very short (up to 0.6 mm high). Peristome fibrillose, lacerate with age, non-delimited to weakly delimited, mammiform to flattened (< 1 mm high), lighter or concolorous with endoperidium. Columella circular, central, white (4A1). Mature gleba greyish brown (5F3). Eucapillitium brownish, thin- walled (< 1 μm diam), 2 – 5 μm diam, surface encrusted, warts absent, lumen seen, branch absent. Basidia clavate to pyriform, 19 – 24.5  8.8 – 6.3 μm, 2 – 3 sterigmata. Basidiospores brown- ish to yellowish in 5 % KOH, globose to subglobose, 6 – 8.5 μm (x = 6.8 ± 0.7, Qm = 1.02, n = 30), densely verrucose, warts long (up to 1.3 μm high), truncate; apiculous reduced. Ecology & Distribution — The specimens were found in the biome Figura 35. The first of three equally most parsimonious trees of the ITS Atlantic Rainforest (Tropical & Subtropical Moist Broad- leaf Forests of nrDNA sequence alignment were obtained from a heuristic search. The Brazil – Pernambuco interior forests ecoregion) (Dinerstein et al. 2017), analysis was conducted with PAUP v. 4.0b10 (Swof- ford 2003) with 10 000 growing on sandy soil, without forest cover (exposed to sun), with bootstrap replicates. The new Geastrum species described here are marked with a coloured box. The accession numbers from EMBL/GenBank gregarious or solitary habit. databases are indi- cated on the tree. Bootstrap support values greater Typus. Brazil, Paraíba, Mamanguape, Reserva Biológica Guaribas, than 50 % for Parsimony and Maximum-Likelihood (ML) are indicated on S6°44'32.1" W35°08'25.8", on sandy soil, 26 June 2014, J.O. Sousa et al. (holotype UFRN Fungos–2312, ITS and LSU sequences GenBank the branches. ML analysis was run with RAxML-HPC2 v. 8.2.10 (Stamatakis MG938496 and MG938497, MycoBank MB824254). 2014) under a GTR model. Geastrum fornicatum was included as outgroup. CorelDRAW ® X8 software was used to edit the final tree. Figura 34. Colour illustrations. Brazil, Paraíba, Reserva Biológica Guaribas, Julieth O. Sousa, Pós-graduação em Sistemática e Evolução, SEMA II, open area of Atlantic rainforest where the type species was Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do collected; expanded basidiomata in situ (UFRN – Fungos 2312, holotype); Norte, Brazil;e-mail: [email protected] expanded basidiomata ex situ (UFRN – Fungos 2312, holotype); basidiospores Bianca D.B. da Silva, Universidade Federal da Bahia, Salvador, Bahia, under LM; basidiospores under SEM; eucapillitium under SEM. Scale bars = Brazil; e-mail: [email protected] 2.5 mm (basidiomata in situ), 2 mm (basidiomata ex situ), 10 μm (basidiospores Paulo Marinho, Departamento de Biologia Celular e Genética, Universidade under LM), 1 μm (basidiospores and eucapillitium under SEM).Additional Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil;e-mail: material examined. Brazil, Paraíba, Mamanguape, Reserva Biológica [email protected] Guaribas, 11 July 2013, J.O. Sousa et al., UFRN Fungos– 2309; ibid., 27 July María P. Martín, Departamento de Micología, Real Jardín Botánico-CSIC, 2012, B.D.B. Silva et al., paratype UFRN Fungos– 2310, ITS and LSU Plaza de Murillo 2, 28014 Madrid, Spain; e-mail: [email protected] sequences GenBank MG938498 and MG938499. Iuri G. Baseia, Departamento de Botânica e Zoologia, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil Notes — Geastrum magnosporum is morphologically close to Geastrum floriforme. However, G. floriforme has strongly 164