Lycoperdon Pers

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE BIOCIÊNCIAS PROGRAMA DE PÓS-GRADUAÇÃO EM SISTEMÁTICA E EVOLUÇÃO

REVISÃO DO GÊNERO LYCOPERDON PERS. (LYCOPERDACEAE, AGARICALES) MEDIANTE ANÁLISES MORFOLÓGICAS E MOLECULARES

DÔNIS DA SILVA ALFREDO ______Tese de Doutorado Natal/RN, março de 2017 DÔNIS DA SILVA ALFREDO

REVISÃO DO GÊNERO LYCOPERDON PERS. (LYCOPERDACEAE, AGARICALES) MEDIANTE ANÁLISES MORFOLÓGICAS E MOLECULARES

Tese de doutorado apresentada ao

Programa de Pós-Graduação em Sistemática e Evolução da Universidade Federal do Rio Grande do Norte, em cumprimento às exigências para a obtenção do título de Doutor em Sistemática e Evolução

Área de concentração: taxonomia e sistemática Orientador: Dr. Iuri Goulart Baseia

Coorientadora: Dra. María Paz Martín Coorientador: Dr. Paulo Marinho

NATAL – RN 2017 0

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

Alfredo, Dônis da Silva. Revisão do gênero Lycoperdon Pers. (Lycoperdaceae, Agaricales) mediante análises morfológicas e moleculares / Dônis da Silva Alfredo. - Natal, 2017. 298 f.: il.

Tese (Doutorado) - Universidade Federal do Rio Grande do Norte. Centro de Biociências. Programa de Pós-Graduação em Sistemática e Evolução. Orientador: Dr. Iuri Goulart Baseia. Coorientadora: Dra. María Paz Martín. Coorientador: Dr. Paulo Marinho.

1. Basidiomycota - Tese. 2. Fungos gasteroides - Tese. 3. ITS barcode - Tese. 4. Puffballs - Tese. 5. Taxonomia - Tese. I. Baseia, Iuri Goulart. II. Martín, María Paz. III. Marinho, Paulo. IV. Universidade Federal do Rio Grande do Norte. V. Título.

RN/UF/BSE-CB CDU 582.284 DÔNIS DA SILVA ALFREDO

REVISÃO DO GÊNERO LYCOPERDON PERS. (LYCOPERDACEAE, AGARICALES) MEDIANTE ANÁLISES MORFOLÓGICAS E MOLECULARES

Tese de doutorado apresentada ao

Aprovado em: 30 de março de 2017

Comissão examinadora

Dr. Luiz Fernando Pascholati Gusmão, UEFS Examinador Externo à instituição

Dra. Tiara Sousa Cabral, INPA Examinadora Externa à Instituição

Dr. Bruno Cavalvanti Bellini, UFRN Examinador Interno

Dra. Raquel Cordeiro Theodoro, UFRN Examinadora Interna

Dr. Iuri Goulart Baseia, UFRN Presidente

Dedico aos meus pais José Benedito Alfredo e Isabel S. Alfredo por sempre acreditarem e me incentivarem a chegar até aqui. 3

AGRADECIMENTOS

Primeiramente gostaria de agradecer à Universidade Federal do Rio Grande do Norte por oferecer o curso de doutorado no Programa de Pósgraduação em Sistemática e Evolução. Também, devo agradecimentos à Coordenação de Aperfeiçoamente de Pessoa de Nível Superior / CAPES, por proporcionar o apoio financeio de bolsa de estudo no Brasil e estágio sanduíche na Espanha (Programa Ciéncias Sem Fronteiras), que viabilizou diversos resultados e parcerias; ao Conselho Nacional de Desenvolvimento Científico e Técnológico / CNPq, pelo suporte financeiro do Projeto Pesquisador Visitante Especial (PVE-MEC/MCTI/CNPq/FAPs nº71/2013), sem o qual as análises moleculares e o intercambio com o/a pesquisador(a) do exterior seria muito difícil de se realizar; também agradecer ao Programa de Pesquisa em Biodiversidade do Semiárido (PPBio/Semiárido) por ter custeado parte das análises em microscópio eletrônico de varredura.

Acima de tudo, não conseguiria encontrar palavras para agradecer ao meu orientador Prof. Dr. Iuri G. Baseia. Pois, desde que nossa parceria se formou a confiança mútua foi aumentando e o respeito por este excelente pesquisador tomou grandes proporções. Professor Iuri, sempre esteve disposto a ajudar, escutar e guiar em todas a situações; como um pai sempre se preocupou não somente com a situação acadêmica de seus alunos, mas também com a vida pessoal. Tenho uma profunda gratidão a esse parceiro profissional e amigo. Se hoje eu alcancei uma situação profissional melhor foi porque seis anos atrás ele meu deu um voto de confiança, e desde então, tenho me esforçado todos os dias para corresponder a confiança dada por ele. Professor muitíssimo obrigado! Claro, não poderia me esquecer de sua adorável esposa Tereza Cristina O. Galvão, pelos momentos alegres e descontraídos em nossas confraternizações. À ambos, que são meus padrinhos de casamento, Obrigado!

Também quero deixar meu profundo agradecimento a minha coorientadora no exterior Dra. Maria P. Martín, que foi fundamental para o sucesso dos estudos moleculares além das preciosas orientações na construção da tese. Profa. MariPaz, saiba que desejo tudo de bom a ti e muito sucesso em sua vida profissional e pessoal. Saiba que tenho por ti um profundo respeito e carinho. Agradeço pelos momentos vividos na Espanha, e por ter acolhido a mim e minha esposa como membros de sua família. Aprendi muito contigo durantes esses anos, ensinamentos que jamais irei me esquecer. Tu és uma

profissional de excelência, uma verdadeira amiga que irei levar no fundo de meu coração. Agradeço também, a Paco, Analí e Eva, todos vocês fizeram a distância e as saudades do Brasil e de nossos familiares ficarem menos perceptíveis. Tenho vocês como parte da família.

Ao meu coorientador Prof. Dr. Paulo Marinho, que me confiou o acesso ao seu laboratório de biologia molecular, também por me aceitar como aluno no estágio a docência, meus eternos agradecimentos, foi ótima nossa parceria na ciência e seus ótimos conselhos.

Aos Professores Doutores Bruno C. Bellini, Raquel C. Theodoro, Tiara Cabral e Luis Fernando P. Gusmão, fico muito agradecido por aceitarem a participarem da banca avaliadora; aos Professores Doutores Bruno T. Goto e a Vagner G. Cortez, obrigado por se disporem a participarem como suplentes da banca avaliadora.

De maneira muito especial, sou eternamente grato aos meus pais José Benedito Alfredo e Isabel da Silva Alfredo, que sempre me incentivaram a continuar meus estudos; por se preocuparem todos os dias pela distância que o desafio de estudar nos impôs; principalmente por terem me ensinado a trabalhar sempre com honestidade e sempre preserverar nos momentos difíceis. Do fundo do meu coração, muito obrigado! Amo vocês!

Aproveito para deixar meus agradecimentos aos meus irmãos, pela preocupação e pelo carinho que sempre tivemos um pelo outro.

Aos colegas do Laboratório de Biologia de Fungos pela companhia e preciosos momentos de convivência, e em especial a Rafaela Gurgel e Cinta Samara, pelo trabalho em enquipe e aprendizado mútuo. Também, agredeço a T. Accioly por ter cedido gentilmente os espécimes de L. exoelongatum.

Aos curadores dos herbários BAFC, CORDC, FH/HUH, LG, VDEMOULIN, MA-Fungi, MCF-Fungi, NY, K. ICN, PACA, UFRN-Fungos, pelos empréstimos das exsicatas. Em especial as pesquisadoras Dra. Laura Domínguez, Dra. María Luciana Hernández-Caffot e Dra. Silvana Longo por me acolherem estupendamente em minha visita ao herbário IMBIV-Museo Botánico (CORD) de Buenos Aires/Argentina.

Ao pesquisador Dr. Francisco de Diego Calonge (Paco), que durante meu doutorado sanduíche no Real Jardím Botánico, Marid/Espanha, me proporcionou ótimos momentos de trocas de experiência, ensinamentos e boas conversas. Da mesma forma, a todos do Real Jardín Botánico, em especial: M. Dueñas, M. Tereza Tellería, Ricarda Riina, Vlademir Sandoval, Javier Fernández-Lopez, Kina Gracia, Irene Villa Machio, Emilio Cabeza, Yolanda León e Yurena Arjona, muito obrigado por proporcionarem uma maravilhosa companhia durante os nove meses que convivemos. Um especial agradecimento à Fátima Duran, por to paciência e disposição em ajudar me nas atividades do laboratório.

Do mesmo modo, deixo meus agradecimentos às pesquisadoras do Iran, Dr. Farideh Moharrek and Tahmineh Shagholi, pelos momentos felizes e divertidos que passamos na Espanha. Também as pesquisadoras Dra. Laura Martinez-Suz e a curadora assistente Dra. Begoña Aguirre-Hudson, que me deram um especial suporte durante minha rápida visita ao herbário do Kew Botanical Gardens. Bem como, a pesquisadora da Macedonia, Dr. Katerina Rusevska, pelo trabalho em equipe realizado, e pela ótima companhia durante as extrações de DNA e alinhamentos das sequências.

Deixo também meus sinceros agradecimentos à Dra. Marian Glenn (Seton Hall University, USA), que exaustivamente realizou as correções da versão em inglês de todos os capítulos da presente tese, um muito obrigado pelo seu tempo e dispocição!

Agradeço aos meus sogros Joaci Lavor e Onelia Lavor pelo apoio durante esses anos, vocês são exemplos a serem seguidos.

Deixo meu agradecimento em especial a minha amada esposa e compamnheira de pesquisa Pâmela Lavor, que esteve comingo em todos os momentos, nas alegrias e nas frustações que às vezes a pesquisa nos proporciona. Também por ser essa maravilhosa pessoa que sempre sabe como me erguer diante das minhas dificuldades não só na pesquisa quanto na vida pessoal. Muito obrigado! Amo você!

Obrigado Deus, por me dar forças para sempre continuar diante das intempéries da vida.

Trabalhe duro e em silêncio. Deixe que seu sucesso faça barulho. (Dale Carnegie).

RESUMO

Em uma estimativa sobre a biodiversidade da terra acredita-se haver entorno de 50 milhões de espécies e para os fungos acredita-se que essa estimativa gira em torno de 5,1 milhões de espécies, o que levaria cerca de 1000 anos para os taxonomistas identificassem todas essas espécies. Os taxonomistas têm empregado muito esforço para identificar essas espécies de fungos, incluindo o gênero representante dos chamados “puffballs” - Lycoperdon Pers. Durante um longo período, a taxonomia do gênero Lycoperdon se baseou fundamentalmente em caracteres morfológicos, e muitos conceitos adotados para nomear espécies tem se mostrado divergentes entre os taxonomistas. Até o final da década de 80, diversos trabalhos de taxonomia morfológica foram publicados e ajudaram a ampliar o conhecimento sobre a diversidade de espécies de Lycoperdon em quase todos os continentes, no entanto, em muitos casos a morfologia não é suficiente para segregar espécies crípticas, ou esclarecer problemas de divergência quanto a sinonimização de táxons. No início da década de 90 houve uma revolução na taxonomia de fungos com o uso de ferramentas moleculares na classificação de espécies, relacões filogenéticas e identificação novos táxons. Representantes do gênero Lycoperdon foram estudados por meio dessa nova ferramenta somente no início do século 21, e logo em seguida em 2008, com os trabalhos de Larsoon e Jeppson, sendo que alguns gêneros como Morganella e Vascellum foram reconhecidos como subgêneros de Lycoperdon. Durante esse período outra ferramenta passou a ganhar espaço na identificação de espécies por meio de um código de barras molecular, e essa ferramenta passou a ser usada na identificação de animais, plantas e fungos, usando dentre outra, a região espaçadora transcrita interna (ITS), embora, até o presente momento, nenhum trabalho de DNA “barcoding” havia sido realizado com as espécies do gênero Lycoperdon. O presente trabalho teve o objetivo de revisar as espécies do gênero Lycoperdon para América do Sul, identificando-as com base nos caracteres morfológicos e no uso da região ITS como DNA “barconding” de fungos e, posteriormente, adicionando a região da subunidade maior (LSU) do nrDNA para avaliar se ao adicionar as espécies sul-americanas, Morganella seguiria como subgênero de Lycoperdon. Para isso, foram realizados empréstimos de herbários nacionais e internacionais; usando literatura especializada na taxonomia do gênero Lycoperdon e com ajuda de renomados especialistas do grupo, as espécies de Lycoperdon foram identificadas. Posteriormente, foi extraído o DNA de pequenas porções internas do basidiomas; logo em seguida o produto extraído foi amplificado e sequenciado; as

sequências foram editadas e submetidas à busca por similaridade no GenBank utilizando o programa BLAST para checar se as sequências se assemelhavam as regiões comparadas; uma vez realizado esse processo as sequências foram corridas em análises de Máxima Parcimônia e distância (K2P) utilizando o programa PAUP; a árvore de distância se obteve por Neighbor-Joining. Além disso, foi realizada a análise Bayesiana no programa MrBayes; as árvores geradas foram visualizadas no FigTree e editadas no programa InkScap. Com base somente no ITS como DNA barcoding do gênero Lycoperdon, obteve-se os seguintes resultados: 65% das amostras obtiveram sucesso de amplificação; foram identificadas 19 espécies, excluindo aquelas que estavam sob Morganella, para América do Sul e Central; quatro táxons são primeiros registros para o continente sul-americacano: Lycoperdon calvescens, L. endotephrum, L. ericaeum e L. eximium; e foi erguido um novo subgênero: Lycoperdon subg. Arenicola. Com base nas análises de morfológia e DNA barcoding foi possível identificar 43 espécies do gênero Lycoperdon, sendo que destas, 30 espécies são reportadas para América do Sul e Central; poucas espécies se encontram nos dois hemisférios (aprox. 30 %). Com estes resultados, concluímos que ITS como DNA “barcoding” de Lycoperdon é promissor, embora alguns grupos necessitem incluir mais espécimes e marcadores. A taxonomia morfológica continua sendo crucial na interpretação de dados geradas pela taxonomia molecular.

Palavras-chave: Basidiomycota, fungos gasteroides, ITS barcode, puffballs, Taxonomia.

ABSTRACT

Earth’s biodiversity is estimated to include about 50 million species. For Fungi this estimate is around of 5.1 million species, and to identify them all, it is believed would take the taxonomists about 1000 yrs. The taxonomists have been making the effort to identify these fungal species, including the puffballs genus, Lycoperdon Pers. Traditionally, the taxonomy of Lycoperdon has been based only on morphological characters, and individual taxonomists have adopted their own distinct concepts to separate the species. Through the end of 1980`s many projects based on morphological taxonomy were published to describe the diversity of species of Lycoperdon encompassing almost all continents, to separate cryptic species, and to solve problems of divergence by synonymizing of taxons. In the beginning of the 90`s there was a revolution in the taxonomy of fungi with the introduction of molecular tools in species classification, as well to infer phylogenetic relationships and to identify new taxa. Lycoperdon taxonomy was hit by these new tools in the beginning of the 21th century; with the 2008 Larsson and Jeppson work, some genera, such as Morganella and Vascellum were recognized as being subgenera of Lycoperdon. During this period, identifying species by a molecular bar coding gained acceptance, opening the era of DNA barcoding to identify animals and plants, and for fungi, using the internal transcribed spaced region (ITS) to distinguish among species. The work described here is the first to focuss on extensive DNA barcoding to classify the species in the Lycoperdon genus. The present work aims to review the species of Lycoperdon from South America, based on morphological features and on the use of ITS as barcode. Also, using the large subunit (LSU) of nrDNA, to check wether the South American species, Morganella could be a subgenus of Lycoperdon. For this work, loans were made from nationals and international herbaria; the specialized literature in the taxonomy of Lycoperdon was studied, and renowned experts of Lycoperdon were consulted. Afterward the DNA was extracted from little portions of the inside of basidiomata; next the product extracted was amplified and sequenced. The sequences were edited and submitted to the search by similarity on the GenBank website using the BLAST software to check whether the sequences matched homologous sequences or were contaminants. Sequences were aligned with the aid of free software for download, such as Geneious Pro v4.8.5. After the editing of the sequences, analyses of maximum parsimony and distance (K2P) were carried out with the PAUP software; the distance tree was constructed by Neighbor-Joining. Also, a Bayesian analysis was

done using MrBayes software. The generated trees were viewed with FigTree software and edited on InkScap software. Based on ITS as barcoding of Lycoperdon genus, the following results were obtained: 65% of the samples were successfully amplified; 19 species, excluding those under Morganella, were identified from Central and South America; four are first records to South American continent: Lycoperdon calvescens, L. endotephrum, L. ericaeum and L. eximium. Adding the LSU region, Morganella is confirmed as a subgenus of Lycoperdon; L. demoulinii is a new species for science; and a new subgenus, Arenicola, emerged. Based on morphological analyses and DNA barcoding, it was possible to identify 43 species of Lycoperdon genus around the world; 30 species from Central and South America; few species are distributed in both Hemisphere (around 30%). Finally, we concluded that the ITS is promising as DNA barcode of Lycoperdon; however, some groups need to include more specimens and markers. Morphological data are crucial for interpretation of the data generated by molecular methods of taxonomy.

Key-words: Basidiomycota, ITS barcode, gasteroid fungi, puffballs, Taxonomy

LISTA DE FIGURAS

Figura 1. Tipos de formas dos basidiomas em Lycoperdon e Morganella. a. L. mammiforme basidioma piriforme (K s/n coletado por M.C. Cook 1885); b. L. pulcherrimum basidioma subgloboso (K 3933 – holotipo !); c. M. compacta, basidiomas depresso globosos (PDD10140 holotipo!); d. M. fuliginea basidioma globoso (UFRN- Fungos 1768); e. L. utriforme, basidioma depresso globose a obovoide (K200231); f. L. pratense, basidiomas subglobose a turbinado (VDEMOULIN 7412). Barras = 10 mm. 37 Figura 2. Tipos de subgleba em espécies de Lycoperdon. a. Subgleba reduzida e com células compactas, L. fuligineum (UFRN-Fungo 1768); b. Subgleba bem desenvolvida, celular e com diafragma L. marginatum (CORD756); c-d, f. Subgleba desenvolvida e celular: (c) L. perlatum (MA-Fungi 29372), (d) L. pratense (K200229), (f) L. utriforme (K200230) em basidioma parcialmente imaturo; e. subgleba bem desenvolvida e lanosa, (e) L. pyriforme (MA-Fungi 16908). Barras = 10 mm...... 37 Figura 3 Tipos de ornamentação do exoperídio e superfície do endoperídio. a, d. Espinhos com as pontas alongadas, delgadas e curvadas; b, e, f. Verrugoso; c. Espinhos piramidais; a-c, e-f. Superfície do endoperídio liso; d. Superfície do endoperídio areolada. Todas as ornamentações apresentadas são caducas com o tempo. Barras = 2 mm...... 40 Figura 4 Tipos de esferocistos e células alongadas do exoperídio apical; micoesclereídeos e hifas infladas do endoperídio apical. a. Células alongadas e dextrinoide de L. atrum; b. Esferocistos em cadeias de L. perlatum; c. Esferocistos com formas irregulares de L. pyriforme; d, f. Micoesclereídeos com formas irregulares de L. marginatum e Lycoperdon sp.; e. Hifas com terminações de L. umbrinum. a, c, e. Barras = 20 µm; b. Barra = 100 µm; d, f. Barras = 10 µm...... 41 Figura 5 Exemplos de basidiosporos quanto a forma e classificação da ornamentação espécies de Lycoperdon. a. basidiosporos lisos [A], L. pyriforme (NY0071979); b-d. Levemente verrugoso [B], L. marginatum (NY00398533), L. eximium (NY0071978), L. umbrinum (MA-Fungi 63394); e-f. Verrugoso [C], L atrum (UFRN-Fungos 832), L. perlatum (NY00793106); g-i. Fortemente verrugoso [D], M. nuda (UFRN-Fungos 1766), M. velutina (NY00839022), L. decipiens (MAF27674); f, g. Basidioporos com o número de ornamentações contadas e diâmetro medido para o cálculo de densidade de ornamentação. Barras = 10 µm...... 42 Figura 6 Exemplos de basidiosporos com as classificações de A-D em microscopia eletrônica de varredura (MEV). a, b. Punctados [A], L. pyriforme e L. compactun (= M.

compacta); c, d. Levemente verrucosos [B], L. arenicola (= M. arenicola), L. marginatum; e, f. Verrucoso [C], L. perlatum, L. atrum; g-i. Fortemente verrucoso [D], L. fuligineum (= M. fuliginea), L. atropurpureum e L. mauryanum. Barras = 1 µm...... 43 Figura 7. Materiais e equipamentos usados durante a extração e amplificação do DNA. a. Colunas (lilás e brancas) do kit de extração; b. Tubos eppendorfs contendo em seu interior uma esfera desidratada com todos os reativos para a amplificação por PCR; c. Termociclador; d. Transluminador acoplado com câmera fotográfica para a visualização e confecção de fotografias dos géis em agarose; e-f. Fotografias dos géis em agarose; e. Gel sem as bandas de amplificação da PCR; f. Gel contendo as bandas dos produtos amplificados da PCR...... 52 Figura 8. Esquema do nrDNA no qual se pode observar as regiões dos marcadores e a posição dos iniciadores. Baseado em http://sites.biology.duke.edu/fungi/mycolab/...... 53 Figure 9. Formação das sequências consensos e busca no megablast para verificar se há contaminação das mesmas. a. Visualização das sequências direto e reverso e formação da sequencias consenso; b-g. Submissão da sequencias consenso no megablast disponível na web site NCBI...... 54 Figura 10. Número de amostras analisadas, e porcentagem de falha e sucesso de amplificação da região ITS...... 58 Figure 11 Agarose gel to verify the PCR products. a. Amplification of ITS5 – ITS4 region: eleven positive PCR products; the eighth sample failed; b. Amplification of ITS3 – ITS4b region: all samples have been successful; c. Amplification of ITS5 – ITS4 have been unsuccessful in all samples; d. Amplification of ITS3 – ITS4b have been unsuccessful in all samples and the control sample is contaminated. M - is the run marker, and N - is the control sample...... 89 Figure 12 Example of search in the UNITE database. a. Set up of run analysis where the query sequences from the user are pasted and blasted; b. Results including the list of homolog sequences to; c. UNITE sequences linked to the SHs with threshold of at least 3% similarity; d. Selected SHs sequence with threshold of 1.5%, name and number access, and statistics on distribution distance...... 91 Figure 13 Neighbor-Joining tree using Kimura 2 Parameter based on sequences of ITS sequences of Lycoperdon species. The support values obtained from parsimony (bs) and Bayesian (pp) analyses are on the branches...... 93 Figure 14 PCI for each sample from each Lycoperdon species of dataset non-GenBank plus GenBank samples...... 100

Figure 15 Average intraspecific and interspecific genetic variability for conspecifics and congeneric of all sequences of dataset excluding Bovista and Morganella...... 100 Figure 16 Percent identity of sequences compared with UNITE Species Hypothesis (SHs)...... 101 Figure 17 Dry basidiomata of Lycoperdon species. a. Lycoperdon altimontanum (MA- Fungi 63392); b. L. atropurpureum (MA-Fungi 63389); c. L. decipiens (MA-Fungi 27674); d. L. echinatum (MA-Fungi 39586); e. L. ericaeum (MA-Fungi 63393); f. L. excipuliforme (MC-Fungi 06:6224); g. L. lividum (MA-Fungi 68348); h. L. mammiforme (MA-Fungi 31251); i. L. marginatum (MA-Fungi 31232); all bars = 10 mm...... 108 Figure 18 Dry basidiomata of Lycoperdon species. a. Lycoperdon molle (MA-Fungi 31259); b. L. nigrescens (MA-Fungi 22012); c. L. niveum (MA-Fungi 21618); d. L. perlatum (MA-Fungi 64953); e. L. pratense (MC-Fungi 08: 10114); f. L. pyriforme (MA- Fungi 16908); g. L. subumbrinum (MA-Fungi 63391); h. L. umbrinoides (MA-Fungi 53530); i. L. umbrinum MA-Fungi 63394 – unsuccessful amplification); all bars = 10 mm...... 116 Figure 19 Scanning microscope electronic of some Lycoperdon species. a. Lycoperdon atropurpureum (MA-Fungi 63389); b. L. ericaeum (MA-Fungi 63393); c. L. lividum (MA-Fungi 74402 – unsuccessful amplification); d. L. mammiforme (MA-Fungi 24103); e. L. marginatum (MA-Fungi 31252); f. L. nigrescens (MA-Fungi 63396 – unsuccessful amplification); g. L. perlatum (MA-Fungi 64953); h. L. subumbrinum (MA-Fungi 63391); i. L. umbrinum (MA-Fungi 73600 – unsuccessful amplification); all bars = 5 µm...... 117 Figure 20 Neighbord-Joining tree obtained after calculating the K2P distance of the ITS sequences included in Table 1. Names and vouchers numbers as mentioned in the text and tables. New sequences in bold. Colours to indicate the geographic origin of the specimens and sequences. Yellow, mainly specimens from Central and South America (it can be some specimens from other part from the Southern Hemisphere); Orange, specimens from the Southern Hemisphere, not Central or South America; Red, specimens from both Hemispheres; Blue, only specimens from the Northern Hemisphere...... 160 Figure 21 Dry basidiomata of Lycoperdon species. a. L. atrum (UFRN-Fungos 832); b. L. calvescens (CORD 997); c. L. curtisii (VDEMOULIN 4073); d. L. endotephrum (K (M): 200182); e. L. eximium (NY0071978); f. L. hyalinum (DSA 178, UFRN-Fungos 2829); g. L. lividum (NY 0071655); h. L. marginatum (CORD 72); i. L. mauryanum (LG 1570). Bars = 10 mm...... 175

Figure 22 Scaning electronic microscopy of basidisporoes. a. L. atrum (UFRN-Fungos 832; b. L. marginatum (CORD 573); c. L. mauryanum (VD 1570); d. L. nigrescens (PACA 13783); e. L. ovoidisporum (UFRN-Fungos 967); f. L. perlatum (UFRN-Fungos 811); g. L. pratense (ICN 154485); h. L. pyriforme (NY 0071971); i. Lycoperdon sp. 5 (UFRN-Fungos 152)...... 186 Figure 23. Dry basidiomata of Lycoperdon species. a. L. nigrescens (NY0071657); b. L. ovoidisporum (13771); c. L. perlatum (VDEMOULIN ex ENCB 1533); d. L. pratense (K(M): 200187); e. L. pyriforme (UFRN-Fungos 2271); f. L. umbrinum (VDEMOULIN 4784); g. L. utriforme (K (M): 2002300); h. Lycoperdon sp. 1 (CORD 167); i. Lycoperdon sp. 4 (UFRN-Fungos 495). Bars = 10 mm...... 194 Figure 24 Comparative between the species of Lycoperdon sp. 5, L. endotephrum and L. pratense. a-c. Lycoperdon sp. 5 (UFRN-Fungos 152); d-f. L. endotephrum (UFRN- Fungos 164); g-i. L. pratense (Basidioma – VDEMOULIN ex AGUCH-ET8; spore – ICN 154485); a, d, g. Dry basidiomata; b, e, h. Sphaerocysts; c, f, i. Basidiospores in scanning eletronic microscopy (SEM). Bars a, g = 10 mm; d = 5 mm; b, e, h = 20 µm; c, f, i = 1 µm...... 195 Figure 25 Phylogenetic tree obtained from parsimony analysis of ITS and LSU combined alignment. Clades and subclades as described in the text. Numbers above branches are parsimony bootstrap (bs) and posterior probability (pp) values. Bovista, Calvatia, Disciseda, Mycenastrum and Tulostoma as outgroups. Vouchers numbers are indicated as in Table 1 and 2...... 234 Figure 26 Lycoperdon albostipitatum (Holotype INPA 239563). a. Dry specimen. b. Detail of apical pore. c. Detail of exoperidium ornamentation. d. Elongated dextrinoid hyphae from exoperidium ornamentation. e. Basidiospores strongly verrucose [D] in LM. f. Scanning electron microscopy of basidiospores. Bars (a) 10 mm; (b) 1 mm; (c) 0.2 mm; (d) 50 µm; (e) 5 µm; (f) 2 µm...... 238 Figure 27 Lycoperdon arenicola (Holotype UFRN-Fungos 1006). a. Basidiomata in situ. b. Dry specimen in cross section. c. Sphaerocysts from exoperidium ornamentation. d. Detail of capillitium and paracapillitium mixed. e. Basidiospores punctate [A] in LM. f. Scanning electron microscopy of basidiospores. Bars (a–b) 10 mm; (c) 50 µm; (d) 20 µm; (e) 5 µm; (f) 2 µm...... 240 Figure 28 Lycoperdon demoulinii (Holotype UFRN-Fungos 655). a. Dry specimen; b. Detail of exoperidium ornamentation; c. Detail of endoperidium surface areolate; d. Details of spines from exoperidium; e. sphaerocysts forming the spines; f. Mycosclerids

15 from apical endoperidium; g. Capillitium and basidiospores punctate in LM. Bars (a) 10 mm; (b) 2 mm; (c) 2 mm; (d) 50 µm; (e–f) 20 µm; (g) 5 µm...... 244 Figure 29 Lycoperdon exoelongatum. a. Basidiome in situ; b. Dry specimen; c. Detail of tufts in exoperidium of young basidiome; d. Detail of exoperidial surface in aged basidiome; e. Paracapillitium covered with glebal membrane; f. Basidiospores strongly verrucose [D] in LM; g. Scanning electron microscopy of basidiospores; h. Exoperidial elements. Bars: (a, b, d) bar = 5 mm.; (c) bar = 0.5 mm; (e) bar = 20 µm; (f) bar = 10 µm; (g) bar = 1 µm; (h) bar = 50 µm (Photos: Accioly, T.)...... 248 Figure 30 Lycoperdon fuligineum. a. Paratype of Morganella mexicana (NY00839022, J.B. Ellis 5013), dried specimens; b. Dry specimens of Morganella mexicana mixed with M. velutina (NY00839023, J.B. Ellis 5013); c. Detail of exoperidium ornamentation; d. Sphaerocysts from exoperidium ornamentation; e. Basidiospores strongly verrucose [D] in LM; f. Scanning electron microscope of basidiospores. Bars: (a–b) 10 mm; (c) 0.2 mm; (d) 50 µm; (e) 5 µm; (f) 2 µm...... 251 Figure 31 Lycoperdon nudum (Holotype UFRN-Fungos 1765). a. Dried specimen; b. Detail of exoperidium falls off; c. Detail chain of exoperidium ornamentation; d. Basidiospores strongly verrucose [D] and paracapillitium in LM; e. Scanning electron microscope of basidiospores. Bars (a) 2 mm; (b) 2 mm; (c) 50 µm; (d) 5 µm; (f) 2 µm...... 254 Figure 32 Lycoperdon velutinum. a. Dried specimens of type (NY00398807); b. Dried specimens of the mixed basidiomes with L. fuligineum (NY00839022); c. Exoperidium ornamentation; d. Elongated hyphae from exoperidium; e. Basidiospores strongly verrucose [D] in LM (type); f. Scanning electron microscope of basidiospores (type). Bars: (a) 10 mm; (b) 5 mm; (c) 0.2 mm: (d) 50 µm; (e) 5 µm; (f) 2 µm...... 257

LISTA DE TABELAS

Tabela 1 Iniciadores empregados na amplificação dos fragmentos ITS e LSU nrDNA. Indicação de suas sequências e temperatura de hibridização...... 51 Table 2 Sequences used in the molecular analysis. In bold new ITS sequences generated in this work ...... 138 Table 3 Identifications success based on ¨Best Match¨ and ¨Best Close Match¨...... 143 Table 4 Sequence names with incorrect identification based on "Best Match/Best Close Match" within threshold. In bold the values that repeat in the “Best Close Match”. .... 143 Table 5 All specimens used in this study with voucher number, country and Genbank Accession Numbers when available. C: Calvatia; L: Lycoperdon; M: Morganella; V: Vascellum. In black, the new sequences generated in this study, from some of them Genbank Accession Numbers (Acc. N.) are not obtained yet. NP: No herbarium permission to molecular studies; NT: Not DNA isolation; C: sequences obtained do not belong to Lycoperdon, different contamination; : not successful amplifications are indicated or not good sequences were not obtained...... 212 Table 6 Distance in percent of all specimens of Lycoperdon perlatum terminal branch based on GenBank sample DQ112630...... 219 Table 7. Distance in percent of all specimens of Lycoperdon pyriforme terminal branch based on GenBank sample DQ112558...... 220 Table 8 Specimens studied in this work with voucher numbers, country, and the main morphological features considered. Collections which ITS and LSU sequences were obtained are indicated with (+), sequences were not obtained (-), sequences were not attempt (0)...... 266 Table 9 All the specimens used in the molecular phylogenetic analyses with voucher number and GenBank Accession Numbers. When ITS and LSU have different numbers, the first number corresponds to ITS and the second to LSU. In black are indicated the new sequences generated in this study...... 270

LISTA DE ABREVIAÇÕES

A seguir serão detalhadas algumas abreviações mais utilizadas ao longo desta tese de doutorado. Não se incluem as abreviaturas dos herbários que estão disponíveis no Index Herbariorum, tão pouco as abreviaturas padronizadas dos diferentes ranques taxonômicos que podem ser consultadas na Recomendação 5ª do Código Internacional de Nomeclatura para Algas, Fungos e Plantas. As abreviaturas dos autores seguem “Author of Fungal Names” que se pode consultar no “Index Fungorum”.

ADN/DNA: Ácido desoxirribonucleico/deoxyribonucleic acid. No brasil é muito comum usar DNA.

ARN/RNA: Ácido ribonucleico/ ribonucleic acid.

CO1: Citocromo c oxidase/cytochrome c oxidose subunit. dNTP: Desoxirribonucleotídeos TriFosfatos/ Deoxynucleotide TriPhosphates.

EDTA: Ácido Etilenodiamino Tetra-acético/ Ethylenediamine Tetraacetic Acid. e.g.: Exempli gratia, por exemplo.

ITS: Espaçador transcrito interno/ internal transcribed spacer.

K2P: Kimura 2 parametros. l: litro, Equivalente a um decímetro cúbico.

LSU: Subunidade maior do DNA ribossômico/large subunit ribossomal. min.: Minuto, equivalente a 60 segundos. ml: Milímetro, equivale a um centímetro cúbico. mm: Milítemtro, equivale a um milésimo do metro. nr: Prefix que indica ribossômo nuclear, pode preceder a ADN, LSU, SSU etc./prefix for “nuclear ribosomal”; it may preced DNA, LSU, SSU, et.

MP: Máxima parcimonia/Maximum parcimony.

MCMC: Métodos de Monte Carlo via cadeias de Markov/Markov chain Monte Carlo.

PCI: Porcentagem de identificação correta/Percentage of correct identification.

PCR: Reação em cadeia da polimerase/Polymerase reaction chain.

RPB 1: Gene da RNA polimerase subunidade 1/RNA polymerase II gene subunit 1.

RPB2: Gene da RNA polimerase subunidade 1/RNA polymerase II gene subunit 2.

SH/plural SHs: Hipóteses de espécies/Species Hypotheis.

SSU: Subunidade menor do DNA ribossômico/small ribosomal subunit.

TAE: Solução Tampão Tris-Acetato-EDTA.

µl: microlitro equivalente a um milímetro cúbico.

µm: micrômetro equivale a um milécimo do metro

SUMÁRIO 1 INTRODUÇÃO GERAL ...... 24

1.1 A identificação de espécies em fungos ...... 24

1.2 Conceito de espécies em fungos ...... 26

1.3 O gênero Lycoperdon Pers...... 28

1.4 Histórico sobre os principais trabalhos do gênero Lycoperdon abordando morfologia e divergências na classificação das espécies...... 29

1.5 Classificação de Lycoperdon com base em análises moleculares de ITS e LSU... 33

1.6 Os subgêneros Morganella e Vascellum e seus históricos como espécies de Lycoperdon………...…………………………………………………………………...34

1.7 Caracteres macro e micromorfológicos em Lycoperdon, Morganella e Vascellum ……………………………………………………………………………………35

Caracteres macroscópicos...... 35

Caracteres microscópicos ...... 39

2 JUSTIFICATIVA, HIPÓTESES E OBJETIVOS ...... 44

3 MATERIAL E MÉTODOS ...... 46

3.1 Coleções estudadas...... 46

3.2 Análises morfológicas ...... 46

3.2.1 Analises dos caracteres macromorfológicos ...... 46

3.2.2 Análise dos caracteres micromorfológicos ...... 47

3.3 Análises moleculares ...... 49

3.3.1 Isolamento do DNA, amplificação e sequenciamento ...... 49

3.3.2 Identificação molecular das espécies de Lycoperdon .…………………………...55

3.4 Nomenclatura ...... 56

4 RESULTADOS ...... 58

5 DISCUSSÃO GERAL ...... 59

6 CONCLUSÕES GERAIS ...... 62

REFERENCES ...... 63

CAPÍTULO 1/ CHAPTER 1 ...... 78

1 INTRODUCTION ...... 81

1.1 Animal, algae, plant and oomycota barcode ...... 82

1.2 Fungi barcode and automatic identification tools ...... 84

1.3 The genus Lycoperdon Pers...... 86

2 MATERIAL AND METHODS ...... 88

2.1 Collections studied ...... 88

2.2 Morphological analysis ...... 88

2.3 Molecular analyses ...... 88

2.3.1 DNA isolation, amplification and sequencing ...... 88

2.3.2 Molecular identification of Lycoperdon species ...... 89

2.3.3 Performance of automatic identification through the unite database ...... 91

3 RESULTS ...... 92

3.1 Molecular analyses ...... 92

3.1.1 DNA, amplification and sequencing ...... 92

3.1.2 Molecular identification of Lycoperdon species ...... 92

3.1.3 Probability correct identification (PCI) of Lycoperdon species ...... 96

3.1.4 Automatic Identification through UNITE ...... 101

4 DISCUSSION ...... 118

5 CONCLUSION ...... 125

REFERENCES ...... 126

CAPÍTULO 2 / CHAPTER 2 ...... 144

1 INTRODUCTION ...... 146

1.1 Problems with the species concept and names in Lycoperdon ...... 148

1.2 Current taxonomy of Lycoperdon ...... 149

1.3 DNA Barcode to identify fungi ...... 150

2 MATERIAL AND METHODS ...... 152

2.1 Collections studied ...... 152

2.2 Morphological analyses ...... 152

2.2.1 Analyses of macrostructures ...... 152

2.2.2 Analyses of microstructures ...... 153

2.3 Molecular Analyses ...... 154

2.3.1 DNA isolation, amplification and sequecing ...... 154

2.3.2 The molecular identification of Lycoperdon species ...... 154

3 RESULTS ...... 157

3.1 Molecular analysis ...... 157

3.1.1 DNA, amplification, and sequencing ...... 157

3.2 Molecular identification of Lycoperdon species ...... 157

3.3 Probability Correct Identification (PCI) of Lycoperdon perlatum and L. pyriforme . …………………………………………………………………………..161

3.4 Taxonomy ...... 162

4 DISCUSSION ...... 196

5 CONCLUSIONS ...... 199

REFERENCES ...... 200

CAPÍTULO 3 / CHAPTER 3 ...... 223

1 INTRODUCTION ...... 225

2 MATERIAL AND METHODS ...... 227

2.1 Material studied ...... 227

2.2 Morphological analysis ...... 227

2.3 Molecular Analyses ...... 228

2.3.1 DNA isolation, amplification and sequencing ...... 228

2.3.2 Sequence alignment and phylogenetic analyses ...... 229

3 RESULTS ...... 231

3.1 Molecular and morphological analyses ...... 231

3.2 Taxonomy ...... 235

4 DISCUSSION ...... 260

REFERENCES ...... 261

CAPÍTULO 4 / CHAPTER 4 ...... 273

1 INTRODUCTION ...... 275

2 MATERIAL AND METHODS ...... 276

3 RESULTS ...... 277

3.1 List of species ...... 277

4 DISCUSSION ...... 285

5 CONCLUSIONS ...... 287

REFERENCES ...... 288

INDEX TAXONÔMICO ...... 292

1 INTRODUÇÃO GERAL 1.1 A identificação de espécies em fungos

A princípio, o planeta terra em uma subestimada previsão de sua biodiversidade teria entorno de 5 a 15 milhões de espécies (Stork, 1993), estimativas menos conservadoras consideram que esta diversidade possa ultrapassar os 50 milhões de espécies em todo planeta (Wilson, 2003; Scheffers et al., 2012). Para fungos a estimativa é que se tenha em torno de 3 a 5 milhões de espécies (Blackwell, 2011). Ao longo do tempo, os taxonomistas tem empregado um valioso tempo em descrever e nomear espécies, aumentando a cada ano a quantidade de nomes de espécies registradas em herbários ou museus (Alberch, 1993; Wilson, 2003).

Tendo isto em vista, a sistemática e a taxonomia tem um papel crucial em nomear e organizar o posicionamento das espécies quanto a seu reino, filo, classe, ordem, família, gênero e espécie. Obviamente, muitos desses nomes trazem consigo uma certa confusão, isto porque, para cada local adotavam-se nomes diferentes para um mesmo grupo de indivíduos e as descrições eram no mínimo imprecisas (Margulis e Sagan, 2002). Para isso, adotou-se o sistema binominal de Linnaeus (Linnaeus, 1753), teve um importante papel no desenvolvimento da taxonomia e classificação das espécies (Hitchcock, 1926; Cain, 1958a; b).

Do mesmo modo, muitos pesquisadores empenharam esforços na taxonomia e classificação de diversos grupos de fungos (Tournefort, 1700; Linnaeus, 1753; Scopoli, 1772; Schaeffer, 1774; Persoon, 1801; Fries, 1829; Vittadini, 1842; Berkeley, 1873). Assim como, outros tantos investiram tempo na taxonomia em grupos específicos, como por exemplo, do gênero Lycoperdon Pers. (Massee, 1887; Lloyd, 1905a; b).

Dessa forma, por muito tempo, a classificação e nomenclatura das espécies de Lycoperdon foram baseadas exclusivamente em caracteres macromorfológicos (Bonorden, 1857; Massee, 1887). Com o desenvolvimento da microscopia, os taxonomistas passaram a considerar as características micromorfólogicas, usando microscópio ótico para investigar as estruturas da gleba, como esporos e capilícios (Cunningham, 1926, 1944; Coker e Couch, 1928; Johnson, 1929; Bottomley, 1948). Posteriormente, a melhoria da precisão dos microscópios de luz e o advento da microscopia eletrônica de varredura, proporcionou descrições mais detalhas do grupo (Cunningham, 1944; Bottomley, 1948; Demoulin, 1968a; Eckblad, 1971; Perreau, 1971;

Calonge, 1975), persistindo até o final da década 80 (Demoulin, 1983; Homrich and Wright, 1988; Domínguez de Toledo, 1989; Jeppson and Demoulin, 1989; Calonge, 1990).

Ao final da década de 80 e início dos anos 90, houve uma revolução na taxonomia não só em Lycoperdon, mas englobando grande parcela dos grupos de fungos conhecidos, foi então o início da era das ferramentas de genética molecular para classificar, identificar e inferir as suas interrelações evolutivas (Bruns et al., 1989, 1991; White et al., 1990; Baldwin, 1992, 1993). O gênero Lycoperdon foi efetivamente estudado por esta nova abordagem de classificação somente com o trabalho de Hibbett e colaboradores que abordaram aspectos evolutivos entre os fungos lamelados e gasteroides utilizando de DNA ribossomal (Hibbett et al., 1997).

A partir de então, muitos taxonomistas tem utilizado das ferramentas moleculares para classificar níveis hierárquicos superiores como famílias ou ordens, (Kurtzman e Robnett, 1998; Martín et al., 2000; Krüger e Kreisel, 2003). Uma nova ferramenta passou a ser usada para elucidação de espécies crípticas, identificação e descrição de novas espécies, o DNA barcode (Hebert et al., 2003), utilizando a mesma ideia de um código de barras usado em supermercados e lojas comercias. O DNA barcode surgiu com a ideia de um sequência universal, de rápida amplificação, curto número de pares de bases (600- 700 pb) e de fácil edição, para identificar especies (Hogg e Hebert, 2004; Hebert e Gregory, 2005; Summerbell et al., 2005).

Consequentemente, o DNA barcode passou a ser usado na taxonomia de vários grupos de fungos (Rehner e Buckley, 2005; Grasso et al., 2006; Schubert et al., 2007; Seifert et al., 2007; Nguyen e Seifert, 2008; Nilsson et al., 2009). Da mesma forma, em fungos gasteroides, como por exêmplo, nos gêneros Astraeus, Pisolithus y Scleroderma, o uso de sequências de barcode tem permitido identificar e descrever novas espécies (Phosri et al., 2014; Rusevska et al., 2014, 2015). Com respeito ao gênero Lycoperdon, somente os trabalhos de Larsson e Jeppson (2008) e Jeppson et al. (2012), utilizaram as sequências barcode, junto com um fragmento da região LSU nrDNA para inferir relações filogenéticas entre Lycoperdon e outros gêneros afins. Até o presente momento nenhum trabalho havia sido idealizado com a proposta de avaliar se a sequência de DNA barcode permitiria identificar as espécies de Lycoperdon.

Com isso, este trabalho tem como proposta integrar a taxonomia morfológica à taxonomia molecular, utilizando a região espaçadora interna (ITS) como barcoding de Lycoperdon e seus subgêneros Morganella e Vascellum. Assim, após detalhada e extenuante revisão literária do gênero e discussões sobre o tema com especialistas do grupo como o Dr. F.D. Calonge (Real Jardín Botánico de Madrid) e o Dr. Vincent Demoulin (Université de Liège – Bélgica), apresenta-se aqui algumas hipóteses: (i) as espécies de Lycoperdon podem ser identificadas com o uso do ITS como DNA “barcoding” quando integrado aos dados morfológicos; (ii) os nomes de espécies adotados para identificar os espécimes da América Central e Sul-americanos de Lycoperdon refletem os conceitos adotados pelos taxonomistas do velho mundo; (iii) ao adicionar as espécies Sul-americanas ao conjunto de dados de Larsson e Jepsson (2008), Morganella continuará sendo subgênero de Lycoperdon.

1.2 Conceito de espécies em fungos

A grande a necessidade do ser humano para nomear e classificar espécies (Stork, 1993), traz consigo um grande questão entre pesquisadores e naturalistas que se dedicam para descobrir sobre a diversidades das espécies; o que é uma espécie? Ou, qual o conceito de espécies? O conceito de espécie, então passou a ser discutido entre muitos pesquisadores do passado com intuito de unificá-lo (Mayr, 1969; Ghiselin, 1974; Ridley, 1989; Mayden, 1997). Ghiselin (1974), cita que o problema do conceito de espécies está no entendimento do status ontológico da fundamentação biológica. Para Mishler e Donoghue (1982), o reconhecimento de espécie que é feito pelos taxonomistas é baseado em suas experiências com a sistemática dos grupos de organismos de seus estudos.

Mayden (1997) comenta que muitas classificações podem existir para o mesmo grupo de organismos, dependendo de qual critério foi adotado para o conceito de espécie; ainda, se os relacionamentos descendem de um hipotético ancestral comum, então a categoria supra específica (gêneros ou famílias) são mais inclusivas do que a categoria de espécies. Para os taxonomistas o conceito de espécies (Taxonomic Species Concept - TSC), consiste de que todos os espécimes que são membros de um único tipo, mostrado por evidência ou suposição, são tão similares quanto sua prole ou parentes hereditários; levando-se em consideração esse critério, pesquisadores que não compartilham desse conceito, por exemplo, os pesquisadores afins do conceito genético de espécies (Genetic

Species Concept – GSC) podem não assumir as entidades consideradas espécies pelos taxonomistas e vice-versa (Mayden, 1997; de Queiroz, 2005).

Paralelamente aos 22 conceitos de espécies abordado por Mayden (1997), um conceito de espécies unificado foi proposto como solução dos problemas conceituais, eliminando rivalidades entres conceitos, onde as espécies não precisam ser feneticamente diferentes, ou diagnosticamente, ou ser monofiléticas, ou ainda estarem isoladas reprodutivamente, mas sim, as espécies tem que estar evoluindo separadamente (de Queiroz, 2005); Este conceito unificado nos leva a dois importantes pontos: o primeiro é que, ele serve como um importante critério operacional ou traços de evidências que são relevantes na separação de espécies, que podem ser fenéticos, grupos monofiléticos isolamento reprodutivo, e que são traços ou atributos adquiridos durante a divergência evolutiva; o segundo é que, estas propriedades permanecem importantes, e podem ser usadas na delimitação de espécies ou subcategorias de espécies (de Queiroz, 2006).

Assim como os demais gruposde organismos, para os fungos, os taxonomistas também tem se preocupado em definir o conceito de espécies (Petersen e Hughes, 1999) e, segundo os autores temos três conceitos de espécies mais usados em fungos, a saber: conceito biológico (Biological Species Concept (BSC)) – no qual os indivíduos se reconhecem como parceiros reprodutivos; o conceito morfológico (Morphological Species Concept (MSC)), onde as espécies são separadas por diferênças na morfologia, refletido pelas diferênças genéticas entre dois grupos; e terceiro conceito, o filogenético (Phylogenetic Species Concept (PSC), no qual populações pode ser ranqueadas como espécies, se elas compartilham linhagens evolutivas comuns, que são expressadas em nós terminais em uma árvore filogenética. Os autores acreditam que considerando as peculiaridades dos fungos como a variabilidade nos caracteres morfológicos intraespecíficos, reprodução clonal alternando para reprodução sexuada, etc., tornando difícil a elaboração de um conceito unificado de espécies, embora, eles sugiram que o conceito deva demonstrar uma coesão fenética, de descendência evolutiva comum e, quando possível isolamento reprodutivo.

Igualmente, Taylor et al. (2000), considerando que os fungos apresentam características insatisfatórias no reconhecimento de espécies, propuseram o reconhecimento de espécies por meio da concordância genealógica, onde as espécies estão em isolamento genético. De fato, este tipo de reconhecimento de espécies somente é possível devido aos avanços nos estudos de filogenia molecular, usando diversos tipos 27 de regiões como ITS e LSU (Bruns et al., 1989; Vilgalys e Hester, 1990; Gardes e Bruns, 1993; Rehner e Samuels, 1994). Assim o conceito filogenético de espécies passou a ser difundido, onde a definição de espécies remeta à um grupo de indivíduos que compartilham no mínimo um caráter derivado em comum (Wheelwe e Meier, 2000; Agapow et al., 2004). Consequentemente, este atualizado conceito, baseado em análises filogenéticas, pode separar em mais de uma espécie, quando anteriormente estavam sob um único nome específico, passando então a ser definido o conceito de espécies crípticas (Bickford et al., 2007); para os autores em uma explicação mais simplista, as espécies crípticas carecem de caracteres morfológicos suficientes para a separação destas espécies de forma eficaz. E isto mostra a fragilidade do conceito morfológico de espécies (Crespo e Pérez-Ortega, 2009), tendo em vista que os fungos apresentam caracteres morfológicos similares entre espécies (Seifert, 2009). Então, Tan et al. (2010), optou por delimitar espécies crípticas, baseando-se nas diversas ferramentas disponíveis (caracteres morfológicos, moleculares, biológicos e ecológicos) para minimizar os problemas intrínsecos a cada conceito de espécie.

Desta maneira, considerando os conceitos mais usados em fungos (conceito biológico, morfológico e filogenético), mesmo com seus tradicionais problemas (Petersen and Hughes, 1999; Taylor et al., 2000), somado aos novos conceitos baseados em ferramentas moleculares (Wheelwe and Meier, 2000; Agapow et al., 2004; Bickford et al., 2007), na presente obra nós optamos pelo conceito da “taxonomia integrativa” (Tan et al., 2010), levando-se em conta todas as ferramentas disponíveis no presente estudo, como dados morfológicos, moleculares e ecológicos, para a delimitação de espécies.

1.3 O gênero Lycoperdon Pers.

O gênero Lycoperdon Pers. é um representante dos fungos chamados de puffballs, referindo-se aos fungos sem um distinto pedicelo (stem), com um perídio flácido abrindo por uma boca definida, esporos sem pedicelos e mesclado com capilícios (Lloyd, 1905a; Cunningham, 1926).

No presente trabalho, será adotado o conceito de definição do gênero conforme propostos nas obras de Demoulin (1970, 1972b, 1973, 1979). Assim para uma geral definição do gênero temos: basidioma com subgleba bem desenvolvida, celular, embora, em alguns casos as células compactas podem ocorrer, apresentando tons esbranquiçada a

acinzentada; deiscência por um poro apical ou ruptura irregular do perídio; gleba composta sempre por capilícios elásticos a subelásticos (em referencias àqueles que podem ocorrer com partes quebradas, mas não por completo), podendo ou não ocorrer poros e septos; paracapilício podendo ocorrer em diferentes proporções; e esporos globosos a subelípticos, sendo punctados a fortemente verrucosos.

Tradicionalmente, o gênero pertencia a família Lycoperdaceae (Coker and Couch, 1928). Além de diversos estudos terem sidos realizados para a compreensão da diversidade de espécies e a distribuição do gênero (Johnson, 1929; Rick, 1930; Cunningham, 1944; Bottomley, 1948). Ainda que, em um contexto atual, baseando em analises de filogenia molecular, o gênero está alocado junto aos Agaricales em Agaricaceae, sendo que o nome Lycoperdaceae não reflete sua atual classificação (Hibbett et al., 1997).

1.4 Histórico sobre os principais trabalhos do gênero Lycoperdon abordando morfologia e divergências na classificação das espécies

Linnaeus (1753), tentou nomear várias espécies de fungos, entre elas, as do gênero Lycoperdon Pers.: L. aurantium L. (= Tubiferaceae), L. carpobulus L. (= Sphaerobulus stellatus Tode), L. epidendrum J.C. Buxb. ex L. (= Lycogala epidendrum Fr.), L. pedunculatum (= Tulostoma brumale Pers.), L. stellatum L. (= Tubiferacea), embora em sua maioria tenham sido transferidas para outros gêneros.

O gênero Lycoperdon foi erguido por Tournefort (1700: pag. 563), logo depois no século XVIII, outros autores usaram este nome para reconhecer fungos com o corpo de frutificação globoso a subgloboso e com o conteúdo interno pulverulento (Scopoli, 1772; Schaeffer, 1774). Embora, muitos táxons não pertencessem ao gênero Lycoperdon, a saber: L. pedunculatum Batsch, L. carpobolus Batsch, L. epidendrum (= Lycogala epidendrum (J.C. Buxb. ex L.) Fr.), L. stellatum L. per Rehl. (=Geastrum coronatum Pers.). No século XIX, Persoon (1801) foi quem validamente publicou o gênero Lycoperdon, inserindo-o em Gasteromycetes. Em seu trabalho intitulado Synopsis Methodica Fungorum”, o autor forneceu uma descrição do gênero: “peridium caulescens, apice demum ruptum, verrucis squamulosis, aut spinulosis obsitum (Pulvis seminalis viridis). O autor listou 14 espécies: Lycoperdon bovista Pers., L. candidum Pers., L. echinatum Pers., L. excipuliforme (Scop.) Pers., L. gigateum Batsch, L. gossypinum Bull.,

L. mammiforme Pers., L. molle Pers., L. perlatum Pers., L. pratense Pers., L. quercinum Pers., L. umbrinum Pers., L. utriforme Bull. e L. pyriforme Schaeff.

Ainda no séc. XIX, Fries (1829), contribuiu com a sistemática do gênero descrevendo nove espécies alocando elas em duas tribos: Bovistoides, composto pelas espécies de L. bovista, L. caelatum e L. pusillum; tribo Proteoides, composto pelas espécies de L. brasiliense (espécie brasileira), L. constellatum, L. gemmatum, L. gossypinum, L. pyriforme e L. saccatum. Ainda que, nenhum outro autor tenha utilizado essas tribos. O autor ainda indica L. album e L. esculentum como espécies duvidosas no gênero. Vittadini (1842), teve uma importante contribuição com a sistemática do gênero com o trabalho “Monographia Lycoperdineorum”. Logo em seguida, Bonorden (1857) em seu trabalho fornece uma lista com 31 espécies, onde muitas delas são hoje consideradas sinônimos: L. laxum Bonorden (= L. mammiforme), L. pistiliforme Bonorden (= L. excipuliforme), L rusticum Bonorden (= L. excipuliforme), etc.

Massee (1887), em sua obra “A Monograph of the genus Lycoperdon (Tournef.) Fr.”, fez uma exaustiva revisão dando descrições de 129 espécies, claro que muitos delas, hoje são sinônimos, ou pertencem a outro gênero, como exemplo: Lycoperdon sculptum Harkn. (= Calvatia sculpta Lloyd), L. serotinum Bonord. (=L. pyriforme), L. mundula Kalch. (= Bovista pusilla Batsch), etc). Ainda que, para todos os autores expostos até aqui, o conceito de caracterização das espécies dentro do gênero tenha sido de forma muito ampla, muitas dessas espécies estudas por eles continuam permanecendo em Lycoperdaceae.

No século XX, muito autores contribuíram exaustivamente para a compreensão do gênero Lycoperdon, e as descrições das espécies passaram a ser melhor caracterizadas e menos amplas (Lloyd 1905a). Lloyd revisou as exsicatas de Persoon (1801), Vittadini (1842), e Bonorden (1857), que foram reportadas para Europa. Subsequente a este trabalho, Lloyd (1905b) publicou um segundo trabalho em continuação as suas revisões de espécies de Lycoperdon, porém com espécimes norte Americanas.

Coker e Couch (1928) contribuíram com um extenso trabalho sobre Gasteromycetes para América do Norte, com descrições de 25 espécies de Lycoperdon, dando chave de identificação e boas figuras com fotos dos basidiomas, ainda que, algumas espécies tratadas por eles hoje são sinônimos em outros gêneros, por exemplo, Lycoperdon polymorphum Vittad. (= Bovista polymorpha Kreisel), L. oblongisporum

Berk. M.A. Curt. (= B. longispora Kreisel) e L. acuminatum Boc. (= B. acuminata Kreisel). Em seguida, Johnson (1929), seguindo os conceitos adotados por Coker e Couch (1928), reportou 25 espécies de Lycoperdon para Ohio, Canada. Como o autor seguiu Coker e Couch, os mesmos sinônimos são encontrados nesse trabalho. Na presente obra consideramos muitas das descrições de espécies como referências para aquelas registradas no Capítulo 1 e Capítulo 2, como por exemplo, L. atropurpureum Vittad., L. eximium Morgan e L. umbrinum.

Dentre muitos trabalhos sobre a diversidade de espécies de Lycoperdon em diversas partes do mundo (Cunningham, 1926, 1944; Kobayasi, 1937; Bottomley, 1948; Perdeck, 1950; Smith, 1951; Dennis, 1953; Garner, 1956; Pilát, 1958; Bowerman, 1961; Dissing e Lange, 1962; Dring, 1964; Herrera, 1964), devemos aqui pontuar os trabalhos de Cunningham, para Nova Zelândia e Austrália; o trabalho com as espécies africanas de Bottomley; e as espécies holandesas de Perdeck. Estes trabalhos oferecem um conceito de delimitação de espécies em Lycoperdon que permite o leitor a compreender a classificação adotada, ainda que não concorde com a sinonimização de uma ou outra espécie, a saber: Cunningham e Bottomley, em seus distintos trabalhos, consideram Lycoperdon excipuliforme um sinônimo de L. perlatum, fato esse, apropriadamente desconsiderado por Perdeck, o qual aqui consideramos o conceito de Perdeck. Kreisel (1973), em seu importante trabalho sobre Lycoperdaceae na Alemanha, inseriu os conceitos de tipos de exoperídio e capilício em Lycoperdaceae, além disso, Kreisel forneceu as localidades tipos de diversas espécies de Lycoperdon, ainda que por muitas vezes a localidade tipo era vagamente informada como Europa, como exemplo, em L. perlatum.

Demoulin (1968a), em seu trabalho de fungos gasteroides para Bélgica, segue os conceitos adotados por Perdeck (1950), e fornece descrições detalhadas de diversas espécies e sua distribuição. Demoulin (1968b) direciona os critérios para identificação das espécies: L. molle, L. muscorum and L. umbrinum, o qual o autor chama de grupo molle-umbrinum-muscorum. Ainda neste trabalho, o autor fornece uma tabela com todas as coleções estudadas por ele, discutindo os caracteres variáveis e os constantes nessas espécies.

Do mesmo modo, o autor seguiu aprofundando seus conhecimentos em Lycoperdon e, em sua principal obra “Le genre Lycoperdon en Europe et en Amérique du Nord Étude taxonomique et phytogéographique”, Demoulin (1972), reporta 30 táxons 31 para Europa e América do Norte. Além de explanar sobre a distribuição geográfica, elaborou uma chave de identificação levando em consideração não somente os caracteres morfológicos, mas também os tipos de substratos em que as espécies ocorriam. Este trabalho foi utilizado como principal meio de definição do gênero e identificação morfológica das espécies. Posteriormente, Demoulin e Schumacker (1972), forneceram um importante trabalho para o problema do grupo de Lycoperdon molle-umbrinum- muscorum, agora baseando-se na técnica de agrupamento, enumerando os caracteres taxonômicos. Nesta obra, por exemplo, os autores relatam que Lycoperdon lambinonii, anteriormente publicado por Demoulin (1972a), é uma espécie intermediário entre L. molle e L. umbrinum.

Outro importante pesquisador que contribuiu com os conhecimentos sobre a diversidade de espécies em Lycoperdon foi o Dr. Francisco Diego Calonge, que em várias oportunidades realizou parcerias com Dr. V. Demoulin. Calonge e Demoulin (1975), reportaram 12 espécies para o gênero e mais três espécies, que no presente trabalho trataremos como pertencentes a Lycoperdon, são elas: Calvatia excipuliformis, C. utriformis Bull. per Pers. e Vascellum pratense F. Smarda. A mais recente obra e que também iremos considera-la por diversas vezes em nossos tratamentos taxonômicos é Calonge (1998), que reportou 13 espécies para a flora Ibérica, ainda que, nesta obra o autor trata L. decipiens como sinônimo de L. atropurpureum, porém, veremos no decorrer desta obra que ambas são espécies distintas. Ainda, para a Inglaterra temos o trabalho de Pegler et al. (1995), seguindo principalmente os conceitos de Demoulin (1972b), Kreisel (1973), Calonge e Demoulin (1975). Pegler e colaboradores reportaram 13 espécies para o território inglês.

Na América do Sul os registros de Lycoperdon são reportados por Fries (1829), com a espécie L. brasiliense. Na sequência, Massee, (1887) reportou L. gardneri Berk., L. pyriforme, L. velutinum Berk. & M.A. Curt., para Venezuela; L. albinum, L. astrocaryi Berk. & M.A. Cooke, L. brasiliense e L. pusillum Fr. para Brasil e Peru; Spegazzini (1898) teve grande influência no conhecimento para a Argentina, com registros e novas espécies L. argentinum, L. asperum, L. bonariense etc. No século 20 temos o registro de alguns trabalhos esporádicos: Hennings (1904) e Sydow e Sydow (1907); sobre as espécies reportadas para o Brasil, Rick (1930, 1961) teve um grande papel no avanço do conhecimento das espécies de Lycoperdon e de outros gêneros e famílias; embora,

diversas espécies reportadas por Rick, hoje sejam consideradas sinônimos (Cortez et al., 2013).

A partir do século 21, muitos trabalhos tem contribuído para o conhecimento de Lycoperdon para o mundo (Calonge e Palacios, 2000; Demoulin, 2000; Martín e Jeppson, 2001; Baseia, 2005; Cortez et al., 2007, 2008, 2011, 2013) Todos estes trabalhos relacionados aqui são baseados exclusivamente em caracteres morfológicos.

Como visto anteriormente, após o trabalho de Hibbett et al. (1997), baseando em análises com sequencias da região espaçadora interna transcrita (ITS) e subunidade maior espaçadora (LSU) do nrDNA, houve uma corrida para compreensão dos relacionamentos filogenéticos em Lycoperdaceae e reclassificação dos gêneros tratados como “puffballs”, entre eles Lycoperdon.

1.5 Classificação de Lycoperdon com base em análises moleculares de ITS e LSU

Inicialmente, Krüger et al. (2001) com suporte de um especialista morfológico de Lycoperdaceae, o Dr. H. Kreisel, selecionaram espécies de Bovista Pers., Bovistella Morgan, Calvatia Fr., Lycoperdon, Morganella Zeller e Vascellum F. Smarda, todos os gêneros da família Lycoperdaceae. Adicionando às regiões usadas por Hibbett et al. (1997), a subunidade espaçadora menor (SSU) do nrDNA, os autores concluíram que Lycoperdon parece ser polifilético na família e que as delimitações genéricas entre ele e os demais gêneros (Bovistella, Morganella e Vascellum) eram indefinidas. Ainda, Krügger e colaboradores indicam que mesmo aliando seus dados moleculares aos morfológicos não podiam definir com precisão a delimitação desses gêneros e que, por isso mais trabalhos com mais adições de táxons e sequências eram necessários.

Krüger e Kreisel (2003), tentaram esclarecer o posicionamento de Lycoperdon pyriforme Schaeff., uma espécie morfologicamente próxima a Morganella por apresentar hábito lignícola e além de apresentar capilício na gleba, apresenta também abundante paracapilício. Para isto, os autores analizaram a região ITS e os caracteres morfológicos da espécie, concluindo que L. pyriforme não pertencia ao gênero Lycoperdon, e transferiram a espécie para Morganella erguendo o novo subgênero denominado de Apioperdon.

Posteriormente, Larsson e Jeppson (2008), adicionaram mais táxons ao conjunto de dados de trabalhos anteriores e realizaram análises com sequencias de ITS e LSU.

Estes autores chegaram aos seguintes resultados: Lycoperdon é parafilético, sendo que o tipo genérico L. perlatum formou agrupamento com Morganella, Vascellum e Bovistella. L. pyriforme não se agrupou com Morganella, mantendo-o então como espécie em Lycoperdon, sendo Apioperdon subgênero de Lycoperdon; as espécies de Calvatia excipuliforme e C. utriforme voltaram a pertencer ao gênero Lycoperdon. Seguindo esses resultados, os autores propuseram algumas mudanças quanto ao nível genérico, a saber: Lycoperdon sugb. Bovistella; L. subg. Lycoperdon; L. subg. Morganela; e L. subg. Vascellum. Embora, neste trabalho, Larsson e Jeppson (2008) tenham utilizado um número limitado de espécimes, principalmente oriundas do Hemisfério Norte.

1.6 Os subgêneros Morganella e Vascellum e seus históricos como espécies de Lycoperdon

O gênero Morganella Zeller foi estabelecido por Zeller (1948) para alocar espécies lignícolas que possuíam paracapilício em abundancia e o capilício estava sempre ausente. Posteriormente, Kreisel e Dring (1967) fizeram uma emendação no gênero e adicionaram os caracteres como: basidioma nunca excedendo os 30 mm de amplitude; subgleba reduzida, podendo ser com células compactadas ou celular; e paracapilício com incrustações da membrana glebal. Os autores transferiram para o gênero seis espécies anteriormente alocadas em Lycoperdon e uma nova espécie. Logo após, Ponce de León (1971), revisou o grupo e adicionou mais duas espécies, e em sua discussão geral excluiu Lycoperdon albidum (= Lycogalopsis solmsii E. Fisch), e L. pisiforme Henn (pode pertencer ao gênero Lycogalospis E. Fisch). Posteriormente, Morales et al. (1974) reportam a ocorrência de três espécies para Costa Rica. Em seguida, Suárez e Wright (1996), fazem um trabalho de revisão para as espécies sul-americanas, reportando espécies para Argentina, Bolívia, Brasil, Paraguai e Uruguai. Com base nos trabalhos elucidados, Cortez et al. (2007), transferiram a espécie brasileira L. benjaminii Rick para M. benjaminii (Rick) Cortez, Calonge & Baseia. Ainda para o Brasil, mais cinco novas espécies foram descritas M. albostipitata Baseia & Alfredo, M. arenicola Alfredo & Baseia M. nuda Alfredo & Baseia, M. rimosa Alfredo & Baseia e M. sulcatostoma C.R. Alves & Cortez (Alfredo et al., 2012, 2014; Alves e Cortez, 2013; Alfredo e Baseia, 2014). Em seguida, Rebriev e Bulakh (2015) descrevem para Rússia a nova espécie M. sosinii Yu. Rebriev & E. Bulakh.

Por outro lado, Vascellum F. Smarda foi erguido para alocar espécies de Lycoperdon que possuíam um diafragma separando a gleba da subgleba; abundante paracapilício e capilício quando presente era escasso (Pilát, 1958). O primeiro trabalho de revisão das espécies de Lycoperdon que apresentavam diafragma foi realizado por Ponce de León (1970), aqui foram transferidas seis espécies de Lycoperdon para o gênero Vascellum. Logo em seguida, Smith (1974), em seu trabalho de Vascellum para os Estados Unidos, discute a dificuldade em delimitar o gênero, tendo em vista que muitos espécimes não apresentavam um definido diafragma. Também, o autor adiciona seis espécies em seu trabalho, embora ele tenha excluído uma espécie reconhecida por Ponce de León (L. curtisii Berk.). Em seguida, L. endotephrum Pat. foi transferida para V. endotephrum (Pat.) Demoulin & Dring (Demoulin e Dring 1975). Do mesmo modo, Homrich (1975), e Homrich e Wright (1988), estudaram espécies Sul-americanas, reportando sete espécies para América do Sul, sendo quatros novas espécies e uma nova combinação, ainda, aqui os autores também consideram o diafragma como carácter de pouco valor taxonômico.

1.7 Caracteres macro e micromorfológicos em Lycoperdon, Morganella e Vascellum

Caracteres macroscópicos

Forma do basidioma – O basidioma em Lycoperdon pode ser encontrado de várias formas: depresso globoso, subgloboso, piriforme a turbinado (Fig. 1); em exsicatas malconservadas esses formatos podem ser alterados e essa característica não é relevante na determinação das espécies, é apenas utilizada de forma tradicional e para se ter uma ideia de como o corpo de frutificação foi encontrado.

Rizomorfas – O basidioma se corretamente coletado, poderá apresentar em sua base as rizomorfas, que são cordões de micélio de coloração esbranquiçada, e essa estrutura deve ser estudada com mais atenção sob microscópio.

Subgleba ou base estéril – Esta zona pode ser encontrada ocupando igual ou mais que um terço do basidioma, nesse caso foi determinado como bem desenvolvida; embora em Morganella essa estrutura seja sempre reduzida, ocupando menos de um terço do basidioma, ou até mesmo não existindo (Fig. 2). A coloração da subgleba pode variar de cor em Lycoperdon atingindo tons de marrons e cinza; em Morganella e Vascellum, pode

35 ser constante em tons de amarelo claro. Ademais, a subgleba quando observada em um corte transversal, pode ser celular, como na maioria das espécies de Lycoperdon; ou compactada, ocorrendo na maioria das espécies de Morganella. Também, ainda que pouco frequente, pode aparecer uma subgleba lanosa, como em L. ovoidisporum e L. pyriforme, ainda no primeiro, os espécimes apresentaram um transição de lanoso a celular (Cortez et al., 2011). O diafragma pode ocorrer tanto em espécies de Lycoperdon quanto em Vascellum, mas não foi observa em nenhuma das exsicatas de Morganella.

Figura 2. Tipos de subgleba em espécies de Lycoperdon. a. Subgleba reduzida e com células compactas, L. fuligineum (UFRN-Fungo 1768); b. Subgleba bem desenvolvida, celular e com diafragma L. marginatum (CORD756); c-d, f. Subgleba desenvolvida e celular: (c) L. perlatum (MA-Fungi 29372), (d) L. pratense (K200229), (f) L. utriforme (K200230) em basidioma parcialmente imaturo; e. subgleba bem desenvolvida e lanosa, (e) L. pyriforme (MA-Fungi 16908). Barras = 10 mm.

Ornamentação do exoperídio – De fato, o trabalho de Kreisel de 1962, citado aqui a obra re-editada (Kreisel, 1973), foi quem iniciou a caracterização em Lycoperdaceae, antes nenhum dos trabalhos tradicionais haviam empregado esforço na caracterização do perídio na família. O exoperídio pode ser composto por verrugas; espinhos com as pontas agudas; espinhos com as pontas alongadas, delgadas e curvadas; espinhos em conjuntos de três ou mais unidos por suas pontas, que dão uma aparência estelar; e espinhos

37 piramidais; essas ornamentações podem ser persistentes; ou cair com tempo, deixando amostra a superfície do endoperídio (Fig. 3). Essas ornamentações podem atingir até 6 mm de comprimento em espinhos, embora este padrão ocorra em espécimes de L. echinatum (Demoulin, 1972b; Calonge e Demoulin, 1975; Calonge, 1998), e que não foi reportado ainda na América do Sul; nas ornamentações em forma de verrugas o comprimento atinge 1 mm. O padrão de coloração pode ser em tons esbranquiçados a enegrecidos.

Superfície do endoperídio – Pode ser liso ou areolado; alguns espécimes, no qual a espécie teria endoperídio areolado, as aréolas não estavam presentes, ou estavam incompletas, mas poder ter ocorrido por duas situações: as más condições do espécime ou, o espécime em seu habitat sofreu pelas intempéries das chuvas; quando o exoperídio não é persistente, a superfície do endoperídio pode apresentar um aspecto furfuráceo; a coloração geralmente é em tons de amarelo. O aspecto enrugado, tem sido usado por exemplo, em espécies de Morganella (Alfredo e Baseia 2014), na descrição de M. nuda, embora na descrição os autores tenham considerado que a superfície do endoperídio varia de liso a enrugado; ao longo das análises morfológicas em Lycoperdon, esse carácter parecia ocasionado pelas condições dos espécimes, e não da natureza do endoperídio portanto, essa característica em Lycoperdon, Morganella e Vascellum é considerado como um carácter não informativo.

Padrão de desenvolvimento da deiscência – Em Lycoperdon, Morganella e Vascellum, a deiscência é sempre na porção apical do basidioma. Se o endoperídio apical é composto por micoesclereídeos ou células com terminações infladas, a deiscência tende a ser por um poro irregular ou por ruptura do perídio (exoperídio + endooerídio); os espécimes em Vascellum apresentam deiscência irregular ou até mesmo por ruptura do perídio; em Lycoperdon, a deiscência irregular pode ocorrer, por exemplo, em espécimes de L. marginatum; quando o endoperídio não é composto por micoesclereídeos e/ou células com terminações infladas, a deiscência sempre será por um poro apical regular; por isso, todas espécies em Morganella apresentam poro apical regular.

Textura da gleba – A gleba pulverulenta é a mais comum; ainda que também pode ser lanosa (somente encontrado em L. umbrinoides). A coloração é em tons de marrom, e algumas vezes pode ser tingida em tons de lilás e cinza.

Pseudocolumela – Não é comumente presente, e quando presente não foi taxonomicamente informativo para a separação das espécies; a coloração sempre acompanhava o tom da gleba.

Coloração das estruturas – Neste estudo usamos Küppers (2002), nos Capítulos 1 e 2; e Kornerup e Wanscher (1967), no Capítulo 3, que são as obras disponíveis no momento de realizar as identificações.

Caracteres microscópicos

Ornamentação do exoperídio – Os espinhos ou verrugas são compostos por: (1) hifas celulares denominadas esferocistos que apresentam formas globosas, subglobosas ou piriformes; também podem ser encontradas hifas com formas irregulares, mas que, tradicionalmente continuam sendo chamadas de esferocistos. Se estas células estão dispostas em cadeias, o termo está correto; e (2) hifas alongadas, podendo ser encontradas em grupos, com formas de clavas ou elípticas. O exoperídio é sempre analisado separando-se uma porção basal e uma porção apical, para verificar se há diferenças quanto a formas, tamanho e reação em Melzer, como por exemplo, em L atrum.

Hifas do endoperídio – O endoperídio é composto por hifas entrelaçadas, podendo ser encontrado hifas com terminações infladas e micoesclereídeos; por isso as análises do endoperídio são feitas separando a porção apical da porção basal. Na porção apical, que pode ser extraído deste o poro até metade do basidioma, podem ser encontrados micoesclereídeos e/ou hifas com terminações infladas. Os micoesclereídeos são células unitárias com formas irregulares, podendo ser sem reação ao Melzer até fortemente dextrinoide. As hifas com terminação infladas, apresentam as terminações distintas das hifas entrelaçadas normais, estas terminações irão apresentar diâmetro de 5 até 30 µm. Estes dois tipos de elementos podem auxiliar na descamação do perídio e formação do poro apical, acredita-se que em alguma fase do desenvolvimento todas as espécies podem apresentar estes elementos, até a formação completa do poro apical; em outras espécies estes elementos seguem persistentes, e isso é observado em espécies nos quais a deiscência é por poro apical irregular ou ruptura do perídio.

Figura 3 Tipos de ornamentação do exoperídio e superfície do endoperídio. a, d. Espinhos com as pontas alongadas, delgadas e curvadas; b, e, f. Verrugoso; c. Espinhos piramidais; a-c, e-f. Superfície do endoperídio liso; d. Superfície do endoperídio areolada. Todas as ornamentações apresentadas são caducas com o tempo. Barras = 2 mm.

Porção da gleba – Nesta região são encontrados os basidiosporos, capilícios e paracapilícios. Os basidiosporos podem ser globosos, subglobosos, ovoides a subelípticos (Fig. 5). A superfície do basidiosporos pode ser punctado, levemente verrugoso, verrugoso a fortemente verrugoso, sendo classificado de A-D seguindo Demoulin (1972a; b) e Moyersoen e Demoulin (1996): [A] ornamentação lisa a punctada; [B] ligeiramente verrucosa; [C] verrucosa e [D] fortemente verrucosa (Fig. 5-6). Os basidiosporos podem ter una reação em Melzer como fortemente dextrinoide; marrom claro a marrom em KOH 5%; e possuírem ou não pedicelos (restos de esterigmas).

As hifas encontradas na gleba são chamadas de capilício ou paracapilício. Os capilícios podem ser asseptados ou septados, possuir poros ou não; quando com poros, podem ocorrer em diferentes quantidades, proporções de tamanho e formas, estas últimas de difícil avaliação, sendo somente possível sob microscópio eletrônico de varredura. As hifas do capilício não apresentam reação ao Melzer ou fracamente dextrinoide; e são amareladas a marrons em KOH 5%. Os paracapilícios, são sempre septados; algumas vezes ramificados; sem reação em Melzer, sempre cianófilos, e sempre hialinos em KOH 5%. 40

Figura 4 Tipos de esferocistos e células alongadas do exoperídio apical; micoesclereídeos e hifas infladas do endoperídio apical. a. Células alongadas e dextrinoide de L. atrum; b. Esferocistos em cadeias de L. perlatum; c. Esferocistos com formas irregulares de L. pyriforme; d, f. Micoesclereídeos com formas irregulares de L. marginatum e Lycoperdon sp.; e. Hifas com terminações de L. umbrinum. a, c, e. Barras = 20 µm; b. Barra = 100 µm; d, f. Barras = 10 µm.

Subgleba – As hifas da subgleba, podem ser septadas, ramificadas, sem reação em Melzer a fortemente dextrinoide.

Pseudocolumela – A hifas da pseudocolumela são, facilmente confundidas com o capilício; quando presente estas estruturas são poucos septadas, pouco ramificadas e desprovidas de poros; sem reação em Melzer, acianófilas e amareladas a marrom em KOH 5%.

Rizomorfas – São compostas de dois tipos de hifas, um tipo mais interno e o outro mais externo. As hifas mais internas podem alcançar até 5–20 µm diâm., são septadas, com septos incompletos frequentes, sem reação em Melzer ou fracamente dextrinoide. As hifas externas são de calibre mais finos do que as internas 1–3 µm diâm., asseptadas, sempre fortemente dextrinoides. Aderidas as hifas externas podem haver cristais de diferentes formatos, de agulhas, bastonetes ou losangos; podem estar em aglomerações ou isolados; geralmente os cristais que estão aglomerados, possuem formatos de agulhas ou bastonetes, enquanto que, aqueles que apresentam formatos de losangos ocorrem isolados. No futuro estes cristais poderão ajudar na separação grupos de espécies ou ainda, separação de espécies. Para isso, é necessário que as coletas do basidiomas sejam

41 realizadas de modo que, estas estruturas permaneçam preservadas juntamente com o basidioma.

Figura 5 Exemplos de basidiosporos quanto a forma e classificação da ornamentação espécies de Lycoperdon. a. basidiosporos lisos [A], L. pyriforme (NY0071979); b-d. Levemente verrugoso [B], L. marginatum (NY00398533), L. eximium (NY0071978), L. umbrinum (MA-Fungi 63394); e-f. Verrugoso [C], L atrum (UFRN-Fungos 832), L. perlatum (NY00793106); g-i. Fortemente verrugoso [D], M. nuda (UFRN-Fungos 1766), M. velutina (NY00839022), L. decipiens (MAF27674); f, g. Basidioporos com o número de ornamentações contadas e diâmetro medido para o cálculo de densidade de ornamentação. Barras = 10 µm.

Figura 6 Exemplos de basidiosporos com as classificações de A-D em microscopia eletrônica de varredura (MEV). a, b. Punctados [A], L. pyriforme e L. compactun (= M. compacta); c, d. Levemente verrucosos [B], L. arenicola (= M. arenicola), L. marginatum; e, f. Verrucoso [C], L. perlatum, L. atrum; g-i. Fortemente verrucoso [D], L. fuligineum (= M. fuliginea), L. atropurpureum e L. mauryanum. Barras = 1 µm.

2 JUSTIFICATIVA, HIPÓTESES E OBJETIVOS

Em Lycoperdon e nos gêneros afins, em muitas ocasiões é difícil delimitar as espécies somente baseado em seus caracteres morfológicos, isto porque pode encontrar- se pouca variabilidade interespecífica, ou porque exista uma grande variabilidade intraespecífica (Demoulin, 1968b, 1972b; Demoulin e Schumacker, 1972; Calonge, 1998). Embora os estudos moleculares baseados em um ou mais loci permitam analisar os especímes independentemente dos dados morfológicos (Phosri et al., 2014; Rusevska et al., 2014, 2015). A partir da informação obtida pelas análises moleculares se pode realizar um segundo estudo morfológico por clados, que permite, em ocasiões, identificar caracteres com valor taxonômico que antes poderiam ter passado por alto (Crespo and Pérez-Ortega, 2009). Entretanto, ainda que seja muito importante encontrar caracteres morfológicos que permitam separar as espécies, às vezes estão tão ocultos que é difícil descobri-los. Existem numerosos exemplos de táxons em fungos gasteroides cuja problemática para delimitar possíveis espécies mediante caracteres morfológicos tem dado resultados graças aos estudos moleculares, por exêmplo, em Phosri et al.( 2014) e Rusevska et al. (2014, 2015); nestes estudos foram incluídos espécimes tanto do Hemisfério Norte como do Hemisfério Sul.

Em fungos, a região compreendida entre os espaçadores de transcrição interna ITS1 e ITS2 do DNA ribossômico nuclear (ITS nrDNA), incluída a subunidade 5.8S, se tem designado como região barcode, já que permite delimitar uma porcentagem muito alta das espécies descritas (Schoch et al., 2012). Nos trabalhos sobre o género Lycoperdon de Larsson e Jeppson (2008) e Jeppson et al. (2012), se analizaram estas regiões junto aos dominios D1-D2 da subunidade maior do DNA ribosómico nuclear (LSU nrDNA); contudo, nestes estudos não foram incluídos espécimes do Hemisfério Sul.

Neste presente trabalho, se idealizaram as seguintes hipóteses:

Hipótese 1: A região barcode de fungos (ITS nrDNA) permite delimitar as espécies de Lycoperdon.

Hipótese 2: Basendo-se nos caracteres morfológicos e moleculares (região barcode ITS nrDNA), se confirma que a maioria dos espécimes sul-americanos constituem táxons distintos daqueles do Hemisfério Sul.

Hipótese 3: O gênero Lycoperdon é um gênero monofilético, e os gêneros Morganella e Vascellum não são gêneros independentes de Lycoperdon.

Para avaliar as hipóteses mencionadas nos propomos analisar se a diversidade de espécies de Lycoperdon do Hemisfério Norte coincide com a diversidade de espécies do Hemisfério Sul. Com isso, propomos os seguintes objetivos específicos:

Objetivo 1. Confirmar se a região barcode (ITS nrDNA) permite identificar as especies de Lycoperdon, do Hemisfério Norte. (Capítulo 1).

Objetivo 2. Identificar as espécies de Lycoperdon do Hemisfério Sul mediante sua região barcode. (Capítulo 2).

Objetivo 3. Confirmar a posição taxonômica das espécies brasileiras do gênero Morganella (Capítulo 3).

Objetivo 4. Revisar os caracteres morfológicos que permitem delimitar as espécies (Capítulo 1, 2 e 3).

Objetivo 5. Descrever as novas espécies com base nos caracteres morfológicos e moleculares. (Capítulo 2 e 3).

Objetivo 6. Propor as mudanças taxonômicas e nomenclaturais para estabelecer uma sistemática revisada que sirva como ponto de partida para estudos futuros (Capítulo 1 ao 4).

3 MATERIAL E MÉTODOS 3.1 Coleções estudadas

Os espécimes estudados foram obtidos por meio de empréstimos dos herbários internacionais e nacionais: Universidad de Buenos Aires (BAFC), IMBIV-Museo Botánico (CORDC), Herbarium Harvard University (FH/HUH), Université de Liège (LG) e herbário pessoal do Dr. Vincent Demoulin (VDEMOULIN), MA-Fungi (Real Jardín Botánico de Madrid), MCF-Fungi (Macedonian Collection of fungi), The New York Botanical Garden (NY), Royal Botanical Gardens (K); Universidade Federal do Rio Grande do Sul (ICN), Instituto Anchietano de Pesquisas/UNISINOS (PACA), Universidade Federal do Rio Grande do Norte (UFRN-Fungos); com isso foi possível analisar 11 espécies tipos (tabela 1). Infelizmente, os herbários da Universidade Federal de Pernambuco (URM), Instituto de Botânica (SP) e Herbarium of U.S. National Fungus Collection (BPI), não enviaram seus espécimes com tempo hábil para serem incorporados no presente trabalho. O herbário FH/HUH, não concedeu permissão para incluir seus espécimes nas análises moleculares.

3.2 Análises morfológicas 3.2.1 Analises dos caracteres macromorfológicos

Os estudos dos espécimes foram realizados em parte no Laboratório de Biologia de Fungos do Departamento de Botânica e Zoologia – Centro de Biociências da Universidade Federal do Rio Grande do Norte; onde foram analisadas as exsicatas dos herbários: ICN, FH/HUH, NY, K, UFRN-Fungos e PACA; as exsicatas do herbário MA- Fungi, foram analisadas no laboratório do Departamento de Micología do Real Jardín Botánico de Madrid; as exsicatas do herbário (K) foram parcialmente analisadas no próprio Royal Botanical Gardens e depois foram concluídas no laboratório de Fungos (UFRN); as exsicatas do herbário LG e VDEMOULIN, foram analisadas no laboratório de micologia da Universidade de Liege (Université de Liège – Bélgica).

As análises das estruturas macroscópicas foram realizadas seguindo a proposta de Silva et al. (2014); assim, os basidiomas foram analisados quanto a sua forma, coloração, e o tamanho foi medido com paquímetro (altura × comprimento) considerando seus extremos; foram observadas as ornamentações do exoperídio, superfície do endoperídio,

a olho nu ou com ajuda do esteromicroscópio; as cores das macroestruturas foram codificadas de acordo com Kornerup e Wanscher (1967) e Küppers (2002).

A subgleba foi observada considerando os seguintes aspectos: (1) celular ou células compactadas – para isso o basidioma foi cortado transversalmente com ajuda de um estile ou lamina de barbear; então foi observada se a textura era de células globosas ou isodiamétricas que possibilitavam a medição de seu diâmetro, caracterizando uma subgleba celular; se as células eram horizontalmente elípticas impossibilitando de se medir seu diâmetro, foi considerada como células compactadas. (2) bem desenvolvida ou reduzida – para isso foram consideradas as literaturas tradicionais que usavam os termos, por exemplo, igual ou maior que um terço ou uma metade para subgleba bem desenvolvida; menor que um terço ou um quarto para subgleba reduzida (Massee, 1887; Cunningham, 1926; Coker and Couch, 1928); assim, fizemos os seguintes cálculos: altura total do basidioma dividido por 3; se o resultado for menor que um terço (< ⅓) do basidioma = reduzida; se o resultado for maior igual a um terço (≥ ⅓) do basidioma = bem desenvolvida (Fig. 2).

3.2.2 Análise dos caracteres micromorfológicos

As microestruturas foram montadas em lâminas e cobertas com lamínulas contendo uma pequena porção do basidioma misturado a uma solução, que pode ser azul de algodão em lactofenol, reagente de Melzer ou hidróxido de potássio (KOH) a 5% de acordo com Cunningham (1944) e Bottomley (1948); seguindo Cunningham (1944), as laminas contendo a solução mais a porção do basidioma foram aquecidas até começar a aparecer bolhas; em seguida esperou-se que esfriasse, para então serem observadas em microscópio óptico; essa ação permite que as estruturas retornem o seu tamanho original, e os capilícios quando apresentarem lúmen, os mesmo ficarem livres de bolhas.

Para analisar as ornamentações do exoperídio(Fig. 3a), e verificar se ocorrem esferocistos, células alongadas ou clavadas (Fig. 4); bem como verificar as hifas do endoperídio, e como elas estão dispostas; o perídio foi seccionado transversalmente com ajuda de laminas de barbear, e logo em seguida, foram montadas lâminas contendo reagente Melzer e partículas do perídio seccionado, novamente aqueceu-se a lamina, sem pressionar para não romper as camadas exo- + endoperídio, e após alguns segundos depois de esfriada, foi observado em microscópio ótico (Fig. 3b); posteriormente a

47 primeira observação a lâmina contendo solução mais o perídio foi pressionada até rompimento das camadas do perídio (Fig. 3 a, c-f) para realizar as medições; tanto o exoperídio quanto o endoperídio foram analisados levando-se em consideração porção apical (do poro apical até os limites da subgleba) separado da porção basal (dos limites da gleba até a base).

Os basidiosporos foram analisados seguindo Demoulin (1972a; b), Moyersoen e Demoulin (1996); foram medidos 10 basidiosporos por espécime; nas descrições das espécies, as ornamentações dos basidiosporos foram incluídas, e quando necessário nós discutiremos o tamanho dos basidiosporos sem ornamentação; também, os basidiosporos foram classificados como: lisos a punctados [A], levemente verrucoso [B], verrucoso [C] e fortemente verrucoso [D] (Fig. 5-6); com o propósito de chegar a uma dessas classificações, primeiramente nós analisamos as lâminas em microscópio sob objetiva de 40x, para checar o padrão de ornamentação; se a ornamentação não foi visualizada os basidiosporos são lisos a punctados [A] (Fig. 5a), se a ornamentação foi facilmente observa em objetiva de 40x, os basidiosporos são fortemente verrucosos [D] (Fig. 5 g-i); as outras duas classificações são mais complicadas, porque o limite entres B-C é mais sensível; se o observador consegue visualizar as ornamentações dos basidiosporos em objetiva de 40x, mas não conta-las, é necessário que mude a objetiva para 100x; já neste objetiva, se o observador pode contar as ornamentações, a classificação é [C] (Fig. 5 e-f); se as ornamentações continuam sem se poder contá-las, a classificação é [B] (Figs. 5 b- d); para checar a densidade de ornamentação dos basidiospores foi usado o seguinte cálculo: número de ornamentação dividido pelo diâmetro do basidiósporo multiplicado por 3,14, assim, nós encontraremos o número de verrugas por uma circunferência reduzida de 10 µm; as fotos das microestruturas foram confeccionadas no MO Nikon “Eclipse Ni” com câmera fotográfica “DSRi” acoplado ao microcomputador, usando o programa de suporte para a captura das imagens – “Software de Imagem de Microscopia NIS-Elements”; a ornamentação dos basidiosporos também foi analisada sob microscopia eletrônica de varredura (Fig. 6) seguindo a metodologia adotada por Cortez et al. (2008) e Silva et al. (2011).

3.3 Análises moleculares 3.3.1 Isolamento do DNA, amplificação e sequenciamento

As extrações de DNA e amplificações das reações em cadeias da polimerase (PCR) foram realizadas em dois laboratórios distintos: (a) Laboratório de Biologia Celular e Genética de Plantas no Departamento de Biologia Celular e Genética – UFRN; (b) Laboratório de Biologia Molecular do Real Jardín Botánico de Madrid – RJB/CSIC; as extrações de DNA foram realizadas com basidiomas herborizados de tecidos extraídos das partes internas do basidioma e colocando-as em tubos eppendorf 1,5 ou 2 ml (Fig. 7 a), usando o kit de extração DNeasy Plant Kit (Qiagen 69106), seguindo as instruções do fabricante; excerto em três modificações: para agregar o tampão AP1 e o RNase A, em vez de incubar 10 min a 65ºC, se incubou toda a noite a 60ºC, para favorecer a ruptura das paredes quitinosas dos fungos; para adicionar por uma segunda vez 500 μl do tampão de limpeza AW, seguido, de realizar a centrifugação de 1 min a 8000 rpm e descartar-se a fração líquida, se realizou uma segunda centrifugação a 14.000 rpm durante 2 min, para assegurar que a coluna ficava completamente seca de álcool, já que este poderia interferir nas posteriores reações de amplificação; e para aumentar a concentração de DNA eluído, o tampão AE foi pré-aquecido a 60ºC. Uma vez finalizado o isolamento se quantificou o DNA utilizando o espectrofotómetro ARN/ADN calculator GeneQuant II (Pharmacia Biotech). As amplificações se realizaram em reações individuais mediante IllustraTM PuReTaqTM Ready-To-GoTM PCR beads (GE Healthcare, UK). Cada tubo contendo em seu interior uma esfera desidratada com todos os reativos para uma amplificação por PCR (Taq polimerasa, dNTPs, cloreto de magnésio e tampão Tris-HCl) (Fig. 7 b), para que se possa adicionar os iniciadores (1 μl a 10 μM), o DNA genômico (1.0-10.0 μl, dependendo da concentração) e água ultrapura estéril (Water 95284 Biochemica, SIGMA), até um volume de 25 μl. Para o marcador ITS, o fragmento que se tentou amplificar compreende os espaçadores internos ITS1 e ITS2, incluindo a subunidade 5.8S do DNA ribossômico nuclear. Para o marcador LSU, o fragmento selecionado compreende os domínios D1-D2 da subunidade maior do DNA ribossômico nuclear (LSU nrDNA). Os iniciadores utilizados para a amplificação destas regiões se indicam na figura 8, e na Tabela 1 se inclui a sequência dos mesmos.

Se realizaram diferentes estratégias de amplificação, já que em geral se realizaram

49 isolamentos de DNA de material de herbário, para conseguir os amplímeros desenhados. Em primeiro lugar, se realizaram amplificações diretas com os iniciadores ITS5 e ITS4 (Fig. 8) (White et al., 1990), no caso de ITS, e com os iniciadores LROR (Rehner e Samuels, 1994) e LR7r (Vilgalys e Hester, 1990), para o de LSU. Para cada série de amplificação foi corrido gel de comprovação para determinar a qualidade e quantidade do produto amplificado. Para isto, se realizou uma electroforese em gel de agarose a 2% (Agarose D-1 low EEO, Conda S.A., Pronadisa) com tampão TAE 1x (tampão TAE 50x: EDTA 0.05M, Tris Acetato 2M). Como agente intercalante de DNA se adicionou 1 μl de SYBR SafeTM por cada 10 ml de agarose. Em cada poço foi preenchido com 5 μl de produto amplificado mesclados com 1 μl de tampão de carga LB diluído 1:10 (LB 6x: azul de bromofenol 0.25 %, xilencianol 0.25 %, glicerol 30 %). Para conhecer a longitude dos possíveis amplímeros foi realizado como padrão de peso molecular o marcador 1 Kb Plus DNA Ladder, diluído 1:19. A eletroforeses foi percorrida a 100V durante 30 min, tendo em vista que os moldes dos géis utilizados são de 10 × 15 cm. A visualização do DNA foi por meio do sistema de documentação de géis G:BOX EF (SynGene) ou Cleaver. Em cada série de amplificações se incluiu sempre um controle negativo. Uma vez preparados os tubos de PCR beads (Fig. 7 b) se procedeu à amplificação utilizando o termociclador MJ Research-PTC-200 (Fig. 7 c). Os programas empregados nas amplificações para cada região correspondem a Martín e Winka (2000) para ITS, e Telleria et al. (2013), para LSU.

Naqueles casos em que não foram visualizadas banda depois da amplificação direta (Fig. 7d), se procedeu a realizar una amplificação por partes. Para o marcador ITS se utilizou os pares de iniciadores ITS1F/ITS2 e ITS3/ITS4 (White et al., 1990), e para o marcador LSU, LROR/LR5 (White et al., 1990) e LR3R /LR7r (Vilgalys e Hester, 1990) (Fig. 8).

Tabela 1 Iniciadores empregados na amplificação dos fragmentos ITS e LSU nrDNA. Indicação de suas sequências e temperatura de hibridização.

Região Primer Sequências (5’...3’) T hibridização

ITS1F CTT GGT CAT TTA GAG GAA GTA A 67ºC

ITS2 GCT GCG TTC TTC ATC GAT GC 57ºC

ITS3 GTC TTG AAA CAC GGA CC 57ºC ITS ITS4 TCC TCC GCT TAT TGA TAT GC 53ºC

ITS4-B CAG GAG ACT TGT ACA CGG TCC AG 67ºC

ITS5 GGA AGT AAA AGT CGT AAC AAG G 69ºC

LROR ACC CGC TGA ACT TAA GC 57.9ºC

LR3 CCG TGT TTC AAG ACG GG 56ºC LSU LR5 TCC TGA GGG AAA CTT CG 58.8ºC

LR7r TAC TAC CAC CAA GAT CT 46.2ºC

Quando tão pouco foi possível visualizar produto das amplificações (Fig. 7 e), se realizaram nested PCR para ITS e seminested PCR para LSU. No caso do ITS se realizou uma primera amplificação com 1 μl de DNA genômico e o par de iniciadores ITS1F/ITS4B (Gardes e Bruns, 1993), e uma segunda amplificação (PCR nested) com os pares de iniciadores ITS5/ITS4, naquela que se incluiu com DNA de 1 μl da primeira amplificação. No caso do LSU, se realizou uma primeira amplificação com os pares de iniciadores LROR/LR7r e 1 μl de DNA genômico, depois se conduziu uma segunda amplificação com 1 μl da amplificação anterior e os pares de iniciadores LROR/LR5 e LR3R/LR7r por separado.

Aquelas amplificações em que se observaram bandas no gel de comprovação se procedeu a purificação. Dependendo da qualidade e concentração do produto amplificado, se utilizou um dos seguintes protocolos:

(1) nas amplificações que se obtiveram bandas duplas ou uma banda muito grossa (Fig. 7 f), se utilizou o kit QIAquick Gel Extraction (QiaGen®). Para esta purificação, em primeiro lugar se realizou uma segunda eletroforese em gel de agarose a 2 %; nos géis adicionou-se os 20 μl restantes de amplificação aos que se adicionou 1 μl de LB. No gel se deixou um poço vazio entre as amostras para evitar arrastrar amostras vizinhas ao cortar. Para assegurar que as bandas se separaram corretamente se deixou correr o gel a 120 V durante 45 min, já que os moldes eram de 10 × 15 cm. Depois se visualizaram as

51 bandas com o transluminador de luz azul SafeImagerTM 2.0 Blue-Light Transilluminator ou Clear View UV Transilluminator Cleaver, e se cortaram utilizando palitos de madeira estéreis. Cada porção de agarosa com sua banda correspondente se introduziu em um tubo eppendorf de microcentrífuga de 1.5 ml, e despois se seguiu o protocolo indicado pelo fabricante.

Figura 7. Materiais e equipamentos usados durante a extração e amplificação do DNA. a. Colunas (lilás e brancas) do kit de extração; b. Tubos eppendorfs contendo em seu interior uma esfera desidratada com todos os reativos para a amplificação por PCR; c. Termociclador; d. Transluminador acoplado com câmera fotográfica para a visualização e confecção de fotografias dos géis em agarose; e-f. Fotografias dos géis em agarose; e. Gel sem as bandas de amplificação da PCR; f. Gel contendo as bandas dos produtos amplificados da PCR.

Figura 8. Esquema do nrDNA no qual se pode observar as regiões dos marcadores e a posição dos iniciadores. Baseado em http://sites.biology.duke.edu/fungi/mycolab/.

(2) o segundo protocolo de purificação que foi empregado para aquelas amplificações em que os géis de comprovação se visualizou uma única banda bem definida e intensa (mais de 20 ng/μl). A purificação foi realizada com a enzima Exosap, IllustraTM ExoStar-1-Step (GE Healthcare, UK) segundo as instruções do fabricante, com a enzima diluída a 1:10. Depois de adicionar 8 μl de Exosap 1:10 aos tubos que continham as amplificações; estes foram introduzidos no termociclador MJ Research- PTC-200 para a eliminação dos restos de dNTPs, dímeros, etc. Se utilizou o seguinte programa: um ciclo à 37ºC durante 30 min (ativação e atuação da enzima), um ciclo a 80ºC durante 15 min (desativação da enzima) e um ciclo de conservação à 4ºC até que as purificações fossem armazenadas ao freezer (4ºC).

Para cada série de purificação se realizou também um gel de comprovação para assegurar que, sobre tudo nos casos em que haviam cortado as bandas, não se havia perdido o DNA. Estas eletroforeses foram realizadas sob as mesmas condições que as dos géis de comprovação das amplificações.

As purificações foram enviadas para serem sequenciadas a serviço de sequenciamento de DNA da Macrogen (Coreia), com os iniciadores (10 μM) que se haviam utilizado nas amplificações, para obter tanto a sequência direta (forward) quanto a reversa (reverse).

Figure 9. Formação das sequências consensos e busca no megablast para verificar se há contaminação das mesmas. a. Visualização das sequências direto e reverso e formação da sequencias consenso; b-g. Submissão da sequencias consenso no megablast disponível na web site NCBI.

Para obter as sequências consenso das regiões analisadas de cada uma das amostras, editaram-se as sequências direta e reversa utilizando o programa Sequencher 4.1.4 ou no programa Genious Pro v4.8.5 (Fig. 9 a). Uma vez obtidos os consensos, para comprovar que estas sequências correspondiam ao gênero de espécie de estudo e que não provinham de uma contaminação, se analisaram uma por uma com a opção de busca megablast (BLAST®) (Fig. 9 b-g) disponível no NCBI (National Center of Biotechnology Information (Altschul et al., 1997).

3.3.2 Identificação molecular das espécies de Lycoperdon

As sequencias de ITS obtidas (Capítulo 1-3) e a de LSU (Capítulo 3) foram alinhadas usando o programa Seaview versão 4.6 (Galtier et al., 1996; Gouy et al., 2010) para múltiplas sequencias. As sequencias foram comparadas como homólogos de Lycoperdaceae do GenBank principalmente as sequencias publicadas por Larsson e Jeppson (2008), Jeppson et al. (2012), Kumla et al. (2013) e Kim et al. (2016). Foram usadas como grupo externo as sequencias de Bovista (Capítulo 1-2), Mycenastrum e Tulostoma (Capítulo 3). Onde ocorreram ambiguidades no alinhamento caracteres informativos foram escolhidos. Onde haviam gaps foram marcados com “–“, nucleotídeos não resolvidos e desconhecidos na sequencia foram indicados com “N”. Para analisar se a sequência ITS é uma boa região “barcode” para identificar as espécies de Lycoperdon, se realizou um agrupamento das sequências, mediante uma análise de distância com o algoritmo de Kimura-2-Parametros (K2P) sob Neighbor- Joining (NJ) (Meier et al., 2006; Chen et al., 2010; Ramadan and Baeshen, 2012). Se obtiveram os valores de PCI (porcentagem de identificação correta) e a variação intraespecífica e interespecífica utilizando o programa TaxonDNA disponível em http://taxondna.sf.net/. Para avaliar se os agrupamentos obtidos por NJ eram apoiados, se realizaram análises de Máxima Parcimônia (MP) e de inferência bayesiana. A análise de Máxima Parcimônia (MP) foi conduzida no programa PAUP versão 4.0a152 (Swofford, 2002). Como medida de apoio para a análises MP, utilizou-se o método de bootstrap (bs) (Felsenstein, 1985), com a opção fast-step, retendo 10000 réplicas por árvore, e se calcularam também os índices de consistência (CI; Kluge e Farris, 1969), de retenção (RI; Farris, 1989) e o índice de consistência reescalado (RC; Farris, 1989).

Para a análise Bayesiana (Larget e Simon, 1999; Huelsenbeck e Ronquist, 2001), 55 foi utilizado o programa MrBayes v. 3.1. A análise se realizou baseada no modelo de tempo reversível (Rodríguez et al., 1990), incluindo a estimação de sítios invariáveis e assumindo uma distribuição gama com seis categorias (GTR+I+G), modelo selecionado com o programa MrModeltest v. 2.3 (Nylander, 2004). Mediante o MrBayes se realizaram as análises independentes e simultâneas, a partir de distintas árvores aleatórias, ao longo de 2.000.000 de gerações correndo quatro cadeias paralelas e guardando-se as árvores e os valores dos modelos de cada 100 gerações. De cada 1.000 árvores geradas em cada uma das duas análises se tomou uma como amostra para mediar a similaridade entre elas e determinar o nível de convergência entre as duas análises. O MrBayes obteve a árvore consenso pela regra da maioria a 50% e os valores de probabilidade dos nós.

No Capítulo 3, para as análises preliminares se realizou um alinhamento combinado das sequências de ITS e LSU, excluindo as sequências de EMBL/GenBank/DDBJ das que não estavam disponíveis as sequências das duas regiões.

A visualização dos dendogramas foi realizado com auxílio do TreeView (Page, 1996) e foram editados com auxílio do programa InkScape Wink, versão 0.92.0 r15299 (http://wiki.inkscape.org/wiki/index.php?title=Release_notes/0.91&oldid=98966).

3.4 Nomenclatura

A nomenclatura micológica e de plantas está de acordo com o Código Internacional de Nomenclatura para Algas, Fungos e Plantas (McNeill et al., 2012). Tendo em vista, que a presente obra de tese de doutorado não possui ISSN e ISBN, e tem como o seu propósito a aquisição de título de doutorado em Sistemática e Evolução, o qual é submetida a Universidade Federal do Rio Grande do Norte, cabe ressaltar que nenhuma das novidades nomenclaturais mencionadas pode-se considerar validamente publicada na própria obra de tese de acordo com o Art. 30.8. De fato, nenhuma destas tais novidades nomenclaturais pretende-se ser publicadas de forma efetiva aqui, a não ser aquelas que foram (no caso dos Capítulos 2 e 3) ou serão publicadas de forma efetiva por meio de aceitação e publicação dos diferentes capítulos em revistas científicas correspondentes. Se incluem, todavia, as diagnoses, citações de material tipo e números de MycoBank tal qual como se deveriam aparecer na efetiva publicação. Por esses motivos, pedimos aos possíveis leitores desta obra que não citem as novidades

nomenclaturais que aqui aparecem em seus respectivos capítulos, excepcionalmente se estas forem citadas a partir de suas efetivas publicações, uma vez que tenha sido realizada.

4 RESULTADOS

A partir dos empréstimos realizados dos herbários nacionais e internacionais supracitados, foi possível revisar 334 exsicatas, totalizando 888 espécimes; houve uma média de 2,7 espécimes (ou basidiomas) por exsicata; com isso, obteve-se um sucesso de amplificação da região ITS em 220 exsicatas (65,9 %), em 96 amostras (28%) não se obteve sucesso de amplificação (Fig. 10); sendo que os resultados de sucesso de amplificação obtidos neste estudo são equivalentes aos encontrados por Schoch et al.

1000 888 900

800

700

600

500

400 334 300 220 200 96 100

0 Número total de Quantidade de exsicatas Números de amostras com Números de amostras sem basidiomas analizados revisadas ITS amplificado amplificação (2012).

Figura 10. Número de basidiomas analisados, considerando o número de exsicatas, as amostras com sucesso de amplificação da região ITS, e as amostras sem sucesso de amplificação da região ITS.

Os resultados se apresentam nos seguintes capítulos: Capítulo/Chapter 1: Is the ITS barcode useful to discriminate Lycoperdon species?

Capítulo/Chapter 2: Integrative taxonomy for the identification of species of Lycoperdon (Basidiomycota) from Central and South America.

Capítulo/Chapter 3: Revision of species previously reported from Brazil under Morganella.

Capítulo/Chapter 4: Updated check-list to Lycoperdon from South America.

5 DISCUSSÃO GERAL

A ideia de usar sequências de DNA igual a um código de barras usado em lojas e supermercados tem se mostrado promissor e de muita ajuda em trabalhos voltados a identificação de espécies em amostras ambientais (solo, pedaços de madeira em decomposição, folhagem, etc.) (Hogg and Hebert, 2004; Blaxter et al., 2005). Assim também, o uso do DNA barcode tem auxiliado os taxonomistas em identificar espécies quando os caracteres morfológicos não são suficientes para segregá-las morfologicamente (Page et al., 2005); e a possibilidade de identificar espécies não é restrita somente aos animais como proposto inicialmente (Hebert et al., 2003), mas também para descriminar espécies em algas (Robba et al., 2006), plantas (Taberlet et al., 2007) ou fungos (Smith et al., 2007). A sequência de DNA para ser usada como barcoding deve possuir um comprimento curto de pares de bases (600 – 700 bp), de fácil amplificação (Hebert et al., 2003). A região ITS como barcoding de fungos passou a ser amplamente aceita pela comunidade científica após o trabalho de Schoch et al. (2012). No presente trabalho, a região ITS mostrou-se ser promissora na identificação de espécies de Lycoperdon. Nesse sentido, nossos resultados mostram 65,9 % de sucesso na amplificação contabilizando todos os capitulos, embora esses valores sobem quando observados somente para o capítulo, onde trabalhou-se com espécimes de somente dois herbários (87%), em contraste aos 28% de falha na amplificação somando-se os espécimes analisados em todos os capítulos, enquanto que somente para o capítulo 1 esse porcentagem cai para 13%; assim como aqueles obtidos por Schoch et al. (2012), onde o sucesso de amplificação da região ITS foi de 90%. Ainda que, a falha na amplificação do ITS em Lycoperdaceae dever ser pelo fato da gleba ser pulverulenta, no qual os esporos são transportados facilmente pelo ar e então sendo depositado em outras espécies, criando um problema durante o processo de extração e amplificação. Usando a identificação baseado na distância entre sequencias, foi obtido um sucesso de 80% na identificação de espécies, embora, em Hebert et al., (2003) que obtiveram 100% de sucesso de identificação de espécies, os resultados aqui são satisfatórios; em Meier et al. (2006) o sucesso de identificação de espécies foi de apenas 67%, porém os autores chamam atenção pelo fato de terem usados sequencias de diferentes tamanhos, onde a maioria eram menores que 500 pares de bases.

Consequentemente, as falhas na identificação de espécies ocorreram justamente no grupo de Lycoperdon molle/L. umbrinum como é tratado por Demoulin (1968b), Demoulin e Schumacker (1972), onde os caracteres morfológicos não são muito eficientes para separá-las. Neste caso, a região ITS não foi suficientemente variável para separar as espécies, assim como esperado para alguns grupos de fungos (Geiser et al., 2004), portanto, (Stielow et al., 2015), propõe a adição de mais marcadores à região ITS como barcoding, com isso, os autores indicam a região TEF1 α como uma região potencialmente promissora. A integração da sequência ITS como barcoding com a taxonomia morfológica demonstrou ser suficiente na discriminação de 19 espécies para a América do Sul; da mesma forma Goldstein e DeSalle (2011) citam que o conhecimento de especialistas morfológicos integrados aos trabalhos de DNA “barcoding” são eficientes na discriminação de espécies. Ainda, três espécies permaneceram como não identificadas; anteriormente essas espécies estavam erroneamente identificadas como espécies europeias ou norte americanas, mas em nossas análises, mostram-se táxons sul- americanos distintos, sendo necessária a adição de mais espécimes e marcadores para esclarecer esse fato. Também outros grupos como L. atropurpureum e L. excipuliforme devem ser estudados em um trabalho direcionado, levando em consideração os problemas históricos taxonômicos empregados a eles (Coker e Couch, 1928; Perdeck, 1950; Demoulin, 1968b; Jeppson e Demoulin, 1989; Calonge, 1998), e que possam ser abordados espécimes de diversas localidades de todos os continentes para onde foram reportados; assim uma comparação mais abrangente dos dados moleculares e morfológicos possam esclarecer esses problemas; como indicado por Osmundson et al. (2013), e mesmo que a região ITS não é eficiente para a separação de alguns grupos de espécies, ela pode indicar quais grupos de espécies ou coleções, necessitam de mais revisão taxonômica e a inserção de análises moleculares com vários marcadores. Com a revisão morfológicas das espécies, revisão de literatura e identificação, a América do Sul apresenta uma diversidade de espécies similar à América do Norte e Europa (Demoulin, 1972b); ou mesmo quando comparada com a estimativa de espécies para o mundo (Kirk et al., 2008). Ainda, o número de espécies registradas para Argentina foi superior em 12 espécies em relação aos números reportados anteriormente por Domínguez de Toledo (1989) e Hernádez- Caffot et al. (2013). Além do mais, Argentina e Brasil registraram juntos o maior número de espécies de Lycoperdon para o continente sul-americano. Embora, em países como

Bolívia, Paraguai ou Uruguai, etc., necessitam de mais trabalhos voltados a inventários de espécies de Lycoperdon; com base em nossos resultados, a América do Sul demonstra possuir uma grande diversidade de espécies em Lycoperdon quando comparado com outros continentes que tiveram mais esforços em trabalhos para inventariar as espécies de Lycoperdon. As novas ferramentas de identificação molecular, tais como ITS “barcoding” tem sido de grande ajuda para taxonomistas acessarem a diversidade de para vários organismos, como demonstrado por Janzen et al. (2005), que obtiveram sucesso no uso do DNA “barcoding” na identificação de espécies em inventários Lepidoptera. Com base nas regiões de ITS e LSU, o gênero Lycoperdon mostrou-se parafilético, e mesmo adicionando as espécies sul-americanas de Morganella, o gênero mostrou-se não independente de Lycoperdon; estes resultados corroboram os resultados obtidos por (Larsson e Jeppson, 2008), no qual os autores confirmam que Lycoperdon é parafilético, e propuseram Apioperdon, Morganella e Vascellum como gêneros não independentes de Lycoperdon; igualmente, aqui foi proposto o novo subgênero Arenicola, com base nas regiões ITS e LSU de espécies exclusivamente sul-americanas.

6 CONCLUSÕES GERAIS

1. A região ITS como “barcoding” na identificação de espécies de Lycoperdon é promissora, embora, seja necessário melhorar alguns protocolos para se obter a sequência “barcoding” para importantes espécimes (e.g. tipos), no qual a sequência “barcoding” ainda não foi obtida; 2. Embora, as análises de “barcoding” tenham ajudado na discriminação entre espécies e a detectar espécies crípticas, a taxonomia morfológica continua sendo essencial na separação de espécies, especialmente quando as sequências são idênticas ou nos casos em que não foi possível obtê-las; 3. A utilização de caracteres de diferentes fontes de informação taxonômica (morfológicos, moleculares e ecológicos) permitiu estabelecer uma sistemática mais robusta do que aquele que é estabelecido sob apenas um tipo de evidência; 4. Em conformidade com os dados moleculares, Lycoperdon é um gênero parafilético, no qual Morganella e Vascellum continuam como táxons não independentes de Lycoperdon; 5. Alguns táxons mostraram-se com a máxima variabilidade intraespecífica maior do que a mínima variabilidade interespecífica, assim o uso apenas de uma região ITS como DNA “barcoding” necessita ser combinada com um ou mais marcadores para resolver problemas nos limites entre alguns grupos de espécies, como no grupo de Lycoperdon molle/L. umbrinum. 6. Como em outros grupos de organismos, o número de espécies de Lycoperdon nos dois Hemisférios é similar; embora, nem todos estão em todas partes, já que o número de espécies com distribuição ampla é baixo. 7. Como em outros grupos de organismos, Argentina e Brasil se confirmam como “hotspots” de diversidade para as espécies de Lycoperdon.

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7. CAPÍTULOS / CHAPTERS

CAPÍTULO 1/ CHAPTER 1

Is the ITS barcode useful to discriminate Lycoperdon Pers. species?

Abstract

The estimate of unknown species of fungi is about 5.1 million and taxonomists would take almost 1000 years to describe them. The Consortium for the Barcode of Life developed the DNA barcode, a method that uses a short DNA sequence (about 800 bp) for species identification. Used in many groups of organisms (animals, plants, oomycetes and fungi), the DNA barcode plus morphological and ecological characters can improve the identification of new species. Moreover, the barcode can help researchers in the identification of herbarium and environmental samples (soil, decaying wood etc.). Many studies with animals, plants and oomycetes have shown high efficiency in species identification. The COI region is a very efficient barcode, with high success of amplification, in the identification of animals; however, in diverse groups, such as plants or fungi, the COI does not discriminate among closely related species. Researchers started to use the ITS nrDNA (ITS) region, initially used for phylogenetic analyses, as the barcode for plants and, afterward, for fungi, with success of amplification and identification from different sources, such as soil samples, plant debris, and cultures. Additionally, in 2012, with the work of Schoch and colleagues, the scientific community accepted the ITS region as a general DNA barcode for fungi. Moreover, the ITS region was used in the automatic identification of fungi in the UNITE web, where a two-tier clustering process is done: the first to the genus level and the second to the species level (the taxa arising are Species Hypothesis, SH). In the genus Lycoperdon some species are easy to identify, such as L. perlatum or L. echinatum; however, others are quite difficult to separate, such as those in the Lycoperdon molle group. The main aims of this work are to: a) test whether the ITS region is useful in the identification of species of Lycoperdon; and b) test whether the UNITE database provides a good tool for automatic identification in this genus. To achieve our aims, specimens were previously identified using macro- and micro-morphological characters. Afterward, genomic DNA was isolated, and the ITS region was amplified and sequenced. Different analyses were performed: a) to evaluate whether specimens of the same species group together, trees were obtained using parsimony and Bayesian inferences; b) to evaluate the percentage of correct identifications (PCI), the intra and interspecific variability based on a distance matrix (K2P) were calculated (a tree was also obtained using a Neighbor-Joining approach); c) to evaluate the automatic identification search in the UNITE database. We obtained 80% successful amplification, with very high quality. The identification based on K2P-NJ and

PCIs were similar with more than 80% correctly identified. Based on morphological analyses, ITS analyses, and automatic identification based on SHs, we have identified eighteen species of Lycoperdon; however, Lycoperdon sp. 1 and Lycoperdon sp. 2 have no match with any SHs. The ITS region showed itself promising as the DNA barcode of Lycoperdon, although another marker is needed to stabilize the terminal branches of L. altimontanum, L. excipuliforme, L. lambinonii, L. molle and L. umbrinum.

Key-words: Agaricales, DNA sequences, Identification, Lycoperdaceae, Taxonomy

1 INTRODUCTION

The Earth is home to an estimated 10 million species of fungi, animals and plants (Wilson, 2003; Mora et al., 2011; Scheffers et al., 2012), and the human brain can learn to identify a few thousand species, a small fraction of life’s diversity. It will take at least 1000 years to discover the world’s unknown fungi to discover 3.5 to 5.1 million unknown fungi (Blackwell, 2011). The names of the species are the framework for organizing the knowledge of all life forms on Earth. The Consortium for the Barcode of Life (CBOL, http:/www.barcoding.si.edu) is an international initiative devoted to developing DNA barcoding as a global standard for identification of species. The DNA barcodes are short DNA sequences (500–800 base-pairs, bp) from standardized and well-known portions of the genome, used as an aid to identify the species to which a specimen of fungus, animal or plant belongs. The idea for DNA barcoding was introduced by Hebert et al. (2003) to develop species identification of animals, in the same way that the barcode black/white lines are useful to discriminate products in a market. The effectiveness of this approach clearly depends on the availability of extensive databases of barcodesequence standards.

Prendini (2005), criticizes barcodes: “DNA is not a panacea for the woes of taxonomy” but he agrees that DNA barcodes should be used as a supplement to species description and diagnosis. Yet, according to the author, this method of identification is not demonstrably more objective, accurate, or useful than morphology or other sources of phenotypic data for species identification or other taxonomic purposes. More, the DNA barcoding as presented, or DNA taxonomy more generally, is just another technique for species identification and can not be utilized as a global identification system. On other hand, Hebert and Barrett (2005) disagree with Prendini, and agree that the barcode approach challenges usual concepts in morphological taxonomy, for example, shape or color, breaking these subjective characters. Moreover, the authors affirm that the addition of morphological and ecological characters with the information on the DNA barcode improves the identification of new species. Finally, collaboration between barcoders and taxonomists is of utmost importance to the advance in understanding of species diversity.

1.1 Animal, algae, plant and oomycota barcodes

In animals, there is an agreement that the 600 bp fragment of the COI1 gene is a good barcode standard. Hebert et al. (2003) obtained 100% success in delimitation of lepidopteran species, one of the most taxonomically diverse orders that also has low sequence divergence. Later, Hebert et al. (2004) showed that the COI1 was of great help to discriminate birds species where the identification of young specimens is difficult; Ben-David et al. (2007), identified tetranychid species of spiders, where morphological characters are not helping to discriminate the species; Hubert et al. (2008), analyzed 1360 specimens of freshwater fishes from Canada and delimited 190 species (about 95% of Canadian freshwater species); Chen et al. (2011), studied species of heterodont bivalve mollusks where not only the morphological characters are insufficient to identify the species, but also taxonomic experts disagree on which morphological characters are valid to delimitate species, Chen et al. (2011) and Morinière et al. (2014), used a DNA barcode approach for fauna inventories. However, in some groups, such as Diptera, it was a very low rate of success (<70%) for delimiting species based on COI1 (Meier et al., 2006): COI1 was unsuccessful because 21% of all species used have identical sequences, and their results showed that the COI1 sequences were variable among individuals of the same species.

In algae, Phaeophyceae (brown algae) and Rhodophyta (red algae), Robba et al. (2006) and McDevit and Saunders (2009), were successful in delimitation of species with the use of DNA barcode (COI1); and the most important was the alliance between the variability observed in COI1 sequences and alpha taxonomy, allowing identification of 29 species from 20 genera in nine families from five orders of Phaeophyceae (there are an estimated 6000 species of red algae worldwide). Le Gall and Saunders (2010), studied 380 specimens of red algae (Phyllophoraceae) from North America, Australia and Europe; they identified 29 species from approximately 100 worldwide species already known. Also, COI1 allowed study of biogeography (McDevit and Saunders, 2009), and helped in discovering new species (Milstein and Saunders, 2012; Dixon and Saunders, 2013).

In plants, the mitochondrial genes (COI1) does not work as a DNA barcode (Stoeckle, 2003). Baldwin (1992, 1993), used the internal transcribed spacer (ITS) region from nuclear ribosomal DNA for species identification and phylogenetic analyses; Álvarez and Wendel (2003) call attention to many concerns in the use of ITS as a locus 82

for phylogenetic analysis: as ITS is not protein-encoding the natural alignment guides are unavailable and the researcher cannot check the changes every three nucleotides as in the sequence alignment protein code, or assess how high GC content and indel accumulation impact the alignment. However, ITS can be used as a barcode because the region can be amplified in two smaller fragments ITS1 and ITS2, with adjoining 5.8S locus that is useful for degraded samples (Kress et al., 2005); and ITS as a barcode of plants can be more effective than the mitochondrial DNA, in fact it evolves more slowly in plants than in animals (Chase et al., 2005; Pennisi, 2007). ITS does not work very well in some groups of plants because of problems of paralogy and other factors associated with the complex evolution of this highly repeated part of the nuclear genome (Chase et al., 2005; Kress et al., 2005). Moreover, in the herbarium specimens the ITS failed in 12% of the samples tested (Kress et al., 2005); that fact is very important because the amplification of dried samples for barcoding is important to confirm many identifications of rare or perhaps extinct taxa. In many studies, it is necessary to combine ITS with another region such as trnh-psbA to improve the results of identification by DNA barcoding (Rubinoff et al., 2006). Despite these problems, Edwards et al. (2008), show that ITS and trnT-trnL are the most effective regions to discriminate among Aspalathus species; and their results were confirmed by Roy et al. (2010), using ITS in the delimitation of Berberis, Ficus and Gossypium species. Also, (Yao et al., 2010), used ITS to discriminate plant taxa and as a complementary locus of COI1: 97% discrimination to the genus and 74% to species levels.

Moreover, the ITS region was proposed by Chen et al. (2010), as a standard DNA barcode for medicinal plants and they justify the use of ITS: (1) it is short, relatively easy to amplify; (2) it possesses high interspecific divergence; (3) the identification accuracy of the ITS region was 92.7% at the species level and 99.8% at the genus level.

In the case of Straminipila, Göker et al. (2009) used ITS to discriminate species of Peronospora Corda and Pseudoperonospora Rostovzev, obligate plant pathogens with very few morphological characters to identify the species. Robideau et al. (2011) compared the ITS region with COI1 as barcode of oomycete species; they concluded that both ITS and COI are efficient as barcodes, although ITS has more interspecific variation than COI1. Sandoval-Sierra et al. (2014), confirmed the use of ITS as a potential region for barcoding of species of Saprolegnia Nees.

1.2 Fungi barcode and automatic identification tools

According to Iwen et al. (2002), in Ascomycota, the ITS region was a good target for the identification of human fungal pathogens to the species level. Druzhinina et al. (2005), used ITS to identify Trichoderma Pers. and Hypocrea Fr. species, since morphological characters were difficult even for taxonomists experts. Also, ITS was very helpful to discriminate fungal species in sample soils (O’Brien et al., 2005). Grasso et al. (2006), compared ITS with COI1 in phylogenetic studies, and showed that ITS evolves faster than COI1, and therefore, the ITS region was more efficient in distinguishing among species, such as in Puccinia P. Michieli where the COI1 sequences were identical in all the species tested. However, the ITS region was not successful in delimiting species in some groups (Geiser et al., 2004), such as in Penicillium Link Seifert et al. (2007), whereas COI1 was useful to discriminate the species of these molds. In some cases, such as Leohumicola N.L. Nick, the ITS and COI1 had similar results to delimit species and both markers could be suitable DNA barcodes (Nguyen and Seifert, 2008): the mean sequence divergence between species was 10 times larger than the mean divergence within species, demonstrating a barcode gap.

In Usnea Dill. ex. Adans (lichenized fungi), the ITS region had a 92% success rate in discriminating among species (Kelly et al., 2011): the success of discrimination is present if the minimum of uncorrected interspecific p distance exceeded the maximum uncorrected intraspecific p distance (a barcode gap exists). Also, Suwannasai et al. (2013), tested the ITS sequences as barcoding for Annulohypoxylon and Hypoxylon (Xylariaceae), suggesting that ITS can be useful as a barcode.

For the Basidiomycota, the ITS has been used with success to delimit species from ecological samples such as soil and plant debris, that are devoid of fruit bodies of the fungi (Nilsson et al., 2008, 2009). Thus, the macroscopic and microscopic interpretation of characters, the morphologically similar species and the speciose nature of the kingdom Fungi make the DNA barcode an essential approach to delimit species of fungi (Seifert, 2009). Bellemain et al. (2010), used the ITS region to identify species of fungi from environmental samples, and they found more basidiomycete amplicons than ascomycetes, mostly in the Agaricales (269 distinct sequences). Dentinger et al. (2011), comparing COI1 and ITS in Agaricomycotina, demonstrated that ITS has a disadvantage as a barcode

due to the inability to produce a reliable alignment of ITS sequences across a divergent set of taxa. However, the ITS regions do work well for identifying mushrooms species.

Although the ITS region is not very successful in discriminating species in all groups of fungi, Schoch et al. (2012), proposed ITS as the first barcode of fungi. They compared ITS with others markers: SSU nrDNA, LSU nrDNA, RPB1, RPB2 and MCM7. The ITS was proposed mainly due to the higher success of PCR amplifications (90%) and the most resolving power of species discrimination, for example, in Basidiomycota with 77% resolution. In Suwannasai et al. (2013), authors assessed the efficacy of automated species identification using the fungal barcode marker (ITS) and the model system of Annulohypoxylon and Hypoxylon (Xylariaceae); they showed that automated species identification is sensitive to a specific choice of evolutionary distance, and statistical methods are available to address the possibility of expert misidentification of species.

In general, for automatic identification of fungi, authors can use the popular BLAST sequence alignment tool provided by NCBI, as well as tools provided in other websites, such as in UNITE (http://unite.ut.ee).

UNITE started in 2003, including several tools, to aid in the identification of unknown sequences of ectomycorrhizal fungi in Northern Europe (Kõljalg et al., 2005). However, as noted in Nilsson et al. (2006), more than 27% of all fungal sequences available in public repositories were insufficiently identified, and the approach adopted by the UNITE database was to generate good sequences obtained from voucher specimens and with good metadata, such as voucher specimens, country, ecology and host (Abarenkov et al., 2010). Another step toward automatic identification was taken by Kõljalg et al. (2013), who address the idea of species hypotheses (SH). To generate a concise set of reference sequences, UNITE applies two clustering levels, one to the subgenus/genus level and the other to the species level; the term “species hypotheses” (SH) is applied to the taxa arising from the second round of clustering. A representative sequence for each SH is chosen automatically, but taxonomic experts may override the choice by designating a reference sequence based on type status, source of isolation and sequence quality.

1.3 The genus Lycoperdon Pers.

The species of Lycoperdon (Agaricales) are mainly distinguished by their subglobose to pyriform and closed basidiomata (Bottomley, 1948), with a cellular subgleba, and a single opening in the apex through which basidiospores are discharged (Demoulin, 1972a; Calonge, 1998). In the taxonomic databases IndexFungorum (http://www.indexfungorum.org/) and Mycobank (http://www.mycobank.org) approximately 786 names (including synonyms) are listed. Demoulin (1972a), in his work of Lycoperdon Pers. from Europe and North America, did an exhaustive review of the genus, with complete taxonomic studies, including a list of more than 550 names validly published under Lycoperdon; although many taxa have been transferred to the other genera, such as Lycoperdon sessile Sowerby (current name Geastrum fimbriatum Fr.), L. tropicale Speg. (current name Calvatia tropicalis (Speg.) Speg.), L. ulmi Bell. (a Myxomycete), etc. In Kirk et al. (2008), 50 Lycoperdon species are recorded, many of these species have been described only from the Northern Hemisphere (Bowerman, 1961; Demoulin, 1971, 1972b; Kreisel, 1976), with very few works describing new species from the Southern Hemisphere (Spegazzini, 1898; Hennings, 1904a, b; Rick, 1961; Cortez et al., 2011). Demoulin (1972a), recognized 30 species of Lycoperdon in Europe and North America, based only on morphological taxonomy. Larsson and Jeppson (2008), based on ITS-LSU nrDNA and morphological data, considered that in Europe the genus comprises about 31 species, including some species that were under other genera (e.g. Calvatia excipuliformis (Scop.) Perdeck, Morganella subincarnata (Peck) Kreisel & Dring. and Vascellum pratense (Pers.) Kreisel). From the Southern Hemisphere, 44 species (including Morganella and Vascellum) have been recorded, although some are considered synonymous e. g. L. gemmatum Batsch (current name L. perlatum Pers.), or transferred to another genus such as L. acuminatum Bosc (current name Bovista acuminata (Bosc) Kreisel). In the UNITE database, 32 species hypotheses (SHs) are under Lycoperdon; again, based mainly on sequenced specimens from Europe and North America.

In Lycoperdon some species are easy to identify even when the basidiomata are immature (e.g. L. perlatum Pers. or L. echinatum Pers), but other species are difficult to distinguish one from another, such as L. atropurpureum Vittad., L. decipiens Durieu & Mont., and L. umbrinum Pers., often misidentified as L. molle Pers. or L. atropurpureum Vittad. (Demoulin, 1972a).

This work combines classical taxonomic methods with sequence analyses and statistics to evaluate the suitability of ITS as a barcode in Lycoperdon. The ITS region is tested as a barcode for Lycoperdon specimens previously identified by morphological data in collaboration with Lycoperdon specialists from Europe (F.D. Calonge, V. Demoulin and M. Jeppson), and, possible biases introduced by incorrect morphological identification is evaluated. Moreover, we tested whether the UNITE database provides a good proxy for automatic identification in this genus. For this, we have expanded the ITS database from Larsson and Jeppson (2008), with sequences from specimens mainly located in MA-Fungi (Real Jardín Botánico de Madrid) and MC-Fungi (Republic of Macedonia).

2 MATERIAL AND METHODS 2.1 Collections studied

Specimens were obtained from MA-Fungi (Real Jardín Botánico, Madrid, Spain) and MCF-Fungi (Macedonian Collection of fungi, Macedonia) herbaria. Also, collections from the ICN (Rio Grande do Sul/Brazil) herbarium were analyzed. Data of collections studied are included in Table 2.

2.2 Morphological analysis

The specimens were analyzed and identified following the specific literature of the genus Lycoperdon (Demoulin, 1972b, 1976). Microstructures were mounted in Melzer’s reagent to examine a dextrinoid reaction. Gleba was examined in a lactophenol cotton blue stain. All slides were boiled following Cunningham (1944), to return specimens to the normal size. Basidiospore measurements were based on ten basidiospores per fruitbody using the classification A to D (punctate to strongly verrucose) (Demoulin, 1972b). The ornamentation density of the spores was calculated using scanning electron microscopy (SEM) spores, following Cortez et al. (2008): number of ornamentation divided by the diameter of the spore multiplied by 3.14, to give a number of warty by a reduced area of 10 µm.

2.3 Molecular analyses 2.3.1 DNA isolation, amplification and sequencing

Total genomic DNA was extracted using DNeasy Plant Kit (Qiagen 69106) following the instructions of the manufacturers; lysis buffer incubation was done overnight at 55º C. The PCR was done in a 25 μl reaction mix using illustraTM PureTaqTM Ready-To-Go-TM PCR Beads (GE Healthcare, Buckinghamishire, UK) as described in Winka et al. (2000). The internal transcribed spacer of ribosomal nrDNA (ITS) was amplified with the primer ITS5/ITS4, the thermal cycling conditions in accordance with Martin and Winka (2000). Negative controls lacking fungal DNA were run for each experiment to check for contamination. The extracted DNA was tested by electrophoresis in a 1.5% agarose gel. From each DNA sample 5 µl were deposited mixed with 2 µl of run buffer plus 1 µl of GelRed buffer to visualize the PCR products (Fig. 11). The PCR products were subsequently purified using the QIAquick Gel PCR Purification (Qiagen) according to the manufacturer's instructions or with 8 μl of 1:10 ExoSAP-IT®

(USB Corporation, OH, USA). The purified PCR products were sequenced using the same amplification primers in Macrogen Inc. (Amsterdam, Netherlands). Sequencher 4.1.1 (Gene Codes Corporation, Ann Arbor, Michigan, USA) was used to edit the resulting electropherograms and to assemble contiguous sequences. To test if the sequences belong to the genus Lycoperdon and not to contaminations with other fungi, BLAST searches with the megablast option were used to compare the sequences obtained against the sequence in the National Center for Biotechnology Information (NCBI) nucleotide databases (Altschul et al., 1997).

Figure 11 Agarose gel to verify the PCR products. a. Amplification of ITS5 – ITS4 region: eleven positive PCR products; the eighth sample failed; b. Amplification of ITS3 – ITS4b region: all samples have been successful; c. Amplification of ITS5 – ITS4 have been unsuccessful in all samples; d. Amplification of ITS3 – ITS4b have been unsuccessful in all samples and the control sample is contaminated. M - is the run marker, and N - is the control sample.

2.3.2 Molecular identification of Lycoperdon species

The ITS nrDNA sequences obtained (Table 2) were aligned using Se-Al v2.0a11 Carbon (Rambaut, 2002) for multiple sequences. The sequences were compared with homologous Lycoperdaceae from GenBank mainly published in Larsson and Jeppson

(2008) and Jeppson et al., (2012). Three sequences of Bovista Pers. were included as outgroup. Where ambiguities in the alignment occurred, the alignment generating the fewest potentially informative characters was chosen. Alignment gaps were marked “–“, unresolved nucleotides and unknown sequences were indicated with “N“.

A maximum parsimony analysis (MP) was carried out, minimum length Fitch trees were constructed using heuristic searches with tree-bisection-reconnection (TBR) branch swapping, collapsing branches if maximum length was zero and with the MulTrees option on in the PAUP*4.0b10 (Swofford, 2002); and a default setting to stop the analysis at 100 trees. Gaps were treated as missing data. Nonparametric bootstrap support (bs) (Felsenstein, 1985) for each clade, based on 10,000 replicates using the fast- step option, was tested. The consistency index CI (Kluge and Farris, 1969), retention index RI, and rescaled consistency RC (Farris, 1989), were obtained.

A second analysis was done by the Bayesian approach (Larget and Simon, 1999; Huelsenbeck and Ronquist, 2001) using MrBayes 3.1 (Ronquist and Huelsenbeck, 2003). The analysis was performed assuming the general time reversible model (Rodríguez et al., 1990) including estimation of invariant sites and assuming a discrete gamma distribution with six categories (GTR+I+G) as selected by MrModelTest v.2.3 (Nylander, 2004). Two independent and simultaneous analyses starting from different random trees were run for 10,000,000 generations with four parallel chains and tree 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 determine 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. Both the 50% majority- rule consensus tree and the posterior probability (pp) of the nodes were calculated from the remaining trees with MrBayes. The phylogenetic tree was viewed with FigTree v1.42 (http://tree.bio.ed.ac.uk/ sofware/figtree/) and edited with free software InkScape Wink, version 0.92.0 r15299 (http://wiki.inkscape.org/wiki/index.php?title=Release_notes/0.91&oldid=98966).

To characterize the intraspecific and interspecific variation (Meyer and Paulay, 2005; Chen et al., 2010; Ramadan and Baeshen, 2012), and to check whether a barcode gap exists, pairwise K2P distances were calculated; a tree based on a Neighboun-Joining

algorithm was obtained. Moreover, the probability of correct identification (PCI) based on distance (CBOL, 2009; Suwannasai et al., 2013), was calculated using the Taxon/DNA following Meier et al. (2006); the probability of correct identification is given for at least two samples from any species showing the barcode gap: if the maximum intraspecific sequence distance is less than its minimum interspecific sequence distance, this is considered “correct identification”.

2.3.3 Performance of automatic identification through the unite database

The ITS nrDNA newly-generated sequences were analyzed through the UNITE database (https://unite.ut.ee) version 7.1, to search for the best matching sequence of the species hypothesis (SHs) in accordance with Kõljalg et al. (2013). The SHs can be viewed and edited in a Web browser using the PlutoF workbench. Our search for SHs was done with minimum distance of 1.5% (Fig. 12).

Figure 12 Example of search in the UNITE database. a. Set up of run analysis where the query sequences from the user are pasted and blasted; b. Results including the list of homolog sequences to; c. UNITE sequences linked to the SHs with threshold of at least 3% similarity; d. Selected SHs sequence with threshold of 1.5%, name and number access, and statistics on distribution distance.

3 RESULTS 3.1 Molecular analyses 3.1.1 DNA, amplification and sequencing

DNA was isolated from one hundred and forty-two specimens, and one hundred and twenty-tree sequences with high quality (Fig. 11a-b) with length of 500–700 bp were generated with a success of amplification of 83%, one sequence is from ICN, 36 from MA-Fungi and 54 from MC-Fungi herbaria. From nineteen samples, amplification was unsuccessful (Fig. 11c-d), representing 13%; even when we retried the amplification by parts with primer pairs ITS1F/ITS2 and ITS3/ITS4B, the failure of amplification was persistent. A blast search showed seven sequences belong to the genus Bovista, four to the genus Calvatia and four sequences were identified as belonging to Trichoderma rosicum, a contamination. From sixteen samples, sequences obtained were contaminated by other Lycoperdon spp. Two samples have been amplified with less than 550 bp and these sequences were not used in barcoding analyses since the length of sequence influences the length of the terminal branches of the Neighbor-Joining tree, leading to a misinterpretation. Also, sequences that belong to other genera, such as Bovista and Calvatia, another phylum, or contaminated by other Lycoperdon species were not used in the molecular analysis. All the sequences were deposited in the EMBL/GenBank/DDBJ databases (Table 2).

3.1.2 Molecular identification of Lycoperdon species

The new ITS sequences were aligned with eighty-five sequences downloaded from GenBank under Bovista Pers., Morganella Zeller, and Lycoperdon species to produce a matrix of 735 unambiguously aligned nucleotide positions. No characters (nucleotide positions) were excluded from the analyses. All characters are of type 'unord' and have equal weight, 69 characters are parsimony-uninformative, 151 parsimony- informative. The 100 most parsimonious trees gave a length of 502 steps, CI = 0.586, HI = 0.890, and RI = 0.414. The trees obtained from the MP (strict consensus tree), Neighbor-Joining K2P and Bayesian analysis show similar topologies (Fig. 13, Figs. Supp. 1–2).

Figure 13 Neighbor-Joining tree using Kimura 2 Parameter based on sequences of ITS sequences of Lycoperdon species. The support values obtained from parsimony (bs) and Bayesian (pp) analyses are on the branches.

Using the tree-based identification of Neighbor-Joining (K2P) (Fig. 13), a success of 80% was obtained (72 sequences identified at species level), in contrast, the 20% considered misidentification,18 sequences, had incorrect identification. Twenty-one species of Lycoperdon were identified and the groups correspond to morphological species: Lycoperdon altimontanum, L. atropurpureum, L. decipiens, L. cf. delicatum, L. echinatum, L. ericaeum, L. excipuliforme, L. lambinonii, L. lividum, L. mammiforme, L. marginatum, L. molle, L. nigrescens, L. niveum, L. perlatum, L. pratense, Lycoperdon sp. 01, L. subumbrinum, L. umbrinoides, L. umbrinum and L. pyriforme. The terminal branches with L. lambinonii and L. lividum are unresolved.

The other 12 terminal branches are formed by sequences found only from GenBank: Morganella fuliginea, M. subincarnata, Lycoperdon caudatum, L. cretaceum, L. dermoxanthum, L. frigidum, L. muscorum, L. norvegicum, L. radicatum, L. rupicola, Lycoperdon sp. 03 and L. utriforme. These species are not discussed in this paper, since we have no access to the study the specimens.

The analyses of Neighbor-Joining tree (K2P) showed the genera Bovista is separated from Lycoperdon, and this result is supported in the Bayesian analyses (pp = 1). Morganella genus is maintained as a subgenus of Lycoperdon with moderate to well support (bs = 83%, pp = 1.0).

The Lycoperdon perlatum terminal branch is well supported (bs = 97%, bpp = 1.0, and included samples from GenBank (1), MCF-fungi (15) and MA-Fungi (4). The Lycoperdon marginatum terminal branch (bs = 100%, pp = 1.0), is a sister group from L. perlatum, and includes samples from GenBank (1) and MA-Fungi (2).

The Lycoperdon sp. 01 terminal branch is well supported (<50%, pp = 1.0). This terminal branch is formed only by samples from the neotropical region (Brazil). The Lycoperdon pratense terminal branch is well supported (bs = 100%, pp = 1.0), and includes samples from GenBank (3) and MC-Fungi (2). Lycoperdon pratense is a sister terminal branch from Lycoperdon sp. 1.

The Lycoperdon ericaeum terminal branch is moderately to well supported (bs = 83%, pp = 1.0), including samples from GenBank (2) and MA-Fungi (1). The Lycoperdon subumbrinum terminal branch (pp = 97%, pp = 1.0) includes samples from GenBank (3), MA-Fungi (4) and MC-Fungi (1); and it is the sister group of L. ericaeum.

The Lycoperdon mammiforme terminal branch is well supported (bs = 97%, pp = 0.98) and includes samples from GenBank (1), MA-Fungi (2) and MC-Fungi (1). The Lycoperdon umbrinum and Lycoperdon atropurpureum groups are closest to L. mammiforme. Lycoperdon umbrinum terminal branch is moderate to well supported (bs = 59%, pp = 0.99%), including samples form GenBank (2) and MC-Fungi (2). The Lycoperdon atropurpureum terminal branch is moderately supported (bs = 50%, pp = 0.84%), and includes samples from GenBank (1), MA-Fungi (2) and MC-Fungi (8);

The Lycoperdon niveum terminal branch with moderate support (bs = 62%, pp = 0.71), includes samples from GenBank (5), MA-Fungi (2) and MC-Fungi (2); the L. decipiens and L. molle are sister groups to L. niveum. Lycoperdon decipiens terminal branch (bs = 82%, pp = 1.0), includes samples from GenBank (1) and MA-Fungi (3). The Lycoperdon molle terminal branch (bs = 100%, pp = 1.0), includes samples from MA- Fungi collected in Spain (3), Portugal (1) and Greece (1). The Lycoperdon lividum terminal branch (bs = 51, pp = 0.78), is closest to L. decipiens, L. molle and L. niveum; and includes samples from GenBank (2), MA-Fungi (3) and MC-Fungi (4).

The Lycoperdon altimontanum terminal branch is weakly supported (bs = < 50%, pp = 0.92), and includes samples from GenBank (2), MA-Fungi (1) and MC-Fungi (1); the Lycoperdon excipuliforme (bs = <50%, pp = 0), terminal branch is the sister group of L. altimontanum, and includes samples from GenBank (1) and MC-Fungi (6).

The Lycoperdon lambinonii terminal branch is weakly supported (bs = 52%, pp = 0.96) and includes eight samples from GenBank: DQ112575, DQ112576 (L. lambinonii); DQ11259, DQ112594 and DQ112595 (L. turneri); DQ112591, DQ112592 and DQ112593 (L. umbrinum); and one MC-Fungi05:10226 (L. lambinonii); this terminal branch is closest to L. altimontanum and L. excipuliforme.

The Lycoperdon echinatum terminal branch is well supported (pp = 96%, bs = 1.0), and includes samples from GenBank (1), MA-Fungi (2) and MC-Fungi (6); the Lycoperdon nigrescens terminal branch (bs = 100%, pp = 1.0) is closest to L. echinatum group, and includes samples from GenBank (1) and MA-Fungi (3); the Lycoperdon radicatum and L. utriforme (GenBank sample) terminal branches are sister groups to L. nigrescens.

The Lycoperdon umbrinoides terminal branch (bs <50%, pp = 1.0) includes a sample from MA-Fungi (1); the Lycoperdon pyriforme terminal branch is closest to L. umbrinoides terminal branch (bs = 100%, pp = 1.0), and includes samples from GenBank (1), MA-Fungi (1) and MC-Fungi (5).

3.1.3 Probability correct identification (PCI) of Lycoperdon species

Our results showed a barcode gap of 50% between species (Fig. 14): Lycoperdon decipiens obtained 0.43 ± 0.06; L. echinatum 1.14 ± 0.12; L. mammiforme 0.89 ± 0.05; L. marginatum 2.95 ± 0.15; 2.02 ± 0.28; L. perlatum 1.62 ± 0.12; L. pratense 3.93 ± 0.19; L. pyriforme 5.19 ± 0.25; L. rupicola 1.73 ± 0.10. Additionally, identification based on sequence distance, gave an 87.58% success of identification based on “best match and best close match”, and the incorrect identification was 11.03%, and 9.45% (“Best Match and Best Close Match”) (Table 3, 4), from our complete dataset (non-GenBank plus Genbank samples).

In the PCI analysis, with the search by the Best Match/Best Close Match using a threshold of 3%, in the terminal branch, that includes the subgenus Lycoperdon (23 sequences, 20 belong to Lycoperdon perlatum and three to L. marginatum), at least one matching sequence was found, and the correct identifications according to “Best Match” was 100%, the ambiguous and incorrect identification was 0%. The average intraspecific distance in L. perlatum and L. marginatum was 0.11% and 0.40%, respectively, while the average interspecific distance between the two species was 3.35%; however, this latter value is smaller than when all species are compared: L. perlatum 4.76% and L. marginatum 5.40% (Fig. 14).

The terminal branch that includes the subgenus Vascellum, in the analysis of the Best Match/Best Close Match using threshold of 3.0% (six sequences: five belonging to L. pratense and one sequence to Lycoperdon sp.1), has at least one matching sequence and the correct identifications according to “Best Match” was of 83.33%, the ambiguous 0%, the incorrect identification was one (16.66%). The correct identifications according to “Best Close Match” was of five (83.33%), the ambiguous and the incorrect identifications were 0%; however, one sequence (16.66%) had no match closer than 3.0%. The average intraspecific distance in L. pratense and Lycoperdon sp. 1 were of 0.27% and N/A, while the average interspecific distance between these species was 4.21%;

however, this last value is smaller than when we compare with all species here L. pratense 5.14% and Lycoperdon sp.1 4.81% (Fig. 15).

The terminal branches that include subgenus Utraria, in the analysis of the Best Match/Best Close Match using a threshold of 3.0% (13 sequences: five belong to L. ericaeum and eight to L. subumbrinum), have at least one matching sequence and the correct identifications according to “Best Match” was 100%, the ambiguous and incorrect identification was 0%. The average intraspecific distance in L. ericaeum and L. subumbrinum were 1.50% and 0.58%, while the average interspecific distance between them was 2.55%, however, this value is smaller than when we compare with all species here: L. ericaeum 2.80% and L. subumbrinum 3.25% (Fig. 15).

In the terminal branches of Lycoperdon mammiforme, L. umbrinum and L. atropurpureum, the analysis of the Best Match/Best Close Match using a threshold of 3.0% (24 sequences: four belong to L. mammiforme, ten to L. umbrinum and L. atropurpureum), they have at least one matching sequence and the correct identifications according to “Best Match” was 95.83%, the ambiguous 0% and the incorrect identification was 4.16%. The correct identifications according to “Best Close Match” were 95.83%, incorrect identifications were 4.16%. The average intraspecific distance in L. mammiforme, L. umbrinum and L. atropurpureum was 0.02%, 0.08% and 1.86%, while the average interspecific distance between these species was 1.50%, 1.63% and 1.79%; however, these last values are smaller than when we compare with all species here: L. mammiforme 2.89%, L. umbrinum 4.47% and L. atropurpureum 2.76% (Fig. 15).

In the terminal branches of Lycoperdon decipiens, L. molle and L. niveum, the analysis of the Best Match/Best Close Match using a threshold of 3.0% (23 sequences: seven belong to L. niveum, four belong to L. decipiens and six belong to L. molle), they have at least one matching sequence and the correct identifications according to “Best Match” was 100%, ambiguous and incorrect identifications were 0%. The correct identifications according to “Best Close Match” were 100%, ambiguous and incorrect identifications 0%. The average intraspecific distance of L. niveum, L. decipiens and L. molle was 0.60%, 0% and 1.38%, while the average interspecific distance between these species was 1.48%, 1.26% and 1.75%, however, these latter values are smaller than when we compare with all species here: L. niveum 2.58%, L. decipiens 2.57% and L. molle 3.09% (Fig. 15).

For the nine sequences identified as belonging to L. lividum, the analysis of the Best Match/Best Close Match using a threshold of 3.0%, have at least one matching sequence and the correct identifications according to “Best Match” was 100%, ambiguous and incorrect identifications was 0%. The correct identifications according to “Best Close Match” were 100%, ambiguous and incorrect identifications were 0%. The average interspecific distance was 2.72% (Fig. 15).

For the four sequences identified as belonging to L. altimontanum and seven as belonging to L. excipuliforme, the analysis of the Best Match/Best Close Match using a threshold of 3.0%, have at least one matching sequence and the correct identifications according to “Best Match” was 90.9%, ambiguous 0% and incorrect identification 9.09%. The correct identifications according to “Best Close Match” were of 90.9%, ambiguous 0%, incorrect identification 9.09%. The average intraspecific distance of L. altimontanum and L. excipuliforme was 0.68% and 0.28%, while the average interspecific distance between these species was 0.815%% and 0.815%, however, these latter values are smaller than when all species here are compared: L. altimontanum 2.76% and L. excipuliforme 2.62% (Fig. 15).

For the nine sequences that are included in the Lycoperdon lambinonii terminal branch the Best Match/Best Close Match using a threshold of 3.0% (three belong to L. turneri and three belong to L. umbrinum), have at least one matching sequence and the correct identifications according to “Best Match” was 77%, the ambiguous 0% and the incorrect identifications was of 22.22%. The correct identifications according to “Best Close Match” were of 77.77%, the ambiguous was 0%, incorrect identification was 22.22%. The average intraspecific distance of L. lambinonii, L. turneri and L. umbrinum was 1.68%, 0.34% and 0.14%, while the average interspecific distance between these species was 1.36%, 0.95% and 0.87%, however, these latter values are smaller than when we compare with all species here: L. lambinonii 3.18%, L. turneri 2.82% and L. umbrinum 2.86% (Fig. 15).

In terminal branch of Lycoperdon echinatum, L. nigrescens, L. radicatum and L. utriforme the analysis of the Best Match/Best Close Match using a threshold of 3.0% (15 sequences: nine sequences belong to L. echinatum, four belong to L. nigrescens and one belongs to L. radicatum and L. utriforme), have at least one matching sequence and the correct identifications according to “Best Match” was 85.71%, the ambiguous sequences

was 0% and incorrect identifications was two sequences (14.28% - GenBank samples). The correct identifications according to “Best Close Match” were 85.71%, the ambiguous sequences were of 0%, and incorrect identifications was two sequences (14.28% - GenBank samples). The average intraspecific distance of L. echinatum and L. nigrescens was 0.07% and 0.11%, while the average interspecific distance between these species was 2.74% and 2.94%, however, these latter values are smaller than when compared with all species here: L. echinatum 3.01% and L. nigrescens 3.68% (Fig. 15).

In the terminal branch of Lycoperdon pyriforme, L. caudatum and L. umbrinoides the analysis of the Best Match/Best Close Match using a threshold of 3.0% (13 sequences: eight belong to L. pyriforme, one belongs to L. caudatum and L. umbrinoides), have at least one matching sequence and the correct identifications according to “Best Match” was 70%, ambiguous sequences were of 0% and incorrect identifications was of three sequences (30% - two GenBank samples and one MA-Fungi 35530). The correct identifications according to “Best Close Match” were 70%, the ambiguous sequences were 0%, the incorrect identifications were 0%. The average intraspecific distance of L. pyriforme 2.27%, while the L. umbrinoides had no valid comparisons. The average interspecific distance between these species was 5.82% and 5.76%, however, these latter values are smaller than when we compare with all species here: L. pyriforme 6.36% and L. umbrinoides 4.47% (Fig. 15).

6,00

5,00

4,00

3,00

2,00

1,00

0,00

Largest intraspecific distance Mininum interspecific distance Standard deviation Intraspecific distance Standard deviation Interspecific Distance

Figure 14 PCI for each sample from each Lycoperdon species of dataset non-GenBank plus GenBank samples.

8,00%

7,00%

6,00%

5,00%

4,00%

3,00%

2,00%

1,00%

0,00%

-1,00%

Lycoperdon species Average intraspecific distance Average interspecific distance

Figure 15 Average intraspecific and interspecific genetic variability for conspecifics and congeneric of all sequences of dataset excluding Bovista and Morganella.

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3.1.4 Automatic Identification through UNITE

The sequences obtained in this study, after a UNITE search, match with the following eighteen SHs (Fig. 16): SH078551.07FU–Lycoperdon altimontanum with mean of 98.5% of identity; SH310925.07FU–L. atropurpureum with mean of 99.5 % of identity; SH226422.07FU–L. decipiens (99.5%); SH175905.07FU–L. echinatum (98.5%); SH175895.07FU–L. ericaeum (98.5%), SH078579.07–L. excipuliforme (99.0%); SH078579.07FU–L. lambinonii (99.0%); SH078533.07FU–L. lividum (99.0%); SH175887.07FU–L. mammiforme (98.5%), SH175899.07FU–L. marginatum (98,5%); SH175919.07FU–L. molle (98.5%), SH175893.07FU–L. nigrescens (98.5%); SH078531.07FU–L. L.niveum (99%), SH175885.07FU–L. perlatum (99.0%); SH175883.07FU–L. pratense (99%), SH078515.07FU–L. pyriforme (98.5%); SH175888.07FU–L. subumbrinum (98.5%) and SH311013.07FU–L. umbrinum (99.5%). The sequence of Lycoperdon sp. 1 and Lycoperdon sp. 2 booth with only one sample have not match in UNITE.

99,80% 99,60% Percent Identity - UNITE 99,40% 99,20% 99,00% 98,80% 98,60% 98,40% 98,20% 98,00% 97,80%

Figure 16 Percent identity of sequences compared with UNITE Species Hypothesis (SHs).

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3.2 Taxonomy

The revision of morphological characters allows us to discriminate the twenty-one Lycoperdon species. However, two species remain unidentified (Lycoperdon sp. 1 and Lycoerdon sp. 2); these species morphologically look like L. umbrinum, however based on molecular analyses, they do not form a group together with GenBank samples of L. umbrinum.

Lycoperdon altimontanum Kreisel, Feddes Repert. Spec. Nov., Beih. 87 (1-2): 97 (1976) – MycoBank: MB 317026. Fig. 17a.

Basidiomata pyriform, 32 mm length × 35 mm width; subgleba cellular, well developed (≤ ⅓ of basidioma) and olive-brown. Exoperidium with whitish to light brown spines 0.1– 0.3 mm long., falling off with the age, leaving a smooth and light brown endoperidium surface. Gleba dark olive to brown. Apical endoperidium without inflated hyphae and mycosclereids. Capillitium with scattered pores. Basidiospores 6.0–8.0 µm, strongly verrucose [D] in LM, ornamentation number 7.3–11.2 for a circumference reduced to 10 µm.

For a complete description: see Kreisel (1976).

Specimens examined – REPUBLIC OF MACEDONIA, Mavrovo National Park, in beech forest, 20 Oct 2005, leg. K. Rusevska and M. Karadelev (MC-Fungi 05-787).

Lycoperdon atropurpureum Vittad., Monogr. Lycoperd.: 42 (1842) – MycoBank: MB 118735. Figs. 17b, 19a.

Basidioma pyriform to turbinate, 26–42 mm length × 15–35 mm wide. Subgleba cellular, well developed (≤ ⅓ of basidioma) and brown. Exoperidium with spines and granular ornamentations, the spines are falling off at maturity. Endoperidium surface smooth and buff colored. Gleba powdery and deep purple. Apical endoperidium without inflated hyphae and mycosclereids. Capillitium elastic with abundant pores. Basidiospores 6.5– 8.0 µm, strongly verrucose [D] in LM and columnar warts becoming flattened at the tips in SEM, 5.7–10.0 for a circumference reduced to 10 µm, surrounded by remains of sterigma and a short pedicel (up to 2.0 µm).

For a complete description: see Demoulin (1972a) and Jeppson and Demoulin (1989).

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Specimens examined – FRANCE, Pyrénées-Orientales, between Corsavy and Batere, habitat near Pinus sylvestris in high herbs, 1200 m, 6 Nov1968, leg. V. Demoulin (MA-

Fungi 63392). REPUBLIC OF MACEDONIA, deciduous forest, 10 Sep 2008, leg. K. Rusevska (MC-Fungi 08-10194); ibidem, Querco-Carpinetum orientalis with Castanea sativa plantings, 05 Oct 2008, leg. K. Rusevska (MC-Fungi 08-10227); ibidem, oak forest (Quercus frainetto), 10 Nov 2008, leg. K. Rusevska (MC-Fungi 08-10122); ibidem, in Quercus frainetto, 20 Oct 2005, leg. K. Rusevska and M. Karadelev (MC-Fungi 05- 2233); ibidem, 11 Jul 1999, leg. BSRS (MC-Fungi 99-10248); ibidem, oak forest mixed of Quercus frainetto and Corylus avellana, 15 Nov 2010, leg. K. Rusevska and M. Karadelev (MC-Fungi 06-10067); ibidem, black locust plantings, 20 Oct 1989, leg. M. Karadelev (MC-Fungi 89-9572); ibidem, Querco-Carpinetum orientalis, 30 Oct 1987, leg. M. Karadelev (MC-Fungi 87-7733).

Lycoperdon decipiens Dur. & Mont., Expl. Sci. Algérie, Bot. 1 Crypt.: 380 (1848) – MycoBank: MB232329. Fig. 17c.

Basidiomata subglobose to turbinate, 12–26 mm length × 24–32 mm wide. Subgleba cellular, well developed (≤ ⅓ of basidioma), grayish pink. Exoperidium with fragile spines, convergent at the apex, at the base becoming brown, and cream at the apical portion, both are falling off with age leaving a furfuraceous aspect. Surface endoperidium is smooth and grayish yellow. Gleba olive brown. Apical endoperidium with inflated hyphae 6.0–19.0 µm, septate and weakly dextrinoid, mycosclereids absent; apical sphaerocysts 13.0–22.0 × 9.5–19.0 µm. Capillitium with abundant pores. Basidiospores 6.0–7.0 µm, strongly verrucose [D] in LM, ornamentation as aculeate form, number of ornamentation 7.0–10.0 for a circumference reduced to 10 µm.

For a complete description: see Demoulin (1972a, 1983).

Specimens examined – FRANCE, Pyrénées-Orientales, between Corsavy and Batere, in naked soil or lawn of beach, 1200 m, 06 Nov 1968, leg. V. Demoulin (MA-Fungi

63390). SPAIN, Monarca, Santa Barbara, 17 Nov 1990, leg. F.D. Calonge (MA-Fungi 27674); Barcelona, Torrelletes, 18 Mar 1971, leg. V. Demoulin (45055).

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Lycoperdon echinatum Pers., Ann. Bot. (Usteri) 1: 147 (1794) – MycoBank: MB414453. Fig. 17d.

Basidiomata turbinate, 32–43 mm length × 32–54 mm wide. Subgleba cellular, well developed (≤ ⅓ of basidioma) and grayish brown. Exoperidium with dense curved black spines 1.0–4.0 (5.0) mm long., falling off with the age, leaving a reticulate and greyish brown endoperidium surface. Gleba brown. apical endoperidium not observed. Capillitium with pores. Basidiospores 5.5–6.5 µm, verrucose [C] in LM, ornamentation number of 8.8–10.7 for a circumference reduced to 10 µm;

For a complete description: see Demoulin (1972a) and Pegler et al. (1995).

Specimens examined – REPUBLIC OF MACEDONIA, beech forest, 23 Oct 2005, leg. M. Karadelev (MC-Fungi 05-782); ibidem, beech forest, 11 Sep 2005, leg. K. Rusevska (MC-Fungi 05-5160); ibidem, beech forest, 19 Aug 2006, leg. K. Rusevska (MC-Fungi 06-5916); ibidem, beech forest, 18 Sep 2006, leg. K. Rusevska (MC-Fungi 06-9676); ibidem, beech forest, 10 Oct 2005, leg. K. Rusevska (MC-Fungi 05-5376); ibidem,16 Aug

2006, leg. K. Rusevska (MC-Fungi 06-5900). SPAIN, Salamanca, Miranda del Costañar, 30TTK4786, humus of madroñal, 620 m, 27 Nov 2002, leg. S.P. Gorjón and P.G. Jimenez SPG 0197 (MA-Fungi 84507); Soria, Vinuesa, under Pinus and Fagus, 15 Oct 1997, leg. F.D. Calonge (MA-Fungi 39586).

Lycoperdon ericaeum Bonord., Bot. Ztg. 15: 628 (1857) – MycoBank: MB 223495. Figs. 17e, 19b.

Basidiomata turbinate, 46 mm length × 35 mm wide. Subgleba cellular, well developed (≤ ⅓ of basidioma) and grayish pink. Exoperidium ornamentation mixed with granules and stellate spines that falling off, leaving a furfuraceous aspect covering the smooth surfaced endoperidium; gleba brown. Apical endoperidium not observed. Capillitium with abundant pores. Basidiospores 4.5–5.0 µm, slightly verrucose in LM, columnar warty, flattened top and sometimes the ornamentation base connected by lines in SEM.

For a complete description: see Calonge and Demoulin, (1975).

Specimens examined – USA, Ohio, Forken Run State Park, Neigs Co., on soil, 21 Oct 1979, leg. W.B. and V.G. Cooke 57994 (MA-Fungi 63393, ex Herb. V. Demoulin 1358). 104

Lycoperdon excipuliforme (Scop.) Pers., Syn. Meth. Fung. (Göttingen) 1: 143 (1801) – MycoBank: MB 223280. Fig. 17f.

Basidiomata pyriform 150 mm length × 30–45 wide. Subgleba well developed (≤ ⅓ of basidioma) and brown. Exoperidium with minute warts or spines grouped in pyramids, exoperidium and endoperidium falling off with the age. Gleba yellowish brown. Apical endoperidium no observed. Capillitium with pores. Basidiospores 4–7 µm, verrucose [C] in LM.

For a complete description: see Calonge and Demoulin, (1975) and Perdeck (1950).

Specimens examined – REPUBLIC OF MACEDONIA, on soil of beech forest, 25 Sep 1998, leg. M. Karadelev (MC-Fungi 98-10138); ibidem, found in Salicetum, 10 Nov 2008, leg. M. Karadelev (MC-Fungi 08-10217); ibidem, Querco-Carpinetum orientalis, 22 Oct 2006, leg. K. Rusevska (MC-Fungi 06-6224); ibidem, Carpinus and Castanea forest, 01 Jan 2003, leg. Daov (MC-Fungi 03-2892); ibidem, found in Corylus association, 20 Sep 2005, leg. K. Rusevska and M. Karadelev (MC-Fungi 06-9715); ibidem, in pine plantings, 12 Oct 2008, leg. P. Atanasovski (MC-Fungi 08-10049).

Lycoperdon lambinonii Demoulin, Lejeunia, n.s. 62: 13 (1972) – MycoBank: MB 317033.

Basidiomata subglobose, pyriform to turbinate, 10 – 45 mm length × 20 – 50 mm wide. Subgleba cellular, well developed (≤ ⅓ of basidioma), olive-brown to grayish pink. Exoperidium with granules or spines (up to 0.8 mm long.), falling off with the age. Endoperidium surface smooth, without scars as in L. perlatum, yellowish brown. Gleba brown. Apical endoperidium not observed. Capillitium with rare pores and paracapillitium occasional. Basidiospores 5.4–5.8 µm, slightly verrucose to verrucose [B- C], in LM.

For a complete description: see (Demoulin, 1972b).

Specimens examined – REPUBLIC OF MACEDONIA, on soil of Beech and Molika pine forest, 20 Oct 2005, leg. K. Rusevska and M. Karadelev (MC-Fungi 05-10226). SPAIN, Tenerife, El Realejo Bajo, on soil of pine forest, 1 Dec 1972, leg. W. Wildpret and E. Beltrán (MA-Fungi 6902).

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Lycoperdon lividum Pers. J. Bot. (Desvaux) 2: 18 (1809) – MycoBank: MB 414454. Figs. 17g, 19c.

Basidiomata pyriform, 28 mm length × 18 mm wide. Subgleba cellular well developed (≤ ⅓ of basidioma), grayish pink, subgleba supported by a pseudorhiza 20 mm length, encrusted with particles of soil and/or sand. Exoperidium granulose, falling off with the age, leaving a smooth and grayish brown endoperidium surface. Gleba olive-brown. Apical endoperidium not observed. Capillitium with pores. Basidiospores 4.5–5.0 µm, slightly verrucose [A-B] in LM, with a shortly pedicel 0.5–1.0 µm long.

For a complete description: see Calonge and Demoulin, (1975).

Specimens examined – REPUBLIC OF MACEDONIA, on soil of oak forest, 22 Oct 2005, leg. M. Karadelev (MC-Fungi 05-10007); ibidem, on soil of meadow, 10 Nov 2005, leg. K. Rusevska (MC-Fungi 05-5323); ibidem, on soil of Digitalis viridiflorae and Pinetum peuces, 30 Sep 2002, leg. K. Rusevska and M. Karadelev (MC-Fungi 02-3594); ibidem, on soil of Quercus coccifera, 21 Oct 2008, leg. M. Karadelev (MC-Fungi 08- 10040). Spain, Almería, Tijola, under Pinus halepensis, 01 Feb 1987, leg. J.A.O. Rueda (MA-Fungi 19219); Guadalajara, Tamajón, 30SVH4859, in humus of Pinus pinea, 850 m, 09 Dec 2004, leg. F. Esteve, A. González, G. Moreno, et al., no. GP477 (MA-Fungi 68346); Ciudad Real, Viso del Marqués, in humus of Pinus pinea, 850 m, 03 Dec 2005, F.D. Calonge et al., no. GP639 (MA-Fungi 68348); Madrid, El Berrueco, in moss, on slate soil, 28 Mar 2002, leg. A. González and G. Sastre (MA-Fungi 74402).

Lycoperdon mammiforme Pers., Syn. Meth. Fung. (Göttingen) 1: 146 (1801) – MycoBank: MB 220868. Figs. 17h, 19d.

Basidioma subglobose, 42 mm length × 32 mm wide. Subgleba cellular and well developed (≤ ⅓ of basidioma), grayish. Exoperidium ornamentation with deciduous withe spines, convergent at the tips, recovering by withe veil, falling off in plates. Endoperidium surface smooth, grayish brown. Gleba powdery brown. Apical endoperidium without inflated hyphae and mycosclereids. Capillitium elastic, pores rare. Basidiospores globose 4.0–5.0 µm, verrucose [C] in LM and with columnar warts becoming flattened at the tips in SEM, number of ornamentation of 8.5–11.0 for a circumference reduced to 10 µm.

For a complete description: see Calonge (1998).

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Specimens examined – REPUBLIC OF MACEDONIA, on soil of pine plantings, 23 Oct

2005, leg. K. Rusevska and M. Karadelev (MC-Fungi 05-773). SPAIN, Huesca, Parque Nacional da Ordesa, Circo de Pineta next to the wall of Monte Perdido, 17 Oct 1989, leg. F.D. Calonge (MA-Fungi 24103); Navarra, Zarranz, on hayedo, 10 Dec 1990, leg. F.D. Calonge (MA-Fungi 31251).

Lycoperdon marginatum Vittad. ex Moris & De Not., Monogr. Lycoperd. 185 (1842) – MycoBank: MB 220937. Figs. 17i, 19e.

Basidiomata globose to subglobose, 10 mm length × 35 mm wide. Subgleba compact to cellular, well developed (≤ ⅓ of basidioma), grayish brown. Exoperidium ornamentation with deciduous white pyramidal spines, falling off in plates; surface of endoperidium smooth. Apical endoperidium with dextrinoid inflated hyphae 6.0–13.0 µm, septate hyphae, mycosclereids 34.0–61.0 × 14.0–39.0 µm, weakly dextrinoid. Capillitium elastic with abundant pores. Basidiospores globose 4.0–4.8 µm, punctate to slightly verrucose [A-B] in LM and verrucose in SEM.

For a complete description: see Demoulin (1972a).

Specimens examined – SPAIN, Gerona, Vilobi d’ Onyar, 19 Oct 1987, leg. J.M.Vidal (MA-Fungi 31252); Teruel, Linares de Mora, in pine forest of Pinus sylvestris, 25 Oct 1995, leg. F.D. Calonge (MA-Fungi 34063).

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Figure 17 Dry basidiomata of Lycoperdon species. a. Lycoperdon altimontanum (MA-Fungi 63392); b. L. atropurpureum (MA-Fungi 63389); c. L. decipiens (MA-Fungi 27674); d. L. echinatum (MA-Fungi 39586); e. L. ericaeum (MA-Fungi 63393); f. L. excipuliforme (MC-Fungi 06:6224); g. L. lividum (MA- Fungi 68348); h. L. mammiforme (MA-Fungi 31251); i. L. marginatum (MA-Fungi 31232); all bars = 10 mm.

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Lycoperdon molle Pers., Syn. Meth. Fung. (Göttingen) 1: 150 (1801) – MycoBank: MB 271483. Fig. 18a. Basidiomata pyriform to turbinate, 32–53 mm length × 27–39 mm wide. Subgleba cellular, well developed (≤ ⅓ of basidioma), white becoming grayish pink. Exoperidium with spines (no more 1 mm long) falling off at maturity; endoperidium surface smooth and grayish brown. Gleba olive to brown. Apical endoperidium with weakly dextrinoid inflated hyphae 5.5–10.0 µm, without mycosclereids. Capillitium with scattered pores. Basidiospores 5.0–6.0 µm strongly verrucose [C] in LM, number 8.2–11.0 for a circumference reduced to 10 µm.

For a complete description: see Calonge and Demoulin, (1975).

Specimens examined – Greece, Stratoniki, Stratoni, in Pinus forest, 26 Apr 2007, leg. F.D. Calonge (MA-Fungi 73601). Portugal, Minho, Gerês, Parque Nacional Peneda- Gerês, 19 Nov 1988, leg. J. Cardoso, E. Descals, C. Lado, I. Melo and M.T. Telleria (MA-

Fungi 31618). SPAIN, Madrid, Guadarrama, La Jarosa, 10 Mar 1991, Leg. A. Garcia (MA- Fungi 31259); Vizcaya, Altube, en hayedo, 23 Sep 1976, leg. E.P. Moral (MA-Fungi 21623); Pontevedra, Villagarcia de Arosa, 18 Nov 1983, leg. E. Valdez (MA-Fungi 21737). Republic of Macedonia, on soil (acid) of beech forest, 19 Oct 2006, leg. K. Rusevska (MC-Fungi 06-5918); ibidem, on soil (acid) of beech forest, 10 Oct 2005, leg. K. Rusevska (MC-Fungi 05-5380).

Lycoperdon nigrescens Pers., Neues Mag. Bot. 1: 87 (1794) – MycoBank: MB 218442. Fig. 18b, 19f. Basidiomata subpyriform to turbinate, 23–50 mm length × 24–46 mm wide. Subgleba cellular, well developed (≤ ⅓ of basidioma), yellow brown becoming pinkish gray. Subgleba supported by a pseudorhiza up to 4.0 mm length. Exoperidium ornamentation mixed with granules and dark spines (up to 1.0 mm long.), spines falling off with age leaving an areolate endoperidium surface. Gleba olive brown. Apical endoperidium with septate and inflated hyphae 8.0–16.0 µm and mycosclereids 35.0–68.0 × 14.0–30.0 µm, both strongly dextrinoid. Capillitium with occasional pores; paracapillitium rare. Basidiospores 4.5–6.5 µm, slightly verrucose [B], short pedicel 0.5–1.0 µm long. in LM, warty with flattened tips in SEM.

For a complete description: see Calonge (1998).

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Specimens examined – LUXEMBURGO, Grand-Duché de Luxemburgo, Oeslebf Espeldange, 10 Oct 1986, leg. J. Lambon (MA-Fungi 63396); Spain, Ávila, Navaluenga, Venero Claro, 12 Nov 1987, leg. F.D. Calonge (MA-Fungi 22012); Burgos, Quintanar de la Sierra, 14 Oct1997, leg. F.D. Calonge (MA-Fungi 39587); Madrid, Montejo de la Sierra, under Quercus pirenaica, 19 Jun 1988, leg. A. Guerra (MA-Fungi 31296).

Lycoperdon niveum Kreisel, Khumbu Himal 6(1): 30 (1969) – MycoBank: MB 317036. Fig. 18c.

Basidiomata subglobose to subpyriform, 14–35 mm length × 34–35 mm wide. Subgleba cellular, well developed (≤ ⅓ of basidioma), grayish. Exoperidium with spiny ornamentation, the spines are falling off at maturity; surface endoperidium smooth and light brown. Gleba powdery, brown. Apical endoperidium not observed. Capillitium elastic with abundant pores. Basidiospores 4.5–5.0 µm, verrucose [D], warts up to 0.3 µm long. in LM, number of 7–9.5 for a circumference reduced to 10 µm.

For a complete description: see Demoulin (1972a).

Specimens examined – NORWAY, Hordaland Virk Furise, Samddalsnuten, in Salix reticulate and Silene actual, 13 Ago 1985, leg. V. Demoulin (MA-Fungi 63397).

REPUBLIC OF MACEDONIA, on soil of meadow, 19 Ago 2006, K. Rusevska (MC-Fungi 06- 5915); ibidem, on soil of deciduous forest of Castanea sativa, 29 Sep 2002, leg. unknown

(MC-Fungi 02-10008). SPAIN, Huesca, San Juan de La Penta, under Pinus sylvestris, 13 Sep 1974, leg. A. Tomas (MA-Fungi 21618).

Lycoperdon perlatum Pers.: Pers., Synops. Meth. Fung. 1: 145 (1801) – MycoBank: MB 220647. Figs. 18d, 19g.

Basidiomata obovate, subpyriform to turbinate, 30–59 mm length × 20–36 mm wide. Subgleba cellular, well developed (≤ ⅓ of basidioma), cream becoming pela orange, grayish to brownish. Apical exoperidium ornamentation with deciduous spines leaving areolate scars on endoperidium surface. Apical endoperidium with inflated hyphae 7.0– 14.0 µm, mycosclereids 26.0–89.0 × 12.0–25.0 µm. Capillitium elastic with occasional pores. Basidiospores globose, 4.0–5.0 µm, verrucose [B-C] in LM and aculeate in SEM, number of ornamentation 7.0–11.5 for a circumference reduced to 10 µm.

For a complete description: see Calonge and Demoulin, (1975).

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Specimens examined – REPUBLIC OF MACEDONIA, on soil of open grassland, 01 Jan 2003, leg. S. Daov (MC-Fungi 03-10225); ibidem, on soil of beech forest, 19 Aug 2006, leg. K. Rusevska (MC-Fungi 06-5917); ibidem, on soil of deciduous forest, 09 Apr 2007, leg. S. Spasikova (MC-Fungi 07-6663); ibidem, on soil of Quercus coccifera, 30 Oct 1987, leg. M. Karadelev (MC-Fungi 87-7735); ibidem, on soil of meadow, 01 Oct 2002, leg. K. Rusevska and M. Karadelev (MC-Fungi 02-2389); ibidem, on soil at roadsides, 29 Sep 1990, leg. M. Karadelev (MC-Fungi 90-10234); ibidem, on soil of Querco- Caarpinetum orientalis, 11 Oct 2002, leg. S. Dimkov (MC-Fungi 02-2995); ibidem, on soil deciduous forest, 11 Oct 2008, leg. K. Rusevska (MC-Fungi 08-10055); ibidem, on soil of edge of oak forest, 14 Nov 2008, leg. K. Rusevska (MC-Fungi 08-10119); ibidem, on soil of Quercus coccifera and Carpinetum orientalis, 21 Oct 2008, leg. M. Karadelev (MC-Fungi 08-10035); ibidem, on soil of meadow in oak forest with Castanea sativa plantings, 01 Jan 2007, leg. M. Karadelev (MC-Fungi 07-10144); ibidem, on soil of planting of pine, 10 Oct 2005, leg. K. Rusevska (MC-Fungi 05-5316); ibidem, 12 Sep 2008, leg. K. Rusevska (MC-Fungi 08-10209); ibidem, on soil of meadow, 16 Jun 2007, leg. M. Karadelev and K. Rusevska (MC-Fungi 07-7207); ibidem, 09 Apr 2008, leg. K. Rusevska (MC-Fungi 05-9247). Spain, Mallorca, Monnaber, in Quercus ilex and Pinus halepensis, 13 Nov 1992, F.D. Calonge (MA-Fungi 29732); Teruel, 05 Jun 1988, leg. F.D. Calonge (MA-Fungi 31233); León, Puebla de Lillo, in Fagus sylvatica, 21 Oct 1992, leg. F.D. Calonge (MA-Fungi 31253); Navarra, Sra. Aralar, in mixed forest of Picea and Fagus, 03 Sep 1977, leg. L.M.G. Bona, no. 965 (MA-Fungi 64953).

Lycoperdon pratense Pers., Neues Mag. Bot.1: 87 (1794): Pers., Syn. meth. fung.: 142 (1801) – MycoBank: MB 174409. Fig. 18e.

Basidiomata pyriform, 30–50 mm length × 30 mm wide. Subgleba cellular, well developed subgleba (≤ ⅓ of basidioma). Exoperidium ornamentation with deciduous small spines or granules; surface endoperidium smooth. Apical endoperidium without inflated hyphae and mycosclereids. Paracapillitium abundant. Capillitium when present without pores. Basidiospores globose 3.0–4.0 µm, slightly verrucose [A-B] in LM.

For a complete description: see Calonge and Demoulin (1975), Moyerson and Demoulin (1996), Calonge (1998).

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Specimens examined – REPUBLIC OF MACEDONIA, on soil of pine plantings, 14 Nov 2008, leg. K. Rusevska (MC-Fungi 08-10114); ibidem, on soil of Querco-Carpinetum orientalis, 12 Mar 2009, leg. S. Stojanov (MC-Fungi 03-3040).

Lycoperdon pyriforme Schaeff., Fung. Bavar. Palat. nasc. 4: 128 (1774): Pers., Syn. Meth. Fung.: 148 (1801) – MycoBank: MB 499404. Fig. 18f.

Basidiomata 27–41 mm length × 36–41 mm wide, subgleba cellular, well developed (≤ ⅓ of basidioma), whitish. Exoperidium with persistent brown warts. Endoperidium yellowish brown; gleba olive brown. Endoperidium apical without inflated hyphae and mycosclereids; exoperidium is composed of spiny cells arranged in regular chains, strongly dextrinoid. Capillitium and paracapillitium abundant. Basidiospores 3.0–4.0 µm, smooth to punctate [A] in LM.

For a complete description: see Demoulin (1970).

Specimens examined – AUSTRALIA, rotten wood of deciduous and coniferous tree,

10 Sep 2008, leg. K. Rusevska (MC-Fungi 08-10196). REPUBLIC OF MACEDONIA, decaying wood in beech forest mixed forest (Fagus, Pinus, Acer, Fraxinus and Quercus), 11 Oct 2001, leg. K. Zimbacova (MC-Fungi 01-745); ibidem, decaying wood of beech forest, 04 Sep 2005, leg. K. Rusevska (MC-Fungi 05-5074); ibidem, growing in decaying wood of beech and Salicetum albae-fragilis, 13 Nov 2008, leg. M. Karadelev (MC-Fungi 08-10128); ibidem, growing in decaying wood of beech forest, 07 Nov 1998, leg. M.

Karadelev (MC-Fungi 98-3045). SPAIN, Austurias, Covadonga, subida a los lagos, growing in decaying wood, 10 Jan 1986, leg. M. Dueñas (MA-Fungi 16908).

Lycoperdon subumbrinum Jeppson & E. Larss., Mycol. Progr. 11: 891 (2012) – MycoBank: MB 563802. Fig. 18g, 19h.

Basidiomata subpyriform to pyriform, 30–38 mm length × 32–35 mm wide, cellular, well developed (≤ ⅓ of basidioma), grayish brown. Exoperidium with tiny spines (0.5–0.8 mm long.) falling off with age exposing a shining surface endoperidium. Gleba yellowish brown. Apical endoperidium with inflated hyphae 11.0–25.0 µm, septate and strongly dextrinoid, without mycosclereids. Capillitium with pores. Paracapillitium occasional. Basidiospores 4.5–5.0 µm, slightly verrucose [B-C], short pedicel 0.5–1.0 µm long., in LM, columnar warty bonded to each other in SEM.

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For a complete description: see Jeppson et al. (2012).

Specimens examined – UK, England, Straud, Cranham, in forest of Fagus sylvatica,

13 Sep 1992, leg. F.D. Calonge (28449). FRANCE, Pyrénées-Orientales, surround Borg- Argental by a road crossing a pine forest, 01 Sep 1972, leg. J. Delangue (MA-Fungi 63391). Spain, Gerona, Bañolas, in soil of holm oaks and jars, 17 Nov 1985 (MA-Fungi 31234); Villaviciosa, Monte Trigo, 26 Dec 1991, leg. A. Castro (MA-Fungi 30564). Republic of Macedonia, on soil of beech forest, 30 Oct 1987, leg. M. Karadelev (MC- Fungi 87-10137).

Lycoperdon umbrinoides Dissing & Lange, Bull. Jard. bot. État Brux. 32: 344 (1962) – MycoBank: MB 333492. Fig. 18h.

Basidiomata subpyriform, 59 mm length × 54 mm wide. Subgleba cellular, well developed (≤ ⅓ of basidioma), reddish brown. Exoperidium ornamentation with dark spines, spines falling off with age, leaving a smooth, brownish endoperidium surface. Gleba olive. Apical endoperidium not observed. Capillitium without pores and septate. Basidiospores 4–5 µm, smooth to punctate [A-B] in LM, pedicel 1.0–1.5 µm long.

For a complete description: see Dissing and Lange (1962).

Specimens examined – SPAIN, Gerona, Sant Sadurní de L’Huera, in riverside forest, under Alnus glutinosa, 08 Nov 1995, leg. J.M. Vidal (MA-Fungi 35530).

Lycoperdon umbrinum Pers., Ann. Bot. (Usteri) 11: 28 (1797): Syn. meth. fung.: 147 (1801) – MycoBank: MB 232824. Figs. 18i, 19i.

Basidiomata pyriform to turbinate, 18–35 mm length × 15–39 mm wide. Subgleba cellular, well developed (≤ ⅓ of basidioma), brown to pinkish grey. Exoperidium ornamentation with persistent spines (up to 1 mm length). Endoperidium surface smooth, orange umber to light brown. Gleba powdery, brown. Apical endoperidium not observed. Capillitium elastic, with pores. Basidiospores globose 3.0–5.0 µm, slightly verrucose [A- B] in LM, pedicelate 0.5–1.5 µm length.

For a complete description: see Demoulin (1972a).

Specimens examined – BELGIUM, Luxemburg, húmus el arguilles d’épigeas en pessieie sur ochiste de Famenne, 23 Feb 1972, leg. V. Demoulin, no. 4342 (MA-Fungi

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63394). GREECE, Stratonik, Stratoni, under Pinus sp., 26 Apr 2007, leg. F.D. Calonge (MA-Fungi 73600).

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Species not determined

Here are the species that need more analysis, or have only one basidioma and require a greater sampling effort.

Lycoperdon sp. 1

Basidioma immature 9 mm length × 23 mm wide, subglobose, yellow. Subgleba white, cellular. Gleba yellowish. Basidioma mature in bad condition, 14–28 mm length × 29–34 mm wide, pyriform to subpyriform. Exoperidium warty, with warts falling off, yellow. Endoperidium papery, smooth, cream. Subgleba well-developed 17 mm length, cellular, gray. Diaphragm concolorous with gleba. Gleba powdery, greyish brown. Apical endoperidium without hyphae with inflated terminations, mycosclereids 35.7–85.7 µm long × 13.0–32.8 µm wide, weakly dextrinoid. Capillitium 3–4.5 µm diam., smooth, branched, aseptate, without pores, yellowish brown, non-dextrinoid. Paracapillitium 2.6– 6.9 µm diam. Basidiospores 3.8–5.3 × 3.7–4.7 µm, slightly verrucose [A-B], pedicel 0.5– 2.5 µm long, or remains of sterigma 1–5 µm long.

Specimens Examined – BRAZIL, Rio Grande do Sul, Santa Maria, Campus Universidade Federal de Santa Maria, on soil of Atlantic forest, 06 Mar 2009, leg. V.G. Cortez (ICN154484).

Lycoperdon sp. 2

Basidiomata 27–45 mm long × 25–28 mm wide, subglobose to turbinate. Exoperidium warty, warts persistent, brown; endoperidium papery, cream. Subgleba well developed, 20 mm length, cellular, yellowish becoming gray. Gleba powdery, brown. Apical endoperidium without hyphae with inflated terminations and mycosclereids. Capillitium 4–6 µm diam., smooth, branched, aseptate, without pores, yellowish brown, non- dextrinoid. Paracapillitium absent. Basidiospores 5.5–6.0 µm, globose, strongly verrucose [D], pedicel occasional 0.5–1.5 µm long.

Specimens examined – INDIA, Uttaranchal, Pauri, 27 Jun 2006, leg. D. Bisht (MA- Fungi 73250).

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Figure 18 Dry basidiomata of Lycoperdon species. a. Lycoperdon molle (MA-Fungi 31259); b. L. nigrescens (MA-Fungi 22012); c. L. niveum (MA-Fungi 21618); d. L. perlatum (MA-Fungi 64953); e. L. pratense (MC-Fungi 08: 10114); f. L. pyriforme (MA-Fungi 16908); g. L. subumbrinum (MA-Fungi 63391); h. L. umbrinoides (MA-Fungi 53530); i. L. umbrinum MA-Fungi 63394 – unsuccessful amplification); all bars = 10 mm.

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Figure 19 Scanning microscope electronic of some Lycoperdon species. a. Lycoperdon atropurpureum (MA-Fungi 63389); b. L. ericaeum (MA-Fungi 63393); c. L. lividum (MA-Fungi 74402 – unsuccessful

amplification); d. L. mammiforme (MA-Fungi 24103); e. L. marginatum (MA-Fungi 31252); f. L. nigrescens (MA-Fungi 63396 – unsuccessful amplification); g. L. perlatum (MA-Fungi 64953); h. L. subumbrinum (MA-Fungi 63391); i. L. umbrinum (MA-Fungi 73600 – unsuccessful amplification); all bars = 5 µm.

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4 DISCUSSION

To use DNA sequences as a barcode of life is a great idea, since it could be very useful, for example, to identify biodiversity from environmental samples (Floyd et al., 2002; Hogg and Hebert, 2004; Blaxter et al., 2005); in cases when the morphological characters are not enough to discriminate species (Druzhinina et al., 2005; Page et al., 2005); in dry specimens which can have degraded DNA (Hajibabaei et al., 2006); or even to use in forensic techniques (Nelson et al., 2007). The web site of Barcode of Life (www.barcoding.si.edu) defines barcoding as “a diagnostic technique for species identification, that uses a short, standardized sequence region”. The use of the DNA barcode to help in species discrimination has been an important approach for expert taxonomists in different groups of organisms such as: algae (Robba et al., 2006), animals (Ben-David et al., 2007), plants (Yao et al., 2010), oomycota (Robideau et al., 2011) and fungi (Bellemain et al., 2010). As proposed by Hebert et al. (2004), the sequences to be selected as a DNA barcode should be short (500–800 bp), and easy to amplify to be used to species identification. The ITS is shown to be promising as DNA barcode for the genus Lycoperdon; the success of amplification was 87% in contrast to the 13% of unsuccessful, a similar result was found by Schoch et al. (2012); they obtained 90% success in amplification of ITS region with high-quality PCR product and a high power for species discrimination in Basidiomycota. Unsuccessful amplification in Lycoperdaceae is due to the powdery gleba, so the spores are easily carried by air and can be deposited in other species. This creates a problem during the process of extraction and amplification.

The success of identification using ITS as barcode obtained here was above 80% in the two techniques used; Hebert et al., (2003) obtained 100% success in the identification at the species level. The incorrect identifications are found in terminal branches such as Lycoperdon excipuliforme, L. altimontanum, L. molle, L. umbrinum, L. subumbrinum or L. lambinonii. Within these terminal branches there are sequences that belong to specimens that we expected to be difficult to determine morphologically, e.g. immature specimens of L. excipuliforme that can be mistaken for L. molle (Perdeck, 1950) or specimens of L. molle that are difficult to separate from L. umbrinum (Demoulin, 1968).

Using the identification based on distance between sequences, 87.58% success of identification was obtained, based on “best match and best close match”, and the incorrect identification was 11.03% and 9.45% (best match and best close match); these results are 118

in contrast to those found by Meier et al. (2006), who obtained low success of identification (67.7%) based on “best match” and 66.3% based on “best close match”, although the authors considered the low success of identification was due to the relatively small number of sequences with more than 500bp of overlap; in the dataset used here all species/sequences are 550 to 700 bp. Another interesting fact is that the TaxonDNA used by Meier et al. (2006) was developed to refute the works that have used the distance methods and Neighbor-Joining tree (for example Hebert et al., 2003, 2004). However, our results with both techniques are similar. Some authors argued against the use of tree- based identifications on Neighbor-Joining trees because (i) the forming of a cluster on a tree is insufficient to identify species, (ii) the large size of the dataset results in ambiguities that are difficult to detect, because most Neighbor-Joining algorithms generate a single tree, in which there is the possibility to have internal attachment points that are ambiguous in any rooted tree (Will and Rubinoff, 2004; Meier et al., 2006).

The results obtained by techniques of tree-based identification of Neighbor- Joining, identification based on distance sequence, and the search performed on UNITE are most similar when treating species morphologically identified as L. altimontanum, L. excipuliforme (if immature species are used), L. molle and L. umbrinum. ITS as barcoding of Lycoperdon genus is promising; however, some groups of Lycoperdon species are not yet well delimited. Geiser et al. (2004), observing that ITSs tend to evolve at a high rate at the species level, tested the translation elongation factor 1-α (TEF) gene as an alternative in the identification of Fusarium species.

The morphological analysis showed that exoperidium ornamentation patterns (macromorphology and microstructures), endoperidium surface, the size and pattern of basidiospore ornamentation, and the capillitium with pores or not, are the most important characters in Lycoperdon taxonomy. This fact has been observed by Demoulin (1972a, 1976), and Homrich and Wright (1988). Demoulin (1972a), observed that those features such as strongly ornamented spores and big size are more ancestral than less ornamented spores and smaller size.

The terminal branch of L. perlatum clusters with L. novergicum and L. marginatum in the major clade of Lycoperdon subg, Lycoperdon. The ITS barcode results are similar to those obtained by Larsson and Jeppson (2008), using phylogenetic analyses of ITS-LSU sequences. In the morphological analysis, Lycoperdon perlatum and L. norvegicum share exoperidium ornamentation with deciduous spines leaving an areolate 119 endoperidium surface; however, the former has verrucose basidiospores [B-C] whereas, L. norvegicum has smooth basidiospores [A] (Demoulin, 1972a; Calonge and Demoulin, 1975). On the other hand, L. marginatum differs by its exoperidium ornamentation with pyramidal spines, also, falling off, but in this case the spines fall in plates leaving a smooth endoperidium surface, and it has basidiospores punctate to slightly verrucose [A-B] (Calonge and Demoulin, 1975; Calonge, 1998).

The terminal branch of Lycoperdon pratense remained with Lycoperdon subg. Vascellum, similar to the results of Larsson and Jeppson (2008) and Bates et al. 2009). The only sample from Brazil (Lycoperdon sp.1) clusters with L. pratense; however, we need more samples and analysis to clarify this branch. As described by Calonge and Demoulin (1975), Moyersoen and Demoulin, (1996), and Calonge (1998), L. pratense showed exoperidium ornamentation with groups of white spines or granules, falling off with age, capillitium absent, and abundant paracapillitium, these characters are distinct from those found in Lycoperdon sp. 1.

Inside the clade Lycoperdon subg. Utraria fifteen species were grouped, in accordance with Larsson and Jeppson (2008) and Bates et al. (2009): L. altimontanum, L. atropurpureum, L. decipiens, L. echinatum, L. ericaeum, L. excipuliforme, L. lambinonii, L. lividum, L. mammiforme, L. molle, L. nigrescens, L. niveum, L. subumbrinum, L. umbrinum and Lycoperdon sp. 2.

With the Utraria clade expert taxonomists disagree about some morphological concepts, mostly when delimiting the species of Lycoperdon atropurpureum, L. decipiens, L. molle, L. umbrinum (Perdeck, 1950; Demoulin 1972a; Calonge and Demoulin 1975). According to Demoulin (1968), due to the morphological variability of L. molle, taxonomists have many problems delimiting the species of L. atropupureum, L. molle and L. umbrinum, and sometimes these species were assembled into the Lycoperdon Molle-group. Calonge and Demoulin (1975) mention that L. atropurpureum specimens can be found under the names of L. molle and L. umbrinum. Calonge (1998) studied L. atropurpureum and L. decipiens and considered that there were not enough morphological features to distinguish both species, and he synonymized them. In the present study, the terminal branch of Lycoperdon atropurpureum (weakly supported) clusters with the terminal branch of L. umbrinum; L. atropurpureum has a deep purple gleba, and basidiospores strongly verrucose [D] while, L. umbrinum showed a brown gleba, and basidiospores mostly slightly verrucose [B]; Perdeck (1950) considered L. 120

atropurpureum Vittad. and L. umbrinum Hollós synonymous with L. molle; on the other hand, L. umbrinum Pers. is considered as a distinct species. He has observed, also, that L. umbrinum subgleba is dark or purplish brown and the gleba is olive or umber brown; Moyersoen and Demoulin (1996), separated L. molle from L. atropurpureum because the first one has distinct characters as well as persistent spines, capillitium with abundant pores, and basidiospores with smaller ornamentation. In our opinion, L. atropurpureum, L. decipiens and L. umbrinum are distinct species; even though weakly supported in the molecular analysis; the ITS sequences together with the morphological analysis helped us to separate these species. According to Hebert and Barrett (2005), the DNA barcode, in addition to the morphological and ecological characters, can improve the identification of species. Perhaps, by adding other markers to the molecular analyses, the support value can improve. Following Stielow et al. (2015), one gene can improve the species discrimination by ITS region; the TEF1α, is considered by Stielow et al. (2015) as likely to be one candidate as a second secondary barcode for fungi.

The terminal branch of Lycoperdon frigidum is close to L. atropurpureum and L. umbrinum. This terminal branch also is weakly supported (bs = 57%, pp = 0.71), however, we have not analyzed these specimens morphologically. The characters distinctive of L. frigidum according Demoulin (1972a) are: exoperidium ornamentation with persistent white spines in cone format, subgleba well developed, brown-purple, gleba brown, basidiospores verrucose [B-C], capillitium septate and abundant pores: these characters differ from those shown by L. atropurpureum and L. umbrinum.

Lycoperdon mammiforme is a well supported branch and is close to L. atropurpureum, L. frigidum and L. umbrinum; however, the characters shown by L. mammiforme are quite different from them: exoperidium ornamentation with deciduous white spines, convergent at the tips, covered by a white veil, falling off in plates, basidiospores verrucose [C] and capillitium with pores; these characters were also found by Pegler et al. (1995) and Calonge (1998). When the specimens of L. mammiforme lose the veil before maturity they can be mistaken for L. molle because the latter also has white spines and verrucose basidiospores [C]. The terminal branch of Lycoperdon sp. 2 is close to L. mammiforme yet with a less derived position, it does not have characters of L. mammiforme.

In our molecular analysis Lycoperdon decipiens, L. molle and L. niveum are close. L. niveum is weakly supported, L. decipiens is moderately to well supported and L. molle 121 is well supported; L. decipiens differs by its exoperidium with fragile spines, falling off with age leaving a furfuraceous aspect, gleba olive brown, basidiospores strongly verrucose [D] and capillitium with abundant pores; while L. molle has spines (no more 1 mm long) falling off, subgleba colored grayish pink, gleba olive brown and basidiospores verrucose [C], in contrast, L. niveum has basidiospores strongly verrucose [D] and capillitium with abundant pores. Following the criteria adopted by Demoulin (1972a, 1983) and Calonge (1998), L. decipiens is synonymous with L. atropurpureum and the morphological features are insufficient to separate them. As discussed above, we believe they are distinct species. Lycoperdon atropurpureum lacks the furfuraceous endoperidium surface when the spines from exoperidium fall off with age, in contrast to the furfuraceous endoperidium surface in L. decipiens.

The terminal branch of Lycoperdon lividum is separated into two weakly supported terminal branches. All morphological features lead us to believe these specimens belong to L. lividum; this species is easily recognizable by its subgleba supported by a pseudorhiza, exoperidium granulose and falling off, endoperidium surface smooth, basidiospores punctate [A-B] and capillitium with abundant pores; these characters follow those found by Pegler et al. (1995) and Calonge (1998).

The terminal branch of Lycoperdon altimontanum and L. excipuliforme are close, but not well supported. Lycoperdon altimontanum was described the first time from alpine species of the Himalaya mountains (Nepal) (Kreisel, 1976); and Calvatia excipuliformis was described by Perdeck (1950) as a comb. nov. to accommodate L. excipuliforme. Later, (Kreisel, 1989) rearranged the species with slit-like pitted capillitium and placed C. excipuliformis into the genus Handkea. However, after work of Larsson and Jeppson (2008), based on molecular data, C. excipuliformis belongs in the Lycoperdon genus. The features that separate these species are: exoperidium ornamentation with whitish spines, falling off, basidiospores 6.0–8.0 µm, strongly verrucose [D], and capillitium with scarred pores in L. altimontanum; whereas, L. excipuliforme has exoperidium ornamentation with minute warts or spines grouped in pyramids, exoperidium and endoperidium falling off with the age, basidiospores 4–7 µm, verrucose [C], capillitium with irregular pores. The bigger basidiospores in L. altimontanum, and the exoperidium and endoperidium falling off in L. excipuliforme make clear the distinction between these species. Yet, L. excipuliforme can be mistaken with L. molle when the specimens are small and the subgleba is poorly developed (Calonge and Demoulin, 1975); moreover, we observed

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when the specimens of L. excipuliforme, L. molle or both species are immature and the basidiospores are not completely formed, they are most difficult to determinate and can be confused; consequently the use of immature specimens to delimitate or to describe species is not advisable because this fact can bring several problems in the identification.

The terminal branch of Lycoperdon lambinonii is separated into two weakly supported terminal branches; one includes the specimen of L. lambinonii (Demoulin4622) and L. turneri (MJ5251), the other branch has L. lambinonii (MJ5245), one specimen from Macedonia (MC-Fungi05-10226), two species of L. turneri (MJ 4265 and Lange 0895), and three specimens of L. umbrinum (MJ4556, MJ4556a and MJ 4559). Lycoperdon lambinonii was described by Demoulin (1972b); the author considers that the species occupies an intermediary position between L. molle and L. umbrinum; In this terminal branch the specimens Demoulin4622 and MC-Fungi05-10226 have been checked by V.D., L. lambinonii has basidiospores [B-C] and capillitium with rare pores, while L. umbrinum has basidiospores [A-B] and capillitium with abundant pores; L. turneri was a surprise here, because the morphological features described by Demoulin and Lange (1990): exoperidium ornamentation with small spines (0.2–0.3 mm length), endoperidium falling off, capillitium septate, distinguish these species; although, the basidiospores are similar (slightly to verrucose [B-C]) in both species, the number of ornamentations for a circumference of 10 µm in L. lambinonii is bigger than L. turneri, 8–11 versus 6–9 (Demoulin 1972a).

The terminal branch of Lycoperdon echinatum is well supported; morphologically this species is characterized by its exoperidium ornamentation with long and dark spines (up to 5.0 mm), falling off, leaving an areolate endoperidium surface, basidiospores verrucose [C] and capillitium with pores. Demoulin (1972a) considered that, depending on the stage of maturation, many specimens can be found with the compact subgleba and can be mistakenly identified. To Pegler et al. (1995), L. nigrescens can be confused with L. echinatum, although the characters analyzed here and in L. nigrescens (see below) are easily distinguished in both species.

The terminal branch of Lycoperdon nigrescens is well supported and close to L. utriforme and L. radicatum. Larsson and Jeppson (2008) found different results: L. utriforme and L. radicatum are sister clades in Lycoperdon subgenus Bovistella; in our analysis, with only ITS as barcode, these two species are grouped into subgenus Utraria. Probably, adding more markers, this terminal branch can be better resolved. According 123 to Pegler et al. (1995) immature specimens of L. echinatum can be mistaken for L. nigrescens; maybe this is because in young specimens the exoperidium ornamentation is not yet fallen off, and the other features, such as basidiospores and capillitium, are not completely formed; in mature specimens, Lycoperdon echinatum has verrucose basidiospores [C], in contrast to the slightly verrucose [B] in L. nigrescens.

Another terminal branch well supported is Lycoperdon pyriforme; our results corroborate the phylogenetic results found by Larsson and Jeppson (2008) and Bates et al. (2009), in which L. pyriforme belongs to Lycoperdon subg. Apioperdon. and Lycoperdon umbrinoides is a sister group of L. pyriforme; however, these species differ in the exoperidium ornamentation that shows persistent warts, basidiospores punctate [A] and sphaerocysts with irregular forms in L. pyriforme as described by (Pelger et al., 1995), and exoperidium ornamentation with deciduous spines, basidiospores verrucose [C] and sphaerocysts with regular forms (perlatum-type) likely described by Dissing and Lange, (1962).

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5 CONCLUSION

In the present work, the use of ITS region as DNA barcode of Lycoperdon genus is promising; although, some terminal branches such as Lycoperdon altimontanum, L. excipuliforme, L. lambinonii, L. molle, L. subumbrinum and L. umbrinum need more attention and the use of other markers is necessary to obtain more resolution. In fact, other empirical results are that the integration of morphological taxonomy and molecular analysis is the best methodology in work about DNA barcoding (Barrett and Hebert, 2005; Hebert and Gregory, 2005).

The method of search by matching on UNITE website can help the researchers around the world who want to use the DNA barcode to identify species, and need a first and faster identification to delimit their samples. The use of several tools can aid in the identification of unknown sequences (Kõljalg et al., 2005). And the idea of Species Hypothesis, adopted by Kõljalg et al. (2013), is a useful concept and provided good data along with fully annotated ITS sequences, ecology, and taxonomic information. The BLASTn tool is the other technique to aid the taxonomist who needs a preliminary identification, and the resolution of the approximation of species identity is interesting and satisfactory, although, more analysis are necessary to confirm any results that you have with this technique.

The neighbor-joining tree discriminating species in barcodestudies have been conventionally used by Herbert et al. (2003); however, Meier et al. (2006) disagreed with the conclusion of Herbert and colleagues. In the present study, the neighbor-joining tree allied with morphological notes proved successful in species discrimination, although some terminal branches are unclear. We believe that adding molecular and morphological data in studies of the DNA barcodecan improve success in delimitation of species, but adding other markers will bring more understanding to the questions left by using the ITS region alone.

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Supplementary Figure 1 Strict consensus maximum parsimony of ITS sequences of Lycoperdon species.

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Supplementary Figure 2The 50% majority-rule consensus tree from the Bayesian phylogenetic analysis of

ITS sequences of Lycoperdon species.

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Table 2 Sequences used in the molecular analysis. In bold new ITS sequences generated in this work

Species Country Herbarium voucher GenBank Acc. Number Lycoperdon altimontanum Nepal Dobremez – holotypus DQ112589 Lycoperdon altimontanum Norway MJ 4270 DQ122588 Lycoperdon altimontanum (as France MA-Fungi 63392 KX686860 L. molle) Lycoperdon altimontanum (as Macedonia MC-Fungi 05-787 KX686859 L. cf molle) Lycoperdon atropurpureum Sweden MJ 3269 DQ112586 Lycoperdon atropurpureum France MA-Fungi 63389 KX686841 Lycoperdon atropurpureum Spain MA-Fungi 38535 KX686842 Lycoperdon atropurpureum Macedonia MC-Fungi 08-10194 KX686834 Lycoperdon atropurpureum Macedonia MC-Fungi 08-10227 KX686838 Lycoperdon atropurpureum Macedonia MC-Fungi 08-10122 KX686840 Lycoperdon atropurpureum Macedonia MC-Fungi 05-2233 KX686843 Lycoperdon atropurpureum Macedonia MC-Fungi 99-10248 KX686835 Lycoperdon atropurpureum Macedonia MC-Fungi 06-10067 KX686837 Lycoperdon atropurpureum Macedonia MC-Fungi 89-9572 KX686836 Lycoperdon atropurpureum Macedonia MC-Fungi 87-7733 KX686839 Lycoperdon caudatum Sweden RG 920818 DQ112633 Lycoperdon cretaceum Iceland MJ 4105 DQ112597 Lycoperdon cretaceum Norway MJ 4302 DQ112598 Lycoperdon decipiens Sweden MJ 7715 DQ112583 Lycoperdon decipiens Spain MA-Fungi 27674 KX686855 Lycoperdon decipiens France MA-Fungi 63390 KX686856 Lycoperdon decipiens Spain MA-Fungi I45055 KX686857 Lycoperdon dermoxanthum Sweden MJ 4568 DQ112579 Lycoperdon echinatum Sweden MJ 6498 DQ112578 Lycoperdon echinatum Spain MA-Fungi 39586 KX686881 Lycoperdon echinatum Spain MA-Fungi 84507 KX686882 Lycoperdon echinatum Macedonia MC-Fungi 05-782 KX686875 Lycoperdon echinatum Macedonia MC-Fungi 05-5160 KX686876 Lycoperdon echinatum Macedonia MC-Fungi 06-5916 KX686879 Lycoperdon echinatum Macedonia MC-Fungi 06-9676 KX686880 Lycoperdon echinatum Macedonia MC-Fungi 05-5376 KX686877 Lycoperdon echinatum Macedonia MC-Fungi 06-5900 KX686878 Lycoperdon ericaeum Sweden MJ 4866 DQ112606 Lycoperdon ericaeum Sweden MJ 5395 DQ112605 Lycoperdon ericaeum USA MA-Fungi 63393 KX686883 Lycoperdon excipuliforme Sweden MJ 6467 DQ112590

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Lycoperdon excipuliforme (as Macedonia MC-Fungi 98-10138 KX686861 L. altimontanum) Lycoperdon excipuliforme Macedonia MC-Fungi 08-10217 KX686862 Lycoperdon excipuliforme Macedonia MC-Fungi 06-6224 KX686863 Lycoperdon excipuliforme Macedonia MC-Fungi 03-2892 KX686864 Lycoperdon excipuliforme Macedonia MC-Fungi 06-9715 KX686865 Lycoperdon excipuliforme Macedonia MC-Fungi 08-10049 KX686866 Lycoperdon frigidum Greenland Lange 901009 DQ112563 Lycoperdon frigidum Iceland MJ 4088 DQ112564 Lycoperdon frigidum Norway MJ 4273 DQ112562 Lycoperdon frigidum Svalbard Lange 191 DQ112559 Lycoperdon frigidum Sweden MJ 7716 DQ112561 Lycoperdon lambinonii Belgium Demoulin 4622 DQ112575 Lycoperdon lambinonii Norway MJ 5245 DQ112576 Lycoperdon lambinonii (as L Sweden MJ 4556 DQ112591 umbrninum) Lycoperdon lambinonii (as L Sweden MJ 4556a DQ112593 umbrninum) Lycoperdon lambinonii (as L Sweden MJ 4559 DQ112592 umbrninum) Lycoperdon lambinonii Macedonia MC-Fungi 05-10226 KX686867 Lycoperdon lividum Nepal Dobremez 19740514 DQ112599 Lycoperdon lividum Sweden MJ 4005 DQ112600 Lycoperdon lividum Spain MA-Fungi 19219 KX686873 Lycoperdon lividum Spain MA-Fungi 68346 KX686871 Lycoperdon lividum Spain MA-Fungi 68348 KX686874 Lycoperdon lividum Macedonia MC-Fungi 05-10007 KX686872 Lycoperdon lividum Macedonia MC-Fungi 05-5323 KX686868 Lycoperdon lividum Macedonia MC-Fungi 02-3594 KX686869 Lycoperdon lividum Macedonia MC-Fungi 08-10040 KX686870 Lycoperdon mammiforme Sweden MJ 4841 DQ112567 Lycoperdon mammiforme Spain MA-Fungi 24103 KX686831 Lycoperdon mammiforme Spain MA-Fungi 31251 KX686827 Lycoperdon mammiforme Macedonia MC-Fungi 05-773 KX686833 Lycoperdon marginatum USA Anderson & Parker DQ112632 750822 Lycoperdon marginatum Spain MA-Fungi 31252 KX686827 Lycoperdon marginatum Spain MA-Fungi 34063 KX686828 Lycoperdon molle Norway MJ 4260 DQ122566 Lycoperdon molle Sweden MJ 4557 DQ112565

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Lycoperdon molle Greece MA-Fungi 73601 KX686853 Lycoperdon molle Spain MA-Fungi 31259 KX686850 Lycoperdon molle Portugal MA-Fungi 31618 KX686851 Lycoperdon molle Spain MA-Fungi 21623 KX686852 Lycoperdon molle Spain MA-Fungi 21737 KX686854 Lycoperdon molle Macedonia MC-Fungi 06-5918 KX686844 Lycoperdon molle Macedonia MC-Fungi 05-5380 KX686845 Lycoperdon muscorum Sweden MJ 7717 DQ112604 Lycoperdon nigrescens Sweden MJ 5376 DQ122577 Lycoperdon nigrescens Spain MA-Fungi 22012 KX686889 Lycoperdon nigrescens (as L. Spain MA-Fungi 31296 KX686891 pyriforme) Lycoperdon nigrescens Spain MA-Fungi 39587 KX686890 Lycoperdon niveum Iceland MJ 4109 DQ112570 Lycoperdon niveum Iceland MJ 4068 DQ112571 Lycoperdon niveum Iceland MJ 484 DQ112568 Lycoperdon cf niveum Sweden MJ 5594 DQ112572 Lycoperdon niveum Norway MJ 5267 DQ112569 Lycoperdon niveum Spain MA-Fungi 21618 KX686848 Lycoperdon niveum Norway MA-Fungi 63397 KX686847 Lycoperdon niveum Macedonia MC-Fungi 06-5915 KX686849 Lycoperdon niveum Macedonia MC-Fungi 02-10008 KX686846 Lycoperdon norvegicum Sweden MJ 5453 DQ112631 Lycoperdon perlatum Sweden MJ 4684 DQ112630 Lycoperdon perlatum Spain MA-Fungi 29732 KX686826 Lycoperdon perlatum Spain MA-Fungi 31233 KX686825 Lycoperdon perlatum Spain MA-Fungi 31253 KX686824 Lycoperdon perlatum Spain MA-Fungi 64953 KX686808 Lycoperdon perlatum Macedonia MC-Fungi 03-10225 KX686814 Lycoperdon perlatum Macedonia MC-Fungi 06-5917 KX686816 Lycoperdon perlatum Macedonia MC-Fungi 07-6663 KX686822 Lycoperdon perlatum Macedonia MC-Fungi 87-7735 KX686812 Lycoperdon perlatum Macedonia MC-Fungi 02-2389 KX686811 Lycoperdon perlatum Macedonia MC-Fungi 90-10234 KX686813 Lycoperdon perlatum Macedonia MC-Fungi 02-2995 KX686815 Lycoperdon perlatum Macedonia MC-Fungi 08-10055 KX686817 Lycoperdon perlatum Macedonia MC-Fungi 08-10119 KX686820 Lycoperdon perlatum Macedonia MC-Fungi 08-10035 KX686821 Lycoperdon perlatum Macedonia MC-Fungi 07-10144 KX686823 Lycoperdon perlatum Macedonia MC-Fungi 05-5316 KX686809

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Lycoperdon perlatum Macedonia MC-Fungi 08-10209 KX686810 Lycoperdon perlatum Macedonia MC-Fungi 07-7207 KX686818 Lycoperdon perlatum Macedonia MC-Fungi 05-9247 KX686819 Lycoperdon pratense Czechia MJ 5880 DQ112556 Lycoperdon pratense Sweden MJ 4864 DQ112554 Lycoperdon pratense Russia SAHanson 20000915 DQ112555 Lycoperdon pratense Macedonia MC-Fungi 08-10114 KX686830 Lycoperdon pratense Macedonia MC-Fungi 03-3040 KX686829 Lycoperdon pyriforme Sweden MJ 4849 DQ112558 Lycoperdon pyriforme Spain MA-Fungi 16908 KX686895 Lycoperdon pyriforme Macedonia MC-Fungi 01-745 KX686892 Lycoperdon pyriforme Australia MC-Fungi 08-10196 KX686897 Lycoperdon pyriforme Macedonia MC-Fungi 05-5074 KX686893 Lycoperdon pyriforme Macedonia MC-Fungi 08-10128 KX686894 Lycoperdon pyriforme Macedonia MC-Fungi 98-3045 KX686896 Lycoperdon radicatum USA Parker 970911 DQ112608 Lycoperdon rupicola Norway MJ 4304 DQ112580 Lycoperdon rupicola Sweden Vetter 407 DQ112581 Lycoperdon sp. 1 Brazil ICN 154484 KX686807 Lycoperdon sp. 2 India MA-Fungi 73250 KX686858 Lycoperdon sp. 3 (as L. Norway MJ 4285 DQ112573 ericaeum) Lycoperdon sp. 3 (as L. Norway MJ 4277 DQ112574 ericaeum) Lycoperdon subumbrinum Sweden MJ 6377 DQ112602 Lycoperdon subumbrinum Sweden MJ 6394 DQ112601 Lycoperdon subumbrinum Sweden LO 148-03 DQ112603 Lycoperdon subumbrinum United Kingdom MA-Fungi 28449 KX686884 Lycoperdon subumbrinum Spain MA-Fungi 30564 KX686888 Lycoperdon subumbrinum France MA-Fungi 63391 KX686885 Lycoperdon subumbrinum Spain MA-Fungi 31234 KX686887 Lycoperdon subumbrinum Macedonia MC-Fungi 87-10137 KX686886 Lycoperdon tuneri Greenland Lange 08-95 DQ11259 Lycoperdon tuneri Norway MJ 5251 DQ112594 Lycoperdon tuneri Norway MJ 4265 DQ112595 Lycoperdon umbrinum Macedonia MC-Fungi 06-5918 KX686844 Lycoperdon umbrinum Macedonia MC-Fungi 05-5380 KX686845 Lycoperdon umbrinoides Spain MA-Fungi 35530 KX686898 Lycoperdon utriforme Sweden MJ 5388 DQ112607 Morganella fuliginea Paraguay TENN59070 AF485065

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Morganella subincarnata Germany REG106/81 AJ237626 Outgroup Bovista furfuracea Sweden MJ 5435 DQ112622 Bovista nigrescens Sweden MJ 7719 DQ112612 Bovista plumbea Sweden MJ 4856 DQ112613

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Table 3 Identifications success based on ¨Best Match¨ and ¨Best Close Match¨.

Best match Best close match

Number Success Ambiguous Incorrect Success Ambiguous Incorrect of identification identification sequences

145 127 2 (1.37%) 16 (11.03%) 127 2 (1.37%) 14 (9.65%) (87.58%) (87.58%)

Table 4 Sequence names with incorrect identification based on "Best Match/Best Close Match" within threshold. In bold the values that repeat in the “Best Close Match”.

Sequence names Incorrect match Lycoperdon frigidum MAF63397 0.00% Lycoperdon molle DQ122566 0.00% Lycoperdon niveum MAF21618 0.00% Lycoperdon umbrinum 05MCF5380Lu 0.00% Lycoperdon umbrinum DQ112591 0.42% Lycoperdon excipuliforme 08MCF10049Lmol 0.43% Lycoperdon radicatum DQ112608 0.71% Lycoperdon utriforme DQ112607 0.71% Lycoperdon ericaeum DQ112605 1.17% Lycoperdon niveum DQ112569 1.19% Lycoperdon atropurpureum 06MCF10067La 1.28% Lycoperdon perlatum DQ112630 1.62% Lycoperdon rupicola DQ112581 1.73% Lycoperdon lividum DQ112600 2.55% Lycoperdon norvegicum DQ112631 3.36% Lycoperdon atropurpureum 87MCF7733La 3.55%

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CAPÍTULO 2 / CHAPTER 2

Integrative taxonomy for the identification of species of Lycoperdon (Basidiomycota) from Central and South America

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Abstract

The species of the genus Lycoperdon (puff ball fungi) are recognized by their basidioma with a cellular subgleba and dehiscence by an apical pore. The genus has a large distribution in North America and Europe, and many works also report Lycoperdon species from New Zealand, Australia, Japan, Mexico and Africa. In Central and South America, 30 species are reported, similar to the number reported from North America and Europe. Traditionally, the taxonomy of Lycoperdon has been based only on morphological characters, and individual taxonomists have adopted their own distinct concepts to separate the species. Recently, based on ITS-LSU sequences, the genus Morganella and Vascellum were included in Lycoperdon as two distinct subgenera. In a previous study (Chapter 1), the ITS barcoderegion was evaluated to be used in the discrimination and identification of Lycoperdon species from the Northern Hemisphere; the identification power was around 80%. The main aim of the present work, that combines morphological taxonomic methods with sequence analyses and statistics to identify species, is to confirm whether or not the Lycoperdon species from Central and South America are the same as those described mainly from the Northern Hemisphere. We have obtained the ITS barcodefrom Lycoperdon specimens previously identified by morphological data; some from types or from specimens from type localities. After the analyses, excluding specimens previously identified as Morganella, nineteen Lycoperdon species were delimitated to Central and South America; from some of the specimens no ITS sequences were obtained (e.g. L. calvescens, L. endotephrum, L. eximium or L. utriforme). More samples are needed before describing three possible new species (Lycoperdon sp. 1, Lycoperdon sp. 4 and Lycoperdon sp. 5). In conclusion, four of the species are first records to Central and South and American. According our dataset, and literature available, only six species are confirmed to be present in countries of the two Hemispheres (L. lividum, L. marginatum, L. nigrescens, L. perlatum, L. pyriforme and L. umbrinum). DNA barcoding is an exciting tool for taxonomic research, but the effectiveness of this approach clearly depends on the availability of an extensive database of barcodesequence standards from specimens previously identified by taxonomists.

Key-words: DNA barcoding, Lycoperdaceae, morphological review, Neotropics.

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1 INTRODUCTION

The genus Lycoperdon Pers. is recognized by its basidiomata subglobose, pyriform to turbinate, with a cellular subgleba and dehiscence by an apical pore (Demoulin, 1970; Calonge, 1998); and it is well distributed around the world (Demoulin, 1973a).

Lycoperdon has a large distribution in North America and Europe; reported by many taxonomists since the 19th century (Bonorden, 1857; Berkeley, 1873; Massee, 1887; Saccardo, 1888; Morgan, 1891). Afterward, Lloyd (1905a) and Coker and Couch (1928) described in detail 25 and 35 species respectively, increasing the knowledge about the Lycoperdon species from United States. In Canada, Bowerman (1961) reported 18 Lycoperdon species, giving new combinations (L. ericaetorum Pers. var. cepiforme (Massee) Bowerman and L. perlatum Pers. var. excoriatum (Lloyd) F. Smarda and a new species (L. flavotinctum Bowerman).

From Europe, diverse works have contributed to the understanding of the Lycoperdon species, here some of them are mentioned. Lloyd (1905b), in his review of the European herbaria began to report taxonomic problems with Bonorden’s descriptions of the species. Lloyd says “…he [Bonorden] observed and recorded many unimportant details such as the color of the plant at different stages of its growth…”. Perdeck (1950) reviewed the Persoon specimens to describe the Lycoperdon species from Netherlands; this is an important work, since the concepts adopted by him help to identify the species of Lycoperdon excipuliforme Pers. (= Calvatia excipuliformis (Pers.) Perdeck), L. Pers. and L. umbrinum Pers. Demoulin (1968a), reported 10 species from Belgium giving detailed description of the macro- and micromorphological features. From Germany, Kreisel (1973), in the reedited work of 1962, contributed to the knowledge of the genus establishing the different kinds of peridium, and the localities of the type species. Pegler et al. (1995), in their work about the British gasteroid fungi, studied 30 Lycoperdon species. From the Iberian Peninsula, Calonge and Demoulin (1975) and Calonge (1998) studied many species of gasteroid fungi including 12 Lycoperdon species. After that Calonge et al. (2006) made an extensive list of gasteroid fungi from Castilla-La Mancha in Spain, with ten Lycoperdon species. From Italy, Sarasini (2005) conducted extensive work about the gasteroid fungi including Lycoperdon species.

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Demoulin (1970) studied deeply the concept of species in Lycoperdon, making the typification of species described by Persoon, and reporting new species (Demoulin, 1971, 1972a). Moreover, in his thesis work (Demoulin, 1972b) updated the definition of the genus, made distribution maps for Europe and North America, and an artificial phylogenetic relationship based on morphological characters. Demoulin (1972b) recognized 30 Lycoperdon species in Europe and North America; whereas Larsson and Jeppson (2008), based on ITS-LSU nrDNA and morphological data, considered that in Europe the genus comprises about 31 species, including some species that were under other genera (e.g. Calvatia excipuliformis (Scop.) Perdeck, Morganella subincarnata (Peck) Kreisel & Dring. and Vascellum pratense (Pers.) Kreisel).

Cunningham (1926, 1944) reported 14 species of Lycoperdon from New Zealand and Australia; however, some species included in their studies are currently considered synonymous, e.g. L. spadiceum Pers. (= L. lividum Pers.) or L. polymorphum Vittad. (= Bovista polymorpha Kreisel).

In South Africa, Bottomley (1948) considered the occurrence of 15 species with two South African species: L. duhiei Bottomley and L. qudenii Bottomley. Afterward, Dissing and Lange (1962) and Dring (1964) reported a total of 15 species from Congo and West of Africa, including three new species: L. angulatum Dissing & Lange, L. umbrinoides Dissing & Lange, and L. ashantiense D.M. Dring.

In the Asian continent, the mycologists Kobayasi (1937), Dennis (1953) and Sharma (1991) worked in Japan and India. Dennis (1953) conducted extensive work in India about the gasteroid fungi describing nine species and indicating the localities of the types. Bisht et al. (2006) described a peculiar new species with basidiospores having a pedicel 22 µm long.

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The Lycoperdon species from Central and South America — Fries (1829) reported Lycoperdon brasiliense Fr. and eight additional species from South America, with deep description and discussion. Massee (1887), Saccardo (1888) and Spegazzini (1898) contributed to the knowledge of Lycoperdon species from Argentina, Brazil, Peru and Venezuela (Saccardo, 1888). In the 20th century taxonomists contributed to the knowledge of Lycoperdon genus in the continent (Hennings, 1904a; b; Sydow and Sydow, 1907). Rick (1930, 1961), Bononi et al. (1981, 1984) and Domínguez de Toledo (1989, 1993) made a great contribution to the study of Lycoperdon from Brazil and Argentina. Dios et al. (2011) added one species from Argentina: Lycoperdon furfuraceous.

In the 1990s there was a gap without publications about Lycoperdon from South America. In the 21th, Baseia (2005), Cortez et al. (2008, 2011, 2013) gave new reports and new species of Lycoperdon from Brazil: L. atrum, L. lividum, L. marginatum, L. nigrescens, L. perlatum, L. pyriforme and L. ovoidisporum. Also, from South America, Siabatto (2005), in a preliminary record of basidiomycetes from Colombia, cited Lycoperdon sp.; and Rocabado et al. (2007) gave two records of Lycoperdon species: L. perlatum and L. wrightii (= L. curtisii). According to the literature, around 42 species of Lycoperdon have been recorded in South America, a similar number to that in Nort America and Europe.

Garner (1956), Herrera (1964) and Rodríguez and Herrera (1970) contributed to the knowledge of the genus from Costa Rica, Panamá and Mexico. Demoulin (1972a), described L. mauryanum from Guatemala and Mexico. Calonge et al. (2005), reported nine species from Guatemala; Moreno et al., (2010) reported six species from Mexico.

1.1 Problems with the species concept and names in Lycoperdon

Many experts in gasteroid fungi (Massee, 1887; Cunningham, 1926; Coker and Couch, 1928; Perdeck, 1950; Demoulin, 1968a; Kreisel, 1973; Pegler et al., 1995; Calonge, 1998) have studied the genus Lycoperdon; however, the current and modern concepts about the delimitation of Lycoperdon species were established by Dr. Vincent Demoulin in his thesis work (Demoulin, 1972b).

A divergence of opinion about the concept and naming of the species has been on- going among various expert taxonomists. For example, Cunningham (1926) and Coker and Couch (1928) considered Lycoperdon excipuliforme Scop. synonymous with L.

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perlatum Pers.; however, Perdeck (1950), in the revision of the species studied by Persoon, treated L. excipuliforme and L. perlatum as distinct species, and, even more, he proposed the new combination, Calvatia excipuliformis (Pers.) Perdeck. Also, Perdeck (1950) expanded the concept of L. molle Pers. and included it as synonymous with L. atropurpureum Vittad. However, Calonge and Demoulin (1975) and Jeppson and Demoulin (1989) disagreed with Perdeck and considered these taxa as two separate species. In more recent work, Calonge (1998) synonymized L. decipiens Durieu & Mont. to L. atropurpureum, disregarding the work of Demoulin (1968a) and Calonge and Demoulin (1975).

1.2 Current taxonomy of Lycoperdon

With the advances of the molecular analyzes based on sequences of nrDNA, specialists also wanted to understand the relationships among the fungi (Bruns et al., 1989; Baldwin, 1992; Gardes and Bruns, 1993). Related to basidiomycete gasteroid fungi, the paper of Hibbett et al. (1997) confirmed that these fungi do not form a monophyletic group: the gasteroid habit has arisen many times, for example from agaricoid or boletoid morphotypes.

Krüger et al. (2001) based on ITS and SSU rDNA regions demonstrated that Lycoperdon was polyphyletic; and L. pyriforme was segregated from the Lycoperdon genus. The positions of genera Bovistella Morgan, Morganella Zeller and Vascellum Smarda in relation to Lycoperdon remained uncertain. Afterward, based on the ITS region, Kruger and Kreisel (2003), following Krüger et al. (2001), transferred L. pyriforme to Morganella, and a new subgenus Apioperdon emerged. In addition, Larsson and Jeppson (2008), based on the ITS and LSU regions, proposed a world taxonomy for Lycoperdon, where species and specimens from the Southern Hemisphere were infrarepresented; they proposed two new subgnera: Lycoperdon subgenus Morganella and Lycoperdon subgenus Vascellum.

The genus Morganella emerged in Zeller (1948), and Kreisel and Dring (1967) ammended the genus to accommodate the species of Lycoperdon with no capillitium in the mature gleba, but abundant paracapillitium encrusted with remains of glebal membrane. Also, they proposed six new combinations and one new species: Morganella fuliginea (Berk. & M.A. Curt.) Kreisel & Dring (≡ L. fuligineum), M. velutina Berk. & M.A. Curti) Kreisel & Dring (≡ L. velutinum), M. purpurascens (Berk. & M.A. Curti)

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Kreisel & Dring (≡ L. purpurascens), M. puiggarii (Speg.) Kreisel & Dring (≡ Bovista puiggarii), M. compacta (G.H. Cunn.) Kreisel & Dring (≡ L. compactum), M. subincarnata (Peck) Kreisel & Dring (≡ L. subincarnatum) and M. afra. Afterward, Ponce de León (1971) added two more species: M. samoensis (Bres. & Pat.) Ponce de León (Globaria samoensis), and M. stercoraria Ponce de León. Suárez and Wright (1996) reviewed the genus from South America and added the first record of M. costaricensis Morales to the continent. In Larsson & Jeppson (2008) only two samples from Morganella were included; in our opinion, more species and specimens need to be studied, using ITS and LSU sequences, to clarify the status of Morganella as a subgenus of Lycoperdon. In this study, some ITS sequences of Morganella species are included, and the taxonomic revision of the samples is done in Chapter 3.

The genus Vascellum emerged in F. Smarda (in Pilát,1958), to include Lycoperdon species that had a diaphragm to separate the gleba from subgleba; Vascellum pratense was erected as the type species of the genus. Later, Ponce de León (1970) added eight more species: V. subpratense (Lloyd) Ponce de León (≡ L. subpratense), V. curtisii (Berk.) Kreisel, V. cruciatum (Rostk.) Ponce de León (≡ L. cruciatum), V. djurense (P. Henn.) Ponce de León (≡ L. djurensis), V. abscissum (Fr.) Kreisel (≡ L. abcissum), V. qudenii (Bottom) Ponce de León (≡ L. qudenii), V. angulatum (Dissing & Lange) Ponce de León (≡ L. angulatum) and V. rhodesianum (Verwoerd) Ponce de León (≡ L. rhodesianum). Next, Homrich (1975) and Homrich and Wright (1988) studied the genus from South America, giving descriptions and distributions of new species. In this present work we follow the taxonomy proposed by Larsson and Jeppson (2008) and consider Vascellum as a subgenus of Lycoperdon.

1.3 DNA Barcode to identify fungi

The DNA barcodeis a fast and practical tool to identify species based on short sequences, around 700 bp long (Hebert et al., 2003, 2004; Kress et al., 2005). This approach, as described in the web site of Barcodeof Life (www.barcoding.si.edu), arose to help taxonomists identify and describe biodiversity; clarify limits between species, and to detect cryptic species (DeSalle et al., 2005; Janzen et al., 2005; Hubert et al., 2008).

For the Basidiomycota, the ITS have been used with success to delimit species from ecological samples such as soil and plant debris, in the absence of fruitbodies (Nilsson et al., 2008, 2009). Although the ITS region is not very successful to discriminate

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species in all groups of fungi (Dentinger et al., 2011), Schoch et al. (2012) proposed ITS region as the first barcodeof fungi, due to mainly the high success of PCR amplifications (90%), and the most resolving power of species discrimination (e.g. Basidiomycota 77% resolution).

Although the genus Lycoperdon has been studied in the Northern Hemisphere by a great number of authors; they do not always agree on the limits among species. This fact leads us to consider the following question: Are the Lycoperdon species from Central and South America correctly identified since they are compared with Northern Hemisphere species? Following the research conducted in Chapter 1, where the ITS DNA barcodeallowed delimiting and identifing 80% of the Lycoperdon species from the Northern Hemisphere, in this study we used the ITS barcode from Lycoperdon specimens from Central and South America (including some types, and specimens from type localities from other parts of the world), to check whether the species reported from Central and South America correspond to Northern Hemisphere species. As mentioned above, ITS barcode sequences were obtained from Morganella and included in the ITS database in this study; however, as other markers are required to clearly state the phylogenetic position of Morganella species, in this chapter these species are not discussed (see Chapter 3).

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2 MATERIAL AND METHODS

2.1 Collections studied

Specimens were obtained from Herbarium Harvard University (FH), personal herbaria Dr. Vincent Demoulin (VDEMOULIN), Université de Liège (LG). Brazilian collections Universidade Federal do Rio Grande do Sul (ICN), Instituto Anchietano de Pesquisas/UNISINOS (PACA), Universidade Federal do Rio Grande do Norte (UFRN- Fungos) were analyzed; as well as from the New York Botanical Garden (NY), Royal Botanical Gardens (K), Universidad de Buenos Aires (BAFC), IMBIV-Museo Botánico (CORDC). Collection data are included in Table 1. Unfortunately, the herbaria of U.S. National Fungus Collection (BPI), Universidade Federal de Pernambuco (URM) and Instituto de Botânica (SP) did not send the specimens to analyze in our studies. The Harvard University herbarium did not permit the use of the specimens for molecular analyses.

2.2 Morphological analyses

2.2.1 Analyses of macrostructures

Macrostructure analyses were done following Silva et al. (2014). The size of basidioma was measured (length × width), with naked eyes or with aid of stereomicroscopy; the exoperidium ornamentation and endoperidium surface, as well as the texture of the gleba and subgleba were observed. According to the traditional works about Lycoperdon (Massee, 1887; Cunningham, 1944; Bottomley, 1948; Perdeck, 1950; Bowerman, 1961), the authors have considered that when the subgleba occupied one- third to one-half of the basidioma then it was well-developed, and when it occupied less than one-third or one-fifth of the basidiome it was considered reduced. Here, the basidioma length was divided by three: if the result is greater than or equal to ⅓ of the basidioma, the subgleba is considered well-developed; and if the result is less than to ⅓ of basidioma, it is reduced. The color of macrostructures such as exoperidium ornamentation, endoperidium surface, gleba and subgleba were coded accordance Küppers (2002).

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2.2.2 Analyses of microstructures

The microstructures were mounted on slides and immersed in lactophenol cotton blue, Melzer´s reagent and/or 5% KOH following (Cunningham, 1944; Bottomley, 1948). In accordance with Cunningham, we warmed the slides containing portion of the gleba and peridium plus solution of lactophenol cotton blue or Melzer´s reagent until bubbles begin to appear. The heating of the solution helps the microstructures return to the normal size. The peridium was cut in cross section to visualize how the layers of endoperidium and exoperidium are organized. After a first visualization, the slides are splashed to measure the hyphae from endoperidium, sphaerocysts, mycosclerids and hyphae with inflated termination (these hyphae can be found in the apical portion of the basidioma). To check if there are differences in the kind of peridium from the apical and the basal part of the basidiome, observations were done to the apical portion (from half basidioma towards the apical portion), and to the basal portion (from half basidioma towards the basal portion).

Ten basidiospores were measured per specimen; the degree of ornamentation was annotated following Demoulin (1972a,b) and Moyersoen and Demoulin (1996). In the description of the species, the size of the spores includes the ornamentation, and when necessary the size of diameter without ornamentation is discussed. The basidiospores were classified as: [A] to smooth to punctate ornamentation; [B] slightly verrucose; [C] verrucose and [D] strongly verrucose. The mounted basidiospores were observed first at 40 , to check the ornamentation: [A] smooth to punctate, when no ornamentation is observed; [D] strongly verrucose, when the ornamentation is easily visualized. These kinds of ornamentations are easy to observe and to discriminate at 40 ; however, to see [B] slightly verrucose, and [C] verrucose, the observation has to be done at 100 . If the observer cannot easily count the ornamentation, the spores are [B] slightly verrucose, and if the ornamentation can be counted, the spores are [C] verrucose. The density of ornamentation was calculated as: number of ornamentations divided by the diameter of the spore multiplied by 3.14, to give a number of warts by a reduced area of 10 µm; spore ornamentation was also analyzed using scanning electron microscopy (SEM); the preparation and mounting of samples was as described by Cortez et al. (2008) and Silva et al. (2011).

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The descriptive terminology of the basidiome shapes, sphaerocysts and basidiospores follows those adopted by (Stearn, 2010).

2.3 Molecular Analyses

2.3.1 DNA isolation, amplification and sequecing

Total genomic DNA was extracted using DNeasy Plant Kit (Qiagen 69106) following the instructions of the manufacturer; lysis buffer incubation was done overnight at 55º C. The PCR was done in a 25 μl reaction mix using illustraTM PureTaqTM Ready- To-Go-TM PCR Beads (GE Healthcare, Buckinghamishire, UK) as described in Martín and Winka (2000). The thermal cycling conditions to amplify internal transcribed spacer of ribosomal nrDNA (ITS) were used as indicated in Martín and Winla (2000) with the primer ITS5/ITS4, or ITS1F/ITS2 and ITS3/ITS4b (White et al., 1990; Gardes and Bruns, 1993). Negative controls lacking fungal DNA were run for each experiment to check for contamination. Also, to check the quality of extracted DNA from each sample, an electrophoresis in agarose gel (1.5%) was made. Five µl of DNA from each sample was mixed with 2 µl of run buffer plus 1 µl of GelRed buffer to visualize the PCR products.

The PCR products were subsequently purified using the QIAquick Gel PCR Purification (Qiagen) according to the manufacturer's instructions or with 8 μl of 1:10 ExoSAP-IT® (USB Corporation, OH, USA). The purified PCR products were sequenced using the same amplification primers in Macrogen Inc. (Amsterdam, Netherlands). Sequencer 4.1.1 (Gene Codes Corporation, Ann Arbor, Michigan, USA) was used to edit the resulting electropherograms and to assemble contiguous sequences. To test if the sequences belong to the genus Lycoperdon and not to contaminations with other fungi, BLAST searches with the megablast option were used to compare the sequences obtained against the sequence in the National Center of Biotechnology Information (NCBI) nucleotide databases (Altschul et al., 1997).

2.3.2 The molecular identification of Lycoperdon species

The ITS nrDNA sequences obtained (Table 5) were aligned using Seaview version 4.6 (Galtier et al., 1996; Gouy et al., 2010) for multiple sequences; these sequences were compared with homologous sequences from GenBank mainly published in Larsson and Jeppson (2008), Jeppson et al., (2012), Kumla et al. (2013) and Kim et al. (2016) (Table

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5); as well as sequences obtained in Chapter 1 (Table 2). Three sequences of Bovista Pers. were included as outgroup. Where ambiguities in the alignment occurred, the alignment generating the fewest potentially informative characters was chosen. Alignment gaps were marked “–“, unresolved nucleotides and unknown sequences were indicated with “N“.

First, a maximum parsimony analysis (MP) was carried out; minimum length Fitch trees were constructed using heuristic searches with tree-bisection-reconnection (TBR) branch swapping, collapsing branches if maximum length was zero and with the MulTrees option on in the PAUP*4.0b10 (Swofford, 2002); and a default setting to stop the analysis when reaching 100 trees. Gaps were treated as missing data. Nonparametric bootstrap support (bs) (Felsenstein, 1985), for each clade, based on 10,000 replicates using the fast-step option, was tested. The consistency index CI (Kluge and Farris, 1969), retention index RI, and rescaled consistency RC (Farris, 1989) were obtained.

A second analysis was done by Bayesian approach (Larget and Simon, 1999; Huelsenbeck and Ronquist, 2001), using MrBayes 3.1 (Ronquist and Huelsenbeck, 2003). The analysis was performed assuming the general time reversible model (Rodríguez et al., 1990), including estimation of invariant sites and assuming a discrete gamma distribution with six categories (GTR+I+G) as selected by MrModelTest v.2.3 (Nylander, 2004). Two independent and simultaneous analyses starting from different random trees were run for 10,000,000 generations with four parallel chains and tree 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 determine 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. Both the 50% majority- rule consensus tree and the posterior probability (pp) of the nodes were calculated from the remaining trees with MrBayes. The dendogram tree was viewed with FigTree v1.42 (http://tree.bio.ed.ac.uk/ sofware/figtree/) and edited with free software InkScape Wink, version 0.92.0 r15299 (http://wiki.inkscape.org/wiki/index.php?title=Release_notes/0.91&oldid=98966).

To check if a barcode gap exists, pairwise K2P distances were used to characterize the intraspecific and interspecific variation (Meyer and Paulay, 2005; Chen et al., 2010; Ramadan and Baeshen, 2012). Moreover, the probability of correct identification (PCI) 155 based on distance (CBOL, 2009; Suwannasai et al., 2013), was calculated using the Taxon/DNA in accordance with (Meier et al., 2006). The probability of correct identification is given for at least two samples from any species showing the barcode gap. If the maximum intraspecific sequence distance is less than its minimum interspecific sequence distance, this is considered “correct identification”. With the K2P distances, a tree was generated using neighbor-joining (NJ) to visualize the specimens under each species.

Trees were viewed with Figtree v. 1.3.1 (http:// tree.bio.ed.ac.uk/sofware/figtree/) and edited with adobe Illustrator CS3 v.11.0.2 (adobe Systems).

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3 RESULTS

3.1 Molecular analysis

3.1.1 DNA, amplification, and sequencing

DNA was isolated from two hundred and thirty-nine samples (Table 5-6) obtaining successful amplification in forty-five samples representing 19%, and seventy- four samples (31%) were not unsuccessfully amplified. From thirty-three samples (13%) no permission from the herbarium was obtained for the molecular analysis. Seventy-one sequences were obtained. After blast search, twenty-six (11%) of them belong to Cryptococcus albidus, Schizophillum commune, and Phoma spp. All new sequences were deposited at the EMBL/GenBank/DDBJ databases.

3.2 Molecular identification of Lycoperdon species

The new ITS sequences were aligned with 73 sequences downloaded from GenBank under Bovista Pers., Morganella Zeller and Lycoperdon Pers., and ninety-one sequences generated in Chapter 1 (Table 2), to produce a matrix with two hundred thirty- eight sequences and 777 unambiguously aligned nucleotide positions (characters). No nucleotide positions were excluded from the analyses. All characters are of type 'unord' and have equal weight, 79 characters are parsimony-uninformative, 240 parsimony- informative. The 100 most parsimonious trees gave a length of 900 steps, CI = 0.518, HI = 0.482 and RI = 0.896. The trees obtained from the MP (strict consensus tree) (Suplement 1, Fig. 1), the Bayesian analyses (Suplement 1, Fig. 2), and the Neighbor- Joining K2P (NJ) (Fig. 20), except in a few branches, showed similar topologies. Here, the clades and single branches from Fig. 20 are described, and the bootstrap (bs) and posterior probabilities (pp) indicated to show the support of each clade.

Twenty-five Lycoperdon terminal branches are formed by sequences retrieved from GenBank or obtained by us from Europe (Chapter 1); these branches correspond to the following morphological taxa: Lycoperdon albiperidium, L. altimontanum, L. atropurpureum, L. caudatum, L. cretaceum, L. decipiens, L. dermoxanthum, L. echinatum, L. ericaeum, L excipuliforme, L. frigidum, L. lambinonii, L. lividum, L. mammiforme, L. molle, L. niveum, L. norvegicum, L. radicatum, L. rupicola, L.

157 subumbrinum, L. turneri, L. umbrinum and L. utriforme; also two unnamed taxa Lycoperdon sp. 2 and Lycoperdon sp. 3.

The Lycoperdon specimens from Central and South America are distributed in 10 terminal clades, mainly related to known morphological species: L. atrum, L. endotephrum, L. marginatum, L. nigrescens, L. ovoidisporum, L. perlatum, L. pratense and L. pyriforme. Based on morphological analyses, some specimens were not possible to identify to the species level, they appear in the tree in single branches (e.g. Lycoperdon sp.5) or forming clades exclusively with Southern Hemisphere specimens, such as Lycoperdon sp. 1, Lycoperdon sp. 4.

From up to down in Fig. 20, the specimens are distributed in the following species: - Lycoperdon sp. 4 terminal branch is well supported (bs = 100%, pp = 1), and includes samples from Brazil (UFRN-Fungos 164 and UFRN-Fungos 485), and Hungary (MA-Fungi 14141). Previously these samples were identified under Lycoperdon pratense (=Vascellum pratense); however, the morphological review showed distinct features.

- Lycoperdon perlatum terminal branch (bs = 77%, pp = 1) includes 35 samples, from GenBank (3), MA-Fungi (6), MC-Fungi (15), UFRN-Fungos (6) and LG (4) and KW (1) herbaria. The samples were collected in Asia (China and South Korea), Europe (Macedonia, Spain and Sweden) and South America (Argentina and Brazil). These sequences were distributed in two subgroups; one formed by specimens only from Europe (ESP, MKD and SWE), and the second with samples from Asia (KOR), Europe (ESP and MKD), South America (ARG and BRA) and Central America (MEX). Some samples from Brazil (DeMeijer 206, UFRN-Fungos 811, UFRN-Fungos TSC70, KW200236 and exU87) identified as L. perlatum did not group here; they are distributed in different branches.

- Lycoperdon marginatum terminal branch is well supported (bs = 97%, pp = 0.99); and the seven specimens are distributed in two subgroups: one composed of North American and European samples, while the second is formed by only South American samples. The Argentinean samples were identified previously under the name Vascellum pampeanum (CORD 72) and V. texense (CORD 573); however, the morphological review showed typical features of L. marginatum. - Lycoperdon sp. 1 terminal branch is well supported (bs = 100%, pp = 1) and includes samples from Argentina and Brazil. The sample ICN154484, in Chapter 1, was

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under L. pratense, and the sample CORD 167 under L. lambinonii. These two samples share the same morphological features, and molecular analysis confirmed that they belong to the same species.

- Lycoperdon sp. 5 terminal branch has two sequences, both from Brazil, are arranged unexpectedly in the different analyses. In the NJ tree, one sample (UFRN- Fungos 152) grouped among some Morganella specimens, whereas the other sample (UFRN-Fungos 300) is basal to the specimens of the subgenus Vascellum (L. endotephrum and L. pratense). However, both in MP and Bayesian analyses, these two samples grouped with L. curtissi, L. endotephrum and L. hyalinum.

- Lycoperdon endotephrum terminal branch is weakly supported in bootstrap (bs = <50%) and highly supported in Bayesian (pp = 1). The group includes Brazilian samples, and the South African sample LG 72RW1006 that Demoulin and Dring (1975) examined to propose Vascellum endotephrum comb. nov. The Brazilian samples belong to different morphological species; moreover, L. curtisii and L. hyalinum grouped here. also, The Lycoperdon pratense terminal branch is sister to L. endotephrum and allies, and is well supported (bs = 96%, pp = 1), including samples from South and Central America, and Europe.

- Lycoperdon ovoidisporum terminal branch (bs = 52%, pp = 1) includes only Brazilian samples.

- Lycoperdon nigrescens terminal branch is well supported (bs = 98%, pp = 1), and includes samples from Asia (South Korea), Europe (Spain and Sweden) and South America (Argentina).

- Lycoperdon atrum terminal branch is moderate to well supported (bs = 74%, pp = 1), and includes samples from Europe (Spain) and South America (Brazil); in Chapter 1, the sample MA-Fungi 35530 was named under L. umbrinoides, but here more samples in a more conserved state, show that it belongs to L. atrum.

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- Lycoperdon pyriforme terminal branch is moderate to well supported (bs = 73%, pp = 1), grouping samples from Europe (Belgium, Macedonia, Spain and Sweden), Oceania (Australia), South America (Argentina and Brazil) and North America (Mexico). Lycoperdon melanesicum, together with the sample VDEMOULIN 3195, is an authentic specimen of L. pyriforme; although the branch length in L. melanesicum is larger than other specimens of L. pyriforme; the morphological features are different between these species.

Figure 20 Neighbord-Joining tree obtained after calculating the K2P distance of the ITS sequences included in Table 1. Names and vouchers numbers as mentioned in the text and tables. New sequences in bold. Colours to indicate the geographic origin of the specimens and sequences. Yellow, mainly specimens from Central and South America (it can be some specimens from other part from the Southern Hemisphere); Orange, specimens from the Southern Hemisphere, not Central or South America; Red, specimens from both Hemispheres; Blue, only specimens from the Northern Hemisphere.

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3.3 Probability Correct Identification (PCI) of Lycoperdon perlatum and L. pyriforme

To the analysis of PCIs, intraspecific and interspecific distances were calculated. In the Lycoperdon perlatum terminal branch, with the search by the Best Match/Best Close Match using threshold of 3%, 32 sequences were found and the correct identifications according to “Best Match” was100%, the ambiguous and incorrect identification was 0%. The correct identification according to “Best Close Match” was 97.05%, the ambiguous and incorrect identifications were 0%. Based on GenBank sample DQ112630, the group of specimens that formed the larger sub-branch obtained the intraspecific distance of 0.43–2.28% (Table 6).

In the terminal branch of L. pyriforme (including L. melanesicum NY00795902 in this terminal branch), with the search by the Best Match/Best Close Match using threshold of 3%, 19 sequences were found (18 L. pyriforme, and one L. melanesicum). The sequences have at least one matching sequence and the correct identifications according “Best Match” was 94.73%, and the incorrect identification 5.26% due to the sample of L. melanesicum. The results of “Best Close Match” were the same as that obtained by the “Best Match”; the intraspecific distance based on GenBank sample DQ 112558, for all specimens in L. pyriforme excluding L. melanesicum was 0–2.58% (Table 7), while L. melanesicum was 6.25%.

From some species/specimens no ITS sequences were obtained due to failure of the amplifications or because, after Blast search, sequences were confirmed as belonging to contaminants, mainly Ascomycetes; however, the morphological characters were enough to identify them as distinct species. Based on the ITS sequences from many of the specimens analyzed, and the study of macro and microstructures, nineteen species from Central and South America are included in the taxonomic part of this paper.

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3.4 Taxonomy

Lycoperdon atrum Pat. Bull. Soc. Mycol. France XVIII (2): 176 (1902). Mycobank: MB 168322. Figs. 21a, 22a.

Basidiomata subpyriform to pyriform, 29–35 length ×12–32 mm width. Subgleba cellular, well-developed (occupying > ⅓ of basidiome), yellowish (N00Y10M00). Exoperidium persistent to falling off with the age, verrucose, with minute (< 0.5 mm long) darkish warty (N99Y00M00) at apex, becoming brown (N70Y90M20) at the base, sometimes velutineous and falling off. Endoperidium papery, smooth, yellowish (N30Y90M20).

Dehiscence by plane pore apical. Gleba grayish brown (N70Y20M20).

Exoperidium basal and apical composed by two types of sphaerocysts: elongated one 60–97 length × 14–25 µm width, wall 2–3.5 µm thickness, weakly dextrinoid, below at the elongated sphaerocysts there are globose to subglobose cells 22–36 µm length × 14–24 µm width, wall 1–1.5 µm thickness, weakly dextrinoid, yellowish in 5% KOH. Endoperidium apical like to the basal, without mycosclerids and hyphae with terminations inflated. Endoperidium basal 3.5–4 µm diam., wall 0.5–1 µm thickness, aseptate, weakly to strongly dextrinoid, yellowish in 5% KOH. Paracapillitium 4–6 µm, septate, 0.5–1 µm thickness, cyanophilous, hyaline in 5% KOH. Capillitium 3.5–6 µm diam., wall 1–1.5 µm thickness, aseptate, branched, absent pore, acyanophilous, yellowish in 5% KOH. Basidiospores, globose 3.5–5 × 3.6–5.2 µm, slightly verrucose to verrucose [B-C] often [C] in LM, number of ornamentation 9.5–12.5 for a circumference reduced to 10 µm.

Habitat and substrate: gregarious, growing on leaf-litter.

Specimens Examined: ARGENTINA, Catamarca, Dpto. Concepción, between leaf- litter, 01 Mar 1995, leg. V.L. Suárez and L.D. Gottlieb (BAFC s.n.). BRAZIL, Rio Grande do Sul, Santa Maria, Morro do Elefante, on soil in forest seasonal deciduous, 30º08’18’’S and 54º19’58’’W, 23 Jun 2006, leg. V.Gd. Cortez, no. V.G. 059 (UFRN-Fungos 832 – as L. juruense); ibidem, 1935, leg. Pe. J. Rick (HUH 00373703 – as L. atropurpureum); ibidem, 1935, growing on soil, leg. Pe. J. Rick (PACA 13773); ibidem, Itaara, Parque do Pinhal, growing on soil on leaf-litter, 30º00’58’’S and 54º16’27’’W, leg. V.G. Cortez, no. 044/06 (UFRN-Fungos 825); ibidem, São Leopoldo, 1907, leg. Pe. J.Rick (HUH 00373794 – as L. juruense); ibidem, 1906, leg. Pe. J. Rick (HUH 00373795 – as L. juruensis); ibidem, 1915, leg. Pe. J. Rick (HUH 00373796 – as L. juruense); ibidem, n.d.,

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leg. Pe. J. Rick (HUH 00373798); ibidem, 1907, leg. Pe. J. Rick (PACA 13788 – as L. juruense); Goiás, n.d., leg. Pe. J. Rick (PACA 13791 – as L. nigrescens). Extralimital collections: SPAIN, Gerona, Sant Sadurní de L’Huera, in riverside forest, under Alnus glutinosa, 08 Nov 1995, leg. J.M. Vidal (MA-Fungi 35530).

Notes: Lycoperdon atrum is characterized by its exoperidium ornamentation composed of sphaerocysts with weakly dextrinoid elongated cells; endoperidium hyphae are dextrinoid; and basidiospores slightly verrucose to verrucose [B-C], frequently [C]. Lycoperdon juruense Henn was synonymized to L. atrum by Demoulin (1976); the author studied the isotypes of L. jurunse, and described that some specimens can have an exoperidium falling off at the apex and velvety spines at the base (at the level of subgleba). From the specimens studied here, some of them, such as HUH 00373703 (labelled by Demoulin 1970), HUH 00373794 and HUH 00373795, have a velutinous basal portion, and the apical exoperidium falling off. The specimens of UFRN-Fungi 832 and UFRN-Fungi 825 have the apical exoperidium darker than other specimens analyzed here; but all specimens have the same microstructures. In Chapter 1, the specimen MA- Fungi 35530 was identified as L. umbrinoides; in our molecular analyses, the specimens MA-Fungi 35530, UFRN-Fungos 825 and UFRN-Fungos 832, grouped in the same terminal branch. These samples are from the type locality of L. juruense, synonymous of L. atrum. Thus, we keep the three specimens under L. atrum.

Lycoperdon calvescens Berk. & M.A. Curtis, Grevillea 2 (16): 50 (1873). Mycobank: MB 218823. Fig. 21b.

Basidiomata subpyriform, pyriform to turbinate, 12–47 mm length × 11–45 mm width. Subgleba compact cell, well-developed (occupying > ⅓ of basidiome), grayish brown

(N30Y50M20) to purplish brown (N50Y60M50). Exoperidium falling off in units, with pyramidal spines with slender tips (< 1 mm long.), cream (N00Y30M00) to brown

(N40Y80M80). Endoperidium papery, grayish yellow (N20Y50M10); in some specimens (NY 0071975) the exoperidium and endoperidium partially fallen off. Gleba powdery, yellowish brown (N20Y80M50) to brown (N50Y88M40).

Exoperidium basal likely to the apical, composed by sphaerocysts subglobose to clavate, 16–30 length × 8–17 µm width, wall 1–2 µm thickness, weakly reaction, acyanophilous, hyaline in 5% KOH. Endoperidium apical composed by mycosclerids and hyphae with inflated terminations: mycosclerids composed by cells of 33.92–63.3 length

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× 12.5–31.5 µm width, wall 0.6–3.2 µm thickness, irregular shape, weakly to dextrinoid, yellowish brown to brown in 5% KOH; hyphae with terminations inflated 8.5–20.5 µm diam., wall 0.9–2 µm thickness, weakly to dextrinoid, brownish in 5% KOH. Endoperidium basal composed by hyphae 3.3–6.9 µm diam., aseptate, weakly dextrinoid. Paracapillitium 1.7–7.1 µm diam., wall 0.4–1.5 µm thickness, septate, cyanophilous, hyaline in 5% KOH. Capillitium 2.7–5.5 µm diam., wall 0.6–1.2 µm thickness, occasional septate, occasional pores, without reaction in Melzer’s reagent, yellowish in 5% KOH. Basidiospores globose, 3–5 length × 3–4.5 µm width, smooth to punctate [A], sometimes slightly verrucose [B] in LM, pedicel 1–3 µm long.

Habitat and substrate: gregarious and cespitose, growing in fields of clay soil, also between Eucalyptus trees.

Specimens examined: ARGENTINA, Córdoba, Pernilla, Cuesta Blanca, Sierra Grande, 09 Mar 1980, leg. L. Dominguéz, no. CORD 997, (CORDC 191). URUGUAY, Montevideo, Miguelete, Jun 1928, leg. W.G. Herter, no. 203a (HUH 00373989, ex. HERB. HERT. 83476); ibidem, (NY 0071974, ex. HERB. HERT. 83476 – as L. pyriforme); ibidem, Candones, Floresta, Jul 1946, leg. W.G. Herter, no. 203d (NY 0071975, ex Herb. HERT. 61021 – as L. pyriforme).

Notes: Lycoperdon calvescens is characterized by its exoperidium with slender spines falling off, and basidiospores smooth [A]; capillitium with occasional pores and septate, hyphae from endoperidium weakly dextrinoid and mycosclerids present. Demoulin (1972b), reported the occurrence of sphaerocysts from exoperidium of big size and brown color, likely found in the specimens studied here. The exoperidium falling off in plates, subgleba not tinged purplish brown and basidiospores [A-B] often B in L. marginatum are distinguished from L. calvescens (Demoulin, 1983). The specimens here studied were misidentified under L. pyriforme; however, L. calvescens is distinct from L. pyriforme because the sphaerocysts do not have a spine shape, and it does not grow in decaying wood. Lycoperdon calvescens was described by Berkeley (1873) for USA, the author reported the presence of basidiospores pedicellate, here the specimens NY0071974 showed pedicel 1–3 µm long., and sometimes has remains of pedicels immersed between basidiospores. Here is the first time reported for South America.

Lycoperdon curtisii Berk., Grevillea 2 (16): 50 (1873). Mycobank: MB 221192. Fig. 21c.

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Synonymous (Homrich and Wright 1988): = Vascellum curtisii (Berk.) Kreisel, Feeds Repert. 68: 87 (1963). = Lycoperdon curtisii Brek., N.A. Fungi no. 333. Grevillea, 2 (16): 50 (1873). = Lycoperdon argentinum Speg., Anal. Mus. Nac. Hist. Nat. Buenos Aires 6: 196 (1899) = Lycoperdon wrightii Berk. & M.A. Curt., N.A. Fungi no. 334. Grevillea, 2 (16): 50(1873).

Basidiomata globose to turbinate, 10–22 mm length × 9–20 mm width. Subgleba compact cellular, well-developed (occupying > ⅓ of basidiome), cream (N00Y10M20); diaphragm present only in one specimen, thin < 0.05 mm thickness. Exoperidium spiny, persistent to falling off in some specimens, with stellate spines < 1.0 mm long., cream (N10Y10M20), can be granulose or slender minute spines (< 0.1 mm long.) around of the stellate spines.

Endoperidium papery, yellowish (N20Y90M00); dehiscence by an irregular pore apical.

Gleba yellowish brown (N30Y99M00).

Exoperidium apical likely to the apical 12.3–30 length × 10–21 µm width, wall 1 – 1.5 µm thickness, without reaction, acyanophilous, yellowish in 5% KOH. Endoperidium apical composed by mycosclerids and hyphae with inflated termination: mycosclerids 50– 68 length × 14.6–26.3 µm width, wall 0.8–2.9 µm thickness, without reaction, acyanophilous, yellowish 5% KOH; hyphae with inflated terminations 7–13.8 µm diam., 1.4–2.2 µm thickness, without reaction, acyanophilous, yellowish in 5% KOH; endoperidium basal composed by hyphae 2–3 µm diam., wall < 0.5 µm thickness, aseptate, without reaction, acyanophilous, yellowish 5% KOH; paracapillitium 2.5–5.5 µm diam., wall 0.5–1 µm thickness, without reaction, cyanophilous, hyaline in 5% KOH; capillitium 3–3.8 µm diam., wall, < 0.5 µm thickness, aseptate, pores absent, without reaction, acyanophilous, yellowish in 5% KOH. Basidiospores globose, 3.5–4 µm × 3.61– 4 µm, slightly verrucose [B] in LM (specimens with incomplete maturation can be found [A]); without reaction, acyanophilous, yellowish in 5% KOH;

Habitat and substrate: gregarious, growing on debris of plants.

Specimens Examined: BRAZIL, Rio Grande do Sul, São Leopoldo, Mar 1933, leg. Pe. J. Rick (HUH 00373747 – as L. wrightii); Pernambuco, Recife, Casa Forte-Recife, on soil, 05 Mar 1955, leg. E. Barros-Wandeley, no. 2005 (K (M): 200237). Extralimital collections: UNITED STATES, New York, Athens, Greene Co., besides the Hudson, alluvial sands to the Hudson with little vegetation (some Rhus typhina), 25 Oct 1970, V.

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Demoulin (LG/V.DEMOULIN 4073); ibidem, North Dakota, Kulm, Sep 1912, leg. J.F. Brenckle, no. 334 (PACA 21636 – as L. wrightii).

Notes: Lycoperdon curtisii is characterized by its exoperidium with stellate spines and basidiospores globose and slightly verrucose. In the morphological analyses, the stellate exoperidium spines and the slightly verrucose basidiospores [B], leads us to think of Lycoperdon marginatum and L. pratense. However, in L. marginatum the exoperidium is falling off in plates and has bigger basidiospores (4–4.8 µm), these features are not found in L. curtisii. Lycoperdon pratense has an exoperidium with slender spines, the capillitium is generally absent, basidiospores slightly verrucose [B] (Calonge and Demoulin, 1975). Unfortunately, the molecular analyses were not possible because no amplifications were obtained for ITS. In agreement with Smith (1974), in all specimens analyzed (except in K(M) 200237), no conspicuous diaphragm was found; however, in K(M) 200237 a thin diaphragm was found as described by Ponce de León (1970).

Lycoperdon endotephrum Pat., Bull. Soc. mycol. Fr. 18(4): 300 (1902), Fig. Mycobank: MB 218055. Fig. 21d, 5d-f.

Synonymous (Demoulin and Dring, 1975): = Vascellum endotephrum (Pat.) Demoulin & Dring, Bull Jard. Bot. Natn. Belg. 45 (3-4): 358 (1975). = Lycoperdon todayense Copel., Ann. Mycol. 3: 25 (1905). = Lycoperdon vanderystii Bres., Ann. Mycol. 9: 274 (1911). = Lycoperdon djurense Auct. non-P. Henn.

Basidioma subglobose, subpyriform to pyriform, 5–13 mm length × 9–18 mm width.

Subgleba cellular, well-developed (≥ ⅓ basidioma), cream (N00Y10M00), sometimes becoming grayish (N20Y30M10). Diaphragm present but not at all specimens, when present < 0.1 mm thickness, concolor to gleba. Exoperidium with warts at the base, becoming spiny at the top, spines < 0.1 mm long., falling off with the age, brown (N40Y70M50).

Endoperidium papery, smooth, cream (N00Y20M10) to gray (N10M30M10); gleba grayish brown (N40Y60M20), powdery.

Exoperidium apical with chain of sphaerocysts composed of cells subglobose to pyriform 14–38 × 14–29 µm, wall up to 1.5 µm thickness, without reaction, acyanophilous, yellowish in 5% KOH. Exoperidium basal with chain of sphaerocysts composed of cells globose, subglobose to pyriform 15.0–31.0 × 14.0–25.0 µm, wall 0.7– 1.3 µm thickness without reaction, yellowish. Endoperidium apical composed of mycosclerids and hyphae with inflated terminations: mycosclerids 40.5–66.2 length ×

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13.2–55 µm width, wall 1.3–2.2 µm thickness, without reaction, yellowish in 5% KOH; hyphae with inflated terminations 6–10 µm diam., wall 0.8–1.5 µm thickness, without reaction, yellowish in 5% KOH. Endoperidium basal composed by hyphae 3.5–5.3 µm diam., wall 0.9–1.5 µm thickness, without reaction to weakly dextrinoid, yellowish in 5% KOH. Paracapillitium 3–5.9 µm diam., septate, wall 0.6–1.2 µm thickness, cyanophilous, hyaline in 5% KOH. Capillitium rare, when present 3–4 µm diam., aseptate, pores absent, without reaction, yellowish in 5% KOH. Basidiospores globose, subglobose to oboval, 3.5–4.8 length × 3.5–4.5 µm width, slightly verrucose [B] in LM, minute warty (<0.5 µm long.) in SEM (ICN 154482 – as V. hyalinum), pedicel 0.35–3.7 µm long.

Habitat and substrate: gregarious, growing in soil.

Specimens examined: BRAZIL, São Paulo, Itapeva, (17 Km Se of), Canyon do Rio Taquari-Guaçu, Oct 1968, leg. D. Aytai (K (M): 19869); ibidem, Goiás, Goiáma, Hospital Veterinário, May1968, s. leg. (K (M) 200182 – as V. curtisii); Rio Grande do Sul, Santa Maria, Bairro Dores, 29º41’03’’S and 53º48125’’W, 23 Feb 2009, leg. V.G. Cortez, no. 007/09 (ICN 154482); Rio Grande do Norte, Natal, 6º18’18’’S and 35º21’40’’, 2008, leg. M.I.M. Cocentino, s.n. (UFRN-Fungos 814 – as Morganella). Extralimital collections: RWANDA, District des Lacs Edouard et Kivu, Forêt de Rugege, Vallée de la rivière, Bizumu (entre le mont Muzimu et le Bigugu, prefect. Cyangugu), 2400 m, pelouse semi- naturelle de versant, à Eragrostis blepharoglumis et Exotheca abyssunuca, 04 Mar 1972, leg. Lambinon, no. 72/1006 (LG 72RW1006).

Notes: Lycoperdon endotephrum can be recognized by its exoperidium falling off with spines < 0.1 mm long., rare capillitium, abundant paracapillitium, endoperidium apical with mycosclerids and hyphae with inflated terminations. Demoulin and Dring (1975) consider that the features such as the presence and thickness of diaphragm, color of gleba, shape of basidiospores and strength of warts are not sufficient to recognize this species. In the specimens studied here, the diaphragm was absent in UFRN-Fungos 814 and ICN 154482; the exoperidium was partially or completely fallen off in the specimen’s K (M) 200182 and K (M)19869. Demoulin and Dring (1975) consider L. endotephrum close to L. curtisii, separating these two species by exoperidium less floccose in L. endotephrum, and absence of capillitium in this species.

Lycoperdon eximium Morgan, J. Cincinnati Soc. Nat. Hist. 14: 15 (1892), Mycobank: MB 227237. Fig. 21e.

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Basidiomata subglobose to pyriform, 10–19 mm length × 11–17 mm width. Subgleba reduced (occupying < ⅓ of basidioma), cellular, yellowish (N10Y70M10). Exoperidium falling off, remains only warts, light brown (N40Y70M30). Endoperidium papery, sometimes with furfuraceous aspect, yellowish (N00Y70M10). Gleba cottony, grayish brown (N30Y60M40).

Exoperidium apical likely to the basal, 13.6 – 32.9 length × 15.7 – 28 µm width, wall 0.7–1.7 µm thickness. Exoperidium basal composed by globose to subglobose cells, 13.8–31.3 length × 10.4–23.7 µm width, wall 0.6–1.2 µm thickness, without reaction, yellowish in 5% KOH. Endoperidium apical composed by mycosclerids and hyphae with inflated terminations: mycosclerids irregular shape, 22.5–77. 8 length × 6.5–46.4 µm width, wall 0.5–1 µm thickness, weakly reaction, yellowish in 5% KOH; hyphae with inflated terminations 2.8–8 µm diam., wall 0.6–1 µm thickness, weakly reaction. Endoperidium basal composed by hyphae 2.1–4.2 µm diam., wall 0.7–1.1µm thickness, septate, strongly dextrinoid, yellowish in 5% KOH. Capillitium elastic, 2.5–4 µm diam., wall 0.5–1 µm thickness, septate, abundant pores, without reaction, yellowish in 5% KOH. Paracapillitium absent. Basidiospores ovoid, oboval to ellipsoid, 4.3–6.1 length × 3.5–4 µm width, often punctate [A] to occasional slightly verrucose [B]in LM, pedicel 0.5–3 µm, strongly dextrinoid, yellowish in 5% KOH.

Habitat and substrate: gregarious, growing on leaf-litter.

Specimens examined: BRAZIL, Rio Grande do Sul, São Leopoldo, Mar 1933, leg. Pe. J. Rick (HUH 374007 – as L. spadiceum). VENEZUELA, Maracay, on the Maracay- Choroni road, Parque Nacional Henry Pittier, Edo. Aragua, on unidentified rotted wood, 12 Jul 1971, leg. K.P. Dumont, J.H. Haines and G.J. Samuels, no. Dumont-VE1961 (NY 0071978 – as L. pyriforme).

Notes: Lycoperdon eximium is recognized by its elastic capillitium with abundant pores, basidiospores ovoid, oboval to ellipsoid, often punctate [A] but can be slightly verrucose [B], dextrinoid, and basal endoperidium strongly dextrinoid. Morgan (1891) described this species from United States. In his description, the basidiospores showed size of 5–6 × 4–4.5 µm, the specimens analyzed here showed difference only in the width size (4.3–6.1 length × 3.5–4 µm width). Also, the exoperidium with slender spines reported by Morgan is not found in the present specimens; may be already fallen off. Demoulin (1972b), discussed that the slender spines are fragile and can be mixed or not

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with granules; in the specimens NY 0071978 the granules are present. These specimens have been misidentified as L. pyriforme. However, L. pyriforme does not have basidiospores ovoid and dextrinoid, and the sphaerocysts from exoperidium are quite different since they do not have globose to subglobose shape (Moyersoen and Demoulin, 1996). Lycoperdon eximium is the first report in the South American continent.

Lycoperdon hyalinum Homrich, Can. J. Bot. 66 (7): 1296 (1988) – Mycobank: MB 134081. Fig. 21f.

Synonymous (Homrich and Wright, 1988): = Vascellum hyalinum Homrich, Can. J. Bot. 66(7): 1296 (1988).

Basidioma subglobose 12 mm length × 8 mm width; exoperidium persistent, warty, warts

< 0.05 mm long., dark brown (N60Y99M70). Endoperidium papery, wrinkly, yellowish brown (N30Y99M60). Subgleba reduced (occupying < ⅓ of basidioma), cellular, yellowish

(N60Y99M60). Gleba yellowish (N00Y30M00), incomplete mature.

Exoperidium basal likely apical composed by sphaerocysts globose to subglobose, 15–34 length × 12–25 µm width, displayed in regular chain, wall 0.6–1.2 µm, weakly dextrinoid, yellowish in 5% KOH. Endoperidium apical composed by only hyphae with inflated termination, 10–16 µm diam., without to weakly reaction, yellowish in 5% KOH. Endoperidium basal composed by hyphae 4–7 µm diam., wall 1–1.5 µm thickness, septate, weakly reaction, hyaline in 5% KOH. Paracapillitium 4–5.7 µm diam., wall 0.7– 1 µm thickness, septate, cyanophilous, hyaline in 5% KOH. Capillitium absent. Basidiospores 3–3.5 length × 3–3.5 µm width, globose to ovoid, smooth [A] in LM, asperulate in SEM, without reaction, yellowish in 5% KOH.

Habitat and substrate: gregarious, growing on soil.

Specimens examined: BRAZIL, Rio Grande do Sul, Santa Maria, 29º41’03’’S and 53º48’25’’W, 04 Mar 2009, leg. V.G. Cortez, no. 009/09 (ICN 154483); Paraíba, Areia, Reserva Ecológica Estadual Mata do Pau-Ferro, 06º59’091’’S and 35º44’398’’W, 592 m, between litter-leaf and piece of wood, 17 Jul 2013, leg. D.S. Alfredo, no. DSA178 (UFRN-Fungos 2829).

Notes: This species is characterized by its cottony gleba, absence of capillitium and presence of mycosclerids in apical endoperidium. The specimens studied were not well preserved; and the presence or absence of diaphragm was not possible to confirm. Cortez

169 et al. (2013), studied this specimen and also identified it as L. hyalinum (= V. hyalinum). Homrich and Wright (1988), described L. hyalinum from Argentina and Brazil, and reported the presence of mycosclerids at the apical pore, although these features were not observed here. The specimens showed hyphae with inflated terminations originating from the apical part of the endoperidium; perhaps, the apical mycosclerids are not present because of the poor conservation of this specimen.

Lycoperdon lividum Pers. J. Bot. (Desvaux) 2: 18 (1809) – Mycobank: MB 414454. Fig. 21g.

Synonymous (Demoulin 1972): = Lycoperdon spadiceum Pers., J. Bot. (Desvaux) 2: 20 (1809) non (Schaeff. 1774). = Lycoperdon fuscum Bonord., Bot. Ztg. 15: 628 (1857). = Lycoperdon calcareum Velen. České Houby 4-5: 826 (1922).

Basidiomata depressed globose to turbinate, 13–25 mm length × 18–24 mm width.

Subgleba well-developed (occupying >⅓ of basidioma), cellular, yellowish (Y40M00C00). Exoperidium falling off, warty, warts < 0.5 mm long., in some specimen’s spines can be found, grayish brown (N30Y70M40). Endoperidium papery, surface sometimes covered by crystals, areolate, pale yellow (N10Y20M10). Gleba powdery, yellowish brown

(N20Y90M40) to brown (N60Y80M40), occurring a transition of color between gleba and subgleba.

Exoperidium basal composed by sphaerocysts 11.2–37. 4 length × 9.5–24. 3 µm diam., wall 0.8–2 µm thickness, without reaction, acyanophilous, yellowish in 5% KOH. Endoperidium apical composed only by hyphae with inflated terminations 10.9–20.8 µm diam., wall 0.9–1.7 µm thickness, dextrinoid, acyanophilous, yellowish in 5% KOH. Endoperidium basal composed by hyphae 4–6 µm diam., wall 0.6–1.3 µm thickness, occasional septate, without reaction, acyanophilous, yellowish in 5% KOH. Paracapillitium absent. Capillitium 3.5–7 µm diam., aseptate, pores commonly found, without reaction, acyanophilous, brown in 5% KOH.Basidiospores globose to subglobose, 4–5.5 length × 4–5.5 µm width, punctate [A] in LM, pedicel 0.5–9 µm long. (appear only in Melzr’s reagent), acyanophilous, without reaction, brown in 5% KOH.

Habitat and substrate: gregarious, growing on soil or leaf-litter.

Specimens examined: BRAZIL, Rio Grande do Sul, São Leopoldo, 1939, leg. Pe. J. Rick (PACA 13775 – as L. umbrinum). COLOMBIA, Dpto Boyacá, Ca. 92 km from

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Aguazul, on the Aguazul-Sogamoso, on identified duff, 14 Jun 1976, leg. K.P. Dumont, S.E. Carpenter, M.A. Sherwood, L.A. Molina, no. Dumont-CO 5388 (NY 0071655 – as L. perlatum).

Notes: Lycoperdon lividum is characterized by its exoperidium warty, falling off, crystals present at the surface of areolate endoperidium, capillitium with pores and basidiospores punctate [A]. Lycoperdon lividum is distinct from L. ericaeum Bonord. because of its capillitium aseptate and basidiospores punctate [A] (Demoulin, 1972a, b). The specimens here studied were misidentified as L. umbrinum (PACA 13775) and L. perlatum (NY 0071655); although, L. umbrinum has basidiospores slightly verrucose [B] and capillitium with pores more abundant than L. lividum. Lycoperdon perlatum has exoperidium with conical spines, paracapillitium abundant and basidiospores verrucose [C] (Calonge and Demoulin, 1975; Moyersoen and Demoulin, 1996). This is the first time that Lycoperdon lividum is reported for South America.

Lycoperdon marginatum Vittad., Monogr. Lycoperd.: 185 (1842), Mycobank: MB 220793. Fig. 21h, 22b.

Synonymous (Demoulin 1972b): = Lycoperdum cruciatum Rostk., Deutschl. Fl., 3 Abt. (Pilze Deutschl.) 5(18): 19 (1839). = Lycoperdon muricatum Bonord., Bot. Ztg. 15: 612 (1857). = Utraria cruciatus Rostk., Quél., Mém. Soc. Émul. Montbéliard, Sér. 2 5: 368 (1873). = Lycoperdon separens Ann. Rep. N.Y. St. Mus. nat. Hist. 26: 73 (1874). = Lycoperdon wrightii Berk et M.A. Curt. var. atropurpureum Peck, [as 'atra-punctum'], Ann. Rep. N.Y. St. Mus. nat. Hist. 32: 67 (1880). = Vascellum cruciatum (Rostk) P. Ponce de León, Fieldiana, Bot. 32(9): 118 (1970).

Basidiomata globose, subglobose, pyriform to turbinate, 13–32 mm length × 10–61 mm width. Subgleba well-developed (occupying > ⅓ basidioma), cellular, cream (N00Y10M00) becoming grayish (N40Y40M10). Diaphragm present, < 1 mm thickness, brown

(N60Y80M60). Exoperidium falling off in plates, white pyramidal spines at the apex 1–2.5 mm long., while at the base persistent slender spines, yellowish (N00Y20M00) to brownish

(N10Y80M20 to N20Y60M30). Endoperidium surface covered by a brown (N50Y60M30) material furfuraceous, papery, yellowish (N00Y40M00) to grayish (N20C00Y20); dehiscence by a circular pore apical or partial fragmentation of endoperidium. Gleba powdery, brown

(N60Y80M60 to N70Y70M40).

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Exoperidium basal likely to the apical, composed by globose, subglobose to pyriform sphaerocysts, 12.5–33 length × 10.5–33.5 µm width, wall 0.5–1 µm thickness, without reaction, acyanophilous, yellowish in 5% KOH. Endoperidium apical composed by mycosclerids and common hyphae: mycosclerids 42–98 length × 8–24 µm width; wall 1–1.5 µm thickness, weakly to strongly dextrinoid, acyanophilous, yellowish in 5% KOH; common hyphae 3.1–5.5 µm, wall 0.8–1 µm, septate, weakly dextrinoid, acyanophilous, yellowish in 5% KOH. Endoperidium basal composed by hyphae aseptate 3–6.5 µm, without reaction to weakly dextrinoid, acyanophilous, yellowish in 5% KOH. Paracapillitium 2.6–7 µm diam., septate, without reaction, cyanophilous, hyaline in 5% KOH. Capillitium 3.8–7.6 µm diam., wall 0.5–1.2 µm, aseptate, commonly with pores, without reaction, acyanophilous, yellowish to brown in 5% KOH. Basidiospores globose, 3.7–4.8 length× 3.7–4.8 µm width, punctate to slightly verrucose [A-B], in LM, pedicel 0.5–1 µm, acyanophilous, without reaction, brown in 5% KOH.

Habitat and substrate: gregarious, growing in sand soil, soil and leaf-litter.

Specimens examined: ARGENTINA, Córdoba, Punilla, Los Gigantes, 1839 m on road, 08 Feb 2007, leg. M.L. Hernádez-Caffot, no. CORD72 (CORDC 374); ibidem, San Javier, Quebrada del Tigre, 11 May 2007, leg. M.L. Hernández-Caffot, no. CORD 272 (CORDC 314); Ciudad Universitaria, 06 Oct 1986, leg. L. Domínguez de Toledo, no. CORD573 (CORDC 839): ibidem, Calamuchita, Villa Alpina near from Cumbrecita, under Pinus sp., 26 Apr 1981, leg. L. Domínguez de Toledo, no. 345 (HUH 00373827). BRAZIL, Rio Grande do Sul, São Leopoldo, 1907, leg. Pe. J. Rick (HUH 00373825 – as C. cruciata); ibidem, 1906, leg. Pe. J. Rick (PACA 12527); ibidem, 1929, leg. L. Braum (PACA 12533); ibidem, 1933, leg. Pe. J. Rick (PACA 12534); ibidem, 1929, leg. L. Braum (PACA 13779 – as L. pulcherrimum); ibidem, Serro Azul, 1935, leg. Pe. J. Rick (PACA 13794); ibidem, Salvador do Sul, São Salvador, 18 Jan 1944, leg. Pe. J. Rick (PACA 20848). COLOMBIA, Boyaca, Paramo de Guantiva, Valle Q. El Desaguadero 4 km al NW de Santa Rosita, en terraza seca a lo largo de la Quebrada con rastrojo de Senecio sp. asociado con Halenia y Acaena cylindrostachya, n. d., leg. A.M. Cleef, no. 9768A (NY00398532 – as L. candidum). ECUADOR, s. d., s. leg., no.7190 (NY 00340572). URUGUAY, Canelones, in sand soil, Jul 1946, W.G. Herter (NY00398533 – ex HERB. HERT. 61104 – as L. cruciatum); ibidem, (HUH 00373829 – ex HERB. HERT. 61104 – as L. cruciatum); ibidem, Artigas, Estancia Timbauba, Barra del Arrago Tres Crucest, Feb 1955, leg. N. Garcia-Zorron, no. 2389 (K (M): 200242).

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Notes: Lycoperdon marginatum is characterized by its exoperidium with white pyramidal spines at the apex of basidioma falling off in units or frequently in plates, surface of endoperidium with a furfuraceous material, basidiospores punctate to slightly verrucose [A-B], capillitium with pores. Lycoperdon calvescens is close to L. marginatum (Demoulin, 1972b, 1983); however the spines are falling off in units and basidiospores always punctate [A] are not found in L. marginatum. The specimens NY00398532 and NY00398533, as well as HUH 00373825 and HUH 00373829 have been labelled under the name L. candidum and L. cruciatum, respectively, considered by Ponce de León (1970), distinct species of L. marginatum. Although these specimens labelled by himself under the name L. candidum and L. cruciatum, share the same features as specimens labelled as L. marginatum; also, specimens have the same features described by (Demoulin, 1972b, 1983). Moreover, the molecular analyses confirm the position of these specimens as L. marginatum.

Lycoperdon mauryanum Pat. ex Demoulin, Leujenia 62: 16 (1972), Mycobank: MB 317034. Fig. 21i, 22c.

Basidioma subglobose, 41 mm length × 45 mm width; subgleba well-developed

(occupying > ⅓ of basidioma), cellular, purplish brown (N50Y60M50). Exoperidium persistent, verrucose, minute warts (< 1 mm long.), brown (N50Y60M30), also slender spines can be found spread in parts of basidioma. Endoperidium papery, smooth, yellowish brown (Y90M60C30). Gleba grayish brown (N50Y80M50).

Exoperidium basal likely to the apical, composed by sphaerocysts globose, subglobose to pyriform, 20.5–33 length × 21–32 µm width, wall 1–1.8 µm thickness, without reaction, acyanophilous, yellowish brown in 5% KOH. Endoperidium basal banal likely to the apical, hyphae aseptate, 2.5–3.5 µm diam., wall 0.5–1 µm thickness, aseptate, without reaction, acyanophilous, yellowish in 5% KOH. Paracapillitium absent. Capillitium 3.5–8.3 µm diam., wall 1–1.5 µm thickness, aseptate, occasional pores, without reaction, acyanophilous, brown in 5% KOH. Basidiospores globose, 3.8–4.5 µm width, strongly verrucose [D], in LM, number of ornamentation 9–11.5 for a circumference reduced to 10 µm.

Habitat and substrate: gregarious or solitary, growing on soil.

Specimens examined: GUATEMALA, Mixco, between Guatemala City and Antigua, on the ground, 22 Jun 1963, leg. B. Lowy (LG 1570!).

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Notes: Lycoperdon mauryanum is characterized by its verrucose exoperidium, mixed with sparse slender spines in parts of the basidioma; basidiospores strongly verrucose [D], capillitium with occasional pores. According to Demoulin (1972b), L. mauryanum belongs to the Molle-group, together with L. molle and L. umbrinum; however, L. mauryanum has capillitium with occasional pores, and basidiospores strongly verrucose as mentioned in Demoulin (1972a). Although L. atropurpureum also has a strongly verrucose basidiospores, L. mauryanum has smaller ones (3.8–4.5 µm vs. 5.5–7 µm in L. atropurpureum), as indicated in Calonge et al. (2005). In accordance with some authors, L. mauryanum is a representative species of L. atropurpureum in America, and would be better as synonymous or a subspecies considering the similar features shared by these taxa. Despite that, the authors said “no somos partidarios de crear entidades taxonómicas a partir de diferencias en distribución geográfica”. Here, we have studied only the specimen LG 1570 (paratype) and more samples should be studied to clarify if L. mauryanum is a distinct species. Demoulin (1972b) preferred to consider this taxon as a good species, even though the morphological features are not enough to separate L. mauryanum from L. atropurpureum, and he said in Demoulin (1972b): “Il s’agit d’un de ces taxons que l’on pourrait considerer comme sous-espèce géographique. Nous préférons toutefois en principe ne pas utiliser de categories infraespécifiques dans l’étrat actuel de la science mycologique”.

Lycoperdon nigrescens Pers., Neues Mag. Bot.1: 87 (1794): Pers. Syn. meth. fung.: 146 (1801) – Mycobank: MB 218442. Figs. 22d, 23a.

Synonymous (Larsson and Jeppson, 2008) = Lycoperdon perlatum var. nigrescens Pers., Syn. meth. fung.: 146 (1801). = Lycoperdon foetidum Bonord., Handb. Allgem. Mykol. (Stuttgart): 253 (1851).

Basidiomata pyriform, subpyriform to turbinate, 20–36 mm length × 18 – 30 mm width.

Subgleba well-developed, cellular, pale orange (N00Y40M10), grayish (N60Y40M10) to grayish purple (N60Y30M30). Exoperidium falling off, spines with slender and convergent tips, yellowish (N00Y10M00), yellowish gray (N10Y30M00), grayish brown (N60Y70M40) to darkish brown (N90Y99M90). Endoperidium papery, areolate, yellowish (N00Y10M00 to

N00C00Y10) to yellowish brown (N20Y60M20); gleba powdery grayish brown (N70Y70M30) to brown (N90Y99M60).

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Exoperidium basal likely to the apical composed by sphaerocysts globose, subglobose to pyriform 13–35 length × 9.5–33.5 µm width, without reaction, acyanophilous, hyaline in 5% KOH. Endoperidium apical composed also by mycosclerids 29.9–78.8 length × 8.6–31 µm width., wall 1–2.3 µm thickness, dextrinoid, acyanophilous, yellowish in 5% KOH. Endoperidium basal likely to the apical composed by hyphae 3–6.5 µm diam., wall 0.5–1.5 µm thickness, weakly reaction to dextrinoid, acyanophilous, hyaline in 5% KOH.

Figure 21 Dry basidiomata of Lycoperdon species. a. L. atrum (UFRN-Fungos 832); b. L. calvescens (CORD 997); c. L. curtisii (VDEMOULIN 4073); d. L. endotephrum (K (M): 200182); e. L. eximium (NY0071978); f. L. hyalinum (DSA 178, UFRN-Fungos 2829); g. L. lividum (NY 0071655); h. L. marginatum (CORD 72); i. L. mauryanum (LG 1570). Bars = 10 mm.

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Paracapillitium 2.5–6.5 µm diam., wall 0.5–1 µm thickness, septate, cyanophilous, hyaline in 5% KOH. Capillitium 2.5–7.5 µm diam., wall 0.5–1 µ, thickness, aseptate, occasional pores, acyanophilous, brownish in 5% KOH. Basidiospores globose, 3.5–5 length × 3.6–5.2 µm width, punctate [A] in LM, remains of sterigmal mixed with the basidiospores, pedicel (cyanophilous) 1–4 µm long., without reaction, acyanophilous, brown in 5% KOH.

Habitat and substrate: gregarious and cespitose, growing on soil or decaying wood.

Specimens examined: ARGENTINA, Bariloche, Cerro Tronador, sobre solo, 2001, leg. M.L. Hernández-Caffot (UFRN-Fungos 2275 – as L. ericaeum); ibidem, Corrientes, Capital, Av. Independencia 5000, em isleta de monte, creciendo em el suelo entre la hojarasca, 21 Jan 1987, leg. Popoff, no. 130 (BAFC 50177 – as L. perlatum); ibidem, Tierra del Fuego, Bahía Ensenada, en suelo y restos de madera, entre Hepáticas, 24 Jan 1992, leg. I. Gamundi (BAFC 50186 – as L. foetidum); ibidem, Punta Lara, 01 May 1966, leg. S. Matteucci (BAFC 51008). BRAZIL, Rio Grande do Sul, São Leopoldo, 1904, leg. Pe. J. Rick (HUH 00373907 – as L. perlatum); ibidem, 1930, leg. Pe. J. Rick (PACA 13774 – as L. perlatum); ibidem, 1931, leg. Pe. J. Rick (PACA 13796 – as L. rimulatum). COLOMBIA, Boyacá, Pantano de Vargas, Vicinity km post 12 from Paipa, on soil, 12 Jun 1976, leg. K.P. Dumont, S.E. Carpenter, M.A. Sherwood, L.A. Molina, no. Dumont- CO 4864 (NY 0071657 – as L. perlatum). URUGUAY, Tacuarembó, Taquarembó, 1936, leg. Pe. J. Rick (PACA 13783 – as L. spacideum).

Notes: Lycoperdon nigrescens is a species with great variation in exoperidium spine color (yellowish to dark brown); basidiospores punctate [A]. Note that as the exoperidium is falling off, L. nigrescens can be misidentified as L. perlatum (e.g. in the specimens BAFC 50177 or PACA 13774), even with L. ericaeum (UFRN-Fungos 2275). However, L. nigrescens has an exoperidium with slender spines and convergent tips, and basidiospores [A] (Demoulin, 1972b). This last author has considered L. nigrescens (Pers. per. Pers.) C.G. Lloyd non (Pers. per Pers.) Poiret and L. perlatum var. nigrescens Pers. per Pers. as synonymous of L. foetidum; afterward he proposed the new combination to L. peckii Morg. as Lycoperdon foetidum var. peckii (Demoulin, 1979), considering that the pale spines are a variant of L. foetidum. After that, in the key to the identification of Lycoperdon species from South Europe, Demoulin (1983) treated L. foetidum and L. perlatum var. nigrescens as synonymous of L. nigrescens. The specimen PACA 13796, has been identified under the name Lycoperdon rimulatum; however this species has an 176

endoperidium not areolate and basidiospores strongly verrucose (Coker and Couch, 1928). This is the first time Lycoperdon nigrescens is reported from Argentina and Uruguay.

Lycoperdon ovoidisporum Cortez, Baseia & R.M.B. Silveira, Sydowia 63(1): 2 (2011), Mycobank: MB 561562. Figs. 22e, 23b.

Basidiomata, pyriform to turbinate, 10–31 mm length × 14–35 mm width. Subgleba well- developed, lanose, in some specimens with compact cell, yellowish brown (N30Y90M20), reddish brown (Y99M80C20) to brown (N60Y99M60). Exoperidium persistent, warty, warts

< 0.5 mm long., yellowish (N10Y99M90) to brown (N70Y99M70). Endoperidium papery, smooth, yellowish (N00Y10M00). Gleba olive brown (N60Y99M40).

Exoperidium basal likely to the apical composed by sphaerocysts 13–28 length × 11–29 µm width, wall 1–2 µm thickness, without reaction, acyanophilous, hyaline in KOH. Endoperidium apical composed by hyphae with inflated termination and mycosclerids: hyphae inflated terminations 6–15 µm diam., wall 0.5–1 µm thickness, septate, weakly reaction to dextrinoid, acyanophilous, hyaline in 5% KOH; mycosclerids 37–110 length × 10–25 µm width, 1–2 µm thickness, weakly reaction, acyanophilous, hyaline in 5% KOH. Paracapillitium absent; endoperidium basal composed by hyphae 3– 6 µm diam., wall 0.5–1 µm thickness, occasional septate, weakly reaction, acyanophilous, hyaline in 5% KOH. Capillitium 2.5–4.5 µm diam., 0.5–1 µm thickness, occasional septate, occasional pores, branched, without reaction, acyanophilous, brownish in 5% KOH. Basidiospores subglobose, ovoid to suboval (often ovoid have been found), 4.2–5 length × 3–4 width smooth, punctate to slightly verrucose [A-B] in LM, pedicel 0.5–1.2 µm thickness, without reaction, acyanophilous, brownish in 5% KOH.

Habitat and substrate: gregarious, growing on soil.

Specimens examined: ARGENTINA, Tucumán, En Quebrada de Lules, 10 Nov 1952, leg. A.T. Hunziker, no. CORD 510 (CORDC 182 – as L. gemmatum). BRAZIL, Rio Grande do Sul, Viamão, Parque Estadual de Itapuã, on soil, 30º11’42’’S and 51º03’18’’W, 22 May 2004, leg. V.G.Cortez (UFRN-Fungos 867); ibidem, São Leopoldo, 1931, leg. V.G. Cortez (PACA 13771); ibidem, leg. Pe. J. Rick (PACA 13805); Paraná, Curitiba, Reserva Biológica Cambuí, forest edge under shrub on the soil, 27 Jul 1979, leg. A.A.R. de Meijer, (VDEMOULIN ex De Meijer 103a).

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Notes: Lycoperdon ovoidisporum is characterized by its exoperidium with persistent warts, gleba olive brown, basidiospores mostly ovoid. This species was described for the first time from Brazil in Cortez et al. (2011); these authors considered the basidiospore shape diagnostic for the species. Lycoperdon eximium also has peculiar ovoid basidiospores (Coker and Couch, 1928; Demoulin, 1972b), and it is close to L. ovoidisporum. However, L. eximium is separated from L. ovoidisporum by its capillitium with abundant pores, bigger basidiospores 4.3–6.1 length × 3.5–4 µm width, strongly dextrinoid (mostly in specimens studied here). Lycoperdon darjeelingense B. M. Sharma and L. ovalicaudatum D. Bisht, J.R. Sharma & Kreisel, are others species with ovoid basidiospores; however, they have a longer pedicel, up to 19 and 22 µm long, respectively (Sharma, 1991; Bisht et al., 2006).

Lycoperdon perlatum Pers., Observ. mycol. 1: 145 (1796): Pers., Syn. Meth. Fung.: 145 (1801), Mycobank: MB 220647. Figs. 22f, 23c.

Synonymous (Demoulin 1972b and the Index Fungorum website) = Lycoperdon gemmatum Schaeff., Fung. Bavar. Palat. Nasc. (Ratisbonae) 4: 130 (1774). = Lycoperdon gemmatum Batsch, Elench. Fung. (Halle): 147 (1783). = Lycoperdon perlatum var. lacunosom Pers., Syn. Meth. Fung. (Göttingen) 1: 146 (1801). = Lycoperdon perlatum var. albidum Alb. & Schwein., Consp. fung. (Leipzig): 80 (1805). = Lycoperdon perlatum var. bonordenii (Massee) Perdeck, Blumea 6: 505 (1950). = Lycoperdon var. dobremezianum Kreisel, Feddes Repert. Spec. Nov., Beih. 87(1-2): 103 (1976). = Lycoperdon perlatum var. lacunosum Pers., Syn. meth. fung. (Göttingen) 1: 146 (1801). = Lycoperdon pseudogemmatum Speg., Anal. Soc. cient. argent. 17(2): 85 [no. 95] (1884).

Basidiomata subglobose, pyriform, subpyriform to turbinate, 8–65 mm length × 10–40 mm width. Subgleba well-developed (occupying > ⅓ of basidioma), cellular, yellowish

(N10Y30M00), grayish (N50Y80M00) to gray (N50Y20M10). Exoperidium falling off (mostly the spines), conical and pointed spines, cream (N00Y10M00 to N00Y30M00) to light brown

(Y80M40C30), mixed with persistent warts brown (N70Y90M60). Endoperidium papery, areolate, in oldest or bad conserved specimen the areoles cannot be there, yellowish gray

(N50Y40M00), gold yellow (N10Y80M20) to shiny gray (N50C00Y10); dehiscence by an apical regular pore. Gleba grayish brown (N50Y60M20 to N50Y80M20), reddish brown

(N30Y99M60) to brown (N70Y90M70).

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Exoperidium basal likely to the apical, composed by sphaerocysts globose, subglobose to pyriform, 19.4–65.1 length × 15.6–43.3 µm width, wall 0.7–2.7 µm thickness, without reaction, acyanophilous, hyaline to yellowish in 5% KOH. Endoperidium basal 3.3–7.7 µm diam., wall 0.5–2 µm thickness, without reaction to weakly reaction, acyanophilous, yellowish in 5% KOH. Paracapillitium 3–6.5 µm diam., wall 0.5–1.5 µm thickness, septate, without reaction, cyanophilous, hyaline in 5%KOH. Capillitium 3–4.5 µm diam., wall 0.5–1 µm thickness, aseptate, occasionally with pores, without reaction, acyanophilous, brown in 5% KOH. Basidiospores globose to subglobose, 3.5–5.5 length × 3.6–5 µm width, verrucose [C] in LM, aculeate, slender aculeus connected each other by lines in SEM, number of ornamentation 6.3–11.8 for a circumference reduced to 10 µm; acyanophilous, yellowish to brown in 5% KOH.

Habitat and substrate: gregarious, cespitose, growing on soil on decaying wood.

Specimens examined: ARGENTINA, Missiones, Parque Nacional Iguazú, bosque de Palo Rosa, 03 Mar 1982, leg. J. Wright (BAFC 28012); ibidem, Dpto. Guarani, prédio Guarani, Fac. Cs. Forest. El Dorado, 06 Sep 1994, leg. O. Popoff, no. 2343 (BAFC 34025); Salta, Campamento Las Heras (Los Tigres), en el suelo, 23 Sep 1958, leg. Morello, Legname y Cuezzo, no. 833 (BAFC 32162); Patagônia, Província Rio Negro, Parque Nacional de Lanín, sobre suelo, 2002, leg. M.L. Hernández-Caffot (UFRN- Fungos 2273 – as L. perlatum var. nigrescens); ibidem, 2005, leg. Hernández-Caffot (UFRN-Fungos 2274); ibidem, 2003, leg. M.L. Hernández-Caffot (UFRN-Fungos 2272). BRAZIL, Amazonas, leg. T.S. Cabral, no. TSC 70 (UFRN-Fungos 2828); Minas Gerais, Santa Rita do Sapucaí, Reserva Biológica Municipal de Santa Rita Mitzi Brandão, 22°15’35.17”S and 45°43’59.41”W, 27 Dec. 2013, leg. D.S. Alfredo and P. Lavor, no. DSA 198 (UFRN-Fungos 2335); Paraná, Curitiba, Reserva Biológica Cambuí, secundar leafy tree on humus path, 02 Nov 1979, leg. A.A.R. de Meijer (VDEMOULIN ex DeMeijer206 – as L. pseudogemmatum); Rio Grande do Sul, Santa Maria, on wood of Enterolobium sp. 30º08’18’’S and 54º19’58’’, 17 May 2003, leg. G. Coellho (UFRN- Fungos 811); ibidem, São Salvador, Jun 1943, Pe. J. Rick (PACA 20088); ibidem, São Leopoldo, 1929, leg. L. Brun (PACA 13789 – as L. gemmatum); ibidem, 1908, Pe. J. Rick (HUH 00373903 – as L. gemmatum); ibidem, 1906, Pe. J. Rick (HUH 00373916 – as L. pseudogematum); São Paulo, Instituto de Botânica, on wood, 23 Nov 1969, leg. D.M. Dring, no. 139 (K (M) 200238); ibidem, Estação Ecológica de Jataí, Mar 2000, leg. I.G. Baseia (UFRN-Fungi 2269 – contaminated amplification). Colombia, Departamento del

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Cauca, Cordillera Central, vertiente occidental, Cabeceras del rio palo, quebrada del rio López, Alto del Duende matorrales y bosquecillo de páramo, 3300-3350 m alt., 1-2 Dec 1944, leg. J. Cuatrecasas (HUH 00373892); ibidem, Vereda Guazabarita, Finca “El Bosque”, El Tambo, on soil, 15 Jul 1974, leg. K.P. Dumont, J.H. Haines, J.M. Idrobo, L.F. Velasquez, no. Dumont-CO 1365 (NY 0071656). ECUADOR, Galapagos Islands, San Cristobal Island (as Chatham), n.d., leg. Capitan Wood, s.n. (KW 200236 – as L. ericaeum). PARAGUAY, Guarapi, Aug 1882, leg. B. Balansa, no. 3921 (K (M) 200241 – Isotype of L. pseudogemmatum!). URUGUAY, Montevideo, n.d., leg. Herter (PACA 13795). VENEZUELA, Caracas, Junquito-Colonia Tovar, Edo. Aragua, on road, on unidentified wood, 22 Jun 1971, leg. K.P. Dumont, J.H. Haines, E. Moreno, no. Dumont- VE 446 (NY 00793106 – as L. pseudogemmatum); Estado de Merida, Sierra Nevada, La Mucuy, Laguna de Coromotto, in soil amongst rocks, 3200 m alt., 07 Aug 1958, leg. R.W.G. Dennis, no. 1797 (K(M):200240); Chichiriviche, Falcon (as Destrito Federal), on soil aroundo of Coffea sp., 500 m alt., 06 Jul 1958, leg. R.W.G. Dennis, no. 1312 (K (M) 200239). Extralimital collections: MEXICO, Falla de Telapón, Rio Frio, bosque de Abies and Pinus, Jul 1958, leg. G. Guzmán (VDEMOULIN ex ENCB 1533); ibidem, Zacatecas, Cerro del Pinal, 20 Sep 1959, leg. G. Guzmán (VDEMOULIN ex ENCB 2223); Hidalgo, Zacualtipan, en bosque subtropical, 1900 m alt., 22 Sep 1960, leg. G. Guzmán (VDEMOULIN ex ENCB 2633). UNITED STATES, Michigan, Pellston, Michigan University, Lawrence lake, NW of Biological Station, deciduous forest, Jun 1965, leg. G. Guzmán (VDEMOULIN ex ENCB - U87 – as L. pyriforme); Washington, Bremerton, on gravelly ground among short grass, 2 Sep 1912, leg. E. Bartholomew (K (I.M.I) 28707).

Notes: Lycoperdon perlatum is characterized by its basidioma mostly pyriform or turbinate, exoperidium spines falling off and remaining warts, endoperidium surface mostly areolate, basidiospores verrucose [C], capillitium with occasional pores and abundant paracapillitium. This species has worldwide distribution growing everywhere (Calonge, 1998), and, as mentioned in Demoulin (1972b) it has a wide morphological variation. Demoulin (1972b) also reported that in North American specimens, in the capillitium the pores are rare. Likewise, in many of the specimens studied here, the capillitium has no or rare pores (PACA 20088, K(M) 200240, etc.). The specimen UFRN- Fungos 2335 was reported from Brazil as first record under the name Lycoperdon lambinonii (Alfredo et al., 2016). Cunningham (1944) considered that L. perlatum has basidiospores [A], but it must be a mistake; because just the basidiospores [C] separates

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L. perlatum from L. lividum and L. nigrescens (both have basidiospores [A]) (Pegler et al., 1995). Also, the exoperidium spines in L. perlatum are more conical and with angular tips, whereas in L. nigrescens slender curved tips are found (Honrubia et al., 1982). The authors found 10-11 ornamentations in a circumference of 10 µm. Here similar number (6.8–11.8) were obtained; the extreme values were slightly different because of our increased sampling. An important factor here was the analysis of the specimen K (M) 200241, isotype of L. pseudogemmatum. After morphological reexamination, we confirmed that L. pseudogemmatum shares the same characters as L. perlatum: warts remain around areoles of endoperidium surface, capillitium with rare pores (Paraguayan specimen) and basidiospores verrucose [C]. Moreover, the description of Saccardo (1888) (“cortice exterior mox evanescente, albo, tenui, cortice interior tenui payraceo- membranaceo, tenacello, laxe minuteque granuloso-verrucoloso, sicco albo vel pallescente…”) fits with L. perlatum. Additionally, the specimens labelled under L. pseudogemmatum (NY 00793106, HUH 00373916 and VDEMOULIN ex DeMeijer206) have no differences with the L. perlatum specimens studied here; therefore L. pseudogemmatum is confirmed as a synonym of L. perlatum.

Lycoperdon pratense Pers., Neues Mag. Bot.1: 87 (1794): Pers., Syn. Meth. Fung.: 142 (1801), Mycobank: MB 174409. Fig. 22g, 23d, 24g-i.

Synonymous (Larsson and Jeppson 2008): = Vascellum pratense (Pers.) Kreisel, Feddes Repert. 64: 159 (1962). = Lycoperdon depressum Bonord., Bot. Zeitung (Berlin) 15: 611 (1857). = Vascellum depressum (Bonord.) F. S ˇmarda, Flora CSR, B1: 305 (1958). Synonymous (here proposed) = Vascellum delicatum Homrich, Canadian Journal of Botany 66 (7): 1292 (1988) [Mycobank: MB134080].

Basidiomata pyriform to turbinate, 12–20 mm length × 13–32 mm width. Subgleba well- developed, cellular whitish, yellowish (N00Y40M00) in specimens not complete mature, brown (N30Y80M30). Diaphragm absent or disappearing; exoperidium falling off in plates, stellate spines with slender tips, pale yellow (N00C00Y10) to yellowish (N00Y20M00).

Endoperidium papery, smooth, yellowish (N00Y30M00) in specimens immature, becoming shiny gray (N80C00Y20) to grayish brown (N50Y50M30). Gleba powdery, whitish becoming light brown (N20Y80M10) to brown (N60Y60M40).

Exoperidium likely to the apical, composed by sphaerocysts globose to subglobose 14.6 – 34.8 length × 13.5 – 29.6 µm width, wall 0.87 – 2.2 µm thickness, weakly reation,

181 acyanophilous, yellowish in 5% KOH. Endoperidium apical composed by mycosclerids and hyphae with inflated terminations: mycosclerids irregular shape, 60–110 length × 12– 50 µm width, without reaction, acyanophilous, yellowish in 5% KOH; hyphae inflated terminations 7.8–14.2 µm diam., wall 1.2–3 µm thickness, without reaction, acyanophilous, yellowish in 5% KOH. Endoperidium basal composed hyphae 3–4 µm diam., wall 0.5–1 µm diam., without reaction, acyanophilous, yellowish in 5% KOH. Paracapillitium 4–6 µm diam., wall 0.5–1 µm thickness, without reaction, acyanophilous, hyaline in 5% KOH. Capillitium can be absent in some specimens mostly in young ones, 3–4.5 µm diam., wall 0.5–1 µm thickness, aseptate, pores absent, branched, without reaction, acyanophilous, yellowish in 5% KOH. Basidiospores globose, 3.5–4.5 µm width, punctate to slightly verrucose [A-B], in LM, minute aculeus in SEM, without reaction, acyanophilous, hyaline to yellowish in 5% KOH.

Habitat and substrate: gregarious, cespitose, growing on soil

Specimens examined: BRAZIL, Rio Grande do Sul, Santa Maria, Campus UFSM, 29º41’03’’S and 53º48’25’’W, 05 Mar 2009, leg. G. Coelho, s.n. (ICN 154485 – as V. hyalinum). Chile, Santiago, Forest close to El Tabo, among the grass at the border of the road, 01 Jul 1966, leg. W. Lazo (VDEMOULIN ex AGUCH-ET8 – as Isotype of V. delicatum!). NICARAGUA, Nueva Segovia, Santa Clara, on the ground, around Pinus caribae var. hondurensis, 700 m alt., Jun 1973, leg. A. Greaves, s.n. (K (M) 200187). URUGUAY, Montevideo, Paysandu, Jul 1934, leg. Herter, no. 2183 (HUH 00373786 ex HERB. HERT. 94793 – as L. hiemale). Extralimital collections: MEXICO, Ciudad de Mexico, Colonia Santo Tomás, calle de Plan de Ayala, jardín con pasto, 2200 m alt., 15 Jul 1969, leg. G. Guzmán, (VDEMOULIN ex ENCB 7412); ibidem, SW de La Presa Guadalupe, carretera a Nicolas Romen, pastizal árido, 2380 m alt., leg. G. Guzmán, no. 4036 (K (M) 200229).

Notes: Lycoperdon pratense is characterized by its exoperidium falling off in plates, stellate spines with slender tips, endoperidium smooth and shiny, basidiospores globose, punctate to slightly verrucose [A-B]. In respect to the absence of capillitium, Smith (1974) reported that in some specimens from North America he did not find capillitium. This feature has been found near the inner surface of the endoperidium or near the subgleba (when present) as indicated by Reid (1977). Although authors have found basidiospores dextrinoid, in the specimens studied here there was no reaction in Melzer’s reagent. Pegler et al. (1995) and Cortez et al. (2008), described an exoperidium furfuraceous, however 182

this feature has not found here, only in the K (M) 20018, in which the powdery gleba has covered the endoperidium surface with masses of basidiospores giving a furfuraceous aspect. Lycoperdon pratense has a worldwide distribution being reported in all continents (Ponce de León, 1970). In Mexico, L. pratense has been found under the name L. hiemale (Herrera, 1964), in Brazil L. pratense has been reported by Bononi et al. (1984) and Cortez et al., 2008, 2013).

Lycoperdon pyriforme Schaeff., Fung. Bavar. Palat. nasc. 4: 128 (1774): Pers., Syn. Meth. Fung.: 148 (1801), Mycobank: MB 123159. Fig. 22h, 23e. Synonymous (Larsson and Jeppson 2008): = Morganella pyriformis (Schaeff.) Kreisel & D. Kruger, Mycotaxon 86: 175 (2003).

Basdiomata pyriform to turbinate, 12–50 mm length × 9–35 mm width. Subgleba well- developed, lanose, whitish, yellowish (N00Y30M00) becoming grayish (N10Y20M00 to

N40Y30M10). Exoperidium persistent, minute spines (< 0.5 mm) brown (N40Y80M80); endoperidium papery, yellowish (N00Y00M00 to N00Y10M00); dehiscence by a regular apical pore or sometimes by rupture of the peridium. Gleba powdery, yellowish brown

(N20Y80M50) to grayish brown (N60Y70M30).

Exoperidium basal likely apical composed by sphaerocysts irregular of spiny aspect 56.4–109.9 length × 38.1–76.7 µm width, wall 0.9–3.2 µm thickness, dextrinoid, acyanophilous, yellowish in 5% KOH. Endoperidium basal likely apical composed by hyphae 2.3–4.5 µm diam., wall 0.5–2.2 µm thickness, aseptate, weakly to dextrinoid, acyanophilous, yellowish in 5% KOH. Paracapillitium 3–6.7 µm diam., wall 0.6–1.82 µm thickness, septate, without reaction, cyanophilous, hyaline in 5% KOH. Capillitium 2.5– 4.7 µm diam., wall 0.5–1.2 µm thickness, aseptate, absent pores, without reaction, acyanophilous, brown in 5% KOH. Basidiospores globose 2.6 – 4.6 µm width, smooth [A] in LM, pedicel 1.5–3.4 µm long., without reaction, acyanophilous, brown in 5% KOH.

Habitat and substrate: gregarious and cespitose, growing on decaying wood.

Specimens examined: ARGENTINA, Provícia de Córdoba, Parque Nacional Quebrada del Condorito, sobre solo, 2007, leg. M.L. Hernádez-Caffot (UFRN-Fungos 2276); Tucumán, Chicligasta, Alpachiri, Piedra Grande, 500 m alt., 02 Feb 1946, leg. A. Garolere, no. 7181 (BAFC 31387). BRAZIL, Pernambuco, solo humoso, 06º37’47’’S and 35º02’15’’W, 13 Feb 2003, leg. T.G. Gibertoni (UFRN-Fungos 155); Rio Grande do Sul, 183

São Francisco, Floresta Nacional, 29º26’52’’S and 50º35’02’’W, 23 May 2015, leg. L. Trierveiler-Pereira, no. LTP236 (ICN 177101); ibidem, Nova Petropolis, 1912, leg. Pe. J. Rick (HUH 00373988); Porto Alegre, Iboti, 17 Jan 1976, leg. Y. Doi, no. AS-166 (VDEMOULIN ex TNSF 226943 – as L. atrum); ibidem, São Leopoldo, 1904, Pe. J. Rick (HUH 00373990); ibidem, 1904, leg. Pe. J. Rick (PACA 13780); ibidem, 1904, (NY 0071972); São Paulo, Instituto de Botânica de São Paulo, leaf-litter, 11 Dec 1999, leg. I.G. Baseia (UFRN-Fungi 2271); ibidem, 27 Oct 1998, leg. I.G. Baseia (UFRN-Fungi 2268 – as Calvatia rubroflava). COLOMBIA, Depto. Magdalena, Sierra Nevada de Santa Maria, Hacienda Cincinati, 1250-1500 m alt., 17 Aug 1935, leg. G.W. Martin, no. 3437 (HUH 00373985); ibidem, (NY 0071971). ECUADOR, n.d., leg. G. Massee (NY 0071973). Extralimital collections: BELGIUM, Liege, between Brachythecium rutabulum, 03 Oct 1965, leg. V. Demoulin (VDEMOULIN 3195). MEXICO, Amecameca, Tlamácas, en Abietum, 300 m alt., 18 Aug 1963, leg. G. Guzmán (VDEMOULIN ex ENCB 5084); ibidem, La Marquesa, El Zarco, en Abietum, 24 Nov 1957, leg. G. Guzmán (VDEMOULIN ex ENCB 1401). UNITED STATES, Alaska, Ptermingan Creek, 14 Sep 1970, leg. W. Kemptn (VDEMOULIN 4593). Venezuela, n.d., leg. Fendler (NY 0071979).

Other specimens examined: Lycoperdon melanesicum — FIJI, Viti Levu, Vicinity of Rewasa, near Vaileka, alt. 50–200 m, on log. on dry forested forehill, 28 May and 17 Jun 1941, leg. O. Degener, no. 15547a (NY00795902 – Type!).

Notes: Lycoperdon pyriforme is characterized by its persistent exoperidium, with minute spines, endoperidium smooth, basidiospores globose and smooth [A], capillitium and paracapillitium abundant, sphaerocysts of irregular shape resembling a spiny shape, dextrinoid. In our analyses, some specimens were labelled under L. pyrifome due to their smooth basidiospores (NY 0071978 – see in L. eximium; NY 0071974 and NY 0071975 – see in L. calvescens). However, it could be a misidentification with L. calvescens and L. eximium; although L. calvescens has exoperidium with pyramidal spines, capillitium with pores and basidiospores can be found as slightly verrucose [B], features not found in L. pyriforme; while in L. eximium the ovoid and dextrinoid basidiospores are diagnostics to separate these two species (Demoulin, 1968a, 1972b, 1983). In South America, Wrigth et al. (2008) reported L. pyriforme from Argentina, and Rocabado et al. (2007), from Bolivia; the authors reported sphaerocysts of big size likely found in the specimens here studied. In Brazil it was cited by Sydow and Sydow (1907), Rick (1961),

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Bononi et al. (1984) and Baseia (2005). López-Quintero et al. (2011) reported L. pyriforme from Colombia, and Herrera (1964), as well as Rodríguez and Herrera (1970) from Mexico. Cunningham (1944) considered that L. pyriforme has a worldwide distribution, and cited the type locality in Europe.

Lycoperdon melanesicum is a peculiar species easy to recognize by its exoperidium falling off, with brown warts, sometimes the exoperidium has fallen off in all basidioma, exoperidium is composed by dextrinoid elongated hyphae with pointed terminations, basidiospores smooth [A], capillitium and paracapillitium are abundant. Lycoperdon pyriforme is close to L. melanesicum; however, L. pyriforme has an exoperidium composed by different cells (like irregular spines), that are also dextrinoid (Demoulin, 1976). According to this author, L. melanesicum can be misidentified with some Morganella species, especially M. samoense. Although, the occurrence of capillitium distinguishes these species.

185

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Lycoperdon umbrinum Pers., Ann. Bot. (Usteri) 11: 28 (1797): Syn. meth. fung.: 147 (1801), Mycobank: MB 232824. Fig. 23f. Basidiomata pyriform to turbinate, 31–64 mm length × 24–60 mm width. Subgleba well- developed (occupying < ⅓ of basidioma), cellular, light brown (N30Y90M50), olive brown

(N60Y80M20) to grayish brown (N50Y50M30). Exoperidium persistent to falling off, warty, warts < 1 mm long., light brown (N20Y60M20 to N50Y90M40). Endoperidium papery, smooth, pale yellow at the base (N00Y40M00) to yellowish green (N20Y30M00), becoming shiny gray (N30Y20M10) to light brown (N00Y80M30) to reddish brown (N30Y90M50). Gleba powdery, light brown (Y99M40C20), olive brown (N60Y99M30), brown (N70Y99M50).

Exoperidium basal likely apical, composed by sphaerocysts globose, subglobose, pyriform to collapsed 13–35 length × 13–25 µm width, wall 1.2–2.3 µm thickness, weakly reaction, acyanophilous, hyaline in 5% KOH. Endoperidium basal likely to the apical, composed by hyphae 3–5 µm diam., wall 0.5–1 µm thickness, weakly to strongly dextrinoid, acyanophilous, hyaline in 5% KOH. Capillitium 3–6.5 µm diam., wall 0.7–1 µm thickness, septate, abundant pores, without reaction, acyanophilous, brown in 5% KOH. Basidiospores globose 4–5.5 × 4.2–5.3 µm width, punctate [A] to slightly verrucose [B], in LM, columnar warts with flattened top in LM, pedicels 0.7–2 µm long., without reaction, acyanophilous, brown in 5% KOH.

Habitat and substrate: gregarious, can be found solitary, growing on soil or leaf- litter.

Specimens examined: ARGENTINA, Tierra del Fuego, Parque Nacional Lapataia, pasando cruce de camino a Cascada Rio Pipo, en base de N. Betuloides, sobre tierra apoyado al árbol, 24 Feb 1988, leg. M. Rajchemberg, no. 4048 (BAFC 31601); ibidem, Dpto. Ushuaia, Ea. Harberton, en bosque de Nothofagus pumilio sobre suelo, 26 Feb 1988, s.leg. (BAFC 32079); ibidem, camino con troncos, 30 Oct 1989, leg. C.L. Leite (BAFC 32183); ibidem, Turbera cercana a La Pataia, sobre suelo, en bosque de

Nothofagus batuloides, 21 Nov 1974, leg. I. Gamundi (BAFC 32178). BRAZIL, Rio Grande do Sul, São Salvador, 17 Jan 1944, leg. Pe. J. Rick (PACA 20701). Extralimital collections: UNITED STATES, Florida, Alachua Co., Sugarfoot Hammock, Lat. 29ºN and Log. 82ºW, 27 Aug 1977, leg. J. Ammirati and Chenes (VDEMOULIN 5119); Michigan, Livingston Co., oak grove, sous Picea, 04 Oct 1973, leg. V. Demoulin (VDEMOULIN 4784).

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Notes: Lycoperdon umbrinum is characterized by its exoperidium persistent but in some specimens can be found falling off, warty, light brown, endoperidium smooth, gleba powdery light brown, mostly olive to brown in mature basidioma, basidiospores slightly verrucose [B] (but specimens with punctate ones [A] can be found), capillitium with abundant pores. The specimens analyzed here have the same features as those described by Coker and Couch (1928). Bottomley (1948) considered the color of gleba (olivaceous to brown) and size of basidiospores (3.7–5.2 µm) as the main characters to identify this species. Bowerman (1961) considered L. umbrinum synonymous of L. molle; however, as discussed by Perdeck (1950), the brown gleba color and the ornamentation pattern of basidiospores [A-B] in L. umbrinum are decisive to separate these species. Herrera (1964) and Rodríguez and Herrera (1970), consider two varieties: L. umbrinum var. umbrinum and L. umbrinum var. floccosum Lloyd, differing because the last one has globose to subglobose basidiomes, exoperidium furfuraceous and persistent, with spines mixed with granules, and the spines partially falling off in flakes; however, the authors said: “la distinción entre L. umbrinum var. umbrinum y L. umbrinum var. floccosum no siempre puede hacerse con facilidad, por la inestabilidad de algunos de sus caracteres fundamentales (forma de la fructificación, color de la gleba, tamaño de la base estéril, evolución del exoperídio, etc)”. Demoulin (1972b), treated L. umbrinum var. floccosum as synonymous of Lycoperdon floccosum Lloyd. Here some specimens (BAFC 32183 and BAFC 32079) show the exoperidium falling off likely described by Bottomley (1948) and Perdeck (1950), however Calonge and Demoulin (1975), Pegler et al. (1995) and Calonge (1998) have not reported this character.

Lycoperdon utriforme Bull., Hist. Champ. France: 153 (1791): Pers., Syn. meth. fung.: 143 (1801), Mycobank: MB 232539. Fig. 23g.

Synonymous (Larsson and Jeppson (2008) and the IndexFungorum website) = Calvatia utriformis (Bull.) Jaap, Verh. Bot. Vereins Prov. Brandenburg 59: 37 (1918). = Handkea utriformis (Bull.) Kreisel, Nova Hedwigia 48: 288 (1989).

Basidiomata subglobose to turbinate 20–45 mm length × 9–28 mm width. Subgleba well- developed (occupying > ⅓ of basidioma), lanose in immature specimens, becoming cellular in mature specimens, white in immature specimens, becoming yellowish

(N10Y10M10) to yellowish brown (N30Y99M00) to brown (N40Y70M50) in mature specimens. Exoperidium echinulate, pyramidal spines 1–1.5 mm long. with convergent extremities, yellowish (N10Y00M10) to grayish brown (N40Y60M20). Endoperidium yellow (N10Y10M10) 188

at base, becoming brown (N70Y80M70), smooth. Gleba powdery, white becoming olive brown (N60Y60M20).

Exoperidium basal likely to the apical composed by sphaerocysts globose to subglobose 22.5–30 length × 20–29 µm width, with reaction, acyanophilous, yellowish in 5% KOH. Endoperdidium basal likely to the apical, composed by interwoven hyphae 3.5 5.3 µm diâm., wall 0.8 – 1.2 µm thickness, weakly dextrinoid, acyanophilous, yellowish in 5% KOH. Capillitium 5–7.2 µm diam., wall 0.6–1.3 µm thickness, septate, abundant pores, pores slit-like, acyanophilous, without reaction, brown in 5% KOH. Basidiospores globose, subglobose, sometimes ovoid, 4.6–5.6 length × 4.5–5.6 µm width, punctate [A] in LM, pedicels 0.5–1 µm, acyanophilous, without reaction, brown in 5% KOH.

Habitat and substrate: gregarious, growing on soil.

Specimens analyzed: ARGENTINA, Falkland Islands (Islas Malvinas), Old Wireless Station, Mar 1942, leg. J.G. Gibbs, s.n. (K (M): 200231 – as L. argentinum); ibibem, Pebble Island, Feb 1942, leg. J.G. Gibbs, s.n. (K (M): 200232 – as L. argentinum). CHILE, Puerto Arturo, Tierra del Fuego, 01 Feb 1946, leg. J.G. Gibbs, s.n. (K (M) 200230 – as L. argentinum).

Notes: Lycoperdon utriforme is characterized by its exoperidium with pyramidal spines and extremities convergent, subgleba becoming yellowish brown at maturity, gleba olive brown at maturity, capillitium with abundant pores slit-like and septate, basidiospores punctate [A]. This species is easily misunderstood as L. excipuliforme; however, the absence of pyramidal exoperidium spines and the verrucose basidiospores [C] in L. excipuliforme separate these two species (Moyersoen and Demoulin, 1996). The specimens studied here were misidentified as L. argentinum; specimen K(M) 200232 has mature gleba, whereas in the others, the gleba is in different degrees of maturation. Lycoperdon argentinum was synonymized to L. curtisii (= Vascelllum curtisii) by (Homrich and Wright, 1988); however, the pyramidal exoperidium ornamentation and capillitium with pores and septae have not been found in L. curtisii. This is the first time Lycoperdon utriforme is reported in South America.

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Lycoperdon sp. 1, Fig. 23h.

Basidiomata immature subglobose to pyriform, 13 mm length × 9 mm width. Basidiomata mature globose, subglobose, pyriform to turbinate, 14–30 mm length × 17–36 mm width.

Subgleba well-developed (occupying > ⅓ of basidioma), cellular, yellowish (N00Y10M00) becoming gray (N30Y20M10); diaphragm < 2 mm thickness, concolor to the gleba.

Exoperidium persistent becoming falling off, warty, warts yellowish (N00Y40M00) becoming grayish brown (N60Y40M10). Endoperidium papery, smooth yellowish

(N00Y10M00) becoming yellowish gray (N10C00Y20); dehiscence by an irregular apical pore or rupture of endoperidium. Gleba powdery, grayish brown (N20Y70M40) to brown

(N70Y80M40).

Exoperidium basal likely to the apical 12–26.5 length × 9.3–22.8 µm width, wall 0.6–1.2 µm thickness, without reaction, acyanophilous hyaline in 5% KOH. Endoperidium apical composed by mycosclerids and banal hyphae: mycosclerids 35.7– 85.7 length × 13–32.8 µm width; wall 1.2–2.5 µm thickness; weakly reaction, acyanophilous, yellowish in 5% KOH; banal hyphae 4.5–12.3 µm diam., wall 0.9–1.6 µm thickness, weakly reaction, hyaline in 5% KOH. Endoperidium basal composed by hyphae 4–6 µm diam., wall 0.5–1.2 µm thickness, without reaction, acyanophilous, hyaline in 5% KOH. Paracapillitium 3–6 µm diam., wall 0.7–1.2 µm thickness, septate, incrusted with remains of membrane glebal. Capillitium 3.2–5.3 µm diam., wall 0.5–1 µm thickness, septate, pores absent, without reaction, acyanophilous, light brown in 5% KOH. Basidiospores globose to subglobose, 3.5–4.4 length × 3.5–4.6 µm width, punctate to slightly verrucose [A-B], pedicel 0.5–7.3 µm long., without reaction, acyanophilous, light brown in 5% KOH.

Habitat and substrate; gregarious, growing on soil.

Specimens examined: ARGENTINA, Córdoba, San Javier, Quebrado El Hueco, 02 Mar 2007, leg. M.L. Hernández-Caffot, no. CORD 167 (CORDC 308). BRAZIL, Rio Grande do Sul, Santa Maria, Campus Universitário UFSM, 29º41’03’’S and 53º48’25’’W, 06 Mar 2009, leg. V.G. Cortez, no. VG010/09 (ICN 154484).

Notes: This species is recognized by its cellular subgleba well-developed and separate from the gleba (shades of brown) by a conspicuous diaphragm, exoperidium falling off with brown warts, irregular dehiscence or rupture of endoperidium, basidiospores punctate to slightly verrucose [A-B], abundant capillitium and

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paracapillitium, mycosclerids weakly reacting covering the apical portion where the exoperidium is falling off. Lycoperdon pratense is distinct from Lycoperdon sp.1 because its exoperidium ornamentation is falling off in plates, with stellate spines of slender tips, capillitium less abundant or absent and the absence of basidiospores with long pedicel (Smith, 1974; Reid, 1977). Lycoperdon endotephrum can be separated from Lycoperdon sp.1 by the endoperidium basal hyphae weakly dextrinoid, and absence of capillitium (Demoulin and Dring, 1975). Lycoperdon marginatum is distinct because it has white pyramidal spines, and the hyphae from the basal part of the endoperidium are weakly dextrinoid, as well as mycosclerids from the apical portion (Demoulin, 1972b, 1983).

Lycoperdon sp. 4, Fig. 23i. Basidiome subglobose, pyriform to turbinate, 26–47 mm length × 17–25 mm width.

Subgleba well-developed (occupying > ⅓of basidioma), cellular, cream (N00Y10M00) becoming purplish gray (N70Y40M40). Diaphragm concolor to subgleba, < 0.1 mm thickness. Exoperidium ornamentation with slender spines falling off, 0.5–1 mm long., yellowish (N00Y20M00), mixed with persistent warts < 0.5 mm long., greyish brown

(N30Y40M20). Endoperidium surface pale yellow (N10Y40M00), areolate (< 0.5 mm width); dehiscence by an irregular apical pore or rupture of endoperidium. Gleba powdery, greyish brown (N50Y40M20) to olive brown (N60Y90M30).

Exoperidium basal likely to the apical, composed by sphaerocysts globose, subglobose to elliptic 15.1–25.5 length × 9.3–21.3 µm width, wall 0.5–1.0 µm thickness, without reaction, acyanophilous, hyaline in 5% KOH. Endoperidium basal likely to the apical, composed by hyphae 3–4.2 µm, wall 0.6–1µm thickness, pale yellow without reaction, mycosclereids and inflated terminations absent. Paracapillitium 3.2–7 µm diam., wall 0.7–1.3 µm thickness septate, without reaction, cyanophilous, hyaline in 5% KOH. Capillitium 4.8–7.7 µm diam., wall 1.3–1.7 µm thickness, septate, branched, pores absent, without reaction, acyanophilous, brown in 5% KOH. Basidiospores globose 3.8– 4.8 × 3.8–4.5, slightly verrucose to verrucose [B-C] in LM, aculeate, aculeus with slender tips in SEM (UFRN 164), number of ornamentation 6.3–11.8 for a circumference reduced to 10 µm; pedicels 0.5–1.5 µm long., without reaction, acyanophilous, light brown in 5% KOH.

Habitat and substrate: gregarious, growing on grassland or leaf-litter.

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Specimens examined: BRAZIL, Pernambuco, Igarassu, Reserva Ecológica Charles Darwin, in soil insight of forest, 03 Apr 2003, leg. I.G. Baseia, s.n. (UFRN-Fungos 164); São Paulo, Instituto de Botânica, on soil, 07 Dec 2000, leg. I.G. Baseia (UFRN-Fungos 495 – as Vascellum pratense). Extralimital collections: HUNGARY, Tolna, Szeskszard, field of Palank, n.d., leg. L. Hollós (MAF-Fungi 14141).

Notes: Lycoperdon sp. 4 is recognized by its exoperidium with spines falling off mixed with persistent warts, endoperidium areolate, basidiospores slightly verrucose [B- C], capillitium without pores and paracapillitium present. These specimens were labelled L. pratense; however, the exoperidium ornamentation of stellate spines with slender tips, endoperidium smooth, basidiospores punctate to slightly verrucose [A-B], and the presence of mycosclerids and hyphae with inflated termination allow to distinguish L. pratense from Lycoperdon sp. 4 (Kreisel, 1973; Reid, 1977). On the other hand, L. perlatum can be confused with Lycoperdon sp. 4, because of its basidiospores [C]; however, the conical and pointed spines plus the capillitium with pores are absent in Lycoperdon sp. 4. These two species are morphologically very close; however, molecular analyses help to separate them. More samples of Lycoperdon sp. 4 are needed to clarify the morphological differences with L. perlatum. For now, we prefer keeping them as distinct unnamed species.

Lycoperdon sp. 5, Figs. 22i, 24 a-c. Basidiomata pyriform to turbinate, 42–52 mm length × 18–26 mm width. Subgleba well- developed, cellular, yellowish (N00Y60M20); exoperidium persistent, warty at the base becoming spiny at the apex, dark brown (N60Y99M70). Endoperidium papery, wrinkle, yellowish (N20Y80M50); dehiscence by a plane pore apical. Gleba powdery, brown

(N60Y99M60); pseudocolumella clavate concolor to the gleba.

Exoperidium basal likely to the apical composed sphaerocysts subglobose, clavate to irregular shape 8–23 length × 12–36 µm width, weakly dextrinoid, acyanophilous, hyaline in 5% KOH; mycosclerids and hyphae with inflated terminations absent. Endoperidium basal likely to the apical composed banal hyphae 5–10 µm diam., wall 1– 2 µm thickness, septate, without reaction, acyanophilous, yellowish in 5% KOH. Paracapillitium 2–4 µm diam., wall 0.5–1 µm thickness, septate, without reaction, acyanophilous, hyaline in 5% KOH. Paracapillitium 2–4 µm diam., wall 0.5–1 µm thickness, septate, without reaction, acyanophilous, hyaline in 5% KOH. Basidiospores

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globose to subglobose, 3.5–4.5 length × 4–5 µm width, smooth [A], in LM, without reaction, acyanophilous, yellowish 5% KOH.

Habitat and substrate: gregarious, growing on soil.

Specimens examined: BRAZIL, Pernambuco, Gurjaú, Cabo Santo Agostinho, sobre solo, 25 Mar 2003, leg. I.G. Baseia, s.n. (UFRN-Fungos 152); Rio Grande do Norte, Natal, Parque Estadual Dunas do Natal, Atlantic forest, on leaf-litter, 6º18’18’’S and 35º21’40’’W, 08 Aug 2004, leg. I.G. Baseia and M.M.B. Barbosa, s.n. (UFRN-Fungos 300).

Notes: Lycoperdon sp. 5 is characterized by its persistent exoperidium, warty at the base and spiny at the apex, basidiospores smooth [A], capillitium and paracapillitium present, mycosclerids and hyphae with inflated terminations absent, and sphaerocysts with irregular shape. Lycoperdon pyriforme remembers Lycoperdon sp. 5 because of the irregular shaped sphaerocysts and abundant capillitium and paracapillitium (Pegler et al., 1995; Calonge, 1998); however, Lycoperdon sp. 5 does not have dextrinoid sphaerocysts, and there are mixed of subglobose to clavate cells. Lycoperdon endotephrum and L. pratense are close to Lycoperdon sp. 5, but the exoperidium ornamentation is quite different from latter: L. endotephrum has an exoperidium falling off and the basidiospores slightly verrucose and suboval; and in L. pratense the exoperidium ornamentation is falling off in plates with stellate spines (Demoulin and Dring, 1975). For now, we prefer to keep these specimens as Lycoperdon sp. 5.

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Figure 23. Dry basidiomata of Lycoperdon species. a. L. nigrescens (NY0071657); b. L. ovoidisporum (PACA13771); c. L. perlatum (VDEMOULIN ex ENCB 1533); d. L. pratense (K(M): 200187); e. L. pyriforme (UFRN-Fungos 2271); f. L. umbrinum (VDEMOULIN 4784); g. L. utriforme (K (M): 2002300); h. Lycoperdon sp. 1 (CORD 167); i. Lycoperdon sp. 4 (UFRN-Fungos 495). Bars = 10 mm.

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Figure 24 Comparative between the species of Lycoperdon sp. 5, L. endotephrum and L. pratense. a-c. Lycoperdon sp. 5 (UFRN-Fungos 152); d-f. L. endotephrum (UFRN-Fungos 164); g-i. L. pratense (Basidioma – VDEMOULIN ex AGUCH-ET8; spore – ICN 154485); a, d, g. Dry basidiomata; b, e, h. Sphaerocysts; c, f, i. Basidiospores in scanning eletronic microscopy (SEM). Bars a, g = 10 mm; d = 5 mm; b, e, h = 20 µm; c, f, i = 1 µm.

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4 DISCUSSION

The DNA barcode s are used around the world to identify species (Hebert et al., 2003, 2004; Kress et al., 2005) from various groups of organisms. The web site of Barcode of Life (www.barcoding.si.edu) was created with the main idea of helping scientists in the identification of organisms through short DNA sequences (around 700 bp). For example, with the DNA barcode , cryptic species can be detected helping taxonomists to describe them (Salle et al., 2005; Janzen et al., 2005; Hubert et al., 2008).

The use of DNA barcodehas expanded recently (Druzhinina et al., 2005; O’Brien et al., 2005; Rehner and Buckley, 2005; Seifert et al., 2007). However, no barcode was designed for fungi untill the creation of the Fungal Barcoding Consortium that involves researchers from around the world, working with different groups of fungi to find the best barcode region. In Schoch et al. (2012), the ITS region was proposed as the first barcode to fungi, because around 80% of the species tested could be identified, and separated from others; however, it was clear that in some groups a second marker would be needed. In our study, the ITS barcode was efficient to discriminate 19 Lycoperdon species in Central and South America; although as stated in Chapter 1, in some species groups (e.g. L. excipuliforme, L. lambinonii, L. molle and L. umbrinum), a second marker is needed to discriminate among species (Stielow et al., 2015).

The NJ-tree based on K2P distances showed some difference of topologies, compared with the strict MP consensus tree and the Bayesian tree. These differences, such as in Lycoperdon perlatum terminal branch are due to the differences in the sequence length. From some specimens, the sequences were shorter than 400 bp, (e.g. deMeijer 206, UFRN-Fungos 811, UFRN-Fungos 2335, UFRN-Fungos 2272, TSC70, KW200236 and exU87). Likewise, in the L. pyriforme terminal branch with specimens NY00795902, UFRN-Fungos 2271, UFRN-Fungos 2268, UFRN-Fungos 2269. From these specimens, only part of the ITS region was amplified and sequenced (ITS1 region with primer pair ITS1F/ITS2, or ITS2 region with primer pair ITS3/ITS4b. Min and Hickey (2007) noted this problem and wrote “…as the length of the molecular sequences is reduced, there is a concomitant loss of resolution at the internal nodes of the phylogenetic tree”. Also, the branch length could be related to the geographic location since, in NJ, MP and Bayesian trees, the Central and South America specimens are distributed in at least two subgroups in the main L. perlatum group; however, the intraspecific variability can attract the

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branches from the same geographic locality while excluding the specimens from others localities (Bergsten et al., 2012).

In L. perlatum the intraspecific distance in all specimens was 0–2.28%, while in the subgroup with long branches was 0.6–2.28%. Meier et al. (2006) concluded that when small length sequences are analyzed together with big ones, the accuracy of the analyses decreases. In this group, specimens are similar. The association of morphological plus molecular taxonomy is strongly recommended in barcode approaches (Hebert and Barrett, 2005; Hebert and Gregory, 2005; Nguyen and Seifert, 2008).

In Lycoperdon sp. 5, specimens UFRN-Fungos 152 and UFRN-Fungos 300 show the same morphological features, and the differences in the tree topologies are not due to sequence length, but in some ambiguous bases that made that these sequences to not group together in the NJ tree. In the MP and Bayesian analyses, these specimens appear together within L. endotephrum and L. hyemale sequences. These species are morphologically different. In these cases, some more markers are needed to discriminate both species. As suggested by Stielow et al. (2015), the TEF1α marker could improve the discrimination of Lycoperdon species.

Demoulin (1972b) using only morphological data proposed an artificial phylogeny of Lycoperdon species. The features such as exoperidium falling off, basidiospores smooth/punctate to slightly verrucose [A-B] placed the species L. marginatum, L. calvescens and Vascellum spp. as closest taxa to L. perlatum and L. norvegicum. Although our study is not related to phylogenetic relationship among Lycoperdon species, the NJ tree based on ITS also includes L. perlatum, L. norvegicum and L. marginatum as nearby species.

The groups Lycoperdon atropurpureum, L. molle and L. umbrinum, have occasioned some disagrements among taxonomists. In the taxonomic section of this paper, only L. umbrinum is described and discussed, since no specimens of L. atropurpureum nor of L. molle were studied from South America, although they were studied in Chapter 1. Lycoperdon atropurpureum has been treated as a good species by many mycologists (Massee, 1887; Lloyd, 1905a; b; Coker and Couch, 1928); however Perdeck (1950) synonymized it to L. molle. Demoulin (1972b) did not agree with Perdeck’s work, and treated L. molle and L. atropurpureum as distinct species, but called caution to L. atropurpureum since it can be confused with L. molle and L. decipiens.

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Additionally, Demoulin, (1968b) and Demoulin and Schumacker (1972) have considered as a Lycoperdon-Molle group the species: L. atropurpureum, L. molle and L. umbrinum. Calonge (1998), has considered Lycoperdon decipiens as synonymous of L. atropurpureum, deciding not to follow the concepts of Jeppson and Demoulin (1989) that treated them as separate species. Afterward, the molecular analyses based on ITS and LSU regions, confirmed those morphological studies that treated them as distinct species (Larsson and Jeppson, 2008). Both in Chapter 1, and in this study, L. atropurpureum, L. molle and L. umbrinum are resolved as distinct species.

The analyses of the ITS barcode also help to find misidentifications, such as in the case of the specimen UFRN-Fungos 2275 that was previously labelled under L. ericaeum; however, the position in the molecular trees, and a second revision of the morphological features, spines of exoperidium with slender and convergent tips, and basidiospores [A] (Demoulin 1972b; Pegler et al. 1995), confirmed this specimen as belonging to L. nigrescens.

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5 CONCLUSIONS

- In the present work, the use of DNA barcode based on ITS region to separate species of Lycoperdon showed promise; however, it would be necessary to improve some protocols to obtain the barcode sequences from those precious specimens (e.g. types) for which no barcode sequence has yet been obtained.

- As in other organisms, the number of Lycoperdon species studied with a world wide distribution is reduced (7); although the number of species studied from both Hemispheres is similar (21 studied from Northern Hemisphere vs. 19 from Southern Hemisphere).

- Even though the barcode analysis helps to discriminate among species, and to detect cryptic species, morphological taxonomy continues being essential in discriminate species when the sequences are identical or when it is not possible to obtain them. As mentioned in Hebert and Barrett (2005) the partnership between “barcoders” and alpha taxonomists has to continue in order to advance the understanding of species diversity.

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