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UNIVERSIDADE FEDERAL DO PAMPA

EHIDY ROCIO PEÑA CAÑÓN

FUNGOS ENDOLIQUÊNICOS ASSOCIADOS À Cladonia curta Ahti & Marcelli NO

PAMPA GAUCHO - BRASIL

São Gabriel

2017
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EHIDY ROCIO PEÑA CAÑÓN

FUNGOS ENDOLIQUÊNICOS ASSOCIADOS À Cladonia curta Ahti & Marcelli NO

PAMPA GAUCHO - BRASIL

Dissertação apresentada ao programa de Pós-graduação

Stricto Sensu em Ciências Biológicas da Universidade
Federal do Pampa, como requisito parcial para obtenção
do Título de Mestre em Ciências Biológicas.

Orientador: Dr. Antônio Batista Pereira

Co-Orientador: Dr. Filipe de Carvalho Victoria

Dr. Jair Putzke

São Gabriel

2017
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C419 PENA-CANON, EHIDY ROCIO

FUNGOS ENDOLIQUÊNICOS ASSOCIADOS À Cladonia curta Ahti &
Marcelli NO PAMPA GAUCHO - BRASIL / EHIDY ROCIO PENA CANON.
84 p.

Dissertação (Mestrado)- Universidade Federal do Pampa,

MESTRADO EM CIÊNCIAS BIOLÓGICAS, 2017.
"Orientação: Antonio Batista Pereira".

1. Micologia. 2. Taxonomia. 3. Filogenia. 4. Botânica.

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EHIDY ROCIO PEÑA CAÑÓN

FUNGOS ENDOLIQUÊNICOS ASSOCIADOS À Cladonia curta Ahti & Marcelli NO

PAMPA GAUCHO - BRASIL

Dissertação apresentada ao programa de Pós-graduação

Stricto Sensu em Ciências Biológicas da Universidade
Federal do Pampa, como requisito parcial para obtenção
do Título de Mestre em Ciências Biológicas.

Área de concentração: Ecologia e Sistemática

Dissertação defendida e aprovada em: 26 de Abril de 2017.

Banca examinadora:

________________________________________________

Prof. Dr. Antônio Batista Pereira

Orientador
UNIPAMPA

_________________________________________________

Prof. Dr. Marcelo A. Sulzbacher

UFSM

_________________________________________________

Prof. Dr. Luiz Fernando Wurdig Roesch

UNIPAMPA
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A ti meu anjo, que desde sempre e em qualquer lugar cuidas de mim, mãe tu é
o meu maior exemplo a seguir, te amo!
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AGRADECIMENTOS

Ao meu pai, Alirio Alfonso Peña, pelo amor, carinho e por estar sempre me apoiando. Aos
meus amados e inseparáveis irmãos, que a pesar da distancia sempre estiveram cuidando de
mim e me dando forças constantemente. Ao meu namorado Rafael, pela compreensão,
incentivo, amor, amizade e paciência no decorrer do tempo!
Ao Professor Dr. Antônio Batista Pereira pela orientação, confiança depositada em mim, por
todo o conhecimento transmitido e ajuda para a realização deste trabalho.
Ao professor Dr. Filipe de Carvalho Victoria pela dedicação disponibilizada ao longo deste
trabalho, pela ajuda, pelos ensinamentos, pela paciência e principalmente pela confiança em
mim depositada.
A Professora Dra. Margeli Pereira de Albuquerque pelo conhecimento transmitido, amizade e
importantes contribuições para á realização deste trabalho.
Ao professor Dr. Jair Putzke, pela disponibilidade, paciência e auxílio, que foram
fundamentais para realização deste trabalho.
Aos meus amigos Gefferson Metz e Vanusa Camargo porque sem vocês a minha adaptação
ao Brasil não tivesse sido a mesma.
As queridas Fabiola Lucini, Bruna Laindorf e Karine Janner pelo apoio nas horas tensas e
pela amizade incondicional e para a vida toda.
Aos colegas do Núcleo de Estudos da Vegetação Antártica (NEVA), Juliana, Guilherme,
Brunno, Raylane, Mariele, Eduardo, Maria, Mônica e Rodrigo pela ajuda e carinho;
especialmente ao Brunno Bragança pelo auxilio constante no laboratório.
A todos os professores que contribuíram ao longo das disciplinas cursadas e que desde o
inicio se mostraram sempre dispostos para o meu aprendizado.
À Universidade Federal do Pampa que permitiu a realização deste trabalho.
À Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) e ao
Instituto Nacional de Ciência e Tecnologia Antártico de Pesquisas Ambientais (INCT-APA)
pelo suporte financeiro ao projeto. À Organização dos Estados Americanos e ao grupo
COIMBRA de universidades brasileiras pelo seu programa de bolsas que me brindou a
oportunidade de fazer o Mestrado.
A todos vocês e aqueles que não foram citados, mas que contribuíram de alguma forma,

MUITO OBRIGADA MESMO!

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“La gente piensa que las historias son moldeadas por la gente.

De hecho, es al revés.”

Terry Pratchett
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RESUMO

Os liquens são considerados uma associação simbiótica composta por um micobionte, um ou

mais fotobiontes e inclusive um terceiro simbionte Basidiomycota, representam um dos estilos
de vida de maior sucesso para os fungos e uma das formas mais bem sucedidas da simbiose
na natureza. Liquens exploram todos os ambientes naturais e se adaptam a condições
extremas como as causadas pelas perturbações humanas, tornando-se importantes
bioindicadores da qualidade ambiental. Cladonia curta Ahti & Marcelli é uma espécie de
líquen escamoso da família Cladoniaceae distribuído em três locais ao sudeste do Brasil, em
florestas mesófilas e no Cerrado e coletado no bioma Pampa durante o presente estudo,
tornando-se um registro de nova ocorrência. Por outro lado, existem estudos que demonstram
a presença de fungos que ocorrem dentro dos talos aparentemente saudáveis dos liquens,
conhecidos como endoliquênicos, associados principalmente ao fotobionte. Durante os
últimos 30 anos, fungos endoliquênicos têm sido estudados com base em métodos
dependentes do cultivo in vitro e o uso de ferramentas moleculares empregadas
principalmente na sua detecção e identificação. Assim o objetivo do presente trabalho foi
conhecer a diversidade de fungos endoliquênicos abrigados no talo de Cladonia curta e suas
relações filogenéticas com outros fungos endófitos. As análises filogenéticas baseadas na
amplificação das regiões ITS do rDNA e do gene da β-Tubulina infere as linhagens de fungos
endoliquênicos no gênero Xylaria. Do mesmo modo, as características morfológicas das
colônias e dos estromas anamórficos obtidos em cultura confirmam esta classificação. Nossos
resultados baseados em marcadores moleculares fornecem evidências de que fungos
endoliquênicos isolados de Cladonia curta estão intimamente relacionados com fungos
endófitos e saprofíticos; no entanto, os isolados se mostram mais estreitamente relacionados
com fungos endófitos, sugerindo que a sua associação ao talo não é puramente acidental e
respaldando a relação filogenética entre estes três estilos de vida, como previamente relatado
por outros autores. Estudos adicionais são necessários para demostrar que os isolados Xylaria
spp. e Xylaria berteri sejam conspecíficos. Além disso, nós sugerimos o uso do meio de
cultura MS como uma alternativa nos esforços para conhecer a grande diversidade de fungos
endófitos que habitam tecidos vivos de liquens. Finalmente, a diversidade e a prevalência dos
fungos endoliquênicos continua sendo estudada sendo este o primeiro relato de isolamento e
identificação de fungos endoliquênicos no Brasil.

Palavras-chave: Xylaria, endosimbiontes, Cladoniaceae, liquens.

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ABSTRACT

Lichens considered as a symbiotic association between a mycobiont, one or more photobiont

and even a third Basidiomycota symbiont represent one of the most successful lifestyles for
fungi and one of the most successful forms of symbiosis in nature. Lichens explore all natural
environments and adapt to extreme conditions such as those caused by human disturbances,
becoming important bioindicators of environmental quality. Cladonia curta Ahti & Marcelli
is squamulose lichen of Cladoniaceae family distributed in three sites in southeastern Brazil,
in mesophyll forests and in the Cerrado and collected in the Pampa biome during the present
study, becoming a record of new occurrence. On the other hand, there are studies
demonstrating the presence of fungi that occur within apparently healthy lichen thalli, known
as endolichenic, associated mainly with the photobiont. During the last thirty years,
endolichenic fungi have been studied based on in vitro culture-dependent methods and the use
of molecular tools mainly used in their detection and identification. Thus, the aim of the
present work was to know the diversity of endolichenic fungi harbored in Cladonia curta
thallus and their phylogenetic relationships with other endophytic fungi. Phylogenetic
analyzes based on the amplification of the ITS regions of the rDNA and the β-Tubulin gene
infers the endolichenic fungi lineages in the genus Xylaria. Likewise, the morphological
characteristics of the colonies and the anamorphic stromatas obtained in culture confirm this
classification. Our results based on molecular markers provide evidence that endolichenic
fungi of C. curta are closely related to endophytic and saprophytic fungi; however, they are
closely related to endophytic fungi, suggesting that their association with thallus is not purely
accidental and supporting the phylogenetic relationship between these three lifestyles, as
previously reported by other authors. Additional studies are needed to demonstrate that
isolates of Xylaria spp. and Xylaria berteri are conspecific. In addition, we suggest the use of
the MS culture medium as an alternative in the efforts to know the great diversity of
endophytic fungi that inhabit living lichen tissues. Finally, the diversity and prevalence of
endolichenic fungi continues to be studied with this, the first report of isolation and
identification of endolichenic fungi in Brazil.

Key Word: Xylaria, Endosymbionts, Cladoniaceae, lichens.

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LISTA DE FIGURAS

Figura 1 - Detalhe dos exemplares de Cladonia Curta...........................................................14

Figura 2 - Fotobionte de Cladonia curta obtido em cultura...................................................66

Manuscrito 1:

Figura 1 - Xylaria endoliquênica cresciendo sobre podécios de Cladonia curta....................54

Figura 2 - Relações filogenéticas entre fungos endoliquênicos isolados de Cladonia curta e

sequencias de fungos endófitos de plantas, hepáticas e liquens inferidas por Máxima
Verossimilhança com base no conjunto de dados da região ITS do rDNA..............................51

Figura 3 - Relações filogenéticas entre fungos endoliquênicos isolados de Cladonia curta e

sequencias de fungos endófitos de plantas, hepáticas e liquen sinferidas pela análise de
Inferência Bayesiana do conjunto de dados da região ITS do rDNA.......................................52

Figura 4 - Relações filogenéticas entre fungos endoliquênicos isolados de Cladonia curta,

sequencias de fungos endófitos de outros liquens e sequencias de correspondência mais
próxima com BLASTn inferidas a partir de Inferência Bayesiana baseada no conjunto de
dados do gene β-tubulina..........................................................................................................53

Figura 5 - Isolado Xylaria sp.1................................................................................................54

Figura 6 - Isolado Xylaria sp.2.…….......................................................................................55

Figura 7 - Isolado Xylaria sp.3................................................................................................55

Manuscrito 2:

Figura 1- Podécios de Cladonia curta.....................................................................................63

Material Suplementar

Figura 8 - Relações filogenéticas entre fungos endoliquênicos isolados de Cladonia curta e

sequencias de fungos endófítos com máxima similaridade utilizando BLASTn. Árvore
inferida pela Máxima Verossimilhança com base no conjunto de dados da região ITS do
rDNA.........................................................................................................................................76
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Figura 9 - Relações filogenéticas entre fungos endoliquênicos isolados de Cladonia curta e

fungos endófítos de plantas e liquens obtidos por Arnold et al. (2009), inferidas pela Máxima
Verossimilhança com base na análise da região ITS do rDNA................................................77

Figura 10 - Relações filogenéticas entre fungos endoliquênicos isolados de Cladonia curta e

fungos isolados de espécies de Cladonia e outros liquens (U'Ren et al., 2016), inferidas pela
Máxima Verossimilhança com base no conjunto de dados da região ITS do rDNA................79

Figura 11 - Relações filogenéticas entre fungos endoliquênicos isolados de Cladonia curta e

sequencias de endófitos de hepáticas obtidos por Davis et al. (2003), inferidas pela Máxima
Verossimilhança com base na análise da região ITS do rDNA ……………………...............80

Figura 12 - Relações filogenéticas entre fungos endoliquênicos isolados de Cladonia curta,

sequencias de maoir similaridade (BLASTn) e sequencias de fungos endófitos de liquens
(U'Ren et al., 2016), inferidas pela máxima verossimilhança e baseada no gene β-
tubulina......................................................................................................................................81

Figura 13 - Aparência das colônias do isolado Xylaria sp. 1 em meio MS ............................82

Figura 14 - Aparência das colônias do isolado Xylaria sp. 2 em meio MS ............................83

Figura 15 - Aparência das colônias do isolado Xylaria sp. 3 em meio MS ............................84

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LISTA DE TABELAS

Manuscrito

Tabela 1 Identificação de fungos endoliquênicos isolados de Cladonia curta baseada na

comparação de sequencias da região ITS do rDNA e do gene β- Tubulina com sequências
disponíveis na base de dados Genbank utilizando o algoritmo BLASTn................................ 48

Material Suplementar

Tabela 2 Números de acesso ao Genbank e referências das sequências da região ITS do

rDNA dos táxons incluídos nas análises filogenéticas……………………………................. 71

Tabela 3 Números de acesso ao Genbank e referências das sequências do gene β-Tubulina

dos táxons incluídos nas análises filogenéticas........................................................................ 73

Tabela 4 Modelos de substituição de nucleotídeos dos grupos de sequências estabelecidas

para proporcionar um contexto filogenético para os fungos endoliquênicos isolados de
Cladonia curta com base na região ITS do rDNA …….......................................................... 74
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SUMARIO

1. INTRODUÇÃO ............................................................................................................................ 13
2. OBJETIVOS ................................................................................................................................. 17
2.1 Objetivo geral ................................................................................................................................ 17
2.2 Objetivos específicos..................................................................................................................... 17
3. ARTIGO 1: Caracterização morfológica e molecular de três isolados de Xylaria (Xylariaceae),
endoliquênicos em Cladonia curta Ahti & Marcelli (Cladoniaceae) ................................................... 18
4. ARTIGO 2: Occurrence of the Cladonia curta Ahti & Marcelli (Cladoniaceae, lichenized
Ascomycota) in the Pampa biome ......................................................................................................... 58
5. CONSIDERAÇÕES FINAIS ........................................................................................................ 65
6. PERSPECTIVAS FUTURAS ....................................................................................................... 66
7. REFERÊNCIAS ............................................................................................................................ 67
8. ANEXOS....................................................................................................................................... 72
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1. INTRODUÇÃO

Os liquens são considerados como uma associação simbiótica entre um micobionte (fungo
filamentoso) e um ou mais fotobiontes (alga ou cianobactéria), representando um dos estilos
de vida de maior sucesso para os fungos (Ahmadjian, 1966; Honegger, 1998; De Priestt,
2004). Inclusive, um terceiro simbionte do filo Basidiomycota formando parte do córtex de
Bryoria tortuosa (G. Merr.) Brodo & D. Hawksw., foi recentemente descoberto (Spribille et
al., 2016). Sem dúvida, os liquens representam uma das formas mais bem sucedidas da
simbiose na natureza, encontrando-se distribuídos em todo o globo, explorando não só os
ambientes naturais, mas em muitos casos adaptando-se a condições extremas, incluindo
algumas provocadas por perturbações humanas (Seaward, 2008). Estes organismos são
reconhecidos por mostrar alta sensibilidade a poluentes já que absorvem metais pesados do ar
e da água (Brodo et al., 2001); moléculas prejudiciais são absorvidas com os nutrientes, e os
liquens são frequentemente incapazes metabolicamente de processar ou de isolar estas
rapidamente, causando não somente a diminuição da sua vitalidade, como também a alteração
do balanço simbiótico entre o fotobionte e o micobionte e sintomas externos característicos
(Valencia e Ceballos, 2002; Martins-Mazzitelli et al., 2006). A relação demostrada
anteriormente torna os liquens importantes bioindicadores da qualidade ambiental
(Hawksworth et al., 2005; O-H NG et al., 2005; Martins et al., 2008; Lijteroff et al., 2009).

Existem três formas principais de crescimento ou de configuração geral dos talos

liquênicos sendo estes: foliáceos, fruticosos e crostosos. Porem, um quarto tipo, os liquens
escamosos também podem ser reconhecidos (Fleig et al., 2008). O talo dos liquens foliosos é
achatado e prostrado com diferenças na cor e nas características das superfícies superior e
inferior; geralmente o talo esta dividido em numerosos ramos chamados lóbulos e ligado ao
substrato por rizinas. Os liquens fruticosos consistem em ramos simples ou divididos,
redondos a aplanados, com pouca diferença entre as superfícies superior e inferior,
desprovidos de rizinas. Nos talos crostosos, a parte central do talo é areolada, apresentando
margens inteiras que às vezes desaparecem no substrato tornam-se indistintas. O talo de
liquens escamosos constitui-se de lóbulos pequenos e separados, sem córtex inferior nem
rizinas (Hale, 1979).

Cladonia Curta Ahti & Marcelli é um líquen escamoso de talo primário persistente que
consiste em escamas esverdeadas, macias e muito pequenas com margens ligeiramente
sorediadas; apresenta podécios muito curtos, espessos, sem ramificações e com apotécios
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vermelhos cónicos; cresce sobre troncos em decomposição na borda da floresta e é

considerada uma espécie muito rara (Fig. 1). No Brasil, C. curta é distribuída em três locais
ao sudeste do país, em florestas mesófilas e no Cerrado nos estados de Minas Gerais
(Município Catas Altas) e São Paulo (Município São Bernardo do Campo, 1970), além do
Distrito Federal (Jardim botânico de Brasília, 0,5 km NW da escola Fazendária e Reserva
ecológica de IBGE), entre 800-1200 metros acima do nível do mar (a.n.m.) (Ahti, 2000).
Recentemente a espécie foi coletada no estado Rio Grande do Sul inserido no bioma Pampa
tornando-se um registro de nova ocorrência (Artigo 2).

Figura 1 - Detalhe dos exemplares de Cladonia Curta Ahti & Marcelli. (A) Habito da espécie. (B)
Aparência do talo.

A B
Fonte: fotos Geferson Metz e Rocio Peña.

No Brasil o bioma Pampa é caracterizado por apresentar uma vegetação típica de

pastagem nativa, com arbustos escassos e formações arbóreas (Overbeck et al., 2007). Porém,
a região principal de formação de pastagens esta principalmente distribuída na região sudoeste
do bioma e é chamada “Campanha Gaúcha”. O bioma Pampa esta localizado na região sul do
país, no estado Rio Grande do Sul, dentro da Zona Temperada, apresentando climas
subtropicais e temperados. Embora seja frequentemente interpretado como um ambiente de
pastagem pura é dividido em cinco ambientes principais: costeiro, cerrado, estepe, estepe-
savana e parque do espinilho (Roesch et al., 2009). Representando cerca do 2,07% do
território nacional é um dos menores, ocupando uma área de 176.496 km, e foi oficialmente
reconhecido em 2004 pelo Instituto Brasileiro de Geografia e Estatística (IBGE, 2004). Com
tais características e o tipo de vegetação que apresenta, este bioma possui um grande valor
ecológico, não obstante, encontra-se em estado de vulnerabilidade (Overbeck et al., 2007).
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O ramo da taxonomia de fungos vem demostrando a presença de espécies do filo

Ascomycota, diferentes ao micobionte, ocorrendo em simbiose nos talos liquênicos (Petrini et
al., 1990; Girlanda et al., 1997; Suryanarayanan et al., 2005; Li et al., 2007). Assim, o talo dos
liquens é considerado um dos nichos ecológicos para fungos (Nash, 2008), abrigando três
tipos de fungos ecologicamente diferentes: liquenícolas, incidentes e endoliquênicos (Lutzoni
et al., 2001; Lawrey e Diederich, 2003; Arnold et al., 2009). Os fungos liquenícolas são um
grupo altamente especializado e bem sucedido de organismos que se desenvolvem e
esporulam sobre liquens (Lawrey e Diederich, 2003), entrando numa relação estável que pode
causar perda de cor, formação de galhas e inclusive a morte dos liquens. Estes, bem como
fungos saprófitos ocorrem em talos em decomposição (Petrini et al., 1990). Assim mesmo,
aqueles fungos que habitam nas superfícies dos talos liquênicos são conhecidos como fungos
incidentes (Lutzoni et al., 2001).

Por outro lado, os fungos endoliquênicos são um grupo de endosimbiontes que

ocorrem dentro dos talos de liquens aparentemente saudáveis, preferencialmente associados á
camada do fotobionte (Suryanarayanan et al., 2005; Arnold et al., 2009; U´Ren et al., 2010;
Tripathi e Joshi, 2015; Suryanarayanan et al., 2017). Estes fungos habitam os espaços
intercelulares dos simbiontes liquênicos (Wu et al., 2011), sua colonização é assintomática,
são hiperdiversos e muitos são transmitidos horizontalmente como análogos aos endófitos das
plantas (Arnold et al., 2009; Kannangara et al., 2009; U´Ren et al., 2012). Nos últimos trinta
anos, os fungos endoliquênicos foram conhecidos de todas as espécies de liquens amostradas
para ecossistemas que vão desde desertos quentes até florestas úmidas e tundras árticas
(U´Ren et al., 2010; Tripathi et al., 2014; Padhi e Tayung, 2015; U´Ren et al., 2016;
Suryanarayanan et al., 2017).

Existem diferentes estudos para estimar a diversidade de fungos endoliquênicos.

Suryanarayanan et al., (2005) e Suryanarayanan et al., (2017) recuperaram um total de 24
espécies e 33 morfoespécies a partir de espécies de liquens foliáceos e fruticosos de regiões
tropicais na Índia pertencentes a cinco famílias (Caliciaceae, Collemataceae, Lobariaceae,
Parmeliaceae, Physiaceae, Ramalinaceae e Roccellaceae). Em contraste, Tripathi e Joshi
(2015) e Tripathi et al. (2014) isolaram 10 espécies a partir de liquens coletados em diferentes
regiões temperadas de Kumaun Himalaya. Na China, foram investigados um total de 32
fungos endoliquênicos isolados desde sete espécies de liquens pertencentes às famílias
Cladoniaceae, Parmeliaceae, Ramalinaceae, Teloschistaceae e Verrucariaceae (Li et al,
 16

2007). No Sri Lanka, 29 morfoespécies foram isoladas de Parmotrema sp., Usnea sp. e
Pseudocyphellaria sp. No entanto, este número é pequeno quando comparado com a
diversidade de fungos endoliquênicos relatados nos Estados Unidos (Arnold et al., 2009;
U´Ren et al., 2010; U´Ren et al., 2012; U´Ren et al., 2016). Da mesma forma, foram feitas
pesquisas sobre a produção de metabólitos (Wu et al., 2011) e a avaliação dos potenciais
antimicrobianos das sustâncias produzidas pelos fungos endoliquênicos (Padhi e Tayung,
2015). Recentemente foi apresentada uma revisão das relações filogenéticas moleculares dos
fungos endófitos e endoliquênicos da família Xylariaceae, mostrando que a grande maioria
dos isolados foi incluída dentro das subfamílias Hypoxyloideae e Xylarioideae (U´Ren et al.,
2016). Após revisão bibliográfica, foi evidenciado que o presente trabalho é o primeiro estudo
na América do Sul sobre a diversidade de fungos endoliquênicos abrigados nos talos de
liquens.

O gênero Xylaria Hill ex Schrank é um dos mais diversos dentro da família

Xylariaceae com cerca de 600 espécies a maioria tropical, tem mostrado como um grupo
parafilético (Hsieh et al., 2010; Rogers e Ju, 2012); junto a outros 85 gêneros da família é
classificado dentro da classe Sordariomycetes (Stadler et al., 2013). Os membros deste gênero
são considerados saprófitos importantes, por vezes desde ligeiramente até fortes parasitas em
madeira de árvores, encontrados em folhas e raramente observados em frutos (Lee et al.,
2000; Rogers 2000; Rogers e Ju, 2012). Xylaria são visíveis durante a esporulação sexual,
formando estromas macroscópicos relativamente grandes (Davis e Shaw, 2008). Apesar da
sua condição saprófita, espécies de Xylaria são típicos fungos endófitos isolados a partir de
fragmentos de plantas e liquens aparentemente saudáveis (Guo et al., 2000; Shibuya et al.,
2003; Arnold et al., 2009; Thomas et al. 2016; U´Ren et al., 2016) e predominantes em
estudos de diversidade tanto de fungos endoliquênicos quanto de endófitos (Bayman et al.,
1998; Vaz et al., 2014; Suryanarayanan et al., 2017).

Fungos endófitos em plantas e liquens têm sido estudados e descritos com base em
métodos dependentes do cultivo in vitro. No entanto, muitas Xylariaceae permanecem
frequentemente estéreis em cultura ou reproduzem-se só assexuadamente sobre meios de
cultura padrão (Stadler et al., 2013). Assim, na ausência de características do teleomorfo, as
culturas podem ser classificadas, em alguns casos, com base em características coloniais e
anamórficas observadas, tais como taxas de crescimento, cor, morfologia superficial das
colônias, bem como na ramificação de conidióforos e a natureza da proliferação celular
 17

conidiogênica (Petrini e Petrini, 1985; Callan e Rogers, 1993; Stadler et al., 2013). Porém,
várias ferramentas moleculares foram adicionadas a esta metodologia utilizada para
caracteriza-los. Regiões ITS do rDNA e o gene da β-Tubulina são atualmente empregados na
detecção e identificação de fungos endoliquênicos para analisar posições ou relações
filogenéticas ao nível de espécie ou interespécies (Lee et al., 2000; Arnold et al., 2009; Hsieh
et al., 2010; U´Ren et al., 2016). Tendo em conta a vasta diversidade ainda pouco explorada
dos fungos endoliquênicos, que ainda há muito a ser entendido e explicado sobre as suas
características, particularidades e as condições da sua associação com os simbiontes dentro
dos talos liquênicos, um esforço imenso e estudos adicionais ainda são necessários para
aumentar seus conhecimentos e compreender sua relação filogenética com outros fungos
endófitos de plantas e líquenes.

2. OBJETIVOS

2.1 Objetivo geral

Conhecer a diversidade de fungos endoliquênicos abrigados no talo de Cladonia curta Ahti &
Marcelli, espécie de líquen registrada no bioma Pampa, RS – Brasil, e suas relações
filogenéticas com fungos endófitos isolados de plantas e liquens.

2.2 Objetivos específicos

 Caracterizar morfologicamente as linhagens de fungos recuperados em cultura desde

podécios do líquen Cladonia curta.
 Identificar o posicionamento taxonômico dos fungos endoliquênicos isolados.
 Inferir relações filogenéticas para os isolados obtidos in vitro a partir de Cladonia
curta usando regiões ITS do rDNA e do gene da β-Tubulina.
 18

3. ARTIGO 1: Caracterização morfológica e molecular de três isolados de Xylaria

(Xylariaceae), endoliquênicos em Cladonia curta Ahti & Marcelli (Cladoniaceae)

(Artigo a ser submetido para a revista Fungal Biology, conforme normas da revista).
 19

Morphological and molecular characterization of three isolates of Xylaria (Xylariaceae),

endolichenic in Cladonia curta Ahti & Marcelli (Cladoniaceae)

Ehidy Rocio Peña Cañón1a; Filipe de Carvalho Victoria1b, 2*; Jair Putzke1c; Margeli Pereira de

Albuquerque2a; Antônio Batista Pereira1d.

1
Antarctic Plants Studies Core - NEVA, Federal University of Pampa (UNIPAMPA), Av. Antônio

Trilha 1847, São Gabriel, RS. Brazil.

2
Antarctic Plants Communities in ice-free areas -INCT/APA,
*
Author for correspondence: Filipe de Carvalho Victoria. filipevictoria@[Link]. Tel.: (55)

55 84157963. São Gabriel, RS. Brazil. CEP: 973000000. (55) 55-84157963. (55) 3232-6075.
1a
erociopc2609@[Link]; 1b filipevictoria@[Link];
1c
jrputzkebr@[Link]; 2a margeli_albuquerque@[Link]; 1d antoniopereira@[Link].

Abstract

The biology of endophytes is a branch of science that is contributing to understand the

diversity and ecology of fungi that live inside plants, fungi and lichen. Considering that this

diversity of endolichenic fungi is little explored and there is still much more to be understood

and explained about its phylogenetic relationship with other lifestyles (endophyte and

saprotrophism), this paper presents data on axenic growing and phylogenetic relationships of

three endolichenic fungi, isolated in MS medium. Cladonia curta Ahti & Marcelli a species of

lichen described in Brazil is distributed in three sites in the southeast of the country, in

mesophilous forests and Cerrado. The initial growth of hyphae of Xylaria spp. on C. curta

podetia started four days after inoculation and continued up to the next 13 days until the

hyphae completely covered the podetia. Stromata formation and differentiation were

observed, occurring approximately one year after of isolation and consecutive subculture of
 20

lineages. The phylogenetic analyses based on ITS rDNA and β-tubulin infers lineages of

endolichenic fungi in the genus Xylaria, even as the morphological characteristics of colonies

and anamorphous confirm this classification. Our preliminary result based in molecular

marker provides evidences that this endolichenic fungi are closely related to both endophytic

and saprophytic fungi, nevertheless, they are more closely related to endophytic fungi

suggesting that associations are not purely incidental. Further studies are needed for accept or

reject the hypothesis that endolichenic fungi isolated Xylaria spp. and X. berteri are

conspecific, specially phylogenetic analyses using robust multi-locus dataset.

Key Word: Anamorph, Fungi, Phylogeny, Lichen, Brazil, Xylaria berteri, Xylaroideae.

1. Introduction

The biology of endophytes is a recent science that is starting to known the diversity and

ecology of endolichenic fungi, a group of endosymbionts which occur inside of symptomless

lichen thalli preferentially associated with the green algal photobiont (Arnold et al., 2009;

Suryanarayanan et al., 2005; Suryanarayanan et al., 2017; Tripathi and Joshi, 2015; U´Ren et

al., 2010). They inhabit the intercellular spaces of the hosts (Wu et al., 2011), their

colonization is asymptomatic, are hyperdiverse and many are transmitted horizontally as

analogous to the plant endophytes (Arnold et al., 2000; Arnold et al., 2009; Kannangara et al.,

2009; U´Ren et al., 2012). In the last thirty years, endolichenic fungi are known from every

lichen species sampled to date in ecosystems ranging from hot deserts to moist forests and

arctic tundra (Arnold et al., 2009; Li et al., 2007; Padhi and Tayung, 2015; Suryanarayanan et

al., 2005; Suryanarayanan et al., 2017; Tripathi et al., 2014; U´Ren et al., 2010; U´Ren et al.,

2012; U´Ren et al., 2016; Wang et al., 2016).

The efforts have been made to compare the different fungi that inhabit the tissues of

lichens and their host tissues (Suryanarayanan et al., 2005; U´Ren et al., 2010) establishing
 21

effective methods for surface sterilization in order to estimate their diversity (Li et al., 2007;

Suryanarayanan et al., 2017; Tripathi et al., 2014). In the same way, some studies try to

explain both the evolution of endophism and the diversification of the Ascomycota

endolichenic (Arnold et al., 2009) and evaluate the biotic, biogeographic and abiotic factors

that structure their communities (U´Ren et al., 2012;Wang et al., 2016). Likewise, research

has been carried out on the metabolites produced (Wu et al., 2011) and the evaluation of the

antimicrobial potentials of endolichenic fungi (Padhi and Tayung, 2015). Recently, a review

of the molecular phylogenetic relationships of the endophytic and endolichenic fungi of the

Xylariaceae family was presented, showing that the great majority of the isolates were

included within the Hypoxyloideae and Xylarioideae subfamilies (U'Ren et al., 2016).

To date, studies on endolichenic fungi in India from foliose and fruticose lichen species

belonging to five families (Caliciaceae, Collemataceae, Lobariaceae, Parmeliaceae,

Physiaceae, Ramalinaceae and Roccellaceae) have recovered a total of 24 species

(Suryanarayanan et al. , 2005) and 33 morphospecies (Suryanarayanan et al., 2017) from

tropical regions. In contrast, Tripathi and Joshi (2015) and Tripathi et al. (2014) worked on

endolichenic fungi of temperate regions of Kumaun Himalaya and isolated a total of 10

species that are reported as true endolichenic fungi. In China endophytic fungi of seven lichen

species belonging to five families (Cladoniaceae, Parmeliaceae, Ramalinaceae,

Teloschistaceae and Verrucariaceae) were investigated, with a total of 32 taxa (Li et al,

2007). In Sri Lanka, 29 endolichenic fungal strains were isolated from the lichens

Parmotrema sp., Usnea sp. and Pseudocyphellaria sp. However, this number is small when

compared with those of some more studied in North American states (Arnold et al., 2009;

Chagnon et al., 2016; U‟Ren et al. 2010; U‟Ren et al. 2012; U‟Ren et al. 2016). After a

bibliographic review, it was evidenced that the present work is the first study in South

America on the diversity of endolichenic fungi harbored in lichen thallus.

 22

The genus Xylaria Hill ex Schrank is one of the most diverse within the family

Xylariaceae, with about 600 species, mostly tropical has been shown to be paraphyletic

(Hsieh et al., 2010; Rogers and Ju, 2012) and along with 85 other genera of family is

classified within the class Sordariomycetes (Stadler et al., 2013). Members of this genus are

considered important saprophytes, sometimes from slightly to strongly parasites in wood of

trees and even found on leaves and rarely on fruits (Lee et al., 2000; Rogers 2000; Rogers and

Ju, 2012). Xylaria are visible during sexual sporulation forming relatively large, macroscopic

stromata (Davis and Shaw, 2008). Despite their condition saprophytic, Xylaria species are an

example of typical endophytic fungi that have been isolated from fragments of apparently

healthy plants and lichens (Arnold et al., 2009; Guo et al., 2000; Shibuya et al., 2003; Thomas

et al., 2016; U´Ren et al., 2016) and predominant in studies of diversity of both endolichenic

and endophytic fungi (Bayman et al., 1998; Suryanarayanan et al., 2017; Vaz et al., 2014).

Xylariaceae are often remaining sterile in culture or reproduce only asexually (Stadler et

al., 2013), therefore, in the absence of teleomorph features, cultures can be classified, in the

some cases, based on colonial and anamorphic features observed, such as growth rates, color,

colony surface morphology, as well as conidiophore branching and the nature of

conidiogenous cell proliferation (Callan and Rogers, 1993; Petrini and Petrini, 1985; Petrini

and Petrini, 1990; Stadler et al., 2013). Additionally, molecular techniques have been

employed in the detection and identification of endolichenic fungi using principally ITS

regions and β-tubulin gene to analyze phylogenetic positions or relationships at a species or

interspecies level (Arnold et al., 2009; Hsieh et al., 2010; Lee et al., 2000; U´Ren et al., 2016).

Cladonia curta Ahti & Marcelli is squamulose lichen that grows on decomposing logs

at the edge of the forest and is considered a very rare or ignored species. In Brazil, this species

is distributed in three sites in the southeast of the country, in mesophilous forests and Cerrado
 23

in the states of Minas Gerais and São Paulo and in the Federal District of Brasília, between

800-1200 msl. (Ahti, 2000). Considering the vast diversity still little explored of endolichenic

fungi, that there is still much to be understood and explained about their features, particularity

and conditions of their association with symbiont within lichen thallus, an immense amount of

effort and additional studies is still required to increase their knowledge and understand the

phylogenetic relationship with other endophytic fungi isolated of plant and lichens. Thus, the

aim of the present work was to know the diversity of endolichenic fungi harbored in Cladonia

curta thallus, collected in the south of Brazil and their phylogenetic relationships with other

endophytic fungi.

2. Materials and Methods

2.1. Collection

The samples of lichen Cladonia curta were identified and collected in the municipality

of São Gabriel, state of Rio Grande do Sul, Brazil at Federal University of Pampa

(UNIPAMPA) campus (30° 20' 06.3" S, 054° 21' 46.5" W) at 124 m asl., on dead wood of an

Eucalyptus camaldulensis Dehnh. (Myrtaceae) trunck. The local of collection lies within the

Pampa biome located in the southern region of Brazil. The lichen sample was deposited in the

Bruno Edgar Irgang herbarium (HBEI- Federal University of Pampa) under the voucher

number HBEI 023.

2.2. Fungal isolation and culturing

The endolichenic fungi were isolated from podetia of Cladonia curta apparently healthy

without symptoms of colonization of other fungi, one day after collected the lichen. The

isolation of endolichenic was achieved following the protocol of the spore-shot method for

mycobionts (McDonald et al., 2013) in which under sterile conditions, the podetia of lichen
 24

were removed, rinsed, disinfested and affixed to the lid of an inverted petri dish with sterile

solid Vaseline (Synth®). Surface sterilization of the fragments was performed by consecutive

immersion in 25% bleach solution for 3 min, ethanol (70%) and rinsed three times for 2 min,

with ultra-pure water and subsequently dried on sterile filter paper, followed a modified

version of the procedure from Arnold et al. (2000). We use the medium Murashige and Skoog

(MS) (Murashige and Skoog, 1962) modified, for the growth and maintained of endolichenic

fungi. The MS medium modified (pH 5.5) contains: 1.650 g. L-1 NH4NO3; 1.9 g. L-1 KNO3;

370 mg. L-1 MgSO4 x H2O; 16.9 mg. L-1 MnSO4 x H2O; 8.6 mg. L-1 ZnSO4 x H2O; 0.025 mg.

L-1 de CuSO4 x H2O; 333 mg. L-1 de CaCl2; 6.2 mg. L-1 H3BO3; 170 mg. L-1 KH2PO4; 0.83

mg. L-1 KI; 0.25 mg. L-1NaMoO4 x 2H2O; 0.025 mg. L-1 CoCl2 x 6H2O; 37.25 mg. L-1

Na2EDTA; 27.85 mg. L-1 FeSO4 x 7H2O; 1 mg. L-1 thiamine; 0.5 mg. L-1de Pyridoxine; 0.5

mg. L-1 nicotinic acid; 2 mg. L-1 glycine; 30 g. L-1 sucrose; 9 g. L-1 Agar and without myo-

inositol content. After 18 days the hyphae from the growth on the podetia were transferred to

new MS plates. The plates were maintained in culture chamber at 20 ± 1°C without light

incidence. A month later the endolichenic fungi isolated were subculture in MS and

maintained at 20 ± 1°C and photoperiod of 16h light and 8h dark until the stromata formation.

For stimulate the abundant formation of stromata in the mycelium, endolichenic fungi were

sub-cultured in 2% malt extract agar (2% MEA) (Persǒh et al., 2009). The cultures were

preserved in sterile water by Castellani method (Hartung et al., 1989) and deposited at HBEI

under the vouchers numbers 001, 002 and 003, together with dry stromata of isolated Xylaria

sp. 1 and Xylaria sp. 2, vouchers HBEI 021 and HBEI 022 respectively.

2.3. Morphological characterization

We described texture, topography, margin, exudates, coloration verse and reverse,

amount of aerial mycelium and stromata production from the colonies formed by the
 25

endolichenic fungi isolated. Stromata size, shape, surface and color were observed and

measurements were based on available stromata in culture. To describe the color

nomenclature was used the dictionary Maerz and Paul (1930). For measuring and

characterizing microscopic reproductive structures, cross-sections were made by hand of the

stromata and the material was mounted in water, Melzer iodine reagent and 5% KOH. The

mean spore width and height was measurement taken from 20 randomly selected spores in

water mounts. Microscopic features were measured (increasing 100x) and examined by

differential interface contrast (DIC) and bright-field microscopy with optical microscope Axio

Imager A2 (ZEISS®) equipped with Axiocam ERc5s (ZEISS®) and software ZEN 2 v 4.0.

2.4. DNA Extraction, Amplification, PCR Purification and Sequencing

Total DNA from mycelia were extracted using DNeasy Plant Mini Kit (Quiagen®)

following the manufacturer's instructions. PCR amplification of the nuclear ribosomal internal

transcribed spacers and the region 5.8S (ITS rDNA), and β-tubulin gene were performed

using primers ITS1_F with ITS4_R (White et al., 1990) and Bt2b_F with Bt2a_R (Glass and

Donaldson, 1995). The following cycle parameters were used for ITS1 and ITS4: initial

denaturation at 94°C for 2 min, 30 cycles of 45 s at 94°C, 30 s at 55°C and 35 s at 72°C and a

final elongation for 7 min at 72°C. For the Bt2b and Bt2a the amplification conditions were: 2

min at 94°C (denaturing), 29 cycles of 10 s at 94°C, 10 s at 58°C (annealing), 20 s at 72°C

and 5 min at 72°C (extension). For the PCR reaction were used 4 μl DNA sample, 0.75 μl

Milli-Q® H2O, 1.25 μl solution of each primer and 1.2 μl of GoTaq® PCR Master Mix (Taq

DNA polymerase, dNTPs and MgCl2). PCR products were purified with the Wizard PCR

Preps DNA Purification System (Promega®) kit according to the manufacturer's protocol and

quantified using NanoVue™ Plus spectrophotometer. All products were sequenced in ABI-

Prism 3500 Genetic Analyzer (Applied Biosystems). The sequences obtained were manually
 26

adjusted using the software Bioedit v. 7.2.5 (Hall, 1999) and a consensus sequence was

obtained using SeqMan package of Lasergene software v [Link] (DNASTAR/Inc.). New

sequences were deposited in Genbank under the accession numbers KY962975, KY962976,

KY962977.

2.5. Phylogenetic analyses

ITS regions of rDNA and β-tubulin consensus sequences were compared to the

Genbank database ([Link]) using the BLASTn to estimate the taxonomic

placement of each isolate. The closest matches sequences with query cover and maximum

identity ≥ 98% and ≥ 97% for sequences ITS and β-tubulin and e-value ≥ 0, were included in

the phylogenetic analysis (Nilsson et al., 2008). Available sequences (November 2016) of

endolichenic and endophytic fungi for ITS regions of rDNA and sequences of endolichenic

fungi provided by U´Rent et al. (2016) for β-tubulin were downloaded from NCBI. In order to

provide a phylogenetic context based in ITS rDNA for the isolated fungi from C. curta, four

clusters of sequences were established: a) BLASTn search; b) endophytic and endolichenic

fungi obtained by Arnold et al. (2009); c) isolated fungi from the interior of Cladonia and

other lichen species (U'Ren et al., 2016) and d) liverwort endophytes fungi cultured by David

et al. (2003). For β-Tubulin, one sequence group was formed including the sequences

resulting from the use of the BLASTn algorithm and sequences of endolichenic isolated by

U'Ren et al. (2016). The Genbank accession numbers for sequences of taxa included in

phylogenetic analyses in this study are showed in Table 2 and Table 3 of supplementary

material. For each data group, the pairs and multiples alignment were performed by using

CulstalW as implemented in MEGA v. 6.06 (Tamura et al., 2013). Models for nucleotide

substitution were estimated and these with the lowest BIC scores (Bayesian Information

Criterion) were considered to describe the best substitution pattern (Supplementary material –
 27

Table 4). Phylogenetic trees for each dataset of sequences were constructed with MEGA

using maximum likelihood with Neighbor-Joining method and bootstrap values calculated

from 1000 replicate (using all sites).

From each phylogenetic tree obtainded, based in ITS regions, the sequences of

endophytes and endolichenic fungi most closely related to the species isolated during this

work were selected (Supplementary material – Fig 8 -11) and the phylogenetic relationships

between these were analyzed using Maximum Likelihood (ML) and Bayesian Inference (BI).

Maximum likelihood as implemented in MEGA was used with bootstrap values calculated

from 1000 replicate, Neighbor-Joining method and the Kimura 2-parameter substitution

model. Bayesian analyses were conducted on the aligned data set using BEAST v. 1.8.3

(Drummond et al., 2012) using the Tamura-Nei model of equal base frequencies and gamma

distribution with five categories. In order to identify the posterior probability tree set

10,000,000 million Markov chains Monte Carlo (MCMC) were run and trees were sampled

every 1000 generations. Tracer v1.6 (Rambaut et al., 2014) was used to evaluate the effective

population size (ESS >100) and TreeAnnotator v1.8.3 (of package BEAST) for condensed the

information of the trees sampled by MCMC. The phylogenies produced from ML and BI

based in ITS region of rDNA were rooted with sequences of Ophiostoma valdivianum (Butin)

Rulamort (NR145317), Ophiostoma eucalyptigena Barber & Crous (NR137979), Ophiostoma

tetropii Math.-Käärik (NR145271) and Peziza fascicularis (LT158418). The phylogenetic

trees of β-tubulin were constructed as describe above for analyses of ML and BI. Maximum

likelihood were estimated using Tamura-Nei substitution model and Tamura-Nei model of

equal base frequencies and gamma distribution was used for Bayesian analyses. Ophiostoma

tetropii (AY305701), Ophiostoma grandicarpum (Kowalski & Butin) Rulamort (KX590762)

and Ophiostoma microsporum Arx (KX590764) were used to rooted. Alignments and trees

are deposited in TreeBase.

 28

3. Results

3.1. Endolichenic fungi isolated

We characterize both for molecular and morphological features three endolichenic fungi

isolated from Cladonia curta. The initial growth of hyphae of isolated Xylaria spp. on C.

curta podetia started four days after inoculation and continued for the next 13 days until the

hyphae completely covered the podetia (Fig 1A-B). The podetia transferred to petri dishes

with fresh culture medium continued the growth and developed mycelium until colony

formation (Fig 1C). The appearance of colonies of isolates Xylaria spp. on MS in photoperiod

and in darkness are present in Material supplementary (Fig 13 – 15). The stromata formation

and differentiation occurred ten months (isolates MS1 and LB1) and one year (isolated MS2)

after of isolation and consecutive subculture of lineages.

3.2. Phylogenetic analyses

The sequences obtained of axenic cultures of the endolichenic fungi from Cladonia

curta resulted in BLASTn hits close for xylariaceous fungi. The isolates were considered as

belonging to the Xylaria genus after a comparison of their nucleotide sequences that revealed

an identity above 98% and 97% for regions ITS of rDNA and β-tubulin, respectively. The

compatibility of species of endolichenic fungi mainly with sequences deposited as Xylaria

berteri and reported as endophytic fungi of angiosperm (Douanla-Meli and Langer, 2012;

Gazis and Chaverri, 2010; Hsieh et al., 2005; Hsieh et al., 2010; De Souza Leite et al., 2013;

Rojas-Jiménez et al., 2016; Shibuya et al., 2003; Smith et al., 2008; Vaz et al., 2014; Vega et

al., 2010), liverworts (Davis and Shaw, 2008) and ferns (Fu et al., 2013) (Table 1).
 29

The total number of sequences of ITS regions of rDNA that were compared with

sequences of endolichenic fungi of Cladonia curta was 52. The maximum likelihood (Fig 2)

and Bayesian Inference (Fig 3) trees based in this dataset were congruent and differ slightly in

their topology. Based on our results four distinct clade are supported (A, B, C and D). The

clade A be comprised of a total of 14 endolichenic fungi sequences including Xylaria

arbuscula Sacc., Xylaria venustula Sacc., 7 species of Xylaria isolated from six species of

lichens (Cladonia evansii, Usnea subscabrosa, Cladonia subradiata, Flavoparmelia caperata

and Cladonia didyma) (U´Rent et al., 2016) and four species sequences of endolichenic fungi

isolated from Peltigera neopolydactyla s.l. lichen by Arnold et al. (2009). The formation of

this clade is few supported using both the maximum likelihood and the Bayesian analyses

with bootstrap values of 1% and posterior probability of 0.25. An endophytic fungus of

Faramea occidentalis (Rubiaceae) (KT289626) was included in this clade using BI and

inferred as sister group of two species sequences reported as endolichenic fungi (PP = 0.21).

The comparison of the phylogenetic trees obtained from analyzes of maximum

likelihood and Bayesian Inference, showed that the clade B formation presents slight

differences in composition and in grouping of the species. Within this clade were grouped

new sequences from this study and sequences of fungi principally reported as endophytic and

some endolichenic. Of these taxa, 9 are identified as Xylaria berteri in Genbank and were

isolated from angiosperms in different studies: 6 sequences were obtained of fungi isolated

from soybean cultivars Glycine max (Fabaceae) by De Souza Leite et al. (2013), two from

dicotyledons by Hsieh et al. (2010) and one from Myrceugenia ovata var. nanophylla

(Myrtaceae) by Vaz et al. (2014). Among the remaining species that comprise the clade B

were included three species of endophytic fungi from liverworts that correspond to two

species of Xylaria isolated from Bazzania sp. (Lepidoziaceae) and Trichocolea tomentella

(Ehrhart) Dumortier (Trichocoleaceae) and an unidentified isolate from Plagiochila

 30

(Plagiochilaceae) (EU686041), as well as, one sequence of endophytic Xylaria in Cinchona

pubescens (Rubiaceae), two endophytic fungi of the Xylariaceae family, grown from the

interior of healthy leaves of Coffea arabica (Rubiaceae), two isolated endophytic fungi from

F. occidentalis and Arundinaria gigantea (Poaceae) respectively and three endolichenic fungi

isolated from Lecanora oreinoides (JQ761659), Usnea mutabilis (JQ760593) and Umbilicaria

mammulata (KT289568) lichens. This clade showed low ML support (1%) and was

moderately supported on BI (PP= 0.49). The Bayesian analysis resulted in a modification of

the clade B showed by ML, excluding F. occidentalis and an endolichenic of P.

neopolydactyla s.l. (KT289550) were grouped with hepatic endophytic species that comprise

the clade C.

The isolated Xylaria sp.1 (LB1) was inferred together with an endophytic fungus

identified as Xylaria berteri cultivated from soybean leaves of the "Conquista" cultivar in a

clade poorly supported by maximum likelihood (7%). In the same way, the posterior

probability that Xylaria sp.1 is closest to soybean endophytic was low (0.08). The culturing

Xylaria sp.2 (MS1) was showed forming a clade with one endophytic fungus of F.

occidentalis (KT289626) and a endolichenic fungi cultured from P. neopolydactyla s.l.

(KT289550) (ML = 3%). Using BI, this isolated shows close relationship with seven

sequences of endophytic fungi from angiosperms identified as X. berteri. The isolated Xylaria

sp.3 (MS2) was inferred as sister of the others lineages (PP= 0.38). Sequence similarities by

ML between isolated Xylaria sp.3 (MS2) and the taxon identified as X. berteri (GU324750)

isolated from bark of a dicotyledonous was 6%. Similarly, this isolate was inferred within the

same clade with the endophytic X. berteri of M. ovata var. nanophylla, supported with

posterior probability value of 0.07. Lower sequences similarities of ML analysis between

Xylaria sp.3 (MS2) and the taxon identified as X. berteri (GU324750) isolated from bark of a

dicotyledonous was found, up to 6% from both homologues. Likewise, this isolate was
 31

inferred within the same clade with the endophytic X. berteri of M. ovata var. nanophylla,

supported with posterior probability value of 0.07.

Seven species of Xylaria isolated by Davis et al. (2003) from liverworts rhizoids of

Bazzania species and three species of endolichenic fungi of P. neopolydactyla s.l. comprises

the clade C. This clade has low support with bootstrap value of 28% and a posterior

probability of 0.18. The clade D it grouped together four endolichenic fungi isolated from P.

neopolydactyla s.l. and a Xylaria isolated from Bazzania sp. with weak bootstrap support

(18%) while that by posterior probability was strongly supported (0.99), however, no include

the sequence of Xylaria sp. (AY315388) in this clade.

A total of 43 sequences from the β-tubulin gene were compared to the sequences

obtained in this study (LB1 and MS1). Of these, two sequences were the result of the

BLASTn search, one sequence of Xylaria berteri cultivated by Hsieh et al. (2010) and the rest

of the sequences correspond to species of endolichenic fungi isolated and cultivated by U'Ren

et al. (2016). The fungal isolate MS2 was not included in the phylogenetic analysis because it

sequence presented a size smaller, low quality and e-value > 0 in the BLASTn query that

generated conflict during the alignment with other sequences of endophytic and endolichenic

fungi. Taking into account the results generated by the ITS regions of rDNA phylogeny, we

consider pertinent to include the sequence of X. berteri (GQ502698) grown in culture from

ascospores. The trees generated by the ML and BI analyses based on β-tubulin dataset were

highly similar in topology. Since these trees are congruent only the tree obtained by Bayesian

inference (Fig 4) is present and described in this document. The tree generated by ML is

provided in the Fig 12 of supplementary material.

The phylogenetic tree inferred showed clearly the formation of two large clades and the

sequence of endolichenic fungus from Pseudevernia consocians outside the groups. One of
 32

them, the clade with high support (PP= 0.83) included the species of endolichenic fungi

isolated from Cladonia curta (LB1 and MS1), three sequences of X. berteri and sequences of

endophytic fungi isolated mainly from lichen species of the genus Cladonia (C. evansii, C.

didyma, C. subradiata, C. subtenius) and other lichens (Usnea subscrabosa and

Flavoparmelia caperata). The species remaining of endolichenic fungi formed a branch with

low posterior probability support (0.35). The sequences of fungal isolates (MS1 and LB1)

were close related to three sequence of X. berteri, two saprophytic and one endophytic (PP=

1) and sequences of Xylaria cubensis (Mont.) Fr. and Xylaria cf. heliscus, endolichenic fungi

in Lecanora oreinoides and Usnea mutabilis respectively (PP= 1). This clade was strong

supported with posterior probability value of 1.

4. Taxonomy

Isolated: Xylaria sp. 1 (LB1 – Fig 5)

Culture: colonies on MS medium cover a 9 cm plate in 18 days. In photoperiod of 16 h

light and 8h dark, at first white, cottony, zoned in the center and irregularly radial toward the

edge; abundant aerial mycelium forming prominent protrusions with flattened topography and

finally forming irregular beige dark spots (M&P K1–Plate 9). Reverse mostly remaining

white for up to four weeks, finally turning yellowish white (M&P G1–Plate 11); radial with

black scores up to one millimeter in diameter forming concentric zones. In the center of the

colonies was observed a primordium of stroma, pyramidal-acute, black with cylindrical stipe

up to 0.8 mm of height with irregular flattened branches. In dark, the colonies were uniformly

white, cottony, zoned in the center, radial towards the margin with elevated topography.

Reverse radial, uniformly yellowish white. Clear droplets were exudate on surface of young

colonies and (M&P B9–Plate 13) droplets were found in mature colonies. On 2% MEA the

primordia grew and the mycelium formed more stromata.

 33

Stromata: 10 stromata solitaries or gregarious were formed; stipe initially cylindrical to

clavate later bifurcate to cerebriform with several small digitate projections, 1.5 – 3 mm

(base) and 2 – 6 mm (apex) wide × 11 – 16 mm high, verruculose to dendroid surface; apex

white and pale orange (M&P B9–Plate 4) at base during the formation of conidia to finally

gray ocher (M&P E1–Plate 8) (Fig 5A-C). Hyphae with thick wall, dark brown, regularly

septated with branches forming 90° angles, without reaction to the Melzer reagent (Fig 5E).

The central axis of the stroma presents thin hyphae immersed in a gelatinous matrix.

Conidiospore: ovoid to clavate, 1.72 – 2.96 (3.47) × 4.30 – 6.35 (8.22) μm (M = 5.66 –

2.41 μm) of thin wall, smooth ornamentation, hyaline with flattened basal scar (Fig 5G-H).

Conidiophores laterally compressed into tight layer or palisade.

Perithecia: the initial formation of primordium perithecium-like subglobose structures,

that reaction with KOH 5% was observed (Fig 5D).

Ascospores: at least two ascospores, reniform, 2.27 – 2.20 × 5.01 – 5.10 μm, dark

brown to blackish brown of thin wall with smooth ornamentation and longitudinal germ slit,

straight were observed (Fig 5F).

Specimens examined: BRAZIL, Rio Grande do Sul – São Gabriel. Endolichenic fungi

isolated from C. curta. February 11th, 2016. Isolator: Peña-Cañón, R. HBEI 001 and HBEI

021.

Commentary: based on synoptic key to Xylaria species of Callan and Rogers (1993) the

isolated presents colonial and anamorphic features observed in cultures that are shared with

Xylaria multiplex (Kunze) Fr. and Xylaria longiana Rehm.

Isolated: Xylaria sp. 2 (MS1 – Fig 6)

Culture: colonies on MS medium cover a 9 cm plate in 21 days. In photoperiod of 16 h

light and 8h dark, the mycelium was uniformly white, cottony, radial non-zoned with flat
 34

topography. Reverse radial and uniformly yellowish white (M&P G1–Plate 11). In the

colonies was observed a primordium of stroma not-central, pyramidal-obtuse, black and

irregular in appearance and shape on the reverse. In dark, colonies were white with center

greyish white (M&P A1–Plate 39), cottony, radial, margin forming wide lobes and irregular

topography. Reverse yellowish white (M&P G1–Plate 11) becoming more pale colored

towards the margin, except in an area where developing partridge brown (M&P L12–Plate 15)

to black patches. Clear droplets were exudate on surface of young colonies, orange (M&P

B12–Plate 13) droplets in mature colonies. Present black immature stromata with cylindrical

stipe up to 0.8 mm high, apex branched and irregularly flattened. On 2% MEA the primordia

of stromata grew and the mycelium formed more stromata.

Stromata: on MS medium, cylindrical stipe of 12 mm high, central and irregularly

branched in the apex; initially orange (M&P E8–Plate 13) later black at base with white apex

(Fig 6A). On MEA 2%, 17 irregular to irregularly clavate stromata, 1 – 4 mm (base) and 1 – 7

mm (apex) wide × 9 – 16 mm high were formed, verrucose to dendroid surface with distal

lobes, irregularly verrucose extended up to 5mm from a base with two to three millimeters of

high. Base of stipe white passing of willow brown color (M&P L7–Plate 15) to smoke brown

(M&P A2–Plate 16) almost black and apex pale orange (M&P B9–Plate 4) (Fig 6B-C).

Hypothecium of ectostroma formed by pseudo-catenulate, erect and varied bifurcated hyphae

of thin wall, probably being the beginning of hymen formation; some hyphae presented

anastomoses (Fig 6E).

Conidiospore: obovoid to clavate, 4.38 – 5.86 × 9.92 – 10.99 (13.35) μm (M = 5.21 –

10.60 μm, n = 20), of thin wall, smooth ornamentation, hyaline with flattened basal scar (Fig

6F-H).

Perithecia: initials formation of perithecium were observed but without ascospores

development (Fig 6D).

 35

Specimens examined: BRAZIL, Rio Grande do Sul – São Gabriel. Endolichenic fungi

isolated from C. curta. February 11th, 2016. Isolator: Peña-Cañón, R. HBEI 002 and HBEI

022.

Commentary: based on synoptic key to Xylaria species of Callan and Rogers (1993) the

isolated shares a greatest number of colonial and anamorphic features with Xylaria longipes

Nitschke, Xylaria polymorpha (Pers.) Grev. and Xylaria schweinitzii Berk. & M.A. Curtis.

Isolated: Xylaria sp. 3 (MS2 – Fig 7)

Culture: colonies on MS medium cover a 9 cm plate in 28 days. In photoperiod of 16 h

light and 8h dark, at first white, cottony, non-zoned, margin forming wide lobes and elevated

topography. The aerial mycelium later varied to pale pink (M&P D1–Pate 1) towards the

margin. Reverse yellowish white with irregular zonation of semicircular brown spots (M&P

H5–Plate 7). The formation of stromata primordia were observed at prolonged incubation

times. Towards the edge of the colony was observed some primordium of stroma with apex

pyramidal-acute, varying of pale orange (M&P B9–Plate 4) to white and cylindrical stipe,

orange (M&P E8–Plate 13), between 0.3 – 0.9 mm in height. In dark, colonies white, cottony,

with finely lobed margins and flat topography. Reverse yellowish white (M&P G1–Plate 11)

and with black spots arranged radially.

Stromata: two stromata of 0.6 – 0.9 mm of height with 1 mm in diameter, smooth and

orange (M&P A12–Plate 4) basal region and apex bifurcated, cottony and whitish covered

with conidiophores (Fig 7A).

Conidiospore: obovoid to clavate, 1.67 – 2.41 (3.65) × (4.10) 4.81 – 6.26 (6.66) μm (M

= 2.19 – 5.45 μm, n = 20), of thin wall, smooth ornamentation, hyaline with flattened basal

scar (Fig 7B). Conidiophores laterally compressed into tight layer or palisade (Fig 7C).
 36

Specimens examined: BRAZIL, Rio Grande do Sul - São Gabriel. Endolichenic fungi

isolated from C. curta. February 11th, 2016. Isolator: Peña-Cañón, R. HBEI 003.

Commentary: based on synoptic key to Xylaria species of Callan and Rogers (1993) the

isolated shares the greatest number of colonial and anamorphic features with Xylaria

polymorpha (Pers.) Grev. and Xylaria longiana Rehm.

5. Discussion

The phylogenetic analyses of ML and BI based on the ITS regions of rDNA and β-

tubulin dataset, infer and support the placement of the three fungal isolated from disinfected

surface of podetia of Cladonia curta within the genus Xylaria. Unifying morphological

characters for the Xylaria genus include conidiophores usually compressed laterally into tight

layer or palisade covering all or part of the stromata surface, conidia hyaline, ovoid to

ellipsoid and a flattened basal scar indicating the former point of attachment to the

conidiogenous cell (Callan and Rogers, 1993). Thus, the morphological characteristics

observed in anamorphic state of endolichenic fungi obtained in culture confirm their

classification into this genus. The presence of Xylaria representatives as endolichenic in C.

curta agreed with previous studies that revealed a particular diversity and richness of

xylariaceous fungi into lichens of the diverse growth form and substrates, in subtropical,

temperate and boreal environments (Arnold et al., 2009; Suryanarayanan et al., 2005;

Suryanarayanan et al., 2017; U´Ren et al., 2016; Wu et al., 2011).

Taking into account that contemporaneously endophytic and endolichenic associations

are two ecologically similar types of interactions that have influence on ecological network

structure (Chagnon et al., 2016), both live within apparently healthy hosts and often represent

the same phylogenetic lineages (Arnold et al. 2009; U‟Ren et al. 2012), the molecular
 37

phylogeny based in ITS region of rDNA was congruent inferring sequences of endolichenic

fungi Xylaria spp., recovered in this work, as close to sequences of the endophytic fungi

cultivated from angiosperms. Likewise, these sequences were grouped together in the major

clades with sequences obtained from other endolichenic fungi showing topologies consistent

with its reported by Arnold et al. (2009) and U´Rent et al. (2016), where endolichenic and

endophytic fungi of order Xylariales were grouped in the same clades. This fact, confirmed

the endolichenic lifestyle of these species as shown by previous work (Arnold et al., 2009;

Petrini and Petrini, 1985; Suryanarayanan et al., 2005; Suryanarayanan et al., 2017; U´Ren et

al., 2016; Wu et al., 2011). Nevertheless, we obtained reduced bootstrap support, as well as

medium supported from posterior probability.

In the phylogenetic trees ITS region of rDNA, isolated Xylaria spp. were clustered with

sequences of Xylaria berteri (Mont.) Cooke (in Index Fungorum ([Link])

the current name is Xylaria berteroi (Mont.) Cook ex J.D. Rogers & Y.M. Ju. [as 'berteri']), a

species of saprophytic fungus reported as endophytic of angiosperms but which before had

not been registered as endolichenic. However, sequences from type material of X. berteri are

no available in Genbank database and in consequence some authors have assigned an

endophytic lifestyle for X. berteri based only in query BLASTn (De Souza Leite et al., 2013;

Vaz et al., 2014; Vega et al., 2010); in Genbank database, 38 sequences of X. berteri were

available: 27 correspond to sequences of 18S, 5.8S and 28S genes and internal transcribed

spacer (ITS 1 and ITS 2) of rDNA, 6 sequences of the β-tubulin gene and four sequence of

genes, Calmodulin, 1-alpha, alpha-actin and DNA-dependent RNA polymerase II (as of

November 2016). In addition, it is important to note that in the case of the endophytic fungi is

recommended that comparisons of sequence data be made using sequences from material type

of species, and if such sequences are no available, then the data must be treated with caution

(Ko Ko et al., 2011).

 38

In accordance with U‟Ren et al. (2010), our analyses based on β-tubulin data set

revealed that endolichenic Xylaria spp. could be phylogenetically related with the saprophytic

X. berteri, nevertheless they are more closely related to an endophytic symbiont isolated from

bark of Cyathea lepifera (Cyatheaceae) (Fu et al., 2013), suggesting that associations of

endolichenic fungi with lichen thalli are not purely incidental. Although endolichenic fungi of

Xylaria are often host-generalists, Thomas et al., (2016) specify that they are not obligated

and suggest that Foraging Ascomycota strategy to be a specialized survival or dispersal

mechanism utilized by a subset of fungi as Xylaria and that the variation in niche or preferred

habitat would modulate the selective advantage of endophytism.

On the other hand, there are a number of indications that support relationship between

isolated Xylaria spp. endolichenic in C. curta and X. berteri with saprophytic lifestyle. First,

the endolichenic fungi identified as Xylaria cubensis and Xylaria cf. heliscus, recovered from

L. oreinoides and U. mutabilis (U‟Ren et al., 2016) were nested within the same in-group

where endolichenic of C. curta were located. Second, the results of the search using BLASTn

algorithm to find sequences highly similar to ours included two sequences of Xylaria

allantoidea (Berk.) Fr. (KR534643 and KR534722) with similarity and coverage values ≥

98%; this species is considered part of the X. cubensis aggregate (Hsieh et al., 2010; Ju et al.,

2012) and were isolated from healthy leaves of Nertera granadensis and Leandra longicoma

(Rojas-Jiménez et al., 2016). In third place, X. allantoidea, X. cf. heliscus and X. cubensis, all

are located in the clade “PO” (the clade containing X. polymorpha and closely related species)

together with X. berteri in the currently phylogenetic status of the subfamily Xylarioideae

(Hsieh et al., 2010; U´Ren et al., 2016). Finally, in the same way also contribute the similarity

in the β-tubulin alignment of our sequences of endolichenic fungi with sequences obtained of

X. berteri, from two saprophytic fungi that grow on wood of C. carlesii var. sessilis (Hsieh et

al., 2005) and on dicot bark (Hsieh et al., 2010), both cultured from ascospores. In
 39

concordance, U´Ren et al. (2016) suggest that in general, temperate and boreal xylariaceous

endophytes, both plant and lichens, have endophytic and saprotrophic life stages. Also,

Xylaria displaying both life stages were found in the endophytic and saprotrophic phases

(Thomas et al., 2016).

The use of anamorphic characters of the cultures often lack sufficient information for

taxonomic identification (Arnold et al., 2007; Petrini and Pertrini, 1985). Furthermore, the

colonial and anamorphic features described in the key of Callan and Rogers (1993) for

Xylaria species of continental United States and Canada can represent an alternative to

approach to identification of lineages, however, in the same way delimits the identification to

the species included. Thus, the endolichenic fungi recovered in this study exhibit similar

morphological appearance of state anamorphic of five species, X. multiplex, X. longiana, X.

longipes, X. polymorpha and X. schweinitzii. Nevertheless, is not possible to corroborate this

information with other authors, since descriptions of Xylaria usually include descriptions of

teleomorphic state (Da Silva and Cortez, 2015; Hladki and Romero, 2010; Medel et al., 2008;

Rogers, 1983; Rogers and Ju, 2012) and this feature has not been obtained in vitro for

endolichenic Xylaria spp.

The morphological features that characterize the teleomorph of X. berteri, including

stromata ± pulvinate to discoid, with stipe or connective narrow and margins usually drooping

(parasol- shaped) (Rogers and Ju, 2012), differs from the features observed in the

anamorphous stromata of endolichenic fungi obtained in culture. Besides, none of taxa of

endolichenic isolated present stromata sessile, a defining feature of all known taxa in the

Xylaria cubensis (Mont.) Fr. aggregate, that is the group that harbors penzigioid species in

which X. berteri is probably the most frequently observed (Ju et al., 2012). This species is

reported throughout the tropics of both hemispheres growing on Acacia koa, Albizia sp.,
 40

Alnus nepalensis, Eucalyptus robusta, Fraxinus uhdei, Macadamia sp., Metrosideros

polymorpha, Psidium sp., Sapindus saponari, Spathodea campanulata (Rogers and Ju, 2012;

Rogers and Ju, 2015). We also encountered that the size (8)12-13.5 x 6-7.5(8) of ascospores

of X. berteri (Rogers and Ju, 2012) was greater than to present by isolated Xylaria sp. 1 (2.27

– 2.20 × 5.01 – 5.10), the unique specie of that only spores were observed. However, this

observation is not conclusive considering the small number of spores found.

In vitro isolates of Xylariaceae specimens can be identified only after comparison with

cultures that was originated from identified teleomorphic material (Callan and Rogers, 1990).

Consequently, is not possible to determine with certainty the identification to specie-level of

the tree isolated and culturing from lichen C. curta from cultural or anamorphic features. As

recommended by Stlader et al. (2013) the teleomorphic material is needed to establish more

teleomorph-anamorphism relations and finally apply a unified nomenclature to the

Xylariaceae in general using a polyphasic taxonomic approach, especially in the tropical

regions of the world and in the southern hemisphere. In the case of a conspecific relationship

between isolated Xylaria spp and X. berteri, the group of taxa reported as endophytic fungi of

angiosperms and species isolated in this work, at first time recorded as endolichenic, could be

a species complex. Considering that the characterization on the anamorph of Xylaria is

required to separate closely related taxa in certain species complexes (Callan and Rogers,

1990) this work provide primordial information for answer this question.

According to our results sequences of endophytic fungi in liverworts (Davis et al., 2003)

that were observed growing inside the rhizoids without penetrating the thalli of their hosts

were grouped in a separate clade of the sequences of endophytic fungi of angiosperms and

lichens used in the present study. At the same time two endophytes in which hyphal growth
 41

was not visible in or near the rhizoids using optical microscopy are shown integrating the

clade that includes the sequences of endolichenic in Cladonia curta.

The most favorable medium for stromatal and conidial production by most Xylaria is 2%

oatmeal agar (Callan and Rogers, 1993), 2% MEA (Persǒh et al., 2009) or PDA (Tripathi and

Joshi, 2015). Nevertheless, based on experimental observations during the progress of this

work, we recommend the use of culture medium MS as an alternative for the colony

development of endolichenic fungi, taking into account that it could favors growth. Despite

this, it is necessary using culture medium established (i.e. MEA 2%) for differentiation and

development of structures of the anamorphic phase these fungi in culture.

6. Conclusion

Our work highlight the less explored diversity of endolichenic fungi reported at present,

including the characterization morphological and molecular of three lineages of fungi that

inhabit inside of symptomless lichen thallus of Cladonia curta. The phylogenetic analyses

based on ITS region of rDNA and β-tubulin infers these endolichenic fungi close to the genus

Xylaria. The morphological characteristics of colonies and anamorphous also confirm this

classification. The phylogenetic relationships among species of endophytic fungi identified as

X. berteri isolated of angiosperms and endolichenic Xylaria spp. is supported. However, the

morphological features of anamorphic stromata obtained in culture provide arguments that

discuss the identification as X. berteri of endolichenic fungi isolated in this work. Given the

current situation, it is premature assign taxonomic names to specie-level for isolated

endolichenic Xylaria spp. Our preliminary data based in molecular marker ITS rDNA and β–

tubulin provides information complementary for evidence that endolichenic fungi are closely

related to endophytic fungi and saprophytic fungi. Further studies are needed for accept or

reject the hypothesis that endolichenic fungi isolated Xylaria spp. and X. berteri are
 42

conspecific, specially phylogenetic analyses using robust multi-locus dataset. Besides, the use

of culture medium MS constitute an alternative in the efforts to know the great diversity of

endophytic fungi that inhabiting living tissues of lichens. Finally, the diversity and prevalence

of the endolichenic fungi have not been studied extensively and this is the first report of

isolation and identification of endolichenic fungi from a species of lichen collected in the

south of Brazil.

Acknowledgements

This work was supported by the Brazilian Antarctic Program through the National Council for

Research and Development – CNPq, Research Foundation of the State of Rio de Janeiro –

FAPERJ, Ministry of Environment – MMA, Ministry of Science and Technology – MCT and

Interministerial Commission for Sea Resources – CIRM, whom the authors thank for the

financial and logistic support needed to perform this study. We thank to scholarship program

of Organization of American States and the Group Coimbra of Brazilian Universities - GCUB

by finance; E. L. Gumboski for identification of lichen material.

Disclosure Declaration

The authors declare that there is no conflict of interest.

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 49

Table 1 Identification of fungal endolichenic isolated from Cladonia curta based on ITS region of rDNA and β-Tubulin gene sequences data comparison with

sequences available in the Genbank database using BLASTn algorithm. Sequences of fungi of accession numbers JF773597, KP133344, JN418792,

KC771483 and HQ117853 were not included; although they met the statistical significance the studies reporting these sequences have not yet been published.

ID Sequences of Genbank
Isolate Region Nearest match Accession Query Max Substrate Host Reference
(Size) cover Identity types species
Xylaria berteri GU324749 99% 98% Bark Dicotyledons Hsieh et al., 2010
LB1 ITS Xylaria berteri JQ936299 98% 99% Leaves Glycine max De Souza Leite et al., 2013
(565bp) rDNA Xylariaceae sp. EU009985 98% 98% Leaves Coffea arabica Vega et al., 2010
Xylaria berteri JQ327861 99% 98% Leaves Myrceugenia ovata var. nanophylla Vaz et al., 2014
Xylaria berteri JQ936295 99% 98% Leaves Glycine max De Souza Leite et al., 2013
Xylariaceae sp. EU009986 98% 98% Leaves Coffea arabica Vega et al., 2010
Xylaria berteri JQ936294 99% 98% Leaves Glycine max De Souza Leite et al., 2013
Xylaria berteri JQ936291 98% 98% Leaves Glycine max De Souza Leite et al., 2013
Xylaria sp. AB743839 99% 98% Stems Cinchona pubescens Shibuya et al., 2003
Xylaria berteri GU324750 99% 98% Bark Dicotyledons Hsieh et al., 2010
MS1 ITS Xylaria berteri GU324749 100% 99% Bark Dicotyledons Hsieh et al., 2010
(566bp) rDNA Xylaria berteri JQ327861 100% 98% Leaves Myrceugenia ovata var. nanophylla Vaz et al., 2014
Xylariaceae sp. EU009985 98% 99% Leaves Coffea arabica Vega et al., 2010
Xylaria berteri JQ936295 100% 98% Leaves Glycine max De Souza Leite et al., 2013
Xylariaceae sp. EU009986 98% 98% Leaves Coffea arabica Vega et al., 2010
Xylaria berteri JQ936294 100% 98% Leaves Glycine max De Souza Leite et al., 2013
Xylaria berteri JQ936291 99% 98% Leaves Glycine max De Souza Leite et al., 2013
Xylaria allantoidea KR534643 98% 98% Leaves Nertera granadensis Rojas-Jiménez et al., 2016
Xylaria berteri GU324750 100% 98% Bark Dicotyledons Hsieh et al., 2010
MS2 ITS Fungal endophyte EU686041 99% 99% leaves Plagiochila sp. Davis and Shaw, 2008
(515bp) rDNA Xylaria berteri GU324749 100% 99% Bark Dicotyledons Hsieh et al., 2010
 50

Table 1 Continued

ID Sequences of Genbank
Isolate Region Nearest match Accession Query Max Substrate Host Reference
(Size) cover Identity types species
Xylaria berteri JQ936300 99% 99% Leaves Glycine max De Souza Leite et al., 2013
Xylaria berteri JQ936295 100% 99% Leaves Glycine max De Souza Leite et al., 2013
Xylaria berteri JQ327861 100% 99% Leaves Myrceugenia ovata var. nanophylla Vaz et al., 2014
Xylaria berteri JQ936294 100% 99% Leaves Glycine max De Souza Leite et al., 2013
Xylaria berteri JQ936291 100% 99% Leaves Glycine max De Souza Leite et al., 2013
Xylaria allantoidea KR534643 100% 98% Leaves Nertera granadensis Rojas-Jiménez et al., 2016
Xylaria berteri JQ936297 100% 98% Leaves Glycine max De Souza Leite et al., 2013
Xylaria sp. AB743839 100% 99% Stems Cinchona pubescens Shibuya et al., 2003
Xylaria berteri JQ936292 100% 99% Leaves Glycine max De Souza Leite et al., 2013
Xylaria berteri JQ936301 98% 98% Leaves Glycine max De Souza Leite et al., 2013
Xylaria berteri JQ936298 98% 98% Leaves Glycine max De Souza Leite et al., 2013
Xylaria allantoidea KR534722 100% 98% Leaves Leandra longicoma Rojas-Jiménez et al., 2016
Xylaria sp. JQ341065 100% 98% Leaves Diospyros crassiflora Douanla-Meli and Langer, 2012
Xylaria allantoidea FJ884194 99% 98% Leaves Hevea brasiliensis Gazis and Chaverri, 2010
Xylaria berteri GU324750 98% 99% Bark Dicotyledons Hsieh et al., 2010
Fungal endophyte EU977315 99% 98% Twigs Angiosperm Smith et al., 2008
LB1 β- Xylaria berteri AY951763 100% 98% On wood Castanopsis carlesii var. sessilis Hsieh et al., 2005
(404bp) tubulin Xylaria berteri KC473561 100% 98% Bark Cyathea lepifera Fu et al., 2013
MS1 β- Xylaria berteri AY951763 100% 97% On wood Castanopsis carlesii var. sessilis Hsieh et al., 2005
(409bp) tubulin Xylaria berteri KC473561 100% 97% Bark Cyathea lepifera Fu et al., 2013
MS2 β- Xylaria berteri FJ904911 90% 77% 1e-21 Leaves Grevillea robusta Unpublished
(242bp) tubulin Xylaria berteri AY951763 90% 77% 1e-21 On wood Castanopsis carlesii var. sessilis Hsieh et al., 2005
 51

99 Fungal endolichenic KT289542

Fungal endolichenic KT289541
Xylaria sp. JQ760210
99 Fungal endolichenic KT289566
Fungal endolichenic KT289545
Xylaria arb uscula JQ760654
54 Xylaria sp. JQ760665
A
Xylaria venustula JQ760209
Xylaria sp. JQ760320
Xylaria sp. JQ761635
95 Fungal endolichenic KT289525
93 Xylaria sp. JQ760970
Xylaria sp. JQ760869
Xylaria sp. JQ760565
LB1 KY962975
Xylaria b erteri JQ936295
Xylaria b erteri JQ936292
Xylaria b erteri JQ327861
Xylaria b erteri JQ936291
MS2 KY962977
Xylaria b erteri GU324750
Xylaria b erteri JQ936294
Xylaria b erteri JQ936299
Xylariaceae sp. EU009985
Xylaria sp. AB743839
Xylaria sp. AY315405
56 B
Fungal endophyte EU686041
Xylariaceae sp. EU009986
Xylaria b erteri JQ936300
Xylaria b erteri GU324749
MS1 KY962976
Fungal endophyte KT289626
Fungal endolichenic KT289550
85 Xylaria sp. AY315404
Fungal endolichenic KT289568
Xylaria cf. heliscus JQ760593
100 Fungal endophyte KT289616
Xylaria cub ensis JQ761659
Fungal endolichenic KT289549
Fungal endolichenic KT289547
Xylariaceae sp. AY315402
Xylaria sp. AY315386
70 Xylaria sp. AY315392
63
Xylaria sp. AY315390
84 Xylaria sp. AY315391 C
Xylaria sp. AY315387
85 Fungal endolichenic KT289579
Xylaria sp. AY315389
Fungal endolichenic KT289531
Xylaria sp. AY315388
Fungal endolichenic KT289523
52 Fungal endolichenic KT289522 D
88 Fungal endolichenic KT289526
Fungal endolichenic KT289546
Fungal endolichenic KT289564
100 Ophiostoma valdivianum
63 Ophiostoma eucalyptigena
Ophiostoma tetropii
Peziza fascicularis

Fig. 2
 52

Fig. 3
 53

Fig. 4
 54

A B C

Fig. 1

A B C

D E H

Fig. 5
 55

A B C

D E H
Fig. 6

A B

C D E
Fig. 7
 56

Figure legends

Fig 1 – Endolichenic Xylaria growing on podetia of Cladonia curta. (A – B). Stereoscope (increasing

1.6); (C). Stereoscope (increasing 3.2); (A). Initial growth of hyphae on podetium after four days of

inoculation; (B). Appearance of hyphae 18 days after isolation; (C). Formation of the mycelium in

culture medium MS. – Scale bars (A – C) = 500 µm.

Fig 2 - Phylogenetic relationships among endolichenic fungi isolated from Cladonia curta and

sequences of endophytic fungi from plants (BLASTn; Arnold et al.,2009), liverworts (Davis et al.,

2003) and lichens (Arnold et al.,2009; U´Ren et al., 2016). MS1, MS2 and LB1 corresponding to

sequences obtained in the present study. The phylogenetic tree inferred by maximum likelihood based

on one-locus ITS region of rDNA dataset. The numbers at each internode indicates bootstrap support

values. Three species of Ophiostoma (O. valdivianum, O. eucalyptigena and O. tetropii) and Peziza

fascicularis were used as outgroup.

Fig 3 - Phylogenetic relationships among endolichenic fungi isolated from Cladonia curta and

sequences of endophytic fungi from plants (BLASTn; Arnold et al.,2009), liverworts (Davis et al.,

2003) and lichens (Arnold et al.,2009; U´Ren et al., 2016). MS1, MS2 and LB1 corresponding to

sequences obtained in the present study. A phylognetic tree generated by using BI analysis from the

ITS region of rDNA dataset. Number at internodes represent posterior probability values of a 95%

majoritty rule consensus tree from 10,000,000 million generation Markov Chain Monte Carlo analysis.

Three species of Ophiostoma (O. valdivianum, O. eucalyptigena and O. tetropii) and Peziza

fascicularis were used as outgroup.

Fig 4 - Phylogenetic relationships among endolichenic fungi isolated from Cladonia curta, sequences

of endophytic fungi from other lichens (U´Ren et al., 2016) and closest match BLASTn. MS1 and LB1

corresponding to sequences of two fungi obtained in the present study. The phylogenetic tree created

by using Bayesian statistical analysis with 10,000,000 million generation Markov Chain Monte Carlo

based on one-locus dataset β-tubulin. The numbers at each internode indicates posterior probability
 57

values of a 95% majoritty rule consensus tree. Ophiostoma tetropii, Ophiostoma grandicarpum and

Ophiostoma microsporum were used to rooted the trees.

Fig 5 – Isolated Xylaria sp. 1. (HBEI 021) (A – C). Stromata obtained in culture on MEA 2% medium;

(D – F). Bright - field microscopy; (D). Initial formation perithecia (arrows); (E). Hyphae regularly

septated (arrow) with branches forming 90° angles (arrowhead); (F). Ascospores with longitudinal

germ slit (arrow); (G – H) DIC microscopy, Conidiospore. – Scale bars (A – C) = 4 mm, (D – H) = 10

µm.

Fig 6 - Isolated Xylaria sp. 2. (HBEI 022) (A).Stroma obtained in culture on MS medium; (B – C).

Stromata on MEA 2% medium; (D). Initial formation of the perithecium (arrows); (E). Bright - field

microscopy. Hyphae of hypothecium with anastomose (arrow a) and basal (arrow b) and irregular

hyphae (arrow c). (D, F – H). DIC microscopy; (F – H). Conidiospore. – Scale bars (A – C) = 4 mm,

(D – H) = 10 µm.

Fig 7 - Isolated Xylaria sp.3. (A). Stromata obtained in culture on MS medium; (B – E). DIC

microscopy (40x); (B). Conidiospore; (C). Palisade of mature conidiophores; (D). Mature

conidiophores; (E). Conidia produced at the tip of a conidiophore. – Scale bars (A) = 1 mm, (B – E) =

10 µm.
 58

4. ARTIGO 2: Occurrence of the Cladonia curta Ahti & Marcelli (Cladoniaceae,

lichenized Ascomycota) in the Pampa biome

(Artigo a ser submetido como Nota Científica para a Acta Botânica Brasílica).
 59

Occurrence of the Cladonia curta Ahti & Marcelli (Cladoniaceae, lichenized Ascomycota)

in the Pampa biome

Emerson Luiz Gumboski1*; Ehidy Rocio Peña Cañón2a; Margéli Pereira de Albuquerque2b;

Jair Putzke2c; Antônio Batista Pereira 2d.

1
Universidade da Região de Joinville, Departamento de Ciências Biológicas. Rua Paulo Malschitzki

10. Zona Industrial Norte 89219710, Joinville, SC - Brasil.

2
Núcleo de Estudos da Vegetação Antarctica - NEVA, Universidade Federal do Pampa

(UNIPAMPA), Av. Antônio Trilha 1847, São Gabriel, RS - Brasil.

1
emersongumboski@[Link]; 2aerociopc2609@[Link];
2b
margeli_albuquerque@[Link]; 2cjrputzkebr@[Link];dantoniopereira@[Link].
*
Author for correspondence

Abstract. Cladoniaceae is a family with more than 450 species registered and distributed in

the most varied environments. It presents species with large thalli, as well as species with

diminutive thalli, which often are neglected even by lichenologists. The aim of this note is to

record a occurrence of Cladonia curta in the Pampa biome, a species previously known only

from Cerrado and mesophyll forest in the states of Minas Gerais, São Paulo and the Federal

District, considered a very rare or ignored species.

Introduction

The Brazilian portion of the Pampa biome is located in the Southern region of the country, in

the state of Rio Grande do Sul, within the Temperate Zone showing both subtropical and

temperate climates. The biome represents about 2.07% of the national territory, occupying an

area of 176,496 km². Although is frequently interpreted as a pure grassland environment, it

comprises at least seven different physiographic formations: savanna, steppe, steppe-savanna,

 60

coast, transition areas and patches of seasonal deciduous and semideciduous forests (Roesch

et al., 2009).

The state of Rio Grande do Sul has a well-documented lichen biota (although probably

underestimated). Spielmann (2006) listed 912 species of lichenized fungi for the state,

distributed among the most diverse environments, among which, the Pampa biome was an

important collection area, with researches carried out mainly by the lichenologists: Gustaf O.

An. Malme, Mariana Fleig and Hector S. Osorio. Fleig et al. (1995) recorded 40 species of

Cladoniaceae for the state, distributed in four different genera: Cladia Nyl., Cladina (Nyl.)

Nyl. (=Cladonia), Cladonia P. Browne and Pycnothelia (Ach.) Dufour. Three species of

Cladonia were described as new (C. maculate, C. paranaensis and C. quiririensis) and two

new records for Brazil, as well as several new records for the state (Charnei et al., 2014).

However, the last initiative to inventoried lichenized fungi species for the Pampa Biome was

carried out punctually in a conservation area, where 254 taxa were recorded, including three

Cladonia species, such Cladonia ceratophylla (Sw.) Spreng., Cladonia chlorophaea (Flörke

ex Sommerf.) Spreng. and Cladonia cf. sphacelata Vain (Käffer et al., 2015).

Cladoniaceae has species very variable morphologically (Fleig et al. 1995; Ahti 2000),

from species with very prominent thalli occupying large areas (e.g., Cladonia crispatula

(Nyl.) Ahti and C. confusa R. Sant.), to very discrete species, with reduced podetia and/or

primary squamules (C. cartilaginea Müll. Arg. and C. nana Vain.), these usually overlooked

in field.

The present article aim to contribute to the knowledge about the distribution of the

family Cladoniaceae in Brazil, reporting the occurrence of the specie Cladonia curta Ahti &

Marcelli in Pampa biome. Until then, according to Ahti (2000), the species endemic from

Brazil, was known in three localities to the Southeast region, from Federal District, state of
 61

Minas Gerais, and state of São Paulo, in mesophilic forests and Cerrado, between 800 and

1200 alt.

Materials and Methods

The samples of lichen were collected in the municipality of São Gabriel, state of Rio Grande

do Sul – Brazil at Federal University of Pampa (UNIPAMPA) campus (30° 20' 06.3" S, 054°

21' 46.5" W) at 124 m asl., on dead wood of an Eucalyptus camaldulensis Dehnh (Myrtaceae)

trunck, in edge of vegetable spot with Eucalyptus understory. Predominant native species of

the generational state include Schinus polygamus (Cav.) Cabrera, Lithraea molleoides (Vell.)

Engl., Saccharum angustifolium (Nees) Trin., and representative of genera Baccharis,

Eugenia, Vachellia and Eryngium. Four samples of material vegetal were collected from

October 2015 to February 2016; rainfall average 196 ±1 mm, humidity was typically medium

(~57 %), and daily temperatures at the site were range from 18°C to 27°C (INMET, 2017).

The lichen samples were deposited in the Bruno Edgar Irgang Herbarium (HBEI-

Federal University of Pampa) under the voucher number HBEI 023 and Joinvillea herbarium

(JOI- University of Joinville Region). The specimens were examined using standard

stereoscopic and light microscopic techniques according to Brodo et al. (2001). Sections of

primary thallus, podetia and ascomata were mounted in water. Chemical constituents were

identified by spot tests, ultraviolet light exposure and thin layer chromatography using solvent

system C (Orange et al., 2001; Elix, 2014).

Results and discussion

Cladonia curta Ahti & Marcelli, Flora Neotropica 78: 192. 2000.

Type. Brazil. Minas Gerais: Mun. Catas Altas, 0.5 km S of Santuario do Caraca, 1100-1200

m, 1993, Marcelli, Ahti, et al. 25916 (holotype, SP; isotype, H).

 62

Description. Primary thallus persistent, irregular, squamules 0.8–3.5 mm wide, 0.5–4.5 mm

long, slightly to clearly arachnoid above, whitish to beige, in part dissolved into soredia,

margins irregularly crenate, slightly sorediate, soredia 200–800µm in diam.; upper surface

greenish, slightly maculate, smooth, epruinose, cortex 60–120 µm thick, algal layer

continuous, 15–25 µm thick, medulla 100–500 µm thick. Pycnidia not seen. Podetia common,

inconspicuous, 0.5–0.8 mm tall, 0.2–1.0 mm thick, greenish, submarginal to marginal,

unbranched, ascyphose; surface verruculose-corticate, in part denuded mainly near the

hymenial discs, esorediate, usually esquamose, not melanotic; cortex 0–50 µm thick, medulla

loose, not clearly differentiated between stereome and central cavity. Pycnidia not seen.

Hymenial disks red, 1.0–1.8 mm diam., hymenium hyaline, 25–30 µm thick, hipohymenium

25–38 µm thick, ascospores not seen (Figure 1).

Chemistry. Spot tests: [primary squamules and podetia] K-, C-, KC-, P-, UV-. TLC: barbatic

and didymic acids, traces of condidymic. Hymenia with rhodocladonic (K+ purple).

Specimens examined: Brazil. State of Rio Grande do Sul, Municipality of São Gabriel,

30°20'06.3"S, 54°21'46.5"W, on dead wood, Pena-Cañón, R. s.n. (HBEI 023; JOI).

Comments. Cladonia curta is characterized by the discrete primary squamules, and by the

short podetia with red hymenial disc. The primary squamules are arachnoid above and slightly

sorediated at margins. The podetia are mainly corticated without symbiotic propagules.

The species is similar to C. pumila Ahti by the tiny podetia, but differs by the

chemistry (sekikaic acid) and byssoid primary squamules in the last (Ahti, 2000). The

constant presence of the red hymenial discs could remind C. didyma (Fée) Vain. or even a

tiny podetia of C. macilenta Hoffm., however, both species have a ecorticate podetia, added

C. didyma present squamules on podetia, and C. macilenta are dense sorediate (Gumboski &

Eliasaro 2012).
 63

Specimens of Cladonia ahtii S. Stenroos and C. miniata G. Mey grows on wood and

could remind C. curta. However, C. ahtii and C. miniata have a well-developed primary

squamules, C. ahtii present a clearly sorediate under surface and margins, while C. miniata

have a reddish medulla on primary thallus (Stenroos 1989; Fleig et al. 1995; Ahti 2000).

Figure 1 - Podetia of Cladonia curta (specimens on JOI). Scale bar = 1.0 cm.

References

Ahti T, 2000. Cladoniaceae. Flora Neotropica 78:1-362.

Brodo, I.M.; Duran Sharnoff, S. & Sharnoff, S. 2001. Lichens of North America. Yale

University Press, New Haven & London. 795 pp.

Charnei, A.M.; Eliasaro, S. & Gumboski, E. L. 2015. Three New Species of Cladonia

(Cladoniaceae, Ascomycota) from Southern Brazil. Anais da Academia Brasileira de

Ciências 87 (1): 101–8.

Elix, J.A. 2014. A catalogue of standardized chromatographic data and biosynthetic

relationships for lichen substances. 3rd Edition. Published by the author, Canberra,

Australia. 320 pp.

 64

Fleig, M., Ahti, T. & Stenroos, S. 1995. A família Cladoniaceae (liquens) no Rio Grande do

Sul, Brasil. Napaea 11: 1-29.

Gumboski, E. L. & Eliasaro, S. 2012. Espécies de Cladonia P. Browne (Cladoniaceae,

Ascomycota) dos Supergrupos Cocciferae, Crustaceae e Perviae em restingas e costões

rochosos dos estados do Paraná e de Santa Catarina, Brasil. Acta Botanica Brasílica, 26:

619-631.

Käffer, M. I.; Martins, S. M. A.; Dantas, R. V. & Maciel, F. C. 2015. Composição da

comunidade liquência em floresta ribeirinha na APA do Ibirapuitâ, RS, Brasil. Hoehnea

42(2): 273-288.

National Institute of Meteorology INMET, 2017. Meteorological database for teaching and

research – BDMEP, Brasília - DF. [Link]

Orange, A.; James, P.W. & White, F.J. 2001. Microchemical Methods for the Identification of

Lichens. - British Lichen Society. 101 pp.

Roesch, L.F.W.; Vieira, F.C.B.; Pereira, V.A.; Schünemann, A.L.; Teixeira, I.F.; Senna,

A.J.T. & Stefenon, V.M. 2009. The Brazilian Pampa: A Fragile Biome. Diversity 1: 182-

198.

Spielmann, A. A. 2006. Checklist of lichens and lichenicolous fungi of Rio Grande do Sul

(Brazil). Caderno de Pesquisa Série Biologia 18 (2): 7-125.

Stenroos, S. 1989. Taxonomic revision of the Cladonia miniata group. Ann. Bot. Fenn. 26:

237-261.
 65

5. CONSIDERAÇÕES FINAIS

Com base nos dados obtidos no presente trabalho pode-se concluir que a caracterização
morfológica e molecular de três linhagens de fungos que habitam dentro do talo assintomático
do líquen Cladonia curta Ahti & Marcelli, fornece informações complementares para
evidenciar que fungos endoliquênicos estão intimamente relacionados com fungos endófitos e
fungos saprófitos. Porém a relação parece ser mais estreita com fungos endófitos em
angiospermas. As análises filogenéticas baseadas na amplificação das sequencias da região
ITS do rDNA e do gene β- Tubulina, infere os fungos endoliquênicos recuperados durante o
presente estudo como próximos ao gênero Xylaria. Da mesma maneira, as características
morfológicas das colônias e dos estromas obtidos in vitro também confirmam esta
classificação.

Por sua parte, a filogenia molecular baseada em sequencias da região ITS do rDNA, suporta a
relação filogenética entre as três linhagens de fungos endoliquênicos isolados com fungos
endófitos de angiospermas identificados na base de dados do Genbank como Xylaria berteri,
uma espécie saprófita antes não citada como endoliquênica. No entanto, o formato cilíndrico a
clavado dos estromas da fase assexuada obtidos em cultura é dissimilar ao estroma séssil, tipo
característico observado nesta espécie penzigioide. Assim, se estabelecem argumentos que
discutem a identificação dos fungos endoliquênicos isolados neste trabalho como Xylaria
berteri (Mont.) Cooke. Com tudo, é prematuro atribuir nomes taxonômicos ao nível de
espécie para os isolados Xylaria spp. Estudos adicionais são necessários para confirmar ou
contradizer que estes fungos sejam conspecíficos com X. berteri.

Considerando que a espécie de líquen objeto de estudo corresponde a um novo registro de

ocorrência para o sul do Brasil, região do país incluída no bioma Pampa, o presente trabalho
contribui a ressaltar a grande importância desde bioma como fonte de biodiversidade que
requer ser explorada. Ao mesmo tempo, a diversidade e a prevalência dos fungos
endoliquênicos continua sendo estudada com este, o primeiro relato de isolamento e
identificação de fungos endoliquênicos no Brasil.
 66

Embora, os meios de cultura previamente estabelecidos para o isolamento de fungos

endoliquênicos não foram usados nas praticas experimentais, o meio Murashige e Skoog (MS)
resultou ser útil na recuperação de fungos endoliquênicos. Sugere-se seu uso como uma
alternativa para o isolamento, crescimento e desenvolvimento deste tipo de fungos, numa
temperatura de 20°C. Do mesmo modo se ressalta a necessidade e importância de usar meios
tradicionais, no caso MEA 2%, para a formação de estromas anamórficos em cultivo.

6. PERSPECTIVAS FUTURAS

- Tendo em conta que fungos endoliquênicos residem dentro do talo dos liquens numa estreita
associação com o fotobionte, se espera utilizar o cultivo dos fungos endoliquênicos Xylaria
spp. e o fotobionte de Cladonia curta Ahti & Marcelli (Fig. 2), isolados durante o presente
estudo, em processos de ressíntese, tentando assim ter uma melhor compreensão dos
processos de reconhecimento e de interação existentes entre eles e identificar os níveis de
expressão de genes relacionados com a simbiose.

Figura 2 – Fotobionte de Cladonia curta obtido em cultura. (A). Aspecto macroscopico da colonia
crescendo em meio trebouxia a 20°C em fotoperiodo 16h luz / 8h osuridad. (B). Aspecto microscopico
do fotobionte em aumento 40x.

A B
Fonte: Autora
 67

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8. ANEXOS

Supplementary Material

Table 2 Genbank accession numbers and references for ITS region of rDNA sequences of taxa
included in phylogenetic analyses in this study.

Accession Reference Accession Reference

AY315386 Davis et al., 2003 KT289545 Arnold et al., 2009
AY315387 Davis et al., 2003 KT289546 Arnold et al., 2009
AY315388 Davis et al., 2003 KT289547 Arnold et al., 2009
AY315389 Davis et al., 2003 KT289548 Arnold et al., 2009
AY315390 Davis et al., 2003 KT289549 Arnold et al., 2009
AY315391 Davis et al., 2003 KT289550 Arnold et al., 2009
AY315393 Davis et al., 2003 KT289551 Arnold et al., 2009
AY315394 Davis et al., 2003 KT289552 Arnold et al., 2009
AY315395 Davis et al., 2003 KT289553 Arnold et al., 2009
AY315396 Davis et al., 2003 KT289554 Arnold et al., 2009
AY315400 Davis et al., 2003 KT289555 Arnold et al., 2009
AY315401 Davis et al., 2003 KT289556 Arnold et al., 2009
AY315402 Davis et al., 2003 KT289557 Arnold et al., 2009
AY315404 Davis et al., 2003 KT289558 Arnold et al., 2009
AY315405 Davis et al., 2003 KT289559 Arnold et al., 2009
AY315406 Davis et al., 2003 KT289560 Arnold et al., 2009
AY315407 Davis et al., 2003 KT289561 Arnold et al., 2009
KT289521 Arnold et al., 2009 KT289562 Arnold et al., 2009
KT289522 Arnold et al., 2009 KT289563 Arnold et al., 2009
KT289523 Arnold et al., 2009 KT289564 Arnold et al., 2009
KT289524 Arnold et al., 2009 KT289565 Arnold et al., 2009
KT289525 Arnold et al., 2009 KT289566 Arnold et al., 2009
KT289526 Arnold et al., 2009 KT289567 Arnold et al., 2009
KT289527 Arnold et al., 2009 KT289568 Arnold et al., 2009
KT289528 Arnold et al., 2009 KT289569 Arnold et al., 2009
KT289529 Arnold et al., 2009 KT289570 Arnold et al., 2009
KT289530 Arnold et al., 2009 KT289571 Arnold et al., 2009
KT289531 Arnold et al., 2009 KT289572 Arnold et al., 2009
KT289532 Arnold et al., 2009 KT289573 Arnold et al., 2009
KT289533 Arnold et al., 2009 KT289574 Arnold et al., 2009
KT289534 Arnold et al., 2009 KT289575 Arnold et al., 2009
KT289535 Arnold et al., 2009 KT289576 Arnold et al., 2009
KT289536 Arnold et al., 2009 KT289577 Arnold et al., 2009
KT289537 Arnold et al., 2009 KT289578 Arnold et al., 2009
KT289538 Arnold et al., 2009 KT289579 Arnold et al., 2009
KT289539 Arnold et al., 2009 KT289580 Arnold et al., 2009
KT289540 Arnold et al., 2009 KT289581 Arnold et al., 2009
KT289541 Arnold et al., 2009 KT289582 Arnold et al., 2009
KT289542 Arnold et al., 2009 KT289583 Arnold et al., 2009
KT289543 Arnold et al., 2009 KT289584 Arnold et al., 2009
 73

Table 2 Continued

Accession Reference Accession Reference

KT289544 Arnold et al., 2009 KT289585 Arnold et al., 2009
KT289586 Arnold et al., 2009 KT289634 Arnold et al., 2009
KT289587 Arnold et al., 2009 KT289635 Arnold et al., 2009
KT289588 Arnold et al., 2009 JQ759362 U´Ren et al.,2016
KT289589 Arnold et al., 2009 JQ759383 U´Ren et al.,2016
KT289590 Arnold et al., 2009 JQ760192 U´Ren et al.,2016
KT289591 Arnold et al., 2009 JQ760209 U´Ren et al.,2016
KT289592 Arnold et al., 2009 JQ760257 U´Ren et al.,2016
KT289593 Arnold et al., 2009 JQ760320 U´Ren et al.,2016
KT289594 Arnold et al., 2009 JQ760565 U´Ren et al.,2016
KT289595 Arnold et al., 2009 JQ760593 U´Ren et al.,2016
KT289596 Arnold et al., 2009 JQ760665 U´Ren et al.,2016
KT289597 Arnold et al., 2009 JQ760666 U´Ren et al.,2016
KT289598 Arnold et al., 2009 JQ761035 U´Ren et al.,2016
KT289599 Arnold et al., 2009 JQ761586 U´Ren et al.,2016
KT289600 Arnold et al., 2009 JQ761635 U´Ren et al.,2016
KT289601 Arnold et al., 2009 JQ761899 U´Ren et al.,2016
KT289602 Arnold et al., 2009 JQ761992 U´Ren et al.,2016
KT289603 Arnold et al., 2009 HM122805 U´Ren et al.,2016
KT289604 Arnold et al., 2009 HM123248 U´Ren et al.,2016
KT289605 Arnold et al., 2009 HM123416 U´Ren et al.,2016
KT289606 Arnold et al., 2009 JQ760181 U´Ren et al.,2016
KT289607 Arnold et al., 2009 JQ760182 U´Ren et al.,2016
KT289608 Arnold et al., 2009 JQ760210 U´Ren et al.,2016
KT289609 Arnold et al., 2009 JQ760306 U´Ren et al.,2016
KT289610 Arnold et al., 2009 JQ760314 U´Ren et al.,2016
KT289611 Arnold et al., 2009 JQ760457 U´Ren et al.,2016
KT289612 Arnold et al., 2009 JQ760469 U´Ren et al.,2016
KT289613 Arnold et al., 2009 JQ760489 U´Ren et al.,2016
KT289614 Arnold et al., 2009 JQ760548 U´Ren et al.,2016
KT289615 Arnold et al., 2009 JQ760549 U´Ren et al.,2016
KT289616 Arnold et al., 2009 JQ760650 U´Ren et al.,2016
KT289617 Arnold et al., 2009 JQ760654 U´Ren et al.,2016
KT289618 Arnold et al., 2009 JQ760728 U´Ren et al.,2016
KT289619 Arnold et al., 2009 JQ760786 U´Ren et al.,2016
KT289620 Arnold et al., 2009 JQ760795 U´Ren et al.,2016
KT289621 Arnold et al., 2009 JQ760869 U´Ren et al.,2016
KT289622 Arnold et al., 2009 JQ760904 U´Ren et al.,2016
KT289623 Arnold et al., 2009 JQ760970 U´Ren et al.,2016
KT289624 Arnold et al., 2009 JQ760995 U´Ren et al.,2016
KT289625 Arnold et al., 2009 JQ761025 U´Ren et al.,2016
KT289626 Arnold et al., 2009 KT289630 Arnold et al., 2009
KT289627 Arnold et al., 2009 KT289631 Arnold et al., 2009
KT289628 Arnold et al., 2009 KT289632 Arnold et al., 2009
KT289629 Arnold et al., 2009 KT289633 Arnold et al., 2009
 74

Table 3 Genbank accession numbers and references for β-Tubulin sequences of taxa included in
phylogenetic analyses in this study.

Accession Reference

GQ502698 Hsieh et al., 2010

KU684111 U´Ren et al.,2016
KU684112 U´Ren et al.,2016
KU684121 U´Ren et al.,2016
KU684122 U´Ren et al.,2016
KU684141 U´Ren et al.,2016
KU684142 U´Ren et al.,2016
KU684143 U´Ren et al.,2016
KU684144 U´Ren et al.,2016
KU684145 U´Ren et al.,2016
KU684146 U´Ren et al.,2016
KU684147 U´Ren et al.,2016
KU684148 U´Ren et al.,2016
KU684153 U´Ren et al.,2016
KU684154 U´Ren et al.,2016
KU684156 U´Ren et al.,2016
KU684157 U´Ren et al.,2016
KU684158 U´Ren et al.,2016
KU684159 U´Ren et al.,2016
KU684164 U´Ren et al.,2016
KU684165 U´Ren et al.,2016
KU684167 U´Ren et al.,2016
KU684168 U´Ren et al.,2016
KU684170 U´Ren et al.,2016
KU684171 U´Ren et al.,2016
KU684173 U´Ren et al.,2016
KU684174 U´Ren et al.,2016
KU684175 U´Ren et al.,2016
KU684176 U´Ren et al.,2016
KU684177 U´Ren et al.,2016
KU684178 U´Ren et al.,2016
KU684179 U´Ren et al.,2016
KU684193 U´Ren et al.,2016
KU684197 U´Ren et al.,2016
KU684198 U´Ren et al.,2016
KU684201 U´Ren et al.,2016
 75

Table 4 Nucleotide substitution models of group of sequences established in order to provide a phylogenetic context for the isolated endolichenic fungi from
Cladonia curta based in ITS region of rDNA. The groups of sequences (G) were: 1) BLASTn search; 2) Arnold et al. (2009); 3) U'Ren et al., 2016 and 4)
David et al. (2003). BIC: Bayesian Information Criterion; AICc: Akaike Information Criterion, corrected; lnL: Maximum Likelihood value; P: number of
parameters including branch lengths. R: Values of transition/transversion bias; f: nucleotide frequencies; r: rates of base substitutions; +G: Gamma
distribution; +I: Invariant sites; HKY: Hasegawa-Kishino-Yano; TN93: Tamura-Nei; T92: Tamura 3-parameter; K2: Kimura 2-parameter.

G Model P BIC AICc lnL (+I) (+G) R f(A) f(T) f(C) f(G) r(AT) r(AC) r(AG) r(TA) r(TC) r(TG) r(CA) r(CT) r(CG) r(GA) r(GT) r(GC)
1 K2+G+I 58 5708.971 5271.012 -2577.263 0.25 1.21 1.56 0.250 0.250 0.250 0.250 0.049 0.049 0.152 0.049 0.152 0.049 0.049 0.152 0.049 0.152 0.049 0.049

K2+I 57 5709.381 5278.965 -2582.248 0.38 n/a 1.47 0.250 0.250 0.250 0.250 0.051 0.051 0.149 0.051 0.149 0.051 0.051 0.149 0.051 0.149 0.051 0.051

K2+G 57 5723.389 5292.973 -2589.252 n/a 1.21 1.34 0.250 0.250 0.250 0.250 0.053 0.053 0.143 0.053 0.143 0.053 0.053 0.143 0.053 0.143 0.053 0.053

T92+G+I 59 5723.673 5278.171 -2579.835 0.25 1.18 1.55 0.256 0.256 0.244 0.244 0.050 0.048 0.148 0.050 0.148 0.048 0.050 0.156 0.048 0.156 0.050 0.048

2 TN93+G+I 242 13900.845 11895.883 -5703.950 0.33 0.50 1.79 0.242 0.256 0.248 0.254 0.046 0.044 0.112 0.043 0.208 0.046 0.043 0.215 0.046 0.106 0.046 0.044

K2+G+I 238 13903.158 11931.270 -5725.709 0.34 0.52 1.80 0.250 0.250 0.250 0.250 0.045 0.045 0.161 0.045 0.161 0.045 0.045 0.161 0.045 0.161 0.045 0.045

T92+G+I 239 13911.795 11931.639 -5724.877 0.34 0.52 1.80 0.249 0.249 0.251 0.251 0.044 0.045 0.161 0.044 0.161 0.045 0.044 0.160 0.045 0.160 0.044 0.045

HKY+G+I 241 13926.847 11930.153 -5722.102 0.34 0.52 1.80 0.242 0.256 0.248 0.254 0.046 0.044 0.163 0.043 0.159 0.045 0.043 0.165 0.045 0.156 0.046 0.044

3 K2+G 93 9811.489 9103.878 -4458.356 n/a 0.26 2.09 0.250 0.250 0.250 0.250 0.040 0.040 0.169 0.040 0.169 0.040 0.040 0.169 0.040 0.169 0.040 0.040

K2+G+I 94 9814.955 9099.748 -4455.278 0.37 0.74 2.11 0.250 0.250 0.250 0.250 0.040 0.040 0.170 0.040 0.170 0.040 0.040 0.170 0.040 0.170 0.040 0.040

T92+G 94 9823.774 9108.568 -4459.688 n/a 0.26 2.09 0.259 0.259 0.241 0.241 0.042 0.039 0.163 0.042 0.163 0.039 0.042 0.175 0.039 0.175 0.042 0.039

T92+G+I 95 9826.986 9104.183 -4456.483 0.37 0.73 2.12 0.259 0.259 0.241 0.241 0.041 0.039 0.164 0.041 0.164 0.039 0.041 0.176 0.039 0.176 0.041 0.039

4 K2+G+I 56 5722.084 5323.500 -2605.402 0.39 0.87 2.00 0.250 0.250 0.250 0.250 0.042 0.042 0.167 0.042 0.167 0.042 0.042 0.167 0.042 0.167 0.042 0.042

K2+G 55 5722.243 5330.764 -2610.046 n/a 0.29 2.02 0.250 0.250 0.250 0.250 0.041 0.041 0.167 0.041 0.167 0.041 0.041 0.167 0.041 0.167 0.041 0.041

T92+G 56 5723.685 5325.102 -2606.203 n/a 0.29 2.05 0.268 0.268 0.232 0.232 0.044 0.038 0.156 0.044 0.156 0.038 0.044 0.181 0.038 0.181 0.044 0.038

T92+G+I 57 5724.678 5318.989 -2602.134 0.39 0.82 2.04 0.268 0.268 0.232 0.232 0.044 0.038 0.156 0.044 0.156 0.038 0.044 0.180 0.038 0.180 0.044 0.038
 76

LB1
MS2
MS1
Xylaria berteri JQ936295
Xylaria berteri JQ936292
Xylaria berteri JQ936291
Xylaria berteri JQ936294
Xylaria berteri GU324750
Xylaria berteri JQ327861
Xylaria berteri JQ936299
Fungal endophyte EU686041
Xylaria berteri JQ936300
Xylariaceae sp. EU009986
Xylariaceae sp. EU009985
Xylaria berteri GU324749
Xylaria sp. AB743839
Xylaria berteri JQ936301
Xylaria berteri JQ936297
Xylaria berteri JQ936298
Xylaria allantoidea KR534722
Xylaria allantoidea KR534643
Xylaria allantoidea FJ884194
Fungal endophyte EU977315
Xylaria sp. JQ341065
Annulohypoxylon cohaerens
Ophiostoma valdivianum
Ophiostoma eucalyptigena
Ophiostoma tetropii
Peziza fascicularis

Fig 8 - Phylogenetic relationships among endolichenic fungi isolated from Cladonia curta and
sequences of endophytic fungi with máximum similarity using BLASTn in GenBank database. MS1,
MS2 and LB1 corresponding to sequences obtained in the present study. The tree was inferred by
Maximum Likelihood based on one-locus dataset ITS region of rDNA. The numbers at each internode
indicates bootstrap support values. Three species of Ophiostoma, Annulohypoxylon cohaerens and
Peziza fascicularis were used as outgroup. Branch leading to clade with sequences of endophytes most
closely related to the isolated fungi during this work are in bold.
 77

LB1
KT289568
MS1
MS2
KT289549
KT289626
KT289525
KT289550
KT289616
KT289542
KT289541
KT289579
KT289531
KT289523
KT289526
KT289546
KT289547
KT289522
KT289566
KT289545
KT289564
KT289538
KT289534
EF026140
KT289528
KT289625
KT289607
KT289556
KT289634
KT289537
KT289581
KT289558
KT289562
KT289543
KT289635
KT289536
KT289530
KT289557
KT289553
KT289532
KT289578
KT289624
KT289561
KT289576
KT289551
KT289548
KT289630
KT289629
KT289582
KT289580
KT289631
KT289632
KT289623
KT289610
KT289622
KT289572
KT289583
KT289535
KT289529
NR137979
NR145317
NR145271
KT289565
KT289540
KT289567
KT289574
KT289555
KT289573
KT289575
KT289577
KT289533
KT289552
KT289599
KT289619
KT289613
KT289560
KT289544
KT289597
 KT289567
KT289574
KT289555
KT289573 78
KT289575
KT289577
KT289533
KT289552
KT289599
KT289619
KT289613
KT289560
KT289544
KT289597
KT289559
KT289620
KT289614
KT289592
KT289571
KT289569
KT289570
KT289539
KT289621
KT289604
KT289527
KT289524
KT289587
KT289593
KT289602
KT289628
KT289627
KT289590
KT289586
KT289521
KT289588
KT289611
KT289598
KT289595
KT289589
KT289563
KT289633
KT289601
KT289612
KT289554
KT289596
KT289594
KT289609
KT289600
KT289608
KT289605
KT289618
KT289617
KT289606
KT289615
Peziza fascicularis

Fig 9 - Phylogenetic relationships of endolichenic fungi isolated from Cladonia curta (MS1, MS2 and
LB1) and endophytic fungi isolated from plant and lichens obtained by Arnold et al. (2009) based on
analysis of ITS region of rDNA sequences. The tree was inferred by Maximum Likelihood and
numbers above branches indicates bootstrap support values. Three species of Ophiostoma (NR145317,
NR137979 AND NR145271), Annulohypoxylon cohaerens (EF026140) and Peziza fascicularis
(LT1558418) were used to root the tree. Branch leading to clade with sequences of endophytes most
closely related to the isolated fungi in this work are in bold.
 79

Hypoxylon rubiginosum JQ760786

Hypoxylon sp. JQ760795
Hypoxylon sp. JQ761992
Hypoxylon sp. JQ760314
Hypoxylon sp. JQ761586
Hypoxylon sp. JQ760666
Hypoxylon sp. JQ760995
Annulohypoxylon stygium JQ760192
Hypoxylon monticulosum JQ760257
Daldinia eschscholtzii JQ761025
Daldinia sp. JQ761035
Daldinia loculata JQ759383
Daldinia loculata HM123248
Daldinia loculata JQ759362
Annulohypoxylon sp. JQ760181
Hypoxylon polyporus JQ760904
Annulohypoxylon cohaerens EF026140
Xylariomycetidae sp. JQ760182
Xylariomycetidae sp. JQ760548
Biscogniauxia sp. JQ760650
Biscogniauxia mediterranea HM122805
Biscogniauxia mediterranea HM123416
Xylariaceae sp. JQ760469
Xylariaceae sp. JQ760728
Xylariaceae sp. JQ760457
Xylariaceae sp. JQ760306
Nemania sp. JQ760549
Xylaria sp. JQ760565
Xylaria sp. JQ760970
Xylaria sp. JQ760869
Xylaria sp. JQ761635
Xylaria sp. JQ760320
Xylaria sp. JQ760665
Xylaria venustula JQ760209
Xylaria arbuscula JQ760654
Xylaria sp. JQ760210
Xylaria cf. heliscus JQ760593
Xylaria cubensis JQ761659
MS1
LB1
MS2
Xylariomycetidae sp. JQ761899
Xylariomycetidae sp. JQ760489
Ophiostoma tetropii
Ophiostoma eucalyptigena
Ophiostoma valdivianum
Peziza fascicularis

Fig 10 - Phylogenetic relationships among endolichenic fungi isolated from Cladonia curta and fungi
isolated from the interior of Cladonia species and other lichens reported by U´Ren et al. (2016). MS1,
MS2 and LB1 corresponding to sequences obtained in the present study. The tree was inferred by
 80

Maximum Likelihood based on one-locus dataset (ITS region of rDNA) and numbers at each
internode indicates bootstrap support values. Three species of Ophiostoma and Peziza fascicularis
were used as outgroup. Branch leading to clade with sequences of endophytes most closely related to
the isolated fungi in this work are in bold.

Xylaria sp. AY315386

Xylaria sp. AY315390
Xylaria sp. AY315392
Xylaria sp. AY315391
Xylaria sp. AY315387
Xylaria sp. AY315389
Xylariaceae sp. AY315402
Xylaria sp. AY31540
Xylaria sp. AY315404
MS2 ITS
LB1 ITS
MS1 ITS
Xylaria sp. AY315388
Xylariaceae sp. AY315407
Xylaria sp. AY315400
Xylaria sp. AY315395
Xylaria sp. AY315396
Xylariaceae sp. AY315401
Daldinia sp. AY315403
Xylariales sp. AY315393
Xylariales sp. AY315394
Xylariaceae sp. AY315406
Ophiostoma valdivianum
Ophiostoma eucalyptigena
Ophiostoma tetropii
Peziza fascicularis

Fig 11 - Phylogenetic relationships of endolichenic fungi isolated from Cladonia curta and
endophytes of liverworts (Bazzania, Odontoschisma, Tricholea, Plagiochila and Metzgeria) obtained
by Davis et al. (2003) based on analysis of ITS region of rDNA sequences. MS1, MS2 and LB1
corresponding to sequences obtained in the present study. The tree was inferred by maximum
likelihood and numbers above branches indicates bootstrap support values. Three species of
Ophiostoma. Branch leading to clade with sequences of endophytes most closely related to the isolated
fungi during this work are in bold.
 81

LB1

MS1

Xylaria berteri AY951763

Xylaria berteri KC473561

Xylaria cubensis KU684198

Xylaria berteri GQ502698

Xylaria cf. heliscus KU684159

Xylaria sp. KU684144

Xylaria venustula KU684143

Xylaria arbuscula KU684165

Xylaria sp. KU684148 A

Xylaria sp. KU684158

Xylaria sp. KU684173

Xylaria sp. KU684177

Xylaria sp. KU684197

Xylariaceae sp. KU684146

Xylariaceae sp. KU684154

Nemania sp. KU684157

Xylariaceae sp. KU684174

Xylariaceae sp. KU684168

Xylariaceae sp. KU684175

Hypoxylon monticulosum KU684145

Hypoxylon polyporus KU684176

Xylariomycetidae sp. KU684156

Biscogniauxia sp. KU684164

Hypoxylon sp. KU684178

Hypoxylon sp. KU684193

Hypoxylon sp. KU684167

Annulohypoxylon sp. KU684141

B
Annulohypoxylon stygium KU684142

Hypoxylon rubiginosum KU684170

Hypoxylon sp. KU684171

Hypoxylon sp. KU684147

Daldinia eschscholtzii KU684179

Daldinia loculata KU684121

Daldinia loculata KU684111

Daldinia loculata KU684112

Biscogniauxia mediterranea KU684122

Xylariaceae sp. KU684153

Ophiostoma tetropii

Xylariomycetidae sp. KU684201

Ophiostoma grandicarpum

Ophiostoma microsporum

Fig 12 - Phylogenetic relationships among endolichenic fungi isolated from Cladonia curta, BLASTn
similarity sequences and sequences of endophytic fungi isolated from other lichens (U´Ren et al.,
2016). MS1 and LB1 corresponding to sequences of two fungi recovered in the present study. The tree
was inferred by maximum likelihood based on β-tubulin gene. The numbers at each internode
indicates bootstrap support values. Ophiostoma tetropii, Ophiostoma grandicarpum and Ophiostoma
microsporum were used to rooted.
 82

A B

C D

E F

Fig 13 - Appearance of isolated Xylaria sp. 1 (HBEI 001), colonies on MS medium. A -B. Pure culture
of the isolated fungi. C – D. Appearance of endolichenic fungi in photoperiod of 16h light / 8h dark. E
- F. Appearance of endolichenic fungi in darkness.
 83

A B

C D

E F
Fig 14 - Appearance of isolated Xylaria sp. 2 (HBEI 002), colonies on MS medium. A -B. Pure culture
of the isolated fungi. C – D. Appearance of endolichenic fungi in photoperiod of 16h light / 8h dark. E
- F. Appearance of endolichenic fungi in darkness.
 84

A B

C D

E F
Fig 15 - Appearance of isolated Xylaria sp. 3 (HBEI 003), colonies on MS medium. A -B. Pure culture
of the isolated fungi. C – D. Appearance of endolichenic fungi in photoperiod of 16h light / 8h dark. E
- F. Appearance of endolichenic fungi in darkness.