Escolar Documentos
Profissional Documentos
Cultura Documentos
Manaus, Amazonas
Abril, 2016
ii
Manaus, Amazonas
Abril, 2016
iii
Ficha catalográfica
CDD 589.2
SINOPSE
Agradecimentos
Agradeço ao Prof. Charles R. Clement por ter aceitado me orientar durante o
doutorado e por ter confiado na realização desse trabalho. Suas inúmeras sugestões e
incentivos foram extremamente valiosas e levaram à plena realização deste projeto. Aos
Profs. Iuri Goulart Baseia pela co-orientação e confiança no trabalho e Kentaro Hosaka por
me aceitar em seu laboratório durante o período de doutorado sanduíche e por ter me
mostrado as maravilhas do Japão. À Maria P. Martín, por estar sempre disposta a esclarecer
minhas inúmeras dúvidas e pela co-orientação não oficial. Também agradeço à Noemia Kazue
Ishikawa pelos preciosos ensinamentos, tanto acadêmicos quanto para a vida, e pelas
oportunidades proporcionadas. À Doriane P. Rodrigues por ceder o laboratório de Evolução
Aplicada para realizar os experimentos de genética molecular.
Aos companheiros de campo e laboratório, com quem pude dividir as frustrações e
alegrias que os experimentos nos proporcionam! Agradeço à Bianca Denise (UFRN), por
sempre estar disposta a ajudar nos estudos morfológicos. Agradeço aos meus amigos por
proporcionarem horas de divertimento e distração! Agradeço a Flocos que sempre parecia
saber quando eu já tinha passado tempo demais em frente ao computador, e me chamava para
passear!
Em especial agradeço a Emanuell Ribeiro, meu companheiro, que participou
ativamente nas diversas fases desse trabalho, que discutiu os resultados com a dedicação de
como se fossem seus resultados! Sobretudo pela paciência que sempre teve, e entendimento
das limitações que a vida que escolhemos nos impõe. E por não me deixar desistir.
Agradeço a minha família, minha mãe por acreditar em mim, meu pai por me
incentivar a seguir nesse caminho, e a meus irmãos que além de acreditarem, nunca
questionaram o caminho escolhido por mim. Sei que sempre estarão lá quando precisar.
Finalmente, agradeço ao Programa de Pós-Graduação em Genética, Conservação e
Biologia Evolutiva do INPA, e às agências de fomento (CNPq, CAPES e FAPEAM) por
terem concedido as bolsas de doutorado e doutorado sanduíche, e pelo financiamento da
pesquisa. Agradeço também a todos os moradores das comunidades por onde passamos
durante as coletas, que nos receberam com tanto carinho e atenção, e com quem aprendi um
pouco da vida na Amazônia!
v
Resumo
O presente trabalho teve como objetivo principal integrar espécies amazônicas à filogenia
molecular da ordem Phallales com sequências diagnósticas padrões, e com este conjunto de
dados inferir sobre a classificação e evolução do grupo. Para isso, coletas foram realizadas ao
longo dos rios Amazonas, Solimões, Negro, Madeira e Tapajós. Os basidiomas coletados
foram herborizados, depositados no Herbário do INPA, e fragmentos foram retirados para
extração de DNA genômico. Foram obtidas sequências de ITS de todos os espécimes e,
dependendo do grupo em estudo, foram obtidas sequências de outras regiões gênicas.
Análises de Máxima Parcimônia e Bayesiana foram utilizadas para inferência filogenética
molecular dos grupos em estudo, além da construção de árvores de espécies. Esta tese
encontra-se dividida em quatro capítulos. No primeiro capítulo, uma espécie nova (Geastrum
inpaense) e novos registros de fungos gasteroides são descritos: seis espécies constituem
novos registros para Amazônia Central (Geastrum lloydianum, G. schweinitzii, Phallus
merulinus, Staheliomyces cinctus), uma para o Brasil (Phallus atrovolvatus) e uma para
América do Sul (Mutinus fleischeri). O segundo capítulo, dois novos registros de fungos
faloides são descritos para o Brasil (Lysurus arachnoideus) e América do Sul (Phallus
cinnbarinus), com descrições morfológicas detalhadas e fotos, além de sequências da região
ITS. No terceiro capítulo, a diversidade observada entre espécimes coletados do gênero
monotípico Xylophallus foi avaliada por meio de análises morfológicas aliadas a métodos de
delimitação de espécies utilizando sequências de cinco regiões gênicas. As análises
mostraram uma diversidade inesperada dentro do gênero ao revelar a presença de três
unidades evolutivas: uma corresponde a X. xylogenus; uma é uma espécie nova denominada
X. clavatus; e a terceira corresponde a uma espécie críptica – o primeiro registro em fungos
faloides. No quarto capítulo, foi avaliada a diversidade morfológica e molecular de Phallus
indusiatus sensu lato coletada na Amazônia brasileira. A árvore filogenética obtida com
sequências de ITS revelou quatro clados distintos que possuiam concordância com os dados
morfológicos. Um clado corresponde a P. indusiatus Vent., cujos espécimes apresentam
caracteres morfológicos iguais ou bem próximos à descrição original. Os outros três clados
possuem características distintas de P. indusiatus e quaisquer outras espécies com indúsio
branco, correspondendo portanto a três novas espécies: P. amazonicus possui também uma
variedade nova, P. amazonicus var. denigricans, diferenciada principalmente pela coloração
escura da volva; P. squamulosus é caracterizada pela volva com superfície escamosa e foi
encontrada na Mata Atlântica; P. purpurascens é a espécie mais distinta, sendo a maior entre
as espécies citadas, possui volva branca a lilás, reticulações do receptáculo estreitas e esporos
menores que as demais espécies. Até 2012, haviam apenas dois registros de Phallales para a
Amazônia brasileira. Atualmente podem ser contabilizadas 13 espécies de fungos faloides
para a Amazônia brasileira, sendo quatro dessas espécies novas para a ciência. Esses
resultados demonstram a importância de estudar a micobiota de florestas Neotropicais,
principalmente de grupos negligenciados como os fungos faloides. Demostra também a
importância da inclusão de dados moleculares no estudo de taxonomia e sistemática de
fungos, e como esses dados podem ser explorados. Testar diferentes regiões gênicas além de
ITS – o barcode proposto de fungos – e métodos alternativos de delimitação de espécies
também devem ser levados em consideração.
vi
Abstract
The present study aimed to integrate Amazonian species to the molecular phylogeny of
the order Phallales using standard DNA sequences, and with this, to make inferences about
the classification and evolution of the group. Specimens were collected along the main rivers
of the Amazon basin: Negro, Madeira, Solimões, Amazonas and Tapajós. The specimens
were herborized, deposited at the INPA herbarium, and small fragments of basidioma were
excised for DNA extraction. ITS sequences were obtained from all specimens, and sequences
from additional DNA regions were obtained depending on the group of study. Maximum
Parsimony and Bayesian analyses were performed for molecular phylogenetic inferences, in
addition to construction of species trees. This thesis is divided in four chapters. In the first
chapter, one new species (Geastrum inpaense) and new records of gasteroid fungi are
described: six species are new records for Central Amazonia (Geastrum lloydianum, G.
schweinitzii, Phallus merulinus, Staheliomyces cinctus), one for Brazil (Phallus atrovolvatus)
and one for South America (Mutinus fleischeri). In the second chapter, two new records of
phalloid fungi are described for Brazil (Lysurus arachnoideus) and South America (Phallus
cinnbarinus), with detailed morphological descriptions and images, in addition to ITS
sequences. In the third chapter, the diversity observed in specimens of the monotypic genus
Xylophallus was evaluated using morphological analysis allied to methods of species
delimitation using five DNA regions. The analyses showed unexpected diversity in
Xylophallus, by revealing three evolutionary units: one corresponds to X. xylogenus; another
is a new species named X. clavatus; and the third corresponds to a cryptic species – the first
record for phalloid fungi. In chapter four, the morphological and molecular diversity found in
Amazonian specimens of Phallus indusiatus sensu lato was evaluated. The phylogenetic tree
obtained with ITS sequences revealed four distinct clades, which agreed with morphological
data. One clade corresponds to P. indusiatus Vent., The other three clades are
morphologically different from P. indusiatus and any other species with white indusium, thus
corresponding to three new species: P. amazonicus, which also has a new variety, P.
amazonicus var. denigricans differentiated by the darker volva; P. squamulosus is
characterized by the squamous surface of the volva and it was found in the Atlantic
Rainforest; P. purpurascens is the most distinct and the largest among these new species, it
has a pinkish volva, smaller reticulations on the receptaculum and smaller spores than the
other species. Until 2012, there were only two records of Phallales for Brazilian Amazonia.
Currently, 13 species are recorded, of which four species are new to science. These results
show the importance of studying mycobiota from Neotropical forests, especially neglected
groups such as phalloid fungi. It also demonstrates the importance of including molecular data
in the study of systematics and evolution of fungi, and how these data should be explored.
Testing different DNA regions other than ITS – the proposed barcode for fungi – and
alternative species delimitation methods should also be considered.
vii
Sumário
Lista de Figuras
Capítulo 01
Figura 1. Phylogenetic tree of Geastrum used to position G. inpaense within the genus
constructed with Bayesian analysis, with posterior probabilities values on nodes. Herbarium
vouchers follow the taxa name. ..................................................................................................9
Figura 2. Geastrum inpaense macrostructures. Expanded basidiomata (a, b, c, d), scale bars
correspond to 20 mm; peristome (e, f), scale bars correspond to 5 mm; hairs of mycelial layer
indicated by arrows (g, h), scale bars correspond to 10 mm and 1 mm. Photographs by T.S.
Cabral........................................................................................................................................11
Figura 3. Geastrum inpaense microstructures. Endoperidium surface (a), spore ornamentation
(b), capillitial hyphae with (c) and without (d) amorphous substance………………………..12
Figura 4. Geastrum inpaense schematic drawing. Mycelial layer hyphae (a), bar = 10 µm;
fibrous layer hyphae (b), bar = 10 µm; fleshy layer hyphae (c), bar = 20 µm; spores (d), bar =
10 µm; basidia (e), bar = 10 µm. Illustration by D.S. Alfredo………………………………..13
Figura 5. Geastraceae species. Geastrum lloydianum (a), Geastrum saccatum (b), Geastrum
schweinitzii (c), Geastrum triplex (d). Photographs by T.S. Cabral………………………….14
Figura 6. Phallaceae species. Phallus merulinus (a), Phallus atrovolvatus (b), Phallus
indusiatus (c), Mutinus fleischeri (d), Staheliomyces cinctus (e). Scale bars correspond to 20
mm. Photographs (a), (b) and (d) by N.K.Ishikawa, photographs (c) and (e) by T.S.
Cabral…………………………………………………………………………………………15
Figura 7. Morganella fuliginea. Expanded basidiomata (a); exoperidium sphaerocyst chains
(b); spores in 5% KOH (c) and under SEM (d). Photograph by R.B. Neto..............................17
Capítulo 02
Figura 1. Lysurus arachnoideus (INPA 256537). A: Expanded basidioma. B:
Basidiospores............................................................................................................................25
Figura 2. Phallus cinnabarinus (INPA 255835). A: Expanded basidioma. B: Basidiospores.
C: Pseudoparenchymatous hyphae of pseudostipe with pinkish pigment droplets…………..26
ix
Capítulo 03
Figura 1. Geographic distribution of specimens of Xylophallus reported in this study. Blue
dots are specimens of Xylophallus clavatus, red dots are specimens of Xylophallus xylogenus
and green dots are the X. xylogenus phylogenetic cryptic
species………………………………….35
Figura 2. Barplots showing the distribution of the absolute number of false positive (grey) and
false negative (black) identifications across a range of pre-set threshold values on the x axis.
(a) Ef-1α, (b) gpd, (c) rpb1, (d) rpb2, (e)
ITS………………………………………………...........41
Figura 3. Species tree based on five genes inferred by *BEAST. Values on nodes indicate
posterior probabilities. Scale represents the estimated site substitution rates, by units of branch
length………………………………………………………………………………………….42
Figura 4. Macromorphology of Xylophallus clavatus. (a, b) Fresh basidiomata; (c) dried
basidiomata with a longitudinal cut through the volva, showing the pseudostipe not attached
to it; (d) dried basidiomata showing a minutely perforated apex; (e, f) immature basidiomata
with protuberances on surface. Photos: Tiara S.
Cabral…………………………………….............45
Figura 5. Micromorphology of Xylophallus clavatus. (a) Basidiospores; (b) basidium at the
yellow arrow; (c) pseudoparenchymatous hyphae of pseudostipe; (d) filamentous hyphae from
volva…………………………………………………………………………………………..46
Figura 6. Xylophallus xylogenus. (a, b) Fresh mature basidiomata; (c) Immature basidiomata
fresh and (d) dried showing the smooth surface; (e) basidiospores; (f) pseudoparenchymatous
hyphae of pseudostipe; (g) clavate basidia; (h) a representative of the Xylophallus cryptic
species. Photos a-g: Tiara S. Cabral; h: Ricardo Braga Neto...................................................49
Capítulo 04
Figura 01. Currently known distributions of the new Phallus species described in this study.
Colored areas are the Brazilian Biomes (IBGE 2016): Amazonian Rainforest (dark green),
Cerrado (brown), Caatinga (beige), Atlantic Rainforest (light green), Pantanal (dark blue),
Pampa (light blue)…………………………………………………………………………….66
Figura 02. Phylogenetic tree obtained by Bayesian analysis. Colored clades correspond to
Brazilian species: in blue, P. amazonicus; in green, P. purpurascens; in red, P. squamulosus;
x
in brown, P. indusiatus sensu stricto. Posterior probability values are on the nodes. The black
dots indicate specimens under Phallus indusiatus deposited in Genbank and downloaded for
this study….…………………………………………………………………………………..67
Figura 03. Phallus amazonicus. (a) fresh basidiome, holotype; (b) volva with small hyphae
projections on surface; (c) receptacle with a prominent pore and pale-yellow color (TSC 248);
(d) spores; (e) hyphae from volva; (f) rhizomorphs hyphae; (g) crystals in globose cells found
on volva……………………………………………………………………………………….71
Figura 04. Phallus amazonicus var. denigricans fresh basidiome. a. whole basidiome; b.
blackish and smooth volva in detail. Bars = 20 mm. (c) spores; (d) pseudoparenchymatous
hyphae of pseudostipe; (e) hyphae from rhizomorphs; (f) hyphae from
volva…………………………..................................................................................................73
Figura 05. Phallus purpurascens (a) fresh basidiome; (b) longitudinal section of an immature
basidiome, showing the purplish volva and rhizomorphs; (c) gregarious immature basidioma,
with purplish pigments on surface. Bars correspond to 20 mm. (d) spores; (e) rhizomorphs
hyphae; (f) pseudoparenchymatous hyphae from pseudostipe; (g) hyphae from volva; (h)
crystals in globose cells found on volva………………………………………………….….76
Figura 06. Phallus squamulosus DT 253 (a) fresh basidiome and (b) immature basidiome with
squamous surface. Bars correspond to 20 mm. (c) spores; (d) pseudoparenchymatous hyphae
from pseudostipe; (e) hyphae from volva; (f) hyphae from rhizomorphs and crystals deposits
on globose cells……………………………………………………………………………….79
Figura 07. Phallus indusiatus fresh basidiome. (a) TSC 148; (b) TSC 135, showing the volva
with pinkish pigments. Bars correspond to 20 mm..................................................................81
1
Introdução Geral
Os fungos constituem um vasto Reino de organismos eucariotos heterotróficos com
morfologia e dimensões muito distintas, variando desde formas microscópicas até formas
macroscópicas. No passado os fungos foram tratados como pertencendo ao Reino das Plantas
devido à semelhanças compartilhadas, e só foram considerados um reino a parte quando
Whittaker (1959) propôs o estabelecimento do Reino Fungi. O filo Basidiomycota possui
mais de trinta mil espécies (Webster & Weber 2007), caracterizado principalmente por
possuírem estruturas chamadas basídios onde são produzidos os esporos.
A classe Agaricomycetes constitui 98% do subfilo Agaricomycotina (Basidiomycota), e é
composta pelos fungos que produzem corpos de frutificação, conhecidos popularmente por
cogumelos (Kirk et al. 2008). As espécies desta classe podem ser divididas em duas grandes
categorias: formas não gasteroides (agaricoides, boletoides, poliporoides, etc.) e gasteroides
(“puffballs, “birds nest fungi”, “stinkhorns”, etc.). A principal diferença entre esses grupos é
a forma de liberação dos esporos, que é por balistosporia (liberação ativa, com gasto de
energia) em não gasteoides, e estatismosporia (liberação passiva, dependente de agentes
externos) em gasteroides (Wilson et al. 2011)
Os fungos gasteroides evoluíram de maneira independente em diferentes linhagens dentro
dos basidiomicetos. Portanto, essa morfologia convergente não é considerada para
classificação, uma vez que a forma gasteroide é considerada uma homoplasia (Moncalvo et al.
2002; Wilson et al. 2011). Os gasteroides então encontram-se desmembrados em várias
ordens dentro de Basidiomycota. Na subclasse Phallomycetidae, porém, das quatro ordens
que a compõem, duas são compostas inteiramente por fungos gasteroides: Phallales e
Geastrales (Hosaka et al. 2006).
Os fungos faloides (ordem Phallales) são caracterizados principalmente por possuírem a
gleba – porção onde são formados os esporos – mucilaginosa com odor fétido que atrai
agentes dispersores, em sua maioria insetos. Possuem uma parede externa denominada
perídio, composta de duas a três camadas; inicialmente o perídio envolve completamente o
receptáculo e a gleba, e na maturidade pode se romper a partir do ápice, como ocorre na
maioria das espécies (Cunningham 1944). Podem apresentar o desenvolvimento hipógeo e/ou
epígeo (abaixo e acima da terra, respectivamente). As famílias Trappeaceae, Claustulaceae,
Protophallaceae e Gastroporiaceae são caracterizadas por não possuírem pseudoestipe, e em
algumas espécies o perídio permanece fechado mesmo após a maturação da gleba, o que
confere um corpo de frutificação oval a piriforme. O basidioma pode variar de um
2
Os faloides são cosmopolitas e efêmeros, com a maioria das espécies sapróbias. Suas
rizomorfas podem ser encontradas se estendendo às raízes enterradas e pedaços de madeira
em decomposição (Miller & Miller 1988), sendo comum também a frutificação em folhiço,
exercendo papel importante na ciclagem de matéria orgânica (Leite et al. 2007). O mau cheiro
exalado pela gleba tem como finalidade a atração de insetos que se alimentam dela,
caracterizando uma importante e diferente estratégia de dispersão de esporos para os fungos
(Tuno 1998; Oliveira & Morato 2000).
Este grupo tem sido estudado por décadas e estes estudos tem revelado uma grande
diversidade ao redor do mundo. Apesar da sua grande diversidade, ainda há lacunas no
conhecimento do grupo, tanto relacionado a taxonomia quanto a distribuição geográfica.
3
Recentemente, Trierveiler-Pereira et al. (2014) propuseram uma nova filogenia para a ordem
Phallales, focando em gêneros sub-representados, incluindo espécies tropicais e sub-tropicais.
Porém, apesar de as relações entre as famílias estarem aparentemente bem estabelecidas, as
relações entre os gêneros dentro de cada família ou ainda a delimitação taxonômica entre os
taxa, ainda são conflitantes, o que resulta em um grande número de sinonímias para a ordem
Phallales. Isso em parte se deve à dificuldade em separar morfologicamente os taxons, devido
à escassez e plasticidade dos caracteres diagnósticos (Begerow et al. 2010). Aliado a isso, o
fato de possuírem frutificação efêmera e a difícil preservação do material também influenciam
nos estudos de taxonomia e sistemática das espécies nessa ordem.
Ao se integrar dados moleculares aos fenotípicos, muitos caracteres morfológicos
mostram-se homoplásicos ou filogeneticamente não informativos (Begerow et al. 2006;
Cabral et al. 2012; Marincowitz et al. 2015), e a evolução morfológica convergente parece ser
bem difundida entre fungos (Hibbett & Hawksworth 2007). Assim, dados de filogenia
molecular e delimitação por DNA barcode podem ser utilizados como uma importante
ferramenta para auxiliar estudos de sistemática e evolução desses fungos. De fato, vários
estudos moleculares tem revelado novas espécies e a existência de complexos de espécies e
espécies crípticas (Zhao et al. 2008; Cabral et al. 2012; Degreef et al. 2013; Li et al. 2014; Li
et al. 2005; Desjardin & Perry 2009). Apesar do número crescente de estudos filogenéticos
em fungos faloides (Hosaka et al. 2006; Degreef et al. 2013; Trierveiler-Pereira et al. 2014;
Trierveiler-Pereira & Meijer 2014; Li et al. 2014; Marincowitz et al. 2015; da Silva et al.
2015), a classificação infragenética de espécies já descritas ainda permanence inconsistente.
Os faloides possuem distribuição geográfica ampla, porém acredita-se que a maior
diversidade deste grupo seja encontrada em regiões tropicais e subtropicais (Hawksworth
2001). De fato, vários registros e novas espécies vêm surgindo nos últimos anos para África
tropical (Dring 1964; Calonge & Kreisel 2002; Desjardin & Perry 2009; Marincowitz et al.
2015), Ásia e Oceania tropicais (Kasuya 2007; Kasuya 2008; Hosaka 2010; Li et al. 2014;
Rebriev et al. 2014), América Central (Saénz & Nassar 1982; Calonge et al. 2005; Hemmes &
Desjardin 2009), e América do Sul (Baseia et al. 2003; Baseia & Calonge 2005; Gómez &
Gazis 2006; Ottoni et al. 2010; Cheype 2010; Cabral et al. 2012; Cortez et al. 2011; da Silva
et al. 2015). No entanto, no Brasil esses estudos estão restritos basicamente à Mata Atlântica,
de forma que dados sobre outras regiões que englobam ecossistemas diferentes são
inexistentes ou escassos.
A floresta Amazônica, que representa 62% da área total do território brasileiro, é
mundialmente reconhecida pela alta biodiversidade, e ainda assim a micobiota é pouco
4
conhecida. Na Amazônia brasileira, até 2012 havia apenas dois registros de Phallales, as
espécies Phallus indusiatus e Mutinus caninus para o Estado de Rondônia (Trierveiler-Pereira
et al. 2009; Trierveiler-Pereira et al. 2011), além de 21 registros no Herbário INPA para a
família Phallaceae. A Amazônia vem sendo continuamente desmatada e a consequente
fragmentação do habitat é preocupante para a manutenção da biodiversidade, uma vez que
contribui para a perda de espécies endêmicas e não descritas, além das espécies descritas
(Fearnside 2005; Haddad et al. 2015). A fragmentação de habitat também é uma ameaça para
a micobiota (Dahlberg et al. 2010; Dahlberg & Mueller 2011), e por isso é importante obter
informações sobre a diversidade e distribuição de fungos em áreas ameaçadas, como áreas de
floresta Amazônica.
A falta de dados sobre a biodiversidade possui implicações significantes em vários
aspectos, como hipóteses filogenéticas, relações coevolutivas e interpretações de padrões
biogeográficos (Mueller & Schmit 2007). O desenvolvimento de estudos de fungos faloides
em áreas sub-amostradas e ameaçadas, como a floresta Amazônica, com uma abordagem
englobando dados morfológicos e de filogenia molecular, possibilitará um maior
entendimento da diversidade e evolução do grupo. Isso permitirá também estudos de revisões
taxonômicas, como também a delimitação confiável de espécies para apoiar posteriores
estudos de ecologia e conservação, assim como a avaliação da biodiversidade (Begerow et al.
2010).
Os quatro capítulos apresentados a seguir contribuem para o conhecimento da diversidade
de fungos faloides da Amazônia brasileira, e demonstram como o conhecimento dessa
diversidade altera o atual entendimento da sistemática e evolução da ordem Phallales em
níveis infragenéricos. Os resultados aqui apresentados consistem nos primeiros levantamentos
de fungos faloides para a Amazônia brasileira, aliando dados morfológicos e moleculares.
5
Objetivos
O objetivo geral deste trabalho foi integrar espécies amazônicas à filogenia molecular
da ordem Phallales com sequências diagnósticas padrões e DNA barcodes.
Os objetivos de cada capítulo foram os seguintes:
• Capítulos 01 e 02 - Caracterizar morfo e molecularmente as espécies de fungos
gasteroides, principalmente da ordem Phallales, coletadas em períodos chuvosos
ao longo dos rios Amazonas-Solimões e Negro-Madeira;
• Capítulo 03 – Analisar a diversidade observada entre espécimes do gênero
monotípico Xylophallus por meio de análises morfológicas aliadas a métodos de
delimitação de espécies utilizando sequências de cinco regiões gênicas.
• Capítulo 04 - Avaliar a diversidade morfológica e molecular de Phallus
indusiatus sensu lato coletados no Brasil, principalmente na Amazônia brasileira.
6
Capítulo 01
___________________________________________________________________________
Cabral, T.S.; da Silva, B.D.B.; Ishikawa, N.K.;
Alfredo, D.A.; Braga-Neto, R.; Clement, C.R.;
Baseia, I.G. 2014. A new species and new
records of gasteroid fungi (Basidiomycota)
from Central Amazonia, Brazil. Phytotaxa,
183(4), pp.239–253.
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Capítulo 02
___________________________________________________________________________
Cabral, T.S., Clement, C.R. & Baseia, I.G., 2015.
Amazonian phalloids: new records for Brazil
and South America. Mycotaxon, 130(2),
pp.315–320.
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Capítulo 03
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Multiple Loci and Morphology Reveal a New
Species and Hidden Diversity in the Genus
Xylophallus (Phallomycetidae, Basidiomycota).
Tiara S. Cabral, María P. Martín, Charles R.
Clement, Kentaro Hosaka, Iuri G. Baseia.
Manuscrito submetido para PLoS ONE
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31
2 Full title: Multiple Loci and Morphology Reveal a New Species and Hidden Diversity in
5 Tiara S. Cabral1*, María P. Martín2, Charles R. Clement3, Kentaro Hosaka4, Iuri G. Baseia5
11 4. Department of Botany, National Museum of Nature and Science, Tsukuba, Ibaraki, Japan.
12 5. Departament of Botany and Zoology, Universidade Federal do Rio Grande do Norte, Natal,
14 *Corresponding author
15 E-mail: ttiara@gmail.com
16
17 Abstract
18 The Amazon rainforest is the largest Neotropical Biome and plays essential roles in terrestrial
19 carbon storage and climate. Although it is a highly biodiverse biome, the diversity and
20 distribution of fungal species are still poorly known. The Amazon rainforest is being
21 deforested and habitat fragmentation is a serious threat for mycobiota. Information about
22 fungal species diversity and distribution in Amazonia is important, as they are important
23 ecosystem members. Xylophallus xylogenus is the smallest phalloid fungi described to date.
25 During field trips in Amazonia, morphological variations within Xylophallus were observed.
26 We evaluated six independent DNA regions and their power for discovering putative species
32
27 in the genus. We also estimated the species trees for the genus and we tested competing
28 models of species delimitation using Bayes factors. The results reveal the presence of three
29 evolutionary units within the genus: one is X. xylogenus; another is a new species; and the
30 third appears to be a cryptic species – the first recorded in phalloid fungi. These findings
32
33 1. Introduction
34 The Amazon rainforest is the largest Neotropical Biome and contains about 40% of
35 the world’s remaining tropical rainforests. The forest plays essential roles in terrestrial carbon
36 storage and climate, and is a highly diverse biome, hosting about 25% of world’s known
37 biodiversity (Malhi et al. 2008). Nonetheless, the diversity and distribution of numerous taxa
38 are still poorly known or unknown in Amazonia (Fearnside 2006). This is particularly true for
39 the megadiverse kingdom Fungi, which is species-rich in tropical forests, and plays a crucial
40 role in ecosystem functions (Hawksworth & Rossman 1997; Arnold & Lutzoni 2007;
42 The Amazon rainforest has been continuously deforested for economic reasons (Fearnside
44 biodiversity, since it contributes to the loss of known, endemic and undescribed species
45 (Fearnside 2005; Haddad et al. 2015). Since habitat fragmentation is one of the main threats to
46 fungal diversity (Dahlberg et al. 2010; Dahlberg & Mueller 2011), it is important to have
47 information about fungal species diversity and distributions in endangered tropical systems,
48 such as the Amazon rainforest. Although fungi receives limited attention in conservation
49 biology, it provides services for other organisms including humans and can act as indicators
51 fungal species diversity and distribution is important for the development of conservation
52 strategies.
54 (Peterson & Navarro-Sigüenza 1999; Agapow et al. 2004; Frankham et al. 2012; Ortega-
55 Andrade et al. 2015). Traditionally, fungal species have been described using morphological
56 characters, but this can be difficult due to the lack and/or plasticity of characters used in
57 taxonomic classification (Begerow et al. 2010; Suwannasai et al. 2013). When integrating
58 molecular and morphological data for taxonomic studies, several morphological characters
60 2012; Zamora et al. 2014), which leads to frequent changes in classification. In this way,
61 different methods of fungal species recognition have been used according to the taxonomic
64 characterized mainly by the mucilaginous and fetid spore mass that attracts dispersal agents,
65 mostly insects. They have been studied for decades and these studies have revealed great
66 diversity all over the world (Li et al. 2002; Leite et al. 2007; Desjardin & Perry 2009; da Silva
67 et al. 2015; Marincowitz et al. 2015). Phalloid fungi are very ephemeral and a small number
68 of morphological characters are used to identify species, such as basidiomata (fruit body)
69 shape and color (Dring 1980). For this reason, infrageneric classification of phalloid species is
70 often inconsistent.
71 In this paper, we describe a new species and report the existence of a geographically
72 restricted cryptic species in the genus Xylophallus (Schltdl.) E. Fisch based on morphological
73 and molecular data. To date, this genus contained only one species, Xylophallus xylogenus
74 (Mont.) E. Fisch., the smallest phalloid yet described (up to 15 mm high). It is always found
75 on rotten wood and is currently restricted to northern South America (French Guiana, Brazil,
34
76 Peru, Ecuador) and southern Central America (Costa Rica) (Trierveiler-Pereira & da Silveira
77 2012). Several attempts were made to properly classify this species: it was treated as section
79 finally a distinct genus Xylophallus (Schltdl.) E. Fisch. was erected (Fischer 1933). In the
80 most recent phylogeny of the order, based on DNA sequences of three independent genes,
81 Xylophallus is in fact an independent genus and it is closely related to the genus Mutinus
82 (Trierveiler-Pereira et al. 2014). Despite these efforts to classify it, there are still few
83 specimen records of this species (Baseia et al. 2003; Gómez & Gazis 2006; Cheype 2010;
88 Xylophallus when evaluating the power of species discovery of six independent DNA regions
89 and estimating their species trees, and combining these with morphological differences. The
90 discovery of such diversity in a rare genus restricted to Neotropical forests demonstrates the
91 importance of studying the mycobiota of under-sampled and threatened areas, and especially
92 the usage of molecular data allied with morphological analysis on the discovering of new
93 fungal taxa.
94
96 a. Taxon sampling
98 season from 2013 to 2015 along the main rivers of the Amazon basin: Negro and Madeira
99 Rivers (North and South axis) and Solimões and Amazonas Rivers (East and West axis).
100 Additional sampling was done along the lower Tapajós River, and in Cayenne (French
35
101 Guiana) (Fig 1). The specimens were collected in ombrophilous dense forests, both in old-
102 growth and secondary upland forests. Collections were authorized by Sisbio 37506/2012,
104
105 Fig 1. Geographic distribution of specimens of Xylophallus reported in this study. Blue
106 dots are specimens of Xylophallus sp. nov., red dots are specimens of Xylophallus xylogenus
107 and green dots are the X. xylogenus phylogenetic cryptic species.
108
109 We collected basidiomata randomly along pre-existing trails. An electric dryer was
110 used at 40°C during 48 hours to herborize material. After identification, the material was
112 Recently, the synonymy of Phallus pygmaeus to Xylophallus xylogenus was proposed
113 (Cheype 2010). Because the type locality of Phallus pygmaeus is the Brazilian Atlantic
114 Forest, additional specimens were requested from the herbaria Padre Camille Torrand (URM)
115 and UFRN-Fungos, and included in analyses. A specimen from Costa Rica was kindly
118 Specimens were studied at the Laboratory of Fungi Biology, Federal University of Rio
119 Grande do Norte, and at the Laboratory of Botany, National Research Institute for Amazonia.
120 Microscopes and stereoscopes were used to analyze microstructures, following traditional
121 methodology for phalloid fungi taxonomy (Baseia et al. 2006; Cortez et al. 2011; Kornerup &
122 Wanscher 1978). We made cuts from the different layers, including gleba, which were
123 mounted on separate slides with 5% KOH. The hyphae were separated until they became
124 homogenous, when they were observed under the microscope. We took 20 measurements
125 from each structure. Species identification as well as descriptions were made by observing the
126 micro and macrostructures, and were based on the specific literature for this group of fungi
127 (Calonge 2005; Kreisel 1996; Trierveiler-Pereira & da Silveira 2012). Mycological
129
130
131
134 DNA extraction was done from immature basidiomata for all specimens, following
135 Hosaka (2009). The DNA sequences were obtained for five independent regions: internal
136 transcribed spacer (ITS) from rDNA, translation elongation factor subunit 1α (EF-1α),
37
137 glyceraldehyde 3-phosphate dehydrogenase (gpd), and genes encoding the two largest
138 subunits of RNA polymerase II (rpb1 and rpb2). First, we amplified and sequenced the four
139 protein genes for part of the specimens to check for the existence of variable regions. Then,
140 we developed specific primers for Xylophallus xylogenus, targeting the variable regions of the
141 protein genes (Table 01), using Primer3 (Rozen & Skaletsky 2000). For ITS, we used the
142 primer pairs ITS5 and ITS4 (White et al. 1990). We obtained the complete data set of all five
143 genes for most of the specimens. PCR reactions had a final volume of 10 µl, each containing
144 5 µl of EmeraldAmp® MAX PCR Master Mix (2X Premix) (Takara), 0.25 µl of each primer
145 (5 mM) and 1 µl of genomic DNA. The PCR cycling parameters for all genes were 1 cycle of
146 94°C for 3 min, 35 cycles of 94°C for 35 sec, 51°C for 30 sec and 72°C for 1 min, and a final
147 extension of 72°C for 10 min. The DNA fragments were visualized in 1 % agarose gels
148 stained with GelRed™ (Biotium) under UV light. The fragments were purified using Ilustra
149 ExoProStar (GE Healthcare) and sequenced using Big Dye Terminator Cycle Sequencing Kit
150 (Applied Biosystems) with the same primer pairs used for PCR for each gene, except for ITS
151 where the primer pair ITS1/ITS4 was used (White et al. 1990). Overall, the protein genes
152 were easily amplified and sequenced; on the other hand, 21 of 78 specimens presented double
153 peaks in ITS sequence electropherograms, even though it was easily amplified. For this
154 reason, we used a clone sequencing protocol aiming to better resolve the ITS sequences for
155 some specimens. After cloning, there were at least two different copies of ITS sequences for
156 some specimens and all ribotype copies were used for gene tree reconstruction and DNA
157 barcoding analyses, but not for species delimitation with Bayes factors.
158
159 Table 1. Specific primers designed for Xylophallus.
EF-Xy2R CAAGGTCTTTCCCTTCACCA
GPD-Xy1F CCTCGACGTCAGTTACATGG
RPB1-Xy1F TAGATGGATTCGCCACACAA
RPB2-Xy1F GAAACACACAAGGAATTCAACC
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161
162 ii. DNA barcoding
163 Eletropherogram visualization, contig assembly, and alignments were made with
164 Geneious R6.1 (Biomatters Ltd.), creating five DNA sequence matrices (available at
165 TreeBase ID 18935). Five markers were analyzed for their potential for species discovery
166 (Schindel & Miller 2005) by investigating the existence of a barcoding gap (Meyer & Paulay
167 2005) in each gene database, testing several genetic distance threshold values. For this, a false
168 positive and false negative identification analysis across a range of possible genetic distance
169 thresholds calculated based on intra- and interspecific distance values, following Stefani et al.
170 (2014), was performed. For this analysis, we used the threshold optimization approach in R
171 package SPIDER (Brown et al. 2012). With this package, the genetic distance matrices using
172 p-distance for each gene were calculated. Based on these matrices, barplot graphs were
173 constructed for each gene showing the false positive (grey bars) and false negative (black
174 bars) rates of species identification according to a range from 0.1% to 2% (3% for ITS) of
175 genetic distance values. When there is no false identification, there is perfect sequence
176 assignment to species, and so there is a threshold value that can be considered as a barcode
180 trees for each gene separately and for the four protein genes concatenated. We used PAUP*
181 (Swofford 1998) for Maximum Parsimony analysis and MrBayes 3.1.2 to perform Bayesian
182 analysis (Huelsenbeck & Ronquist 2001). Substitution models were chosen in MrModelTest
183 2.3 (Nylander 2004). The outgroup was defined according to the most recent Phallales
184 phylogeny (Trierveiler-Pereira et al. 2014). In both analyses, the phylogenetic reconstruction
185 with protein genes resulted in trees with basically the same topology, where two
186 monophyletic clades could be observed, while ITS analysis showed no congruence with either
187 morphological data or protein genes phylogenetic trees (Fig S1 and S2). Interestingly, with
188 the ITS analysis, a monophyletic clade composed only of specimens from the Madeira River
189 basin was recovered. Because of this incongruence we estimated species trees using *BEAST
190 implemented in BEAST v.1.8.2 (Drummond & Rambaut 2007; Heled & Drummond 2010).
191 We also ran species delimitation analysis using Bayes factors, with the marginal likelihoods
193 For *BEAST analyses, the model K80 was chosen for EF-1α and rpb1 partitions,
194 TrNef for gpd, K80+G for ITS and rpb2, all chosen with jModelTest (Darriba et al. 2012). We
195 selected the Yule speciation process with a strict clock model (fixed rate for EF-1α and the
196 rates for other partitions estimated relative to this gene) (Drummond et al. 2006), and
197 population size as piecewise linear and constant root. We used the default values for the rest
198 of the prior settings. The analysis was run for 50 million generations, sampling at every 5000.
199 For species delimitation through model testing using Bayes factor, we used the marginal
200 likelihood estimator through a stepping-stone process with chain length of 10 million
201 generations for 100 path steps. Two different analyses were run, one considering three species
202 (model M0: X. xylogenus, X. sp. nov. and a third phylogenetic species composed of specimens
203 from Madeira river basin), and another considering of two species (model M1: X. xylogenus
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204 and X. sp. nov.). Then the calculated Bayes factors (2lnBf) were used to evaluate which model
205 best explains the data, following Grummer et al. (2014) and Kass and Raftery (1995).
206 The convergence was calculated with TRACER v1.6 (Rambaut et al. 2014) and trees
207 were summarized with TREEANNOTATOR 1.7.0 (Rambaut & Drummond 2013). All
208 phylogenetic trees were visualized and edited with FigTree v1.4.2 (Rambaut 2012) and the
210
211 Nomenclature
212 The electronic version of this article in Portable Document Format (PDF) in a work with
213 an ISSN or ISBN will represent a published work according to the International Code of
214 Nomenclature for algae, fungi, and plants, and hence the new names contained in the
215 electronic publication of a PLOS article are effectively published under that Code from the
216 electronic edition alone, so there is no longer any need to provide printed copies.
217 In addition, new names contained in this work have been submitted to MycoBank from
218 where they will be made available to the Global Names Index. The unique MycoBank number
219 can be resolved and the associated information viewed through any standard web browser by
220 appending the MycoBank number contained in this publication to the prefix
222 available from the following digital repositories: PubMed Central and LOCKSS.
223
224 3. Results
225 A total of 73 specimens were collected in Brazilian Amazonia and French Guyana,
226 and 4 more specimens from the Brazilian Atlantic rainforest and 1 from Costa Rica were
227 borrowed from herbaria. Collection localities and herbarium vouchers are described in Table
41
228 S3. We discovered one new species belonging to the genus Xylophallus based on
229 morphological and molecular data, and one new cryptic species.
231 A total of 76 specimens were used for phylogenetic analyses. We obtained 364 DNA
232 sequences in this study (72 EF-1α, 72 gpd, 76 ITS, 75 rpb1 and 69 rpb2), which were
233 deposited at Genbank – the accession numbers can be found in Table S3. The alignment
234 lengths for each gene were 303 bp for EF-1α, 791 bp for gpd, 533 bp for ITS, 525 bp for rpb1
236 The barplot graphs (Fig 2) that resulted from threshold optimization analysis show both
237 the absolute number of false positive (in grey) and false negative (in black) species
238 identifications for all five genes, considering the existence of three distinct evolutionary units.
239 In other words, this analysis showed the absence of universal barcode gaps; therefore it was
240 not possible to clearly identify the species limits through standard genetic distance
242
243 Figure 2. Barplots showing the distribution of the absolute number of false positive
244 (grey) and false negative (black) identifications across a range of pre-set threshold
245 values on the x axis. (a) Ef-1α, (b) gpd, (c) rpb1, (d) rpb2, (e) ITS.
42
246
248 The species tree, estimated based on the five-genes dataset in *BEAST, clearly
249 supports (PP>0.95) a hypothesis of three distinct evolutionary units, herein considered
250 different species (Fig 3). One of the clades is composed of the specimens distributed along the
251 upper Solimões, Amazonas and Negro Rivers, Amanã Lake, and the lower Tapajós River,
252 here named Xylophallus clavatus (blue clade in Fig 3, Fig S1 and S2; blue distribution in Fig
253 1). Its sister clade is composed of specimens of Xylophallus xylogenus sensu lato (red clade in
254 Fig 3, Fig S1 and S2; red distribution in Fig 1), distributed along the middle and lower
255 Solimões River, lower Negro River, Cayenne and the Brazilian Atlantic rainforest. Specimens
256 distributed along the Madeira River compose the third and sister clade of X. xylogenus (green
257 clade in Fig 3, Fig S1 and S2; green distribution in Fig 1). When the Bayes factor 2lnBf > 10,
258 there is strong evidence of M0 over M1 (Kass & Raftery 1995). Hence, our data set is more
259 likely to be composed of three phylogenetic species than two (Table 2).
260
261 Table 2. Xylophallus species delimitation based on five gene sequences using model
262 selection with the Bayes factor approach.
263
264
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265
266 Fig 3. Species tree based on five genes inferred by *BEAST. Values on nodes indicate
267 posterior probabilities. Scale represents the estimated site substitution rates, by units of branch
268 length.
269
270
271
272 c. Taxonomy
273 For the morphological analyzes we used the mature basidiomata of 47 Xylophallus
274 specimens. Remarkable morphological differences were found on the basidiomata and in
275 spore sizes of Xylophallus clavatus sp. nov. and Xylophallus xylogenus. On the other hand, no
276 clear morphological distinctions were observed between Xylophallus xylogenus and the
277 cryptic species. In this latter case, our assumption is exclusively based on the phylogenetic
278 tree topology and the geographic distribution of the phylogenetic cryptic species.
279
280 Xylophallus clavatus Cabral T.S., Baseia, I.G., Hosaka K., sp. nov. Fig. 4, 5.
283 Holotype: BRAZIL, Pará, Belterra, National Forest of the Tapajós (W54.92998 S2.94187), 29
284 Mar 2014, T.S. Cabral & D.L. Komura (INPA 264901). DNA sequence data (GenBank
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285 accession numbers): ITS = KU871795, rpb1 = KU871689, rpb2 = KU871723, Ef-1α =
287 Diagnosis: Xylophallus clavatus has an immature basidiome with protuberances on the
288 surface and rhizomorfs on the base. The mature basidiome has a clavate shape, up to 38 mm
289 high; receptacle is campanulate with an umbilicated depression on the apex, the pseudostipe
290 has reticulations that are deeper closer to the receptacle. Gleba is olive brown, with
292 Description: Immature basidiome globose to subglobose, with protuberances on the surface,
293 up to 8 × 6 mm, light brown (N40A99M20) on base to brown to the apex (N90A99M99),
294 rhizomorphs on the base. Mature basidiome up to 38 × 7 mm in its thickest portion when
295 fresh, clavate shape. Receptacle campanulate, smooth, with an umbilicated depression or
297 mm, cylindrical, hollow, not attached to the volva, reticulated surface with reticulations
298 deeper when closer to receptacle, white (N00A00M00), composed of ovoid to pyriform
299 pseudoparenchymatous hyphae 20–35 × 20–27 µm, hyaline in 5 % KOH (same hyphae of
300 receptacle). Volva light brown (N40A99M20) to brown (N90A99M99), with irregular
301 dehiscence, rhizomorfs at base forming a net spreading through substrate, interconnecting
302 basidiomes; external layer composed of filamentous hyphae 2.3–3.5 µm wide, hyaline in 5 %
303 KOH, sinuous, septate and with clamp connections; internal gelatinous layer composed of
305 composed of filamentous hyphae, 1.5–3.6 µm wide, thick walled, septate, hyaline in 5 %
306 KOH. Gleba olive brown (N99A50M10), gelatinous. Basidium clavate bearing 6–8 spores.
307 Basidiospores bacilar, smooth, 4.5–4.9 × 1.6–2.1 µm, greenish to hyaline in 5 % KOH.
308
45
309 Habitat and distribution: found on rotten wood, in the state of Amazonas (municipalities of
310 São Gabriel da Cachoeira, São Paulo de Olivença, Maraã, Parintins and Barcelos) and Pará
312 Specimens examined: BRAZIL, Pará, Belterra, National Forest of Tapajós (W54.92998
313 S2.94187), 25 Mar 2014, INPA 264900; 26 Mar 2014, INPA 264931; 30 Mar 2014, INPA
314 264902, INPA 264903, INPA 264904, INPA 264905, INPA 264906, INPA 264932; 31 Mar
315 2014, INPA 264933; Amazonas, São Paulo de Olivença, Monte Santo Community
316 (W68.99939 S3.49752), 7 Feb 2014, T.S. Cabral & R. Braga-Neto, INPA 264893, INPA
317 264894; Maraã, Sustainable Development Reserve Amanã (W64.62007 S2.48734), 14 Feb
318 2014, T.S. Cabral & R. Braga-Neto, INPA 264896, INPA 264897, INPA 264898; São Gabriel
320 Cabral, INPA 264927; Parintins, Açaí Community, (W2.6475 S56.5484), 5 Feb 2015, T.S.
321 Cabral, INPA 271636, INPA 271637, INPA 271638, INPA 271639, INPA 271640, INPA
322 271641, INPA 271642, INPA 271643, INPA 271644, INPA 271645, INPA 271646, INPA
323 271647; 6 Feb 2015, INPA 271648, INPA 271649, INPA 271650, INPA 271651, INPA
325
46
326
327 Fig 4. Macromorphology of Xylophallus clavatus. (a, b) Fresh basidiomata; (c) dried
328 basidiomata with a longitudinal cut through the volva, showing the pseudostipe not attached
329 to it; (d) dried basidiomata showing a minutely perforated apex; (e, f) immature basidiomata
331
47
332
333 Fig 5. Micromorphology of Xylophallus clavatus. (a) Basidiospores; (b) basidium at the
334 yellow arrow; (c) pseudoparenchymatous hyphae of pseudostipe; (d) filamentous hyphae from
335 volva.
336
337 Xylophallus xylogenus (Mont.) E. Fisch., in Engler & Prantl, Nat. Pflanzenfam., Edn 2
339 Basyonym: Phallus xylogenus Mont., Annales des Sciences Naturelles Botanique 3: 137, t.
342 broad, light brown (N40A99M20) on base to brown to the apex (N90A99M99), rhizomorphs
343 on the base. Mature basidiome up to 13 × 3 mm broad in its thickest portion when fresh.
345 apex, adnate to pseudostipe, 4.5 × 3 mm. Pseudostipe 4 × 2.5 mm, cylindrical, hollow, not
48
346 attached to the volva, reticulated surface with deep reticulations all over pseudostipe, white
348 31 µm, hyaline in 5 % KOH (same hyphae of receptacle). Volva light brown (N40A99M20)
349 to brown (N90A99M99), with irregular dehiscence, rhizomorfs at base forming a net
351 filamentous hyphae 2.1–3.2 µm wide, hyaline in 5 % KOH, sinuous, septate and with clamp
353 18–28 µm, hyaline in 5 % KOH. Rhizomorphs composed of filamentous hyphae, 1.5-3.6 µm
354 wide, thick walled, septate, hyaline in 5 % KOH. Gleba olive brown (N99A50M10),
355 gelatinous. Basidium clavate bearing 6–8 spores. Basidiospores bacilar, smooth, 3.9–4.4 ×
357
358 Habitat and distribution: found on rotten wood. Xylophallus xylogenus sensu lato is known
359 from French Guiana (Montagne 1855; Cheype 2010), French Antilles (Cheype 2010),
360 Suriname (Fischer 1933), Brazil (Baseia et al. 2003), Peru and Ecuador (Gómez & Gazis
361 2006). In this study we also found it in the state of Amazonas (municipalities of Tefé, Novo
362 Airão, Manaus, Autazes and Presidente Figueiredo), Brazil (Fig 1), and these are the first
365 (W59.712514 S3.394185), 7 Feb 2013, T.S. Cabral INPA 264875, INPA 264876, INPA
366 264877; Presidente Figueiredo (W59.987902 S2.046274), 20 Jun 2013, T.S. Cabral INPA
367 264889; Manaus, Botanical Garden (W59.947141 S3.008101), 7 Aug 2013, T.S. Cabral INPA
368 264890; Ducke Reserve (W59.966424 S2.964135), 14 Jan 2014, T.S. Cabral INPA 264892;
369 Novo Airão, São Sebastião Community (W60.47490 S2.82801), 12 Apr 2014, T.S. Cabral
370 INPA 264907, INPA 264908, INPA 264909, INPA 264910; 13 Apr 2014, T.S. Cabral INPA
49
371 264911, INPA 264912, INPA 264935, INPA 264913, INPA 264914, INPA 264915; Centro
373 Apr 2014, T.S. Cabral INPA 264916, INPA 264917, INPA 264918, INPA 264919, INPA
374 264920, INPA 264921; 16 Apr 2014, T.S. Cabral INPA 264922, INPA 264923, INPA
375 264924, INPA 264925; Tefé (W64.623400 S3.482733), 11 Mar 2015, T.S. Cabral INPA
376 271653; Pernambuco, Recife, Trierveiler-Pereira URM 80262, URM 44245, URM 44244;
377 Gurjaú, Estação Ecológica de Gurjaú (W35.569445 S8.597222), 28 Jun 2002, Baseia, I.G.;
378 Gibertoni, T.B.; UFRN-Fungos 458, as Phallus pygmaeus. FRANCE, French Guiana, Rémire-
379 Montjoly (W52.286944 N4.93925), 13 Mar 2013, T.S. Cabral INPA 264878.
380 Additional specimens examined from the cryptic species: BRAZIL, Amazonas, Humaitá, Road
381 to Ipixuna (W063,11626 S07,57440), 4 Apr 2013, T.S. Cabral & R. Braga-Neto INPA
382 264879; Barreira do Tambaqui Community (W062,88771 S07,85109), 5 Apr 2013, T.S.
383 Cabral & R. Braga-Neto INPA 264880; Acará Lake - Terra Preta Community (W062,42154
384 S06,37516), 8 Apr 2013, T.S. Cabral & R. Braga-Neto INPA 264881; Acará Lake – São
385 Francisco Community (W062,45149 S06,35173), 9 Apr 2013 T.S. Cabral & R. Braga-Neto
386 INPA 264882; Manicoré, Barro Alto Community (W061,48618 S05,97782), 11 Apr 2013,
387 T.S. Cabral & R. Braga-Neto INPA 264883; Jatuarana Lake - Castanhal do Galeno
388 (W061,43864 S05,73451), 12 Apr 2013, T.S. Cabral & R. Braga-Neto INPA 264884; Boca
389 do Rio Community (W061,32956 S05,87370), 13 Apr 2013, T.S. Cabral & R. Braga-Neto
390 INPA 264885, INPA 264886; Novo Aripuanã, São Félix Community, 16 Apr 2013, T.S.
392
393 Fig 6. Xylophallus xylogenus. (a, b) Fresh mature basidiomata; (c) Immature basidiomata
394 fresh and (d) dried showing the smooth surface; (e) basidiospores; (f) pseudoparenchymatous
51
395 hyphae of pseudostipe; (g) clavate basidia; (h) a representative of the Xylophallus cryptic
397
398 4. Discussion
399 This study aimed to investigate infrageneric diversity of Xylophallus through
400 morphological and molecular phylogenetic methods. The results reveal the presence of three
401 evolutionary units belonging to the genus. One is the known species X. xylogenus, and
402 another corresponds to a morphologically different and new species in the genus, named
403 Xylophallus clavatus. A third clade can also be distinguished by the ITS rDNA marker, with
404 geographic distribution restricted to the lower Madeira River basin, although specimens of
406 The morphological analysis revealed two different morphospecies, with one
408 confusing (see Trierveiler-Pereira & da Silveira (2012) for details), this is a valid name,
409 following the International Code of Nomenclature for Algae, Fungi, and Plants. Since the
410 holotype is from Cayenne, French Guiana (Montagne 1855), and in our study the specimen
411 from this type locality clustered in the red clade (Fig 3, Fig S1 and S2), we believe that this
412 clade corresponds to Xylophallus xylogenus sensu lato. In fact, comparisons between
413 specimens of X. xylogenus obtained in this study and those published earlier (Cheype 2010;
414 Trierveiler-Pereira & da Silveira 2012; Baseia et al. 2003; Fischer 1898) confirm that they all
415 belong to the same species, with a congruence between morphological characters and
416 geographic distribution. Cheype (2010) proposed the synonymy of Phallus pygmaeus to
417 Xylophallus xylogenus, which was sustained by Trierveiler-Pereira & da Silveira (2012). In
418 our analysis we also included specimens from the Atlantic rainforest published as Phallus
52
419 pygmaeus (Baseia et al. 2003), and they clustered in the X. xylogenus clade (Fig S1 and S2),
420 which confirms that both taxa belong to the same species.
422 macroscopically characterized by its larger basidiomata size, the immature basidiomata
423 surface with protuberances, the clavate shape of the mature basidiomata and the pseudoestipe
424 formed by relatively shallow reticulations (Fig 4). On the other hand, X. xylogenus is smaller,
425 the immature basidiomata has a smooth surface, the mature basidiomata is fusiform, and the
426 pseudostipe is formed by deeper reticulations (Fig 6). Microscopically, they differ mainly by
427 basidiospore sizes: in X. clavatus the basidiospores are 4.5-4.9 µm in length, while in X.
428 xylogenus basidiospores are 3.9-4.4 µm (Figs 5 and 6). Sáenz et al. (1972) provided a very
429 detailed description of specimens from Costa Rica. In our analysis, the Costa Rican specimen
430 grouped in the new species clade. We found morphological similarities between the author’s
431 description and the specimens of X. clavatus analyzed here, such as mature and immature
432 basidiomata sizes, immature basidiomata surface with protuberances and basidiospore sizes.
433 In fact, the authors claim that their results are somewhat different from those previously
434 published, which now can be explained since previous papers were dealing with X. xylogenus
437 between X. xylogenus and the third clade (green on Fig 3), which corresponds to a cryptic
438 species. So far, the only noticeable difference between these two species is their distributions.
439 X. xylogenus can be found both in the Atlantic and Amazon forests, while the cryptic species
440 seems to be restricted to the lower Madeira River basin in the Amazon forest domain.
441 Bickford et al. (2007) consider kingdom Fungi as a group with high probabilities of harboring
442 cryptic species. Indeed, several examples of cryptic speciation in Fungi can be found in the
443 literature with many of them in Basidiomycota (Ramirez-Lopez et al. 2015; Sheedy et al.
53
444 2013; Harder et al. 2013; Stefani et al. 2014; Geml et al. 2006), but the present one is the first
445 example of a cryptic species in phalloid fungi recorded to date. The discovery of cryptic
446 diversity in fungi has significance in species and habitat conservation, and this specific case
447 shows the importance of studying little-studied taxa and under-sampled areas. In addition to
448 the already known X. xylogenus, a new species and a cryptic one were found, all endemic to
449 Neotropical forests, more specifically to old-growth dense forests. The destruction of habitats
450 caused by deforestation could lead to the loss of the Neotropical fungal species yet to be
451 discovered, especially those with very specific habitats. Therefore, more efforts should be
452 spent to study the mycobiota of Neotropical forests so that conservation programs can take
453 into account the existence of this so far hidden fungal diversity.
454 DNA barcode methodology is an elucidative tool for taxonomists. There are two different
455 goals when using DNA barcodes: species identification, when the goal is to assign a name to
456 an unknown specimen using DNA data from correctly identified species; and species
457 discovery, when the goal is to sort collections into species-like units (Schindel & Miller 2005;
458 Collins & Cruickshank 2013). One of our aims in this study was to investigate the resolution
459 power of the barcoding methodology in our dataset, which could be used to avoid
460 misidentification of specimens collected in the future. The threshold optimization approach
461 showed only false negative and positive identifications for all genes used herein, which means
462 that these genes did not present a clear barcoding gap (Brown et al. 2012). Several reasons
463 can be suggested for the lack of a barcoding gap, such as incorrect taxonomy, hybridization,
464 radiation and incomplete lineage sorting (Pino-Bodas et al. 2013). Considering the recent
465 speciation in Xylophallus, as evidenced by the short branch lengths, the lack of a barcoding
466 gap can be explained by incomplete lineage sorting of the gene sequences analyzed, which
467 can also explain the inconsistency between gene trees and species trees (van Velzen et al.
468 2012). Schoch et al. (2012) tested 6 DNA regions and showed that the nuclear ribosomal
54
469 internal transcribed spacer (ITS) was the most suitable region for species delimitation in
470 Fungi, but the authors acknowledge that supplementary barcodes can be developed for
471 specific cases. In fact, there are several studies demonstrating that ITS by itself might fail to
472 delimitate species in some fungi (Pino-Bodas et al. 2013; Bellanger et al. 2015; Li et al. 2013;
473 Lücking et al. 2014; Lindner & Banik 2011), just as it fails to define boundaries between the
474 Xylophallus species based on genetic distance. The lack of a barcoding gap for each gene
475 makes it impossible to define a threshold for delimiting species in Xylophallus; therefore an
476 alternative methodology that is not based on genetic distances was used.
477 Using independently evolving markers in a species tree approach with selection of the best
478 model of species delimitation using Bayes factors seems to be useful in understanding the
480 was recently proposed and estimates marginal likelihoods for each competing model, from
481 which Bayes factors are calculated and compared (Grummer et al. 2014). Although this
482 method can test only a limited number of hypotheses (Yang & Rannala 2014), it still has great
483 advantages, like the ability to compare models with different numbers of species or different
484 assignments of individuals to species. In our dataset, the species delimitation model of three
485 species was selected over the model of two species, partially agreeing with morphological
486 data and fully with geographical data. In the same way, Bryson et al. (2014) were able to
487 delimit species in a group of snakes using model selection with Bayes factors, where two
488 groups were recognized as new species in concordance with morphological data. This
489 methodology is elucidative using either DNA sequences or genome-wide SNP data (Leaché et
490 al. 2014), indicating that it can be a promising method for species delimitation based on
492 The species tree inferred indicates that the previous taxonomy of Xylophallus does not
493 reflect its evolutionary history. This genus is actually composed of at least three species,
55
494 where X. xylogenus is a sister species of a cryptic species, and X. clavatus is a sister clade of
495 these two. The cryptic species shares the same morphological characters with X. xylogenus,
496 probably because of its recent speciation. On the other hand, the speciation between X.
497 xylogenus and X. clavatus is older, so morphological differences accumulated over time.
498 Although it seems to be a true species, we have decided to not provide a description of the
499 cryptic species using the available data, until more specimens can be added for future
500 analysis.
501 The speciation event that gave rise to X. clavatus and X. xylogenus is older than the split
502 between the latter and the cryptic species. Since it is not possible to reliably date the
503 phylogenetic trees, it is hard to infer the most probable geological scenario in which this
504 speciation may have occurred. Still, the lower Madeira River basin might have played an
505 important role in the speciation between Xylophallus cryptic species and X. xylogenus, since
506 the cryptic species is so far restricted to that area. The distribution of Xylophallus cryptic
507 species is on both sides of Madeira River and no other large river seems to be a barrier for
508 other Xylophallus species’ distributions. Given the statimospory nature of Xylophallus spore
509 liberation, its dependence on insects for dispersal and the lack of barriers due to major rivers,
510 we do not yet have a hypothesis to explain the divergence of the cryptic species from X.
512
513 5. Conclusions
514 The species tree allied with methods of species delimitation and morphological data
515 analyses provide strong evidence for the existence of more than one species in the genus
516 Xylophallus. Besides the already known X. xylogenus, this genus harbors a new species
517 named X. clavatus, and a third cryptic species with a very restricted geographic distribution.
518 We provide enough morphological characters to differentiate the newly described species, but
56
519 we have decided to not formally describe the cryptic species until more collections from un-
520 sampled areas are available. All four genes show the same phylogenetic history with the
521 presence of two species, but the ITS region shows the existence of a third species. This
522 difference certainly reflects the lower mutation rates of the protein coding genes. Due to the
523 recent diversification, it was not possible to delimit species in the genus based only on genetic
524 distances of the five DNA regions used in this paper, so a method of species delimitation
525 model selection was used. This shows the importance of testing different DNA regions along
526 with the ITS region, the proposed barcode for fungi, and to test alternative methods of species
527 delimitation. This study demonstrates the importance of studying fungal diversity of
529
530 Acknowledgments
531 We thank Dr. Doriane Picanço Rodrigues for coordination of the Laboratory of Applied
532 Evolution at Federal University of Amazonas, where part of the molecular data were obtained.
533 We thank Dr. Rupert Collins for his insights on species delimitation methods, and Ricardo
534 Braga Neto and Dirce Leimi Komura for collecting support. We are extremely grateful for all
535 residents of the communities visited along the rivers of the Amazon basin, who supported us
537
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63
841 (D) rpb1, (E) rpb2, (F) concatenated protein genes tree. Only PP>0.9 is shown on nodes.
842 Trees A, E and F were rooted using sequences available on Genbank; B, C and D were
844 Figure S2. Phylogenetic trees obtained with Parsimony analysis. (A) EF-1α, (B) gpd, (C)
845 ITS, (D) rpb1, (E) rpb2, (F) concatenated protein genes tree. Only PP>80 is shown on nodes.
846 Trees A, E and F were rooted using sequences available on Genbank; B, C and D were
848 Table S3. Specimens used in this study. Their localities, herbarium vouchers and Genbank
850
65
Capítulo 04
________________________________________________________________
Phallus indusiatus sensu lato from Brazilian
rainforests
Tiara S. Cabral, Bianca D. B. da Silva, María P.
Martín, Charles R. Clement, Kentaro Hosaka, Iuri G.
Baseia.
Manuscrito em preparação
66
35 within the genus (Kreisel 1996; Calonge 2005). As a reflex of these taxonomic uncertainties,
36 a great number of synonyms are reported for several species of this clade. Phallus indusiatus
37 is an emblematic example, where at least nineteen synonyms and several distinct forms have
38 being described for this species (Liu 1984; Kreisel 1996; Calonge 2005; Cheype 2010; Lloyd
39 1909; Guzmán et al. 1990; Das et al. 2007).
40 Due to lack of resolution when using morphological characters to identify Phallus
41 species, we believe that several specimens that have been identified as P. indusiatus might
42 actually consist of independently evolving entities. In fact, some new species with minimal,
43 yet noticeable morphological differences from P. indusiatus, have been proposed. For
44 instance, P. serrata, recently described for China, differs by the meshes of the indusium with
45 serrate edges (Li et al. 2014); P. echinovolvatus has a whitish volva with mycelioid
46 projections on the surface (Zang et al. 1988); and P. flavidus, described for the Seychelles, has
47 yellowish pigments on the receptacle and indusium (Kreisel & Hausknecht 2009). At least
48 three species resembling P. indusiatus were described for Brazil, P. moelleri, P. callichrous
49 and Dictyophora phalloidea; however, they are considered synonyms by some authors (Lloyd
50 1909; Calonge 2005; Kreisel & Hausknecht 2009). Some of these species hypotheses were
51 made in concordance with molecular data, which reinforce the importance of this kind of
52 analysis to solve these taxonomic uncertainties.
53 Phallus indusiatus was described by Étienne Pierre Ventenat in 1798, based on a
54 specimen from Suriname. In 1809, Desvaux created a new genus, Dictyophora, mainly
55 characterized by the presence of an indusium, a skirt-like structure that expands from the
56 receptacle toward the ground. Ventenat’s species was transferred to Dictyophora, and named
57 D. indusiata. Kreisel (1996) considered that the importance of an indusium for the taxonomy
58 of the genus was overestimated, hence he downgraded Dictyophora as a section of Phallus.
59 More recently, with the introduction of molecular data to the systematics and taxonomy of
60 fungi, studies have shown that the indusium is a recurrent character, which independently
61 emerged several times during the evolution of the group (Hosaka et al. 2006; Cabral et al.
62 2012; Marincowitz et al. 2015). Today, Phallus indusiatus is the valid name for Ventenat’s
63 species. P. indusiatus seems to be widespread in Brazil, with records from four of the six
64 Brazilian biomes (Magnago et al. 2013), but information concerning its diversity and
65 distribution is still insufficient.
66 In this study, we examined the P. indusiatus-like species diversity in Brazilian
67 Amazonia. We show a clear congruence between detailed morphological data and ITS based
68 phylogenetic analysis, and describe three new species within the indusiate clade. These results
68
69 highlight the importance of more detailed investigation with the inclusion of non-traditional
70 molecular information in Neotropical fungi.
71
72 MATERIAL & METHODS
73 Morphological data — A total of 26 specimens of Phallus sp. with white indusium were
74 collected during the rainy seasons of 2013 to 2015 in various areas of the Amazon Rainforest
75 domain. We included in the analyses four additional specimens with the name “Phallus
76 indusiatus” borrowed from the Herbarium of the Instituto de Botânica (São Paulo) and the
77 Universidade Federal de Rio Grande do Norte-Fungos, which were collected in various areas
78 of the Atlantic Rainforest domain. A specimen identified as P. indusiatus f. rosea collected in
79 southern Brazil was also included to determine its phylogenetic placement and if it should be
80 considered as a separate species, as Kasuya et al. (2008) observed with P. luteus. Other
81 Phallus species were included in molecular analysis to increase taxon coverage (Table 1).
82 Species were morphologically described based on fresh and dried material. Macroscopic
83 characters were described based on field notes and photographs, while microscopic data were
84 obtained by mounting slides with fragments from different layers and structures of dried
85 basidioma in 5% KOH. We followed the specific literature for species identification (Lloyd
86 1909; Calonge 2005; Kreisel & Hausknecht 2009; Kreisel 1996).
87 DNA extraction, amplification and sequencing — DNA extraction followed Hosaka (2009).
88 The internal transcribed spacer (ITS) region was amplified using ITS4/ITS5 primers, and
89 DNA fragments were visualized in 1 % agarose gel stained with GelRed™ (Biotium) under
90 UV light. The fragments were purified using Ilustra ExoProStar (GE Healthcare), and then
91 sequenced using Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems), with
92 ITS1/ITS4 primers (White et al. 1990). After sequencing, some electropherograms presented
93 double peaks; in order to resolve these, the PCR fragments were cloned. All ribotypes were
94 included in the phylogenetic analysis.
95 Molecular phylogenetic analyses — We submitted each sequence to a BLAST search to
96 identify the closest relatives and to check for possible contamination. The closest sequences
97 resulting from the BLAST search and sequences with genus names of Phallus or Dictyophora
98 were retrieved from Genbank and added to the dataset. All sequences were aligned and
99 manually edited with Geneious R6.1 (Biomatters Ltd.). The final aligned matrix contained
100 538 characters and was used to infer phylogenetic trees. Species of the genus Mutinus were
101 chosen as outgroups, based on previous phylogenies (Trierveiler-Pereira et al. 2014).
102 Maximum Parsimony (MP) analysis was performed under heuristic searches with the TBR
69
103 branch-swapping algorithm; the initial tree was obtained by stepwise addition of random
104 additional sequences repeated 100 times and 1000 replicates as bootstrap (bs) settings. For
105 Bayesian analysis (BA), the substitution model of evolution was chosen with MrModelTest
106 (Nylander 2004). Two parallel runs were executed with four incrementally-heated
107 simultaneous MCMC simulations over 3 million generations, with trees sampled every 1000
108 generations. The consensus tree was reconstructed with the remaining trees after the burn-in
109 stage, which was defined based on the average standard deviation of split frequency values.
110 The confidence values were estimated with posterior probabilities (pp). Trees were visualized
111 and edited in FigTree version 1.4.2. All data are available in TreeBASE under ID XXXX.
112
113 RESULTS
114 A total of 30 recently collected specimens of Phallus sp. with white indusium were
115 studied, 26 of which were collected in Brazilian Amazonia, while four other specimens were
116 collected from the Brazilian Atlantic Rainforest (SP and UFRN-Fungos herbaria) (Fig. 01).
117 The collection localities, herbarium vouchers and Genbank accession numbers can be found
118 in Table 01.
119 Phylogenetic analyses - The DNA extraction, and ITS amplification and sequencing were
120 performed for all specimens, but only 25 specimens yielded good clean sequences. Both
121 Maximum Parsimony and Bayesian analyses resulted in trees with similar topology (Fig. 02).
122 For Maximum Parsimony analysis, of the 538 characters, 299 were informative and resulted
123 in a most parsimonious tree with 1392 steps (CI = 0.518, RI = 0.881, RC = 0.457). The
124 Brazilian specimens of Phallus with white indusium grouped in a monophyletic clade (PP=1,
125 BT=96). This clade could be divided into five clades with strong support values (PP>0.95,
126 BT>92), which correspond to the morphospecies identified and described here. These five
127 clades are closely related to P. cinnabarinus, also found in Amazonia. Based on both
128 morphological and molecular analyses, we described three new species and one new variety.
129 Sequences under the name P. indusiatus (and Dictyophora indusiata) retrieved from
130 Genbank, all from Asia (China and Japan), as well as those collected by us in this study, form
131 a paraphyletic clade with intercontinental disjunct distributions. We believe that the Brazilian
132 clade actually corresponds to the P. indusiatus sensu stricto (brown in Fig. 02), based on
133 morphological similarities and the geographical proximity to the type locality (Suriname) of
134 the Amazonian specimens collected that nested in the clade.
135
70
136
137 Fig. 01. Currently known distributions of the new Phallus species described in this study.
138 Colored areas are the Brazilian Biomes (Anon n.d.)(Anon n.d.)(Anon n.d.)(IBGE 2016):
139 Amazonian Rainforest (dark green), Cerrado (brown), Caatinga (beige), Atlantic Rainforest
140 (light green), Pantanal (dark blue), Pampa (light blue).
71
141
72
142 Fig. 02. Phylogenetic tree obtained by Bayesian analysis. Colored clades correspond to
143 Brazilian species: in blue, P. amazonicus; in green, P. purpurascens; in red, P. squamulosus;
144 in brown, P. indusiatus sensu stricto. Posterior probability values are on the nodes. The black
145 dots indicate specimens under Phallus indusiatus deposited in Genbank and downloaded for
146 this study.
147
148 TAXONOMY
149 Phallus amazonicus T.S.Cabral, B.D.B.Silva & Baseia, sp. nov. Fig. 03
150 Mycobank XXX
151 Diagnosis: Basidiome with completely epigeous development. Receptacle campanulated, but
152 slightly constricted at the base, pale yellow, reticulated, with a prominent apical pore.
153 Indusium extending to 2/3 of pseudostipe, poorly developed, white. Volva with small
154 projections on surface, white, epigeous, hyphae with clamp connections. Gleba olive-brown,
155 mucilaginous.
156 Typification: BRAZIL. Amazonas: Barcelos, Bacabal Community (S0.49004 W62.93089), 7
157 April 2015, Cabral TS (holotype INPAXXXX TSC266). Isotype UFRN-FungosXXX.
158 Genbank accessions: XXX.
159 Immature basidiomes not observed. Fresh expanded basidiome 120 mm high.
160 Receptacle 25 × 25 mm, campanulate but slightly constricted at the base, with a prominent
161 apical pore, deeply reticulated surface, reticulations up to 3.9 x 2.4 mm, whitish to pale
162 yellow (N00A20M00) when gleba is absent. Pseudostipe 81 × 22 mm, cylindrical, spongy,
163 white (N00A00M00), composed of globose to elongate-ovoid pseudoparenchymatous hyphae,
164 20.5–60.8 × 17.5–51.2.5 µm, hyaline. Indusium extending to 2/3 of pseudostipe, white
165 (N00A00M00), 57 mm in length, poorly developed, with polygonal to irregular meshes up to 12
166 × 4 mm, attached to the apex of the pseudostipe. Volva white (N00A00M0), with small hyphae
167 projections on surface, epigeous, formed by filamentous hyphae, septate, branched, hyaline,
168 clamp connections present, 2.5–3.5 μm diameter. Rhizomorphs composed of at least two
169 types of hyphae: filamentous, thin walled hyphae, with clamp connections, and thicker hyphae
170 (2.5-4.5 µm) that seem to communicate with each other by pores on the inflated tips. Crystals
171 in globose cells were found distributed among the hyphae of volva and rhizomorphs,
172 measuring 8.2–11.5 × 6.8–10.6 μm. Gleba olive brown (N99A50M10), mucilaginous.
173 Basidiospores elongated, smooth, 3.4–4 × 1.9 – 2.4 µm, hyaline in KOH 5%.
73
174 Habitat and Distribution: on soil, in a fragment of upland old-growth forest. So far restricted
175 to Amazon rainforest, found in the municipalities of Barcelos and Parintins (State of
176 Amazonas, Brazil) and Belterra (State of Pará, Brazil).
177 Etymology: in reference to the Amazonian biome, to which the species is currently restricted.
178 Other specimens examined: BRAZIL. Amazonas: Novo Airão, São Sebastião Community, 13
179 April 2014, Cabral TS (INPA264935); Parintins, Açaí Community (S2.62665 W56.54041), 5
180 March 2015, Cabral TS (TSC248, TSC250); 6 March 2015 (TSC251, TSC253, TSC255,
181 TSC258); Barcelos, Bacabal Community (S0.49004 W62.93089), 7 April 2015, Cabral TS
182 (TSC264, TSC265); Pará: Belterra, Floresta Nacional do Tapajós, Jamaraqua Community
183 (S2.812667 W55.033083), 31 April 2014, Cabral TS (INPA264933, INPA264934,
184 INPA264928).
185 Commentary: This species is characterized by the shape and color of receptacle, the
186 conspicuous pore, the volva with protruding hyphae on the surface and the poorly developed
187 indusium. P. duplicatus Bosc. is similar to P. amazonicus by having a strongly developed
188 pore and pale yellow receptacle, and by basidiome and spore sizes (Lloyd 1909; Kreisel &
189 Hausknecht 2009); however, the indusium and volva can be white to pinkish, and the
190 indusium threads are thicker than in P. amazonicus (Kreisel & Hausknecht 2009; Martín &
191 Tabarés 1994; Coker & Couch 1928; Kibby & McNeil 2012). P. flavidus Kreisel & Hauskn.
192 can be comparable to P. amazonicus by the pale orange and conical receptacle, and the
193 indusium size; yet, P. flavidus has smaller spores (up to 3.6 x 1.8 µm), the surface of the volva
194 is light grey with an orange flush, and the indusium is cream to yellow (Kreisel & Hausknecht
195 2009). P. impudicus var. obliteratus (Malençon) Kreisel has a reticulate white receptacle and
196 a rudimentary white indusium; P. amazonicus has a poorly-developed indusium, but it is very
197 different from P. impudicus var. obliteratus, where the indusium is hidden under the
198 receptacle (Kreisel & Hausknecht 2009; Calonge 2005). P. callichrous (Möller) Lloyd is a
199 species described for Brazil, with white indusium and differs from P. amazonicus by having
200 an orange to pink receptacle and reddish-violet rhizomorphs; currently, it is considered a
201 synonym of P. indusiatus (Calonge 2005). P. echinovolvatus (M. Zang, D.R. Zheng & Z.X.
202 Hu) Kreisel is another white-indusiate species, characterized mainly by the volva covered
203 with echinulate hyphae projections; in P. amazonicus hyphae projections on the volva surface
204 can also be found, but they are smaller than in P. echinovolvatus (Zang et al. 1988). Finally,
205 in P. indusiatus the receptacle is campanulated, the immature basidiome is hypogeous, so that
206 the volva is buried under the ground when the basidiome is fully developed, the indusium is
207 completely developed reaching the ground, and the volva and rhizomorphs have pinkish
74
208 pigments (Ventenat 1798). On the other hand, in P. amazonicus the campanulated receptacle
209 is constricted at the base, the basidiome has a completely epigeous development, the indusium
210 is poorly-developed and reaches only 2/3 of the basidiome, the rhizomorphs and volva are
211 white, and the latter has small hyphae projections on the surface. P. amazonicus is so far
212 restricted to Brazilian Amazonia, and it was found in sympatry with P. indusiatus specimens
213 collected in the region of the Tapajós River. In both the Bayesian and Maximum Parsimony
214 phylogenetic trees, specimens of P. amazonicus grouped in a monophyletic clade with high
215 support values (pp=1, bs=95), in concordance with morphological data. Hence, based on
216 morphological and phylogenetic analyses, and geographic distribution, we believe that P.
217 amazonicus is in fact a new species.
75
218
219 Fig. 03. Phallus amazonicus. (a) fresh basidiome, holotype; (b) volva with small hyphae
220 projections on surface; (c) receptacle with a prominent pore and pale-yellow color (TSC 248);
221 (d) spores; (e) hyphae from volva; (f) rhizomorphs hyphae; (g) crystals in globose cells found
222 on volva.
76
223 Phallus amazonicus var. denigricans T.S.Cabral, B.D.B.Silva & Baseia, var. nov. Fig. 04.
224 Mycobank: XXX
225 Diagnosis: Basidiome with completely epigeous development. Receptacle campanulated but
226 slightly constricted at the base, pale yellow, reticulated, with a prominent apical pore.
227 Indusium extending to 2/3 of pseudostipe, poorly developed, white. Blackish volva with
228 smooth surface, epigeous, hyphae with clamp connections. Gleba olive-brown, mucilaginous.
229 Typification: BRAZIL. Amazonas: Maraã, Reserva de Desenvolvimento Sustentável do
230 Amanã, Ubim Community (S02.50500 W064.66039), 15 Feb 2014, Cabral TS (holotype
231 INPAXXXX TSC115). Isotype UFRN-FungosXXX. Genbank accessions: XXX.
232 Immature basidiomes not observed. Fresh expanded basidiome 98 mm high. Receptacle
233 26 × 19 mm, campanulate but slightly constricted at the base, with a prominent apical pore,
234 deeply reticulated surface. Pseudostipe 54 × 10 mm, cylindrical, spongy, white (N00A00M00),
235 composed of globose to elongate-ovoid pseudoparenchymatous hyphae, 18.5–65.5 × 19–52.5
236 µm, hyaline. Indusium extending to 2/3 of pseudostipe, white (N00A00M00), 53 mm in length,
237 polygonal to irregular meshes up to 13 × 8 mm, attached to the apex of the pseudostipe. Volva
238 brownish (N60A60M50), with smooth surface, epigeous, formed by filamentous hyphae,
239 septate, branched, hyaline, clamp connections present, 1.8–5 μm diameter, with inflated ends
240 up to 15.5 μm diameter. Rhizomorphs composed of at least two types of hyphae: filamentous,
241 thin-walled hyphae, with clamp connections, and thicker hyphae (7-16 µm) that seem to
242 communicate with each other by pores on the inflated tips. Gleba olive brown (N99A50M10),
243 mucilaginous. Basidiospores elongated, smooth, 3.6–4.6 × 2.2-2.5 µm, hyaline in KOH 5%.
244 Habitat and Distribution: on soil, in a fragment of upland old-growth forest. Found in
245 municipalities of São Gabriel da Cachoeira and Maraã (State of Amazonas, Brazil)
246 Etymology: in reference to the blackish volva.
247 Other specimens examined: BRAZIL. Amazonas: São Gabriel da Cachoeira, Itacoatiara
248 Mirim Community, (S0.304167 W66.8403), 1 April 2013, Komura DL (INPAXXXX
249 DLK1119).
250 Commentary: The main differences between this variety and P. amazonicus sensu stricto are
251 the blackish volva with smooth surface, the larger spores (up to 4.6 x 2.5 µm), and no crystals
252 were found on the volva or rhizomorphs. The two specimens with white indusium and
253 blackish volva from our collection grouped within the P. amazonicus clade with high support
254 values (pp=1, bs=98). For this reason and because there are few differences between the two
255 groups, we decided to describe it as P. amazonicus var. denigricans. A similar case can be
256 found between P. merulinus (Berk.) Cooke and P. atrovolvatus Kreisel & Calonge. These two
77
257 species are very similar, differing by the volva color, that is black in P. atrovolvatus and white
258 in P. merulinus (Francisco Diego Calonge et al. 2005). So far, there are no studies with DNA
259 data that allows determining the species boundaries between P. atrovolvatus and P.
260 merulinus.
261
262 Fig. 04. Phallus amazonicus var. denigricans fresh basidiome. a. whole basidiome; b.
263 blackish and smooth volva in detail. Bars = 20 mm. (c) spores; (d) pseudoparenchymatous
264 hyphae of pseudostipe; (e) hyphae from rhizomorphs; (f) hyphae from volva.
265
266 Phallus purpurascens T.S.Cabral, B.D.B.Silva & Baseia, sp. nov. Fig. 05
267 Mycobank: XXX
268 Diagnosis: Basidiome with partially epigeous development. Receptacle conic, thimble-like,
269 flat at the apex, white, strongly reticulated, with an apical pore. Indusium extending to 2/3 of
270 pseudostipe, white. Volva white, becoming purplish when exposed, with a smooth surface,
78
271 semi-hypogeous, hyphae with clamp connections; purplish rhizomorphs at the base. Gleba
272 olive-brown, mucilaginous.
273 Typification: BRAZIL. Amazonas: Manaus (S3.0615 W60.0111), 27 Feb 2014, Cabral TS
274 (holotype INPAXXXX TSC234). Genbank accessions: XXX. Mato Grosso: Sinop, Parque
275 Florestal de Sinop (S11.8359 W55.5008), 7 Nov 2013, Cabral TS (paratype SINOP27).
276 Immature basidiomes whitish (N60A60M50) with purplish pigments (A10M10C10), globose
277 to subglobose, 56 x 43 mm, growing gregariously. Fresh expanded basidiome up to 200 mm
278 high. Receptacle 45 × 29 mm, thimble like, flat at the apex, with an apical pore, strongly
279 reticulated surface, shallow reticulations up to 3.2 x 1.7 mm, white (N00A00M00). Pseudostipe
280 122 × 21 mm, cylindrical, spongy, white (N00A00M00), composed of globose to elongate-ovoid
281 pseudoparenchymatous hyphae, 37–65.5 × 22.5–48 µm hyaline. Indusium extending up to 2/3
282 of the pseudostipe, white (N00A00M00), 100 mm in length, polygonal meshes up to 10 × 5 mm,
283 attached to the apex of the pseudostipe. Volva white (N00A00M00) becoming purplish
284 (A10M10C10) when exposed, with a smooth surface, semi-hypogeous, formed by filamentous
285 hyphae, septate, branched, hyaline, clamp connections present, 3.1–6.6 μm diameter, with
286 crystal deposits in globose cells widely distributed among the hyphae, 17.5–38 × 20.5–35.7
287 μm. Rhizomorphs composed of at least two types of hyphae: filamentous, thin-walled hyphae,
288 with clamp connections, and thicker hyphae (3-6.5 µm) that seem to communicate with each
289 other by pores on the inflated tips. Gleba olive-brown (N99A50M10), mucilaginous.
290 Basidiospores cylindrical, smooth, 4.4–5 × 2.5-3.4 µm, hyaline in KOH 5%.
291 Habitat and Distribution: on soil, in a fragment of upland secondary forest. It was found in
292 municipalities of Manaus (State of Amazonas, Brazil) and Sinop (State of Mato Grosso,
293 Brazil).
294 Etymology: in reference to the volva becoming purple.
295 Other specimens examined: BRAZIL. Mato Grosso: Sinop, Parque Florestal de Sinop
296 (S11.8359 W55.5008), 7 Nov 2013, Cabral TS (SINOP27, SINOP28, SINOP30).
297 Commentary: This species is the most distinct among our collections. It is characterized by its
298 large basidiome (up to 200 mm), the indusium reaching 2/3 of the basidiome, the purplish
299 volva and rhizomorphs, and the thimble-like and strongly reticulated receptacle. P.
300 rubrovolvatus (M. Zang, D.G. Ji & X.X. Liu) Kreisel is one of the largest white-indusiate
301 species (up to 330 mm); it differs from P. purpurascens by the deep red volva, the fragile
302 indusium, by larger reticulations on the receptacle and smaller spores (3.7-4 x 2-2.5 µm) (Liu
303 1984; Calonge 2005). P. callichrous has reddish-violet rhizomorphs (Kreisel & Hausknecht
304 2009), which differ from the purplish volva and rhizomorphs of P. purpurascens;
79
305 unfortunately, there is little information available for this Brazilian species (Calonge 2005). P.
306 multicolor (Berk. & Broome) Cooke is similar to P. purpurascens in the purplish volva and
307 rhizomorphs, but it differs by the cream to orange indusium and the light yellow pseudostipe
308 (Lloyd 1909; Calonge 2005; Kreisel & Hausknecht 2009). P. indusiatus differs from P.
309 purpurascens by the smaller basidiome, the hypogeous development of the immature
310 basidioma, and smaller spores (4.1 × 2.2 µm), the well-developed indusium reaching the
311 ground, and the campanulate receptacle with wider reticulations (Ventenat 1798). The
312 phylogenetic analyses show specimens of P. purpurascens grouping in a monophyletic clade
313 with high support values (pp=0.96, bs=98), as a sister clade of the newly proposed P.
314 amazonicus. Although they are closely related, they are morphologically distinct: P.
315 purpurascens has larger basidiome and spores, an inconspicuous pore, the thimble-like
316 receptacle and purplish volva and rhizomorphs. P. purpurascens was found in a fragment of
317 secondary forest, in an extremely threatened area of the Amazonian forest domain in the State
318 of Mato Grosso, Brazil. This State was the second most deforested in Brazil in 2015
319 (PRODES – INPE), meaning that species in this area may be suffering the consequences of
320 habitat fragmentation, which is one of the main causes of decline in fungi species
321 (Courtecuisse 2008). Thus, this new species record shows the urgency of cataloging fungal
322 biodiversity of threatened areas, such as Neotropical forests.
80
323
81
324 Fig 05. Phallus purpurascens (a) fresh basidiome; (b) longitudinal section of an immature
325 basidiome, showing the purplish volva and rhizomorphs; (c) gregarious immature basidioma,
326 with purplish pigments on surface. Bars correspond to 20 mm. (d) spores; (e) rhizomorphs
327 hyphae; (f) pseudoparenchymatous hyphae from pseudostipe; (g) hyphae from volva; (h)
328 crystals in globose cells found on volva.
329
330 Phallus squamulosus T.S.Cabral, B.D.B.Silva & Baseia, sp. nov. Fig. 06.
331 Mycobank XXX
332 Diagnosis: Basidiome with completely epigeous development. Receptacle campanulate to
333 thimble like, with a wide apical pore, and strongly reticulated surface. Indusium extending to
334 2/3 of pseudostipe, white. Volva whitish to pale yellow, with squamous surface, epigeous.
335 Gleba olive-brown, mucilaginous.
336 Typification: BRAZIL. Rio Grande do Norte: Baía Formosa, Reserva Particular do
337 Patrimônio Natural Mata Estrela (W35.000365 S6.383307), 27 Feb 2014, Silva BDB
338 (holotype DT 253 UFRN-FungosXXXX). Genbank accessions: XXX.
339 Immature basidiomes whitish (N60A60M50), ovoid, with squamous surface, 39 × 34 mm.
340 Fresh expanded basidiome up to 95 mm high. Receptacle 20 × 28 mm, campanulate to
341 thimble like, with a wide apical pore, and a strongly but shallow reticulated surface,
342 reticulations 1.6-2 x 0.8-1.2 mm. Pseudostipe 60 × 12 mm, cylindrical, spongy, white
343 (N00A00M00), composed of globose to elongate-ovoid pseudoparenchymatous hyphae, 18–71
344 × 10.5–35 µm, hyaline. Indusium extending to 2/3 of pseudostipe, white (N00A00M00), 44 mm
345 in length, polygonal to rounded meshes up to 6 × 3 mm, attached to the apex of the
346 pseudostipe. Volva whitish (N00A00M00) to pale yellow (N00C00A30), with squamous surface,
347 epigeous, formed by filamentous hyphae, septate, branched, hyaline, clamp connections
348 present, 2.5–4.5 μm diameter; base with whitish (N00A00M00) rhizomorphs composed of
349 filamentous, thin-walled hyphae, with clamp connections, with crystal deposits in globose
350 cells distributed among the hyphae, 15–17.9 × 14–17 μm. Gleba olive-brown (N99A50M10),
351 mucilaginous. Basidiospores elongated, smooth, 3.5–4.4 × 1.8-2.2 µm, hyaline in KOH 5%.
352 Habitat and Distribution: found in sandy soil, in an area of the Atlantic Rainforest domain.
353 Etymology: in reference to the volva covered with small scales.
354 Commentary: Only one specimen of this species has been found to date in the northern
355 Atlantic Rainforest domain, but it is quite distinct from other species in Brazil. This species is
356 well characterized by the immature basidiome and volva with a squamous surface, the white
357 receptacle with shallow reticulations and a wide pore. We could not find white-indusiate
82
358 species records with squamous exoperidium in the available literature. However, P.
359 duplicatus, described in Martin and Tabarés (1994), presents an immature basidiome with fine
360 scales on the exoperidium, but this character is not found in other described P. duplicatus
361 (Kibby & McNeil 2012; Liu 1984; Kreisel & Hausknecht 2009; Lloyd 1909). Nevertheless,
362 the material described by Martin and Tabarés (1994) differs from P. squamulosus mainly by
363 having a conic-campanulate receptacle with crenulate disc on the apex. P. amazonicus
364 presents small hyphae projections on immature exoperidium surfaces, but these projections
365 are arranged differently in P. squamulosus, where they appear as scales. P. indusiatus is
366 different from P. squamulosus by the campanulate receptacle with a smaller pore and deeper
367 reticulations, the indusium reaches the ground, the immature basidiome is hypogeous, with
368 smooth surface and pinkish pigments.
83
369
370 Fig. 06. Phallus squamulosus DT 253 (a) fresh basidiome and (b) immature basidiome with
371 squamous surface. Bars correspond to 20 mm. (c) spores; (d) pseudoparenchymatous hyphae
84
372 from pseudostipe; (e) hyphae from volva; (f) hyphae from rhizomorphs and crystals deposits
373 on globose cells.
374
375 Phallus indusiatus Vent., Mém. Inst. Natl. Sci., Sci. Math. 1: 520, 1798. Fig. 07.
376 Immature basidiomes not observed. Fresh expanded basidiome 120 mm high. Receptacle
377 25 × 25 mm, campanulate, with an apical pore, reticulated surface. Pseudostipe 67 × 12 mm,
378 cylindrical, spongy, white (N00A00M00), composed of globose to elongate-ovoid
379 pseudoparenchymatous hyphae, X–X × X–X µm, hyaline. Indusium in full development
380 extending to the ground, white (N00A00M00), 74 mm in length, polygonal to rounded meshes
381 up to 7 × 4 mm, attached to the apex of the pseudostipe, composed of pseudoparenchymatous
382 hyphae, X–X × X–X µm. Volva white (N00A00M0), with pinkish pigments (N00M10C00),
383 hypogeous, outer layer papery, composed of filamentous hyphae, X–X µm, yellowish,
384 septate, with clamp connections; inner layer gelatinous, composed of thin hyphae (X–X µm
385 wide), hyaline; base with rhizomorphs composed of filamentous, thin-walled hyphae, with
386 clamp connections. Gleba olive-brown (N99A50M10), mucilaginous. Basidiospores elongated,
387 smooth, 3.6–4.1 × 1.5 – 2.2 µm, hyaline in KOH 5%.
388 Habitat and Distribution: found on sandy soil, in old-growth dense forest. It has a
389 circumtropical distribution, with records for South and Central America, Mexico, Africa, Asia
390 and Australia.
391 Specimens examined: BRAZIL. Pará: Belterra, Floresta Nacional do Tapajós, Jamaraqua
392 Community (S2.812667 W55.033083), 25 March 2014, Cabral TS (INPA264929,
393 INPA264930, INPA264931); São Paulo, Parque Estadual das Fontes do Ipiranga (S23.54
394 W46.63), Jan. 2011, Oliveira, J.J.S. (SP416389); Mar 2011, Ventura, P.O. (SP416393);
395 Capelari, M. (SP416087).
396 Commentary: According to Ventenat’s original description, P. indusiatus is characterized by
397 the hypogeous volva, the campanulate and reticulated receptacle, and by the indusium
398 reaching the ground. The indusium is white, but it can become reddish as it matures. Ventenat
399 does not give information on color of volva and rhizomorphs, but some authors state that the
400 volva can be light pinkish and rhizomorphs can be pinkish to violet (Kreisel & Hausknecht
401 2009; Calonge 2005). Our collection presents the same characteristics of the original
402 description and those in the key for indusiate species presented by Kreisel & Hausknecht
403 (2009); in addition, some of the specimens are from State of Pará, which is geographically
404 close and with the same forest domain as the type locality (Suriname). For these reasons, we
405 believe that the specimens that are nested in the same monophyletic clade in phylogenetic tree
85
406 (Fig. 02), all collected in the Brazilian Amazonian and Atlantic rainforests, correspond to P.
407 indusiatus sensu stricto. We also included a specimen with pink indusium, identified as P.
408 indusiatus f. rosea (Cesati) Kobayasi, which groups in the P. indusiatus clade. Three colored
409 indusiate forms of P. indusiatus are known, with the pink, white and yellow indusium. The
410 yellow indusiate form P. indusiatus f. lutea was erected to species by Kasuya (2008). In our
411 analysis, the pinkish and white indusiate forms are nested in the same clade. This indicates
412 that the species can present either a white or pinkish indusiate forms.
413
414 Fig. 07. Phallus indusiatus fresh basidiome. (a) TSC 148; (b) TSC 135, showing the volva
415 with pinkish pigments. Bars correspond to 20mm.
416
417 DISCUSSION
418 Molecular and morphological analyses and geographical distributions support the
419 hypothesis of three different species and a variety of Phallus indusiatus-like species with
420 partially overlapping distributions in Brazil. Our results suggest that a great number of species
421 might be hidden within the circumtropical P. indusiatus, since the samples we obtained from
422 Genbank are not monophyletic, but clearly polyphyletic with different relationships with other
423 Phallus species. In a similar way, several studies have unveiled fungal diversity hidden under
424 cryptic speciation and species complexes, especially after the integration of molecular and
425 phenotypic data (Geml et al. 2006; Jargeat et al. 2010; Kasuya et al. 2012). For instance,
426 Geml et al. (2008) reveled that at least eight phylogenetic species are found in the worldwide
427 distribution of Amanita muscaria (L.) Lam, with strong intercontinental genetic disjunctions
428 and intracontinental phylogeographic structure.
86
429 Phallus indusiatus is a paraphyletic clade in our phylogenetic tree (Fig 02). The
430 specimens identified as P. indusiatus (as Dictyphora indusiatus in the figure) do not share a
431 common ancestor; therefore the P. indusiatus-like clade is here considered an artificial group.
432 We suggest that species within this group are in fact divergent entities that maintained the
433 general ancestral phenotype due to high levels of morphological stasis throughout their
434 evolutionary history. This would explain the high frequency of taxonomic uncertainties,
435 which generates a great number of species synonyms for this group.
436 The maintenance of a conserved morphology due to slow rates of phenotypic variation
437 has been widely discussed in evolution (Davis et al. 2014). Two main mechanisms have been
438 proposed to explain the small levels of morphological change through time: genetic and
439 developmental constraints may restrict the appearance of phenotypic variation; or strong
440 stabilizing selection for a phenotype (Geml et al. 2008; Davis et al. 2014; Lee & Frost 2002).
441 Phallus indusiatus sensu lato has a circumtropical distribution; in our hypothesis, this putative
442 species with intercontinental disjunct distributions (as evidenced in phylogenetic tree – Fig
443 02) might occupy similar niches in different continents. Therefore, they are likely to be under
444 similar environmental conditions and selective pressures. A similar pattern was found by
445 Mueller (2001) between two disjunct and paraphyletic populations of Suillus spraguei (Berk.
446 & M.A. Curtis) Kuntze, that presented no noticeable morphological differences, probably as a
447 result of stabilizing selection.
448 When studying phalloid species it is noticeable that macro-characters are more variable
449 than micro-characters. For instance, spores are often cylindrical to bacilloid and smooth
450 throughout the order (except for Gastrosporiaceae), probably as an adaptation for dispersal,
451 since they are dispersed through the gut and do not adhere on the body of insects (Oliveira &
452 Morato 2000; Tuno 1998). The presence of rounded crystals in globose cells among hyphae
453 of volva and rhizomorphs was reported for Phallales species (Iofisidou & Agerer 2002), but it
454 is not a commonly used character in species descriptions. We believe these crystals are
455 formed by calcium oxalate, as found in other Phallomycetidae species, such as Geastrum and
456 Gastrosporium simplex (Zamora et al. 2013; Iofisidou & Agerer 2002), but further studies
457 about function and composition in Phallus are needed. These rounded crystals are present in
458 all species described here, except for P. amazonicus var. denigricans. However, we found
459 these structures only on the volva of P. purpurascens, on rhizomorphs of P. squamulosus, and
460 on both volva and rhizomorphs in P. amazonicus. Further studies are needed in order to
461 evaluate the taxonomic value of the presence of crystals in phalloid fungi. For instance, the
87
462 presence, shape and the arrangement of oxalate crystals were found to be important characters
463 to delimit species in Geastrum (Zamora et al. 2013).
464 On the other hand, macro-characters, such as the shape, surface and color of the main
465 structures (receptacle, pseudostipe, indusium, volva and rhizomorphs), are important
466 characters for infrageneric classification (Kreisel 1996). In this study, the phylogenetic clades
467 of P. indusiatus-like species were differentiated based on these features, confirming their
468 importance as diagnostic characters. Given that these diagnosable characters are lost once
469 phalloid specimens are dehydrated, it is extremely important that the newly described species
470 and new records should be well illustrated with colored pictures of fresh material. In addition,
471 we believe that molecular data seems to be indispensable for delimiting and describing
472 species in Phallus.
473
474 ACKNOWLEDGMENTS
475 We thank CNPq (473422/2012-3), FAPEAM (3137/2012), PPBio and the INCT-
476 CENBAM project for financial support. We thank Dr. Doriane Picanço Rodrigues for
477 coordination of the Laboratory of Applied Evolution at Federal University of Amazonas,
478 where part of the molecular data was obtained. We thank Ricardo Braga Neto, Dr. Dirce
479 Leimi Komura and Leonardo Kumagai for collecting support, and the curators of SP and
480 UFRN-Fungos Herbaria for specimen loan.
481
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782
783
784
785 TABLE 01. The collection localities, herbarium vouchers and Genbank accession numbers of
786 specimens collected in this study.
Species Locality Herbarium voucher Genbank accession
number
TSC121
TSC202
TSC188
Dictyophora KHCAM10143
98
Dictyophora KHCAM10033
Phallus NR_119579
787
99
788
789
790
791
792
100
Síntese
Em uma abordagem envolvendo delimitação de espécies e filogenia molecular baseada em
DNA, aliados a dados morfológicos e de distribuição geográfica, os capítulos aqui
apresentados revelaram uma fração da diversidade de fungos faloides encontrados
principalmente em ambientes de floresta Amazônica, em sua maioria em áreas de floresta
primária.
No primeiro capítulo uma espécie nova (Geastrum inpaense) e novos registros de fungos
gasteroides são descritos: seis espécies constituem novos registros para Amazônia Central
(Geastrum lloydianum, G. schweinitzii, Phallus merulinus, Staheliomyces cinctus), uma para
Brazil (Phallus atrovolvatus) e uma para América do Sul (Mutinus fleischeri). O segundo
capítulo descreve dois novos registros de fungos faloides para o Brasil (Lysurus
arachnoideus) e América do Sul (Phallus cinnbarinus), disponibilizando descrições
morfológicas detalhadas e fotos, além de sequências da região ITS.
No terceiro capítulo foi estudada a diversidade dentro do gênero Xylophallus. A
reconstrução filogenética utilizando genes de proteínas mostraram a presença de duas
unidades evolutivas, o que foi compatível com os dados morfológicos que mostravam a
existência de duas morfoespécies. Por outro lado, o barcode de fungos (ITS) revelou a
presença de um terceiro clado, com espécimes com distribuição restrita à bacia do rio
Madeira. Já o método de delimitação de espécies por distância genética mostrou-se falho para
os cinco genes testados, e por isso outro método foi aplicado, o teste de modelos por fator de
Bayes. A árvore de espécies foi construída a partir das árvores de genes para dois cenários:
um considerando duas espécies e outro considerando três espécies. Esses modelos foram
comparados utilizando fatores de Bayes, e o modelo com 3 espécies apresentou maior
probabilidade de ser o correto. Esses resultados revelaram que o gênero Xylophallus é
composto por no mínimo três espécies, sendo uma espécie nova (X. clavatus) e uma
conhecida (X. xylogenus). A terceira espécie forma um clado irmão desta última, mas por não
terem sido encontradas diferenças morfológicas entre as duas, acredita-se tratar de uma
espécie críptica. Xylophallus xylogenus e a espécie críptica apresentam diferenças na
distribuição geográfica. Enquanto X. xylogenus parece estar bem distribuído pelo norte da
América do Sul, a espécie críptica parece estar restrita à bacia do baixo rio Madeira.
Entretanto, não foi possível estabelecer uma hipótese biogeográfica para explicar os eventos
de especiação dentro do gênero.
101
Staheliomyces cinctus, um fungo faloide que também está restrito a florestas Neotropicais.
Duas espécies novas de Phallus também parecem ser restritas a Amazônia até o momento.
Essas espécies podem estar susceptíveis aos efeitos da fragmentação de habitat, que é uma
ameaça constante na Amazônia.
O presente trabalho também evidencia a importância da inclusão de dados moleculares no
estudo de taxonomia e sistemática de fungos. Apesar das diferenças encontradas entre as
espécies aqui descritas, uma delimitação clara só pôde ser atingida ao se utilizar dados de
filogenia molecular. Um exemplo é a presença de uma espécie críptica em Xylophallus, que
difere apenas na sua distribuição geográfica, e portanto só pôde ser detectada analisando
regiões gênicas. Também, foi possível delimitar a espécie Phallus indusiatus, que, por ter sido
descrita em 1798, ainda deixava dúvidas quanto a identidade da espécie. Da mesma forma,
outras 3 espécies e uma variedade de Phallus com indúsio branco também foram detectadas, e
que poderiam ser morfologicamente confundidas com P. indusiatus. Entretanto, é importante
levar em consideração o uso de métodos alternativos de delimitação de espécies, e testar
diferentes regiões gênicas além de ITS.
103
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Anexos
Capítulo 03: Supporting Information
Figure S1. Phylogenetic trees obtained by Bayesian analysis. (A) EF-1α, (B) gpd, (C) ITS,
(D) rpb1, (E) rpb2, (F) concatenated protein genes tree. Only PP>0.9 is shown on nodes.
Trees A, E and F were rooted using sequences available on Genbank; B, C and D were
midpoint rooted.
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Figure S2. Phylogenetic trees obtained with Parsimony analysis. (A) EF-1α, (B) gpd, (C)
ITS, (D) rpb1, (E) rpb2, (F) concatenated protein genes tree. Only PP>80 is shown on nodes.
Trees A, E and F were rooted using sequences available on Genbank; B, C and D were
midpoint rooted.
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Table S3. Specimens used in this study. Their localities, herbarium vouchers and Genbank
accession numbers.
Xylophallus São Paulo de Olivença, INPA26 KU87 KU87 KU87 KU87 KU87
clavatusc Amazonas, BR 4893 1499 1593 1784* 1710 1722
KU87
1785*
KU87
1787*