Molecular ecology of Fonsecaea sibling species isolated from

Molecular ecology of Fonsecaea sibling species isolated from endemic areas of

chromoblastomycosis inBrazil

• Vania Vicente• Javad Najafzadeh• Jiufeng Sun• Sybren de Hoog

Chromoblastomycosis

• Chronic, progressive cutaneous and subcutaneous fungal infection

• Muriform cells

• Common agents:Fonsecaea pedrosoi,F. monophora, F.nubica,Cladophialophora carrionii

Current hypothesis: traumatic inoculation

Plant-inhabiting fungus Human host

Queiroz –Telles et al., 2009

trauma by plant

spine

Frequencies plant / human of sibling species

Species 1:

Species 2:

Plant host Human host

Fernández-Zeppenfeldt et al. (1994)

Sclerotic bodies in human skin Sclerotic bodies from cactus

muriform cellsin cactus spines

Cadophialophora carrionii Cladophialophora yegresii

TEF1ABT2ITS

de Hoog et al. – Stud. Mycol. 58, 2007

C. carrionii

C. yegresii

Cladophialophora in plant and human

humancactus debrisliving cactus


3C. yegresii:

C. carrionii:

0

Human host

32

Cactus

0Debris

2

Plant

Human

Sibling species

C. yegresii

C. carrionii

Debris

Fonsecaea pedrosoi: a similar plant origin

• Isolation from thorns of plant: Mimosa pudica

• Supposed to be Fonsecaea pedrosoi

Salgado et al. Rev. Inst. Med. trop. S.P , 46: 2004

Isolation from the natural environment

• Endemic area– Maranhão state

in Northeast Brazil

Isolation - Flotation method (Vicente et al. 2008,Studies in Mycology 61: 137–144)

• Samples: 210→ replications 50

→ 400 isolates of black fungi

• Morphology screening for clinically relevant species:Fonsecaea-like, Exophiala spp., Cladophialophora spp.– 100 isolates morphologically looking like clinical species– 38 isolates were Fonsecaea-like

Pairwise (OG:100%,UG:0%) (FAST:2,10) Gapcost:0%ITS

10095908580757065605550454035

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Exophiala sp. n. 3 (turtle)

Exophiala sp. n. 3 (Seadrago

Exophiala pisciphila

Exophiala pisciphila (potbelly

Exophiala superficiale (sp. n

Exophiala superficiale (cf. sa

Exophiala superficiale (sp. n

Exophiala superficiale (twig

Exophiala cf. salmonis

Exophiala equina (drinking w

Exophiala equina (stool)

Exophiala equina (water DE)

Exophiala equina (drinking w

Exophiala equina (dialyse)

Exophiala salmonis

Exophiala salmonis (drinking

Exophiala cancerae-Ex- Exo

Exophiala cancerae (sp. n. 1

Exophiala cancerae. n. 1 (toa

Exophiala cancerae- Crab

Exophiala cancerae. n. 1 (Cl

Exophiala cancerae. n. 1 (wa

Exophiala canceraea

Exophiala sp (E.cancerae)

Exophiala canceraea

Exophiala psychrophila (Salm

Exophiala opportunistica. n. 4

Exophiala opportunistica sp.

Veronea like

Veronaea botryosa (Eucalyp

Veronaea botryosa

Veronea botryosa

Veronea botryosa

Veronea botryosa

Veronea botryosa

Veronaea botryosa (human)

Veronea like

Veronea like

Exophiala brunnea (litter Aca

Exophiala dermatitidis

E. dermatitidis


C.carrionii

Cladophialophora carrionii






Cladophialophora sp.


Phialophora like

Phialophora like

Phialophora

Ramichloridium_sp Cladoph

Ramichloridium sp. (wood) C

Cladophialophora boppii

Cladophialophora boppii (sc

cladophialophora boppii

Cladophialophora chaetospi



Cladophialophora carpetanu



Exophiala bergeri

Exophiala sp

Exophiala bergeri

Exophiala alcalophila (soil)

Exophiala alcalophila (ver %

Cladophialophora sp. "gasst

Cladophialophora sp. 1



Cladophialophora sp

Cladophialophora??

Cladophialophora ??

Cladophialophora sp (enviro

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Cladophialophora devrisei

Cladophialophora cf. devries

Cladophialophora sp

Cladophialophora

Cladophialophora sp

Cladophialophora cf. devries

Cladophoalophora

Cladophialophora

Cladophoalophora

Cladophialophora species


Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Cladophialophora saturnica

Cladophialophora elegans

Cladophialophora elegans (a

Cladophialophora saturnica (

Cladophialophora arxii (trach

Cladophialophora arxii (brain

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Fonsecaea / Clado

Fonsecaea nubica

Fonsecaea nubica

Fonsecaea nubica

Fonsecaea nubica

Fonsecaea nubica

Fonsecaea monophora

Fonsecaea pedrosoi

Fonsecaea pedrosoi

Fonsecaea pedrosoi

Fonsecaea pedrosoi

Fonsecae pedrosoi

Fonsecaea pedrosoi

Fonsecae pedrosoi

Fonsecae pedrosoi

Fonsecaea pedrosoi (chrom

Fonsecae pedrosoi

Fonsecaea pedrosoi

Fonsecaea pedrosoi

Fonsecae pedrosoi

Fonsecae pedrosoi

Fonsecae pedrosoi

Fonsecaea monophora

Fonsecaea monophora

Fonsecaea monophora (chro

Fonsecaea monophora

Fonsecae monophora

Fonsecae monophora

Cladophialophora bantiana (m

Cladophialophora bantiana (m

Cladophialophora bantiana (p

Cladophialophora bantiana


Cladophialophora emmonsii




Exophiala sp

Exophiala sp


Exophiala spinifera

Exophiala spinifera

Exophiala spinifera

Exophiala spinifera

Exophiala spinifera

Exophiala spinifera

Exophiala oligospoerma

Exophiala sp

Cladophialophora

Cladophialophora

Cladophialophora sp

Cladophialophora


Cladophialophora

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Cladophialophora

Cladophialophora sp

Cladophialophora

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Phialophora sp

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp1

Fonsecaea

Exophiala

VeronaeaExophialaCladophialophora

Exophiala

Cladophialophora

Cladophialophora

PhialophoraCladophialophora

Exophiala

Cladophialophora

38 isolatesFonsecaea-like

ITS – Tree: UPGMA

100 isolates

Fonsecaea species

F. pedrosoi F. monophora F. nubica

Fonsecaea-like

Fonsecaea-like

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Fonsecaea multimorphosa ,Ramichoridium sp. Rhin. sp. 1 (trunk)

Fonsecaea multimorphosa (wood Grevillea)

Fonsecaea multimorphosa, Ramichloridium_sp Rhin. sp. 1

Fonsecaea multimorphosa

Fonsecaea multimorphosa. (palm wood) Ram sp 1

Fonsecaea multimorphosa (wood Podocarpus lambertii) Rhin sp 1

Cladophialophora sp. "gasstation" 1

Cladophialophora sp. 1

Cladophialophora sp



Cladophialophora ( new Fons)

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp


Cladophialophora immunda ( Hamid, UK)

Cladophialophora immunda

Cladophialophora immunda sp. 1 (cutaneous Padhye)

Cladophialophora immunda

Cladophialophora sp

Cladophialophora sp (environmental)

Cladophialophora sp

Cladophialophora devriesii (disseminated)

Cladophoalophora

Cladophialophora sp

Cladophialophora sp

Cladophialophora sp

Cladophialophora cf. devriesii (Crab)




Cladophialophora cf. devriesii

Cladophialophora sp


Cladophialophora cf. devriesii

Cladophialophora sp

Cladophoalophora

Fonsecaea minima

Fonsecaea minima

Fonsecaea minima

F. minima

F. minima

Fonsecaea minima

Fonsecaea minima

F.erecta

Fonsecaea erecta

Fonsecaea erecta

Fonsecaea erecta

Fonsecaea erecta

F. pedrosoi

Cladophialophora saturnica (human skin)

Cladophialophora saturnica (dead tree)

Cladophialophora saturnica (litter)

Cladophialophora saturnica (vegetable cover/soil)

Cladophialophora saturnica (dead tree)

Fonsecaea nubica

Fonsecaea nubica

Fonsecaea nubica

Fonsecaea pedrosoi

Fonsecaea pedrosoi

Fonsecaea pedrosoi

Fonsecaea pedrosoi

Fonsecaea pedrosoi

Fonsecaea pedrosoi

Fonsecaea pedrosoi(chromo, Falcon State)

Fonsecaea monophora

Fonsecaea monophora

Fonsecaea monophora (soil, Brazil)

Fonsecaea monophora

F. monophora

Fonsecaea monophora

Cladophialophora arxii

Cladophialophora arxii (trachea)

Cladophialophora arxii (brain, Miami)

Cladophialophora arxii (hamid)

Cladophialophora minourae

Cladophialophora minourae

Cladophialophora minourae (oil p. soil)

Cladophialophora minourae (oil p. soil)

Cladophialophora emmonsii (brain)

Cladophialophora emmonsii (subcutaneous cat)

Cladophialophora emmonsii (subcutaneous)


Cladophialophora bantiana (New)

Cladophialophora bantiana (skin graft)

Cladophialophora bantiana (brain)

Cladophialophora bantiana (brain)

Cladophialophora bantiana (cutaneous cat)

Cladophialophora bantiana (liver dog)


Cladophialophora potulentorum



Cladophialophora australiensis

Capronia pilosella

Capronia pilosella / spin

Capronia pulcherrima Vitis

capronia moravica

capronia pulcherrima

Capronia acutiseta

Capronia acutiseta Dactydium

Cladophialophora mycetomatis

Cladophialophora mycetomatis

ITS

999999999988888888

Fonsecaea-like species 1



Fonsecaea monophora

Fonsecaea pedrosoi

ITS – Tree: UPGMA

Fonsecaea spp.

Multilocus Analysis: ITS, ACT, CDC 42, BT2

CBS119719 Cladophialophora bantianaCBS678 79 Cladophialophora bantianaCBS102586 Cladophialophora bantianaCBS100429 Cladophialophora bantianaCBS444 96 Cladophialophora bantiana

CBS648 96 Cladophialophora bantianaCBS117890 Cladophialophora bantiana

CBS640 96 Cladophialophora emmonsiidH13029 Cladophialophora emmonsii

CBS979 96 Cladophialophora emmonsiiCBS102594 Cladophialophora emmonsii

dH20517 Cladophialophora immunda CBS109797 Cladophialophora immundaCBS834 96 Cladophialophora immundaCBS110551 Cladophialophora immundaCBS123977 Cladophialophora immunda

dH11601 Cladophialophora immundaCBS102237 Cladophialophora immunda

CBS102249 Cladophialophora immundadH21185dh21188dh21172dH21178dH11588dH20537dH20501dH20464dH20474CBS102227

dH20478dH21011 Cladophialophora devriesiidH21170 Cladophialophora devriesii

CBS147 84 Cladophialophora devriesiidH21196 Cladophialophora brasiliensis

dH20490 Cladophialophora brasiliensisdH20497 Cladophialophora brasiliensis




CBS119710 Cladophialophora brasiliensisCBS120532 Cladophialophora brasiliensisCBS120418 Cladophialophora brasiliensisCBS119718 Cladophialophora brasiliensisCBS120419 Cladophialophora brasiliensisCBS118724 Cladophialophora saturnicaCBS109628 Cladophialophora saturnica

CBS102228 Cladophialophora saturnicaCBS102230 Cladophialophora saturnica

CBS109630 Cladophialophora saturnicadH20492 Fonsecaea erecta

dH20513 Fonsecaea erectadH20457 Fonsecaea erectadH20502 Fonsecaea erecta

dH21010 Fonsecaea minimadH20511 Fonsecaea minimadH20485 Fonsecaea minimadH20463 Fonsecaea minimadh21195 Fonsecaea minima

CBS102224 Fonsecaea multimorphosaCBS102240 Fonsecaea multimorphosa

CBS102235 Fonsecaea multimorphosaCBS102226 Fonsecaea multimorphosa

CBS102253 Fonsecaea multimorphosadH20510 Fonsecaea multimorphosa

CBS556 83 Cladophialophora minouraeCBS987 96 Cladophialophora minouraedH18466 Cladophialophora minouraedH18460 Cladophialophora minourae

CBS409 96 Cladophialophora arxiiCBS102461 Cladophialophora arxiiCBS306 94 Cladophialophora arxii

FM52022 Cladophialophora arxiiCBS444 62 Fonsecaea nubicaCBS269 64 Fonsecaea nubica

CBS125191 Fonsecaea nubicaCBS102238 Fonsecaea monophoraCBS102229 Fonsecaea monophoraCBS269 37 Fonsecaea monophoraCBS102225 Fonsecaea monophora

dH20512 Fonsecaea monophoraCBS121721 Fonsecaea monophoradH20500 Fonsecaea pedrosoidH13279 Fonsecaea pedrosoidH20488 Fonsecaea pedrosoi

CBS271 37 Fonsecaea pedrosoiCBS671 66 Fonsecaea pedrosoiCBS253 49 Fonsecaea pedrosoiCBS102245 Fonsecaea pedrosoi

0.01

100

100

92

99

96

Fonsecaea erecta (sp. 1)Fonsecaea minima (sp. 2)

Fonsecaea species (sp. 3)

Fonsecaea monophoraFonsecaea pedrosoi

Tree: RaxML version 7.0.4

96

100

100

92


F. monophora:

F. pedrosoi:

Human host

12

10

DebrisPlant0

0

2

1

Plant

Human

Fonsecaea: sibling species

Debrisplant

Debrisplant

Conclusions

• Relative frequency host / environment is a useful parameter to predict virulence of the fungus

• Sibling species can be very different in ecology

• Living plant-associated species are different from their human-pathogenic siblings

Thank you for your attention !!!

Isolation method

Flotation method – Vicente et al. 2008,Studies in Mycology 61: 137–144

Mineral oil

Saline solution: • Penicillin• Chloramphenicol• Streptomycin• Cycloheximide

Documents

Molecular ecology of Fonsecaea sibling species isolated from