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Submitted 19 July 2014, Accepted 31 December 2014, Published online 10 January 2015
Corresponding Author: Larissa B. Moro – e-mail – larissamoro@hotmail.com 13
Polylobatispora setulosa, a new freshwater hyphomycete from
Ilhabela, Sao Paulo state, Brazil
Moro LB1, Delgado G2 and Schoenlein-Crusius IH1
1 Instituto de Botânica de São Paulo, CEP 04301-902, São Paulo, Brazil.
2EMLab P&K North Phoenix, Phoenix, Arizona, 85027U.S.A.
Moro LB, Delgado G, Schoenlein-Crusius IH 2015 – Polylobatispora setulosa, a new freshwater
hyphomycete from Ilhabela, Sao Paulo state, Brazil. Mycosphere 6(1), 13–18, Doi
10.5943/mycosphere/6/1/3
Abstract
Polylobatispora setulosa sp. nov. is described and illustrated from submerged mixed leaf
litter samples collected at streams in Ilhabela, São Paulo state, Brazil. The fungus is distinct in
having setulae at the tip of the conidial lobes. Acumispora verruculosa, isolated from identical
substrate and characterized by the presence of fusiform, acuminate, rostrate, verrucose, 2-septate,
hyaline to greenish conidia, is recorded for the third time from the Americas. They are compared
with morphologically similar species and identification keys are provided.
Keywords – anamorphic fungi – freshwater fungi – systematics – tropical fungi
Introduction
Among freshwater hyphomycetes, the so called typical aquatic or Ingoldian fungi have been
studied extensively in Brazil, mainly in well-preserved areas of the “cerrado” region (Schoenlein-
Crusius 2002), the Atlantic rainforest of the state of São Paulo (Schoenlein-Crusius & Grandi,
2003) and also in a few urban waters (Schoenlein-Crusius et al. 2009, 2014). Recently, however,
increasing attention has been paid to other groups of freshwater fungi, particularly those
hyphomycete genera frequently reported from terrestrial as well as freshwater habitats and known
as facultative-aquatic (Goh & Hyde 1996) or inmigrants (Shearer et al. 2007). While the semi-arid
“caatinga” biome in the northeast Brazil has proven to be rich in freshwater fungi (Barbosa &
Gusmão 2011, Barbosa et al. 2013, Carneiro et al. 2012), other poorly studied or unexplored areas
are also believed to harbour a high diversity of this group of fungi.
Between the years 2012 and 2013, an extensive survey of freshwater hyphomycetes
inhabiting submerged materials was carried out in the islands off the coast of the state of São Paulo,
including the island of São Sebastião in the municipality of Ilhabela. Two interesting anamorphic
taxa were collected on mixed submerged leaf litter samples. They are morphologically similar to
the genera Acumispora and Polylobatispora, but the latter was found distinct enough from
previously described species to be considered as a new taxon. Taxonomic descriptions and
illustrations are presented here along with identification keys for both genera.
Mycosphere 6 (1): 13–18 (2015) ISSN 2077 7019
www.mycosphere.org Article Mycosphere
Copyright © 2015 Online Edition
Doi 10.5943/mycosphere/6/1/3
14
Materials and Methods
Sampling and morphology
Samples of submerged mixed leaf litter (approx. 10g) and local water were collected in sterile
polyethylene bottles (about 200 ml) and brought to the laboratory. The leaves were cut into pieces
in approximately 1 cm diam. and incubated in Petri dishes containing sterile distilled water. They
were kept in climatic chambers at around 20oC following Ingold (1975). From the seventh day of
incubation and for at least one month, slides of leaf fragments were prepared using polyvinyl
alcohol lacto glycerol resin as mounting medium (Morton et al.1993). Fungal structures were
observed and measured using an Olympus BX50 light microscope and images were taken with a
Leica DM LB2 microscope fitted with a Leica DFC 280 camera.
Isolates
In order to obtain pure cultures of fungi, water droplets containing conidia of the target fungi
were captured with a Pasteur pipette and spread over the surface of Malt Extract Agar (MEA) Petri
dishes containing Chloramphenicol (final conc. 25mg/L). After 24 h of incubation in climatic
chambers at 21oC, the reverse side of the dishes was observed using a stereoscope to verify hyphal
growth the position of the conidia was marked with a permanent marker pen. Under aseptic
conditions and with the aid of a sterile stainless steel needle, the conidia were removed from the
surface of the medium, inoculated onto a new MEA Petri dish and incubated under similar
conditions. Permanent slides are deposited in the Herbarium "Maria P. Eneyda Kauffmann Fidalgo"
(SP) and fungal cultures are deposited in the Fungal Collection of the Instituto de Botânica, São
Paulo (CCIBt).
Scanning electron microscopy
For scanning electron microscopy (SEM), mycelia with conidiophores and conidia grown on
MEA were prepared according to the modified method described by Pan et al. (1994) They were
fixed overnight in 2.5% glutaraldehyde in 0.1M sodium phosphate buffer (pH 7.4), washed
thoroughly with distilled water, dehydrated through an ethanol series (10%, 40%, 60%, 80% and
100%), each series lasting 30 minutes and dried in silica gel. After preparation, the stubs were
covered with double-sided carbon tape, and the specimens were mounted on stubs, sputter-coated
with gold, and examined using a PHILIPS XL series XL 20, S/W, 5.21. scanning electron
microscope at 10KV.
Taxonomy
Polylobatispora setulosa L.B. Moro, G. Delgado &I.H. Schoenlein-Crusius sp. nov Fig. 1–6
MycoBank MB811045
Etymology – Latin, setulosa, provided with setulae.
Colonies on natural substrate inconspicuous. Conidiophores macronematous, septate, hyaline,
forked, bottle-shaped, 10.0–25.74 × 1.43–2.5 m. Conidiogenous cells lageniform, enteroblastic-
phialidic, hyaline to greenish. Conidia hyaline, triangular, 3-lobed, 8.6–14.3 m diam., with thick
dark walls and a hyaline setula at each tip, 5.7–12.87m long; with a central, fuscous halo, 5.7–7.0
m diam.
Teleomorph – unknown.
Material examined – Brazil, São Paulo, Ilhabela, São Sebastião island,“Cachoeira do Gato”
stream, on submerged mixed leaf litter, 14 May 2013, L.B. Moro (Holotype: SP445-978).
Note – Pure culture of Polylobatispora setulosa was not possible to obtain.
Acumispora verruculosa Heredia, R.F. Castañeda & R.M. Arias. Mycotaxon 101: 90, 2007.
Fig. 7–12
15
Figs 1–6 – Polylobatispora setulosa. SP 445-978 (holotype). 1-5 Developmental stages of
conidiophores and conidia. 6 Mature conidium with setulae at the tip of each lobe and central halo.
Scale bar = 10µm.
Colonies on MEA dark gray to greenish, velvety, slow growing, reaching less than 2 cm
diam. after 2 weeks of incubation at 21 ºC. Mycelium composed of branched, septate, hyaline to
pale brown hyphae. Conidiophores macronematous, mononematous, erect, simple or branched, 0–
4-septate, light brown to brown, 9.0–51.5 × 2.86–4.30 µm. Conidiogenous cells mono- or
polyblastic, integrated, terminal and intercalary, proliferating sympodially, denticulate;
conidiogenous loci apical or lateral, often protuberant and denticle-like; conidial secession
rhexolytic. Conidia fusiform, 2-septate, constricted at the septa, with rounded basal cell and slender
apical cell, acuminate, rostrate, hyaline to greenish, 30–45 (–50) × 5.0–8.75 µm, verruculose, with
the basal cell often bearing a denticle-like detachment scar.
Teleomorph – unknown.
Material examined – Brazil, São Paulo, Ilhabela, São Sebastião island, “Cachoeira do Gato”
stream, on submerged mixed leaf litter, 14 May 2013, L.B. Moro (SP445-979, CCIBt 4066).
16
Discussion
The genus Polylobatispora was originally described from leaves collected in Malaysia
(Matsushima 1996) with two species: P. deltoidea Matsush., the type species, and P.
quinquecornuta Matsush., based on the presence of cylindrical or bottle-shaped (doliiform),
conidiophores and enteroblastic-phialidic, hyaline, conidiogenous cells that produce single, light
brown, stauroconidia with a small scar formed after detachment from the conidiophore.
Polylobatispora deltoidea presents characteristic triangular conidia with three lobes and P.
quinquecornuta 5-lobed conidia. New records of these species are unknown. The main difference
between Polylobatispora deltoidea, P. setulosa and P. quinquecornuta is the number of lobes: the
first two species possess 3 lobes and the latter 5 lobes. The 3-lobed P. setulata differs from P.
deltoidea by slightly larger conidia and the presence of flexible setulae, one at the tip of each lobe.
Figs 7–12 – Acumispora verruculosa. 7-9 Conidiophores, conidiogenous cells and conidia. 10
Mature conidium. 11 SEM image of mature conidium. 12 Details of the conidia wall. Scale bar =
10µm.
Identification key to species of the genus Polylobatispora
1a Conidia 5-lobed....................................................................................................P. quinquecornuta
1b Conidia 3-lobed...............................................................................................................................2
2a Conidia 6–8 m diam., setulae absent............................................................................P. deltoidea
2b Conidia 8.6–14.3 m diam., setulae present.....................................................................P.setulosa
17
Table 2 Conidial morphology in species of Polylobatispora
Species
Conidia
References
Shape
Diameter
(m)
Presence\absence of
setulae
P. deltoidea
3-lobed
6-8
absent
Matsushima
(1996)
P. quinquecornuta
5-lobed
11-15
absent
Matsushima
(1996)
P. setulosa
3-lobed
8.6-14.3
present
this paper
Matsushima (1980) described the genus Acumispora to accommodate three species
collected on dead leaves in Taiwan: A. uniseptata Matsush., the type species, A. biseptata Matsush.,
and A. phragmospora Matsush., on the basis of a peculiar acuminate conidial shape and conidia,
with different number of septa that secede rhexolytically from monoblastic or polyblastic,
sympodially proliferating, denticulate conidiogenous cells on simple or irregularly branched
conidiophores. Heredia et al. (2007) described A.verruculosa Heredia, R.F. Castañeda & R.M.
Arias from submerged leaves collected at the waterfall “Los Tuxtlas” in Veracruz, Mexico, with
distinctly rough-walled conidia. This fungus was recently reported for the first time from South
America on submerged leaves and branches collected in aquatic environments at the urban area of
Belém do Pará (“Ilha do Combu”, “Parque Ecológico Gunma” and “Parque Estadual do Utinga”)
located in the Amazon biome (Silva et al. 2013). Our collection is the third from the continent with
the conidia slightly larger than in earlier collections indicating that there are variations in
conidialsize: earlier collections are 25.4–33.5 × 4.4–5.5 μm whereas the present collection has
conidia measuring 30–45 × 5.0–8.75 µm. Taxonomic key and diagnostic features of described
species in Acumispora are given below.
Identification key to species of Acumispora
1a. Conidia with verruculose walls.................................................................................A. verruculosa
1b. Conidia smooth-walled...................................................................................................................2
2a. Conidia 1-septate.........................................................................................................A. uniseptata
2b. Conidia more than 1-septate...........................................................................................................3
3a. Conidia 3-6 septate...............................................................................................A. phragmospora
3b. Conidia 2-septate...........................................................................................................A. biseptata
Table 1 Diagnostic characteristics of the genus Acumispora
Species
Conidia
References
Nº of
septa
Length
(µm)
Width
(µm)
Color
Wall
texture
A. uniseptata
1
23-28
5.5-6.5
Hyaline to very light
brown
smooth
Matsushima
(1980)
A. biseptata
2
16-25
3.8-5
Hyaline to very light
brown
smooth
Matsushima
(1980)
A. phragmospora
3-6
20-35
4-5
Brown to subhyaline
smooth
Matsushima
(1980)
A. verruculosa
2
25.4–33.5
4.4–5.5
Pale brown
verruculose
Heredia et al.
(2007)
A. verruculosa
(present
collection)
2
30-45
5.0-8.75
Hyaline to greenish
verruculose
This paper
18
Acknowledgments
The authors are grateful to the director of the Ilhabela State Park and to the Instituto Florestal,
Secretaria de Estado do Meio Ambiente de São Paulo, for permission and support during field work
and to the Instituto de Botânica, São Paulo, for laboratory facilities. The first author acknowledges
CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the award of a Ph.D.
scholarship. The third author thanks the CNPq for the fellowship (CNPq, Process nº 304526/2009-
6), and Alex Almeida Alcântara for designing the plates.
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