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A new species of Lichtheimia (Mucoromycotina, Mucorales) isolated from

Brazilian soil

Article in Mycological Progress · April 2014

DOI: 10.1007/s11557-013-0920-8

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Mycol Progress (2014) 13:343–352
DOI 10.1007/s11557-013-0920-8

ORIGINAL ARTICLE

A new species of Lichtheimia (Mucoromycotina, Mucorales)

isolated from Brazilian soil
André L. C. M. de A. Santiago & Kerstin Hoffmann & Diogo X. Lima &
Rafael J. V. de Oliveira & Helder E. E. Vieira & Elaine Malosso & Leonor C. Maia &
Gladstone A. da Silva

Received: 16 April 2013 / Revised: 29 July 2013 / Accepted: 13 August 2013 / Published online: 15 September 2013
# German Mycological Society and Springer-Verlag Berlin Heidelberg 2013

Abstract During studies on Mucorales in semiarid and littoral Introduction

dune areas in the northeast of Brazil, two cultures of an Absidia-
like species were isolated from soil. They were characterized The genus Absidia Tiegh. (Mucoromycotina, Mucorales) in-
based on morphological, physiological and molecular data cludes species commonly isolated from soil, characterized by
(5.8S and LSU rDNA sequences). The phylogenetic analyses the production of stolons, rhizoids and apophysate sporangia
of the isolates revealed that they belong to the Lichtheimiaceae with deliquescent walls. One single septum is formed in the
and are closely related to species of Lichtheimia. The two sporangiophore and the zygospores are formed from suspen-
isolates produced simple or branched, erect and circinate spo- sor cells that contain several appendices (Benny 2010).
rophores, occasionally with a septum under the sporangia, Sporophores of Absidia are never produced opposite to the
characteristics also common in Lichtheimia species. However, rhizoids like those of Apophysomyces P.C. Misra and
different from the described Lichtheimia species, the columel- Rhizopus Ehrenb. (Misra et al. 1979; Zheng et al. 2007). In
lae of our isolates were mainly short hemispherical, never general, taxa of Absidia present fast growth between 25 and
spatulate or elliptical and without projections. Sometimes, a 34 °C, although some representatives are capable of growing
long conical or bell shaped apophysis was found. Both isolates between 12 and 37 °C (Hoffmann et al. 2007).
grew better at 30–35 °C, with no development at 42 °C, and Since Absidia was described (van Tieghem 1876), some
giant cells were not observed. Based on the evidence of the species of this genus were transferred to other genera proposed
analyzed datasets a new species of Lichtheimia is proposed. later, such as Tieghemella Berl. & De Toni, Mycocladus
Beauverie, Proabsidia Vuill., Pseudo-Absidia Bainier,
Lichtheimia Vuill. and Protoabsidia Naumov. However, in
Keywords Lichtheimiaceae . Phylogeny . Taxonomy . time, with the exception of Lichtheimia, the other five genera
Temperature were synonymyzed with Absidia (Hesseltine and Ellis 1964;
Schipper 1990; Kirk et al. 2008).
A. L. C. M. de A. Santiago (*)
Universidade Federal Rural de Pernambuco—Unidade Acadêmica Until 2007, the delimitation of Absidia species was mainly
de Serra Talhada, Fazenda Saco, s/n, 56900-000 Serra Talhada, based on morphology criteria, such as size and form of veg-
Pernambuco, Brasil etative structures and those of sexual and asexual reproduc-
e-mail: [email protected]
tion. Beauverie (1900) proposed the genus Mycocladus
K. Hoffmann Beauverie, containing only M. verticillata, which is morpho-
Institute of Microbiology, Department of Microbiology and logically identical to some species of Absidia. Mirza et al.
Molecular Biology, University of Jena and Leibniz-Institute for (1979) also recognized the genus Mycocladus to group a few
Natural Product Research and Infection
species of Absidia that grew well at 37 °C, never produced
Biology—Hans-Knoell-Institute, Jena Microbial Resource
Collection, Jena, Germany sporophores in whorls and had optimum temperature to
form zygospores at 31 °C. Thus, A . blakesleeana Lendn., A.
D. X. Lima : R. J. V. de Oliveira : H. E. E. Vieira : E. Malosso : corymbifera (Cohn) Sacc. & Trotter, A . hyalospora (Saito)
L. C. Maia : G. A. da Silva
Lendn., A. parricida Muskat and A. ramosa (Zopf) Lendn.
Programa de Pós Graduação em Biologia de Fungos. Departamento
de Micologia, Universidade Federal de Pernambuco, Av. Prof. were transferred to Mycocladus. However, these species (al-
Nelson Chaves, s/n, 50670-420 Recife, PE, Brasil ready within Mycocladus) were considered invalid, according
 Author's personal copy
344 Mycol Progress (2014) 13:343–352

to Art. 36.1 of the International Code of Botany (ICBN) Despite several proposed concepts for species recognition
(Benny 2010). Therefore, in 2007, 27 species and nine varie- (Mayden 1997), the genealogical concordance phylogenetic
ties of Absidia were accepted (Benny 2010). Hoffmann et al. species recognition (Taylor et al. 2000) seems to be the one to
(2007), in a study that combined molecular techniques (with a most likely recognize natural species (e.g., Alastruey-Izquierdo
phylogenetic approach) with physiology and micromorphol- et al. 2010). One additional interesting finding is the correlation
ogy studies of Absidia, showed that known species presented between sexual incompatibility and the occurrence of compen-
differentiated growth under different temperatures, and divid- satory base changes (CBC) in the secondary structure of ITS2
ed them into three groups: thermotolerant—optimum growth sequences (Müller et al. 2007). CBCs are changes of paired
temperatures above 37 °C (between 37 and 45 °C); mesophilic— nucleotides in the double-stranded regions of the secondary
optimum growth temperatures between 25 and 34 °C; myco- structure. With a confidence of 93 %, a single observed CBC
parasite—species potentially mycoparasitic on other Mucorales seems to be meaningful in the detection of species boundaries
with optimum growth temperatures below 30 °C. Thus, (Coleman and Vacquier 2002; Müller et al. 2007).
Hoffmann et al. (2007) reintroduced the genus Mycocladus During diversity studies of Mucoromycotina in soils from
and proposed that those thermotolerant species of Absidia pre- the semiarid region (Caatinga) and the Atlantic Forest in
senting zygospores with suspensor cells without appendices, Brazil, we isolated two specimens of Lichtheimia presenting
and forming equator rings on the zygospores (Absidia-like optimum growth temperatures below 37 °C. The isolates
species), were transferred again to this genus, as follows: M. varied morphologically in relation to the other species within
blakesleeanus (Lendn.) J.H. Mirza, M. corymbifer (Cohn) J.H. this genus. Based on the morphophysiology and molecular
Mirza, M. ramosus (Zopf) J.H. Mirza and M. hyalospora (LSU and 5.8S rDNA) analyses carried out, a new species of
(Saito) J.H. Mirza. The two mycoparasite species (A. parricida Lichtheimia is being proposed.
Renner & Muskat ex Hesseltine & J.J. Ellis and A. zychae
Hesseltine & J.J. Ellis) were transferred to the genus
Lentamyces Kerst. Hoffm. & K. Voigt by Hoffmann and Voigt Materials and methods
(2009), including L. parricida (Renner & Muskat ex Hesselt. &
J.J. Ellis) Kerst. Hoffm. & K. Voigt and L. zychae (Hesselt. & Sampling sites
J.J. Ellis) Kerst. Hoffm. & K. Voigt.
After a meticulous review of the original description of M. Soil samples were collected in the municipalities of Araripina
verticillata, Hoffmann et al. (2009) verified that this species is (7º27′58″S, 40º24′53″W), Pernambuco State, and Mataraca
not thermotolerant, as it does not grow above 40 °C, having its (6°28′20″S, 34°57′10″W), Paraíba State, both located in the
optimum growth at 30 °C. Besides, the zygosporangia’s suspen- Northeast of Brazil. In Araripina, soils were collected in a
sor cells of M . verticillata present appendices and the preservation area of Caatinga belonging to the Instituto
zygosporangial wall is ornamented with scales (or tubercles), Agronômico de Pernambuco, in September 2008. The area is
sexual characteristic corresponding to Lentamyces parricida covered by hyperxerophilic Caatinga vegetation with stretches
(Hoffmann and Voigt 2009). Therefore, Hoffmann et al. of deciduous forest and the climate is tropical semiarid. The
(2009), considering the elevated probability of the M. verticillata rainy season begins in November and ends in April. The
culture observed by Beauvérie to be a mesophilic co-culture of average annual rainfall is 431.8 mm (MME 2005a). In
Absidia sp. with Lentamyces sp., transferred the thermotolerant Matacara, soil sampling was carried out in July 2010, in sand
species to the genus Lichtheimia Vuill. (Vuillemin 1903), of dune areas of the Millennium Inorganic Chemicals Mining, a
which the type species is L . corymbifera (Cohn) Vuill. Cristal Company. The climate in this area is tropical rainy with
(Vuillemin 1903). Lichtheimia blakesleeana (Lendn.) Kerst. dry summer. The rainy season extends from February to
Hoffm., Walther & K. Voigt, L. corymbifera, L. hyalospora October. The average annual rainfall is 1634.2 mm. The veg-
(Saito) Kerst. Hoffm., Walther & K. Voigt and L. ramosa etation is predominantly sub-evergreen forest, with sub-
(Zopf) Vuill. have been validated and included in the new deciduous forest and Cerrado inclusions (MME 2005b).
Lichteimiaceae K. Hoffm., G. Walther & K. Voigt family. Next,
Alastruey-Izquierdo et al. (2010) transferred A. ornata A.K. Isolation of Lichtheimia
Sarbhoy to Lichtheimia (L . ornata [A.K. Sarbhoy] A.
Alastruey-Izquierdo & G. Walther), described L. sphaerocystis A volume of 5 mg of soil were added to Petri dishes containing
A. Alastruey-Izquierdo & G. Walther and placed L. blakesleeana culture medium wheat germ agar (Benny 2008) amended with
in synonymy with L. hyalospora. chloramphenicol (NeoFenicol—Neo Química) (100 mg.L−1).
Although morphology and mating behaviour are important The dishes were left over the laboratory bench at room tem-
characteristics in traditional concepts of species recognition, perature (26±2 °C) for 7 days, in alternate periods of light and
delimitation of species within mucoralean fungi needs to be dark. Sporangiospores were transferred directly from the col-
combined with molecular data to ensure a reliable delimitation. onies by touching a single sporangium using a sterile needle
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Mycol Progress (2014) 13:343–352 345

on a stereomicroscope (Leika EZ4), and placing the spores Phylogenetic analyses

onto a fresh malt extract agar (MEA) plate (Benny 2008).
The phylogeny was reconstructed using sequences of the 5.8S
and partial sequences of the LSU rDNA gene. Fungal sequences
Experiments were aligned in CLUSTALX ( Larkin et al. 2007) and edited using
BioEdit (Hall 1999). Prior to phylogenetic analysis, the model of
Pure/axenic cultures from specimens were cultured in triplicate, nucleotide substitution was estimated using Topali 2.5 (Milne
in MEA and PDA, and incubated at 15, 20, 25, 30, 35, 37, 40 et al. 2004). Bayesian (two runs over 1×106 generations with a
and 42 °C, during 15 days. Fragments of mycelia were re- burning value of 2500) and maximum likelihood (1000 boot-
moved from cultures, placed on microscope slides with KOH strap) analyses were performed, respectively, in MrBayes 3.1.2
(3 %) and observed under a light microscope (Carl Zeiss (Ronquist and Huelsenbeck 2003) and PhyML (Guindon and
Axioscope 40). The color designation of colonies was Gascuel 2003), launched from Topali 2.5, using the K2P+G
established according to Maerz and Paul (1950). Mating ex- model for the matrix generated for the 5.8S rDNA and GTR+G
periments were carried out on three SMA and PDA plates at 25, model for the matrix generated for LSU rDNA. Neighbor-
33 and 35 °C, on which one 5 mm colony disk of each isolate joining (established with the models cited above) and maximum
was placed in opposite sides of the plates. parsimony analyses were performed using PAUP *4b10
(Swofford 2003) with 1000 bootstrap replications. The sequence
alignment files were deposited in TreeBASE and are available at
Molecular analyses http://purl.org/phylo/treebase/phylows/study/TB2:S14094 and
http://purl.org/phylo/treebase/phylows/study/TB2:S14099.
Fungal biomass was obtained from MEA cultures in test tubes Sequence similarities based on alignments of the LSU,
kept at 28 °C for up to 6 days. All mycelia was removed from the 5.8S rDNA or ITS1-5.8S rDNA-ITS2 sequences were
test tube with a platinum loop and transferred to 2 mL screw calculated using BioEdit (Hall 1999) after shortening them
caped microtubes containing 0.5 g of glass beads (1:1 [w:w], to same length and exclusion of sequences containing
acid-washed, 150–212 μm and 425–600 μm; Sigma, U.S. indefinite bases.
sieve). The fungal material was macerated by vigorous agitation
using a FastPrep homogenizer. Genomic DNA extraction was Prediction of secondary structures and CBC estimations
carried out with previously macerated material according to
Góes-Neto et al. (2005), that includes a washing in chloroform/ Analyses for compensatory base changes (CBCs), as indicators
isoamyl alcohol (24:1) followed by homogenization of the ma- for species boundaries, were performed after prediction of the
terial in 2 % CTAB buffer, isopropanol precipitation, 70 % putative secondary structures of the ITS2 sequences. Secondary
ethanol washing and re-suspension in 50 μL of ultrapure water. structures for L. brasiliensis and L. ramosa were predicted using
To amplify the ITS region, the primers ITS1 and ITS4 RNAfold (Vienna RNAwebsite) (Gruber et al. 2008). Structures
(White et al. 1990) were used and the LR1 (van Tuinen et al. were used as templates for the other sequences of Lichtheimia
1998) and LSU2 (5′- GGTCCGTGTTTCAAGACGGGTCG- using the ‘model’ option in the ITS2 Database (Schultz et al.
3′) were employed to amplify the LSU rDNA. PCR reactions 2006; Koetschan et al. 2012). CBCs were estimated using 4SALE
were carried out in a 50 μL volume containing 75 mM Tris– (Seibel et al. 2006, 2008). Since no experimentally proven
HCl pH 8.8, 200 mM (NH4)2SO4, 0.01 % Tween 20, 2 mM secondary structure for Lichtheimiaceae is known, the predicted
MgCl2, 200 μM each dNTP, 1 μM of each primer and 2 units structures used for CBC estimation remain highly putative.
of TaqTM DNA polymerase (Fermentas, Maryland, USA); Applying the same parameters to different predictions may
cycling parameters were 5 min at 95 °C (1 cycle), 45 s at result in comparable structures and CBC values.
94 °C, 1 min at 60 C, 1 min at 72 °C (39 cycles), and a final
elongation of 7 min at 72 °C followed the last cycle.
The final amplicons were purified with the PureLink PCR Results
Purification Kit (Invitrogen), sequenced directly or cloned
with a CloneJETTM PCR Cloning Kit (Fermentas; Carlsbad, Phylogenetic analyses
USA), following the manufacturer’s instructions, and se-
quenced. Sequencing was provided by the Human Genome Phylogenetic analyses of the 5.8S and LSU rDNA sequences
Research Center (São Paulo, Brazil). Sequence data were placed the two isolates in the family Lichtheimiaceae (bootstrap
compared to gene libraries (EMBL and GenBank) using values>80 %). The specimens of Lichtheimia form a separate
BLAST n. The sequences derived from the new species were clade close to Dichotomocladium Benny & R.K. Benj.
deposited in the NCBI database under the accession numbers Lichtheimia and Dichotomocladium were supported with boot-
KC740484–KC740489. strap values above 90 % in all analyses (Figs. 1 and 2).
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346 Mycol Progress (2014) 13:343–352

Fig. 1 Phylogenetic tree of the Lichtheimiaceae constructed using 5.8S from this study are in bold letters. Consistency index=0.86; Retention
rDNA sequences. Fennellomyces linderi was used as the outgroup. index=0.96. Right site shows sequence similarity matrices for ITS1-5.8S
Sequences are followed by their respective access number. Bootstrap rDNA-ITS2 (left matrix) or 5.8S rDNA (right matrix) alone. Similarities
values are for neighbor-joining (NJ), maximum parsimony (MP), maxi- are summarized in Table 2
mum likelihood (ML) and Bayesian analysis, respectively. Sequences

In the BLAST n analysis of the LSU rDNA sequences, sp. AA-2009 (CBS 958.68) and Lichtheimia sp. AA-
the closest related species to the two isolates were L . 2009a (CBS 291.66) with 90 % identity. In relation to
hyalospora (CBS 102.36 and CBS 173.67), Lichtheimia the 5.8S rDNA sequences, several isolates of L.
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Mycol Progress (2014) 13:343–352 347

Fig. 2 Phylogenetic tree of the Lichtheimiaceae and related taxa (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayes-
constructed using LSU rDNA sequences. Representatives of the genus ian analysis, respectively. Sequences from this study are in bold letters.
Absidia were used as the outgroup. Sequences are followed by their Consistency index=0.5r; Retention index=0.91. Right: Sequence simi-
respective access number. Bootstrap values are for neighbor-joining larity matrix for LSU sequences. Similarities are summarized in Table 1

corymbifera and L. hyalospora showed 96 % identity species and genera, resulting in difficulties to properly
with the new species. The 5.8S rDNA is a highly con- align the sequences. Based on LSU sequences, similarities
served region and can be used to separate genus and between the different species of Lichtheimia and other
above, but does not give good resolution at the species genera include ranges overall from 60 % to 77 % and
level. The ITS 1 and ITS 2 regions were not used in the 81–100 % within Lichtheimia itself (Table 1). Sequence
analyses due to the high level of variation between similarities of full-length ITS sequences (including 5.8S
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348 Mycol Progress (2014) 13:343–352

Table 1 LSU-sequence similarities (%) of the source sequences for the phylogenetic analyses shown in Fig. 2. Similarities were summarized for the
single clades

Mucorales A F D Lb Lh Ls Lo & Lc Lr

Absidia 78–100 59–63 59–63 61–64 62–65 60–64 60–65 60–64

Fennellomyces 59–63 100 69 69 70 70 70–71 71
Dichotomocladium 59–63 69 87–100 73–75 74–77 72–75 74–76 73–75
Lichtheimia brasiliensis 61–64 69 73–75 100 85 82 83–84 81–82
L. hyalospora 62–65 70 74–77 85 99–100 93–95 93–94 92–93
L. sphaerocystis 60–64 70 72–75 82 93–95 99–100 93–94 93–94
L. ornata & L. corymbifera 60–65 70–71 74–76 83–84 93–94 93–94 98–100 94–95
L. ramosa 60–64 71 73–75 81–82 92–93 93–94 94–95 94–100

rDNA) range from 38 % to 45 % identity between the Taxonomy

investigated genera Fennellomyces, Dichotomocladium
and Lichtheimia and 44–65 % between species within the Lichtheimia brasiliensis A.L. Santiago, Lima & Oliveira, sp.
genus Lichtheimia itself (Table 2). Lichtheimia brasiliensis nov. Fig. 3
shows similar, but slightly lower interspecies similarities (44– MycoBank803795
52 %) when compared to the other Lichtheimia species (50– White colonies at first then turning light grey (MP 11A1),
65 %). Yet, the highly conserved 5.8S rDNA shows 87–90 % higher in the central portion, covering the entire Petri plate and
intergeneric and 93–100 % interspecies similarities (Table 2) . touching the plate lid in the central region and in some points
Comparing the secondary structures of ITS2 and analyzing in the periphery of the colony after 15 days in MEA at 25 °C.
them for CBCs, the species appears to be well delimited Reverse yellow (MP 11I2). Coenocytic or irregularly septate
(Table 3). The number of observed CBCs for L. brasiliensis stolons, with few septa, mainly in proximity to the place of
is comparable to other CBCs observed in this genus, sporophore origin. Rhizoids branched and with bulbous dila-
supporting its proposed unique status as new species. The tation in some points. Sporophores simple, few branched,
observed CBCs should be considered carefully, since different erect, bent or circinate, raising from aerial hyphae or from
parameters used for the prediction of the secondary structures stolons (the latter, in several points and terminally), solitary or
will lead to different structures and therefore to different values in pairs, never in whorls, occasionally with one septum below
of CBCs, although in similar comparable relations (data not the sporangium, colorless and becoming grey near the colu-
shown). Nevertheless, estimation of CBCs could be considered mella (2.6)3.6–7.2(12) μm diameter (diam.). Yellowish
as supportive information in full accordance with all observed sporangia, turning brown, multispored, sphaerical or sub-
morphological features, including the phylogenetic analyses pyriform, apophysate, sometimes with long conical or bell
and the so far unsuccessful crossings. shaped apophysis, 19–55 μm diam. with a smooth,

Table 2 Sequence similarities (%) of the source sequences for the phylogenetic analyses shown in Fig. 1. Similarities are displayed for ITS1-5.8S
rDNA-ITS2 (lower triangle) and 5.8S rDNA alone (upper triangle, grey). Similarities were summarized for the single clades

ITS 5.8S F D Lb Lr Lo & Lc Ls Lh

Fennellomyces 100 90 90-91 87-89 88-89 89 88-90
100
Dichotomocladium 100 91-92 87-88 86-88 88 87-88
38 100
Lichtheimia 99 93-94 93-95 95 94-96
brasiliensis 44-45 40 99
L. ramosa 99-100 97-99 98-99 97-99
41-42 42-43 50-51 89-100
L. ornata & 99-100 99-100 98-99
L. corymbifera 43-45 40 49-52 57-60 81-100
L. sphaerocystis 100 99-100
40-41 41 44-45 54-55 50-53 93-100
L. hyalospora 99-100
41-43 39-41 49-52 55-56 54-57 63-65 85-100
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Mycol Progress (2014) 13:343–352 349

Table 3 Compensatory base changes (CBCs) summarized for ITS2 Discussion

sequences used in this study

Species Lb Lr Lo Lc Ls Lh Several studies have reported the variability of nucleotide

sequences of the ITS region (Iwen et al. 2002; Schwarz et al.
L. brasiliensis 0 3–4 2 2 1 3–4 2006; Hoffmann et al. 2009) and LSU rDNA (Álvarez et al.
L. ramosa 3–4 0–1 1–2 2–5 1–2 3–4 2011; de Souza et al. 2012) as reliable indication of taxon
L. ornata 2 1–2 0 0 1–2 1–2 differentiation in Mucorales. According to our phylogenetic
L. corymbifera 2 2–5 0 0 2–3 1–3 and morphophysiological analysis, L. brasiliensis is geneti-
L. sphaerocystis 1 1–2 1–2 2–3 0 1–2 cally distinct from other described species within the genus.
L. hyalospora 3–4 3–4 1–2 1–3 1–2 0–1 Evidences from the analyzed datasets support the delimitation
of a new species. The analysis of either the 5.8S region or the
LSU rDNA showed the two sequenced specimens grouping
transparent wall. Columellae short hemisphaerical, sometimes strongly in the family Lichtheimiaceae, in the genus
almost non-existing, subglobose, never spatulate nor conical, Lichtheimia and close to Dichotomocladium. Topology and
smooth and without projections, 7.2–26.5×2.5–26.5 μm. statistical support of the phylogenetic trees, as well as genetic
Collar present or absent. Sporangiospores hyaline with differences (maximum identity) between the studied speci-
smooth wall, ellipsoid, subglobose, 2.8–5.0×2.4–3.4. Giant mens and representatives in Lichtheimiaceae confirm the pro-
cells absent. Zygospores not observed. posal of a new species for this family.
Holotype URM 6910. Morphologically, L. brasiliensis resembles the described
Media, temperature tests and mating experiments: On species of Lichtheimia as it presents erect or circinate sporo-
MEA. At 15 °C—slow growth (4.2 cm in 120 h). Few phores emerging from aerial hyphae or from stolons, occa-
sporophores formed; poor sporulation. At 20 °C—slow sionally with a septum below the sporangium, besides pro-
growth (5.3 cm in 96 h); medium sporulation. At 25 °C—faster ducing apophysed sporangia that are common characteristics
growth than at 20 °C (9 cm in 96 h); good sporulation. At of the genus Lichtheimia (Hoffmann et al. 2007, 2009).
30 °C—better growth than at 25 °C (9 cm in 72 h); excellent However, the presence of short and very short hemispheric
sporulation. At 35 °C—similar growth to 30 °C (9 cm in 72 h); columellae that are never spatulate and lack projections, be-
excellent sporulation. At 37 °C—slower growth than at 35 °C sides the absence of giant cells in the mycelium also differen-
(9 cm in 96 h); good sporulation. At 40 °C—limited growth tiate L. brasiliensis from the described Lichtheimia species.
(6.5 cm in 168 h); rare sporophores produced and sporulation Some bell shaped apophyses, sometimes very dilatated can
poor; colonies with irregular borders and many peripheral eventually be observed in L. brasiliensis.
colonies. At 42 °C—lack of growth and sporulation. The According to Santos et al. (2003) and Hoffmann et al. (2007,
growth of L . brasiliensis on PDA was similar to that on 2009), production of giant cells is considered an important
MEA for the temperatures of 15, 20, 25, 30, 37 and 40 °C. morphological characteristic in Lichtheimia. However, giant
At 35 °C the growth was faster in PDA than in MEA (9 cm in cells are not produced by all isolates of this genus. Alastruey-
48 h). The sporangiospores of URM 6911 are mostly Izquierdo et al. (2010) cited that on MEA, at 24 °C, many
subglobose or subglobose to ellipsoid, differing from URM samples (not all) of all known species of Lichtheimia devel-
6910 that are mostly ellipsoid. Colonies in the former are gray oped giant cells. These structures were also common on PDA,
to deep grey, differing from the latter that is light grey. Plates with higher degree of differentiation at 24 °C and lower degree
from mating experiments were analyzed at 5, 7, 14, 21, 28 and of differentiation at 33 and at 37 °C. Hoffmann et al. (2007)
35 d and zygosporangia were not observed. reported the presence of giant cells as an important character-
Influence of light: not detected. istic together with thermotolerance to include L. hyalospora in
Specimen examined: BRAZIL, Pernambuco, Araripina, the family Mycocladiaceae K. Hoffmann, S. Discher & K.
Instituto Agronômico de Pernambuco (IPA), 7º27′58″S and Voigt (= Lichtheimiaceae), as the zygospores in this species
40º24′53″W. Soil, 09. 2009, R.J. Oliveira. Holotype (URM 6910). were never observed. These structures were cited by Ellis and
Other examined specimen. BRAZIL , João Pessoa, Mataraca, Hesseltine (1966) to L. ramosa (cited as A. ramosa [Lindt]
dune areas of the Millennium Inorganic Chemicals Mining, a Lendn) and to L. corymbifera (cited as A. corymbifera [Cohn]
Cristal Company, 6°30′00″S and 34°57′10″W, Soil, 03. 2010, Sacc. & Trotter). In the former species, the giant cells were
D.X. Lima, (URM 6911). common on PDA and absent on Synthetic Mucor Agar (SMA)
Etymology: Brasiliensis. Referring to country where the and, to the latter, giant cells were common on SMA and PDA.
species was first isolated. Hesseltine and Ellis (1966) cited the presence of giant cells in
Habitat: Soil L. hyalospora (cited as A. hyalospora [Saito] Lendn. and as A.
Distribution: Araripina (Pernambuco, Brazil) and Mataraca blakesleeana Lendn.). In this latter species, giant cells were
(Paraíba, Brazil). observed in cultures grown on SMA and MEA. Alastruey-
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350 Mycol Progress (2014) 13:343–352
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Mycol Progress (2014) 13:343–352 351

ƒFig. 3 Lichtheimia brasiliensis. a Colony surface; b Sporophores and 35 °C, and very limited growth at 40 °C. The growth temper-
sporangia; c Simple sporangiophore with a bell-shaped apophysis (arrow) atures observed to L. sphaerocystis and L . brasiliensis indi-
under the sporangium; d, e Simple sporangiophore with a sporangium; f
Branched sporangiophore with sporangium and columella; g, h Circinate
cate that these species of Lichtheimia do not exhibit strict
sporangiophore; i Two sporangiophores with short hemispherical thermotolerance.
columellae and collars (arrow); j Rhizoids; k Sporangiospores. Bars: B, In conclusion, none of the described species of Lichtheimia
C, D, E, K=50 μm; F, G, H, I=25 μm; J =100 μm have all the characteristics as found in L. brasiliensis, there-
fore it is described as new. Although both specimens of L.
Izquierdo et al. (2010) observed the presence of giant cells in L. brasiliensis have been grouped in a subclade slightly distant
sphaerocystis and in L. ornata on MEA and yeast extract agar from the other species of the genus, when considering the two
(YEA), respectively, and used the globose form of these struc- phylogenetic trees shown (5.8S region and the LSU rDNA),
tures, associated with molecular information, to separate L. we did not find morphological characteristics sufficiently
sphaerocystis from the other species within the genus. Thus, strong to propose a new genus.
as some isolates of Lichtheimia do not produce giant cells, the
total absence of these structures in L. brasiliensis, either on Acknowledgments The authors express their gratitude to Conselho
MEA or PDA, at the tested temperatures, cannot be seen as an Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and to
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
indication to exclude it from Lichtheimia. for Master scholarships provided to Rafael José Vilela de Oliveira and
The presence of projections in the columellae has been Diogo Xavier de Lima, respectively. We are also thankful to Fundação de
cited to all known species of Lichtheimia . Hesseltine and Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) and
Ellis (1966) reported projection up to 3.5 μm diam. in colu- to CNPq for a Post Doctoral fellowship and research grants (Protax Proc.
562330/2010-0, INCT-Herbario Virtual Proc. 573883/2008-4, Sisbiota
mellae of L. hyalospora, while Ellis and Hesseltine (1966) Proc. 563342/2010-2) to the first author and to Leonor C. Maia,
observed that columellae of L. ramosa and L. corymbifera respectively.
presented, frequently, one to several projections of 2.5 and
4.5 μm length, respectively. Alastruey-Izquierdo et al. (2010)
cited occasional production of projections of 8.5–29 μm References
length in L . sphaerocystis. As these structures were not ob-
served in L. brasiliensis, the absence can be considered as
[MME] Ministério das Minas e energia (2005a) Projeto cadastro de fontes
distinctive characteristic between our isolate and the other de abastecimento por água subterrânea. Pernambuco. Diagnóstico
Lichtheimia species. Likewise, globose, elliptical, elongated do Município de Araripina. Secretaria de Geologia, Mineração e
and spatulate columellae, besides the hemispheric, were de- Transformação Mineral, Brasil
scribed for Lichtheimia species. In L . brasiliensis only hemi- [MME] Ministério das Minas e energia (2005b) Projeto cadastro de fontes
de abastecimento por água subterrânea. Paraíba. Diagnóstico do
spheric (short and very short) columellae and few subglobose Município de Mataraca. Secretaria de Geologia, Mineração e
(never spatulate) were observed. Moreover, some columellae Transformação Mineral, Brasil
are so small that they seem inconspicuous, differently from the Alastruey-Izquierdo A, Hoffmann K, de Hoog GS, Rodriguez-Tudela JL,
observed in Lichtheimia species so far. Voigt K, Bibashi E, Walther G (2010) Species recognition and
clinical relevance of the Zygomycetous Genus Lichtheimia (syn.
Besides the presence of giant cells, growth temperature has Absidia Pro Parte, Mycocladus). J Clin Microbiol 48:2154–2170
been an important physiological characteristic to separate Álvarez E, Cano J, Stchigel AM, Sutton DA, Fothergill AW, Salas V,
Lichtheimia species from other “Absidia -like” species. Rinaldi MG, Guarro J (2011) Two new species of Mucor from
According to Hoffmann et al. (2007), growth capacity at clinical samples. Med Mycol 49:62–72
Beauverie J (1900) Mycocladus verticillatus (gen. nov. sp. nov.). Ann
37 °C, or at temperatures above this, distinguish species Univ Lyon Sér 2 Sci Méd 3:162–180
included in the group of thermolerant (Lichtheimia ) from the Benny GL (2008) The methods used by Dr. R. K. Benjamin, and other
mesophilic (Absidia) group, being permissive to the former mycologists, to isolate Zygomycetes. Aliso 26:37–61
and suppressive to the latter group. These authors showed that Benny GL (2010) Zygomycetes. Published on the internet at www.
zygomycetes.org
L. hyalospora (as A. hyalospora and A . blakesleeana), and Coleman AW, Vacquier VD (2002) Exploring the phylogenetic utility of
L . corymbifera (as A . corymbifera ) presented optimum ITS sequences for animals: a test case for abalone (Haliotis). J Mol
growth temperatures between 37 and 45 °C, and were capable Evol 54:246–257. doi:10.1007/s00239-001-0006-0
of growing at 42, 50 and 53 °C, respectively. However, de Souza JI, Pires-Zottarelli CLA, dos Santos JF, Costa JP, Harakava R
(2012) Isomucor (Mucoromycotina): a new genus from a Cerrado
Alastruey-Izquierdo et al. (2010) observed that specimens of reserve in state of São Paulo, Brazil. Mycologia 104:232–241. doi:
L. sphaerocystis presented optimum growth temperatures on 10.3852/11-133
MEA at 33 °C and, among the three studied specimens, two Ellis JJ, Hesseltine CW (1966) Species of Absidia with ovoid
presented maximum growth temperatures at 37 °C and only sporangiospores. II. Sabouraudia 5:59–77
Góes-Neto A, Loguercio-Leite C, Guerrero RT (2005) DNA extraction
one was capable of growing at 40 °C. Regarding L . from frozen field-collected and dehydrated herbarium fungal
brasiliensis, the two specimens studied in this work were able basidiomata: performance of SDS and CTAB-based methods.
to grow at 37 °C, with optimum growth temperature at 30– Biotemas 18:19–32
 Author's personal copy
352 Mycol Progress (2014) 13:343–352

Gruber AR, Lorenz R, Bernhart SH, Neubock R, Hofacker IL (2008) The Mirza JH, Khan SM, Begum S, Shagufta S (1979) Mucorales of Pakistan.
Vienna RNA websuite. Nucl Acids Res 36:70–74. doi:10.1093/nar/ University of Agriculture, Faisalabad
gkn188 Misra PC, Srivastava KJ, Lata K (1979) Apophysomyces, a new genus of
Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to the Mucorales. Mycotaxon 8:377–382
estimate large phylogenies by maximum likelihood. Syst Biol 52: Müller T, Philippi N, Dandekar T, Schultz J, Wolf M (2007) Distinguishing
696–704. doi:10.1080/10635150390235520 species. RNA 13:1469–1472. doi:10.1261/rna.617107
Hall TA (1999) BioEdit: a user-friendly biological sequence alignment Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic
editor and analysis program for Windows 95/98/NT. Nucleic Acids inference under mixed models. Bioinformatics 19:1572–1574. doi:
Symp Ser 41:95–98 10.1093/bioinformatics/btg180
Hesseltine CW, Ellis JJ (1964) The genus Absidia: Gongronella and Santos MJS, de Oliveira PC, Trufem SFB (2003) Morphological obser-
cylindrical-spored species of Absidia. Mycologia 56:568–601. doi: vations on Absidia corymbifera and Absidia blakesleeana strains
10.2307/3756362 preserved under mineral oil. Mycoses 46:402–406. doi:10.1046/j.
Hesseltine CW, Ellis JJ (1966) Species of Absidia with ovoid 0933-7407.2003.00923.x
sporangiospores. I. Mycologia 58:761–785. doi:10.2307/3756851 Schipper MAA (1990) Notes on Mucorales—I. Observations on Absidia.
Hoffmann K, Voigt K (2009) Absidia parricida plays a dominant role in Persoonia 14:133–149
biotrophic fusion parasitism among mucoralean fungi (Zygomycetes): Schultz J, Müller T, Achtziger M, Seibel PN, Dandekar T, Wolf M (2006)
Lentamyces, a new genus for A. parricida and A. zychae. Plant Biol The internal transcribed spacer 2 database–a web server for (not
10:537–554 only) low level phylogenetic analyses. Nucleic Acids Res 34:704–
Hoffmann K, Discher S, Voigt K (2007) Revision of the genus Absidia 707. doi:10.1093/nar/gkl129
(Mucorales, Zygomycetes) based on physiological, phylogenetic, Schwarz P, Bretagne S, Gantier JC, Garcia-Hermoso D, Lortholary O,
and morphological characters; thermotolerant Absidia spp. form a Dromer F, Dannaoui E (2006) Molecular identification of
coherent group, the Mycocladiaceae fam. nov. Mycol Res 111: Zygomycetes from culture and experimentally infected tissues. J
1169–1183. doi:10.1016/j.mycres.2007.07.002 Clin Microbiol 44:340–349. doi:10.1128/JCM.44.2.340-349.2006
Hoffmann K, Walther G, Voigt K (2009) Mycocladus vs. Lichtheimia: a Seibel PN, Müller T, Dandekar T, Schultz J, Wolf M (2006) 4SALE—a
correction (Lichtheimiaceae fam. nov., Mucorales, Mucoromycotina). tool for synchronous RNA sequence and secondary structure align-
Mycol Res 113:277–278 ment and editing. BMC Bioinformatics 7:498
Iwen PC, Hinrichs SH, Rupp ME (2002) Utilization of the internal Seibel PN, Müller T, Dandekar T, Wolf M (2008) Synchronous visual
transcribed spacer regions as molecular targets to detect and identify analysis and editing of RNA sequence and secondary structure
human fungal pathogens. Med Mycol 40:87–109. doi:10.1080/ alignments using 4SALE. BMC Res Notes 1:91. doi:10.1186/
mmy.40.1.87.109 1756-0500-1-91
Kirk PM, Cannon PF, Minter DW, Stalpers JA (2008) Ainsworth & Swofford DL (2003) PAUP*. Phylogenetic analysis using parsimony
Bisby’s dictionary of the fungi, 10th edn. CAB International, (*and Other Methods). Sinauer Associates, Sunderland
Wallingford Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS,
Koetschan C, Hackl T, Müller T, Wolf M, Förster F, Schultz J (2012) ITS2 Fisher MC (2000) Phylogenetic species recognition and species
database IV: Interactive taxon sampling for internal transcribed concepts in fungi. Fungal Genet Biol 31:21–32. doi:10.1006/fgbi.
spacer 2 based phylogenies. Mol Phylogenet Evol 63:585–588. 2000.1228
doi:10.1016/j.ympev.2012.01.026 van Tieghem P (1876) Troisième mémoire sur les Mucorinées. Ann Sci
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, Nat Bot sér VI 4:312–399
McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson van Tuinen D, Zhao B, Gianinazzi-Pearson V (1998) PCR in studies of
JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version AM fungi: from primers to application. In: Varma AK (ed) Mycor-
2.0. Bioinformatics 23:2947–2948. doi:10.1093/bioinformatics/btm404 rhizal manual. Springer, Berlin, pp 387–399. doi:10.1007/978-3-
Maerz A, Paul MR (1950) A dictionary of color. McGraw-Hill book 642-60268-9_24
Company, USA Vuillemin P (1903) Le genre Tieghemella et la série de Absidées. Bull
Mayden RL (1997) A hierarchy of species concepts: the denouement in Soc Mycol France 19:119–127
the saga of the species problem. In: Claridge MF, Dawah HA, White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct
Wilson MR (eds) Species: the units of biodiversity. Chapman & sequencing of fungal ribosomal RNA genes for phylogenetics. In:
Hall, London, pp 381–424 Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a
Milne I, Wright F, Rowe G, Marshal DF, Husmeier D, McGuire G (2004) guide to methods and applications. Academic, San Diego, pp 315–
TOPALi: software for automatic identification of recombinant se- 322
quences within DNA multiple alignments. Bioinformatics 20:1806– Zheng R-Y, Chen G-Q, Huang H, Liu X-Y (2007) A monograph of
1807. doi:10.1093/bioinformatics/bth155 Rhizopus. Sydowia 59:273–372

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