Cryptococcus depauperatus, a close relative of the human-pathogen C. neoformans, associated with coffee leaf rust (Hemileia vastatrix) in Cameroon

Abstract

The genus Cryptococcus is well known for its two species —Cryptococcus neoformans and C. gatii— that are etiological agents of cryptococcosis, an important fungal disease of mammals, including humans, and which is particularly common in immunocompromised patients. Nevertheless, Cryptococcus is a large and widely distributed genus of basidiomycetes occupying a broad range of niches, including mycoparasitism. One such mycoparasitic species is Cryptococcus depauperatus, which was firstly mistakenly described as a pathogen of scale insects under the name Aspergillus depauperatus. The “Aspergillus” conidiophores were later shown to be basidia of a Cryptococcus and the new combination C. depauperatus was proposed. Additionally, instead of an entomopathogen, the fungus was found to be a mycoparasite growing on the entomopathogen Akanthomyces (Lecanicillium) lecanii. Recently, during surveys for mycoparasites of coffee leaf rust (Hemileia vastatrix) in the context of a biocontrol project, white colonies covering rust pustules were observed in Cameroon. Upon close examination, instead of a member of the “white colony forming complex” of Ascomycetes, commonly collected growing on H. vastatrix, such colonies were found to represent a basidiomycete fungus with basidia-bearing chains of basidiospores, typical of the genus Cryptococcus. Morphological and molecular evidence was generated supporting the identification of the fungus on rust pustules as C. depauperatus. This is the first record of C. depauperatus from Africa and of its association with coffee leaf rust.

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Vol.:(0123456789)
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Brazilian Journal of Microbiology
https://doi.org/10.1007/s42770-021-00592-2
ENVIRONMENTAL MICROBIOLOGY – RESEARCH PAPER
Cryptococcus depauperatus, aclose relative ofthehuman‑pathogen
C. neoformans, associated withcoffee leaf rust (Hemileia vastatrix)
inCameroon
DéboraC.Guterres1 · MiraineK.Ndacnou1,2 · LauraM.Saavedra‑Tobar1 · SaraSalcedo‑Sarmiento1 ·
AdansA.Colmán1 · HarryC.Evans3 · RobertW.Barreto1
Received: 18 June 2021 / Accepted: 4 August 2021
© Sociedade Brasileira de Microbiologia 2021
Abstract
The genus Cryptococcus is well known for its two species —Cryptococcus neoformans and C. gatii—that are etiological
agents of cryptococcosis,an important fungal disease of mammals, including humans, and which is particularly common in
immunocompromised patients. Nevertheless, Cryptococcus is a large and widely distributed genus of basidiomycetes occupy-
ing a broad range of niches, including mycoparasitism. One such mycoparasitic species is Cryptococcus depauperatus, which
was firstly mistakenly described as a pathogen of scale insects under the name Aspergillus depauperatus. The “Aspergillus”
conidiophores were later shown to be basidia of a Cryptococcus and the new combination C. depauperatus was proposed.
Additionally, instead of an entomopathogen, the fungus was found to be a mycoparasite growing on the entomopathogen
Akanthomyces (Lecanicillium) lecanii. Recently, during surveys for mycoparasites of coffee leaf rust (Hemileia vastatrix)
in the context of a biocontrol project, white colonies covering rust pustules were observed in Cameroon. Upon close exami-
nation, instead of a member of the “white colony forming complex” of Ascomycetes, commonly collected growing on H.
vastatrix, such colonies were found to represent a basidiomycete fungus with basidia-bearing chains of basidiospores, typi-
cal of the genus Cryptococcus. Morphological and molecular evidence was generated supporting the identification of the
fungus on rust pustules as C. depauperatus. This is the first record of C. depauperatus from Africa and of its association
with coffee leaf rust.
Keywords Africa· Basidiomycota· Coffea arabica· Cryptococcosis· Filobasidiella· Tremellales
Introduction
Coffee leaf rust (CLR) caused by Hemileia vastatrix is
one of the major threats to coffee production worldwide.
Since the first record of it causing severe epidemics in Sri
Lanka (formerly Ceylon) in 1860, CLR has spread to all
coffee growing regions in the world [40]. Although widely
recognized as a major cause of losses to coffee, it has gained
additional relevance since 2012 when severe outbreaks dev-
astated plantations in northern South America and Central
America, where coffee is often the only source of income
for farmers and local communities [4, 18].
Management of CLR relies on the use of fungicides,
resistant cultivars, or on escaping climatic conditions that
favor the development of the disease by cultivating coffee
at high altitudes [3, 7, 13]. Biological control is an alterna-
tive method based on the use of natural enemies to control
noxious organisms [42]. Several species of bacterial and fun-
gal antagonists of CLR have been described and studied for
their potential as biocontrol agents [8, 9, 28, 56], including
mycoparasites that are able to penetrate, colonize, and feed
on the rust spores, thereby reducing the inoculum loads and
consequently the incidence and severity of the disease [11,
15, 27, 31].
Communicated by Rosana Puccia.
* Robert W. Barreto
rbarreto@ufv.br
1 Universidade Federal de Viçosa, Viçosa,
MinasGeraisCEP36570-900, Brazil
2 Institute ofAgricultural Research forDevelopment (IRAD),
PO Box. 2067, Yaounde, Cameroon
3 CAB International, Bakeham Lane, EghamTW209TY,
Surrey, UK

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A study was initiated aimed at finding non-pesticide alter-
natives to tackle the coffee rust crisis in Central America,
funded by World Coffee Research. It included surveys in
both Africa and Latin America for antagonistic fungi—
endophytic fungi growing inside coffee tissues and myco-
parasites of H. vastatrix pustules—in a search for potential
biocontrol agents. White colony-forming mycoparasites
were commonly found. Although such colonies have often
been referred to in the literature as Lecanicillium lecanii (in
the earlier literature as Verticillium lecanii and now trans-
ferred to Akanthomyces [34]), our work has revealed that
this treatment grossly overlooked a significant diversity of
mycoparasitic fungi belonging to a range of genera, in addi-
tion to the Lecanicillium/Akanthomyces complex, includ-
ing Pleurodesmospora, Simplicillium, Acremonium, Saro-
cladium, Ijuhya, Ovicillium [16], and Fusarium [43]. One
fungus, in particular, was noted within this “white colony
forming complex” because of the presence of basidia instead
of conidiogenous cells. Here, we describe and discuss this
unusual fungal record and its association with H. vastatrix.
Material andmethods
Collections andmorphological characterization
The sample examined in detail during this study was
obtained from a semi-wild small Arabica coffee farm at
Ekonjo village in the Fako department of south-west Cam-
eroon—on the slope of Mt. Cameroon (Fig.1)—in 2015,
during a survey search for mycoparasites of H. vastatrix.
The focus of this visit to the area was on wild Coffea spp., in
this case C. brevipes, but smallholder plantations were also
sampled. Specimens were dried in a plant press and trans-
ferred to the laboratory for later processing. Unfortunately,
the samples were left in storage for too long (over 6months)
and attempts at isolating it in pure culture, after that time,
either by transfer of spores onto potato dextrose-agar (PDA)
plates of potato carrot-agar (PCA) plates repeatedly failed.
Dried leaves were deposited in the herbarium of the
Universidade Federal de Viçosa (Herbarium VIC) with the
accession number VIC47429. Macromorphological exami-
nation and documentation were done using a SZX7 Zoom
stereo microscope (Olympus, Tokyo, Japan). A BX51 light
microscope coupled to an Olympus Qcolor 3 digital cam-
era (Olympus, Tokyo, Japan) was used for more detailed
observations. Image capture and editing were processed
using cellSens Dimension 1.17 software. Portions of dry
leaves containing rust pustules colonized by the white-col-
ony forming fungus were mounted on copper stubs, gold
sputter-coated, and subjected to scanning electron micros-
copy using a Zeiss Leo 1430 VP microscope (Carl Zeiss AG,
Jena, Germany).
DNA extraction, amplification, andsequencing
In order to obtain a representative sample of the fungal
DNA, two leaves bearing groups of uredinia extensively
covered by white mycelium were examined under a ster-
eomicroscope to check for possible contamination by other
fungi and clean colonies were marked with a felt tip pen.
Selected portions of the white mycelial mat were removed
from leaves with a sterile fine pointed needle and placed
into a microcentrifuge tube (1.5ml) containing 5µl of
double distilled water and zirconium spheres and placed
in a grinder (L-Beader-3, Loccus Biotecnologia). After
5-s grinding, the resulting suspension was drained into a
sterile plastic tube and used for DNA extraction. Genomic
DNA extraction was made using a Wizard Genomic DNA
Purification Kit (Promega, Madison, WI, USA), follow-
ing the manufacturer’s instructions. DNA quantification
was performed by comparison with a Low DNA100 Mass
Ladder (Invitrogen, Carlsbad, CA, USA) following elec-
trophoresis on 1% agarose gels. Polymerase chain reaction
(PCR) amplifications were performed on a Peltier-based
Thermal Cycler A200 (Biopeony Beijing, China).
The D1–D2 domains of nuclear large subunit ribosomal
rDNA (28S) were amplified with primers LR0R [64] and
LR5 [63]. PCR thermocycling was performed with initial
DNA denaturation at 94°C for 1min 30s, followed by
38 cycles of DNA denaturation at 94°C for 30s. Primer
annealing was performed at 53°C for 30s, extension at
72°C for 45s, with a final extension at 72°C for 5min,
followed by storage at 4°C.
PCR products were checked on 1% agarose gels and
then treated with ExoSAP-IT PCR Product Cleanup (Affy-
metrix, Cleveland, OH) before sequencing, which was con-
ducted at Macrogen (Seoul, South Korea). Electrophero-
grams were manually checked, with ambiguous positions
clarified by comparing forward and reverse sequences.
Newly generated sequences were assembled and annotated
using Geneious 9.0.5 [33] and then deposited in GenBank
(http:// www. ncbi. nlm. nih. gov) (Table1).
Preliminary identification of the fungus based on
nuclear rDNA 28S sequences was conducted through que-
ries against the National Center for Biotechnology Infor-
mation (NCBI) nucleotide database with a standard nucle-
otide BLASTn search using the megablast algorithm. After
examining the preliminary results, our taxon sampling was
based on Findley [22] to include the Filobasidiella clade
within the Cryptococcaceae (Tremellales).
Nucleotide sequences were aligned in MAFFT 7.271
[32] using the E-INS-i refinement strategy. Maximum
likelihood analysis using RAxML 8.2.9 [58] started with
randomized stepwise addition parsimony tree, assuming a
GTR + CAT model. Branch support values were calculated

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based on 1000 bootstrap (BS) pseudoreplicates under the
same model.
Bayesian inference (BI) was performed using MrBayes
3.2.6 [50], with appropriate evolutionary model estimated
in jModelTest 2 [20] and chosen according to the corrected
Akaike information criteria. Two independent runs, each one
initiating from random trees with four metropolis-coupled
Markov chains per run for 5 × 107 generations. Trees were
sampled every 1000th generation. Four rate categories
were used to approximate the gamma distribution. Average
standard deviation of split frequencies (ASDSF) and effec-
tive sample size (ESS) were used as convergence criteria for
Bayesian analyses. A total of 25% of all sampled trees were
discarded as burn-in, whereas the remaining 75% trees were
employed to estimate the Bayesian posterior probabilities
(BPPs) for branches. All analyses, RAXML, jModelTest
2, and MrBayes, were conducted on the CIPRES Science
Gateway 3.1 [41].
Results
The morphological examination revealed basidia and basidi-
ospores typical of Cryptococcus depauperatus (Crypto-
coccaceae: Tremellales; see Fig.2c–e). This is described
as follows: Hymenium composed of loose basidial stalks
with no sterile filaments. Basidia aseptate, composed of a
long cylindrical stalk (58–106 × 2–2.5μm), often irregularly
Fig. 1 Site of collection of
Cryptococcus depauperatus in
Ekonjo village, Fako Depart-
ment, Cameroon. (a) Cameroon.
(b) Localization of Ekonjo,
Buéa district in South-west
region of Cameroon; (c) coffee
cultivated in semi-wild, mixed
cropping system at forest edge

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Table 1 Sequences of the nuc
28S rDNA used in the present
study
Isolates and GB accession numbers in bold are from isolates sequenced in this study
Species Isolate or specimen GB accession number
Cryptococcus cuniculi T-26 DQ333885
Cryptococcus amylolentus CBS 6039 AF105391
Cryptococcus depauperatus CBS 7841 FJ534911
VIC47429 a MW209041
VIC47429 b MW209040
Cryptococcus gattii AFLP5_CBS6955 JN939485
CBS 10,514 FJ534907
CBS 6289 AF075526
Cryptococcus neoformans var. grubii CBS 8710 FJ534909
Cryptococcus sp. CBS 12,705 KC894161
Cryptococcus sp. CBS 7712 AJ311450
Cryptococcus sp. CECT 11,955 AY167602
Cryptococcus sp. UFMG-BRO443 JX280388
Cryptotrichosporon anacardii CBS 9551 AY550002
Cryptotrichosporon tibetense XZ 20A4 KP020115
Cystofilobasidium bisporidii CBS 6346 EU085532
Cystofilobasidium capitatum CBS 6358 AF075465
Cystofilobasidium infirmominiatum CBS 323 AF075505
Cystofilobasidium lacus-mascardii CRUB 1046 AY158642
Cystofilobasidium macerans A006 EU082225
Derxomyces wuzhishanensis AS 2.3760 EU517063
Derxomyces yunnanensis AS 2.3562 EU517064
Dimennazyma cisti-albidi 1CSF5; PYCC 5851; CBS
10,049
AY562135
Dioszegia antarctica ANT 03–116 FJ640575
Dioszegia athyri CB 159; AS 2.2559 EU070931
Dioszegia zsoltii AS 2.2089 AF544245
Effuseotrichosporon vanderwaltii CBS 12,124 JF680903
Fellomyces penicillatus CBS5492 AF177405
Fellomyces polyborus CBS 6072 AF189859
Filobasidium chernovii CBS8679 AF181530
Filobasidium elegans CBS7640 AF181548
Filobasidium floriforme CBS 6241 AF075498
Filobasidium globisporum CBS 7642 AF075495
Filobasidium magnum CBS140 AF181851
Filobasidium oeirense CBS8681 AF181519
Filobasidium stepposum PTZ 139VKM-2918 DQ222456
Filobasidium uniguttulatum CBS 1730 AF075468
Filobasidium wieringae CBS1937 AF181541
Mrakia aquatica CBS 5443 AF075470
Mrakia blollopis 124a; CBS 8921 AY038814
Mrakia frigida CBS 5270 AF075463
Sterigmatosporidium polymorphum IGC 5647 AY032662
Udeniomyces kanasensis XJ 6E2 JQ002681
Udeniomyces megalosporus CBS 7236 AF075510
Udeniomyces puniceus CBS 5689 AF075519
Udeniomyces pyricola CBS 6754 AF075507
Vanrija albida JCM 1460 AB126584

Brazilian Journal of Microbiology
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curved, smooth, expanding terminally into an apical head,
obpyriform-clavate, and slightly flattened, 9–10 × 5–6μm,
bearing four short, rounded sterigmatous protuberances—
termed sporogenous loci [39] or apical loci [47] producing
basidiospores in basipetal chains. Basidiospores hyaline,
smooth, obovoid to elliptic, 4.5–5μm × 2.5–3μm, described
by Roberts [47] as statismospores because they are passively
released instead of violently discharged from sterigmata (as
in ballistospores).
BLASTn searches of the newly generatedsequences
pointed out towards ahigh identity of VIC47429 with Cryp-
tococcus species, especially those human-pathogenic species
within the Filobasidiella clade delimited by Findley [22].
Therefore, maximum likelihood (ML) and Bayesian infer-
ence (BI) analyses were performed to infer the phylogenetic
position and accurate identification. The sequence alignment
of rDNA 28S included 660 aligned sites, including gaps.
This matrix had 392 conserved sites, 265 variable sites, and
51 singletons. For BI, the substitution model selected was
the general-time-reversible (GTR) with an unequal propor-
tion of invariable sites (+ I) and a gamma distribution on
substitution rates across sites (+ G). Bayesian inference
sampled 100,002 trees, of which 75,002 were retained after
burn-in and were used to compose the 50% majority-rule
consensus tree. The nucleotide sequence alignments, the
analyses, and phylogenetic trees are available in TreeBASE
(study S28031). In both ML and BI analyses of the rDNA
28S, sequences of isolate VIC47429 clustered with C. dep-
auperatus strain CBS 7841 with maximum support (Boot-
strap (BS) = 100%; posterior probabilities (PB) = 1.00), con-
firming that the fungus retrieved from H. vastatrix pustules
on C. arabica belongs to this species and sits next to the
C. neoformans-gattii complex in the Filobasidiella clade
(Fig.3).
Discussion
Aspergillus depauperatus was first proposed by Petch for a
fungus that he mistakenly interpreted as an entomopatho-
genic species of Aspergillus parasitizing scale insects in
England and Sri Lanka (formerly Ceylon) [44]. The species
epithet referred to his interpretation that this fungus was
an unusual species of Aspergillus having a much-reduced
number of “phialides” on the apical vesicle of its conidio-
phores. These structures were illustrated showing an “empty
head with two-four minute conical projections” [44]. Dur-
ing an examination of entomopathogenic fungi collected on
aphids in the Netherlands, Samson etal. [54] recognized that
one of the cultures, isolated together with Akanthomyces
lecanii, was identical to the fungus described much earlier
by Petch from Sri Lanka but also showed similarities to a
recently described fungus on a spider host in Canada, and
which was identified as a new species of the basidiomycete
Fig. 2 Cryptococcus depauper-
atus on coffee leaf rust colonies.
a Coffea arabica leaf bearing
coffee leaf rust pustules partly
covered by white colonies of
C. depauperatus. b Image from
scanning electron microscopy
(SEM) of C. depauperatus
mycelium growing on uredinia
of H. vastarix. c Basidia and
basidiospores of C. depaupera-
tus mounted in lactoglycerol
(note very long aseptate basidia
with somewhat flattened head).
d Cryptococcusdepauperatus
basidium and basidiospores in a
lactofuchsin mount. e Basidium
and basidiospores (SEM)
(recognizable even if collapsed
during sample drying). Scale
bars (c) = 20μm, (d) = 10μm (e,
f) = 5μm

Brazilian Journal of Microbiology
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genus Filobasidiella, F. arachnophila [39]. A taxonomic
study showed them to be morphologically identical and the
new combination Filobadisiella depauperata (Petch) Sam-
son, Stalpers & Weijman was proposed [54]. In addition,
the results of a carbohydrate profile established its basidi-
omycete nature, confirming those of an earlier ultrastructural
study of the hyphal septa which showed them to be of the
dolipore type, typical of the Basidiomycota [35], and dispel-
ling any doubts that it could be an Ascomycete.
More recently, under the new nomenclatural rules (one
fungus = one name; [61]), the name was changed to Cryp-
tococcus depauperatus (Petch) Boekhout, Xin Z., Liu, F.Y.
Bai & M. Groenew, as the earlier name for the asexual yeast
stage, Cryptococcus took priority over the sexual stage,
Filobasidiella [37, 38].
Malloch etal. [39] isolated C. depauperatus in pure cul-
ture and demonstrated that it grows as a mycelial colony in
contrast to the closely related yeast-forming species, Crypto-
coccus neoformans [36]. They further showed that the poor
sporulation of invitro cultures of C. depauperatus could be
enhanced in the presence of colonies of Akanthomyces leca-
nii and, moreover, that these colonies were eventually over-
run by C. depauperatus [39], providing circumstantial evi-
dence of its mycoparasitic ability. This association reflected
the earlier observations by Petch [44], which were later
endorsed by Samson etal. [54], who isolated it from aphids
heavily infested with A. lecanii and posited that “This may
indicate hyperparasitism on other entomogenous fungi rather
than pathogenicity of the arthropod hosts” [54]. The mecha-
nism of mycoparasitism by C. depauperatus has been shown
to be through the formation of haustorial branches attached
to the mycelium of A. lecanii[24]. The presence of these
haustoria had been overlooked in the original description
of the fungus by Malloch etal. [39], but a re-examination
of the type specimen (on a spider host over-run by A. leca-
nii) revealed their presence. Similarly, haustorial branches
attached to the hyphae of its basidiomycete host have also
been reported for C. luteus [23]. Here, careful examina-
tion of our specimen did not reveal either the presence of
Fig. 3 Phylogenetic tree based
on maximum likelihood analy-
sis of nuc 28S rDNA sequences
of Cryptococcus species. Values
at branches denote support
values of Bootstrap with 1000
replicates followed by posterior
probabilities of Bayesian
Inference analysis. Thickened
branches denote maximum sup-
port in both analyses. Sequences
from specimen obtained from
overgrown Hemileia vastatrix
pustules indicated in shaded
square

Brazilian Journal of Microbiology
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Akanthomyces spp. colonies or the formation of haustorial
branches on H. vastatrix structures.
Although the genus Cryptococcus is infamous for
including the important pathogenic Cryptococcus
neoformans/C. gatii species complex—implicated in res-
piratory and neurological diseases of both humans and
animals [49], as well as HIV-associated cryptococcal men-
ingitis [45]—many of the species closely related to this
complex, such as C. amylolentus, C. depauperatus, and C.
luteus [25, 47], occupy different niches and are saprobes or
mycoparasites [5, 53, 57]. Another member of this genus,
the phylogenetically distant, C. laurentii, has been studied
as a biocontrol agent against the attack of Botrytis cinerea
and Penicillium expansum in stored agricultural products
[14]. However, warnings were made that caution should be
exercised when promoting basidiomycetous yeasts, such
as C. laurentii, as biocontrol agents because of potential
human-health issues [22], and mammals, in general [12].
CLR caused by H. vastatrix is the most important dis-
ease of this globally important and lucrative crop [18,
40]. A number of natural enemies of this plant pathogen
have already been described and studied, with the aim of
developing biocontrol methods which may complement or
substitute chemical control, particularly for organic farm
systems [10, 11, 13, 15, 17, 29, 31, 56, 62].
Surveys for coffee rust mycoparasites usually rely
on direct isolation and cultivation of fungi, followed by
morphological identification [28]. In a recent innovative
approach, pustules of H. vastatrix on coffee leaves col-
lected in Mexico and Puerto Rico were investigated with
single-molecule DNA sequencing resulting in 15 putative
mycoparasitic fungi, mostly concentrated in the family
Cordycipitaceae, but also including specimens of dimor-
phic yeasts such as Bullera sp. and Kockovaella schimae
of the order Tremellales [31]. However, neither this study,
nor any previous studies of mycoparasites of H. vasta-
trix, has reported Cryptococcus depauperatus or any other
member of the genus Cryptococcus from this microhabitat.
Akanthomyces lecanii is probably the most widely
known mycoparasite of H. vastatrix [26, 62], besides its
role as an important natural enemy of scale insects [21, 30,
46]. However, using this ubiquitous, pantropical fungus
as a successful biocontrol agent in the traditional coffee
system production has yet to be achieved [26]. Additional
fungal species known as mycoparasites of the coffee rust
fungus are Acremonium byssoides, Calcarisporium arbus-
cula, C. ovalisporum, Sporothrix guttuliformis, Fusarium
pallidoroseum [11], Talaromyces wortmannii [62], and,
more recently, Calonectria hemileiae and Digitopo-
diumtectonae [15, 19, 52]. However, evidence concerning
the mechanism of colonization by most of the aforemen-
tioned fungi is rare, except for Akanthomyces lecanii and
T. wortmannii, for which hyphae penetrating H. vastatrix
urediniospores has been well documented [62].
Although photographic evidence of penetration and colo-
nization of H. vastatrix is lacking for most putative myco-
parasites, and the label “mycoparasite” may include several
kinds of associations, the consensus is that such fungi are
likely to affect the establishment of plant pathogens, thereby
having significant implications for plant protection [1, 59].
An earlier study involving the direct isolation and culturing
of endophytic and epiphytic fungi on coffee leaves revealed
a surprisingly rich mycobiota comprising 131 morphospe-
cies belonging to genera such as Pestalotia, Botryosphaeria,
Xylaria, Colletotrichum, Guignardia, Aspergillus, Clad-
osporium, Coprinus, Fusarium, Penicillium, Mucor, Rhizo-
pus, and Trichoderma, along with several non-identifyed
taxa [55].
Most research on the natural enemies of CLR is from
Central and South America, although both coffee and cof-
fee rust originated in Africa [2, 6, 60]. Hemileia vastatrix
is commonly found on Coffea species in their native for-
est habitats but is kept under control, probably due to the
action of natural enemies which co-evolved with the coffee
rust. Recent efforts to assess the diversity of mycoparasites
of H. vastatrix in its African center of origin revealed an
impressive fungal diversity, with many records of species
new to science [10, 15, 16, 48, 51, 52]. Cryptococcus dep-
auperatus was found only twice during the surveys (once in
Cameroon and once in Ethiopia). Unfortunately, the speci-
men from Ethiopia contained very little C. depauperatus
and was exhausted while attempts at DNA extraction were
being performed.
These are new records of C. depauperatus both geo-
graphically and occupying the mycoparasite niche on H.
vastatrix. Unfortunately, it was not possible to obtain pure
cultures from the small amount of dried herbarium material
available. Without living cultures, evaluation of the actual
role of C. depauperatus within the H. vastatrix-mycoparasite
complex could not be undertaken and future progress will
depend on recollecting the fungus. It is striking that this
little-known mycoparasitic species occupies such a differ-
ent ecological niche from the infamous human-pathogenic
species to which it is so closely related within the restricted
Filobasidiella clade of the genus Cryptococcus.
Acknowledgements We thank staff from the Botanical Garden in
Limbé for guidance during the survey, and the Institut de Recherche
pour le Developpement (Yaoundé, Cameroon) for arranging the col-
lecting and export permits.
Author contribution All authors contributed to the study conception
and design. Conceptualization, funding acquisition, supervision, and
final writing were performed by R. W. Barreto. Material preparation,
data collection, and morphological analysis were L. M. Saavedra Tobar,
and M. K. Ndacnou. Molecular data analysis and the first draft and
final editing of the manuscript were performed by D. C. Guterres and

Brazilian Journal of Microbiology
1 3
all authors critically revised the work. All authors read and approved
the final manuscript.
Funding This study was financed by the Conselho Nacional de Desen-
volvimento Científico e Tecnológico (CNPq), by the Fundação Arthur
Bernardes (Funarbe), by the Coordenação de Aperfeiçoamento de Pes-
soal de Nível Superior – Brasil (CAPES), and by the World Coffee
Research (WCR).
Data availability All DNA sequence data used in the study are available
in public repository of GenBank. Phylogenetic trees, aligments, and
analyses are available in TreeBase study S28031.
Declarations
Ethics approval Not applicable.
Consent to participate Not applicable.
Consent for publication Not applicable.
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