Phylogenetic analysis of Monascus and new species from honey, pollen and nests of stingless bees

Abstract

The genus Monascus was described by van Tieghem (1884) to accommodate M. ruber and M. mucoroides, two species with non-ostiolate ascomata. Species delimitation in the genus is still mainly based on phenotypic characters, and taxonomic studies that include sequence data are limited. The genus is of economic importance. Species are used in fermented Asian foods as food colourants (e.g. ‘red rice’ (ang-kak, angka)) and found as spoilage organisms, and recently Monascus was found to be essential in the lifecycle of stingless bees. In this study, a polyphasic approach was applied combining morphological characters, ITS, LSU, β-tubulin, calmodulin and RNA polymerase II second largest subunit sequences and extrolite data, to delimit species and to study phylogenetic relationships in Monascus. Furthermore, 30 Monascus isolates from honey, pollen and nests of stingless bees in Brazil were included. Based on this polyphasic approach, the genus Monascus is resolved in nine species, including three new species associated with stingless bees (M. flavipigmentosus sp. nov., M. mellicola sp. nov., M. recifensis sp. nov., M. argentinensis, M. floridanus, M. lunisporas, M. pallens, M. purpureus, M. ruber), and split in two new sections (section Floridani sect. nov., section Rubri sect. nov.). Phylogenetic analysis showed that the xerophile Monascus eremophilus does not belong in Monascus and monophyly in Monascus is restored with the transfer of M. eremophilus to Penicillium (P. eremophilum comb. nov.). A list of accepted and excluded Monascus and Basipetospora species is given, together with information on (ex-)types cultures and barcode sequence data.

Full text

Phylogenetic analysis of Monascus and new species from honey,
pollen and nests of stingless bees
R.N. Barbosa
1
,
2
, S.L. Leong
3
, O. Vinnere-Pettersson
3
,
4
, A.J. Chen
1
,
5
, C.M. Souza-Motta
2
, J.C. Frisvad
6
, R.A. Samson
1
,
N.T. Oliveira
2
, and J. Houbraken
1
*
1
Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands;
2
Departamento de Micologia Prof. Chaves Batista, Universidade Federal de
Pernambuco, Av. Prof. Moraes Rego, s/n, Centro de Bioci^
encias, Cidade Universit
aria, CEP: 50670-901 Recife, PE, Brazil;
3
Swedish University of Agricultural Sciences,
Department of Molecular Sciences, Box 7015, SE-750 07 Uppsala, Sweden;
4
National Genomics Infrastructure-Sweden, Science for Life Laboratory, Department of
Immunology, Genetics and Pathology, Uppsala University, BMC, Box 815, SE-752 37 Uppsala, Sweden;
5
Institute of Medicinal Plant Development, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China;
6
Department of Biotechnology and Biomedicine, Technical University
of Denmark, 2800 Kongens Lyngby, Denmark
*Correspondence: J. Houbraken,
j.houbraken@westerdijkinstitute.nl
Abstract: The genus Monascus was described by van Tieghem (1884) to accommodate M. ruber and M. mucoroides, two species with non-ostiolate ascomata. Species
delimitation in the genus is still mainly based on phenotypic characters, and taxonomic studies that include sequence data are limited. The genus is of economic
importance. Species are used in fermented Asian foods as food colourants (e.g. ‘red rice’(ang-kak, angka)) and found as spoilage organisms, and recently Monascus
was found to be essential in the lifecycle of stingless bees. In this study, a polyphasic approach was applied combining morphological characters, ITS, LSU, β-tubulin,
calmodulin and RNA polymerase II second largest subunit sequences and extrolite data, to delimit species and to study phylogenetic relationships in Monascus.
Furthermore, 30 Monascus isolates from honey, pollen and nests of stingless bees in Brazil were included. Based on this polyphasic approach, the genus Monascus is
resolved in nine species, including three new species associated with stingless bees (M. flavipigmentosus sp. nov., M. mellicola sp. nov., M. recifensis sp. nov.,
M. argentinensis,M. floridanus,M. lunisporas,M. pallens,M. purpureus,M. ruber), and split in two new sections (section Floridani sect. nov., section Rubri sect. nov.).
Phylogenetic analysis showed that the xerophile Monascus eremophilus does not belong in Monascus and monophyly in Monascus is restored with the transfer of
M. eremophilus to Penicillium (P. eremophilum comb. nov.). A list of accepted and excluded Monascus and Basipetospora species is given, together with information on
(ex-)types cultures and barcode sequence data.
Key words: Aspergillaceae, Extrolites, Fungal ecology, Phylogeny, Taxonomy.
Taxonomic novelties: New sections: Monascus section Floridani R.N. Barbosa & Houbraken, Monascus section Rubri R.N. Barbosa & Houbraken; New species:
Monascus flavipigmentosus R.N. Barbosa, Souza-Motta, N.T. Oliveira & Houbraken, Monascus mellicola R.N. Barbosa, Souza-Motta, N.T. Oliveira & Houbraken,
Monascus recifensis R.N. Barbosa, Souza-Motta, N.T. Oliveira & Houbraken; New combination: Penicillium eremophilum (A.D. Hocking & Pitt) Houbraken, Leong
& Vinnere-Pettersson.
Available online 12 April 2017; http://dx.doi.org/10.1016/j.simyco.2017.04.001.
INTRODUCTION
Van Tieghem (1884) introduced the genus Monascus for species
that produce non-ostiolate ascomata and introduced two species,
M. ruber and M. mucoroides. The position of Monascus (and the
Monascaceae) has been the subject of discussion in various
papers and it was often placed outside the order Eurotiales
(Benny & Kimbrough 1980, von Arx 1987, Stchigel & Guarro
2007), but phylogenetic analyses confidentially places this
genus in Aspergillaceae (Eurotiales)(Berbee et al. 1995, Ogawa
et al. 1997, Ogawa & Sugiyama 2000, Peterson 2008,
Houbraken & Samson 2011, Vinnere-Pettersson et al. 2011).
The genus Basipetospora was found to be the anamorph of
Monascus and is characterized by the production of aleur-
ioconidia in a basipetal manner from undifferentiated con-
idiogenous cells that progressively shorten (retrogression, Cole &
Samson 1979). The conidia have a truncated base and resemble
chlamydospores. These features set this genus apart from the
phylogenetically related genera Aspergillus and Penicillium.
After the description of the genus, more than 20 species have
been introduced and many of them are considered to be syno-
nyms (Shao et al. 2011). Classification of Monascus has primary
been based on macro- and microscopic features, such as the
pigmentation of the cleistothecial walls and conidia and growth
rates on agar media. Hawksworth & Pitt (1983) revised the genus
based on physiological and morphological characteristics and
reduced the number of accepted species to three: M. pilosus,
M. ruber and M. purpureus. Since that study, ten new species
were introduced: M. albidulus,M. argentinensis,M. aurantiacus,
M. eremophilus,M. floridanus,M. fumeus,M. lunisporas,
M. pallens,M. rutilus and M. sanguineus (Barnard & Cannon
1987, Hocking & Pitt 1988, Cannon et al. 1995, Udagawa &
Baba 1998, Stchigel et al. 2004,Li & Guo 2004). With the
description of those species, the genus became morphologically
and physiologically more diverse, suggesting a large genetic
diversity. For example, Monascus ruber grows rapidly on agar
media, M. lunisporas and M. pallens grow restrictedly and
M. eremophilus is a strict xerophile and only grows on low water
activity media. The phenotype-based identification schemes in
Monascus were difficult to match with the results obtained by
ITS, partial LSU and/or β-tubulin gene sequencing (Park & Jong
2003, Park et al. 2004). Nowadays, species can be delimited on
the genotype, for example based on the Genealogical Concor-
dance Phylogenetic Species Recognition (GCPSR) concept. The
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© 2017 Westerdijk Fungal Biodiversity Institute. Production and hosting by ELSEVIER B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
available online at www.studiesinmycology.org STUDIES IN MYCOLOGY 86: 29–51 (2017).
29
Studies in Mycology

application of this concept in Monascus has yet not been per-
formed and the results of such an analysis will give insight on the
species boundaries.
The genus Monascus has economic importance in several
areas, and several species have been widely used for over years
in the production of yellow and red food colourants and Asian
fermented foods, particularly red rice (ang-kak, angka, ‘red kojic
rice’). Red rice is of particular interest because of its health pro-
moting effects (Lee & Pan 2011, 2012, Hsu & Pan 2012, Shi & Pan
2012) and indeed, production of compounds with antibacterial
propertiesand cholesterol-lowering statins of the monacolin K-type
(= mevinolin = lovastatin) are reported in the species M. pilosus,
M. pubigerus,M. purpureus,M. ruber and M. vitreus (Negishi et al.
1986, Jůzlova et al. 1996, Vendruscolo et al. 2014). However,
Monascus species such as M. anka,M. aurantiacus,M. kaoliang,
M. pilosus,M. purpureus,M. ruber and M. sanguineus have been
reported to produce the mycotoxin citrinin (Blanc et al. 1995,
Dietrich et al. 1999, Wang et al. 2003, Wang et al. 2005,
Pisareva et al. 2005, Shimizu et al. 2005, Huang et al. 2007,
Pattangul et al. 2008, Kim et al. 2010, Li et al. 2012, Li et al.
2015), and the presence of this mycotoxin in food, including red
rice, should be avoided. Among these reports on citrinin produc-
tion by Monascus species, Wang et al. (2005) also reported on
citrinin production by M. floridanus,M. lunisporas and M. pallens,
but this has not been confirmed by any other authors working on
citrinin and Monascus. Besides their beneficial properties for hu-
man, Monascus species can also cause spoilage, for example of
silage, bakery (tortillas), pasteurized products (olives) and dried
prunes (M. eremophilus). Species are also rarely associated with
human infections, and an invasive gastric infection case was
linked to the consumption of Monascus contaminated dried and
salted fish (Moreau 1971, Iriart et al. 2010, Samson et al. 2010).
Specific fungi and other micro-organisms live in close asso-
ciation with social and solitary bees. This association is
mandatory, and investigations on the biology, ecology and evo-
lution have been undertaken (Wynns 2015). Recently, a study
described a symbiosis between Scaptotrigona postica bees and
a fungus (Menezes et al. 2015). The fungus was identified by
morphology and ITS sequencing as being closely related to
M. ruber and M. pilosus. The study showed that the Monascus
biomass on the food inside the brood cells is essential for the
larvae of the S. postica bees, and without the consumption of this
biomass, only a few larvae can continue their life cycle.
Monascus was one of the predominant genera during the
study of fungi associated with honey, pollen and nests of Meli-
pona scutellaris bees living in the Atlantic Forest in Pernambuco,
Brazil. The phylogenetic relationship of those strains with other
species of the genus was determined by the analysis of ITS,
LSU, β-tubulin (BenA), calmodulin (CaM) and RNA polymerase II
second largest subunit (RPB2) sequences. Furthermore, three
new species from honey, pollen and the inside of the nest are
described based on a polyphasic approach combining sequence
data, macro- and microscopic characters and extrolites.
MATERIALS AND METHODS
Fungal isolation
Samples were collected from honey, pollen and inside nests of
Melipona scutellaris bees in the Brazilian Tropical Forest in
Pernambuco state (8°7ʹ30ʺS, 34°52ʹ30ʺW and 8°4
0
36
00
S,
34°57
0
34
00
W) between January and June 2014. For the honey
and pollen samples, 25 g of each specimen was suspended in
225 mL peptone water (0.1 %) and decimal dilutions were made
until 10
−3
. Subsequently, 0.1 mL of each dilution was spread
plated on the agar media dichloran 18 % glycerol agar (DG18)
and malt extract agar supplemented with chloramphenicol. The
plates were incubated at 25 °C for 7 –14 d in darkness. For
collection of the samples inside nests, a sterile cotton swab was
used to sample the surface of the pollen and honey pots, and
brood cells. The swab was soaked in 3 mL peptone water
(0.1 %) and vortexed vigorously. The samples were subse-
quently analysed as described above. All fungal colonies were
isolated and purified prior identification.
Cultivation and morphological analyses
Thirty Monascus strains were obtained from honey, pollen and
inside nests of Melipona scutellaris bees (Table 1). The colony
characteristics of these strains were compared with represen-
tative and type cultures of currently accepted Monascus species.
For this purpose, the strains were cultivated in three points in
creatine agar (CREA), cornmeal agar (CMA), Czapek yeast
extract agar (CYA), CYA supplemented with 5 % NaCl (CYAS),
dichloran 18 % glycerol agar (DG18), malt extract agar (MEA,
Oxoid), oatmeal agar (OA), potato dextrose agar (PDA) and
yeast extract sucrose agar (YES) incubated at 25 °C for 7 d.
Additional CYA and MEA plates were incubated at 30 and 37 °C.
Monascus eremophilus was inoculated on the malt agar 20 %
sucrose (MA20S) and malt yeast extract 50 % glucose agar
(MY50G). All media were prepared according to Samson et al.
(2010). Colony diameters were measured after 7 d of incuba-
tion and colony characteristics (e.g. presence of soluble pig-
ments, exudates, obverse and reverse colony colours, colour of
mycelium) were recorded. Microscopic observations of the
asexual stage were made from colonies grown on MEA. The
presence of a sexual stage was determined on MEA, CMA, PDA
and OA, and PDA was used for illustrations and measurements.
Lactic acid (60 %) was used as mounting fluid and 96 % ethanol
was used to remove the excess of conidia. The size, shape and
pigmentation of conidia, conidiophores, ascomata, asci and as-
cospores were recorded. A Zeiss Stereo Discovery V20 dis-
secting microscope and Zeiss AX10 Imager A2 light microscope
equipped with Nikon DS-Ri2 cameras and software NIS-
Elements D v4.50 were used to capture digital images. New
species names and associated information were deposited in
MycoBank. All strains were deposited in the culture collection of
Micoteca URM (Federal University of Pernambuco, Recife,
Brazil) and the ex-type strains were also deposited in the CBS
culture collection housed at the Westerdijk Fungal Biodiversity
Institute (formerly known as Centraalbureau voor Schimmelcul-
tures), Utrecht, The Netherlands (under Material Transfer
Agreement –MTA No. 01/2016/Micoteca URM).
Molecular characterization
Genomic DNA of 7 d old cultures was extracted using the Ultra-
Clean Microbial DNA kit (MoBio Laboratories, Solana Beach, CA,
USA) and processed according to the manufacturer's instructions.
Polymerase chain reaction (PCR) amplification of the ITS region
(ITS1, 5.8S rDNA and ITS2) was performed using the primers
BARBOSA ET AL.
30

Table 1. Strains and sequences used in the morphological and molecular study.
Species Strain numbers Substrate; location GenBank accession no.
ITS BenA LSU CaM RPB2
Leiothecium ellipsoideum CBS 607.74
T
= ATCC 32453 Soil, between rocks;
Pelopennesos, Greece
KF732839 KY709178 FJ358285 KY611939 JN121541
Monascus argentinensis CBS 109402
T
= DTO 138-
C5 = FMR 7393
Soil sample; Tucum
an province,
Argentina
JF922046 KY709174 KY645974 KY611935 JN121423
M. eremophilus CBS 123361
T
= DTO 122-
C7 = FRR 3338
Mouldy prunes; New South
Wales, Australia
GU733347 KY709170 KY645973 KY611931 KY611970
M. flavipigmentosus URM 7536
T
= CBS 142366 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511751 KY709168 KY511781 KY611929 KY611968
M. flavipigmentosus URM 7535 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511752 KY709169 KY511782 KY611930 KY611969
M. flavipigmentosus URM 7534 Pollen of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511750 KY709167 KY511780 KY611928 KY611967
M. floridanus CBS 142228
T
= DTO 360-
E7 = CGMCC 3.5843 = IMI
282587 = UAMH 4180
Sand pine roots; USA KY635848 KY709172 KY635856 KY611933 KY611972
M. lunisporas CBS 142230
T
= DTO 360-
E9 = CGMCC 3.7951 = ATCC
204397
Mouldy feed for race horses;
Japan
KY635847 KY709171 KY635855 KY611932 KY611971
M. mellicola URM 7510
T
= CBS 142364 Honey of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511726 KY709143 KY511756 KY611904 KY611943
M. mellicola URM 7507 Honey of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511723 KY709140 KY511753 KY611901 KY611940
M. mellicola URM 7508 Honey of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511724 KY709141 KY511754 KY611902 KY611941
M. mellicola URM 7509 Honey of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511725 KY709142 KY511755 KY611903 KY611942
M. mellicola URM 7511 Honey of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511727 KY709144 KY511757 KY611905 KY611944
M. mellicola URM 7512 Honey of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511728 KY709145 KY511758 KY611906 KY611945
M. mellicola URM 7513 Honey of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511729 KY709146 KY511759 KY611907 KY611946
M. mellicola URM 7514 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511730 KY709147 KY511760 KY611908 KY611947
M. mellicola URM 7515 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511731 KY709148 KY511761 KY611909 KY611948
M. mellicola URM 7516 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511732 KY709149 KY511762 KY611910 KY611949
M. mellicola URM 7517 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511733 KY709150 KY511763 KY611911 KY611950
M. mellicola URM 7518 Honey of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511734 KY709151 KY511764 KY611912 KY611951
M. mellicola URM 7519 Honey of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511735 KY709152 KY511765 KY611913 KY611952
M. mellicola URM 7520 Pollen; Recife, Pernambuco,
Brazil
KY511736 KY709153 KY511766 KY611914 KY611953
M. mellicola URM 7521 Honey of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511737 KY709154 KY511767 KY611915 KY611954
M. mellicola URM 7522 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511738 KY709155 KY511768 KY611916 KY611955
(continued on next page)
PHYLOGENY OF MONASCUS
www.studiesinmycology.org 31

Table 1. (Continued).
Species Strain numbers Substrate; location GenBank accession no.
ITS BenA LSU CaM RPB2
M. pallens CBS 142229
T
= DTO 360-
E8 = CGMCC 3.5844 = ATCC
200612 = IMI 356820
River sediment; Iraq KY635849 KY709173 KY635857 KY611934 KY611973
M. pilosus CBS 286.34
T
= DTO 165-
B1 = ATCC 16363 = FRR
2194 = IFO 4480
Fermented grain, Sorghum
vulgare; Japan
KY635852 JF922085 KY635860 KY849968 KY849967
M. purpureus CBS 109.07
T
= DTO 364-
D8 = ATCC 16365 = IFO
4513 = IMI 210765 = NRRL
1596
Fermented rice grain (‘ang-
quac’); Java, Indonesia
KY635851 KY709176 KY635859 KY611937 JN121422
M. recifensis URM 7524
T
= CBS 142365 Pollen of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511740 KY709157 KY511770 KY611918 KY611957
M. recifensis URM 7523 Pollen of Melipona scutellaris;
Recife, Pernambuco, Brazil
KY511739 KY709156 KY511769 KY611917 KY611956
M. ruber URM 7525 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511741 KY709158 KY511771 KY611919 KY611958
M. ruber URM 7526 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511742 KY709159 KY511772 KY611920 KY611959
M. ruber URM 7527 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511743 KY709160 KY511773 KY611921 KY611960
M. ruber URM 7528 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511744 KY709161 KY511774 KY611922 KY611961
M. ruber URM 7529 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511745 KY709162 KY511775 KY611923 KY611962
M. ruber URM 7530 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511746 KY709163 KY511776 KY611924 KY611963
M. ruber URM 7531 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511747 KY709164 KY511777 KY611925 KY611964
M. ruber URM 7532 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511748 KY709165 KY511778 KY611926 KY611965
M. ruber URM 7533 Inside nest of Melipona
scutellaris; Recife, Pernambuco,
Brazil
KY511749 KY709166 KY511779 KY611927 KY611966
M. ruber CBS 135.60
NT
= DTO 359-
E8 = ATCC 15670 = IFO
8451 = IMI 081596
Soil; India KY635850 KY709175 KY635858 KY611936 KY611974
M. sanguineus IMI 356821
T
= ATCC 200613 River sediment; Iraq JF922055 JF922088 AF364968 KY611938 n/a
Penicillium polonicum CBS 222.28
T
= IBT 12821 = IMI
291194 = NRRL 995
Soil, Poland AF033475 AF001206 JN939272 KU896848 JN985417
P. verrucosum CBS 603.74
NT
= IMI
200310 = ATCC 48957 = FRR
965 = IBT 4733 = NRRL 965
Unknown source, Belgium AB479317 AF001205 AB479285 DQ911138 JN121539
Talaromyces purpurogenus CBS 286.36 = IMI 091926 Unknown source; Japan JX315671 JX315639 KY635863 KF741947 JX315709
T. ruber CBS 132704 = IBT 10703 Aircraft fuel tank; UK NR111780 JX315629 KY635864 KF741938 JX315700
Xerochrysium dermatitidis CBS 132.31
T
= IMI
096729 = UAMH 802
Skin, man; Italy KY635853 n/a KY635861 n/a JN121443
Xeromyces bisporus CBS 236.71
T
= IMI 063718 Mouldy stick of liquorice; New
South Wales, Australia
KY635854 JF922089 KY635862 741987712
1
JN121612
Abbreviations: T = type strain; NT = neotype strain; URM, URM Culture Collection (www.ufpe.br/micoteca), Brazil; CBS, Culture collection of the Westerdijk Fungal
Biodiversity Institute (formerly known as Centraalbureau voor Schimmelcultures), The Netherlands; DTO, Internal culture collection at Westerdijk Fungal Biodiversity
Institute.
1
Sequence from genome sequenced strain; n/a: no sequence available.
BARBOSA ET AL.
32

V9G and LS266 and a part of the Large SubUnit (LSU) rDNA was
amplified using the primers LR0R and LR5. Partial β-tubulin
fragments were generated using the primer combination Bt2a and
Bt2b, for calmodulin the primers Cmd5 and Cmd6 were used and
for RPB2 the primers RPB2-5F and RPB2-7CR. Details on the
primer sequences, PCR mixtures and conditions are previously
described (Samson et al. 2010, Houbraken et al. 2012).
The PCR products were sequenced in both directions with the
same primers using the BigDye
®
Terminator v. 3.1 Cycle
Sequencing Kit (Applied Biosystems Life Technologies, Carls-
bad, CA, USA) and were purified with Sephadex, according to
the manufacturers'recommendations. Contigs were assembled
using the forward and reverse sequence with the SeqMan v.
10.0.1 program. Newly generated sequences were deposited in
GenBank. Sequence datasets were generated by combining the
newly generated sequences with sequences from GenBank
(Table 1). The sequences were aligned using MAFFT (Katoh
et al. 2005) and were manually optimized using MEGA 5
(Tamura et al. 2011). The most suitable substitution model was
determined using FindModel (Posada & Crandall 1998). Phylo-
genetic trees were constructed using maximum likelihood (ML)
analysis in RAxML-VI-HPC v. 7.0.3 (Stamatakis 2006) using the
GTRGAMMA substitution model and 1 000 bootstrap replicates.
Bayesian inference (BI) in MrBayes v.3.2.1 (Ronquist et al. 2012)
was performed using Markov Chain Monte Carlo (MCMC) al-
gorithm and the best scoring substitution model is indicated in
the results section. Trees were visualized in FigTree v. 1.1.2
(Rambaut 2009) and edited in Adobe Illustrator v.CS5.1. Indi-
vidual alignments were concatenated by using Mesquite v3.04
(Maddison & Maddison 2016). The quality of final alignment was
evaluated using Transitive Consistence Score (TCS) by the T-
Coffee web server (Chang et al. 2015).
Extrolite analysis
Extrolites were extracted from fungal strains grown on CYA,
YES, MEA, OA at 25 °C for 14 d and PDA and DG18 at 25 °C for
20 d. Three agar plugs of each culture were extracted as pre-
viously described (Smedsgaard 1997, Houbraken et al. 2012).
After extraction, the liquid was transferred to a clean screw-cap
vial and evaporated to dryness. Prior analysis, the dried extracts
were re-dissolved in methanol by ultrasonication and filtered
through a 0.45 μmfilter. The extracts were analysed by ultra-high
performance liquid chromatography with diode-array detection
(UHPLC-DAD) (Houbraken et al. 2012). The detected eluted
compounds were identified by comparing the retention time,
retention index and UV spectra measured at 200 –600 nm. The
UV spectra were compared to a database of UV spectra and
data from literature (Nielsen et al. 2011, Klitgaard et al. 2014).
RESULTS
Phylogeny and GCPSR
The phylogenetic relationship of the commonly accepted Monascus
species and the isolates obtained from honey, pollen and nests of
Melipona scutellaris bees were studied using concatenated five-
gene data set (ITS, BenA,CaM, LSU and RPB2). The Transitive
Consistence Score (TCS) evaluated the robustness of the five-
gene with the high score of 929. The total length of the aligned
data set was 2930 characters (ITS, 583 bp; BenA, 505 bp; CaM,
490 bp; RPB2, 784 bp; LSU, 568 bp) including alignment gaps. The
GTR+G model was the most optimal and selected for the ITS and
LSU data sets, the HKY+G model for BenA and K80+G for the
CaM and RPB2 data sets. A similar topology was observed in the
five single gene phylogenies and no significant incongruence were
found (Fig. 2A–E). A total of 1692 trees were generated during the
Bayesian inference from which 422 trees were discarded after 25
per cent of the generations in ‘burn-in phase’and posterior prob-
abilities were calculated from the remaining 1 270 trees. The results
of the Bayesian analysis were similar to the results of the ML
analysis, and differences were only in the degree of support for
some branches. The Bayesian consensus tree is presented here
with the relevant bootstrap percentages (>70 %) and posterior
probability values (>0.95) (Fig. 1).
Monascus eremophilus is positioned outside the main Mon-
ascus clade and proved to be related to Penicillium species
(100 % bs, 1.00 pp) (Fig. 1). Our analysis revealed two well-
supported groups in Monascus, referred here to as the
M. floridanus- and M. ruber-clades. Seven well-supported line-
ages are present in the M. floridanus-clade and these lineages
are treated as separate species. Four are known species
(M. lunisporas,M. argentinensis,M. floridanus,M. pallens), and
three are proposed as newly described below (Monascus mel-
licola,M. recifensis and M. flavipigmentosus). Monascus melli-
cola is phylogenetically distinct and is with moderate bootstrap
and posterior probability support (82 % bs, 0.96 pp) related to
M. argentinensis,M. lunisporas,M. recifensis and
M. flavipigmentosus. The latter three species are resolved as
close relatives in a distinct, well-supported clade. In our
concatenate phylogenetic analysis these species are separated
in three well-supported groups, with M. lunisporas and
M. flavipigmentosus being sister species and M. recifensis taking
a basal position. Similar clustering was obtained in the single
gene analyses; however, the species were unresolved in the
LSU phylogram (Fig. 2).
The (neo)type strains of M. pilosus (CBS 286.34
T
),
M. purpureus (CBS 109.07
T
), M. ruber (CBS 135.60
NT
) and
M. sanguineus (ATCC 200613
T
) are located in the M. ruber-
clade. Two lineages are present within the M. ruber-clade
(Fig. 1). The (neo)type strains of M. pilosus (CBS 286.34
T
) and
M. ruber (CBS 135.60
NT
) are together on a well-supported
branch (97 % bs; 1.00 pp), and the branch containing the
types of M. purpureus (CBS 109.07
T
) and M. sanguineus (ATCC
200613
T
) has weak statistical support (78 % bs; <0.95 pp). In our
single gene analyses, M. pilosus (CBS 286.34
T
) and M. ruber
(CBS 135.60
NT
) always cluster together with high (ITS: 99 % bs,
1.00 pp; LSU: 98 % bs, 1.00 pp; CaM: 88 % bs, 0.98 pp) or
moderate (RPB2: 96 % bs, <0.95 pp, BenA 89 % bs, <0.95 pp)
statistical support. The branch with M. purpureus (CBS 109.07
T
)
and M. sanguineus (ATCC 200613
T
) is well supported in the ITS
phylogram (87 % bs, 0.99 pp), and no support was found in the
BenA,CaM and LSU analyses (<70 %, <0.95 pp). Following the
GCPSR concept, we keep two lineages in the M. ruber-clade.
Monascus pilosus and M. sanguineus are treated here as syn-
onym of M. ruber and M. purpureus, respectively.
Morphology
Monascus can also be split into two groups based on morpho-
logical characters. The majority of the species belonging to the
PHYLOGENY OF MONASCUS
www.studiesinmycology.org 33

0.2
Monascus lunisporas CBS 142230 T
Penicillium verrucosum CBS 603.74 T
Monascus mellicola URM 7519
Monascus recifensis URM 7524 T
Monascus mellicola URM 7510 T
Monascus flavipigmentosum URM 7534
Monascus mellicola URM 7517
Monascus flavipigmentosum URM 7535
Monascus mellicola URM 7508
Monascus floridanus CBS 142228 T
Monascus sanguineus ATCC 200613 T
Xerochrysium dermatitidis CBS 132.31 T
Monascus mellicola URM 7518
Monascus mellicola URM 7514
Monascus mellicola URM 7520
Monascus ruber URM 7531
Penicillium eremophilus
(
M. eremophilus) CBS 123361 T/ FRR 3338 T
Monascus mellicola URM 7507
Monascus ruber URM 7527
Monascus mellicola URM 7516
Leiothecium ellipsoideum CBS 607.74 T
Monascus argentinensis CBS 109402 T
Monascus ruber URM 7532
Monascus ruber URM 7525
Monascus mellicola URM 7509
Monascus mellicola URM 7515
Monascus ruber CBS 135.60 NT
Monascus pilosus CBS 286.34 T
Monascus purpureus CBS 109.07 T
Monascus ruber URM 7530
Monascus mellicola URM 7513
Monascus ruber URM 7533
Talaromyces ruber CBS 132704 NT
Monascus mellicola URM 7511
Monascus mellicola URM 7521
Monascus recifensis URM 7523
Xeromyces bisporus CBS 236.71 T
Talaromyces purpurogenus CBS 286.36 T
Monascus pallens CBS 142228 T
Monascus ruber URM 7529
Monascus flavipigmentosum URM 7536 T
Penicillium polonicum CBS 222.28 T
Monascus mellicola URM 7522
Monascus ruber UMR 7526
Monascus mellicola URM 7512
Monascus ruber URM 7528
Monascus
Combined
ITS+BenA+CaM+LSU+RPB2
Bayes/RAXML
Section Floridani
Section Rubri
0.87/78
1/97
1/88
0.96/82
0.98/73
Fig. 1. Concatenated phylogeny of the ITS, BenA,CaM, LSU and RPB2 gene regions showing the relationship in Monascus. Branches with posterior probability values of 1.00
and >95 % are thickened.
BARBOSA ET AL.
34

0.2
M. recifensis
M. pallens
M. ruber
M. floridanus
M. flavipigmentosum
Talaromyces ruber
M. ruber
M. flavipigmentosum
M. recifensis
M. lunisporas
Xerochrysium dermatitidis
Penicillium verrucosum
M. sanguineus
M. eremophilus
M. flavipigmentosum
M.ruber
M. pilosus
M. ruber
M. ruber
M. purpureus
M. ruber
M. argentinensis
M. ruber
Leiothecium ellipsoideum
Talaromyces purpurogenus
Xeromyces bisporus
M.ruber
Penicillium polonicum
M. ruber
M. ruber
M. mellicola
0.99/-
-/*
0.99/*
0.99/87
ITS
0.2
M. flavipigmentosum
M. lunisporas
M. recifensis
M. ruber
Penicillium polonicum
Talaromyces ruber
M. eremophilus
M. flavipigmentosum
Xeromyces bisporus
M. ruber
M. floridanus
Talaromyces purpurogenus
M. ruber
M. ruber
M. ruber
M. argentinensis
Penicillium verrucosum
M. flavipigmentosum
M. ruber
Leiothecium ellipsoideum
M. ruber
M. ruber
M. pilosus
M. ruber
M. pallens
M. purpureus
M. sanguineus
Xerochrysium dermatitidis
M. ruber
M. recifensis
M. mellicola
0.99/-
-/86
1/86
0.98/-
-/89
BenA
0.2
M. floridanus
M. recifensis
M. ruber
M. ruber
Talaromyces purpurogenus
Penicillium polonicum
Talaromyces ruber
M. recifensis
M. ruber
M. flavipigmentosum
M. ruber
M. flavipigmentosum
M. flavipigmentosum
M. purpureus
M. ruber
M. ruber
Penicillium verrucosum
M. eremophilus
Xeromyces bisporus
M. sanguineus
M. ruber
M. pallens
M. ruber
Leiothecium ellipsoideum
M. ruber
M. lunisporas
M. ruber
M. pilosus
M. argentinensis
M. mellicola
0.99/-
1/-
1/88
0.98/88
1/88
1/83
CaM
0.2
M. ruber
M. flavipigmentosum
Leiothecium ellipsoideum
M. eremophilus
M. ruber
M. ruber
M. recifensis
M. ruber
M. floridanus
M. recifensis
M. lunisporas
Penicillium polonicum
Penicillium verrucosum
M. flavipigmentosum
M. ruber
Talaromyces ruber
M. pilosus
M. ruber
M. pallens
M. ruber
M. flavipigmentosum
M. ruber
M. argentinensis
M. ruber
Xeromyces bisporus
M. ruber
M. sanguineus
M. purpureus
Talaromyces purpurogenus
Xerochrysium dermatitidis
M. mellicola
LSU
0.97/-
0.96/-
0.95/84
M. ruber
M. ruber
Leiothecium ellipsoideum
Penicillium verrucosum
M. floridanus
M. ruber
M. lunisporas
M. ruber
Penicillium polonicum
M. eremophilus
M. ruber
M. flavipigmentosum
M. ruber
M. pilosus
Xeromyces bisporus
M. argentinensis
Talaromyces purpurogenus
M. ruber
M. recifensis
M. purpureus
M. pallens
M. recifensis
M. flavipigmentosum
Talaromyces ruber
Xerochrysium dermatitidi
s
M. ruber
M. ruber
M. ruber
M. flavipigmentosum
M. mellicola
RPB2
0.99/-
0.99/-
0.1
Fig. 2. Single gene phylogenetic trees of the ITS, BenA,CaM, LSU and RPB2 gene regions of species from Monascus. Branches with posterior probability values of 1.00 and
>95 % are thickened.
PHYLOGENY OF MONASCUS
www.studiesinmycology.org 35

M. floridanus-clade grow restricted on MEA, PDA, CYA, CMA,
OA and YES and have no or restricted growth on CYA incubated
at 37 °C. Furthermore, the colonies are in shades of brown, the
conidia are brown pigmented and the mycelium is white to
olivaceous-brown (Fig. 3). The isolates belonging to the
M. ruber-clade can be differentiated from the M. floridanus-clade
species by their faster growth on MEA, PDA, CYA, CMA, OA and
YES at 25 °C and CYA incubated at 37 °C. The colonies of
Fig. 3. Cultural characters of Monascus species on different agar media and incubation conditions. Left to right: M. lunisporas,M. flavipigmentosus,M. recifensis,M. mellicola,
M. pallens,M. floridanus,M. argentinensis,M. ruber and M. purpureus.
BARBOSA ET AL.
36

M. ruber-clade species are in shades of white when young, and
turn to shades of brownish-red, orange to red after
7–10 d incubation (Fig. 3). The species can be differentiated
using phenotypic characters; however, for most species only the
type strain or a representative was available and examined. The
most important macro- and microscopic characters are given in
Tables 2, 3 and Fig. 4.
Extrolites
Monascus species are good producers of known and unchar-
acterized extrolites and an overview is given in Table 4. The
majority of the species produced a species specific profile of
extrolites. Monascus recifensis produced asterric acid, methyl-
asterrate, secalonic acid, (−)-bisdechlorogeodin, questin, seca-
lonic acid D and a compound with an orthosporin-chromophore
(orthosporin-like), M. argentinensis produced rubratoxin A and B
or similar nonadrides (rubratoxin-like extrolites) and
M. flavipigmentosus produced a series of extrolites that are to
our knowledge never detected in any other filamentous fungus
until now. Monascus floridanus produced an orthosporin and
other extrolites only found in this species. The extrolite called
“GULLA”is produced by M. mellicola and M. recifensis, while
M. pallens produces curvularin and dehydrocurvularin (Tables 4
and 5). It is interesting that a series of extrolites with charac-
teristic chromophores (metabolite families M, N, O, Y) (see
Supplementary material for UV spectra) have only been found in
Monascus species so far. In the examination of thousands of
extrolite extracts from species in Penicillium,Aspergillus,Pae-
cilomyces,Talaromyces,Fusarium,Trichoderma,Alternaria,
Curvularia,Chaetomium and other genera, those compounds
have never been detected (JC Frisvad, personal observations).
These extrolites are unique for Monascus species may have
ecological roles in the interaction with bees.
Identification of Monascus isolates associated
with Melipona scutellaris
Thirty isolates were obtained from honey, pollen and the inside of
the nest of Melipona scutellaris, representing three new
(M. flavipigmentosus,M. mellicola and M. recifensis) and one
described species (M. ruber). Monascus mellicola was predom-
inantly present (16 isolates), followed by M. ruber (9),
M. flavipigmentosus (3) and M. recifensis (2). Nine M. mellicola
isolates were isolated from honey, five from the inside of the nest
and two from pollen. Sequence variation is observed among the
investigated M. mellicola isolates, showing that the isolates don't
have a clonal distribution. The M. ruber isolates were all from the
inside of the nest and their identity was in agreement with the
results of the morphological examination (Fig. 5). Monascus fla-
vipigmentosus was isolated from inside nests (2 isolates) and
pollen (1 isolate) and both M. recifensis isolates were from pollen.
DISCUSSION
Monascus belongs to the order Eurotiales, and this genus is
characterized by the production of stalked cleistothecial asco-
mata that are non-ostiolate and have hyaline to brown walls.
The ascomatal cavity is filled with unicellular ascospores.
Asexual reproduction takes place on basipetospora-type
Table 2. Growth rate comparison of Monascus species after 7 d (in mm) and most important colony characters.
Species CYA MEA DG18 CYAS OA CREA YES CMA PDA MEA
30 °C
CYA
30 °C
MEA
37 °C
CYA
37 °C
Colour
mycelium
on MEA
Soluble
pigments
Monascus argentinensis 8–911–13 13 –15 ng 10 –11 ng 14 –15 9 –10 10–11 ng 5–6 ng ng White Absent
M. flavipigmentosus 7–10 10–12 8 –10 ng 4 –5ng 10–11 10–12 6 –810–11 9 –10 0–20–3 White Yellow
M. floridanus 9–10 9–10 3 –5ng 10–11 ng 9 –10 9–10 10–11 10 –11 8 –10 2 –43–4 White Absent
M. lunisporas 15–17 24 –25 20 –22 ng 14–15 3–520–23 19 –20 18 –20 24 –25 15–17 9–10 12–13 Brownish Absent
M. mellicola 8–10 11–12 7–10 ng 9–10 5 –710–11 9–10 9–10 21 –22 11 –12 11 –12 5–7 White Absent
M. pallens 10–11 8 –10 3 –43–414–15 9–10 10 –11 9–11 12–13 11–15 17 –18 26 –30 21–22 White Absent
M. purpureus 19 –20 20–22 3–5ng 16–20 ng 13 –18 18 –20 11–15 39–40 20–21 52 –55 20 –21 Red to orange Orange
M. recifensis 12–14 16 –18 20 –21 ng 3–51–214–15 10 –12 10 –11 19–20 10–11 9–10 3 –4 White to brownish Absent
M. ruber 17–19 25 –26 18–20 ng 18–20 8 –10 17 –28 15 –20 26–30 47–48 35–37 49 –50 35 –40 White Absent
PHYLOGENY OF MONASCUS
www.studiesinmycology.org 37

conidiophores. These conidiophores are erect, variable in
length, and the conidia are hyaline to brown and produced singly
or in short basipetal chains (up to 15–20 conidia). Phenotypic
identification of Monascus species largely depends on shape,
size and pigmentation of the cleistothecia and ascospores
(Hawksworth & Pitt 1983). No cleistothecia and only the
basipetospora-state was observed in the two newly described
species M. mellicola and M. recifensis; however, these species
do phylogenetically belong to the Monascus clade. They pro-
duce a basipetospora-state, which is the characteristic asexual
stage of this genus. Following the latest International Code of
Nomenclature for algae, fungi and plants (McNeill et al. 2012), in
respect to the principle of priority, and that nomenclature has
economic and social implications, particularly for old, important
genera, we give priority to Monascus over Basipetospora, even
when no sexual state is observed in those species. This is in line
with the recommendations of Rossman et al. (2016),whoalso
recommended giving priority to the name Monascus over
Basipetospora.
In the last years numerous new genera have been proposed
primary based on phylogenetic data and sometimes with only a
few distinctive morphological features. Phenotypic and phylo-
genetic analysis revealed two well-supported clades in Mon-
ascus. Following the guidelines proposed by Vellinga et al.
(2015), these differences would justify splitting Monascus into
two separate genera. On the other hand, Monascus species do
share various characters, such as similar basipetospora-type
conidiophores and stalked cleistothecia. The majority of Mon-
ascus species produce indole alkaloids (possibly gypsetins) and
this study shows that various Monascus species are also
associated with stingless bees, indicating that they are also
ecologically related. We therefore give preference to introduce
two new sections instead of two small genera. A sectional
classification system is commonly applied in genera related to
Monascus, such as Penicillium,Aspergillus and Talaromyces
and this is in line with that approach (Gams et al. 1985,
Houbraken & Samson 2011, Yilmaz et al. 2014). The two sec-
tions have few extrolites in common (Table 5). The Rubri section
contains species that produce mevinolins, citrinin and other
yellow and red azaphilone pigments, including the red pigments
(rubropunctamine, PP-V, PP-R etc.) that are colouring red rice,
while the species in section Floridani do not produce any of these
bioactive extrolites at all. Isolates in each species in section
Floridani produce species specific combinations of extrolites, and
few are in common between those species. One example is the
red compound “GULLA”, which was detected in both M. mellicola
and M. recifensis, but the latter species produce several extro-
lites that are not produced by M. mellicola, including secalonic
acid D, asterric acid, questin, (−)-bisdechlorogeodin and some
red anthraquinone extrolites not related to the azaphilones pro-
duced by M. purpureus and M. ruber. Strains of
M. flavipigmentosus produce a high number of unique as yet not
structure elucidated extrolites, including some yellow coloured
extrolites (Y1 and Y2) and an anthraquinone (Table 5). The red
extrolite “GULLA”has previously been found in Penicillium
species, including Penicillium oxalicum and P. mononematosum
(Frisvad, personal communication).
Several morphological features are shared between Mon-
ascus species; however, there are also various characters that
can be used for identification (Tables 2 and 3). For example, the
conidial size can differ between species. All species except two
(M. mellicola,M. recifensis) produce a sexual state and the size
and shape of the ascospores can differ among species. The
species also differ in their growth rates, and for example
M. flavipigmentosus,M. pallens and M. floridanus grow more
restrictedly on agar media than M. ruber and M. purpureus. Most
species do not produce soluble pigments; however, the pro-
duction of red (soluble) pigments is a character of M. purpureus
and M. ruber (Hawksworth & Pitt 1983) and M. flavipigmentosus
produces yellow pigments on CMA and PDA (and old cultures on
Table 3. Most important micromorphological characters for species recognition.
Species Colour and
size (μm)
ascomata on
PDA
Shape
ascospores
on PDA
Size
ascospores
(μm)
Shape and
colour conidia
Size of
conidia (μm)
Number of
conidia per
phialide
Monascus argentinensis* Dark olivaceous-brown,
20–75
Ellipsoidal to
subglobose
3–4 × 2.5–3 Globose to obovoid
or obpyriform
Globose, 5–15; obpyriform,
7–15 × 5–9
Single or formed in
short chains
M. flavipigmentosus Hyaline to brown,
40–60
Lunate 4–5 × 1.7 –2.5 Globose to subglobose,
hyaline to brown
5.5–7.5 Single or formed in
short chains
M. floridanus* Dark brown, 22–58 Ellipsoidal 3.5–4.5 × 2 –3 Globose to obovoid or
obpyriform, pale brown
4–9 × 3.5–9 Single or formed in
short chains (up to 6?)
M. lunisporas* Brown, 25 –60 Lunate 6–7×2–2.5 Globose to obpyriform,
hyaline to brown
Globose, 6–11; obpyriform,
5–7×7–10
Single or formed in
short chains
M. mellicola –––Globose to subglobose,
hyaline to brown
2.5–5.0 × 3.5 –5.0 Single or up to
17 conidia
M. pallens* Hyaline, 23–38 Ellipsoidal 3.5–4 × 2.5 –3 Usually pyriform, hyaline 3.5–10 (–13) × 2.5–8 Short terminal or
intercalary basipetal
M. purpureus** Hyaline, (25–)
45 × 60 (–70)
Ellipsoidal (5.5–)6–7×4–5 Globose to obpyriform 8 –11 × 8 –10 Single or in
short chains
M. recifensis –––Globose to subglobose,
hyaline to brown
4.0–7.0 Single or in
short chains
M. ruber** Brown, 30–50 (–60) Ellipsoidal 5–6(–7.5) ×
(3.5–)4–5
Globose to obpyriform 10 –18 × 8 –14 Single or up to
10 conidia
Abbreviations: *Data from original description; **Data from Hawksworth & Pitt (1983);–: not observed.
BARBOSA ET AL.
38

DG18). Also the growth rate at 37 °C is diagnostic. M. pallens,
M. ruber and M. purpureus grow equally or even faster at 37 °C
than at 30 °C. On the other hand, M. floridanus and M. mellicola
and M. recifensis grow slowly at 37 °C, and M. argentinensis and
M. flavipigmentosus did not grow at this temperature at all.
All species except M. floridanus produced a species-specific
series of extrolites, consistent with the phenotypic classification
and the results obtained in the phylogenetic study. Monascus
lunisporas,M. recifensis and M. flavipigmentosus are phyloge-
netically closely related. Their extrolite profiles are distinct.
Monascus flavipigmentosus produces metabolites of biosynthetic
family M and M. recifensis secalonic acid, asterric acid,
sulochrin, questin and an anthraquinone with the same UV
spectrum as physcion (physcion-like in Table 4). None of these
extrolites were found in the closely related species M. lunisporas
(CBS 142230
T
). An indole alkaloid (probably gypsetin) was
produced by 6 of the 9 species (Table 4) and was the only
metabolite found in section Floridani and Rubri. The metabolites
mevinolins and xanthomonasin A were only detected in M. ruber-
clade species. An important characteristic of Monascus ruber is
its ability to produce citrinin, a compound with both antibiotic and
toxic activity. According literature, this extrolite is also produced
by M. purpureus,M. pallens,M. lunisporas and M. floridanus
(Wang et al. 2005). In our study, citrinin was detected only in the
Fig. 4. Conidial shapes and colours of Monascus species. A. M. lunisporas.B. M. flavipigmentosus.C. M. recifensis.D. M. mellicola.E. M. pallens.F. M. floridanus.G.
M. argentinensis.H. M. ruber.I. M. purpureus. Scale bars = 10 μm.
PHYLOGENY OF MONASCUS
www.studiesinmycology.org 39

type of M. purpureus. More strains in addition to other culture
conditions stimulating citrinin production should be investigated
to find out if other species besides M. purpureus can produce
citrinin.
Based on the results of our study combined with data from
previous studies, we accept nine species in Monascus:
M. argentinensis,M. floridanus,M. lunisporas,M. mellicola,
M. pallens,M. purpureus,M. ruber,M. recifensis and
M. flavipigmentosus (Hawksworth & Pitt 1983, Park et al. 2004).
Monascus pilosus,M. sanguineus are also often mentioned in
literature as accepted species in Monascus. Phenotypically,
M. pilosus is very similar to M. ruber and according literature,
Hawksworth & Pitt (1983) indicated that they can differentiated
by the size of ascomata (25–55 vs 30–50 (–60) μm), asco-
spores (5–7(–8.5) × 3–3.5 (–4) vs 5–6.5 (–7.5) × (3.5 –)
4–4.5 μm) and the presence of a brownish pigment in the
cleistothecial walls and conidia. These sizes and colours are
overlapping and during the study of the M. ruber isolates asso-
ciated with bees, we also found considerable variation in
pigmentation among the studied strains. Previous studies
showed that M. pilosus shares ITS and partial LSU and β-tubulin
sequences with M. ruber (Park & Jong 2003, Park et al. 2004),
suggesting that these are conspecific. Monascus pilosus clusters
together with M. ruber in all of our single gene phylogenies,
confirming these results. Additionally, M. ruber and M. pilosus
are similar also in their metabolite profiles and share the pro-
duction of mevinolins, rubropunctamine and xanthomonascin.
Subsequently, there is no basis to accept M. pilosus as a
separate species. Based on sequence data, M. sanguineus is
treated here as a synonym of M. purpureus. Analysis of partial
β-tubulin sequences (another part of the gene than used in this
study) showed that M. sanguineus and M. purpureus are
phylogenetically closely related and distinct from M. ruber (Park
et al. 2004). These results are confirmed in our BenA,CaM, ITS
and LSU phylograms, though statistical support was only found
in the ITS phylogram. Based on the GCPSR concept, these
species are treated as separate species. Phenotypically,
M. sanguineus is differentiated from M. purpureus by its inability
to grow on G25N and colour of ascomata and conidia; however,
these characters might not be stable among a larger set of
isolates, and this needs further investigation.
Many other species are described in Monascus:M. albidulus
(= M. albidus nom. inval.), M. araneosus,M. aurantiacus,
M. fumeus (= M. fuliginosus nom. inval.), M. kaoliang,M. major,
M. paxii,M. pilosus nom. inval., M. pubigerus,M. rubiginosus,
M. rutilus (= M. anka nom. inval.), M. rubropunctatus,
M. serorubescens,M. vitreus. All these species belong to
M. ruber-clade (Hawksworth & Pitt 1983, Park & Jong 2003). A
detailed study is needed to determine the species diversity within
the M. ruber-clade and to resolve the placement of the M. ruber/
M. purpureus synonyms. Six Basipetospora species
(B. chlamydospora,B. denticola,B. halophila,B. rubra,
B. variabilis,B. vesicarum) are described and those might
compete with the new species that are described here, especially
those that lack a sexual state. However, Basipetospora rubra
was described as the asexual state of M. ruber and is in the
single name nomenclature system regarded as a synonym of this
species. Basipetospora halophilica phylogenetically belongs to
Aspergillus and was recently transferred to this genus (Samson
et al. 2014, Kocsub
eet al. 2016). Basipetospora chlamydospora,
B. variabilis and B. denticola represented by CBS 228.84 (16S
rRNA, AB024045), CBS 995.87 (16S rRNA, AF437892) and
CBS 132.78 (ITS, LN850801), respectively, belong to Micro-
ascales. The first two species might represent a novel genus in
this order (J. Woudenberg, pers. comm.) and the latter is a
synonym of Scopulariopsis candida (Jagielski et al. 2016).
Basipetospora vesicarum can be considered a synonym of
M. ruber. This species was introduced based on examination of
the type specimen of Sporotrichum vesicarum and analysis of
this specimen revealed the presence of the Basipetospora
anamorph of M. ruber (Stalpers 1984).
When Monascus eremophilus was described, Hocking & Pitt
(1988) noted the unique features of this species. Based on col-
ony colour and the mode of ascospore production they decided
that the species could best be classified in Monascus. After its
description, Monascus eremophilum was included in various
phylogenetic studies; however, results concerning its placement
inferred from different DNA regions were inconclusive. Park &
Jong (2003) evaluated the use of D1/D2 sequences of the LSU
rRNA for species differentiation in Monascus, and simultaneously
performed a phylogenetic analysis. In their study, M. eremophilus
was found in the clade containing the type of M. ruber; however,
the bootstrap support of that clade was low (61 %). In 2004, Park
et al. studied the genus Monascus by using ITS and partial beta-
tubulin gene sequences. The position of M. eremophilus was
unresolved in their ITS phylogram, and the species grouped
together with M. lunisporas and M. pallens with less than 50 %
bootstrap support. Moreover, when the beta-tubulin sequences
were used, M. eremophilus was placed outside the ingroup. The
authors commented that such an inconclusive placement of
M. eremophilus might indicate: ‘… a unique and unpredictable
genetic combination for this species. It might reflect enormous
and extreme environmental stress and subsequent drastic ge-
netic changes to adapt to extremely dry conditions’(Park et al.
2004). More recently, based on D1/D2 sequence data, Vinnere-
Pettersson et al. (2011) showed that M. eremophilus does not
Table 4. Extrolites detected in Monascus.
Species Extrolites
Monascus argentinensis Anthraquinone Z, indole alkaloid (possibly
gypsetin), rubratoxin-like
M. flavipigmentosus Anthraquinone X (possibly atrochrysone), indole
alkaloid (possibly gypsetin), unknown and
unique metabolite biosynthetic family M and Y
(Y1 and Y2)
M. floridanus “ENDI”, orthosporin-like, “JOPS”
M. lunisporas Citrinadin-like, indole alkaloid (possibly
gypsetin), metabolite N series, metabolite O
series, shamixanthone-like
M. mellicola Indole alkaloid (possibly gypsetin), “GULLA”
M. pallens Curvularin, dehydrocurvularin, indole alkaloid
(possibly gypsetin)
M. purpureus Citrinin, mevinolins, monascin, PP-V, PP-R,
rubropunctamine, rubropunctatin,
xanthomonasin A
M. recifensis Anthraquinone X (= atrochrysone?), asterric
acid, (−)-bisdechlorogeodin, “GULLA”,
orthosporin-like, anthraquinone W (physcion-
like), questin, red anthraquinone pigments,
secalonic acid D, sulochrin
M. ruber Indole alkaloid (possibly gypsetin), mevinolins,
monascin, PP-V, PP-R, xanthomonasin A,
rubropunctamine, rubropunctatin, rubratoxin-like
BARBOSA ET AL.
40

belong to Monascus, and appears to be related to Penicillium.In
order to clarify the difference placements of M. eremophilus in
literature, we re-analysed the LSU data set of Park & Jong (2003)
and Vinnere-Pettersson et al. (2011) together with the data set
generated in this study (data not shown). These results show that
the sequence (AF365023) used in the study of Park et al. (2004)
does not match with the other sequences generated from
M. eremophilus, explaining the various phylogenetic placements
of this species. Based on a 4-gene phylogeny, Houbraken et al.
(2014) confirmed its placement in Penicillium.Theyconfidently
place the species on a branch together with members of section
Charlesia (P. charlesii CBS 304.48
T
,P. fellutanum CBS 229.81),
though there is sufficient genetic distance that would warrant
placement of this species in a new section. Based on this liter-
ature review and additional (sequence) data generated in this
study, we propose to transfer M. eremophilus in Penicillium. The
placement of this species in Penicillium is unexpected. Penicillium
eremophilum is, unlike any other Penicillium (and Monascus)
species, an obligate xerophile. The species is not known to
produce an asexual state and there were until now no strictly
sexually reproducing species within Penicillium, though co-
nidiophores can sometimes be sparsely produced in sexually
reproducing Penicillium species. The formation of two-spored asci
is also not shared with other Penicillium species. This feature,
together with its xerophily, is shared with the phylogenetically
distant species Xeromyces bisporus.
Table 5. Retention index and absorption maxima for extrolites dectected in Monascus (the UV spectra of the unknown compounds are
shown in the Supplementary data).
Extrolite Retention index Absorption maxima (nm) Extrolite by section
Anthraquinone X (= atrochrysone?) 1207 220, 261, 282sh, 427 Floridani
Anthraquinone Z 948 223, 271, 298, 433 Floridani
Asterric acid 921 207, 220sh, 252, 317 Floridani
Asterric acid derivative 825 207, 220sh, 252, 317 Floridani
(−)-bisdechlorogeodin 868 203, 224sh, 278, 336sh Floridani
Citrinadin-like 783, 821, 830 200, 227sh, 246, 265sh, 325 Floridani
Citrinin 907 221, 242sh, 328, 415sh Rubri
Curvularin 881 200, 223, 270, 301 Floridani
Dehydrocurvularin 861 202, 225, 283, 334sh Floridani
ENDI 745 End-absorption Floridani
GULLA 1007 202, 258, 286, 328, 369, 428 Floridani
Indole alkaloid (= gypsetin-like) 967 224, 278, 288, 295 Floridani,Rubri
JOPS 1098 208, 248, 275, 353 Floridani
Metabolite M series 845, 854, 865, 881, 906, 946 291, 242sh, 283, 318 Floridani
Metabolite N series 917, 1048 203, 236, 251sh, 326, 381 Floridani
Metabolite O series 905, 982, 993 202, 226sh, 254, 272sh, 335 Floridani
Metabolite Y series 1097 (Y1), 1273 (Y2) 200, 228sh, 274, 375 Floridani
Methyl asterrate 934 200, 227sh, 246, 265sh, 325 Floridani
Mevinolin 1232 230sh, 240, 250sh Rubri
Mevinolin, open acid form 1121 230sh, 240, 250sh Rubri
Monascin 1251 230, 282, 397 Rubri
Rubratoxin-like (Nonadrides, provisionally
identified as rubratoxins)
1033, 1066 215sh, 263 Floridani
Orthosporin-like 721 241sh, 248273, 282, 324 Floridani
Physcion-like (anthraquinone W) 1079 221, 250sh, 264, 282, 331, 440 Floridani
PP-V 943 250, 296, 420, 524 Rubri
PP-R 981 252, 306, 417, 524 Rubri
Questin 958 223, 247sh, 280, 428 Floridani
Red anthraquinone series 1316, 1326, 1387, 1412, 1422 227, 268, 330, 442 Floridani
Rubratoxin-like 1198 202, 251 Floridani,Rubri
Rubropunctamine 1417 218, 250, 279, 298sh, 447sh, 475, 512sh Rubri
Rubropunctatin 1252 218sh, 235, 279, 394475sh, 521 Rubri
Secalonic acid D 1104 200, 215sh, 258, 331, 388sh Floridani
Shamixanthone-like 1121 201, 228, 263, 301, 366 Floridani
Sulochrin 873 203, 224sh, 278, 324sh Floridani
Xanthomonascin A 1143 230, 282, 397 Rubri
Asterric acid, methyl asterrate and (−)-bisdechlorogeodin are all part of the geodin biosynthetic family; sh: shoulder.
PHYLOGENY OF MONASCUS
www.studiesinmycology.org 41

Fig. 5. Monascus ruber URM 7525 isolated during the course of this study. A. Colonies from left to right (first row) MEA, CYA, OA, CMA; (second row) MEA reverse, CYA
reverse, OA reverse, CMA reverse; (third row) PDA, YES, DG18, CREA; (forth row) PDA reverse, YES reverse, DG18 reverse, CREA reverse. B–C. Typical ascoma and
ascospores. D. Conidiophores with conidia chain. Scale bars = 10 μm.
BARBOSA ET AL.
42

The ITS region is the official DNA barcode for fungi, and is
good practice to include ITS sequences whenever new species
are described (Schoch et al. 2012). However, not all species can
be identified using this marker because certain species share
identical ITS sequences (e.g. Houbraken et al. 2014, Chen et al.
2016). All Monascus species can be recognized on their ITS
sequence only, even though the interspecific differences are low,
especially between M. ruber and M. purpureus. Whether these
barcode gaps remain present when a larger set of isolates is
investigated remains unknown. A larger sequence variation was
observed in the BenA gene. This gene is used as secondary
barcode for the related genera Penicillium and Talaromyces and
we propose the same for Monascus (Visagie et al. 2014, Yilmaz
et al. 2014). The BenA gene is easy to amplify in Monascus and
can distinguish all species. LSU has limited resolving power and
RPB2 is more difficult to amplify and is therefore only recom-
mended in phylogenetic studies.
Stingless beekeeping, or meliponiculture, is an ancient ac-
tivity and many species of stingless bees are managed in the
Americas, Africa, Asia and Australia; however, it remains a
largely under-exploited business and technical knowledge is
scarce. Much practical and academic work is being done about
the best ways of keeping these bees, multiplying their colonies,
and exploring the honey they produce (Cortopassi-Laurino et al.
2006, Villanueva-Guti
errez et al. 2013, Jaff
eet al. 2015).
Melipona scutellaris is most known in the Northeast of Brazil.
Furthermore, these bees are important pollinators in agricultural
and natural ecosystems. Recently, a fungus cultivation mutu-
alism in a social bee (Scaptotrigona postica) was reported for
the first time (Menezes et al. 2015).ThelarvaeofS. postica
have a higher survival rate when they were fed with food grown
with Monascus mycelium. The symbiotic relationships between
microorganisms and stingless bees have been poorly explored,
and during our investigation of fungi associated with Melipona
scutellaris bees, we frequently isolated M. ruber from the inside
of nests. This indicates that also other bee species, like Meli-
pona scutellaris, might also have an (obligatory) relationship
with M. ruber.BesidesM. ruber, also M. mellicola was
frequently isolated from honey, pollen and the inside of nests,
followed M. recifensis and M. flavipigmentosus. This associa-
tion with bees might be a novel unexplored ecological niche of
Monascus species and can be the subject of future studies. The
antibiotic and antifungal activity of some Monascus strains
might play a role in the protection of the larvae food from mi-
crobial contaminations (Jůzlova et al. 1996, Menezes et al.
2015) and our discovery of many Monascus-unique extrolites
in these species (metabolite families M, N, O, and Y) invites
structure elucidation and bioactivity testing of those com-
pounds. Stchigel & Guarro (2007) studied several cleistothecial
ascomycetes and they concluded that the criterion of the pro-
duction of closed ascomata without a predefined opening and
with an irregular arrangement of asci at the centre is of little
systematic value. A recent study about fungi living with asso-
ciation with solitary bees collected in Denmark suggest the
convergent evolution of reduced fruiting bodies in Pezizomy-
cotina is adaptive for spore dispersal to the bee habitat (Wynns
2015). Interesting to note in this context is that Monascus forms
smaller cleistothecia than those produced in the related genera
Aspergillus and Penicillium.
In the past, taxonomic studies on Monascus were solely
based on phenotypic characters, or when sequence data was
used, these were mostly applied for identification purposes. With
the transfer of M. eremophilus to Penicillium, monophyly in
Monascus is restored. The presented 5-gene phylogeny is a
good robust starting point for future taxonomic studies in Mon-
ascus. Furthermore, a list of accepted species is provided,
including information on (ex-)type strains and molecular markers
(see Taxonomy section).
TAXONOMY
Phylogenetically, two well-supported clades (M. floridanus-clade
and M. ruber-clade) are present in Monascus and these groups
can also be differentiated on phenotypic characters. Two
sectional names are introduced for these clades and information
on this taxonomic decision can be found in the Discussion. Our
polyphasic approach revealed the presence of three new species
and these are described below. Furthermore, a new combination
for Monascus eremophilus is proposed.
Section Floridani R.N. Barbosa & Houbraken sect. nov.
MycoBank MB820076.
Typus:Monascus floridanus P.F. Cannon & E.L. Barnard,
Mycologia 79: 480. 1987. MycoBank MB132123.
Diagnosis: Colony diameter on MEA, PDA, CYA, CMA, OA,
YES generally below 20 mm, no or restricted growth (<10 mm)
on CREA and CYAS, and colony diameter less than 30 mm on
MEA incubated at 30 and 37 °C. Colonies in shades of brown;
conidia brown pigmented; mycelium white or in shades of
brown.
Section Rubri R.N. Barbosa & Houbraken sect. nov. MycoBank
MB820077.
Typus:Monascus ruber Tiegh., Bulletin de la Soci
et
e Botanique
de France 31: 227. 1884. MycoBank MB234876.
Diagnosis: Colony diameter on MEA, PDA, CYA, CMA, OA, YES
generally above 15 mm, no or restricted growth on CREA and
CYAS, good growth (>30 mm) on MEA incubated at 30 and
37 °C. Colonies in shades of brown to red; conidia brown pig-
mented; mycelium white or in shades of red or orange.
Monascus flavipigmentosus R.N. Barbosa, Souza-Motta,
N.T. Oliveira & Houbraken sp. nov. MycoBank MB820072.
Fig. 6.
Etymology:flavipigmentosus is referring to yellow pigment pro-
duced on CMA and PDA.
Diagnosis:Monascus flavipigmentosus is phylogenetically
distinct by BenA,CaM and ITS sequencing and characterized by
the absence of growth on CREA 25 °C, and MEA and CYA
incubated at 37 °C. Yellow soluble pigments present on CMA
and PDA (and old cultures on DG18).
PHYLOGENY OF MONASCUS
www.studiesinmycology.org 43

In: Monascus section Floridani
Typus:Brazil, Recife, isolate inside nests of Melipona scutellaris
Jun 2014, isolated by R.N. Barbosa (holotype URM 90064;
culture ex-type URM 7536 = CBS 142366 = DTO 353-A2).
Barcodes: ITS barcode: KY511751 (alternative markers:
BenA = KY709168; CaM = KY611929; RPB2 = KY611968).
Colony diam, 7 d (mm): MEA 10–12; CYA 7–10; CMA 10 –12;
PDA 6–8; YES 10–11; OA 4 –5; DG18 8 –10; CYAS No growth;
CREA No growth; CYA 30 °C 7–9; CYA 37 °C 0–2; MEA 30 °C
10–12; MEA 37 °C 0–3.
Description: Colonies characters after 7 d. MEA, 25 °C: colony
texture velvety to floccose, pulvinate, mycelium white; sporulation
absent; exudates absent; soluble pigments absent; reverse yel-
low. CYA, 25 °C: colony texture floccose low, mycelium white;
sporulation absent; exudates absent; soluble pigments absent;
reverse white to cream. CMA, 25 °C: colony texture lanose, low,
mycelium inconspicuously white at the margin; sporulation weak
at centre, conidia en masse dull brown; exudate absent; soluble
pigments present, yellow; reverse yellow; ascomata abundantly
produced, brown. PDA, 25 °C: colony texture floccose to lanose,
low, mycelium white; sporulation absent; exudates absent; soluble
pigments present, light yellow; colony reverse yellow. YES, 25 °C:
colony texture floccose, low, mycelium white; sporulation absent;
exudates absent; soluble absent; colony reverse yellow to
brownish. OA, 25 °C: colony texture not determinate, mycelium
white; sporulation absent, exudates absent; soluble pigments
absent; colony reverse white to cream. DG18, 25 °C: colony
texture velvety to floccose, low, mycelium white; sporulation ab-
sent; exudates absent; soluble pigments absent; reverse white to
light yellow. CYAS, 25 °C: no growth. CREA, 25 °C: no growth.
MEA, 30 °C: colony texture velvety, umbonate, mycelium white,
sporulation absent, exudates absent; soluble pigments absent;
reverse light brownish. CYA, 30 °C: mycelium brownish, sporu-
lation weak, conidia en masse brownish; ascomata sparsely
produced, brown; exudates absent; soluble pigments absent;
reverse brownish. MEA, 37 °C: no growth. CYA, 37 °C: no growth.
Mycelium abundant, hyphae irregularly branched, hyaline to pale
brown when old, smooth-walled, 1.8–3μm wide. Conidiophores
variable in length, smooth, 3–30 × 1.5–2.5 μm. Conidia single or
formed in short basipetal chains, usually terminal, rarely inter-
calary, 5.5–7.5 × 5.5 –7.5 μm diam, at first hyaline and pale
brown to brown with age. Ascomata, stalked when young, non-
ostiolate, globose to subglobose, 40–60 μm diam, initially light
brown and dark brown in the age; peridium brown, developing
irregularly polygonal plates, surrounded by short hyaline areas,
in time filled with a compact mass of ascospores. Asci
evanescent or no observed. Ascospores hyaline, 1-celled, reni-
form or allantoid, 4–5 × 1.7–2.5 μm, smooth-walled.
Notes: This species shares morphological features with
M. lunisporas, but can be distinguished by the production of
yellow soluble pigments on CMA and PDA, shorter co-
nidiophores (3–28.5 × 1.5–2.5 μmvs 5–500 × 3–5μm), smaller
conidia (5.5–7.5 × 5.5–7.5 μmvs 6–11 μm) and ascospores
(4–5 × 1.7–2.5 μmvs 6–7×2–2.5 μm).
Monascus mellicola R.N. Barbosa, Souza-Motta, N.T. Oliveira
& Houbraken sp. nov. MycoBank MB820073. Fig. 7.
Etymology:mellicola refers to honey, the substrate from which
the type species was isolated.
Diagnosis:Monascus mellicola is phylogenetically distinct by
BenA,CaM and ITS sequencing, a sexual state is not observed
in culture, and the species grows restricted on CREA incubated
at 25 °C. No exudates and soluble pigments are produced on the
agar media used in this study.
In: Monascus section Floridani
Typus:Brazil, Recife, honey from Melipona scutellaris Jun 2014,
isolated by R.N. Barbosa (holotype URM 90065, culture ex-type
URM 7510 = CBS 142364 = DTO 350-E6).
Barcodes: ITS barcode: KY511726 (alternative markers:
BenA = KY709143; CaM = KY611904; RPB2 = KY611943).
Colony diam, 7 d (mm): MEA 11–12; CYA 8–10; CMA 9–10;
PDA 9–10; YES 10–11; OA 9 –10; DG18 7–10; CYAS No
growth; CREA 5–7; CYA 30 °C 10–11; CYA 37 °C 6–8; MEA
30 °C 14–15; MEA 37 °C 5–6.
Description: Colonies characters after 7 d. MEA, 25 °C: colony
texture floccose, raised in centre; mycelium white; sporulation
strong, conidia en masse brown; exudates absent; soluble pig-
ments absent, reverse brown. CYA, 25 °C: colony texture
velvety, low; mycelium white, sometimes inconspicuously brown;
sporulation weak, conidia en masse brown; exudates absent;
soluble pigments absent, reverse dark brown at centre to
brownish at margins. CMA, 25 °C: colony texture velvety, low;
mycelium white sometimes inconspicuously greyish olive, spor-
ulation moderate, conidia en masse brown; exudates absent;
soluble pigments absent; reverse dark brown. PDA, 25 °C:
colony texture velvety, low; mycelium white sometimes incon-
spicuously brown; sporulation strong, conidia en masse brown;
exudates absent; soluble pigments absent; reverse brownish.
YES, 25 °C: colony texture velvety to floccose, low; mycelium
white; sporulation strong, conidia en masse brownish; exudates
absent; soluble pigments absent; reverse dark brown. OA,
25 °C: colony texture velvety, low; mycelium white, sporulation
weak, conidia en masse brown; exudates absent; soluble pig-
ments absent, reverse brown. DG18, 25 °C: colony texture
velvety to floccose, low, mycelium white; sporulation absent;
exudates absent; soluble pigments absent; reverse white in the
margins and dark brown at centre. CYAS, 25 °C: no growth.
CREA, 25 °C: mycelium white, sporulation absent; no acid
production. CYA, 30 °C: colony texture velvety to floccose, low;
mycelium brown, sporulation weak, conidia en masse brownish;
exudates absent; soluble pigments absent; reverse brown. CYA,
37 °C: mycelium white, sporulation absent; exudates absent;
soluble pigments absent, reverse brown. MEA 30 °C: mycelium
white, sporulation moderate to strong, conidia en masse in
shades of brown; exudates absent; soluble pigments absent;
reverse brownish. MEA, 37 °C: mycelium white, sporulation in
centre, weak, conidia en masse in shades of brown; exudates
absent; soluble pigments absent; reverse yellow-brownish.
BARBOSA ET AL.
44

Fig. 6. Monascus flavipigmentosus,URM7536.A. Colonies from left to right (first row) MEA, CYA, OA, CMA; (second row) MEA reverse, CYA reverse, OA reverse, CMA reverse; (third
row) PDA, YES, DG18, CREA; (forth row) PDA reverse, YES reverse, DG18 reverse, CREA reverse. B–C. Conidiophores. D. Ascoma. E. Ascospores. F. Conidia. Scale bars = 10 μm.
PHYLOGENY OF MONASCUS
www.studiesinmycology.org 45

Fig. 7. Monascus mellicola, URM 7510. A. Coloniesfrom left to right (first row) MEA, CYA, OA, CMA; (second row) MEA reverse, CYA reverse, OA reverse, CMA reverse; (third row)
PDA, YES, DG18, CREA; (forth row) PDA reverse, YES reverse, DG18 reverse, CREA reverse. B–E. Conidiophores with conidia chain. D. Conidia. Scale bars = 10 μm.
BARBOSA ET AL.
46

Mycelium abundant, hyphae irregularly branched, hyaline to pale
brown when old, smooth-walled, 2.5–3μm wide. Conidiophores
basipetospora-type, variable in length, smooth,
16–32 × 1.5–2.0 μm. Conidia formed basipetally, in long chains,
up to 17 conidia, globose to subglobose, smooth-walled,
2.5–5.0 × 3.5–5.0 μm diam, hyaline when young, becoming
pale brown to brown with age. Agglomeration of conidia with
variable size observed, 45–65 × 55–65 μm diam. Sexual morph
not observed.
Monascus recifensis R.N. Barbosa, Souza-Motta, N.T. Oliveira
& Houbraken sp. nov. MycoBank MB820074. Fig. 8.
Etymology:recifensis refers to the Brazilian city Recife, the
location of the type strain of this species.
Diagnosis:Monascus recifensis is phylogenetically distinct by
BenA,CaM and ITS sequencing. The species is characterized
by restricted growth on agar media, a sexual state is not
observed, and the species doesn't produce exudates and soluble
pigments on the agar media used in this study.
In: Monascus section Floridani
Typus:Brazil, Recife, isolated from pollen inside nests of Meli-
pona scutellaris Jun 2014, isolated by R.N. Barbosa, (holotype
URM 90066; culture ex-type URM 7524 = CBS 142365 = DTO
350-G6).
Barcodes: ITS barcode: KY511740 (alternative markers:
BenA = KY709157; CaM = KY611918; RPB2 = KY611957).
Colony diam, 7 d (mm): MEA 16–18; CYA 12–14; CMA 10 –12;
PDA 10–11; YES 14–15; OA 3 –5; DG18 20 –21; CYAS not
growth; CREA 1–2; CYA 30 °C 13–15; CYA 37 °C 7–8; MEA
30 °C 17–20; MEA 37 °C 3–4.
Description: Colonies characters after 7 d. MEA, 25 °C: colony
texture floccose to lanose, pulvinate, mycelium white, sporulation
strong, conidia en masse brown; exudates absent; soluble pig-
ments absent; reverse brownish. CYA, 25 °C: colony texture
lanose, pulvinate; mycelium white sometimes inconspicuously
brown; sporulation weak to moderate, conidia en masse
brownish; exudates absent; soluble pigments absent; reverse
dark brown to light brown close at margins. CMA, 25 °C: colony
texture velvety, low; mycelium brown; sporulation moderate to
strong at centre, conidia en masse dark brown; exudates absent;
soluble pigments absent; reverse black. PDA, 25 °C: colony
texture velvety to floccose; mycelium white; sporulation strong,
en masse brownish; exudates absent; soluble pigments absent;
reverse white to cream close at margins, dark brown at centre.
YES, 25 °C: colony texture velvety, mycelium white sometimes
inconspicuously brownish; sporulation strong, conidia en masse
in shades of brown; exudates absent; soluble pigments present
after 10 d. incubation, in shades of brown; reverse dark brown to
light brown close at margins. OA, 25 °C: colony texture velvety;
mycelium white; sporulation weak, conidia en masse dark brown;
exudates absent; soluble pigments absent; reverse dark brown.
DG18, 25 °C: colony floccose, mycelium white; sporulation weak
to moderate, conidia en masse brownish; exudates absent;
soluble pigments absent; reverse white close to margins and
dark brownish at centre. CYAS, 25 °C: no growth. CREA, 25 °C:
growth very poor. MEA, 30 °C: colony texture velvety; mycelium
white; sporulation moderate to strong, conidia en masse brown;
exudates absent; soluble pigments absent; reverse brownish.
CYA, 30 °C: colony texture velvety, mycelium brownish; sporu-
lation moderate, conidia en masse brown; exudates absent;
soluble pigments absent; reverse dark brown, white at margins.
MEA, 37 °C: colony texture velvety; mycelium white; sporulation
absent; exudates absent; soluble pigments absent; reverse
cream. CYA, 37 °C: colony texture velvety to floccose; mycelium
brownish; sporulation moderate, conidia en masse in shades of
brown; exudates absent; soluble pigments absent; reverse dark
brown.
Mycelium abundant, hyphae irregularly branched, hyaline to pale
brown when old, smooth-walled, 1.8–2.5 μm wide. Co-
nidiophores variable in length, smooth, 4.5–21.0 × 1.8–2.5 μm,
sometimes with additional branch, Conidia single, globose,
4.0–7.0 × 4.0–7.0 μm diam, at first hyaline, pale brown to brown
with age. Sexual morph not observed after 60 d incubation.
Notes:Monascus lunisporas and M. flavipigmentosus are
phylogenetically closely related to M. recifensis and the latter
species doesn't produce ascomata, exudates and soluble pig-
ments. These species can also be differentiated by their unique
extrolite profiles (Table 4).
Penicillium eremophilum (A.D. Hocking & Pitt) Houbraken,
Leong & Vinnere-Pettersson comb. nov. MycoBank MB820075.
Basionym:Monascus eremophilus A.D. Hocking & Pitt, Myco-
logia 80: 84. 1988. MycoBank MB132383.
Typus:Australia, New South Wales, Sydney, isolated from
mouldy prunes, isolated by A.D. Hocking, 1986 (Herb.: FRR
3338; Ex-type: IMI 313774 = CBS 123361 = ATCC 62925).
Barcodes: ITS barcode: GU733347 (alternative markers:
BenA = KY709170; CaM = KY611931; RPB2 = KY611970).
Notes: The colony morphology was identical to that described by
Hocking and Pitt in 1998. Monascus eremophilus is indeed an
obligate xerophile. No growth was observed on either MEA or
MA20S at any temperature afterincubation of one year. Monascus
eremophilus grew well on MY50G within the range 10–25 °C.
Good growth at 30 °C and absence of growth at 37 °C has been
previously reported (Leong et al. 2011). Upon microscopy, asco-
matal initials were observed after approximately a month of culti-
vation. However, these cleistothecia never matured and thus no
ascospores were observed. No anamorph was observed during
the time of cultivation or mentioned in the original description. The
fact that we did not observe any fertile cleistothecia may indicate
that the type strain (FRR 3338) is deteriorating. Molecular data
shows that this species is related to Penicillium (Park et al. 2004,
Vinnere-Pettersson et al. 2011, Houbraken et al. 2014)andis
transferred to Penicillium (this study).
List of accepted species in Monascus
Monascus argentinensis Stchigel & Guarro, Stud. Mycol. 50: 301. 2004.
[MB500076]. —Herb.: FMR 6778. Ex-type: CBS 109402 = FMR 6778.
Section Floridani. ITS barcode: JF922046 (Alternative markers:
BenA = KY709174; CaM = KY611935; RPB2 = JN121423).
PHYLOGENY OF MONASCUS
www.studiesinmycology.org 47

Fig. 8. Monascus recifensis, URM 7524. A. Colonies from left to right (first row) MEA, CYA, OA, CMA; (second row) MEA reverse, CYA reverse, OA reverse, CMA reverse;
(third row) PDA, YES, DG18, CREA; (forth row) PDA reverse, YES reverse, DG18 reverse, CREA reverse. B–D. Conidiophores. E. Conidia. Scale bars = 10 μm.
BARBOSA ET AL.
48

Monascus flavipigmentosus R.N. Barbosa,Souza-Motta, N.T. Oliveira & Houbraken
(this study). [MB820072]. —Herb.: URM 90064. Ex-type: URM 7536 = CBS
142366 = DTO 353-A2. Section Floridani. ITS barcode: KY511751 (Alternative
markers: BenA = KY709168; CaM = KY611929; RPB2 = KY611968).
Monascus floridanus P.F. Cannon & E.L. Barnard, Mycologia 79: 480. 1987.
[MB132123]. —Herb.: IMI 282587. Ex-type: FLAS F54662 = CBS
142228 = CGMCC 3.5843 = BCRC 33310 = UAMH 4180. Section Floridani.
ITS barcode: KY635848 (Alternative markers: BenA = KY709172;
CaM = KY611933; RPB2 = KY611972).
Monascus lunisporas Udagawa & H. Baba, Cryptogamie Mycol 19: 270. 1998.
[MB446999]. —Herb.: SUM 3116. Ex-type: CBS: 142230 = CGMCC
3.7951 = ATCC 204397 = NBRC 33241 = BCRC 33640. Section Floridani.
ITS barcode: KY635847 (Alternative markers: BenA = KY709171;
CaM = KY611932; RPB2 = KY611971).
Monascus mellicola R.N. Barbosa, Souza-Motta, N.T. Oliveira & Houbraken (this
study). [MB820073]. —Herb.: URM 90065. Ex-type: URM 7510 = CBS
142364 = DTO 350-E6. Section Floridani. ITS barcode: KY511726 (Alter-
native markers: BenA = KY709143; CaM = KY611904; RPB2 = KY611943).
Monascus pallens P.F. Cannon, Abdullah & B.A. Abbas, Mycol. Res. 99: 659.
1995. [MB413476]. —Herb.: IMI 356820. Ex-type: BSRA 10266 = CBS
142229 = CGMCC 3.5844 = ATCC 200612 = BCRC 33641. Section Floridani.
ITS barcode: KY635849 (Alternative markers: BenA = KY709173;
CaM = KY611934; RPB2 = KY611973).
Monascus purpureusWent, Ann. Sci. Nat., Bot. Ser. 8, 1, 1–18. 1895. [MB235390].
—Herb.: IMI 210765.Ex-type: CBS 109.07 = IF0 45 13 = ATCC 16426 = NRRL
1596 = FRR 1596. SectionRubri. ITS barcode: KY635851 (Alternativemarkers:
BenA = KY709176; CaM = KY611937; RPB2 = JN121422).
Monascus recifensis R.N. Barbosa, Souza-Motta, N.T. Oliveira & Houbraken (this
study). [MB820074]. —Herb.: URM 90066. Ex-type: URM 7524 = CBS
142365 = DTO 350-G6. Section Floridani. ITS barcode: KY511740 (Alter-
native markers: BenA = KY709157; CaM = KY611918; RPB2 = KY611957).
Monascus ruber Tiegh, Bull. Soc. Bot. France. 31: 227. 1884. [MB234876]. —
Herb.: IMI 81596. Ex-type: CBS 135.60 = IFO 8451 = ATCC 15670. Section
Rubri. ITS barcode: KY635850 (Alternative markers: BenA = KY709175;
CaM = KY611936; RPB2 = KY611974).
Overview and status of Basipetospora species
Basipetospora chlamydospora Matsush., Icones Microfungorum
a Matsushima lectorum 13. 1975. [MB309463]. —Herb.: MFC
2307. Ex-type: CBS 228.84 = MFC 2409. 18S rDNA: AB024045.
Note: BLAST analysis of the 18S rDNA sequences shows that
this species belongs to Microascales.
Basipetospora denticola (C. Moreau) C. Moreau, Bull. Soc.
Mycol. France 87: 43. 1971. nom. inval., (Art. 6.10, 41.1 & 41.5)
[MB309464]. Basionym: Chrysosporium keratinophilum var.
denticola C. Moreau [as ‘denticolum’], Mycopathologia et
Mycologia Applicata. 37: 37. 1969. nom. inval., (Art. 39.1 & 40.1)
[MB353354]. —Herb.: n/a. Representative culture: CBS 132.78.
ITS barcode: LN850801. Note: Basipetospora denticola is based
on the invalidly described species C. keratinophilum var. denti-
cola. A representative culture of B. denticola (CBS 132.78) be-
longs to Microascales and is a synonym of Scopulariopsis
candida (Jagielski et al. 2016).
Basipetospora halophila (J.F.H. Beyma) Pitt & A.D. Hocking,
Mycotaxon 22: 198. 1985. [MB105087]. Basionym: Oospora
halophila J.F.H. van Beyma Zentralblatt für Bakteriologie und
Parasitenkunde Abteilung, Abt. II 88: 134. 1933. [MB266778]. —
Herb.: n/a. Representative culture: CBS 232.32 = VKM F-204.
Note: This species was formerly described as Oospora halophila
by van Beyma (1933) and was recently transferred to Aspergillus
under the new name A. baarnensis (Samson et al. 2014,
Kocsub
eet al. 2016).
Basipetospora rubra G.T. Cole & W.B. Kendr., Canadian
Journal of Botany 46: 991. 1968. [MB326938]. —Herb.: ATCC
18199. Ex-type: FRR 2452. Note: The herbarium and ex-type
culture of B. rubra and M. ruber differ. Basipetospora rubra was
described as the asexual state of M. ruber and is in the single name
nomenclature system regarded as a synonym of this species.
Basipetospora variabilis Matsush., Icones Microfungorum a
Matsushima lectorum 13. 1975. [MB309465]. —Herb.: MFC
2428. Ex-type: CBS 995.87. 18S rDNA: AF437892. Note:
Comparison of the publically available 18S rDNA sequence on
GenBank shows that this species belongs to Microascales.
Basipetospora vesicarum (Link) Stalpers, Studies in Mycology
24: 91. 1984. [MB106627]. —Herb.: n/a. Ex-type: n/a. Note: This
fungus was originally described as Sporotrichum vesicarum by
Link (Sprengel et al. 1818). Stalpers (1984) examined a her-
barium specimen from B and this specimen contained the ana-
morph of M. ruber, which he named B. vesicarum. This species is
tentatively placed in synonymy with M. ruber.
ACKNOWLEDGEMENTS
We would like to thank National Council for Scientific and Technological Devel-
opment (CNPq) (Process 201478/2015-3 –SWE) and Coordination for the
Improvement of Higher Education Personnel (CAPES) for financial support and
scholarship for R.N. Barbosa and Associaç~
ao Pernambucana de Apicultores e
Meliponicultores (APIME) is thanked for their help in collecting the honey/pollen
samples. We would like to acknowledge Martin Meijer, Xuewei Wang and Jadson
Bezerra for their support and Konstanze Bench for nomenclatural assistance.
APPENDIX A. SUPPLEMENTARY DATA
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.simyco.2017.04.001.
REFERENCES
Arx JA von (1987). A re-evaluation of the Eurotiales. Persoonia 13: 273 –300.
Barnard EL, Cannon PF (1987). A new species of Monascus from pine tissues
in Florida. Mycologia 79: 479–484.
Benny GL, Kimbrough JW (1980). Synopsis of the orders and families of
Plectomycetes with keys to genera. Mycotaxon 12:1–91.
Berbee ML, Yoshimura A, Sugiyama J, et al. (1995). Is Penicillium mono-
phyletic? An evaluation of phylogeny in the family Trichocomaceae from
18S, 5.8S and ITS ribosomal DNA sequence data. Mycologia 87: 210–222.
Beyma FH van (1933). Beschreibung einiger neuer Pilzarten aus dem Cen-
traalbureau voor Schimmelcultures –Baarn (Holland). Zentralblatt für
Bakteriologie und Parasitenkunde Abteilung 88: 132–141.
Blanc PJ, Loret MO, Goma G (1995). Production of citrinin by various species of
Monascus.Biotechnology Letters 17: 291–294.
Cannon PF, Abdullah SK, Abbas BA (1995). Two new species of Monascus from
Iraq, with a key to known species of the genus. Mycological Research 99:
659–662.
Chang JM, Di Tommaso P, Lefort V, et al. (2015). TCS: a web server for multiple
sequence alignment evaluation and phylogenetic reconstruction. Nucleic
Acids Research 43:W3–W6.
Chen AJ, Sun BD, Houbraken J, et al. (2016). New Talaromyces species from
indoor environments in China. Studies in Mycology 84:119–144.
Cole GT, Samson RA (1979). Patterns of development in conidial fungi. Pitman,
London.
Cortopassi-Laurino M, Imperatriz-Fonseca VL, Roubik DW, et al. (2006). Global
meliponiculture: challenges and opportunities. Apidologie 37: 275–292.
Dietrich R, Usleber E, Martlbauer E, et al. (1999). Detection of the nephrotoxic
mycotoxin citrinin in foods and food colorants derived from Monascus spp.
Archiv für Lebensmittelhygiene 50:17–21.
Gams W, Christensen M, Onions AH, et al. (1985). Infrageneric taxa of
Aspergillus. In: Advances in Penicillium and Aspergillus systematics
(Samson RA, Pitt JI, eds). Plenum Press, New York: 55–62.
Hawksworth DL, Pitt JI (1983). A new taxonomy for Monascus species based on
cultural and microscopical characters. Australian Journal of Botany 31:51–61.
PHYLOGENY OF MONASCUS
www.studiesinmycology.org 49

Hocking AD, Pitt JI (1988). Two new species of xerophilic fungi and a further
record of Eurotium halophilicum.Mycologia 80:82–88.
Houbraken J, de Vries RP, Samson RA (2014). Modern taxonomy of bio-
technologically important Aspergillus and Penicillium species. Advances in
Applied Microbiology 86: 199–249.
Houbraken J, Samson RA (2011). Phylogeny of Penicillium and the segregation
of Trichocomaceae into three families. Studies in Mycology 70:1–51.
Houbraken J, Spierenburg H, Frisvad JC (2012). Rasamsonia, a new genus
comprising thermotolerant and thermophilic Talaromyces and Geosmithia
species. Antonie Van Leeuwenhoek 101: 403–421.
Hsu W-H, Pan T-M (2012). Monascus purpureus-fermented products and oral
cancer: a review. Applied Microbiology and Biotechnology 93: 1831–1842.
Huang Z-B, Li Y-P, Wang Y-H, et al. (2007). Studies on citrinin and pigments in
fermented rice of Monascus aurantiacus (As3.4384) and its mutant strains
by high performance liquid chromatography. Chinese Journal of Analytical
Chemistry 35: 474–478.
Iriart X, Fior A, Blanchet D, et al. (2010). Monascus ruber: invasive gastric
infection caused by dried and salted fish consumption. Journal of Clinical
Microbiology 48: 3800–3802.
Jaff
e R, Pope N, Carvalho AT, et al. (2015). Bees for development: Brazilian
survey reveals how to optimize stingless beekeeping. PLoS ONE 10:
e0121157.
Jagielski T, Sandoval-Denis M, Yu J, et al. (2016). Molecular taxonomy of
scopulariopsis-like fungi with description of new clinical and environmental
species. Fungal Biology 120: 586–602.
Jůzlova P, Martinkova L, Kren V (1996). Secondary metabolites of the fungus
Monascus: a review. Journal of Industrial Microbiology 16: 163–170.
Katoh K, Kuma K, Toh H, et al. (2005). MAFFT version 5: improvement in
accuracy of multiple sequence alignment. Nucleic Acids Research 33:
511–518.
Kim JY, Kim H-J, Oh J-H, et al. (2010). Characteristics of Monascus sp. isolated
from Monascus fermentation products. Food Science and Biotechnology 19:
1151–1157.
Klitgaard A, Iversen A, Andersen MR, et al. (2014). Aggressive dereplication
using UHPLC-DAD-QTOF –screening extracts for up to 3000 fungal
secondary metabolites. Analytical and Bioanalytical Chemistry 406:
1933–1943.
Kocsube
́S, Perrone G, Magista
̀D, et al. (2016). Aspergillus is monophyletic:
evidence from multiple gene phylogenies and extrolites profiles. Studies in
Mycology 85: 199–213.
Lee C-L, Pan T-M (2011). Red mold fermented products and Alzheimer's dis-
ease: a review. Applied Microbiology and Biotechnology 91: 461–469.
Lee C-L, Pan T-M (2012). BenefitofMonascus-fermented products for hyper-
tension prevention: a review. Applied Microbiology and Biotechnology 94:
1151–1161.
Leong SL, Vinnere-Pettersson O, Rice T, et al. (2011). The extreme xerophilic
mould Xeromyces bisporus –growth and competition at various water
activities. International Journal of Food Microbiology 145:57–63.
Li Y, Zhou Y-C, Yang M-H, et al. (2012). Natural occurrence of citrinin in widely
consumed traditional Chinese food red yeast rice, medicinal plants and their
related products. Food Chemistry 132: 1040–1045.
Li Y-P, Tang XC, Wu W, et al. (2015). The ctnG gene encodes carbonic
anhydrase involved in mycotoxin citrinin biosynthesis from Monascus aur-
antiacus.Food Additives & Contaminants: Part A 32: 577–583.
Li ZQ, Guo F (2004). A further studies on the species of Monascus. Myco-
systema 23:1–6.
Maddison WP, Maddison DR (2016). Mesquite: a modular system for evolu-
tionary analysis. Version 3.11. http://mesquiteproject.org.
McNeill J, Barrie FR, Buck WR, et al. (eds) (2012). International Code of
Nomenclature for algae, fungi, and plants (Melbourne Code). Regnum
Vegetabile 154. Koeltz Scientific Books, Königstein.
Menezes C, Vollet-Neto A, Marsaioli AJ, et al. (2015). A Brazilian social bee
must cultivate fungus to survive. Current Biology 25: 2851–2855.
Moreau C (1971). Presence du Monascus purpureus Went dans du mais ensile.
Remarques sur la forme imperfecte Basipetospora Cole et Kendrick. Bulletin
trimestriel de la Soci
et
e mycologique de France 87:39–44.
Negishi S, Huang Z-C, Hasumi K, et al. (1986). Productivity of monacolin K
(mevinolin) in the genus Monascus.Hakko Kogaku Kaishi 64: 584–590 (in
Japanese).
Nielsen KF, Månsson M, Rank C, et al. (2011). Dereplication of microbial natural
products by LC-DAD-TOFMS. Journal of Natural Products 74: 2338–2348.
Ogawa H, Sugiyama J (2000). Evolutionary relationships of the cleistothecial
genera with Penicillium,Geosmithia,Merimbla and Sarophorum anamorphs
as inferred from 18S rDNA sequence divergence. In: Integration of modern
taxonomic methods for Penicillium and Aspergillus classification
(Samson RA, Pitt JI, eds). Plenum Press, New York: 149–161.
Ogawa H, Yoshimura A, Sugiyama J (1997). Polyphyletic origins of species of
the anamorphic genus Geosmithia and the relationships of the cleistothecial
genera: evidence from 18S, 5S and 28S rDNA sequence analyses. Myco-
logia 89: 756–771.
Park HG, Jong SC (2003). Molecular characterization of Monascus strains
based on the D1/D2 regions of LSU rRNA genes. Mycoscience 44:25–32.
Park HG, Stamenova EK, Jong SC (2004). Phylogenetic relationships of
Monascus species inferred from the ITS and the partial beta-tubulin gene.
Botanical Bulletin of Academia Sinica 45: 325–330.
Pattangul P, Pinthong R, Phianmongkhol, et al. (2008). Mevinolin, citrinin and
pigments of adlay angkak fermented by Monsacus sp. International Journal
of Food Microbiology 126:20–23.
Peterson SW (2008). Phylogenetic analysis of Aspergillus species using DNA
sequences from four loci. Mycologia 100: 205–226.
Pisareva E, Savov V, Kujumdzieva A (2005). Pigments and citrinin biosynthesis
of fungi belonging to genus Monascus.Zeitschrift für Naturforschung 60c:
116 –120.
Posada D, Crandall KA (1998). MODELTEST: testing the model of DNA sub-
stitution. Bioinformatics 14: 817–818.
Rambaut A (2009). FigTree v. 1.3.1. Computer program and documentation
distributed by the author at. http://tree.bio.ed.ac.uk/software/.
Ronquist F, Teslenko M, van derMark P, et al. (2012). MrBayes 3.2: Efficient
Bayesian phylogenetic inference and model choice across a large model
space. Systematic Biology 61: 539–542.
Rossman AY, Allen WC, Braun U, et al. (2016). Overlooked competing asexual
and sexually typified generic names of Ascomycota with recommendations
for their use or protection. IMA Fungus 7: 289–308.
Samson RA, Houbraken J, Thrane U, et al. (2010). Food and indoor fungi. In:
CBS Laboratory manual series; No. 2. CBS-KNAW Fungal Biodiversity
Centre, Utrecht, The Netherlands.
Samson RA, Visagie CM, Houbraken J, et al. (2014). Phylogeny, identification and
nomenclature of the genus Aspergillus.Studies in Mycology 78: 141–173.
Schoch CL, Seifert KA, Huhndorf S, et al. (2012). Nuclear ribosomal internal
transcribed spacer (ITS) region as a universal DNA barcode marker for
Fungi. Proceedings of the National Academy of Sciences 109: 6241 –6246.
Shao Y, Xu L, Chen F (2011). Genetic diversity analysis of Monascus strains
using SRAP and ISSR markers. Mycoscience 52: 224–233.
Shi Y-C, Pan T-M (2012). Red mold, diabetes, and oxidative stress: a review.
Applied Microbiology and Biotechnology 94:47–55.
Shimizu T, Kinoshita H, Ishihara S, et al. (2005). Polyketide synthase gene
responsible for citrinin biosynthesis in Monascus purpureus.Applied and
Environmental Microbiology 71: 3453–3457.
Smedsgaard J (1997). Micro-scale extraction procedure for standardized
screening of fungal metabolite production in cultures. Journal of Chroma-
tography A 760: 264–270.
Sprengel K, Schrader AH, Link HF (1818). Jahrbücher der Gew€
achskunde 1.
G.C. Nauck's Buchhandlung, Berlin und Leipzig, Germany.
Stalpers JA (1984). A revision of the genus Sporotrichum.Studies in Mycology
24:1–105.
Stamatakis A (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic
analyses with thousands of taxa and mixed models. Bioinformatics 22:
2688–2690.
Stchigel AM, Cano JF, Abdullah SK (2004). New and interesting species of
Monascus from soil, with a key to the known species. Studies in Mycology
50: 299–306.
Stchigel AM, Guarro J (2007). A reassessment of cleistothecia as a taxonomic
character. Mycological Research 111: 1100–1115 .
Tamura K, Peterson D, Peterson N, et al. (2011). MEGA5: molecular evolutionary
genetics analysis using maximum likelihood, evolutionary distance, and
maximum parsimony methods. Molecular Biology and Evolution 28: 2731–2739.
Tieghem P van (1884). Monascus, genre nouveau de l'ordre des Ascomyc
etes.
Bulletin De La Soci
et
e Botanique De France 31: 226–231.
Udagawa S, Baba H (1998). Monascus lunisporas, a new species isolated from
mouldy feeds. Cryptogamie Mycologie 19: 269–276.
Vellinga EC, Kuyper TW, Ammirati J, et al. (2015). Six simple guidelines for
introducing new genera of fungi. IMA Fungus 6:65–68.
Vendruscolo F, Schmidell W, Ninow JL, et al. (2014). Antimicrobial activity of
Monascus pigments produced in submerged fermentation. Journal of Food
Processing and Preservation 38: 1860–1865.
Villanueva-Guti
errez R, Roubik DW, Colli-Uc
an W, et al. (2013). A critical view of
colony losses in managedMayan honey-makingbees (Apidae: Meliponini) inthe
heart of Zona Maya. Journal of the Kansas Entomological Society86:352–362.
BARBOSA ET AL.
50

Vinnere-Pettersson O, Leong SL, Lantz H, et al. (2011). Phylogeny and intra-
specific variation of the extreme xerophile, Xeromyces bisporus.Fungal
Biology 115: 1100–1111.
Visagie CM, Houbraken J, Frisvad JC, et al. (2014). Identification and nomen-
clature of the genus Penicillium.Studies in Mycology 78: 343–371.
Wang JJ, Lee CL, Pan TM (2003). Improvement of monacolin K, γ-aminobutyric
acid and citrinin production ratio as a function of environmental conditions of
Monascus purpureus NTU 601. Journal of Industrial Microbiology and
Biotechnology 30: 669–676.
Wang YZ, Ju XL, Zhou YG (2005). The variability of citrinin production in
Monascus type cultures. Food Microbiology 22: 145–148.
Wynns AA (2015). Convergent evolution of highly reduced fruiting bodies in
Pezizomycotina suggests key adaptations to the bee habitat. BMC Evolu-
tionary Biology 15: 145.
Yilmaz N, Visagie CM, Houbraken J, et al. (2014). Polyphasic taxonomy of the
genus Talaromyces.Studies in Mycology 78: 175–341.
PHYLOGENY OF MONASCUS
www.studiesinmycology.org 51