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.

Keywords: Aspergillaceae; Extrolites; Fungal ecology; 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; Monascus section Floridani R.N. Barbosa & Houbraken; Monascus section Rubri R.N. Barbosa & Houbraken; Penicillium eremophilum (A.D. Hocking & Pitt) Houbraken, Leong & Vinnere-Pettersson; Phylogeny; Taxonomy.

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.

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.

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.

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.

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.

Fig. 6

Monascus flavipigmentosus, URM 7536. 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.

Fig. 7

Monascus mellicola, URM 7510. 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–E. Conidiophores with conidia chain. D. Conidia. Scale bars = 10 μm.

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.