Rhodosporidiobolus Geoffroeae Sp. Nov., a Basidiomycetous Yeast Isolated from the Waste Deposit of the Attine Ant Acromyrmex Lundii

Home , Acromyrmex lundii, Sporidiobolales

NOTE Masiulionis and Pagnocca, Int J Syst Evol Microbiol 2017;67:1028–1032 DOI 10.1099/ijsem.0.001738

Rhodosporidiobolus geoffroeae sp. nov., a basidiomycetous yeast isolated from the waste deposit of the attine ant Acromyrmex lundii

Virginia E. Masiulionis* and Fernando C. Pagnocca

Abstract A novel basidiomycetous yeast was isolated from the waste deposit of the attine ant Acromyrmex lundii (Hymenoptera: Formicidae). The field colony was located in Santurce town, Santa Fe province, Argentina. The description of the novel species was based on strain LLU043T. Analysis of the D1/D2 domains of the LSU rRNA gene sequences in GenBank demonstrated that strain LLU043T, belongs to the Rhodosporidiobolus clade and is closely related to Rhodosporidiobolus lusitaniae and Rhodosporidiobolus colostri with 97 % similarity to the two species. The novel species differs from R. lusitaniae and R. colostri in some physiological characteristics such as the lack of assimilation of cellobiose, salicin, succinate, citrate and ethylamine. The name Rhodosporidiobolus geoffroeae sp. nov. is proposed, with LLU043T (=CBS 12828T=CBMAI 1618T) as the type strain.

Attine ants are limited to the New World [1] and they are Sporidiobolus and the mixed Rhodosporidium/Sporidiobolus known as ‘fungus growing ants’ because they cultivate [14, 15]. The genus Rhodosporidiobolus was recently pro- basidiomycetous fungi (order Agaricales) as a food source posed by Wang et al. [16] in order to apply the rule ‘One for the colony [2, 3]. This amazing symbiosis has been Fungus=One Name’ [17–19]. This new genus currently extensively studied [3] and many different groups of micro- includes both the sexual and the asexual forms of species in organisms have been frequently isolated from the colonies the lusitaniae clade [16] that were previously classified as [4–6]. Some of them are considered harmful to the symbio- Rhodosporidium/Rhodotorula and Sporidiobolus/Sporobolo- sis, such as Escovopsis sp., whereas others are beneficial, myces. The genus Rhodosporidiobolus contains nine species, such as the actinobacteria [7, 8]. However, the specific role Rhodosporidiobolus fluvialis, R. azoricus, R. microsporus, R. that yeasts can play in this system remains unclear. It is con- nylandii, R. ruineniae, R. lusitaniae, R. colostri, R. odoratus sidered that they can be useful to the ants and to their mutu- and R. poonsookiae [15], that have been isolated from the alistic fungus, providing nutrients for both [9]. Depending soil, plants, freshwater or litter [20–23]. on the ant species, different materials are collected as sub- This study reports on the phenotypic characteristics and the strate for fungal growth [10]. A particular group is known T as ‘leaf-cutting ants’ because they cut plant material (leaves, phylogenetic position of strain LLU043 , which is consid- flowers, fruits, seeds) which is carried to the nest and pre- ered to represent a novel species belonging to the genus pared for the inoculation of the mutualistic fungi, forming a Rhodosporidiobolus, isolated from the waste deposit of the structure called a ‘fungus garden’ [2]. The ‘fungus garden’ is attine ant Acromyrmex lundii (tribe Attini: Formicidae) continually renovated and the exhausted substrate is dis- nest. charged in a waste deposit that can be found inside or out- Material from the waste deposit of the A. lundii (Hymenop- side of the nest, depending on the ant species [11, 12]. tera: Formicidae) nest was collected in a field in Santurce, Acromyrmex lundii from Santa Fe province (Argentina) is a Santa Fe province, Argentina (30 10¢ 52.40† S 61 10¢ leaf-cutting ant that disposes off exhausted substrate outside 05.15¢¢ W) during September 2009 and mixed to form a sin- the nest [13]. gle composite sample. One gram of this sample was homog- Within the order Sporidiobolales, the family Sporidiobola- enized in 9.0 ml of sterile saline solution (0.85 %, w/v) and ceae is divided into three clades: Rhodosporidium, serial dilutions were prepared. Aliquots of 150 µl of each

Author affiliation: Sao~ Paulo State University (UNESP), Institute of Biosciences, Rio Claro, Brazil. *Correspondence: Virginia E. Masiulionis, [email protected] Keywords: Basidiomycota; sporidiobolales; Acromyrme; fungus growing ant; hymenoptera. Abbreviation: ITS, internal transcribed spacer. The GenBank/EMBL/DDBJ accession numbers for the D1/D2 region of the LSU rRNA gene and ITS sequences of LLU043T are KX245044 and KX245045, respectively. The MycoBank number for Rhodosporidiobolus geoffroeae sp. nov. LLU043T is MB 817691.

001738 ã 2017 IUMS Downloaded from www.microbiologyresearch.org by IP: 186.217.236.551028 On: Tue, 23 Apr 2019 17:04:31 Masiulionis and Pagnocca, Int J Syst Evol Microbiol 2017;67:1028–1032 dilution were spread (triplicate) on plates containing MYP manually with the software BioEdit Sequence Aligment Edi- medium [24] supplemented with chloramphenicol at tor v.7.0.5.3 [29]. Sequences of closely related taxa were À 150 mg l 1 and pH adjusted to 4. After incubation for 6 downloaded from GenBank (www.ncbi.nlm.nih.gov) and days at 20 C, colonies were isolated and stored in glucose/ Leucosporidium scottii (CBS 5930T/AF070419) was used as malt extract/yeast extract/NaH2PO4 (GYMP agar) tubes at an outgroup. For multiple sequence alignment, MAFFT v.7 8 C and in GYMP broth plus 15 % glycerol at À80 C. Mor- [30] was used and the analysis of data was performed phological, physiological and biochemical analyses of the using MEGA v.6 [31]. For the neighbour-joining method, dis- isolates were carried out according to Kurtzman et al. [25]. tances between the sequences were based on the Kimura DNA extraction was done as described by Sampaio et al. two-parameter model [32]. Bootstrap analysis was per- [26] and PCR amplification was done using the primer pairs formed to assess the confidence limits of the branching ITS1 (5¢-TCCGTAGGTGAACCTGCGG-3¢) and ITS4 (5¢- (1000 replicates) [33]. ¢ TCCTCCGCTTATTGATATGC-3 ) for the internal tran- Thirty-nine yeast colonies were recovered from the plates. ¢ scribed spacer (ITS) region [27] and NL1 (5 -GCATA Twenty-three were ascomycetous yeasts belonging to the ¢ ¢ TCAATAAGCGGAGGAAAAG-3 ) and NL4 (5 -GGTCCG genera Candida (eleven), Meyerozyma (eight), Trichomo- ¢ TGTTTCAAGACGG-3 ) for the D1/D2 domains of the nascus (yeast-like fungus) (four) and the 16 remaining were LSU rRNA gene [28]. The amplification products were puri- basidiomycetous yeasts belonging to Papiliotrema (six), fied using an Illustra GFX PCR DNA and Gel Band Purifi- Cutaneotrichosporon (six) and Rhodosporidiobolus sp. nov. cation Kit (GE Healthcare). The sequencing reaction was (four). The four isolates of Rhodosporidiobolus sp. nov. were performed with the same primers used during the amplifi- recovered from four different plates. Sequence analysis of cation and ABI Prism Big Dye Terminator v3.1 Cycle the D1/D2 domains of the LSU rRNA gene sequences and Sequencing Kits (Applied Biosystem) and purified using the ITS regions showed that they are identical. The length of 125 mM EDTA, 3 M sodium acetate and ethanol. The the sequences obtained from strain LLU043T was 509 bp for resulting products were placed in a 3130 Genetic Analyzer the D1/D2 domain (GenBank accession no. KX245044) and (Applied Biosystems). Sequences were assembled and edited 574 bp for the ITS region (KX245045).

Fig. 1. Phylogenetic analysis based on the D1/D2 domain of strain LLU043T (CBS 12828T=CBMAI 1618T) and related species taken from GenBank (accession numbers in parentheses). Tree building was performed with the neighbour-joining method using the MEGA v.6 software package and evolutionary distances data were calculated according to Kimura’s two-parameter correction [32]. Bootstrap values of less than 50 % are not shown. Bar, 0.01 substitutions per nucleotide position.

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Phylogenetic analysis of the D1/D2 domain showed that the Table 1. Physiological properties of strain LLU043T (CBS 12828T) that novel species belongs to the Rhodosporidiobolus clade differentiate it from closely related species of the genus in which the closest relatives are R. lusitaniae, R. colostri, R. Rhodosporidiobolus poonsookiae and R. ruineniae (Fig. 1). Comparison of the Strains: 1, LLU043T; 2, R. lusitaniae CBS 7604T; 3, R. colostri CBS 348T; T T sequences of the D1/D2 domain from strain LLU043T with 4, R. poonsookiae CBS 9095 ; 5, R. ruineniae CBS 5001 . +, Positive; À, the sequences of closest related type strains retrieved from negative; W, weakly positive; s, positive but slow; V, variable; N, no data. Data for the reference species were taken from Kurtzman et al. [40] the GenBank database showed 97 % similarity with R. lusitaniae (CBS 7604T/AF070423) and R. colostri (CBS 348T/ Characteristic 1 2 3 4 5 AY372177). In both cases the difference was 3.1 % (16 substitutions without indels) in 509 nt whereas the divergence Assimilation of: ÀÀ À of strain LLU043T with R. poonsookiae and R. ruineniae Sucrose + + ÀÀ À was 3.7 % (19 substitutions and 19 indels) and 4.7 % (24 Maltose + + substitutions and 15 indels) in 509 nt, respectively. Compar- Melezitose ÀÀ V + + ing the ITS region between strain LLU043T and R. lusitaniae Methyl a-D-glucoside ÀÀ V À V and R. colostri revealed 4.5 % divergence (26 substitutions Soluble starch W ÀÀ W À and three indels) and 5 % divergence (29 substitutions and Cellobiose À V + À + five indels) in 574 nt, respectively. Salicin À + + + + L-Sorbose + + À s + The comparative results of the physiological and biochemical L-Arabinose s ÀÀ + + characterization of strain LLU043T and the four most closely Galactitol + + À + + related species are shown in Table 1. Previous studies have Succinate À + + s + described new species of yeasts isolated from the microenvi- À ronment surrounding different attine species. They belong to Citrate + + W + À ÀÀ the genera Cutaneotrichosporon (former Cryptococcus haglero- D-Glucosamine v N À rum, [34]), Blastobotrys (former Sympodiomyces attinorum, Saccharate + v N + [35]), Haglerozyma (former Trichosporon chiarellii, [36]), L-Arabinitol WNNNN Starmerella [37] and Wickerhamomyces [38]. In addition, Ethylamine À + N + N novel melanized fungi have been isolated from the cuticle of Growth on: attine ants such as Phialophora and Ochroconis species [5, 39]. Vitamin-free medium + + À + + Based on the analysis of the D1/D2 domain, ITS region, and the morphological, biochemical and physiological proper- and propane 1, 2 diol (slow) are assimilated. Inulin, sucrose, ties, we conclude that strain LLU043T isolated from the raffinose, melibiose, lactose, maltose, melezitose, methyl waste deposit of an A. lundii nest represents a novel basidio- a-D-glucoside, cellobiose, salicin, L-rhamnose, D-arabinose, mycetous yeast species, for which the name Rhodosporidio- D-ribose, methanol, erythritol, myo-inositol, DL-lactate, suc- bolus geoffroeae sp. nov. is proposed. The origin is cinate, citrate, D-glucosamine, D-gluconate, N-acetyl-D-glu- unknown, but given that it was isolated from waste deposit outside the colony, we can infer that the species is part of cosamine, 2-keto-D-gluconate, 5-keto-D-gluconate and 2,3- the plant tissues discarded, or was vectored by insects that visit the dump.

DESCRIPTION OF RHODOSPORIDIOBOLUS GEOFFROEAE SP. NOV. Rhodosporidiobolus geoffroeae (ge.of.froe¢ae. N.L. gen. fem. geoffroeae refering to Geoffroea, the plant genus in which field workers of the leaf-cutting ant Acromyrmex lundii were foraging during sample collection). After 6 days at 20 C, cells are cylindrical to bacilliform, 2– 3Â6–9 µm, occur singly or in pairs and budding is polar (Fig. 2). The streak culture is pink, butyrous, shiny, smooth and with an entire to slightly lobate margin. After 3 weeks no conjugation is observed. Assimilation of carbon compounds is as follows: glucose, galactose, trehalose, soluble starch (weak), L-sorbose, D- xylose (weak), L-arabinose (slow), ethanol (weak), glycerol Fig. 2. Cell morphology of strain LLU043T after 6 days at 20 C on 5 % (slow), ribitol, galactitol, D-mannitol (weak), D-glucitol, D- malt extract agar. Bar, 20 µm. gluconate, saccharate, xylitol (weak), L-arabinitol (weak)

Downloaded from www.microbiologyresearch.org by IP: 186.217.236.551030 On: Tue, 23 Apr 2019 17:04:31 Masiulionis and Pagnocca, Int J Syst Evol Microbiol 2017;67:1028–1032 butanediol are not assimilated. Nitrate and nitrite (weak) possible roles of yeasts in leaf-cutting ant nests. Insects 2012;3: – are assimilated and growth on vitamin-free medium is posi- 228 245. 10. De Fine Licht HH, Boomsma JJ. Forage collection, substrate prep- tive. No growth occurs on cadaverine, creatinine, L-lysine, aration, and diet composition in fungus-growing ants. Ecol ethylamine, 50 % glucose or 10 % NaCl/5 % glucose. No fer- Entomol 2010;35:259–269. mentation is detected and no growth occurs in the presence 11. Forti LC, Alves Moreira A, Castellani MA, Caldato N. Nidificaçao~ e of 0.01 % cycloheximide. Production of starch-like com- arquitetura de ninhos de formigas-cortadeiras. In: Della Lucia pounds is negative. Reactions with diazonium blue B and TMC (editor). 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