The PhyloCode applied to Cintractiellales, a new order of smut fungi with unresolved phylogenetic relationships in the Ustilaginomycotina

Abstract

The PhyloCode is used to classify taxa based on their relation to a most recent common ancestor as recovered from a phylogenetic analysis. We examined the first specimen of Cintractiella (Ustilaginomycotina) collected from Australia and determined its systematic relationship to other Fungi. Three ribosomal DNA loci were analysed both with and without constraint to a phylogenomic hypothesis of the Ustilaginomycotina. Cintractiella did not share a most recent common ancestor with other orders of smut fungi. We used the PhyloCode to define the Cintractiellales, a monogeneric order with four species of Cintractiella, including C. scirpodendri sp. nov. on Scirpodendron ghaeri. The Cintractiellales may have shared a most recent common ancestor with the Malasseziomycetes, but are otherwise unresolved at the rank of class.

INTRODUCTION

Higher taxonomic ranks of smut fungi and their asexual yeast-like morphs (Ustilaginomycotina, Basidiomycota) were first classified by patterns of teliospore germination (Tulasne & Tulasne 1847), later by ultrastructural characters (Bauer et al. 1997), and most recently by their evolutionary relationships based on genes (Begerow et al. 2006, Wang et al. 2015) and genomes (Kijpornyongpan et al. 2018). Confidence in the phylogenetic relationships and taxonomy of the Ustilaginomycotina has increased as genomes of more taxa are sequenced (Kijpornyongpan et al. 2018).

The smut fungi are evolutionary divergent and polyphyletic (Begerow et al. 2006, McTaggart et al. 2012). The sexual phenotype of smut fungi, when known, replaces the reproductive or other organs (leaves, roots) of their host plants with powdery masses of fungal spores. Phylogenetic studies have re-classified some traditional groups of smut fungi into higher taxonomic ranks outside of the Ustilaginomycotina, e.g. the Entorrhizomycota (Entorrhizomycota) (Aime et al. 2006, Bauer et al. 2015) and Microbotryales (Pucciniomycotina) (Aime et al. 2006). The unresolved relationships between some taxa in the Ustilaginomycotina can be explained by (i) undiscovered biodiversity (missing data in phylogenetic analyses); (ii) few speciation events, and (iii) many extinctions between extant species and their most recent common ancestors. Several monogeneric classes and orders are known in the Ustilaginomycotina, namely, Malasseziomycetes, Ceraceosorales, Golubeviales, Robbauerales, Uleiellales and Violaceomycetales (Begerow et al. 2006, Wang et al. 2014, Albu et al. 2015, Wang et al. 2015, Riess et al. 2016).

Hibbett et al. (2018) used the PhyloCode to define higher taxonomic ranks of Fungi with known phylogenetic relationships. The PhyloCode transcends descriptive taxonomy in that it delimits taxa by their most recent common ancestor in a phylogenetic analysis, rather than by characters that may be homoplasious or difficult to recognise. Phylogenetic taxon definitions are suited to monotypic yeasts of the Ustilaginomycotina, as there are few apomorphies, whether morphological or biological, and their current taxonomies are based on phylogenetic hypotheses (Wang et al. 2014, Wang et al. 2015).

Species of Cintractiella (Ustilaginomycotina) form foliar pseudo-spikelets or modify the reproductive units (see Prychid & Bruhl 2013) of some tropical sedges (Cyperaceae, subfamily Mapanioideae). Three species of Cintractiella have been described, the type, C. lamii on an unidentified species of Hypolytrum in Irian Jaya, Indonesia (Boedijn 1937); C. diplasiae on Diplasia karataefolia in South America (Piepenbring 2001); and C. kosraensis on Mapania pacifica in the Caroline Islands, Federated States of Micronesia (Aime et al. 2018).

Boedijn (1937) did not propose a higher taxonomic rank for Cintractiella. Vánky (2003) established the Cintractiellaceae in the Ustilaginales for two species of Cintractiella. Begerow et al. (2018) were uncertain of the relationship of Cintractiella to other genera of the Ustilaginomycotina and treated the group with an unknown taxonomic affiliation. Our aim was to resolve the systematic placement of Cintractiella within Fungi. We used the PhyloCode to place and classify a new species of Cintractiella in relation to other taxa in the Ustilaginomycotina.

METHODS

Plant and fungal material

Specimens of Scirpodendron ghaeri and its parasitic smut fungus were collected using standard botanical methods. Associated material was dried on silica gel for DNA sequencing and material fixed in FAA (formaldehyde 10 mL, acetic acid 5 mL, 100 % ethanol 60 mL and distilled water 25 mL) and stored in 70 % ethanol. Plant and fungal specimens were lodged in the N.C.W. Beadle Herbarium and fungal specimens were sent to the Queensland Plant Pathology Herbarium (BRIP).

Microscopy

Spores were mounted in lactic acid (100 % v/v) for examination by light microscopy. Ranges were expressed as min–max with values rounded to 0.5 μm. Images were captured with a Leica DFC 550 camera attached to a Leica DM2500 compound microscope with Nomarski differential interference contrast.

DNA extraction, PCR and sequencing

DNA was extracted from single sori (pseudo-spikelets) with a MoBio Microbial DNA extraction kit as specified by the manufacturer. Three ribosomal DNA (rDNA) loci, the internal transcribed spacer (ITS) and the large and small subunit regions (LSU and SSU), were amplified from BRIP 59246, and the ITS region was amplified from BRIP 60160. The ITS was amplified with primers ITS1/ITS4 (White et al. 1990), the LSU with LR0R/LR7 (Vilgalys & Hester 1990) and the SSU with NS1/NS4 (White et al. 1990). All reactions were amplified with high fidelity Phusion proof reading polymerase as specified by the manufacturer. The annealing temperatures in each PCR were 60 °C for ITS and SSU, and 62 °C for LSU. Amplified products were cleaned and sequenced with the primers from PCR by Macrogen (Korea).

Phylogenetic hypotheses

We added the rDNA sequences of Cintractiella to the dataset of Kijpornyongpan et al. (2018), excluding Cystobasidium minutum and Mixia osmundae (Table 1; alignments publicly available at TreeBASE, accession S24092). The rDNA loci were aligned separately with MAFFT (Katoh & Standley 2013) and concatenated in Geneious Prime. Non-homologous parts were curated from the ITS alignment using Gblocks (Castresana 2000). The most likely tree was searched in IQTree v. 1.7 beta (Nguyen et al. 2015) with a GTR gamma FreeRate heterogeneity model of evolution and a different rate for each partition (command -spp -m GTR+R), 10 000 ultrafast bootstraps (Hoang et al. 2018), an approximate likelihood ratio test with 10 000 replicates (Guindon et al. 2010) and genealogical concordance factors calculated from each locus (Minh et al. 2018).

Table 1.

Taxa and their corresponding GenBank numbers used in phylogenetic analyses based on Kijpornyongpan et al. (2018).

Taxon	GenBank numbers			References
	SSU	ITS	LSU
Anthracoidea karii	DQ875376	–	DQ875358	Begerow et al. (2006)
Calocera viscosa	DQ520102	DQ520102	DQ520102	Garnica & Weiss unpubl.
Ceraceosorus bombacis	DQ875377	KT984941	DQ875361	Begerow et al. (2006); Kijpornyongpan & Aime (2016)
Ceraceosorus guamensis	KT984925	KT984926	KT984924	Kijpornyongpan & Aime (2016)
Cintractia axicola	DQ875378	AY344967	AF009847	Stoll et al. (2003); Begerow et al. (2006)
Cintractiella scirpodendri (BRIP 59264)	MK584805	MK584806	MK584752	Present study
Cintractiella scirpodendri (BRIP 60160)	–	MK584807	–	Present study
Conidiosporomyces ayresii	DQ363303	–	AF009848	Begerow et al. (1997); Begerow et al. (2006)
Cryptococcus neoformans	AJ560316	HQ851403	AJ551296	Boekhout unpubl.; Kidd unpubl.
Doassansia hygrophilae	DQ198788	–	AF007524	Begerow et al. (1997); Oberwinkler et al. (2006)
Doassansiopsis limnocharidis	–	DQ875344	AF009850	Begerow et al. (1997); Begerow et al. (2006)
Entyloma arnoseridis	DQ645529	DQ911609	DQ645528	Matheny et al. (2006)
Entyloma calendulae	DQ663688	DQ663689	DQ663687	Matheny et al. (2006)
Erratomyces patelii	DQ663693	DQ663692	AY818966	Castlebury et al. (2005); Matheny et al. (2006)
Exobasidium pachysporum	DQ875379	AB180352	AF487392	Begerow et al. (2002); Begerow et al. (2006); Nagao et al. unpubl.
Exobasidium vaccinii	DQ198792	AB180362	AF009858	Begerow et al. (1997); Oberwinkler et al. (2006); Nagao et al. unpubl.
Fereydounia khargensis	KJ490646	KJ490645	KJ490644	Nasr et al. (2014)
Georgefischeria riveae	DQ363312	–	AF009861	Begerow et al. (1997); Begerow et al. (2006)
Golubevia pallescens	D83191	AY259059	AJ235291	Takashima & Nakase (2001); Boekhout et al. (2006)
Graphiola phoenicis	DQ363306	–	AF009862	Begerow et al. (1997); Begerow et al. (2006)
Ingoldiomyces hyalosporus	–	AF399891	AF133576	Begerow et al. (2000); Zhang et al. unpubl.
Jaminaea rosea	KR912076	KR912071	KR912073	Kijpornyongpan & Aime (2017)
Laurobasidium hachijoense	–	AB180359	AB177562	Nagao et al. unpubl.
Malassezia euphorbiae	JN367342	JN367289	JN367314	Kellner et al. (2011)
Malassezia furfur	KF706457	AY743634	AF063214	Guillot & Guého (1995); Cabañes et al. (2005); Wang et al. (2014)
Malassezia globosa	EU192364	AY743630	AY743604	Cabañes et al. (2005); Xu et al. (2007); Paulino & Blaser unpubl.
Malassezia restricta	EU192367	AY743636	AF064026	Guillot & Gueho (1995); Cabañes et al. (2005); Paulino & Blaser unpubl.
Malassezia sympodialis	KF706460	AY743632	AY387283	Cabañes et al. (2005); Wang et al. (2014)
Meira miltonrushii	JX432964	NR_120190	JQ906774	Rush & Aime (2013)
Melanopsichium pennsylvanicum	JN367341	JN367288	JN367313	Kellner et al. (2011)
Melanotaenium endogenum	DQ789980	DQ789981	DQ789979	Matheny et al. (2006)
Microstroma bacarum	AJ496257	DQ317629	AF190002	Fell et al. 2000; de Beer et al. (2006)
Moesziomyces bullatus	DQ831012	DQ831013	DQ831011	Matheny et al. (2006)
Mycosarcoma maydis	KJ081758	KF278464	KF278480	Amed & Holmstrom unpubl.; Gujjari et al. unpubl.
Phragmotaenium flavum	D82819	AB025708	AJ235285	Boekhout et al. (1995); Takashima & Nakase (2001); Tamura et al. unpubl.
Pseudomicrostroma glucosiphilum	KR912075	KR912070	KR912072	Kijpornyongpan & Aime (2017)
Pseudomicrostroma juglandis	DQ363313	DQ317634	AF009867	de Beer et al. (2006); Begerow et al. (2006)
Pseudozyma hubeiensis	XR001099829	DQ008954	AB566327	Wang et al. (2006); Konishi et al. (2013); Yarita et al. unpubl.
Quambalaria cyanescens	KF706440	DQ317623	DQ317616	de Beer et al. (2006); Wang et al. (2014)
Rhamphospora nymphaeae	DQ363311	DQ831034	AF007526	Begerow et al. (1997); Begerow et al. (2006); Matheny et al. (2006)
Robbauera albescens	KP322968	KP322986	AJ235289	Boekhout et al. (1995); Wang et al. (2015)
Sporisorium reilianum	KF706441	KF706438	KF706430	Wang et al. (2014)
Sporisorium scitamineum	JN367349	JN367296	JN367321	Kellner et al. (2011)
Testicularia cyperi	KU147241	KU147240	KU147242	Kijpornyongpan & Aime unpubl.
Thecaphora amaranthi	DQ875383	EF200013	EF200038	Begerow et al. (2006); Vánky & Lutz (2007)
Tilletia caries	U00972	AF398447	AY819007	Berbee & Taylor (1993); Castlebury et al. (2005); Zhang et al. unpubl.
Tilletiaria anomala	AY803752	DQ234558	AY745715	Matheny et al. 2006
Tranzscheliella hypodytes	JN367351	JN367298	JN367323	Kellner et al. (2011)
Tremella mesenterica	KF036705	AF042448	AF042266	Liu et al. unpubl.; Chen (1998)
Uleiella chilensis	KF061293	KF061293	KF061293	Riess et al. (2016)
Urocystis colchici	DQ839595	DQ839596	DQ838576	Matheny et al. (2006)
Ustilago hordei	JN367357	JN367303	JN367329	Kellner et al. (2011)
Ustilago tritici	DQ846895	DQ846894	DQ094784	Matheny et al. (2006)
Violaceomyces palustris	KM591583	KM591585	KM591584	Albu et al. (2015)
Volvocisporium triumfetticola	DQ317637	–	AF352053	Begerow et al. (2001); de Beer et al. (2006)
Wallemia sebi	FJ641905	AY328917	DQ847518	Matheny et al. (2006); Garnica et al. unpubl.

We used two approaches to determine the phylogenetic relationships of Cintractiella. A maximum likelihood search on the concatenated alignment i) without constraint, and ii) constrained to the class rank topology recovered by Kijpornyongpan et al. (2018). The constrained tree had monophyletic Exobasidiomycetes and Ustilaginomycetes, but relationships below class rank were non-constrained. The topologies of the constrained and non-constrained trees were compared in IQTree v. 1.7 beta, using log-likelihoods, an approximate unbiased test (Shimodaira 2002) and the Kishino-Hasegawa test (Kishino & Hasegawa 1989).

RESULTS

Specimen identification

The eight specimens of Cintractiella on Scirpodendron ghaeri examined in this study differed in host specificity and morphology to the three known species of Cintractiella. A new species is proposed for this taxon. Smut fungi belonging to Cintractiella were also found on one herbarium specimen of Hypolytrum nemorum from Thailand and on ten specimens of Diplasia karatifolia from South America.

Phylogenetic hypotheses

Cintractiella was recovered as sister to the Malasseziomycetes in both the non-constrained (Fig. 1) and constrained (Fig. 2) trees. The Exobasidiomycetes was non-monophyletic in the non-constrained tree, which was congruent with the findings of Wang et al. (2015). The Entylomatales, Georgefischeriales, Golubeviales, Microstromatales, Robbaurerales and Tilletiales were paraphyletic with the Exobasidiomycetes in respect to the Malasseziomycetes, Monilellomycetes and Ustilaginomycetes, and the Ceraceosorales was sister to the Ustilaginomycetes in the non-constrained tree. The non-constrained tree also differed to the phylogenomic hypothesis of Kijpornyongpan et al. (2018), as the Monilellomycetes was sister to the Ustilaginomycetes. The orders of Ustilaginomycotina defined by Wang et al. (2015) were monophyletic in the non-constrained tree, although their relationships to each other were not well-supported or concordant between the three loci.

Fig. 1.

Phylogram obtained from a maximum likelihood search in IQTree v. 1.7 beta, with a GTR gamma FreeRate heterogeneity model of evolution and a different rate for each partition of ribosomal DNA. aRLT values (≥ 0.9) and ultrafast bootstrap values (≥ 95 %) from 10 000 replicates, and genealogical concordance factors for three ribosomal DNA loci above nodes. Dashed lines indicate incongruence between the topology obtained from a phylogenomic dataset by Kijpornyongpan et al. (2018).

Fig. 2.

Phylogram obtained from a maximum likelihood search in IQTree v. 1.7 beta constrained to the class rank topology of Kijpornyongpan et al. (2018). aRLT values (≥0.9) and ultrafast bootstrap values (≥ 95 %) from 10 000 replicates above nodes.

The constrained tree supported the sister relationship of Cintractiella with the Malasseziomycetes. However, the constrained tree had a lower log-likelihood than the non-constrained tree (−23712.10 compared to −23691.44), was less significant with the Kishino-Hasegawa test (p-KH 0.07 compared to 0.93, significant topology ≥0.05) and was less significant with the approximate unbiased test (p-AU 0.057 compared to 0.94, significant topology ≥0.05). Based on these tests, the non-constrained tree is a better hypothesis of evolution based on rDNA loci. We expected the topologies recovered from rDNA loci to differ to the phylogenomic dataset, as there is less phylogenetic signal in rDNA markers when compared to the 910 single copy orthologs used by Kijpornyongpan et al. (2018).

Cintractiella was not recovered in any of the described orders of the Ustilaginomycotina. We propose a new order for these taxa, based on the PhyloCode.

TAXONOMY

Cintractiellales McTaggart & R.G. Shivas, ord. nov. MycoBank MB830085.

Diagnosis: The smallest clade containing Cintractiella scirpodendri but not Malassezia furfur, Ceraceosorus bombacis, Entyloma calendulae, Exobasidium vaccinii, Georgefischeria riveae, Golubevia pallescens, Robbauera albescens, Tilletia caries, Uleiella chilensis, Ustilago hordei, Violaceomyces palustris.

Diagnostic apomorphies: Sexual morph on Cyperaceae subfamily Mapanioideae, forms hypertrophied, pseudo-spikelets filled with reticulate spores.

Notes: The Cintractiellales has an unresolved class affiliation in the Ustilaginomycotina, but does not to belong to the Exobasidiomycetes s. lat. or the Ustilaginomycetes. The Cintractiellales is a monogeneric and monofamilial order of four species.

Included family: Cintractiellaceae.

Cintractiellaceae Vánky, Fungal Diversity 13: 172. 2003.

Sori in adventitious spikelets on vegetative or generative organs of the host. Spore mass black, formed within a hyaline, sporogenous fungal matrix, enclosed in the distal part of sterile spikelets. Spores single, relatively large, darkly pigmented, ornamented. Host-parasite interaction by intracellular haustoria. On Cyperaceae, subfamily Mapanioideae.

Included genus: Cintractiella.

Cintractiella Boedijn, Bull. Jard. bot. Buitenz, 3 Sér. 14: 368. 1937.

Description: See Vánky (2011).

Sori in adventitious spikelets on vegetative or generative organs of host plants in Cyperaceae, subfamily Mapanioideae, in groups, forming witches’ brooms or galls. Spore mass black, initially agglutinated, enclosed in the distal part of sterile spikelets, at maturity exposed at the opened tip of the spikelet. Spores develop embedded in hyaline, sporogenous fungal matrix, when mature solitary, relatively large and thick-walled, reddish brown, without tint of orange-red, ornamented. Host-parasite interaction by intracellular haustoria.

Included species: C. diplasiae, C. kosraensis, C. lamii and C. scirpodendri.

Cintractiella diplasiae (Henn.) M. Piepenbr., Perspect. Pl. Ecol. Evol. Syst. 4: 120. 2001. Fig. 3J.

Fig. 3.

A–E. Cintractiella scirpodendri on Scirpodendron ghaeri. A–B. Spikelet-like growths (BRIP 60160). C–D. Spores (BRIP 59264). E. Spore germination (BRIP 60160). F–I. Cintractiella lamii on Hypolytrum nemorum. F. Illustration by Boedijn (1937). G. Sori (BRIP 59678), H–I. Spores (BRIP 59678). J. Cintractiella diplasiae on Diplasia karatifolia, spores (BRIP 59671). Scale bars: G = 2 mm, all others = 10 μm.

Basionym: Ustilago diplasiae Henn., Hedwigia 43: 155. 1904.

Material examined: Brazil, Diplasia karatifolia, 24 Oct. 1977, Keel, BRIP 59673; 11 Feb. 1992, M. Nee, BRIP 66022. Guyana, D. karatifolia, 18 Jun. 1921, H.A. Gleason, BRIP 59670; Amatuk, Potaro River, 16 Aug. 1959, BRIP 59676; 18 Apr. 1993, T.W. Henkel & R. Williams, BRIP 59671. Peru, D. karatifolia, 22 Aug. 1980, Vasquez, BRIP 59677. Suriname, D. karatifolia, 10 Sep. 1963, H.S. Irwin, BRIP 59672. Venezuela, D. karatifolia, 25 Sep. 1928, G.H.H. Tate, BRIP 59669; 23 Mar. 1976, J.R.A. Lister, BRIP 59674; 10 May 1982, M. Morillo & R. Liesner, BRIP 59675.

Note: Spores of C. diplasiae differ in the ornamentation on the spore walls to other species of Cintractiella (Piepenbring 2001).

Cintractiella scirpodendri Prychid & J.J. Bruhl, sp. nov. MycoBank MB830084. Fig. 3A–E.

Etymology: Named after the host genus, Scirpodendron.

Type in sterile foliar spikelet-like growths on Scirpodendron ghaeri.

Sori (Fig. 1A–B) form in adventitious shoots that form in clusters of 1–15 on mature leaves, naked around the apex of all adventitious shoots, develop from around the shoot apex as a cylindrical column, ca. 5 × 1 mm, hidden by the narrow bracts of the adventitious shoots, semi-agglutinated to granular mass of spores visible at shoot apex. Spores (Fig. 3C–D) globose to subglobose, 14–22 μm, hazel to pale brown; wall 1–2 μm thick, minutely reticulate with up to 30 meshes per spore diam, circumference smooth to minutely dentate in profile. Spore germination (Fig. 1E) on PDA after 48 h produce three-celled phragmobasidia, fusiform or cylindrical, 13–20 × 7.5–10 μm, with lateral and terminal basidiospores. Basidiospores fusiform, 7.5–15 × 1.5–3.5 μm, hyaline, easily separate and continue to produce basidiospores.

Typus: Australia, Queensland, Cape Tribulation Road, 12 May 2013, C.J. Prychid & J.J. Bruhl, NE 99776 (BRIP 59264 holotype), SSU: MK584805, ITS: MK584806, LSU: MK584752.

On Cyperaceae (subfamily Mapanioidea): Scirpodendron ghaeri. Known only from the type locality in northern Australia.

Additional materials examined: Australia, Queensland, Cape Tribulation Road, Scirpodendron ghaeri, 7 Dec. 2012, C.J. Prychid & J.J. Bruhl, NE 99315; Croquette Point Road, C.J. Prychid & J.J. Bruhl, NE 99319; Cape Tribulation, Dubuji Boardwalk, 12 May 2013, C.J. Prychid 67 & J.J. Bruhl, NE 99777; Croquette Point Road, 14 May 2013, C.J. Prychid 71 & J.J. Bruhl, NE 99781; Cape Tribulation, Dubuji Boardwalk, 4 Jan. 2014, R.G & M.D.E Shivas & J. & L. Marsh, BRIP 60160, ITS: MK584807; Cape Tribulation Road, 18 Jun. 2014, C.J. Prychid 79, J.J. Bruhl & I.R. Telford, NE 101800; Croquette Point Road, 18 Jun. 2014, C.J. Prychid, J.J. Bruhl & I.R. Telford, NE 101804.

Notes: Cintractiella scirpodendri differs from C. kosraensis and C. lamii, which have much larger spores 29 × 36 μm diam. Sori of C. scirpodendri develop similarly to C. lamii as described and illustrated by Boedijn (1937), although C. scirpodendri lacks an enveloping fungal membrane.

Cintractiella kosraensis Aime et al., MycoKeys 42: 3. 2018.

Cintractiella lamii Boedijn, Bull. Jard. bot. Buitenz, 3 Sér. 14: 368. 1937. Fig. 3F–I.

Material examined: Thailand, Hypolytrum nemorum, 15 Sep. 1985, C. Niyondham, BRIP 59678.

Notes: The specimen examined morphologically resembled the type description of C. lamii (Boedijn 1937) and occurred on the same host, Hypolytrum. We have not designated a neotype because there is the possibility of cryptic speciation. Furthermore, the specimen that we examined was collected in Thailand and the type location is in Indonesia.

DISCUSSION

The Cintractiellales (Ustilaginomycotina) is a monofamilial and monogeneric order with four species of Cintractiella. We defined the Cintractiellales with the PhyloCode as this accounts for phylogenetic uncertainty within the Ustilaginomycotina (Hibbett et al. 2018), as well as provides flexibility for future descriptions of taxa, such as yeasts, that may belong to this order. Our study is the first to show the systematic placement of Cintractiella in the Fungi.

An advantage of the PhyloCode is that a taxon can be defined by exclusion of taxa that do not share a most recent common ancestor. The phylogenetic relationships of Cintractiella to other orders of smut fungi were unresolved in our phylogenetic analyses. However, all orders in the Ustilaginomycotina had phylogenetic support, and were excluded from the definition of Cintractiellales as they did not share a most recent common ancestor at the rank of order.

The class rank of the Cintractiellales was not defined in the present study, as the Exobasidiomycetes were paraphyletic with regard to the Ustilaginomycetes, similar to the phylogenetic hypothesis recovered by Wang et al. (2015). The boundaries of known classes recovered with rDNA loci were not congruent with the phylogenomic dataset of Kijpornyongpan et al. (2018), which may be a consequence of less data in our study. Based on the trees recovered from rDNA loci, it is possible the Cintractiellales belong to another (monogeneric) class in the Ustilaginomycotina.

We examined two of the four known species of Cintractiella, and a specimen that closely resembled the type species, C. lamii. Based on morphology, C. diplasiae may not be congeneric with Cintractiella. Cintractiella diplasiae forms sori in the inflorescence rather than in pseudo-spikelets on leaves, and its spores have a tuberculate rather than reticulate ornamentation.

Our research is the first report of a species of Cintractiella from Australia. Images and a morphological description are provided on the Smut Fungi of Australia Lucid Key (available at http://collections.daff.qld.gov.au/web/key/smutfungi/) (Shivas et al. 2014).