Mycological Progress (2021) 20:191–201 https://doi.org/10.1007/s11557-020-01664-7 ORIGINAL ARTICLE Broad and narrow host ranges in resolved species of Cintractia limitata s. lat. (Anthracoideaceae, Ustilaginomycotina) on Cyperus J. Kruse 1,2 & A. R. McTaggart 3 & K. Dhileepan 4 & P. M. Musili 5 & F. M. Mutie 5 & J. E. Ntandu 6 & P. R. O. Edogbanya 7 & E. C. Chukwuma 8 & R. G. Shivas 1,3,4 Received: 30 June 2020 / Revised: 18 December 2020 / Accepted: 21 December 2020 # German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature 2021 Abstract Cintractia (Anthracoideaceae, Ustilaginomycotina) is a widespread genus of smut fungi that parasitizes species of Cyperaceae. Specimens of Cintractia spp. on species of Cyperus s. lat. were examined to resolve their taxonomy, including a species on Cyperus aromaticus, which is an invasive weed in Australia. A phylogenetic species concept was used to show that Cintractia limitata s. lat. contains three different species, Cintractia limitata, C. tangensis, and a new species C. kyllingae. The proposed taxonomy is based on phylogenetic analyses of the ITS rDNA region and on the position of the sori in diverse parts of inflorescences examined in approximately 100 specimens of Cintractia. A morphological synapomorphy in C. tangensis is sori with firmly agglutinated spore masses around the base of the peduncles, rarely in the spikelets. Cintractia limitata forms powdery spore masses between flowers and is known only to occur on Cyperus species with spikelets in capitate heads. Cintractia kyllingae was found on Cy. aromaticus and may have potential as a biological control agent in Australia. Keywords Cintractia kyllingae . Host specificity . Mariscus . New species . Taxonomy . Ustilaginales Introduction Species of Cintractia (Anthracoideaceae, Ustilaginales, Ustilaginomycotina) are known to cause smut disease in the inflorescences of 13 sedge (Cyperaceae) species worldwide (Vánky 2012). The type species, Cintractia axicola, was described from Fimbristylis dichotoma collected in the Dominican Republic (Berkeley 1852). Cintractia was often a catch-all for smut fungi on Cyperaceae. Furthermore, some species of Cintractia have variable morphology and overlapping host ranges that complicate their identification. The taxonomic boundaries of Cintractia have been refined by the description of related genera and reclassification of species into Anthracoidea (Piątek 2012, 2013; Vánky 2012), Bauerago (Vánky 2012), Leucocintractia (Piepenbring et al. 1999), Pilocintractia (Vánky 2004), Stegocintractia (Piepenbring et al. 1999) and Trichocintractia (Piepenbring 1995). Cyperus (Cyperaceae) is a pantropical genus of about 950 species (Muasya et al. 2002; Reid et al. 2014, 2017), some of which are economically important as food (Cy. esculentus) or weeds. Cyperus rotundus was considered one of the worst Section Editor: Dominik Begerow * R. G. Shivas roger.shivas@usq.edu.au 1 2 3 Centre for Crop Health, Institute for Agriculture and the Environment, University of Southern Queensland, Toowoomba, Queensland 4350, Australia Natural History Museum of the Palatinate – Pollichia Museum, Hermann-Schäfer-Street 17, 67098 Bad Duerkheim, Germany Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Ecosciences Precinct, Dutton Park, Queensland 4102, Australia 4 Department of Agriculture and Fisheries, Biosecurity Queensland, Ecosciences Precinct, Dutton Park, Queensland 4102, Australia 5 East African Herbarium, National Museums of Kenya, P.O. Box 40658, Nairobi 00100, Kenya 6 National Herbarium of Tanzania, P.O. Box 3024, Arusha, Tanzania 7 Department of Plant Science and Biotechnology, Kogi State University, Anyigba, Nigeria 8 Forest Herbarium Ibadan (FHI), Forestry Research Institute of Nigeria, P.M.B. 5054, Jericho Hills, Ibadan, Nigeria 192 agricultural weeds in the world (Holm et al. 1977), and Cy. aromaticus (Navua sedge) is an introduced, aggressive perennial weed that forms dense stands and replaces palatable tropical pasture species in northern Queensland, Australia. The current taxonomic concept of Cyperus amalgamated smaller genera, including Kyllinga and Mariscus, into a large monophyletic group (Larridon et al. 2011, 2014; Bauters et al. 2014). Two species, Cintractia limitata and C. lipocarpha, and three other smut fungi, Dermatosorus cyperi, Ustanciosporium cyperi, and U. kuwanoanum, have been recorded from Cyperus (Vánky 2012). Cintractia limitata is cosmopolitan to the tropics and subtropics (Vánky et al. 2011; Denchev and Denchev 2012; Vánky 2012) and has been reported on 41 species of Cyperus from Australia (Vánky and Shivas 2008; Shivas et al. 2014), Africa (Piepenbring 2000; Vánky and Vánky 2002), North and South America (Ling 1950; Piepenbring 2000), and Asia (Tai 1979; Guo 2000; Vánky 2007). Cintractia limitata was first described by Clinton (1904) based on a collection on Cyperus ligularis in Puerto Rico. Cintractia lipocarphae has been reported on three different host genera (Bulbostylis barbata, Cy. squarrosus, and Lipocarpha microcephala (type host)) and is restricted to Australia (Vánky and Shivas 2008; Vánky 2012). The aim of the study was to resolve the taxonomy of Cintractia species on Cy. aromaticus and closely related sedges, in order to identify a possible biological control agent for release in Australia. Cyperus aromaticus is an aggressive weed in northern tropical Queensland, affecting the beef, dairy, and sugarcane industries (Vitelli et al. 2010). It is currently managed by mechanical and chemical methods that are expensive and impractical for pastures. Barreto and Evans (1995) considered that C. limitata may have a role as part of an integrated biological control of Cy. rotundus by reducing the number of viable seeds that facilitate long-term recolonization. An understanding of the taxonomy and potential host range of Cintractia spp. on sedges is a step towards ecological control of Cyperus spp. that are environmental weeds. Materials and methods Taxonomic sampling Specimens of Cintractia spp. on Cyperus spp. were collected in Kenya (May 2018), Nigeria (June 2019) and Tanzania (July 2018) (Table 1). Specimens from other herbaria (BRIP, HUV, and M) were also examined (Tables 1, 2). The names of the host plants and fungi were applied according to the latest version of The International Plant Names Index (www.ipni.org) and Vánky (2012), respectively. Specimens were deposited either in the East African Herbarium (EA), Nairobi, Kenya, or in the Forest Herbarium Ibadan (FHI), Mycol Progress (2021) 20:191–201 Ibadan, Nigeria, with duplicates in the Queensland Plant Pathology Herbarium (BRIP), Dutton Park, Australia. DNA extraction and PCR DNA was extracted from specimens either by the methods of Kruse et al. (2018) or using the DNeasy UltraClean Microbial Kit (Qiagen, Hilden) (Table 1). About 2–20 mg of infected plant tissue was taken from dried fungarium samples, placed in 2-mL plastic reaction tubes and homogenized in a TissueLyser (Qiagen, Hilden) using various sizes (0.5–6.35mm diam.) of glass beads at 25 Hz for 90 s. The complete nrITS of all DNA extracts was amplified using PCR with the primer pair M-ITS1 (Stoll et al. 2003) and smITS-R2 (Kruse et al. 2017). Genomic DNA was amplified and sequenced by Macrogen (Korea) or at the Senckenberg Biodiversity and Climate Research Centre (BiK-F) (Germany) using the MITS-1/ITS4 primer set. Forward and reverse sequences were assembled using Geneious Prime 2019 (https://www.geneious.com). Phylogenetic hypotheses The ITS sequences of 41 specimens, including 34 that belong to C. limitata s. lat., were aligned in MAFFT v.7 (Katoh and Standley 2013) using the L-INS-i algorithm. The leading and trailing gaps of the alignment were removed manually. DNA sequence data from four specimens of Tolyposporium, which is a sister genus to Cintractia (Wang et al. 2015), were used as an outgroup. Three methods were used for the phylogenetic reconstruction. A minimum evolution (ME) method using MEGA 7.0.26 (Kumar et al. 2016) with the Tamura-Nei substitution model and assuming complete deletion at 80% cut-off with 1000 bootstrap replicates and all other parameters set to default. A maximum likelihood (ML) method using RAxML (Stamatakis 2014) with parameters set to default values. A Bayesian method using MrBayes 3.2 (Ronquist and Huelsenbeck 2003) with a GTR model of evolution using four incrementally heated chains for 10 million generations, sampling every 1000th tree, discarding the first 30% of the obtained trees, and all other parameters set to default on the TrEase webserver (http://www.thines-lab.senckenberg.de/trease). Morphological examination Teliospores were removed from selected herbarium specimens, mounted on glass slides in 100% lactic acid and gently heated before microscopic examination. Measurements were calculated from at least 30 teliospores. Images were captured with a Leica DFC 500 camera attached to a Leica DMLB compound microscope with Normarski differential interference contrast. The size, shape, and position of sori of Specimens of Cintractia examined in the phylogenetic analysis Accession no.a Host Country Collection date Collectors GenBank no. (ITS) References Cintractia amazonica UNA (MP 2008) Rhynchospora barbata Nicaragua 21 Nov 1995 M. Piepenbring, M. Garth & M. Müller DQ875342 Piepenbring (2000); Begerow et al. (2006) C. axicola MP 3490 M. Piepenbring DQ631908 Matheny et al. (2006); Wang et al. (2015) AY344967 Stoll et al. (2003) C. fimbristylidis-miliaceae HUV 17460 C. kyllingae C. limitata C. tangensis BRIP 67667T BRIP 67669 – Fimbristylis tetragona Cy. aromaticus Cy. aromaticus Panama – India 28 Oct 1995 N.D. Sharma, R. Berndt, C. & K. Vánky Tanzania Tanzania 4 Jun 2018 4 Jun 2018 P.M. Musili, J. Elia, K. Dhileepan, M.D.E. & R.G. Shivas MN121112 This study P.M. Musili, J.E. Ntandu, K. Dhileepan, M.D.E. & R.G. MN121113 This study Shivas C. & K. Vánky MN121114* This study M-0215533 (Vánky, Cy. aromaticus Ust. Exs. No. 952) BRIP 71082 Cy. aromaticus Reunion Is. 30 Nov 1994 Nigeria 13 Jun 2019 BRIP 71086 Nigeria 14 Jun 2019 M-0215531 (Vánky, Cy. cyperoides subsp. Ust. Exs. No. Pseudoflavus 1071) BRIP 71068 Cy. cyperoides Zimbabwe 28 Feb 1999 Nigeria 10 Jun 2019 HUV 21459 Cy. cyperoides M-0215535 (Vánky, Cy. ligularis Ust. Exs. No. 904) BRIP 67647 Cyperus alternifolius M-0235317 Cy. articulatus M-0215534 (Vánky, Cy. compressus Ust. exs. no. 811) BRIP 57998 Cy. compressus Cameroon Venezuela 9 Mar 2007 13 Dec 1993 P.R.O. Edogbanya, E.C. Chukwuma, K. Dhileepan, M.D.E MT478170* This study & R.G. Shivas J. & M. Piatek, A.L. Njouonkou, C. & K. Vánky MN121110 This study C. & K. Vánky MN121111* This study Tanzania 2 Jun 2018 P.M. Musili, J. Elia, K. Dhileepan, M.D.E. & R.G. Shivas MN121093 Nicaragua Indonesia 14 Nov 1995 5 Apr 1992 M. Piepenbring & P. Moreno C. Menge & K. Vánky MN121108* This study MN121102* This study Australia 13 Mar 2012 R.S. James MN121085 BRIP 47773 M-0215520 (Vánky, Ust. exs. no. 1322) BRIP 49163 BRIP 49161 BRIP 54190 Cy. corymbosus Cy. corymbosus Thailand Thailand 19 Dec 2005 19 Dec 2005 MN121086 This study MN121103* This study Cy. digitatus Cy. distans Cy. distans Australia Australia Australia 2 May 2007 2 May 2007 17 Mar 2011 BRIP 48844 BRIP 67629 Cy. distans Cy. distans Australia Kenya 14 Mar 2007 28 May 2018 R.G. & M.D.E. Shivas R.G., M.D.E., A.J. & G.F. Shivas, P. Athipunyakom, S. Likhitekaraj, W. Butranu, C. & K. Vánky A.R. McTaggart & R.G. Shivas A.R. McTaggart & R.G. Shivas D.R. Beasley, C.M. Horlock, M. Krysinska-Kaczmarek, R.J. Southwell & R.G. Shivas not known P.M. Musili, F.M. Mutie, K. Dhileepan, M.D.E. & R.G. Shivas Cy. aromaticus Mycol Progress (2021) 20:191–201 Table 1 P.R.O. Edogbanya, E.C. Chukwuma, K. Dhileepan, M.D.E MT478172* This study Shivas & R.G. Shivas P.R.O. Edogbanya, E.C. Chukwuma, K. Dhileepan, M.D.E MT478171* This study & R.G. Shivas C. & K. Vánky MN121109* This study This study This study MN121106 MN121094 MN121095 This study This study This study MN121107 MN121091 This study This study 193 194 Table 1 (continued) Accession no.a Host Country Collection date Collectors GenBank no. (ITS) References BRIP 67614 Cy. esculentus Kenya 25 May 2018 MN121087 This study BRIP 67643 Cy. esculentus Tanzania 2 Jun 2018 MN121088 This study BRIP 47774 Cy. mitis Thailand 13 Dec 2005 P.M. Musili, F.M. Mutie, K. Dhileepan, M.D.E. & R.G. Shivas P.M. Musili, J.E. Ntandu, K. Dhileepan, M.D.E. & R.G. Shivas R.G. & M.D.E. Shivas MN121096 This study BRIP 57460 Cy. polystachyos Philippines 28 Jun 2012 MN121105 This study BRIP 67592 Cy. rotundus Kenya 24 May 2018 MN121092 This study BRIP 67619 Cy. rotundus Kenya 26 May 2018 MN121089 This study BRIP 67622 Cy. rotundus Kenya 27 May 2018 MN121098 This study BRIP 67624 Cy. rotundus Kenya 27 May 2018 MN121090 This study BRIP 67626 Cy. rotundus Kenya 27 May 2018 MN121099 This study BRIP 65852 Cyperus sp. Australia 27 Apr 2017 BRIP 65869 Cyperus sp. Australia 27 Apr 2017 BRIP 70061 Cyperus sp. Thailand 5 Feb 2018 BRIP 60423 Cyperus sp. Australia 12 Apr 2014 HAJB 10488 Cyperus sp. Cuba K.L. Lancetta, V.A. Felices, T.U. Dalisay, A.I. Llano, A.R. McTaggart, M.D.E. & R.G. Shivas P.M. Musili, F.M. Mutie, K. Dhileepan, M.D.E. & R.G. Shivas P.M. Musili, F.M. Mutie, K. Dhileepan, M.D.E. & R.G. Shivas P.M. Musili, F.M. Mutie, K. Dhileepan, M.D.E. & R.G. Shivas P.M. Musili, F.M. Mutie, K. Dhileepan, M.D.E. & R.G. Shivas P.M. Musili, F.M. Mutie, K. Dhileepan, M.D.E. & R.G. Shivas B. Lemana, K. Vánky, M.J. Ryley, S.M. Thompson, M.D.E. & R.G. Shivas B. Lemana, K. Vánky, M.J. Ryley, S.M. Thompson, M.D.E. & R.G. Shivas J. Kruse, M. Sudanguan, C. Doungsa-ard, M.D.E. & R.G. Shivas, M.J. Ryley, P. Athipunyakom, & S. Likhitekaraj J. Brands, L.S. Shuey, A.R. McTaggart, M.D.E. & R.G. Shivas – Tolyposporium isolepidis HUV 14720 T Isolepis nodosa New Zealand 4 Jan 1986 R.E. Beever T. junci HUV 17169 Juncus bufonius Poland Muntentis AY344994 T. neillii HUV 18533 I. nodosa New Zealand 14 Feb 1998 E.H.C. McKenzie, C. & K. Vánky EU246951 T. piluliforme HUV 15732 J. planifolius New Zealand 13 Jan 1991 E.H.C. McKenzie DQ875345 – 4 Sep 1891 MN121100* This study MN121101* This study MN121104 This study MN121097 This study DQ645508 Matheny et al. (2006); Wang et al. (2015) EU246950 Vánky and Lutz (2008) Stoll et al. (2003) a BRIP Queensland Plant Pathology Herbarium, Brisbane, Australia; HAJB Jardín Botánico Nacional Universidad de La Habana, Cuba; HAJB Jardín Botánico Nacional, Universidad de La Habana, Cuba; HUV Herbarium Ustilaginales Vánky (held in BRIP); M Botanische Staatssammlung München; USJ Universidad Nacional Agraria, Nicaragua; MP Herbarium Meike Piepenbring * DNA extracted using the methods of Kruse et al. (2018). DNA from other specimens extracted using the DNeasy Ultra Clean Microbial Kit (Qiagen, Hilden) T Type specimen Mycol Progress (2021) 20:191–201 Vánky and Lutz (2008) Begerow et al. (2006) Additional specimens of Cintractia on Cyperus examined morphologically Cintractia limitata C. tangensis Host Country Coll. Date Collectors HUV 19687 HUV 18944 HUV 873 HUV 17203 HUV 17201 HUV 17202 HUV 14124 HUV 16005 HUV 18434 Cy. cyperoides Cy. cyperoides Cy. ligularis Cy. ligularis Cy. ligularis Cy. ligularis Cy. ligularis Cy. ligularis Cyperus articulatus Malawi Zimbabwe Dominican Republic Belize Belize Belize Mexico Venezuela India 20 Apr 2001 28 Feb 1999 26 Mar 1930 28 Mar 1995 18 Mar 1995 27 Mar 1995 2 Dec 1971 11 Dec 1993 2 Jan 1997 T., C. & K. Vánky C. & K. Vánky E.L. Ekman M. Piepenbring M. Piepenbring A. Isenberg & M. Piepenbring R. Durán R. Berndt, C. & K. Vánky S. Jsutin HUV 18501 BRIP 25455 HUV 18955 HUV 19270 BRIP 26966 HUV 19484 BRIP 26856 HUV 9491 HUV 8884 HUV 15531 HUV 6650 HUV 21225 HUV 21224 HUV 8890 BRIP 54185 BRIP 54197 HUV 15461T1 Cy. articulatus Cy. compressus Cy. compressus Cy. compressus Cy. compressus Cy. compressus Cy. compressus Cy. compressus Cy. compressus Cy. compressus Cy. compressus Cy. compressus Cy. compressus Cy. digitatus Cy. distans Cy. distans India Australia Australia Australia Australia East Timor East Timor India India Indonesia Sri Lanka Thailand Thailand India Australia Australia 14 Nov 1995 10 Mar 1998 10 Mar 1998 12 Mar 2000 12 Mar 2000 29 Jan 2000 29 Jan 2000 27 Jan 1980 27 Jan 1980 5 Apr 1992 29 Jun 1913 13 Dec 2005 19 Dec 2005 28 Jan 1980 18 Mar 2011 17 Mar 2011 N.D. Sharma, R. Berndt, C. & K. Vánky R.G. Shivas R.G. Shivas R.G. Shivas, I.T. Riley, C. & K. Vánky R.G. Shivas, I T. Riley, C. & K. Vánky A.A. Mitchell A.A. Mitchell K. Vánky K. Vánky C. Menge, C. & K. Vánky – R.G., M.D.E., A.J. & G.F. Shivas, P. Athipunyakom, S. Likhitekaraj, W. Butranu, C. & K. Vánky R.G., M.D.E., A.J. & G.F. Shivas, P. Athipunyakom, S. Likhitekaraj, W. Butranu, C. & K. Vánky K. Vánky R.G. & M.D.E. Shivas, D.R. Beasley, R.J. Southwell, M. Krysinska-Kaczmarek & C.M. Horlock D.R. Beasley, M. Krysinska-Kaczmarek, R.J. Southwell, C.M. Horlock & R.G. Shivas HUV 16381T2 Cy. distans Cy. esculentus India Congo Aug 1940 19 May 1906 P. Maheshwari H.J. Vanderyst HUV 17403T3 HUV 18801 BRIP 7999 BRIP 8000 BRIP 8001 BRIP 8002 BRIP 23350 BRIP 24506 Cy. polystachyos Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Philippines Australia Australia Australia Australia Australia Australia Australia Aug 1906 25 Mar 1996 10 Apr 1964 21 Feb 1962 Mar 1958 Mar 1947 16 Apr 1996 14 Jan 1997 E.D. Merrill A.A. Mitchell I.C. Tommerup R.F.N.Langdon R.F. N.Langdon – K.R.E. Grice R.I. Davis 195 Accession no.a Mycol Progress (2021) 20:191–201 Table 2 196 Table 2 (continued) a Accession no.a Host Country Coll. Date Collectors BRIP 27398 HUV 3659 HUV 15918 HUV 15826 BRIP 26857 HUV 19485 HUV 18474 HUV 18475 HUV 18499 HUV 15704 BRIP 66839 HUV 25510 BRIP 25582 Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Australia Costa Rica Costa Rica Costa Rica East Timor East Timor India India India Indonesia Laos Mexico Papua New Guinea 25 Mar 1996 26 Mar 1991 27 Jul 1991 5 Nov 1992 30 Jan 2000 30 Jan 2000 5 Sep 1992 3 Oct 1992 24 Oct 1995 5 Apr 1992 23 Oct 2015 25 Nov 2003 2 Apr 1998 A.A. Mitchell T. & K. Vánky C. Gerlinde & P. Dobbeler V. Mora & M. Piepenbring A.A. Mitchell A.A. Mitchell T. & K. Vánky C. & K. Vánky C. & K. Vánky C. Menge & K. Vánky R.G. Shivas & P. Sibounnavong T. & K. Vánky R.G. Shivas, G.R. Kula, V. Gavali, C. & K. Vánky HUV 18525 HUV 9492 HUV 8880 BRIP 53230 BRIP 19697 HUV 11640 HUV 11615 BRIP 7567 HUV 20975 HUV 17131 HUV 20078 Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Cy. rotundus Papua New Guinea Sri Lanka Sri Lanka Thailand Thailand Thailand Thailand Australia Venezuela Venezuela Zambia 2 Apr 1998 12 Feb 1980 12 Feb 1980 2 Aug 2006 Nov 1990 10 Nov 1983 29 Oct 1985 16 May 1973 18 Sep 1991 25 Nov 1993 30 Apr 2001 R.G. Shivas, G.R. Kula, V. Gavali, C. & K. Vánky H. Nybom & K. Vánky H. Nybom & K. Vánky S. Likhitekaraj & S. Trinop U. & N. Farungsang M. Kakishima K. Amnuaykit – C.E. Garcia R. Berndt, C. & K. Vánky C. & K. Vánky BRIP Queensland Plant Pathology Herbarium, Brisbane, Australia; HUV Herbarium Ustilaginales Vánky (held in BRIP) T1 Isotype of Cintractia distans; T2 Isoyype of C. congensis; T3 Isotype of C. cyperi-polystachyi Mycol Progress (2021) 20:191–201 Mycol Progress (2021) 20:191–201 197 Cintractia spp. on Cyperus spp. were determined by examination under a stereomicroscope. aromaticus, with a wide distribution extending across equatorial Africa from Nigeria to Tanzania and Reunion Is. in the Indian Ocean (Table 1). Results Cintractia limitata G.P. Clinton, Proc. Boston Soc. Nat. Hist. 31(9): 399. 1904. Fig. 2c–d. Holotype: PUERTO RICO, Mayaguez, on Cyperus ligularis, 23 Apr. 1904, G.P. Clinton (BPI 171857). = Cintractia togoensis Henn., Bot. Jb. 38: 119 (1905). Type: TOGO, Lome, on Cyperus sp., Apr. 1900, Warnecke 118 (BPI 172250). (syn. by Ling 1950). = Ustilago mariscana Zundel, Mycologia 35: 165 (1943). Type: SOUTH AFRICA, KwaZulu-Natal, Melmoth, Mfulazane River, on Cyperus cyperoides (cited as Mariscus sieberianus), 1 Dec. 1919, A.O.D. Mogg 6096 (PREM 33062). (syn. by Ling 1950). Hosts: Cyperus cyperoides (L.) Kuntze, Cy. ligularis L. Specimens examined: See Tables 1 and 2. Notes: The sori of C. limitata replace some flowers of individual spikelets by agglutinated, powdery masses of teliospores (Fig. 2c). Clinton (1904) described the teliospores from the type specimen on Cy. ligularis as ovoid to globose or slightly polyhedral, 9.5–14 μm. A specimen (BRIP 71068) of C. limitata on Cy. cyperoides from Nigeria had teliospores that were flattened, in plain view circular or subcircular, 9– 12 μm, in side view elliptical, 6–8 μm wide, yellowish brown; wall even, 0.7–1 μm thick, smooth (Fig. 2d). The specimen (M-0215535) (Table 1) from Venezuela, South America, on Cy. ligularis (host of the type of C. limitata) differed by one base pair in the ITS sequence from corresponding sequences obtained from three African specimens (Table 1). Cintractia limitata is apparently restricted to species of Cyperus that were formerly classified in Mariscus, a polyphyletic genus that grouped Cyperaceae with deciduous spikelets (Larridon et al. 2013). Based on morphology, C. limitata has a wide host range on Cyperus, with eight host species (Vánky 2012). According to data presented here, C. limitata appears restricted to Cy. cyperoides and Cy. ligularis. One other species, C. minor (syn. C. axicola (Berk.) Cornu var. minor in Clinton 1902, p. 143), forms sori at the base of peduncles of Cy. grayi, which has a North American distribution restricted to the eastern USA. The spores of C. minor measure 10–13 μm (Clinton 1904) and are indistinguishable from other species in C. limitata s. lat. (Ling 1950). We have excluded C. minor as a synonym of C. limitata based on soral morphology and host range. Sequence analysis is required to determine whether C. minor is a distinct species. The specimens of Cintractia spp. on Cyperus spp. formed three well-supported monophyletic clades. The largest clade (ME: 100, ML: 97 and BA: 1) contained the type specimen of Cintractia tangensis. This clade was composed mostly of sequence data obtained from African specimens and was sister to a clade containing two smaller subclades. One subclade contained a specimen on Cy. ligularis the type host of Cintractia limitata, and the other subclade contained five specimens on Cy. aromaticus from equatorial Africa (Nigeria, Reunion Is. and Tanzania). Taxonomy The following nomenclatural and taxonomic changes based on phylogenetic analyses combined with morphological examination and biology (host range) are proposed for Cintractia spp. in the spikelets and inflorescences of Cyperus. We treated taxa as phylogenetic species if they shared morphological apomorphies and a most recent common ancestor in analyses of the ITS region. Synonymies are provided for the species discussed. Cintractia kyllingae J. Kruse & R.G. Shivas, sp. nov. Fig. 2a–b. MycoBank no.: MB835937. Etymology: Named after Kyllinga, a tropical genus of sedges that included K. aromatica (now included in Cyperus), which is the only known host species of this fungus. Holotype: TANZANIA. Hale, S 05°18′ 05″, E 38° 35′ 48″, on Cyperus aromaticus, 4 Jun. 2018, P.M. Musili, J. Elia, K. Dhileepan, M.D.E. & R.G. Shivas (BRIP 67667). Sori replace the flowers in some or all spikelets, spherical, ovoid or fusiform, 0.5–1 mm long, at first covered by a whitish brown fungal peridium, early rupturing, exposing the blackish brown, agglutinated, superficially powdery mass of teliospores. Teliospores flattened, in plain view circular or subcircular, 10–14 μm, in side view elliptical, 7–10 μm wide, yellowish brown; wall even, 0.7–1 μm thick, smooth. Known host species: Cyperus aromaticus (Ridl.) Mattf. & Kük. Additional specimens examined: See Tables 1 and 2. Notes: The spore mass of C. kyllingae is agglutinated, superficially powdery, and replaces individual flowers (often all the flowers of a spikelet or of an inflorescence). Cintractia kyllingae has to date only been found on Cy. Cintractia tangensis Henn., Bot. Jb. 38: 103. 1905. Fig. 2e–f. Holotype: TANZANIA, Usambara, Tanga, on Cyperus sp. (= Cyperus rotundus, det. P.M. Musili 2018), 3 July 1903, F. Eichelbaum 89 (HBG-024610!). 198 Mycol Progress (2021) 20:191–201 = Cintractia congensis Henn., Ann. Mus. Congo Belge, Bot. Sér. 5 2: 87 (1907). CONGO, at km 340 on the Matadi–Léopoldville [Kinshasa] Railway, on Cyperaceae (= Cyperus esculentus, det. Ling 1950: 651), 19 May 1906, H. Vanderyst 105 (BR 5020048751578). (syn. C. limitata by Ling 1950). = Cintractia cyperi-polystachyi Henn., Philipp. J. Sci., Ser. C., Bot. 3: 41 (1908). Type: PHILIPPINES, Luzon, Manila, on Cyperus polystachyos, Aug. 1906, E.D. Merrill 5195 (BPI 171504, K 120, HUV 17403!). (syn. C. limitata by Ling 1950). = Ustilago chacoensis Hirschh., Not. Mus. La Plata 6: 479 (1941). Type: ARGENTINA, Chaco, Resistencia, on Cyperus rotundus, May 1940, C. Carrera (LPS 4923). (syn. C. limitata by Ling 1950). = Cintractia distans Mundkur [as 'disvans'], Indian J. Agric. Sci. 14: 50 (1944). Type: BANGLADESH, Dhaka [Bengal, Dacca], on Cyperus [as ‘Carex’] distans, Aug. 1940, P. Maheshwari (HCIO 7727). (syn. C. limitata by Ling 1950). = Cintractia limitata var. congoensis Zambett., Bull. Trimest. Soc. Mycol. Fr. 95: 410 (1980 ['1979']). (nom. Inval., Art 39.1). Type: CONGO, Kilamita, on Cyperus esculentus, May 1906, H. Vanderyst 105 (BR). (syn. C. limitata by Vánky 2012). 0.02 Known host species: Cyperus alternifolius L., Cy. articulatus L., Cy. compressus L., Cy. corymbosus Rottb., Cy. digitatus Roxb., Cy. distans L.f., Cy. esculentus L., Cy. mitis Steud., Cy. polystachyos Rottb., Cy. rotundus L. Specimens examined: See Tables 1 and 2. Notes: Cintractia tangensis produces oval or subglobose sori with firmly agglutinated black spore masses mostly around the base of the peduncles of partial inflorescences. (Fig. 2e). Hennings (1905) described the teliospores of C. tangensis on Cyperus rotundus [Cyperus sp.] as subglobose (8–11 μm) or ellipsoidal (7–8 × 8–12 μm). A specimen of C. tangensis on Cy. alternifolius from Nigeria (BRIP 67647) had teliospores that were flattened, in plain view circular or subcircular, 9–11 μm, in side view elliptical, 5–7-μm wide, yellowish brown; wall even, 0.7–1-μm thick, smooth to minutely roughened (Fig. 2f). Cintractia tangensis differs from Cintractia limitata, which produces sori in and around some flowers in the spikelets but never around the peduncle. Discussion Cintractia kyllingae, C. limitata, and C. tangensis are differentiated molecularly (Fig. 1), morphologically (Fig. 2), and MN121107 BRIP48844 C. tangensis ex Cy. distans MN121108 M0235317 C. tangensis ex Cy. articulatus MN121105 BRIP57460 C. tangensis ex Cy. polystachyos MN121104 BRIP70061 C. tangensis ex Cy. sp. MN121103 M0215520 C. tangensis ex Cy. corymbosus MN121102 M0215534 C. tangensis ex Cy. compressus MN121101 BRIP65869 C. tangensis ex Cy. sp. MN121100 BRIP65852 C. tangensis ex Cy. sp. MN121099 BRIP67626 C. tangensis ex Cy. rotundus MN121098 BRIP67622 C. tangensis ex Cy. rotundus MN121097 BRIP60423 C. tangensis ex Cy. sp. MN121096 BRIP47774 C. tangensis ex Cy. mitis MN121095 BRIP54190 C. tangensis ex Cy. distans MN121094 BRIP49161 C. tangensis ex Cy. distans MN121093 BRIP67647 C. tangensis ex Cy. alternifolius MN121092 BRIP67592 C. tangensis ex Cy. rotundus MN121091 BRIP67629 C. tangensis ex Cy. distans 100/100/1.0 MN121090 BRIP67624 C. tangensis ex Cy. rotundus MN121089 BRIP67619 C. tangensis ex Cy. rotundus MN121088 BRIP67643 C. tangensis ex Cy. esculentus MN121086 BRIP47773 C. tangensis ex Cy. corymbosus MN121085 BRIP57998 C. tangensis ex Cy. compressus MN121087 BRIP67614 C. tangensis ex Cy. esculentus 95/–/– MN121106 BRIP49163 C. tangensis ex Cy. digitatus DQ645508 C. tangensis MN121110 HUV21459 (ex BRIP) C. limitata ex Mariscus sp. –/70/0.96 FHI006 C. limitata ex Cy. cyperoides 100/100/1.0 MN121109 M0215531 C. limitata ex Cy. cyperoides MN121111 M0215535 C. limitata ex Cy. ligularis 84/88/0.99 MN121112 BRIP67667 C. kyllingae ex Cy. aromaticus MN121113 BRIP67669 C. kyllingae ex Cy. aromaticus MN121114 M0215533 C. kyllingae ex Cy. aromaticus 100/100/1.0 FHI024 C. kyllingae ex Cy. aromaticus FHI020 C. kyllingae ex Cy. aromaticus DQ631908 C. axicola 89/99/1.0 DQ875342 C. amazonica ex Rhynchospora barbata AY344967 C. fimbristylidis-miliaceae ex Fimbristylis tetragona EU246951 Tolyposporium neillii 100/100/1.0 EU246950 Tolyposporium isolepidis AY344994 Tolyposporium junci DQ875345 Tolyposporium piluliforme 83/89/1.0 Fig. 1 Phylogenetic tree based on minimum evolution analysis of nrITS DNA sequences of Cintractia spp., rooted with Tolyposporium. Numbers on branches denote bootstrap support in minimum evolution, maximum likelihood, and Bayesian posterior probabilities. Values below 55% are not included. The bar indicates expected substitutions per site. GenBank numbers precede taxa Mycol Progress (2021) 20:191–201 199 Fig. 2 Cintractia spp. on Cyperus spp. a–b Cintractia kyllingae on Cyperus aromaticus (BRIP 67667, holotype), Tanzania. c Cintractia limitata on Cyperus rotundus, Benin (photo M. Piepenbring). d Cintractia limitata on Cyperus cyperoides (BRIP 71068), Nigeria. e Cintractia tangensis on Cyperus rotundus (MP 5311), Benin (photo M. Piepenbring). f Cintractia tangensis on Cyperus alternifolius (BRIP 67647), Tanzania. a, c, e Sori. b, c, f Teliospores. Scale bars: b, c, f = 10 μm biologically (Tables 1 and 2). Cintractia kyllingae is only known to form sori in the flowers and spikelets of Cy. aromaticus, which is native to tropical Africa and the western Indian Ocean region. Cintractia limitata forms sori in some flowers of individual spikelets of Cy. cyperoides (native to Africa and Asia) and Cy. ligularis (native to Africa and tropical America). Cintractia tangensis forms sori at the base of peduncles in inflorescences of several species of Cyperus, with most collections from Cy. compressus and Cy. rotundus, which are both widespread throughout the tropics and subtropics. The three species, C. kyllingae, C. limitata, and C. tangensis, have teliospores that are similar in shape and size. Teliospore morphology does not allow differentiation of these species. Prior to this study, C. limitata s. lat. was considered to have a wide host range across continents. We have applied the name C. limitata s. str. to a monophyletic clade of specimens that includes a specimen on Cy. ligularis, which is the host of the type of Cintractia limitata. Cintractia limitata appears host specific to Cyperus spp. formerly classified in Mariscus, which has spikelets that detach entirely when mature. Molecular variation between specimens of Cintractia on Cy. rotundus and Cy. ligularis was reported by Piepenbring et al. (1999). Cintractia kyllingae is a newly described species that appears restricted to Cy. aromaticus, which is widespread in tropical and southern Africa and the western Indian Ocean region. Cintractia kyllingae may have potential for the 200 Mycol Progress (2021) 20:191–201 biological control of Cy. aromaticus in Australia, pending an assessment of its host range. Host range testing should include other Australian Cyperus spp. that have been described in Kyllinga or are closely related to these species. There are about 125 native Cyperus spp. in Australia, of which two, C. sesquiflorus and C. sphaeroideus, have been classified in Kyllinga (Australian Plant Names Index, https://biodiversity. org.au/nsl/services/APNI). Acknowledgments The authors wish to thank Prof. Dr. Meike Piepenbring for providing images of Cintractia spp. on Cyperus rotundus and Cyperus cyperoides in the field. Marjan Shivas is thanked for collecting specimens. Permits for the collection and export of specimens from Africa were obtained from the National Museums of Kenya, the Forestry Research Institute, Ibadan, Nigeria, and the Commission for Science and Technology, Tanzania. Authors’ contributions The first draft of the manuscript was written by Julia Kruse and all authors commented on subsequent versions of the manuscript. Julia Kruse, Alistair Ross McTaggart, and Roger Graham Shivas contributed to the phylogenetic and taxonomic analyses. Paul Mutuku Musili organized field work, collected specimens, and identified host plants in Kenya. Fredrick Munyao Mutie collected specimens and identified host plants in Kenya. John Elia Ntandu organized field work, collected specimens, and identified host plants in Tanzania. Ocholi Edogbanya and Emmanuel C. Chukwuma organized field work, collected specimens, and identified host plants in Nigeria. Kunjithapatham Dhileepan and Roger Shivas collected specimens in Kenya, Nigeria, and Tanzania. All authors read and approved the final manuscript. All authors contributed to the study conception, design, preparation of material, data collection, and analysis. Funding This project was supported by the AgriFutures Australia (formerly Rural Industries Research and Development Corporation), through funding from the Australian Government Department of Agriculture, Water and the Environment as part of its Rural Research and Development for Profit Program and the Queensland Department of Agriculture and Fisheries. Data availability Sequence data have been deposited in GenBank. Compliance with ethical standards Conflict of interest interest. 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