Phytotaxa 375 (3): 189–202 http://www.mapress.com/j/pt/ Copyright © 2018 Magnolia Press ISSN 1179-3155 (print edition) Article PHYTOTAXA ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.375.3.1 Crossopsorella, a new tropical genus of rust fungi ERICA S. C. SOUZA1, M. CATHERINE AIME2, SAMUEL G. ELIAS1, DANILO B. PINHO3, ROBERT. N. G. MILLER1,3 & JOSÉ C. DIANESE1,3 1 Departamento de Biologia Celular/Biologia Microbiana, Universidade de Brasília, 70910-900, Brazil. Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, 47907-2054, USA. 3 Departamento de Fitopatologia, Coleção Micológica, Universidade de Brasília, 70910-900, Brazil. 1 Corresponding author: jcarmine@gmail.com 2 Crossopsorella gen. nov.; Crossopsorella byrsonimae comb. nov. 2 Abstract Prior phylogenetic studies of rust fungi have shown the Phakopsoraceae as polyphyletic. However, most of the ca. 13 genera currently placed in Phakopsoraceae s.l. have not been the subject of phylogenetic analyses. In this study we examine the placement of several species of Crossopsora (Phakopsoraceae) from newly generated nuc 28S rDNA (28S) sequence data. While the type species, C. ziziphi, cannot be excluded from the Phakopsoraceae s.s., several other species, including C. byrsonimae, are not congeneric with the type. Herein we describe the new genus Crossopsorella, based on C. byrsonimae as the type, to accommodate specimens of this species found in four different Byrsonima species. Key words: Brazil, Cerrado fungi, molecular phylogeny, neotropical Pucciniales Introduction The family Phakopsoraceae Cummins & Hirats. (type genus: Phakopsora Dietel) was originally established in the second edition of the Illustrated Genera of Rust Fungi (Cummins and Hiratsuka 1983) to accommodate ten genera of rusts including Arthuria H.S. Jacks., Cerotelium Arthur, Crossopsora Syd. & P. Syd., Dasturella Mundk. & Khesw., Monosporidium Barclay, Nothoravenelia Dietel., Phragmidiella Henn., Phakopsora, Physopella Arthur, Pucciniostele Tranzschel & K.L. Kom., and Uredopeltis Henn.. In later editions Cummins & Hiratsuka (2003) added Batistopsora Dianese, R.B. Medeiros & L.T.P. Santos, Kweilingia Teng, and Scalarispora Buriticá & J.F. Hennen to the family. The genus Crossopsora [type species: C. ziziphi (Syd., P. Syd. & E.J. Butler) Syd. & P. Syd.], was established in 1919 (Sydow and Sydow 1919) and contains species that form visible hair-like telia shown as extruded columns. Although 26 names have been applied in the genus, as cited from Index Fungorum (http://www.indexfungorum.org, accessed 17 Dec 2017), nine of these are now treated as members of the Cronartiaceae, and only 12 to 15 Crossopsora species are currently accepted (Cummins and Hiratsuka 2003; Kirk et al. 2008). Members of Crossopsora are only known from dicotyledonous hosts. Although the unrelated genus, Cronartium (Cronartiaceae), produces extruded telial columns that may superficially resemble those formed in Crossopsora, but are embedded in a common matrix. Additionally, Cronartium species are distributed in temperate to cold regions where their alternate Pinaceae hosts occur (Cummins and Hiratsuka 1983), whereas Crossopsora species are tropical to subtropical, and known as being also heterocyclic, but differ in being autoaecious, as seen in several comments and descriptions in Hennen et al. (2005). Finally, the uredinia in Crossopsora species have strong peripheral paraphyses, in contrast to Cronartium species. On the other hand, species of Crossopsora are readily distinguished from those of Phakopsora, the type genus of the family Phakospsoraceae, primarily by their telia, which in the former consist of long extruded columns but in the latter form immersed (generally subepidermal) clusters of single-celled teliospores. Aime (2006) demonstrated the non-monophyly of genera currently placed within Phakopsoraceae, which highlighted the necessity for a thorough systematic revision of the family. As no exemplars from the genus Crossopsora were included in that study, the purpose of this investigation was to infer the phylogenetic placement for this genus. Here we analyze the nuc 28S rDNA (28S) D1-D2 domains from the type species, C. ziziphi, and from additional Accepted by Kevin Hyde: 25 Oct. 2018; published: 13 Nov. 2018 189 neotropical Crossopsora to evaluate their position within the framework of the broader Pucciniales tree of life. Based on these analyses, a new genus, Crossopsorella, is proposed to accommodate species that are not monophyletic with C. ziziphi. Materials and methods Morphological studies.—Herbarium specimens were obtained from the Arthur Fungarium (PUR) and the Mycological Collection of Herbarium UB (Universidade de Brasília), MCHUB. Specimens deposited at MCHUB comprised leaf samples from Byrsonima species with Crossopsora-like infection, showing mostly uredinia and telia. All specimens were collected from the Brazilian savanna, known to be a Neotropical biodiversity hotspot (Myers et al. 2000), the largest and most diverse in the neotropics, designated as Cerrado. Spermogonia, aecio-, uredinio-, and teliospores were free hand excised under stereomicroscopy or sectioned to 20–30 μm thickness with a freezing microtome (Leica CM 1850) and mounted in colorless lactoglycerol for visualization in Nomarski interference microscopy (Leica DM 2500 coupled to a Leica DFC 490 digital camera and microcomputer). Image capture, editing, and structural measurements were conducted using Leica QWin software, version 3, with sizes based on at least 20 measurements. Measurements in parentheses indicated extreme measurements detected. Portions of dry leaves containing uredinia and telia were fixed on copper stubs, gold spluttered, and scanned under electron microscopy (JEOL JSM–700 1F). DNA extraction, PCR amplification, and DNA sequencing.—The 28S sequence of the Crossopsora type species, C. ziziphi, was obtained following the protocols and primers in Aime (2006). In brief individual sori were excised and DNA extracted with the MoBio UltraClean Plant DNA Isolation kit (Qiagen Inc., Germantown, Maryland). The target locus was PCR-amplified and sequenced using primers Rust-2inv and LR6 (Aime 2006) and sent to Beckman Coulter, Inc. (Danvers, Massachusetts, USA) for sequencing. Urediniospore and teliospore masses of our specimens were newly sequenced in this study (TABLE 1) after sampling from host leaves using a needle and placing them separately in 1.5 mL micro-centrifuge tubes, total DNA was extracted using a standard CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle 1990). PCR amplification of 28S was performed in 25 μL reaction volumes containing Taq DNA Polymerase Platinum (0.5 U), dNTPs (0.2 mM), 10X buffer (5 mL), MgCl2 (1.5 mM), forward and reverse primers (0.4 mM), a maximum of 10 ng/mL genomic DNA, and nuclease-free water to complete the total volume. Primer pairs Rust2inv and LR6 (Vilgalys and Hester 1990; Aime 2006), and LR0R and Rust1 (Moncalvo et al. 1995; Kropp et al. 1997) were employed to amplify the target region of the 28S. Thermal cycling was conducted using an initial denaturation of 4 min at 94 C, followed by 30 cycles of 94 C for 1 min (denaturation), 54 C for 1 min (primer annealing), 72 C for 1 min (elongation), followed by 72 C for 5 min (final extension). PCR products were purified using the ExoSAP-IT® PCR Product Cleanup kit (ThermoFisher Scientific, Massachusetts) and forward and reverse-sequenced on an ABI 3730 Genetic Analyzer (Applied Biosystems) using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California) following the manufacturer’s instructions. Electropherograms were edited using the program GENEIOUS version 9.0.5 (Kearse et al. 2012). For the phylogenetic analyses, taxon sampling was based on a 28S dataset compiled by McTaggart et al. (2016) and Aime (2006) that included a total of nine recognized families of Pucciniales: Coleosporiaceae, Melampsoraceae, Mikronegeriaceae, Phakopsoraceae, Phragmidiaceae, Pucciniastraceae, Pucciniaceae, Raveneliaceae and Sphaerophragmiaceae, and an incertae sedis clade composed exclusively by Australian Uromycladium McAlpine species. As the polyphyletic family Phakopsoraceae consists of Phakopsoraceae s.s. on dicots, and Phakopsoraceae p.p. on monocotyledonous hosts (Aime 2006), we included Kweilingia divina Teng. on Bambusa spp. as a representative of the latter group, plus Uredo chusqueae and Stomatisora psychotriicola, which were shown to be monophyletic with K. divina (Wood et al. 2014). Given the morphological similarity of the genus Crossopsora with Cronartiaceae (Cummins and Hiratsuka 2003), Endocronartium harknessii, Cronartium quercuum, and C. flaccidum were included, as well as sequences of Pucciniastrum and Thekopsora (Pucciniastraceae). Eocronartium muscicola and Helicobasidium purpureum were selected as outgroups, in accord with Aime (2006) and Aime et al. (2006). GenBank-derived sequence information, together with data for specimens generated in this study, are listed in TABLE 1. Phylogenetic analysis.—Eighty-five sequences in total were aligned using MAFFT 7.305 (Katoh and Standley 2013) using the X-INS-i strategy (Katoh and Toh 2008). Bayesian inference (BI) was carried out using the program MRBAYES 3.2.6 (Ronquist et al. 2012) using the 4by4 mode of the GTR model. Four rate categories were used to approximate the gamma distribution. Two independent runs were employed, each one starting from random trees and four simultaneous 190 • Phytotaxa 375 (3) © 2018 Magnolia Press SOUZA ET AL. independent chains for one million generations. Trees were sampled every 1000th generation. Average standard deviation of split frequencies (ASDSF) were checked as a chain convergence criterion. From all sampled trees, a total of 25% were discarded as the burn-in while the remaining trees were used to estimate Bayesian posterior probabilities (BPP) of the branches. Maximum likelihood (ML) was carried out using RAXML 8.2.9 (Stamatakis 2014). The analysis first involved 100 ML searches, each one starting from one randomized stepwise addition parsimony tree under a GTRGAMMA model. Branch support was calculated with 1000 bootstrap (BS) replicates under the same model. Both BI and ML analyses were implemented on the CIPRES Science Gateway 3.1 (Miller et al. 2010). TABLE 1. Genbank acession number of specimens included in the phylogenetic analysis. Taxon Achrotelium ichnocarpi Syd. Caeoma torreyae Bonar Ceratocoma jacksoniae (Henn. ex McAlpine) Buritica & J.F. Hennen Chrysomyxa cassandrae (Gobi) Tranzschel Chrysomyxa ledi (Alb. & Schwein.) de Bary Chrysomyxa ledicola Lagerh. Chrysomyxa nagodhii P.E. Crane Chrysomyxa pyrolae Rostr. Coleosporium tussilaginis (Pers.) Lev. Cronartium flaccidum (Alb. & Schwein.) G. Winter Cronartium quercuum (Berk.) Miyabe ex Shirai Crossopsora ziziphi (Syd., P. Syd. & E.J. Butler) Syd. & P. Syd. Crossopsorella byrsonimae (P. Henn.) E.S.C. Souza, Aime, Galvão-Elias, Dianese. Crossopsorella byrsonimae Crossopsorella byrsonimae Crossopsorella byrsonimae Crossopsorella byrsonimae Cystopsora notelaeae Syd. Dasyspora amazonica Beenken Dasyspora echinata Beenken & Berndt Dasyspora gregaria (Kunze) Henn. Dasyspora guianensis Beenken Dasyspora mesoamericana Beenken Dasyspora nitidae Beenken Dasyspora segregaria Beenken Dasyspora winteri (Pazschke) Beenken Endocronartium harknessii (J.P. Moore) Y. Hirats Endocronartium harknessii (J.P. Moore) Y. Hirats. Endoraecium acaciae Hodges & D.E. Gardner Endoraecium auriculiforme McTaggart & R.G. Shivas Endoraecium carnegiei McTaggart & R.G. Shivas Endoraecium disparrimum McTaggart & R.G. Shivas Specimen BRIP 55634 BRIP 57762 Host Plant Ichnocarpus frutescens Torreya californica Davesia sp. Genbank # KT199393 AF522183 KT199394 Reference McTaggart et al. (2015) Direct Submission McTaggart et al. (2015) - Picea glauca FJ666455 Vialle et al. (2009) DAOM 149959 Rhododendron palustre FJ666468 Vialle et al. (2009) BRIP 56944 WM 1182 Rhododendron groenlandicum Picea mariana Pyrola sp. Senecio sp. Vincetoxicum hirundinaria FJ666446 FJ666461 FJ666466 KT199395 AF426239 Vialle et al. (2009) Vialle et al. (2009) Vialle et al. (2009) McTaggart et al. (2015) Mair et al. (2003) U-432 Quercus muehlenbergii DQ190732 Aime (unpublished) - Ziziphus aenoplia MG744558 This work UB22259 Byrsonima coccolobifolia MG250382 This work UB22384 UB23344 UB22347 UB22202 BRIP 58325 BPI 0116382 PUR N6196 Byrsonima crassa Byrsonima pachyphylla Byrsonima verbascifolia Byrsonima laxiflora Notelaea microcarpa Xylopia amazonica Xylopia emarginata MG250386 MG250384 MG250385 MG250383 KT199396 JF263460 JF263462 This work This work This work This work McTaggart et al. (2015) Beenken et al. (2012) Beenken et al. (2012) ZT Myc 3397 ZT Myc 3413 PUR 42390 JF263477 JF263479 JF263480 Beenken et al. (2012) Beenken et al. (2012) Beenken et al. (2012) ZT Myc 3409 PMA MP4941 S F30078 Xylopia cayennensis Xylopia benthamii Xylopia frutescens var. frutescens Xylopia nitida Xylopia aromatica Xylopia sericea JF263484 JF263488 JF263492 Beenken et al. (2012) Beenken et al. (2012) Beenken et al. (2012) TDB152 - AF522175 Direct Submission AFTOL-ID 456 - AY700193 BPI 871098 Acacia koa DQ323916 Matheny et al. (unpublished) Scholler and Aime (2005) BRIP 56548 Acacia auriculiformis KJ862298 McTaggart et al. (2015) BRIP 57924 Acacia dealbata KJ862301 McTaggart et al. (2015) BRIP 55626 Acacia disparrima KJ862304 McTaggart et al. (2015) ......continued on the next page CROSSOPSORELLA GEN. NOV. Phytotaxa 375 (3) © 2018 Magnolia Press • 191 TABLE 1. (Contined) Taxon Endoraecium falciforme McTaggart & R.G. Shivas Endoraecium irroratum McTaggart & R.G. Shivas Endoraecium koae (Arthur) M. Scholler & Aime Endoraecium maslinii McTaggart & R.G. Shivas Endoraecium parvum Berndt Endoraecium peggii McTaggart & R.G. Shivas Endoraecium phyllodiorum (Berk. & Broome) Berndt Endoraecium podalyriifolium McTaggart & R.G. Shivas Endoraecium tierneyi (J. Walker & R.G. Shivas) M. Scholler & Aime Endoraecium tropicum McTaggart & R.G. Shivas Endoraecium violae-faustiae Berndt Eocronartium muscicola (Pers.) Fitzp. Gerwasia rubi Racib. Hamaspora acutissima P. Syd. & Syd. Helicobasidium purpureum (Tul.) Pat. Hemileia sp. Hemileia vastatrix Berk. & Broome Maravalia cryptostegiae (Vestergr.) Y. Ono Masseella capparis (Hobson bis ex Cooke) Dietel Melampsora abietis-canadensis C.A. Ludw. Melampsora aecidioides (DC.) J. Schr€ot. Melampsora medusae f.sp. tremuloides Shain Melampsora pinitorqua Rostr. Phakopsora annonae-sylvaticae Beenken Phakopsora cherimoliae (Lagerh.) Cummins Phakopsora crucis-filii (Dianese, R.B. Medeiros & L.T.P. Santos) Beenken Phakopsora myrtacearum McTaggart, Maier, Jol. Roux, M.J. Wingf. Phakopsora pistila (Buritica & J.F. Hennen) Beenken Phakopsora rolliniae (W.T. Dale) Beenken Phragmidium barnardii Plowr. & G. Winter Phragmidium potentillae (Pers.) P. Karst. Puccinia lagenophorae Cooke Puccinia myrsiphylli (Th€um.) G. Winter Puccinia psidii G. Winter Puccinia stylidii McAlpine Specimen BRIP 57583 Host Plant Acacia falciformis Genbank # KJ862306 Reference McTaggart et al. (2015) BRIP 57286 Acacia irrorata KJ862312 McTaggart et al. (2015) BPI 871071 Acacia koa DQ323918 Scholler and Aime (2005) BRIP 57872 Acacia daphnifolia KJ862314 McTaggart et al. (2015) BRIP 57524 BRIP 55602 Acacia leiocalyx Acacia holosericia KJ862316 KJ862308 McTaggart et al. (2015) McTaggart et al. (2015) BRIP 57516 Acacia aulacocarpa KJ862324 McTaggart et al. (2015) BRIP 57576 Acacia podalyriifolia KJ862334 McTaggart et al. (2015) BRIP 27071 Acacia harpophylla KJ862335 McTaggart et al. (2015) BRIP 56557 Acacia tropica KJ862337 McTaggart et al. (2015) BRIP 55601 BRIP 58369 BRIP 55606 TUB 011542 BRIP 57470 BRIP 61233 BRIP 56898 Acacia aulacocarpa Rubus sp. Rubus moluccanus Carpinus betulus Rubiaceae Coffea robusta Cryptostegia grandiflora KJ862338 AF014825 KT199397 KT199398 AY254180 KT199400 KT199399 KT199401 McTaggart et al. (2015) Direct Submission McTaggart et al. (2015) McTaggart et al. (2015) Lutz et al. (2004) McTaggart et al. (2015) McTaggart et al. (2015) McTaggart et al. (2015) BRIP 56844 Flueggea virosa JX136798 Ligerato et al. (2014) - Tsuga canadensis FJ666512 Vialle et al. (2009) - Populus alba FJ666520 Vialle et al. (2009) - Populus tremuloides FJ666517 Vialle et al. (2009) PUR 87311 Pinus sylvestris Annona sylvatica FJ666523 KF528008 Vialle et al. (2009) Beenken (unpublished) - Annona cherimola KF528011 Beenken (unpublished) ZT Myc 48990 Annona paludosa KF528016 Beenken (unpublished) PREM 61155 Eucalyptus grandis KP729473 Mair et al. (2015) ZT Myc 48992 Annona sericea KF528026 Beenken (unpublished) ZT Myc 49000 Annona exsucca KF528034 Beenken (unpublished) BRIP 56945 Rubus multibracteatus KT199402 McTaggart et al. (2015) BRIP 60089 Acaena novae-zelandiae KT199403 McTaggart et al. (2015) BRIP 57563 BRIP 57782 Emilia sonchifolia Asparagus asparagoides KF690696 KM249854 McTaggart et al. (2014) McTaggart et al. (2016) BRIP 57793 BRIP 60107 Rhodamnia angustifolia Stylidium armeria KF318449 KJ622215 Pegg et al. (2014) McTaggart et al. (2014) ......continued on the next page 192 • Phytotaxa 375 (3) © 2018 Magnolia Press SOUZA ET AL. TABLE 1. (Contined) Taxon Puccinia ursiniae R.G. Shivas Pucciniastrum circaeae (Schumach.) Speg. Pucciniastrum epilobii (Pers.) G.H. Otth Pucciniastrum guttatum (J. Schröt.) Hyl., Jørst. & Nannf. Ravenelia evansii Syd. & P. Syd. Ravenelia macowaniana Pazschke Ravenelia neocaledoniensis B. Huguenin Sphaerophragmium acaciae (Cooke) Magnus Sphenorchidium polyalthiae (Syd. & P. Syd.) Beenken & A.R. Wood Thekopsora minima (Arthur) P. Syd. & Syd Thekopsora symphyti (DC.) J. Müll Uromyces lomandracearum J. Walker & van der Merwe Uromycladium falcatarium Doungsaard, McTaggart & R.G. Shivas Uromycladium fusisporum (Cooke & Massee) Savile Uromycladium notabile (F. Ludw.) McAlpine Uromycladium simplex McAlpine Uromycladium tepperianum Uromycladium tepperianum (Sacc.) McAlpine Specimen BRIP 57993 RB 2098 Host Plant Ursinia anthemoides Circaea lutetiana Genbank # KF690705 AF426227 Reference McTaggart et al. (2014) Mair et al. (2003) WM 1099 Epilobium angustifolium AF426226 Mair et al. (2003) WM 1203 Galium odoratum AF426231 Mair et al. (2003) WM3538 WM3577 BRIP:56907 Vachellia hebeclada Vachellia karroo Vachellia farnesiana KP661595 KP661596 KJ862347 McTaggart et al. (2015) McTaggart et al. (2015) McTaggart et al. (2015) BRIP 56910 Albizzia sp. KJ862350 McTaggart et al. (2015) ZT HeRB 251 Polyalthia longifolia JF263493 Beenken et al. (2012) BRIP 57654 Vaccinium corymbosum KC763340 McTaggart et al. (2013) HeRB 4732 BRIP 59022 Symphytum officinale Lomandra sp. AF426230 KM249862 Mair et al. (2003) McTaggart et al. (2016) BRIP 57447 Falcataria moluccana KJ632973 Doungsa-ard et al. (2015) BRIP 57526 Acacia salicina KJ632991 Doungsa-ard et al. (2015) BRIP 59234 Acacia dealbata KJ632992 Doungsa-ard et al. (2015) BRIP 59214 BRIP 57860 BRIP 56928 Acacia pycnantha Acacia saligna Acacia leiocalyx KJ632990 KJ632988 KJ632981 Doungsa-ard et al. (2015) Doungsa-ard et al. (2015) Doungsa-ard et al. (2015) Results Phylogeny.—Six new sequences were produced for this study. Our final nucleotide matrix contained 1186 aligned positions, of which 533 were variable and 436 parsimony informative (37% of variable sites). The alignment was deposited in TreeBase (http://www.herbaria.harvard.edu/treebase/), study accession number 21504. A stationary distribution of a Markov Chain was reached after ca. 280 000 generations (ASDS > 0.01). After discarding the first 25% of sampled trees, the 17 002 remaining trees were used to construct the 50% majority-rule consensus tree presented in Fig. 1. Neither the position of C. ziziphi or C. byrsonimae could be completely ascertained with these data and analyses, although both are supported as members of the Uredinineae, one of three suborders of Pucciniales sensu Aime (2006) (Fig. 1). However, what is clear is that Crossopsora byrsonimae is not monophyletic with C. ziziphi. Three suborders are indicated in Fig. 1, although bootstrap support is lacking for clade C. Within clade C the Crossopsora species shown fail to cluster with one sequence of C. ziziphi, which forms a weakly supported sister lineage to the families Phakopsoraceae and Raveneliaceae. Taxonomy Crossopsorella E.S.C. Souza, Aime, Galvão-Elias, Dianese, gen. nov. MycoBank MB823952 Typification: Crossopsorella byrsonimae (P. Henn.) E.S.C. Souza, Aime, Galvão-Elias, Dianese, comb. nov. Etymology: Crossopsorella referring to a genus similar previously established. CROSSOPSORELLA GEN. NOV. Phytotaxa 375 (3) © 2018 Magnolia Press • 193 FIGURE 1. BI 50% majority-rule consensus tree of the Pucciniales reconstructed from 28S sequences. Thickened branches represent BPP >0.98 and BS>95%. Tip labels in red represent sequences from our new Crossopsorella samples. Blue labels indicate the names of plant hosts. 194 • Phytotaxa 375 (3) © 2018 Magnolia Press SOUZA ET AL. Description: Spermogonia subcuticular. Aecia subepidermal, peridium persistent with hexagonal elongated verrucose peridial cells. Aeciospores catenate, verruculose, 1-equatorial germ pore. Uredinia subepidermal in origin, erumpent, usually with slightly curved peripheral paraphyses, showing as segmented when seen under SEM. Urediniospores borne singly, echinulate, pores scattered, obscure. Telia subepidermal in origin, erumpent, forming hair-like columns, brown. Teliospores 1-celled, originated from immerse group of closely set sporogenous cells that form spores in basipetal chain, resulting in a solidly adhered column of thick walled teliospores, showing one lateral germ pore, germinating externally, resulting in 3-septate metabasidia. Notes: The genus Crossopsorella is established to allocate species previously allocated in Crossopsora, but were shown to be non congeneric with C. ziziphi, the genus type species. The new genus differs from Crossopsora by spermogonia and aecial types. Although both produce Group VI spermogonia, in Crossopsora they are type 7, while spermogonia of Crossopsorella belong to type 5. Crossopsora produces Caeoma-type aecia, whereas in Crossopsorella they are characteristically of the Aecidium-type (Hennen et al. 2005). Crossopsorella byrsonimae (P. Henn.) E.S.C. Souza, Aime, Galvão-Elias, Dianese, comb. nov. Figs. 2–7. MycoBank MB823953 Basionym: Cronartium byrsonimae (as ‘byrsonimatis’) Henn., Hedwigia 48:2. 1908. ≡ Crossopsora byrsonimae (P. Henn) P.S. Peterson, Rept. Tottori Mycol Inst Japan 10:210. 1973. FIGURE 2. Crossopsorella byrsonimae on Byrsonima coccolobifolia (UB Mycol. Col. 22259). A–B. Spermogonia. C. Inner surface of the aecial peridium. D. Aeciospores seen at a superficial focus in light microscopy in contrast with a group of peridial cells. E. Same aeciospores as in D, but seen at a deeper focus in light microscopy, contrasting with a group of peridial cells. Typification: BRAZIL. SÃO PAULO, Morro Pellado, Jul 1904, Puttemans # 1140 on Byrsonima coccolobifolia. DISTRITO FEDERAL: Brasilia, Close to the Olympic Center of the Universidade de Brasília, Byrsonima coccolobifolia, Jun 21 2012, M. Sanchez (UB Mycol. Col. 22259). Spermogonia (Group VI, Type 5), densely distributed on both sides of leaves and young stems on deformed systemically infected shoots, subcuticular, light brown, fertile showing hyaline receptive hyphae and spermatia. Aecia scattered among the spermogonia, cylindrical, sometimes up to 1–4 mm high × 400 μm diam, white; peridial cells firmly united mostly hexagonal, 36–65 × 20–30 μm, outer smooth, inner facing wall coarsely verrucose. Aeciospores CROSSOPSORELLA GEN. NOV. Phytotaxa 375 (3) © 2018 Magnolia Press • 195 35–55 × 35–50 μm, light brown, angular ovoid, ellipsoid, or oblong, thick walled at the base and apex. Uredinia 130– 190 μm wide, hypophyllous, subepidermal, sparse or gregarious, paraphysate; paraphyses smooth, segmented when seen in SEM, apiculate. Urediniospores 32–38 × 22–24 μm, brown to light golden brown, echinulate. Telia generated as a basipetal chain of teliospores from a compact set of sporogenous cells deeply implanted in the host parenchyma, resulting in hair-like stable columnar brown telia (3–4 mm long × 96–120 μm diam. Teliospores (86–)79–40(–24) μm long × (25–)23–14(–9) μm wide, cylindrical, brown showing one circular lateral germ pore; germination external. Metabasidia 3-septate, 50 × 15 μm, 4–sterigma. Basidiospores hyaline, globose, four per metabasidium. FIGURE 3. Aecia of Crossopsorella byrsonimae on Byrsonima coccolobifolia (UB Mycol. Col. 22259) in stereomicroscopy. Additional specimens examined: BRAZIL. DISTRITO FEDERAL: Brasilia, Arboretum of the Universidade de Brasília, Asa Norte, on Byrsonima coccolobifolia, 7 May 2007, Z.M. Chaves (UB20577). ibid. 14 May 2007, Z.M. Chaves (UB20585). ibid. 14 May 14, M. Sanchez (UB20594). ibid, Campus Universitário Darcy Ribeiro next to the CESPE Building, Asa Norte, on B. coccolobifolia, 21 Jun 2013, E.S.C. de Souza (UB22483). ibid, Cerrado by the Minas Tênis Club, Asa Norte, on B. coccolobifolia, 30 Aug 1992, J.C. Dianese (UB1800). ibid. J. C. Dianese (UB1801). ibid. J. C. Dianese (UB1802). ibid, Península Norte cycle route, B. verbascifolia, Aug 30 1992, J. C. Dianese (UB1773). ibid, J. C. Dianese (UB1774). ibid, J. C. Dianese (UB1775). ibid, J. C. Dianese (UB1776). ibid, J. C. Dianese (UB1777). ibid, J. C. Dianese (UB1778). ibid, J. C. Dianese (UB1779). ibid, J. C. Dianese (UB1780). ibid, J. C. Dianese (UB1781). ibid, J. C. Dianese (UB1783). ibid, J. C. Dianese (UB1784). ibid, J. C. Dianese (UB1785). ibid, J. C. Dianese (UB1786). ibid, J. C. Dianese (UB1787). ibid, J. C. Dianese (UB1788). ibid, J. C. Dianese (UB1789). ibid, J. C. Dianese (UB1790). Brasília, Biological Experiment Station, University of Brasília, B. coccolobifolia, 22 Aug 2007, M. Sanchez (UB20691). ibid. M. Sanchez (UB20697). ibid, M. Sanchez (UB20701). ibid, M. Sanchez (UB20702). ibid, Olhos D’água Ecological Park of Brasília SQN 413 Asa Norte -47.884246 -15.743185, B. crassa, Sep 12 2012, E. S. C. de Souza (UB22383). ibid, E. S. C. de Souza (UB22384). ibid, Península Norte, B. coccolobifolia, May 31 1992, J. C. Dianese (UB1026). ibid, Jun 28 1992, J. C. Dianese (UB2355). ibid, J. C. Dianese (UB2356). ibid, J. C. Dianese (UB2357). ibid, J. C. Dianese (UB2358). ibid, Água Limpa Farm University of Brasília, B. verbascifolia, Sep 3 2012, E. S. C. de Souza (UB22347). ibid, Brasília’s Botanical Garden, B. laxiflora, May 30 2012, E. S. C. de Souza (UB22202). ibid: Brazlândia, Palestina Farm, Área de Proteção Ambiental da Cafuringa, B. verbascifolia, Aug 27 1992, J. C. Dianese & C. Furlaneto (UB1663). ibid, J. C. Dianese & C. Furlaneto (UB1664). ibid, J. C. Dianese & C. Furlaneto (UB1665). ibid, J. C. Dianese & C. Furlaneto (UB1667). ibid: Planaltina, Águas Emendadas Ecological Station. B. verbascifolia, Jun 11 2007, V. R. Rodrigues (UB20643). ibid. BAHIA: Una, Bolandeira farm, next to road I Comandatuba, B. verbascifolia, Aug 26 1995, M. Sanchez (UB9889). ibid. MARANHÃO: Carolina, Parque Nacional Serra das Mesas, B. verbascifolia, Sep 3 2012, D. B. Pinho (UB23344). 196 • Phytotaxa 375 (3) © 2018 Magnolia Press SOUZA ET AL. FIGURE 4. Uredinium of Crossopsorella byrsonimae on Byrsonima coccolobifolia (UB Mycol. Col. 22259) seen in cross section under a light microscope. FIGURE 5. Uredinia of Crossopsorella byrsonimae on Byrsonima coccolobifolia (UB Mycol. Col. 22259) under SEM. A–B. Paraphysate uredinium. C. Detail of segmented paraphyses. D. Urediniospores. CROSSOPSORELLA GEN. NOV. Phytotaxa 375 (3) © 2018 Magnolia Press • 197 FIGURE 6. Telia of Crossopsorella byrsonimae on Byrsonima coccolobifolia (UB Mycol. Col. 22259) under SEM. A. Residual paraphyses around the telial base (arrow). B. Details of apically broken paraphyses. C–D. Telial surface with one germ pore per teliospore as indicated by arrows. Notes: Crossopsorella byrsonimae was originally described from the Brazilian Cerrado on Byrsonima coccolobifolia (Hennings 1908). The holotype with telium, teliospores, and urediniospores were illustrated by Hennings, and can be seen in Fig. 8B. Urediniospores, telia, and teliospores from our recent collections are similar in size to those from the type (Fig. 8B). The type material shows columnar telia at the abaxial face of a leaf of B. coccolobifolia (Fig. 8A). However, Hennings (1908) did not illustrate the spermogonial and aecial states for this rust. Morphologically, the characteristics of spermogonia and aecia of Crossopsorella segregate it from C. ziziphi, in addition to results from molecular analyses (Fig. 1). The spermogonia of Crossopsora ziziphi are subcuticular belonging to Group VI (type 7) sensu Cummins and Hiratsuka (2003), whereas in Crossopsorella the spermogonia are Group VI (type 5) showing well developed receptive hyphae. Additionally, the aecia of C. ziziphi, according to Hennen et al. (2005), are subepidermal, opening by a pore-like rupture of the epidermis without a peridium, i.e., of the Caeoma-type. In contrast, the aecia of Crossopsorella byrsonimae are Aecidium-like, shown as a cylindrical structure with well defined peridium and containing chains of aeciospores that are released with the rupture of the peridium. Discussion Cummins and Hiratsuka (1983, 2003) consistently placed Crossopsora and Cronartium, in separate families (Phakopsoraceae vs. Cronartiaceae, respectively) despite the morphological similarity of the telial phase. Our phylogenetic analyses were unable to resolve the placement of the Crossopsora type species (C. ziziphi) with 28S sequence data although a relationship with Phakopsoraceae cannot be ruled out (Fig. 1). However, our results also shown in Fig. 1, confirm that the Asiatic C. ziziphi and the species previously included in Crossopsora found on Byrsonima species from the Cerrado, now accepted as Crossopsorella species, clearly do not form a monophyletic group and are unlikely to be confamilial (Fig. 1). Therefore, the need for description of the new genus Crossopsorella to accommodate the Neotropical specimens collected in Brazil. 198 • Phytotaxa 375 (3) © 2018 Magnolia Press SOUZA ET AL. FIGURE 7. Crossopsorella byrsonimae on Byrsonima coccolobifolia (UB Mycol. Col. 22259). A. Columnar telium (arrow) under a stereomicroscope. B. Structural details of a columnar telium. C. Sporogenous cells (arrow) generating teliospores in basipetal succession. D. Metabasidium originated by germination of a teliospore. The overall topology of our phylogenetic reconstruction is congruent with previous analyses (Maier et al. 2003; Wingfield et al. 2004; Aime 2006; McTaggart et al. 2016). The anamorphic taxon Caeoma torreyae appears as sister to all other sampled Pucciniales. The three suborders proposed in the two-locus analysis of Aime (2006) were fully supported by our single locus BI analysis and partially supported by the ML analysis (Fig. 1, clades A, B and C). The ten families of Pucciniales and the Uromycladium clade included in our analysis were mostly highly supported by BI and ML (Fig. 1). The monophyly of Coleosporiaceae, Melampsoraceae, Mikronegeriaceae, Phakopsoraceae, Phragmidiaceae, Pucciniastraceae, Pucciniaceae, Raveneliaceae, Sphaerophragmiaceae, and the Uromycladium clade mirror the results of the more extensive three-gene phylogeny of McTaggart et al. (2016). In a similar fashion, CROSSOPSORELLA GEN. NOV. Phytotaxa 375 (3) © 2018 Magnolia Press • 199 Phakopsoraceae p.p. was recovered as a monophyletic group and distantly related to Phakopsoraceae s.s. as observed by Aime (2006). Cronartiaceae formed a sister clade with Coleosporiaceae (BPP = 1.00, BS = 89). A close relationship between these families was observed in previous studies but without significant phylogenetic support (Maier et al. 2003; Wingfield et al. 2004; Aime 2006). With the establishment Crossopsorella, the genus Crossopsora now contains 15 species (Kirk et al. 2008). Thirteen of these are described from neotropical hosts (C. angusta Jørst., C. asclepidiaceae Buriticá & J.F. Hennen, C. bixae Buriticá, Crossopsora caucensis (Mayor) F. Kern, Thurst. & Whetzel, C. crassa Buriticá & J.F. Hennen, C. hymenaeae Dianese, Buriticá & J.F. Hennen, C. mateleae W.T. Dale, C. notata (Arthur & J.R. Johnst.) Arthur, C. opposita Syd., C. piperis Berndt, F.O. Freire & C.N. Bastos, C. stevensii Syd., C. uleana (Syd. & P. Syd.) R.S. Peterson, C. wilsoniana (Arthur & J.R. Johnst.) Arthur. In terms of host association, neotropical Crossopsora have been documented from nine families of angiosperms (Fabaceae, Malpighiaceae, Piperaceae, Solanaceae, Vitaceae, Apocynaceae, Asclepiadaceae, Bignoniaceae and Bixaceae) (Hennen et al. 2005). Within Crossopsorella two strongly supported sub-clades can be distinguished. The first contains Crossopsorella species on Byrsonima laxiflora and B. coccolobifolia, herein treated as Crossopsorella byrsonimae, and the other comprising species infecting B. verbascifolia and B. pachyphylla. A specimen on B. crassa formed a sister lineage with that on B. verbascifolia and B. pachyphylla (BPP = 0.98; BS = 76). At this point, Crossopsorella byrsonimae on B. coccolobifolia and B. laxiflora is the basis for the establishment of the new genus, and the remaining species will be treated in a separate paper. FIGURE 8. Image of the holotype of Cronartium byrsonimae deposited at Herbarium B on July 1904 by Puttemans under number 1140. A. Leaf of Byrsonima coccolobifolia; arrow indicates a group of telia. B. Original drawings by Paul Hennings including measurements for a telium (3 mm × 100 μm), teliospores (40–70 × 12–18 μm), and urediniospores (30–40 × 22–30 μm). C–D. Copies of the original labels of the material deposited at Herbarium B. Reproduced by permission of Dr. Robert Vogt, Curator of Herbarium B, Botanischer Garten und Botanisches Museum Berlin-Dahlem, Freie Universität Berlin. 200 • Phytotaxa 375 (3) © 2018 Magnolia Press SOUZA ET AL. Acknowledgments The authors thank CAPES, Ministry of Education-Brazil for graduate fellowships to the first and third authors; and acknowledge the institutional support from CNPQ, FAP-DF, and PPBIO (MCTI)-Cerrado through grants to the last three authors. 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