Mycoscience VOL.62 (2021) 364-372 Full Paper Phylogenetic relationships among fern rust fungi and Desmella lygodii comb. nov. Izumi Okanea,*, Yoshitaka Onob, Katsura Ohmachia, M. Catherine Aimec, Yuichi Yamaokaa a Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, JAPAN College of Education, Ibaraki University, Mito, Ibaraki 310-8512, JAPAN c Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA b ABSTRACT The rust fungi (Pucciniales) that infect ferns, early diverging vascular plants, are neither “primitive” nor monophyletic, as once hypothesized. The neotropical fern pathogen, Puccinia lygodii (Pucciniaceae), specializes on species of Lygodium. Lygodium is believed to have evolved in a period ca. 211 mya, which is after the evolution of the temperate fern rust fungi that parasitize later diverged ferns. Puccinia lygodii is the only rust species in the genus Puccinia known to infect ferns, the majority of which infect flowering plants. In this study we examined multiple new and herbarium specimens of P. lygodii and reconstructed its phylogenetic history with data generated from the 28S nuclear rDNA repeat. Puccinia lygodii is the sister species to another neotropical fern rust, Desmella aneimiae (Pucciniaceae), which also infects early diverged leptosporangiate fern species, and the new combination D. lygodii is made. Interestingly, P. lygodii and D. aneimiae differ primarily in sorus structure, i.e., subepidermal in the former vs. suprastomatal in the latter fungus. Characters such as suprastomatal sori and probasidia that germinate without dormancy are now known to represent a suite of adaptations that have been derived multiple times within Pucciniales, most likely in response to tropical climates. Key Words: Desmella aneimiae, Neotropical fern, Pucciniales, Schizaeaceae, 1 new taxon Article history: Received 9 February 2021, Revised 28 June 2021, Accepted 29 June 2021, Available online 20 November 2021. 1. Introduction Rust fungi are biotrophic plant parasites. Individual rust species parasitize a variety of plants of lycopods, ferns, gymnosperms, and angiosperms, with narrow host preference. The rust fungi are classified in the order Pucciniales in the Pucciniomycotina of Basidiomycota and comprise about 7,800 species, 166 genera and 14 families worldwide (Kirk, Cannon, Minter, & Stalpers, 2008). Additionally, Aime and McTaggart (2021) proposed 18 families including seven new families and four new genera, newly proposing four new suborders based on phylogenetic analyses of a wide range of rust fungi using the 28S, 18S rDNA and cytochrome c-oxidase subunit 3 (CO3) gene of the mitochondrial DNA sequences. One early hypothesis about rust fungus evolution was that early divergent rust genera and species occur on early divergent host family and genera due to coevolution of rusts and their hosts (Cunningham, 1931; Leppik, 1953, 1965; Savile, 1976). However, multiple studies have shown this not to be the case (Sjamsuridzal, Nishida, Ogawa, Kakishima, & Sugiyam, 1999; Maier, Begerow, Weiss, & Oberwinkler, 2003; Aime, 2006). Accumulated evidence from host * Corresponding author. Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan. E-mail address: okane.izumi.fw@u.tsukuba.ac.jp (I. Okane). relationships, morphology, and molecular phylogenetics have demonstrated that the evolution of rust fungi is more complex, i.e., coevolution with the gametothallus hosts (Aime, Bell, & Wilson, 2018), specialization to new host plants closely related to those of the parental rust species followed by radiation by host-shift or host-jump to distantly related or not related host plants (Leppik, 1965, 1967; Hart, 1988; Roy, 2001; de Vienne et al., 2013; McTaggart et al., 2016). Most rust fungi that parasitize ferns belong to three genera in Melampsorineae, the second major rust radiation, which occurred ca. 100 mya (Aime, 2006; Aime, Bell, & Wilson, 2018). The Melampsorineae fern rusts belong to two families, Pucciniastraceae (Hyalopsora) and Milesinaceae (Milesina and Uredinopsis), which was newly proposed by Aime and McTaggert (2021). With a few exceptions, these species are heteroecious, alternating between ferns and Pinaceae, and found in temperate climates. By contrast, very few species of rust fungi are known to infect tropical ferns; namely, Desmella aneimiae Syd. & P. Syd. (Sydow & Sydow, 1918) and Puccinia lygodii (Har.) Arthur (Arthur, 1924), in the neotropics, and Milesina thailandica Y. Ono, Unartngam & Okane (Ono et al., 2020) in the paleotropics. Of the fern rusts, only D. aneimiae and P. lygodii belong to the most recently radiated suborder and family of rust fungi, Uredinineae (Pucciniaceae). Desmella is a monotypic genus. The type species D. aneimiae parasitizes 45 species in 25 genera and 13 families of Polyodiopsida (= This is an open-access paper distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivative 4.0 international license (CC BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/). doi: 10.47371/mycosci.2021.06.006 ― 364 ― I. Okane et al. / Mycoscience VOL.62 (2021) 364-372 Polypodiophyta) (Farr, D. F., & Rossman, A. Y. Fungal Databases, U.S. National Fungus Collections, ARS, USDA. Retrieved August 5, 2020, from https://nt.ars-grin.gov/fungaldatabases/) in Mexico to Argentina (Hennen, Figueiredo, Carvalho Jr., & Hennen, 2005), Hawaii (Ono, 2020), and Australia (McTaggart, Geering, & Shivas, 2014). The genus Puccinia contains thousands of species, of which only P. lygodii is known to infect ferns. Puccinia lygodii occurs on four species of Lygodium (Schizaeaceae, Schizaeales) and has been recorded in the Gulf States of the U.S, Central America, Colombia, Venezuela, Guyana, and Brazil (Hariot, 1900; Arthur, 1924; Sydow, 1925; Hennen, Figueiredo, Carvalho Jr., & Hennen, 2005). Milesina thailandica is only known from L. flexuosum (L.) Sw. in Thailand (Ono et al., 2020). These three rust fungi are distinguished by sorus type and spore morphology. Desmella aneimiae is characterized by two-celled, pedicellate teliospores and pedicellate urediniospores produced on a fascicle of sporogenous cells emerging through a stoma, while P. lygodii by twocelled, pedicellate teliospores and pedicellate urediniospores produced in a subepidermal, milesia-type sorus. Milesina thailandica produces almost sessile urediniospores in subepidermal, milesia-type sori. Occurrence of P. lygodii on an early divergent fern Lygodium and of D. aneimiae on a wide variety of ferns including an early divergent fern Anemia in the same geographic distribution range and ecological niches, indicate close phylogenetic and taxonomic relationship of the former to the latter fungus. In this study we recollect and examine eleven P. lygodii specimens and apply morphological and molecular data to reveal accurate taxonomic placement of P. lygodii in the Pucciniales, with special reference to another neotropical fern rust, Desmella aneimiae. Our results showed phylogenetic placement of P. lygodii in a monophyletic group together with D. aneimiae. Thus, a new name, D. lygodii, is proposed to replace P. lygodii. 2. Materials and methods 2.1. Specimens examined The specimens examined are listed in Table 1. The plant samples on which presence of rust fungus was confirmed were dried and preserved as the herbarium specimens and deposited in the Herbarium of Systematic Mycology, Ibaraki University (IBAR) or the Arthur Fungarium, Purdue University (PUR). We examined the uredinial-telial stage of P. lygodii on Lygodium plants collected in Brazil, Guyana, and the U.S.A. Type materials relating to the species were loaned from the National Fungal Collections, U.S. Department of Agriculture (BPI) and the Arthur Fungarium, Purdue University (PUR). In addition to this rust species, specimens of D. aneimiae collected in Australia, Brazil, Panama, and U.S.A. (Hawaii) were examined. 2.2. Phylogenetic analysis Sori were excised from dried specimens using a sharp-pointed surgical knife. Several uredinial or telial sori from a single leaf were excised for each sample. Genomic DNA was extracted with the UltraClean Plant DNA Isolation Kit (MoBio Laboratories Inc., Solana Beach, CA, USA) following manufacturer’s protocols, or else suspended in 20 μL of DNA extraction buffer [10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.01% sodium dodecyl sulfate, and 0.01% Proteinase K], incubated at 37 °C for 60 min followed by 95 °C for 10 min, centrifuged at 15,000 rpm for 2 min, and suspended in 30 μL of sterilized distilled water. A region of the nuclear rDNA repeat that included the internal transcribed spacer 2 and the D1-D3 region of the 28S rDNA was amplified using the following primer set: Rust2inv (Aime, 2006) as a forward primer and NL4 (O’Donnell, 1993) or LR6 (Vilgalys & Hester, 1990) as a reverse primer. The PCR reactions were performed in 25 μL reaction volumes, each containing: 1 μL genomic DNA, 12.5 μL of Gene RED PCR Mix Plus (NIPPON GENE, Tokyo, Japan), 2.5 μL (0.2 μM) of each primer, and an additional 6.5 μL of distilled water to obtain 25 μL reaction volumes. PCR was performed in a TaKaRa PCR Thermal Cycler Dice® Touch (TaKaRa, Tokyo, Japan) with the following protocol: 5 min at 95 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 53 °C, 1 min at 72 °C, and a final step of 8 min at 72 °C. PCR products were electrophoresed in 1% agarose gels stained with ethidium bromide, then visualized under UV light. PCR products were purified using MinElute PCR Purification Kit (QIAGEN, Maryland, USA) following the manufacturer’s instructions. Sequencing was consigned to Eurofins Genomics (Tokyo, Japan), using the same primers used for PCR amplification. Sequences were assembled with ATGC ver. 7 software (Genetyx Co., Tokyo, Japan). Sequence data of other rust fungi were chosen based on those previously examined for the phylogeny of rust fungi (McTaggart, Geering, & Shivas, 2014; Beenken, 2017; Aime, Bell, & Wilson, 2018; Bubner, Buchheit, Friedrich, Kummer, & Scholler, 2019; Martins Jr., Sakuragui, Hennen, & Carvalho Jr., 2019; Ono et al., 2020; Aime & McTaggart, 2021) and included to infer the taxonomic relationships among other rust fungi (Table 2). In these analyses, Cae- Table 1 - List of Desmella lygodii and D. aneimiae collections analyzed for this study. Species a Specimen number b Host plant Collection site Collection date Accession number References Desmella aneimiae D. aneimiae D. aneimiae D. aneimiae Desmella lygodii (T) D. lygodii (T) D. lygodii (T) D. lygodii D. lygodii D. lygodii D. lygodii D. lygodii (T) D. lygodii D. lygodii D. lygodii BRIP 60995 BPI 864108 IBAR 4262 IBAR 10001 BPI 20025 BPI 148715 BPI 155242 BPI 878109 IBAR 10744 IBAR 10751 IBAR 10753 PUR F3717 PUR N15340 PUR N16672 PUR N16678 Nephrolepis hirsutula Thelypteris Polypodiacea Nephrolepis exaltata Lygodium venustum Lygodium sp. Lygodium sp. Lygodium japonicum L. japonicum L. japonicum L. japonicum L. venustum Lygodium flexuosum L. japonicum Lygodium sp. Queensland, Australia Chiriqui, Panama Sap Paulo, Brazil Hawaii, USA Vicinity of Bahia, Brazil Tumatumari, Guyana Pernambuco, Brazil Mississippi, USA Florida, USA Georgia, USA Georgia, USA Vicinity of Bahia, Brazil Region 9, Guyana Florida, USA Florida, USA − 29 Nov, 2004 26 Jul, 1976 24 Dec, 2007 28 May, 1915 11 Jul, 1922 − 20 Oct, 2006 7 Oct, 2015 11 Oct, 2015 11 Oct, 2015 28 May, 1915 28 Dec, 2013 12 May, 2009 18 Oct, 2009 KM249867 LC575057 − LC498521 − − − MG907211 LC498523 − LC498525 − LC575054 LC575055 LC575056 McTaggart et al. (2014) This study − This study − − − Aime et al. (2018) This study − This study − This study This study This study a T in parentheses means type material. BPI: The U.S. National Fungus Collections, U.S. Department of Agriculture; BRIP: Department of Agriculture and Fisheries, Australia; IBAR: Mycological Herbarium of Ibaraki Univ.; PUR: The Arthur Fungarium, Purdue University. b doi: 10.47371/mycosci.2021.06.006 ― 365 ― I. Okane et al. / Mycoscience VOL.62 (2021) 364-372 Table 2 - Source of DNA sequence data used in phylogenetic reconstructions. Species Host plants Voucher specimens Collection sites Accession No. References Allodus podophylli Austropuccinia psidii Caeoma torreyae Ceratocoma jacksoniae Chardoniella gynoxidis Chrysocelis lupini Cionothrix praelonga Cronartium harknessii Cronartium ribicola Cumminsiella mirabilissima Dasyspora amazonica Dasyspora gregaria Didymopsora solani-argentei Diorchidium woodii Dipyxis mexicana Edythea quitensis Endophylloides portoricensis Hapalophragmium derridis Hemileia vastatrix H. vastatrix Hyalopsora hakodatensis Hyalopsora polypodii H. polypodii Leptopuccinia malvacearum Macruropyxis fraxini Melampsoridium betulinum Milesina blechni M. blechni Milesina exigua Milesina kriegeriana M. kriegeriana Milesina murariae M. murariae Milesina polypodii Milesina scolopendrii M. scolopendrii Milesina thailandica Milesina vogesiaca Milesina whitei Neopuccinia bursa Nyssopsora echinata Nyssopsora thwaitesii Phakopsora cherimoliae Phakopsora pachyrhizi Phakopsora pistila Phakopsora rolliniae Phragmidium mucronatum Phragmidium tormentillae Puccinia caricis Puccinia convolvuli Puccinia coronata Puccinia graminis Puccinia physalidis Pucciniastrum epilobii Puccorchidium polyalthiae Puccorchidium popowiae Sphaerophragmium acaciae Sphaerophragmium longicorne Sphenorchidium deightonii Sphenorchidium xylopiae Sphenospora smilacina Tegillum scitula Trachyspora intrusa Tranzschelia discolor Triphragmium ulmariae Uredinopsis filicina Uredinopsis filicina Uredinopsis osmundae Uredinopsis pteridis Uromyces appendiculatus U. appendiculatus Uromyces ari-triphylli Podophyllum peltatum Syzygium jambos Torreya californica Davesia sp. Gynoxys sp. Lupinus sp. Eupatorium sp. Pinus sp. Rubus sp. Mahonia aquifolium Xylopia cf. amazonica Xylopia cayennensis Solanum argentum Millettia grandis Adenocalymna sp. Berberis hallii Mikania micrantha Fabaceae sp. Coffea arabica C. arabica Deparia pycnosora Cystopteris fragilis Deparia petersenii Malva parviflora Fraxinus platypoda Alnus sp. Struthiopteris spicant S. spicant Polystichum braunii Dryopteris dilatata D. dilatata Asplenium ruta-muraria A. ruta-muraria Polypodium vulgare Asplenium scolopendrium A. scolopendrium Lygodium flexuosum Polystichum aculeatum Polystichum setiferum Protium heptaphyllum Meum athamanticum Schefflera wallichiana Annona exsucca Glycine ma Annona sericea A. exsucca Rosa x damascena f. trigintipetala Duchesnea sp. Grossularia sp. Calystegia sepium Rhamnus cathartica − Physalis lancelata − Polyalthia longifolia Monanthotaxis caffra Albizia sp. Dalbergia hostillis Xylopia aethiopica X. aethiopica Smilax sp. Vitex doniana Alchemilla vulgaris Prunus domestica undetermined Rosaceae Phegopteris connectilis P. connectilis Athyrium sp. Pteridium esculentum Phaseolus vulgaris − Arisaema triphyllum BPI 842277 115012-Mr DV29.1 BRIP 57717 R15 PUR N11562 PUR 90104 CFB22250 BPI 871660 BPI 871101 BPI US116382 (T) ZT Myc 3397 PUR N3728 ZT Myc 582 BPI 871906 QCAM6453 BPI 844288 PUR N16494 BPI 843642 RAC010 TSH-R25518 FO 47825 PDD 71999 BRIP 57522 ZT Myc 56551 BPI 871107 KR-M-0038516 KR-M-0038519 KR-M-0050247 KR-M-0039321 KR-M-0043165 KR-M-0048133 KR-M-0048134 KR-M-0043190 KR-M-0025400 KR-M-0043186 IBAR11436 KR-M-0043187 KR-M-0049177 RB:757071 KR0012164 AMH:9528 ZT:Myc 49000 BPI 871755 BPI 863563 ZT:Myc 48999 TFS01 BPI 843392 BPI 871515 BPI 871465 BPI 844300 ECS BPI 844306 ECS353 ZT HeRB 251 ZT Myc 1976 BRIP 56910 PUR N16513 PC 0096730 NY s.n. ZT Myc 44038 BPI 871108 BPI 843828 KR-0010966 BPI 881364 KR-M-0050249 USA: Maryland Australia − Australia: Western Australia − − − − USA: Virginia Germany Brazil: Paraiso − − − − Ecuador: Quito Costa Rica − Mexico Peru Japan: Ibaraki − New Zealand Australia: Queensland Japan Costa Rica − − − − − − − − − − Thailan: Chiang Mai − − Brazil − India − Zimbabwe − − Saudi Arabia USA: Maryland USA: North Dakota USA: Maryland USA: North Dakota − USA: North Dakota − − − Australia: Western Australia − − − − Zambia Switzerland Iran Italy − − USA: New York Australia: Tasmania − − USA: Maryland DQ354543 KF792096 AF522183 KT199394 MW049250 MW049251 MW049252 AY700193 DQ354560 DQ354531 JF263460 JF263477 MW049254 KM217352 MW049256 MG596499 DQ354516 MW049263 DQ354566 MN386221 LC576607 AF426229 KJ698627 KU296888 KP858145 DQ354561 MK302193 MK302189 MK302211 MK302192 MK302191 MK302194 MK302195 MK302190 MK302199 MK302198 LC498526 MK302202 MK302212 MH047186 MW049272 KF550283 KF528034 DQ354537 KF528028 KF528032 KJ867552 DQ354553 DQ354514 DQ354512 DQ354526 AF522177 DQ354522 AF522178 JF263493 JF263495 KJ862350 MW147053 KM217350 KM217355 KM217354 DQ354541 DQ354550 DQ354542 JF907676 MK302213 AF426237 MG907244 KM249869 AY745704 AF522182 DQ354529 Aime (2006) Tan et al. (2014) Szaro & Bruns (2002)* McTaggart et al. (2016) Aime & McTaggart (2021) Aime & McTaggart (2021) Aime & McTaggart (2021) Matheny et al. (2004a)* Aime (2006) Aime (2006) Beenken et al. (2012) Beenken et al. (2012) Aime & McTaggart (2021) Beenken & Wood (2015) Aime & McTaggart (2021) Barnes & Ordonez (2017)* Aime (2006) Aime & McTaggart (2021) Aime (2006) Gamarra-Gamarra et al. (2019)* This study Maier et al. (2003) Padamsee & McKenzie (2014) McTaggart et al. (2016) Beenken & Wood (2015) Aime (2006) Bubner et al. (2019) Bubner et al. (2019) Bubner et al. (2019) Bubner et al. (2019) Bubner et al. (2019) Bubner et al. (2019) Bubner et al. (2019) Bubner et al. (2019) Bubner et al. (2019) Bubner et al. (2019) Ono et al. (2020) Bubner et al. (2019) Bubner et al. (2019) Martins et al. (2019) Aime & McTaggart (2021) Baiswar et al. (2014) Beenken (2014) Aime (2006) Beenken (2014) Beenken (2014) El-Deeb et al. (2014)* Aime (2006) Aime (2006) Aime (2006) Aime (2006) Szaro & Bruns (2002)* Aime (2006) Szaro & Bruns (2002)* Beenken et al. (2012) Beenken et al. (2012) McTaggart et al. (2015) Aime & McTaggart (2021) Beenken & Wood (2015) Beenken & Wood (2015) Beenken & Wood (2015) Aime (2006) Aime (2006) Aime (2006) Yun et al. (2011) Bubner et al. (2019) Maier et al. (2003) Aime et al. (2018) McTaggart et al. (2014) Matheny et al. (2004b)* Szaro & Bruns (2002)* Aime (2006) PUR N16024 BRIP 60091 Ua39 TDB BPI 871111 BPI: The National Fungal Collections, U.S. Depertment of Agriculture, USA. BRIP: Plant Pathology Herbarium, Indooroopilly, Australia. HMJAU: Mycological Herbarium of Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, China. IBAR: The Herbarium of Systematic Mycology, Ibaraki University, Japan. RB: R. Bauer (private collection); TSH-R: Rust collection of mycological herbarium of University of Tsukuba, Japan. WM, W. Maier (private collection). Other acronyms are not registered. *Direct submission. doi: 10.47371/mycosci.2021.06.006 ― 366 ― I. Okane et al. / Mycoscience VOL.62 (2021) 364-372 oma torreyae Bonar (AF522183) was chosen as the outgroup (McTaggart, Geering, & Shivas, 2014; Beenken, 2017). The DNA sequences were aligned using MAFFT 7 multiple sequence alignment software with the G-INS-I option (Katoh & Standly, 2013). The aligned sequence data were manually checked using BioEdit ver. 7.1.9 software (Hall, 1999). All the sequences analyzed in this study were deposited in the DNA Data Bank of Japan as LC575054–LC575057 (Table 1). Sequence alignment data were provided in Electronic Supplementary Material S1. Phylogenetic analysis was performed with Bayesian inference (BI) using MrBayes v.3.2.1 software (Ronquist et al., 2012) and Maximum likelihood (ML) using raxmlGUI ver. 1.5b2 mounting RAxML ver. 8.1.2 (Silvestro & Michalak, 2012) and MEGA X (Kumar, Stecher, Li, Knyaz, & Tamura, 2018). The neighbor joining (NJ) and maximum parsimony (MP) methods were also conducted using MEGA X, and MP analysis was performed using the heuristic search option with the bootstrap (BS) test (1,000 replicates) and the tree-bisection-regrafting (TBR) algorithm. ML analysis was performed using the GTR+G+I model with 1,000 BS replicates, which was performed also in NJ analysis. All characters were equally weighted, and gaps were treated as missing data. In Bayesian inference analysis, the best-fit substitution models for different datasets were estimated using MrModeltest ver. 2.3 software (Nylander, 2004) based on the implementation of the Akaike information criterion. Four Markov chains were each run twice for 5,000,000 generations from random starting trees; the trees were sampled every 500 generations. The first 25% of all generations was discarded as burn-in, and a majority rule consensus tree of all remaining trees was constructed to determine the posterior probabilities (PP) for individual branches. The NJ plot (Perrière & Gouy, 1996) was used for constructing the phylogenetic tree. 2.3. Morphological observations The uredinia and/or telia on the host plants were included in our morphological observations (Table 1). Spores were mounted in a drop of lactophenol solution on glass slides for morphological observations and size measurements (30–50 spores per specimen) under a light microscope. Morphological observations of the surface structure of the urediniospores were obtained by scanning electron microscopy (SEM). For our SEM observations, we attached the sori or spores obtained from dry specimens to specimen holders using double-sided adhesive tape, after which we coated the specimens with platinum-palladium under a high vacuum using an E-1030 Ion Sputter (Hitachi, Tokyo, Japan). Prepared specimens were examined with an S-4200 scanning electron microscope (Hitachi) operated at 10 kV. 3. Results 3.1. Molecular phylogeny In this study, while 665–1029 bp contig sequences of the rust fungi were generated (LC575054–LC575057), 28S rDNA region sequences of those sequences were adopted to examine additional sequences chosen based on those previously examined for the phylogeny of rust fungi in phylogenetic analyses. All positions containing gaps and missing data were eliminated from 445 sites in the initial dataset, and then, phylogenetic analyses were conducted based on 326 sites of 81 sequences as the final dataset, including 153 parsimony-informative characters. Parsimony analysis yielded one parsimonious tree with tree length (TL) = 568, consistency index (CI) = 0.359, retention index (RI) = 0.724, and rescaled consistency index (RC) = 0.259. Bayesian analysis resulted in average standard deviation of split frequencies: 0.008504. The results of sequence analyses based on the partial 28S rDNA revealed that samples of P. lygodii and D. aneimiae constituted a monophyletic clade supported by high PP and BS values (Fig. 1). This linkage was pictured in all phylograms generated by BI, ML, MP and NJ analyses with high coefficients. 3.2. Taxonomy Desmella lygodii (Har.) Y. Ono, Okane & Aime, comb. nov. Figs. 2, 3 and Table 3 MycoBank No.: MB 837572. Basionym: Uredo lygodii Har., J. Bot. 14: 117. 1900. [TYPE on Lygodium sp. from Brazil, Pernambuco, date not reported, Gardener-1229 (holotype in P; isotype BPI 155242)] Synonyms: Milesia lygodii (Har.) Buriticá, in Buriticá & Pardo-Cardona, Revista Acad. colomb. cienc. exact. fís. nat. 20 (no. 77): 234 (1996) Puccinia lygodii (Har.) Arthur, Bull. Torrey Bot. Club 51: 55 (1924) Description: Spermogonia and aecia unknown. Sori milesia-type, scattered or in loose groups on the abaxial leaf surface, dome-shaped, surrounded by dark brown or almost blackish epidermis, subepidermal in origin, covered by a layer of thin-walled peridium (Fig. 2A), without ostiolar cell, aparaphysate, and becoming erumpent by a central aperture. Urediniospores borne singly on a short pedicel, appearing almost sessile, obovoid, obovoid-ellip- Table 3 - Morphological characters of Desmella lygidii and D. anemiae. Desmella lygodii Urediniospores Teliospores Size (μm) Wall thickness (μm) Surface Size (μm) Wall thickness (μm) Apicl thickness (μm) Pedicel (μm) 21–33 × 15–24 (Ave: 27.5 × 19.9) 1.0–2.3 (Ave: 1.59) Echinulate; partly smooth 24–30 × 21–26 (Ave: 27.4 × 24.2) 1.1–2.2 (Ave: 1.70) 2.8–6.0 (Ave: 4.23) 9–65 (Ave: 26.0) Table 3 continued Desmella anemiae Urediniospores Teliospores Size (μm) Wall thickness (μm) Surface Size (μm) Wall thickness (μm) Apical thickness (μm) 23–31 × 20–28 (Ave: 26.1 × 24.1) 1.0–2.6 (Ave: 1.98) Echinulate 27–32 × 21–27 (Ave: 29.6 × 25.4) 0.7–1.9 (Ave: 1.22) 1.9–8.1 (Ave: 4.63) doi: 10.47371/mycosci.2021.06.006 ― 367 ― I. Okane et al. / Mycoscience VOL.62 (2021) 364-372 Fig. 1 – A phylogenetic tree for Desmella lygodii and other rust fungi, including Desmella aneimiae and template fern rust fungi, constructed from a Bayesian inference (BI) analysis of partial large-subunit rDNA gene regions. The outgroup is Caeoma torreyae. The support values for the nodes are shown: upper left, Bayesian inference posterior probabilities (BI PP); upper right, maximum likelihood bootstrap (ML BS), lower left, neighbor joining bootstrap (NJ BS); lower, right, maximum parsimony bootstrap (MP BS). Branches supported by high values of all analyses are shown in bold line. doi: 10.47371/mycosci.2021.06.006 ― 368 ― I. Okane et al. / Mycoscience VOL.62 (2021) 364-372 Fig. 2 – Sorus structure and spores of Desmella lygodii (A–C: IBAR 10744, D, E: BPI 20025, the lectotype) and spores of D. aneimiae (F: IBAR 4262). A: Vertical section of uredium. The sorus is covered with a layer of thin-walled peridium (arrowhead). B: Urediniospores with germ pores on equatorial zone (arrowheads). C: Pedicellate teliospores and echinulate urediniospores. D: Pedicellate teliospores of the lectotype. E: Echinulate urediniospores of the lectotype. F: Teliospores of D. aneimiae. Bars: A–F 20 μm. Fig. 3 – Scanning electron micrographs of echinulate urediniospore surfaces of Desmella lygodii with a lateral smooth spot (arrowheads). A: IBAR 10744. B: PUR F3717. Bars: 7.5 μm. soid, or broadly ellipsoid, and 21–33 × 15–24 μm ( x = 27.5 × 19.9 μm) in size; the wall 1–2.3 μm ( x = 1.59 μm) thick, yellowish or pale yellowish brown, with 2 (3) germ pores on equatorial zone (Fig. 2B), echinulate with a lateral smooth spot (Figs. 2C, 3). Teliospores produced together with urediniospores, pedicellate, twocelled by a vertical or oblique septum, ovoid, ovoid-ellipsoid or oblate, and 24–30 × 21–26 μm ( x = 27.4 × 24.2 μm) in size; the wall 1–2.2 μm ( x = 1.7 μm) thick at side, 2.8–6 μm ( x = 4.2 μm) at the apex, yellowish, pale yellowish or almost colorless, brown, smooth, and germinating in situ; the pedicel persistent, colorless, and 9–65 μm ( x = 26.0 μm) long (Fig. 2C, D). Specimens examined: II on Lygodium sp. [later identified as L. polymorphum (Cav.) Kunth], BRAZIL: Pernambuco, date not reported, Gardener-1229, BPI 155242 (isotype of Uredo lygodii); SUdoi: 10.47371/mycosci.2021.06.006 RINAM: Tumatumari, 11 Jul 1922, Sydow, BPI 148715 (the type of Milesina lygodii Syd.), II, III on Lygodium flexuosum (L.) Sw., GUYANA, 28 Dec 2013, M. C. Aime, PUR N15340; on L. japonicum (Thunb.) Sw., U.S.A.: Florida, 12 May 2009, M. C. Aime, PUR N16672; Mississippi, 20 Oct 2006, R. S. Peterson, BPI 878109; Florida, 7 Oct 2015, Y. Ono, IBAR 10744; Georgia, 11 Oct 2015, Y. Ono, IBAR 10751; 11 Oct 2015, Y. Ono, IBAR 10753; on L. venustum Sw., BRAZIL: Vicinity of Bahia, 28 May 1915, J. N. Rose & P. G. Russell, PUR F3717 and BPI 20025 (the specimen cited by Arthur 1924 and designated as the lectotype by Hennen, Figueiredo, Carvalho Jr., & Hennen, 2005); on Lygodium sp., U.S.A.: Florida, 18 Oct 2009, M. C. Aime, PUR N16678. Additional specimens examined: Desmella aneimiae, II, III on Thelypteris sp., PANAMA: Chiriqui, 29 Nov 2004, J. R. Hernandez, BPI 864108; on Polypodiaceae, BRAZIL: Sao Paulo, 26 Jul 1976, J. F. ― 369 ― I. Okane et al. / Mycoscience VOL.62 (2021) 364-372 Hennen, M. B. Figueiredo & Y. Ono, IBAR 4262; II on Nephrolepis exaltata (L.) Schott, U.S.A: Hawaii, 24 Dec 2007, Y. Ono. Note: The Lygodium rust fungus under discussion was first named as Uredo lygodii Har. based on a fungus parasitizing an unidentified Lygodium collected in Pernambuco, Brazil (Hariot, 1900, holotype in BPI). Arthur (1924) found two-celled, pedicellate teliospores on a specimen on L. venustum (originally reported as L. polymorphum) collected in Bahia, Brazil in 1915 by Rose and Russell (19664a in PUR) placing the species in Puccinia, where it shared the characteristic of 2-celled teliospores with other members of the genus. Buriticá and Pardo-Cardona (1996) described Milesia lygodii (Har.) Buriticá as anamophic stage of P. lygodii. Hennen, Figueiredo, Carvalho Jr., and Hennen (2005) proposed to typify the Rose and Russell’s collection (19664a: PUR F3717 and BPI 20025) and to treat P. lygodii as a new name attributed Arthur as a sole author of the fungus name, because he was the first to discover the teleomorph and named the fungus based on it. This nomenclatural procedure is, however, not compatible with the current International Code of Nomenclature for algae, fungi, and plants (https://www.iapt-taxon.org/nomen/main.php, accessed 8 Aug 2020). Milesina lygodii Syd. was once described and named based on an unidentified Lygodium from Guyana (types in BPI, ILL, and NY) (Sydow, 1925). He did not observe teliospores but concluded so apparently because of its thin cellular-peridium bound uredinia, which was characteristic of a milesia-type anamorph. After careful examination of the type materials, Faull (1932) assumed that it was conspecific with P. lygodii and, thus, excluded from the genus Milesia (≡ Milesina). A fungus on L. japonicum in Florida was once reported as Uredinopsis sp. (Alfieri Jr., Langdon, Wehlburg, & Kimbrough, 1984), but it was later determined as P. lygodii (McCain, Hennen, & Ono, 1990). 4. Discussion Our analyses place the Lygodium rust fungus in a monophyletic group with D. aneimiae (Fig. 1). Desmella lygodii occurs only on Lygodium (Schizaeaceae, Schizeaeales), while D. aneimiae infects at least 25 genera of leptosporangiate Polypodiopsida including Anemia (Anemiaceae, Schizaeales). The Desmella clade was sister to Sphaerophragmiaceae sensu Beenken (2017) in the phylogenetic analyses based on the 28S rDNA region sequences (Fig. 1), while D. aneimiae was included in Pucciniaceae in phylogenetic analyses using the 28S, 18S rDNA and CO3 gene coding regions, e.g., Aime & McTaggart (2021). In the phylogram using the 28S region by McTaggart, Geering and Shivas (2014), D. aneimiae which was the same collection examined by Aime and McTaggart (2021) was positioned between Pucciniaceae and the Sphaerophragmium sp.-Austropuccinia psidii (as Puccinia psidii) clade. Although the results of 28S rDNA-based analyses seem to place Desmella between Pucciniaceae and Sphaerophragmiaceae, assignment of Desmella to Pucciniaceae would be demonstrated by multiple DNA locus-based analyses. Desmella aneimiae is characterized by suprastomatal uredinia and telia (wardia-type) and diorchidioid teliospores, i.e., 2-celled pedicellate teliospores with vertical or oblique septa. Wardia-type uredinia have also been reported from Hemileia (Mikronegeriineae, Zaghouaniaceae) and Edythea (Uredinineae, Pucciniaceae) species (Cummins & Hiratsuka, 2003). In contrast, D. lygodii produces milesia-type uredia with a dome-shaped cellular peridium. Milesia-type sori are also produced in the uredinial stages of Hyalopsora, Milesina, and Uredinopsis species (Melampsorineae) among others (Cummins & Hiratsuka, 2003). doi: 10.47371/mycosci.2021.06.006 The diorchidioid teliospores are diagnostic for Desmella species (Fig. 2C, D, F). The teliospores pedicels in D. lygodii are longer than those of D. aneimiae (Fig. 2C, D, F). While both Desmella species share host and teliospore similarities, the differences in sorus development would have traditionally been used to segregate these as different genera. However, several recent studies (e.g., Aime & McTaggart, 2021) have shown that characteristics of sorus development such as suprastomatal sori and probasidia that germinate without dormancy observed in distantly related rust fungi are not homologous and that many rust fungi adapted to tropical habitats have acquired a suite of homoplasious traits that are not indicative of phylogenetic relatedness. Desmella lygodii has been found on L. japonicum, L. microphyllum (Cav.) R. Br., L. venustum, and L. volubile Sw. in the neotropics to date (Arthur, 1924; Sydow, 1925; Kern & Thurston, 1943; Hennen & McCain, 1993; Hennen, Figueiredo, Carvalho Jr., & Hennen, 2005; Hernández, Aime, & Henkel, 2005; Berndt, 2013), while L. flexuosum is newly reported as a host plant of the rust species in this study. Lygodium japonicum (Japanese climbing fern; native from India, east through southeastern Asia and China to Japan and Korea, and south to eastern Australia) (Singh & Panigrahi, 1984; Global Invasive Species Database, Retrieved June 11, 2021, from http://issg.org/ database/species/distribution.asp?si=999&fr=1&sts=&lang=EN) and L. microphyllum (Small leaf climbing fern; native to tropical and subtropical areas of Africa, India, southeastern Asia and China to Japan and Korea, northern and eastern Australia, and the Pacific islands) (Ferriter ed., 2001; Global Invasive Species Database, Retrieved June 11, 2021, from http://issg.org/database/species/ distribution.asp?si=999&fr=1&sts=&lang=EN) have been introduced from original regions to North and Central America in early 1900’s to early 1950’s (Clute, 1903 as cited in Pemberton & Ferriter, 1998; Langeland, Enloe, & Hutchinson, 2016; Global Invasive Species Database, Retrieved June 11, 2021, from http://issg.org/ database/species/distribution.asp?si=999&fr=1&sts=&lang=EN). Lygodium japonicum has become naturalized in Florida and Texas and is also cultivated as an ornamental, and L. microphyllum has naturalized in the southern United States where locally it has become a nuisance (Lygodium, Retrieved July 26, 2020, from https:// uses.plantnet-project.org/en/Lygodium). However, no fern rust fungus has been reported from those two Lygodium species in their native distribution regions. In addition, L. flexuosum is distributed from Sri Lanka and the Himalayas to southern China, Hong Kong, the Ryukyu Islands in Japan, throughout Southeast Asia to northern Queensland in Australia, and Central America (Singh & Panigrahi, 1984; Plants of the World online, Retrieved June 11, 2021, from http://plantsoftheworldonline.org/taxon/urn:lsid:ipni. org:names:17142930-1). However, no rust fungus has been found from L. flexuosum in the Americas, except for D. lygodii, while M. thailandica only on this plant in Thailand (Ono et al., 2020). Apparently D. lygodii and D. aneimiae have evolved from a most recent common ancestor in the neotropics. It is likely that D. lygodii had specialized only to Lygodium species, while D. aneimiae to a wide range of leptosporangiate fern genera and species. Thus, any Lygodium species, which had never exposed to D. lygodii but become invasive to the neotropics would be highly susceptible to D. lygodii, as in the case of L. japonicum and L. flexuosum. An old leptosporangiate fern genus, Osmunda (Osmundaceae), harbors two temperate fern rust fungi, Uredinopsis osmundae Magnus in North America (Faull, 1938; Anonymous, 1960) and Northeast Eurasia (Kuprevich & Tranzcshel, 1957) and Hyalopsora hakodatensis Hirats. f., in China (Zhuang, 1983). Uredinopsis osmundae on Athyrium sp. (Athyriaceae) and H. hakodatensis on Deparia ― 370 ― I. Okane et al. / Mycoscience VOL.62 (2021) 364-372 pycnosora (Athyriaceae) were added in the dataset of this study and confirmed their positions in a clade including the temperate fern rust groups which were assigned to the suborder Melampsorineae, in which the family Milesinaceae including Milesina, Uredinopsis and Naohidemyces was newly proposed (Aime & McTaggart, 2021) (Fig. 1). By the way, no rust fungi have been discovered in the near basal leptosporangiate ferns in Gleicheniaceae and Hymenophyllaceae. Disclosure The authors declare no conflict of interests. All the experiments undertaken in this study comply with the current laws of Japan. Acknowledgments We thank the National Fungus Collections, USDA-ARS and the Arthur Fungarium, Purdue University for loan specimens, and Mr. M. Mori for his support in DNA sequencing. 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