Phylogenetic relationships among fern rust fungi and Desmella lygodii comb. nov.

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.

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 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 (= 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 two-celled, 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.

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	BRIP 60995	Nephrolepis hirsutula	Queensland, Australia	−	KM249867	McTaggart et al. (2014)
D. aneimiae	BPI 864108	Thelypteris	Chiriqui, Panama	29 Nov, 2004	LC575057	This study
D. aneimiae	IBAR 4262	Polypodiacea	Sap Paulo, Brazil	26 Jul, 1976	−	−
D. aneimiae	IBAR 10001	Nephrolepis exaltata	Hawaii, USA	24 Dec, 2007	LC498521	This study
Desmella lygodii (T)	BPI 20025	Lygodium venustum	Vicinity of Bahia, Brazil	28 May, 1915	−	−
D. lygodii (T)	BPI 148715	Lygodium sp.	Tumatumari, Guyana	11 Jul, 1922	−	−
D. lygodii (T)	BPI 155242	Lygodium sp.	Pernambuco, Brazil	−	−	−
D. lygodii	BPI 878109	Lygodium japonicum	Mississippi, USA	20 Oct, 2006	MG907211	Aime et al. (2018)
D. lygodii	IBAR 10744	L. japonicum	Florida, USA	7 Oct, 2015	LC498523	This study
D. lygodii	IBAR 10751	L. japonicum	Georgia, USA	11 Oct, 2015	−	−
D. lygodii	IBAR 10753	L. japonicum	Georgia, USA	11 Oct, 2015	LC498525	This study
D. lygodii (T)	PUR F3717	L. venustum	Vicinity of Bahia, Brazil	28 May, 1915	−	−
D. lygodii	PUR N15340	Lygodium flexuosum	Region 9, Guyana	28 Dec, 2013	LC575054	This study
D. lygodii	PUR N16672	L. japonicum	Florida, USA	12 May, 2009	LC575055	This study
D. lygodii	PUR N16678	Lygodium sp.	Florida, USA	18 Oct, 2009	LC575056	This study

^a T in parentheses means type material.

^b 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.

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, Caeoma torreyae Bonar (AF522183) was chosen as the outgroup (McTaggart, Geering, & Shivas, 2014; Beenken, 2017).

Table 2 - Source of DNA sequence data used in phylogenetic reconstructions.

Species	Host plants	Voucher specimens	Collection sites	Accession No.	References
Allodus podophylli	Podophyllum peltatum	BPI 842277	USA: Maryland	DQ354543	Aime (2006)
Austropuccinia psidii	Syzygium jambos	115012-Mr	Australia	KF792096	Tan et al. (2014)
Caeoma torreyae	Torreya californica	DV29.1	−	AF522183	Szaro & Bruns (2002)*
Ceratocoma jacksoniae	Davesia sp.	BRIP 57717	Australia: Western Australia	KT199394	McTaggart et al. (2016)
Chardoniella gynoxidis	Gynoxys sp.	R15	−	MW049250	Aime & McTaggart (2021)
Chrysocelis lupini	Lupinus sp.	PUR N11562	−	MW049251	Aime & McTaggart (2021)
Cionothrix praelonga	Eupatorium sp.	PUR 90104	−	MW049252	Aime & McTaggart (2021)
Cronartium harknessii	Pinus sp.	CFB22250	−	AY700193	Matheny et al. (2004a)*
Cronartium ribicola	Rubus sp.	BPI 871660	USA: Virginia	DQ354560	Aime (2006)
Cumminsiella mirabilissima	Mahonia aquifolium	BPI 871101	Germany	DQ354531	Aime (2006)
Dasyspora amazonica	Xylopia cf. amazonica	BPI US116382 (T)	Brazil: Paraiso	JF263460	Beenken et al. (2012)
Dasyspora gregaria	Xylopia cayennensis	ZT Myc 3397	−	JF263477	Beenken et al. (2012)
Didymopsora solani-argentei	Solanum argentum	PUR N3728	−	MW049254	Aime & McTaggart (2021)
Diorchidium woodii	Millettia grandis	ZT Myc 582	−	KM217352	Beenken & Wood (2015)
Dipyxis mexicana	Adenocalymna sp.	BPI 871906	−	MW049256	Aime & McTaggart (2021)
Edythea quitensis	Berberis hallii	QCAM6453	Ecuador: Quito	MG596499	Barnes & Ordonez (2017)*
Endophylloides portoricensis	Mikania micrantha	BPI 844288	Costa Rica	DQ354516	Aime (2006)
Hapalophragmium derridis	Fabaceae sp.	PUR N16494	−	MW049263	Aime & McTaggart (2021)
Hemileia vastatrix	Coffea arabica	BPI 843642	Mexico	DQ354566	Aime (2006)
H. vastatrix	C. arabica	RAC010	Peru	MN386221	Gamarra-Gamarra et al. (2019)*
Hyalopsora hakodatensis	Deparia pycnosora	TSH-R25518	Japan: Ibaraki	LC576607	This study
Hyalopsora polypodii	Cystopteris fragilis	FO 47825	−	AF426229	Maier et al. (2003)
H. polypodii	Deparia petersenii	PDD 71999	New Zealand	KJ698627	Padamsee & McKenzie (2014)
Leptopuccinia malvacearum	Malva parviflora	BRIP 57522	Australia: Queensland	KU296888	McTaggart et al. (2016)
Macruropyxis fraxini	Fraxinus platypoda	ZT Myc 56551	Japan	KP858145	Beenken & Wood (2015)
Melampsoridium betulinum	Alnus sp.	BPI 871107	Costa Rica	DQ354561	Aime (2006)
Milesina blechni	Struthiopteris spicant	KR-M-0038516	−	MK302193	Bubner et al. (2019)
M. blechni	S. spicant	KR-M-0038519	−	MK302189	Bubner et al. (2019)
Milesina exigua	Polystichum braunii	KR-M-0050247	−	MK302211	Bubner et al. (2019)
Milesina kriegeriana	Dryopteris dilatata	KR-M-0039321	−	MK302192	Bubner et al. (2019)
M. kriegeriana	D. dilatata	KR-M-0043165	−	MK302191	Bubner et al. (2019)
Milesina murariae	Asplenium ruta-muraria	KR-M-0048133	−	MK302194	Bubner et al. (2019)
M. murariae	A. ruta-muraria	KR-M-0048134	−	MK302195	Bubner et al. (2019)
Milesina polypodii	Polypodium vulgare	KR-M-0043190	−	MK302190	Bubner et al. (2019)
Milesina scolopendrii	Asplenium scolopendrium	KR-M-0025400	−	MK302199	Bubner et al. (2019)
M. scolopendrii	A. scolopendrium	KR-M-0043186	−	MK302198	Bubner et al. (2019)
Milesina thailandica	Lygodium flexuosum	IBAR11436	Thailan: Chiang Mai	LC498526	Ono et al. (2020)
Milesina vogesiaca	Polystichum aculeatum	KR-M-0043187	−	MK302202	Bubner et al. (2019)
Milesina whitei	Polystichum setiferum	KR-M-0049177	−	MK302212	Bubner et al. (2019)
Neopuccinia bursa	Protium heptaphyllum	RB:757071	Brazil	MH047186	Martins et al. (2019)
Nyssopsora echinata	Meum athamanticum	KR0012164	−	MW049272	Aime & McTaggart (2021)
Nyssopsora thwaitesii	Schefflera wallichiana	AMH:9528	India	KF550283	Baiswar et al. (2014)
Phakopsora cherimoliae	Annona exsucca	ZT:Myc 49000	−	KF528034	Beenken (2014)
Phakopsora pachyrhizi	Glycine ma	BPI 871755	Zimbabwe	DQ354537	Aime (2006)
Phakopsora pistila	Annona sericea	BPI 863563	−	KF528028	Beenken (2014)
Phakopsora rolliniae	A. exsucca	ZT:Myc 48999	−	KF528032	Beenken (2014)
Phragmidium mucronatum	Rosa x damascena f. trigintipetala	TFS01	Saudi Arabia	KJ867552	El-Deeb et al. (2014)*
Phragmidium tormentillae	Duchesnea sp.	BPI 843392	USA: Maryland	DQ354553	Aime (2006)
Puccinia caricis	Grossularia sp.	BPI 871515	USA: North Dakota	DQ354514	Aime (2006)
Puccinia convolvuli	Calystegia sepium	BPI 871465	USA: Maryland	DQ354512	Aime (2006)
Puccinia coronata	Rhamnus cathartica	BPI 844300	USA: North Dakota	DQ354526	Aime (2006)
Puccinia graminis	−	ECS	−	AF522177	Szaro & Bruns (2002)*
Puccinia physalidis	Physalis lancelata	BPI 844306	USA: North Dakota	DQ354522	Aime (2006)
Pucciniastrum epilobii	−	ECS353	−	AF522178	Szaro & Bruns (2002)*
Puccorchidium polyalthiae	Polyalthia longifolia	ZT HeRB 251	−	JF263493	Beenken et al. (2012)
Puccorchidium popowiae	Monanthotaxis caffra	ZT Myc 1976	−	JF263495	Beenken et al. (2012)
Sphaerophragmium acaciae	Albizia sp.	BRIP 56910	Australia: Western Australia	KJ862350	McTaggart et al. (2015)
Sphaerophragmium longicorne	Dalbergia hostillis	PUR N16513	−	MW147053	Aime & McTaggart (2021)
Sphenorchidium deightonii	Xylopia aethiopica	PC 0096730	−	KM217350	Beenken & Wood (2015)
Sphenorchidium xylopiae	X. aethiopica	NY s.n.	−	KM217355	Beenken & Wood (2015)
Sphenospora smilacina	Smilax sp.	ZT Myc 44038	−	KM217354	Beenken & Wood (2015)
Tegillum scitula	Vitex doniana	BPI 871108	Zambia	DQ354541	Aime (2006)
Trachyspora intrusa	Alchemilla vulgaris	BPI 843828	Switzerland	DQ354550	Aime (2006)
Tranzschelia discolor	Prunus domestica	KR-0010966	Iran	DQ354542	Aime (2006)
Triphragmium ulmariae	undetermined Rosaceae	BPI 881364	Italy	JF907676	Yun et al. (2011)
Uredinopsis filicina	Phegopteris connectilis	KR-M-0050249	−	MK302213	Bubner et al. (2019)
Uredinopsis filicina	P. connectilis		−	AF426237	Maier et al. (2003)
Uredinopsis osmundae	Athyrium sp.	PUR N16024	USA: New York	MG907244	Aime et al. (2018)
Uredinopsis pteridis	Pteridium esculentum	BRIP 60091	Australia: Tasmania	KM249869	McTaggart et al. (2014)
Uromyces appendiculatus	Phaseolus vulgaris	Ua39	−	AY745704	Matheny et al. (2004b)*
U. appendiculatus	−	TDB	−	AF522182	Szaro & Bruns (2002)*
Uromyces ari-triphylli	Arisaema triphyllum	BPI 871111	USA: Maryland	DQ354529	Aime (2006)

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.

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.

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.

3.2. Taxonomy

Desmella lygodii (Har.) Y. Ono, Okane & Aime, comb. nov. Figs. 2, 3 and Table 3

MycoBank No.: MB 837572.

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.

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)

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-ellipsoid, or broadly ellipsoid, and 21-33 × 15-24 μm ($\overline{x}$ = 27.5 × 19.9 μm) in size; the wall 1-2.3 μm ($\overline{x}$ = 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, two-celled by a vertical or oblique septum, ovoid, ovoid-ellipsoid or oblate, and 24-30 × 21-26 μm ($\overline{x}$ = 27.4 × 24.2 μm) in size; the wall 1-2.2 μm ($\overline{x}$ = 1.7 μm) thick at side, 2.8-6 μm ($\overline{x}$ = 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 ($\overline{x}$ = 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); SURINAM: 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. 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).

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 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. This study was partly supported by the Institute for Fermentation, Osaka, Japan (Grant Number L-2015-1-005 to YY and G-2018-1-019 to YO) and a Grant-in-Aid for Scientific Research No. 25450056 from the Japan Society for the Promotion of Sciences (YO).

References

• Aime, M. C. (2006). Toward resolving family-level relationships in rust fungi (Uredinales). Mycoscience, 47, 112-122. https://doi.org/10.1007/S10267-006-0281-0
• Aime, M. C., Bell, C. D., & Wilson, A. W. (2018). Deconstructing the evolutionary complexity between rust fungi (Pucciniales) and their plant hosts. Studies in Mycology, 89, 143-152. https://doi.org/10.1016/j.simyco.2018.02.002
• Aime, M. C., & McTaggart, A. R. (2021). A higher-rank classification for rust fungi, with notes on genera. Fungal Systematics and Evolution, 7, 21-47. https://doi.org/10.3114/fuse.2021.07.02
• Alfieri Jr., S. A., Langdon, K. R., Wehlburg, C., & Kimbrough, J. W. (1984). Index of Plant Diseases in Florida (Revised). Florida Department of Agriculture and Consumer Services, Division of Plant Industry Bulletin, 11, 1-389.
• Anonymous. (1960). Index of plant diseases in the United States. U.S.D.A. Agric Handb, 165, 1-531.
• Arthur, J. C. (1924). New species of Uredinales - XV. Bulletin of the Torrey Botanical Club, 51, 51-59.
• Baiswar, P., Ngachan, S. V., & Chandra, S. (2014). Identification of Nyssopsora thwaitesii on Schefflera in northeast India. Australasian Plant Disease Notes 9, 124. http://dx.doi.org/10.1007/s13314-014-0124-3
• Beenken, L. (2014). Pucciniales on Annona (Annonaceae) with special focus on the genus Phakopsora. Mycological Progress, 13, 791-809. http://dx.doi.org/10.1007/s11557-014-0963-5
• Beenken, L. (2017). Austropuccinia: a new genus name for the myrtle rust Puccinia psidii placed within the redefined family Sphaerophragmiaceae (Pucciniales). Phytotaxa, 297, 53-61. https://doi.org/10.11646/phytotaxa.297.1.5
• Beenken, L., & Wood, A. R. (2015). Puccorchidium and Sphenorchidium, two new genera of Pucciniales on Annonaceae related to Puccinia psidii and the genus Dasyspora. Mycological Progress, 14, 49. https://doi.org/10.1007/s11557-015-1073-8
• Beenken, L., Zoller, S., & Berndt, R. (2012). Rust fungi on Annonaceae II: the genus Dasyspora Berk. & M. A. Curtis. Mycologia, 104, 659-681. https://doi.org/10.3852/11-068
• Berndt, R. (2013). First catalogue of the rust fungi of French Guiana, northern South America. Mycological Progress, 12, 193-211. https://doi.org/10.1007/s11557-012-0826-x
• Bubner, B., Buchheit, R., Friedrich, F., Kummer, V., & Scholler, M. (2019). Species identification of European forest pathogens of the genus Milesina (Pucciniales) using urediniospore morphology and molecular barcoding including M. woodwardiana sp. nov. MycoKeys, 48, 1-40. https://doi.org/10.3897/mycokeys.48.30350.
• Buriticá, C. P., & Pardo-Cardona, V. M. (1996). Flora Uredineana Colombiana. Revista de la Academia Colombiana de Ciencias Exactas Físicas y Naturales, 20, 183-236.
• Cummins, G. B., & Hiratsuka, Y. (2003). Illustrated Genera of Rust Fungi (3rd edition). St. Paul: APS Press.
• Cunningham, G. H. (1931). The rust fungi of New Zealand: together with the biology, cytology and therapeutics of the Uredinales. Dunedin, New Zealand: John McIndoe.
• de Vienne, D. M., Refregier, G., Lopez-Villavicencio, M., Tellier, A., Hood, M.E., & Giraud, T. (2013). Cospeciation vs host-shift speciation: methods for testing, evidence from natural associations and relation to coevolution. New Phytologist, 198, 347-385. https://doi.org/10.1111/nph.12150
• Faull, J. H. (1932). Taxonomy and geographical distribution of the genus Milesia. Contributions from the Arnold Arboretum of Harvard University, 2, 1-138.
• Faull, J. H. (1938). Taxonomy and geographical distribution of the genus Uredinopsis. Contributions from the Arnold Arboretum of Harvard University, 11, 1-120.
• Ferriter, A. (Ed.). (2001). Lygodium management plan for Florida: A report from the Florida Exotic Pest Plant Council's Lygodium Task Force. Florida: Florida Exotic Pest Plant Council.
• Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95-98.
• Hariot, P. (1900). Urédinées et Ustilaginées nouvelles. Journal de Botanique (Paris), 14, 115-118.
• Hart, J. A. (1988). Rust fungi and host plant coevolution: do primitive hosts harbor primitive parasites? Cladistics, 4, 339-366. https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1096-0031.1988.tb00519.x
• Hennen, J. F., Figueiredo, M. B., Carvalho Jr., A. A., & Hennen, P. G. (2005). Catalogue of species of plant rust fungi (Uredinales) of Brazil. http://www.jbrj.gov.br
• Hennen, J. F., & McCain, J. W. (1993). New species and record of Uredinales from the neotropics. Mycologia, 85, 970-986.
• Hernández, J. R., Aime, M. C., & Henkel, T. W. (2005). The rust fungi (Uredinales) of Guyana. Sydowia, 57, 189-222.
• Katoh, K., & Standly, D. M. (2013). MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution, 30, 772-780. https://doi.org/10.1093/molbev/mst010
• Kern, F. D., & Thurston Jr., H. W. (1943). Additions to the Uredinales of Venezuela. Mycologia, 35, 434-445.
• Kirk, P. M., Cannon, P. F., Minter, D. W., & Stalpers, J. A. (2008). Dictionary of the Fungi. 10th Ed. CABI, UK.
• Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35, 1547-1549. https://doi.org/10.1093/molbev/msy096
• Kuprevich, V, & Transhel, V. (1957). Cryptogamic plants of the USSR, Vol. IV, Rust fungi. No. 1, Family Melampsoraceae. Moscow: The Academy of Science U.S.S.R.
• Langeland, K. A., Enloe, S. F., & Hutchinson, J. (2016). Natural area weeds: Old world climbing fern (Lygodium microphyllum). Florida: the Agronomy Department, UF/IFAS Extension, University of Florida. http://edis.ifas.ufl.edu.
• Leppik, E. E. (1953). Some viewpoints on the phylogeny of rust fungi. I. Coniferous rusts. Mycologia, 45, 46-74. https://doi.org/10.1080/00275514.1953.12024250
• Leppik, E. E. (1965). Some viewpoints on the phylogeny of rust fungi. V. Evolution of biological specialization. Mycologia, 57, 6-22. https://doi.org/10.1080/00275514.1965.12018189
• Leppik, E. E. (1967). Some viewpoints on the phylogeny of rust fungi. VI. Biogenic radiation. Mycologia, 59, 568-579. https://doi.org/10.1080/00275514.1967.12018449
• Maier, W., Begerow, D., Weiss, M., & Oberwinkler, F. (2003). Phylogeny of the rust fungi: an approach using nuclear large subunit ribosomal DNA sequences. Canadian Journal of Botany, 81, 12-23. https://doi.org/10.1139/b02-113
• Martins Jr., A. S., Sakuragui, C.M., Hennen, J. F., & Carvalho Jr., A. A. (2019). Neopuccinia (Pucciniales): a new Puccinia-like genus from the Brazilian Cerrado. Phtotaxa, 406, 169-179. https://doi.org/10.11646/phytotaxa.406.3
• McCain, J. W., Hennen, J. F., & Ono, Y. (1990). New host species and state distribution records for North American rust fungi (Uredinales). Mycotaxon, 39, 281-300.
• McTaggart, A. R., Doungsa-Ard, C., Geering, A. D., Aime, M. C., & Shivas, R. G. (2015). A co-evolutionary relationship exists between Endoraecium (Pucciniales) and its Acacia hosts in Australia. Persoonia, 35, 50-62. http://dx.doi.org/10.3767/003158515X687588
• McTaggart, A. R., Geering, A. W. D., & Shivas, R. G. (2014). Uredinopsis pteridis and Desmella aneimiae, the first rust fungi (Pucciniales) reported on ferns (Pteridophyta) in Australia. Australasian Plant Disease Notes, 9, 149. https://doi.org/10.1007/s13314-014-0149-7
• McTaggart, A. R., Shivas, R. G., van der Nest, M. A., Roux, J., Wingfield, B. D., & Wingfield, M. J. (2016). Host jumps shaped the diversity of extant rust fungi (Pucciniales). New Phytologist, 209, 1149-1158. https://doi.org/10.1111/nph.13686
• Nylander, J. A. A. (2004). MrModeltest version 2. Program distributed by the author. Uppsala: Evolutionary Biology Centre, Uppsala University.
• O'Donnell, K. (1993). Fusarium and its near relatives. In: D. R. Reynolds, & J. W. Taylor (Eds.), The fungal holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematics (pp. 225-233). Wallingford: CABI.
• Ono, Y. (2020). Additional records of rust fungi (Pucciniales) in the Hawaiian Islands. Bulletin of the College of Education, Ibaraki University (Natural Science), 69, 61-73.
• Ono, Y., Ohmachi, K., Unartngam, J., Okane, I., Ayawong, C., & Janruang, P. (2020). Milesina thailandica, a second rust fungus on an early diverged leptosporangiate fern genus, Lygodium, found in Thailand. Mycological Progress, 19, 147-154. https://doi.org/10.1007/s11557-019-01549-4
• Padamsee, M., & McKenzie, E. H. C. (2014). A new species of rust fungus on the New Zealand endemic plant, Myosotidium, from the isolated Chatham Islands. Phytotaxa, 174, 223-230. https://dx.doi.org/10.11646/phytotaxa.174.4.3
• Pemberton, R. W., & Ferriter, A. P. (1998). Old world climbing fern (Lygodium microphyllum), a dangerous invasive weed in Florida. American Fern Journal, 88, 165-175.
• Perrière, G., & Gouy, M. (1996). WWW-Query: an on-line retrieval system for biological sequence banks. Biochimie, 78, 364‒369.
• Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Hohna, S., Larget, B., Liu, L., Suchard, M. A., & Huelsenbeck, J. P. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61, 539-542. https://doi.org/10.1093/sysbio/sys029
• Roy, B. A. (2001). Patterns of association between crucifers and their flower-mimic pathogens: host jumps are more common than coevolution or cospeciation. Evolution, 55, 41-53. https://doi.org/10.1111/j.0014-3820.2001.tb01271.x
• Savile, D. B. O. (1976). Evolution of the rust fungi (Uredinales) as reflected by their ecological problems. In: M. Hecht, W. Steere, & B. Wallace (Eds.), Evolutionary biology (pp. 137-207). New York, NY: Springer.
• Silvestro, D., & Michalak, I. (2012). raxmlGUI: a graphical front-end for RAxML. Organisms Diversity and Evolution, 12, 335-337; https://doi.org/10.1007/s13127011-0056-0
• Singh, S., & Panigrahi, G. (1984). Systematics of genus Lygodium Sw. (Lygodiaceae) in India. Proceedings of Indian Academy of Science, 93, 119-133.
• Sjamsuridzal, W., Nishida, H., Ogawa, H., Kakishima, M., & Sugiyama, J. (1999). Phylogenetic positions of rust fungi parasitic on ferns: evidence from 18S rDNA sequence analysis. Mycoscience, 40, 21-27. https://doi.org/10.1007/BF02465669
• Sydow, H. (1925). Rusts of British Guiana and Trinidad. Mycologia, 17, 255-262. https://doi.org/10.2307/3753892
• Sydow, H., & Sydow, P. (1918). Mykologische Mitteilungen. Annales Mycologici, 16, 240-248.
• Tan, M. K., Collins, D., Chen, Z., Englezou, A., & Wilkins, M. R. (2014). A brief overview of the size and composition of the myrtle rust genome and its taxonomic status. Mycology, 5, 52-63. https://doi.org/10.1080/21501203.2014.919967
• Vilgalys, R., & Hester, M. (1990). Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology, 172, 4238-4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990
• Yun, H. Y., Minnis, A. M., Kim, Y. H., Castlebury, L. A., & Aime, M. C. (2011). The rust genus Frommeella revisited: a later synonym of Phragmidium after all. Mycologia, 103, 1451-1463. https://doi.org/10.3852/11-120
• Zhuang, J. -Y. (1983). A provisional list of Uredinales of Fujian Province, China. Acta Mycologica Sinica, 2, 146-158.