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Novel fungi from an ancient niche: cercosporoid and related sexual morphs on ferns

The fern flora of the world (Pteridophyta) has direct evolutionary links with the earliest vascular plants that appeared in the late Devonian. Knowing the mycobiota associated to this group of plants is critical for a full understanding of the Fungi. Nevertheless, perhaps because of the minor economic significance of ferns, this niche remains relatively neglected by mycologists. Cercosporoid fungi represent a large assemblage of fungi belonging to the Mycosphaerellaceae and Teratosphaeriaceae (Ascomycota) having cercospora-like asexual morphs. They are well-known pathogens of many important crops, occurring on a wide host range. Here, the results of a taxonomic study of cercosporoid fungi collected on ferns in Brazil are presented. Specimens were obtained from most Brazilian regions and collected over a 7-yr period (2009–2015). Forty-three isolates of cercosporoid and mycosphaerella-like species, collected from 18 host species, representing 201 localities, were studied. This resulted in a total of 21 frond-spotting taxa, which were identified based on morphology, ecology and sequence data of five genomic loci (actin, calmodulin, ITS, LSU and partial translation elongation factor 1-α). One novel genus (Clypeosphaerella) and 15 novel species (Cercospora samambaiae, Clypeosphaerella sticheri, Neoceratosperma alsophilae, N. cyatheae, Paramycosphaerella blechni, Pa. cyatheae, Pa. dicranopteridis-flexuosae, Pa. sticheri, Phaeophleospora pteridivora, Pseudocercospora brackenicola, Ps. paranaensis, Ps. serpocaulonicola, Ps. trichogena, Xenomycosphaerella diplazii and Zasmidium cyatheae) are introduced. Furthermore, 11 new combinations (Clypeosphaerella quasiparkii, Neoceratosperma yunnanensis, Paramycosphaerella aerohyalinosporum, Pa. dicranopteridis, Pa. gleicheniae, Pa. irregularis, Pa. madeirensis, Pa. nabiacense, Pa. parkii, Pa. pseudomarksii and Pa. vietnamensis) are proposed. Finally, nine new host associations are recorded for the following known fungal species: Cercospora coniogrammes, Cercospora sp. Q, Ps. abacopteridicola, Ps. lygodiicola and Ps. thelypteridis.


INTRODUCTION
Cercosporoid fungi are well-known plant pathogens that are etiological agents of leaf spot diseases of many important crops (Agrios 2005). Major diseases include angular leaf spot of bean (Pseudocercospora griseola), black leaf streak of banana (Ps. fijiensis) and leaf spots on many other hosts including grapevine (Ps. vitis), celery (Cercospora apii ) and sugarbeet (C. beticola), to name but a few (Braun et al. 2013).
The tradition of naming fungi in the absence of molecular data remains dominant in published literature, despite the limitations of this approach rendering data-driven comparisons difficult to impossible, especially in groups with known wide host ranges. Of the fungal species described in 2013, 65 % still lacked DNA data (Crous et al. 2015a). The lack of DNA barcodes is still further complicated by the lack of ex-type cultures, which are frequently not deposited in publicly available biological resource centres. This is true for fungi in general, but in the case of the cercosporoid fungi in particular, the situation is further complicated by the fact that they are often only found as asexual morphs (Goodwin et al. 2001). When the sexual morph is present, cercosporoid taxa have traditionally been classified in entirely different genera, with few morphological characters that can be used to facilitate accurate identification (Braun et al. 2013(Braun et al. , 2014(Braun et al. , 2015. Moreover, many species (especially in the tropics and subtropics) are known only from their asexual morphs, and may exhibit considerable morphological variation due to environmental conditions, encouraging mycologists to mistakenly recognise them as distinct genera. As a result, numerous asexual genera, which may eventually prove to be artificial, have been introduced e.g. Cercodeuterospora, Centrospora, Heterosporium and others (Chupp 1954). On the other hand, once these groups are subjected to molecular phylogenetic comparisons, it has frequently also led to a high number of generic lineages that previously were not discernable based on morphology alone, e.g. Paracercospora, Phaeocercospora and in the Teratosphaeriaceae (Crous et al. 2013b.
With DNA sequencing becoming widely available for use by mycologists as a reliable source of information (Taylor et al. 2000), a more concrete classification of fungi was initiated, and several studies have since been published on cercosporoid fungi (Arzanlou et al. 2007, Crous et al. 2007a, 2009b, d, 2013a, Braun et al. 2013, Groenewald et al. 2013, Bakhshi et al. 2014, Nguanhom et al. 2015. These studies have shown that some morphology-based genera were largely monophyletic, e.g. Pseudocercospora and Ramularia (Crous et al. 2013a, Groenewald et al. 2013, Bakhshi et al. 2014, Videira et al. 2015 whereas others like Passalora and other genera not recognised as cercosporoids, were clearly polyphyletic, e.g., Phloeospora, Phoma, Pseudocercosporella, Septoria and Stagonospora (Aveskamp et al. 2010, Frank et al. 2010, De Gruyter et al. 2013, Quaedvlieg et al. 2013. Despite the intense effort by mycologists over the last two centuries at describing the world's mycobiota, this task is far from being complete (Crous et al. 2015a). Several niches harbouring unique fungi that may be of relevance for understanding fungal phylogeny, have been mostly neglected. One case in point is fungi associated with ferns. Ferns are members of the division Pteridophyta (= 'Monilophyta'). In recent classifications (e.g., Smith et al. 2008) the division includes 37 families, approximately 300 genera and more than 9 000 species. Although there are presently c. 1 110 species known from Brazil, it has been estimated that this number may be far greater (Forzza et al. 2015). Approximately 60 different species of fungi have been recorded on ferns in Brazil, from which two are cercosporoid (Viégas 1961, Farr & Rossman 2015, Mendes & Urben 2015. In Brazil and elsewhere, ferns have probably been poorly collected because of the lack of economic importance of most species. One exception in the general absence of monographic treatments of fungi on ferns is the recent publications by Braun et al. (2013Braun et al. ( , 2014Braun et al. ( , 2015, a series of works aiming at congregating all cercosporoid taxa by host. Braun et al. (2013) redescribed and discussed 44 cercosporoid species occurring on 47 different fern hosts. One of these (Pseudocercospora davalliicola) was originally described from Brazil. Such significant morphological revisions based on previously published species, provide a solid foundation to facilitate future DNA phylogenetic studies.
Early results of the survey for plant pathogenic fungi occurring on ferns in Brazil indicated a plethora of novel taxa to exist in this niche. Two of the preliminary findings, namely two taxa in the Parmulariaceae, have already been published: the new genus Rhagadolobiopsis (Guatimosim et al. 2014a) and the new species Inocyclus angularis (Guatimosim et al. 2014b). Similarly, another research group in Asia has been studying fungi on ferns and have recently described the new species Venustosynnema reniformisporum and Zasmidium dicranopteridis (Kirschner & Liu 2014). Furthermore, the phylogenetic placement of the monotypic class Mixomycetes was recently elucidated based on the study of Mixia osmundae, which is an intracellular parasite of ferns (Toome et al. 2014).
The present work aims to present part of the results of a broad survey of the mycobiota of ferns in Brazil, with particular reference to the cercosporoid and related fungi which were collected in association with frond spots on members of the Pteridophyta collected in Brazil. Additionally, this work aims at partially supplementing the initiative of Braun et al. (2013) with robust DNA data, in order to promote a precise taxonomic classification of the cercosporoid fungi within Mycosphaerellaceae. In a recent study, Quaedvlieg et al. (2014) proposed employing a Consolidated Species Concept, aiming to integrate ecology, morphology, cultural characteristics and multilocus DNA phylogenetic data in order to appropriately verify species boundaries. The same approach was adopted in the present publication for the cercosporoids occurring on ferns in Brazil.

Specimens and isolates
Frond samples bearing fungal colonies were collected in Brazil from different biomes, including natural ecosystems in the Amazon, the Atlantic rainforest, the Caatinga and the Cerrado, as well as ruderal areas and gardens between 2009 and 2015. These were dried in a plant press and later examined under a dissecting microscope to detect fungal structures. Such fungal structures, preferably spores, were scraped from a single frond spot, and whenever possible, single conidial colonies were established on potato carrot agar (PCA) (Crous et al. 2009e). In the case of ascospores-producing structures being present, excised lesions were placed in distilled water for approximately 2 h, after which they were placed at the bottom of Petri dish lids, over which the plate containing PCA was placed. Ascospore germination patterns were recognised using the different modes of ascospore germination proposed by Crous (1998). Freehand sections of fungal colonies were prepared and fungal structures mounted in clear lactic acid, lactophenol, lactofuchsin, and/or Melzer᾿s reagent. When necessary, sections were made using a Microm HM 520 freezing microtome. Observations were made with a Nikon SMZ1500 stereo-microscope and with a Nikon Eclipse 80i light microscope using differential interference contrast (DIC) illumination and a Nikon DS-Fi1 camera and NIS-Elements imaging software. Colony descriptions were made on 2 % malt extract agar (MEA), potato dextrose agar (PDA), PCA and oatmeal agar (OA) (Crous et al. 2009e), in the dark at 25 °C and under a 12 h light/dark regime. Colony colours (surface and reverse) were rated according to the colour charts of Rayner (1970). Representative fungarium specimens were deposited in the Fungarium of the Universidade Federal de Viçosa (VIC) and the Fungarium of the CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands (CBS H). Axenic cultures were deposited in the working collection of P.W. Crous (CPC), housed at CBS, and in the Coleção Octávio de Almeida Drumond (COAD), housed at the Universidade Federal de Viçosa. A complete list of the species and isolates included in this study is presented in Table 1.

Scanning electron microscopy
Samples of dried material containing fungal structures were mounted on stubs with double-sided adhesive tape and goldcoated using a Balzer's FDU 010 sputter coater. A Carl-Zeiss Model LEO VP 1430 scanning electron microscope (SEM) was used to analyse and generate images from the samples.

DNA isolation, amplification and sequencing
Isolates were grown on MEA plates for 20 d at 25 °C. Genomic DNA was extracted from mycelium using the Wizard ® Genomic DNA Purification Kit (Promega Corporation, WI, USA) following the manufacturer's instructions. The  subsequently diluted 50-100 times in preparation for further DNA amplification reactions. Four partial nuclear genes were initially targeted for PCR amplification and sequencing, namely 28S nrRNA gene (LSU), internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS) of the nrDNA operon, actin (act), and translation elongation factor 1-α (tef1). Additionally, for the Cercospora strains, a part of the calmodulin gene (cmdA) was amplified. The primers employed are listed in Table 2. The PCR amplifications were performed in a total volume of 12.5 μL solution containing 10-20 ng of template DNA, 1× PCR buffer, 0.63 μL DMSO (99.9 %), 1.5 mM MgCl 2 , 0.5 μM of each primer, 0.25 mM of each dNTP, 1.0 U BioTaq ® DNA polymerase (Bioline GmbH Luckenwalde, Germany). PCR conditions for ITS and LSU were set as follows: an initial denaturation (95 °C; 5 min), 35 cycles amplification (95 °C, 30 s; annealing (

Phylogenetic analyses
The three datasets consisted of 1  After topological convergence of the Bayesian runs, the following numbers of trees were generated and subsequently sampled (using a burn-in fraction of 0.25 and indicated after the slash) in order to generate the three Bayesian phylogenies: 2948/2140 for Cercospora ( Fig. 1), 4465/3572 for Pseudocercospora ( Fig.  2) and 1710 /1368 for mycosphaerella-like taxa (Fig. 3). The resulting phylogenetic trees of all three individual combined datasets showed consistent clustering of all taxa over each one of the trees, and the results are treated below. Bayesian posterior probabilities (PP) are presented on the left of each node, on each tree.

TAXONOMY
The Consolidated Species Concept was employed in this study to distinguish species, revealing a rich diversity among the cercosporoid fungi on ferns in Brazil. Forty-three isolates of cer-cosporoid and mycosphaerella-like species, collected from 18 host species representing 201 localities, were studied. The Bayesian analysis resulted in a total of 20 frond-spotting taxa, which belong to eight genera including Cercospora, Clypeosphaerella, Neoceratosperma, Paramycosphaerella, Phaeophleospora, Pseudocercospora, Xenomycosphaerella and Zasmidium. Three of these were assigned to an existing species name, one more could not be named unequivocally, a further 15 were described as new, and one novel species, as well as one new genus, are introduced below for the remaining taxon. Etymology. Name refers to the common name used for ferns in Brazil, or of native Indian Tupi language origin -samambaia.
Description in planta -Frond spots irregular, starting on the edges of the pinnulets, extending to encompass whole pinnulets and sometimes leading to the necrosis of the entire pinnule. Starting centrally, pale brown, becoming pale brown to red at the edges, coalescing, turning dark brown to black. Caespituli   Table 2 Details of primers used in this study for the PCR amplification and sequencing of different genes.      Notes -In the tef1, and cmdA phylogeny, isolates of C. samambaiae and Cercospora sp. F (sensu Groenewald et al. 2013) cluster together in a distinct well-supported clade. In the act phylogeny, C. samambaiae forms a distinct clade, whereas Cercospora sp. F cannot be distinguished from Cercospora sp. Q (sensu Groenewald et al. 2013), nor from C. coniogrammes (data not shown). The different act sequences explain the basal position of Cercospora sp. F to the C. samambaiae clade in the combined phylogeny (Fig. 1). Two Cercospora species are known to cause frond spots on species of Thelypteridaceae, namely C. abacopteridis and C. cyclosori. Cercospora abacopteridis is morphologically quite distinct from C. samambaiae in having much smaller and narrower conidiophores (15-120 × 4-5 µm), rising directly from the internal hyphae. Additionally, C. abacopteridis is only known from Singapore, causing leaf spots on Abacopteris urophylla (Braun et al. 2013). Cercospora cyclosori, described on Cyclosorus spp. from India and Taiwan, is even more distinct from C. samambaiae in having shorter and wider conidia (50 -110 × 3 -4 µm) and shorter and narrower conidiophores (25 -160 × 4 -5 µm) (Braun et al. 2013).

Cercospora sp. A
Culture characteristics -Colonies on PCA slow-growing, 60 mm diam after 28 d; flat, with sparse aerial mycelium, pale mouse grey centrally, mouse grey to olivaceous grey at periphery; reverse leaden black. Notes -Fungarium specimens of this fungus were in poor condition and no conidia were seen. Isolation was performed by conidiophore transfer only. Phylogenetically, this specimen has C. cf. physalidis (CBS 765.79) as sister clade (Fig.1), but differs from the latter by having the following number of variable sites: 11 for act, 5 for cmdA and 1 for tef1. Once no conidia were seen and all attempts to promote sporulation in vitro proved to be unsuccessful, it is not possible to determine the species boundaries of this isolate.
Culture characteristics -Colonies on MEA slow-growing, 22 mm diam after 24 d; raised, aerial mycelium velvety, laven-der grey centrally and pale vinaceous at periphery, vinaceous buff reverse. On OA, aerial mycelium sparse, mouse grey centrally, buff periphery; dark mouse grey with rosy buff periphery reverse. On PDA pale mouse grey centrally, white periphery; smoke with rosy buff periphery reverse; cultures sterile. Notes -Morphologically, Cl. sticheri is most similar to Cl. quasiparkii described on Eucalyptus sp. from Thailand (Cheewangkoon et al. 2008), but can be distinguished from it by having smaller and wider asci (45-50 × 8.5-9 µm in the later), larger ascospores (10-11 × 3-3.5 µm in the latter) and by the germination of the ascospores -following the main axis, regular in width, not distorted in Cl. sticheri (Type F, Crous 1998) whereas in Cl. quasiparkii germ tubes arise from the polar ends, develop firstly parallel to the main axis, and later grow perpendicularly, becoming distorted (Type D, Crous 1998) (Cheewangkoon et al. 2008). Additionally, it is also phylogenetically distinct (Fig. 3).
Neoceratosperma Crous & Cheew., Persoonia 32: 255. 2014 -MycoBank MB808935 Notes -Neoceratosperma has thus far been known only from its type species, N. eucalypti, isolated on Eucalyptus sp. (Myrtaceae) from Thailand . Neoceratosperma eucalypti is asexual and zasmidium-like in morphology. In the present study, we expanded the generic concept by including three additional species, two of which are known from their sexual morphs, being mycosphaerella-like in morphology. Guatimosim, R.W. Barreto & Crous, sp. nov. -MycoBank MB812816;Fig. 8 Etymology. Name refers to the host genus from which it was isolated, Alsophila.
Culture characteristics -Colonies on MEA, slow-growing, 26 mm diam after 24 d; centrally raised, with lobate, smooth margins, aerial mycelium velvety, olivaceous grey centrally, and mouse grey in the outer region; leaden black in reverse. On OA, colony radially striate with lobate margins, aerial mycelium cottony, pale mouse grey centrally and mouse greenish grey in the outer region; leaden black in reverse. On PDA colony centrally elevated, aerial mycelium sparse to absent, mouse grey centrally and producing a black halo in the outer region; leaden black in reverse; cultures sterile. Notes -Morphologically and phylogenetically, N. alsophilae is closely related to N. yunnanensis described on Eucalyptus urophylla, restricted to the southwest of China (Burgess et al. 2007). It can be distinguished from N. yunnanensis by having narrower, obclavate to broadly ellipsoidal asci (ovoid to obclavate, 27-38 × 7-11 µm in N. yunnanensis) and ascospores (10-12.5 × 2.5 -3 µm in N. yunnanensis). Moreover, N. yunnanenis is phylogenetically distinct from N. alsophilae (Fig. 3). Guatimosim, R.W. Barreto & Crous, sp. nov. -MycoBank MB812817;Fig. 9 Etymology. Name refers to the host genus from which it was isolated, Cyathea.
Culture characteristics -Colonies on MEA and OA slowgrowing, 20 mm diam after 24 d; raised, with lobate, feathery margins and velvety aerial mycelium, lavender grey centrally, leaden black mixed with lavender grey areas at periphery; irongrey reverse. On PDA, colony humid centrally, pale mouse grey centrally, mouse grey periphery; green-black reverse; cultures sterile. Notes -Neoceratosperma cyatheae is phylogenetically different from all other species in this clade (Fig. 3). It was not possible to compare N. cyatheae with N. alsophilae and N. yunnanensis since the latter species are only known from their sexual morphs (Burgess et al. 2007, this study). In contrast for N. cyatheae only the asexual morph was found, which resembles zasmidium-like fungi, which are known to be polyphyletic (Crous et al. 2009a, b  genus. In the present study, we expanded the genus by also including additional phylogenetically related taxa. (Crous & Fig. 10 Etymology. Name refers to the host genus from which it was isolated, Blechnum.
Culture characteristics -Colonies on MEA and PDA slowgrowing, 42 mm diam after 24 d; raised with lobate margins, sparse feathery aerial mycelium in centre, immersed mycelium at periphery, humid, lavender grey to white in centre, iron-grey at periphery; reverse iron-grey. On OA, colony entirely lavender grey; leaden grey with amber zones in reverse; cultures sterile.

Notes -Morphologically, Pa. blechni is rather similar to
Pa. dicranopteridis-flexuosae described on Dicranopteris flexuosa from Brazil (this study), but can be distinguished from it by having narrower obpyriform to ovoid asci (pyriform to narrowly ellipsoid, 10 -18 µm wide in Pa. dicranopteridis-flexuosae). Phylogenetically, Pa. blechni is related to Pa. dicranopteridis, which is only known from its asexual morph. Both species differ from other species within this clade (Fig. 3). Paramycosphaerella dicranopteridis is presently only known from its ITS DNA sequence data (Kirschner & Liu 2014). Nevertheless, the two species differ on 33 bp for the ITS region. Guatimosim Fig. 11 Etymology. Name refers to the host genus from which it was isolated, Cyathea.
Culture characteristics -Colonies on MEA, OA and PDA slow-growing, 23 mm diam after 24 d; raised, with lobate, undulate, feathery margins, and cottony aerial mycelium, iron-grey centrally, lavender grey at periphery; leaden black in reverse; On OA and PDA, slightly pigmenting the media, rosy vinaceous; cultures sterile. be distinguished from the latter by having longer and wider asci (24 -51 × 10 -18 μm in Pa. dicranopteridis-flexuosae and 18-33 × 9-15 µm in Pa. gleicheniae) (Ramakrishnan & Ramakrishnan 1950). In fact, the two hosts, D. flexuosa and D. linearis, are also very similar and retained as two geographical entities: the former occurring only in the Neotropics, and the latter in the Paleotropics (Mickel & Smith 2004, Bingyang et al. 2013. Phylogenetically, only ITS sequence data is available for Pa. gleicheniae (Kirschner & Liu 2014), from which only 5 bp are different from Pa. dicranopteridis-flexuosae. Nevertheless, the tree produced in this study (Fig. 3) demonstrated that Pa. gleicheniae is quite distinct from Pa. dicranopteridis-flexuosae. Additional loci should be sequenced for the former species, aiming at clarifying the true species boundaries. At present, based on the host species, geographical distribution, and until additional loci have been studied, we decided to maintain them as distinct taxa. An asexual stigmina-like morph was observed on different specimens, collected in different seasons at the same place, being associated with similar symptoms to those caused by Pa. dicranopteridis-flexuosae. However, no cultures were obtained from this fungus and the connection between these two morphs needs to be confirmed. Notes -Paramycosphaerella gleicheniae was described from India, the holotype of which has presumably been lost (Aptroot 2006). The specimens examined here are from the same host, but from a different country (Taiwan), therefore inadequate to be used as neotype. However, despite the ascospores from the Taiwanese material being somewhat different from the type (Kirschner & Liu 2014), it is probable that they are conspecific. Paramycosphaerella gleicheniae still awaits neotypification. Notes -The link between the sexual (PREM 50668, culture CBS 387.92) and asexual morph (PREM 51713) was based on morphology, and never corroborated by DNA sequence data. Because subsequent studies have revealed 'Mycosphaerella' parkii to be a species complex (Crous et al. 2006b, Cheewangkoon et al. 2008 Guatimosim, R.W. Barreto & Crous, sp. nov. -MycoBank MB812777;Fig. 13 Etymology. Name refers to the host genus from which it was isolated, Sticherus.
Culture characteristics -Colonies on MEA slow-growing, 46 mm diam after 24 d; undulated, spreading, with lobate, fea-thery margins and sparse aerial mycelium, mouse grey centrally, pale mouse grey at periphery with a distinct narrow white external rim; greenish grey reverse. On OA, cream with a honey to buff periphery; iron-grey centrally with amber periphery reverse. On PDA, mouse grey with lavender grey periphery; mouse grey reverse centrally, amber periphery; cultures sporulating moderately on OA, producing conidia. Notes -The genus Phaeophleospora, which is based on Ph. eugeniae, was collected from Eugenia uniflora (Myrtaceae) in Brazil (Crous et al. 1997) and clusters within Mycosphaerellaceae (Crous et al. 2007a). In the past, this genus included species that are presently accommodated in Teratosphaeria (= Kirramyces) and have pycnidial asexual morphs (Walker et al. 1992, Andjic et al. 2007). The new species described on Serpocaulon triseriale (Polypodiaceae) was based on material producing both the sexual and asexual morphs. Surprisingly, its asexual morph is a sporodochial hyphomycete (Fig. 3). Given the recent conidiomatal species with aseptate conidia described from ferns collected in Thailand (Crous et al. 2015b), the genus Phaeophleospora as presently defined based on DNA phylogeny, is morphologically rather diverse.  Culture characteristics -Colonies on MEA slow-growing, 26 mm diam after 20 d in the dark; surface smooth, raised with dense aerial mycelium and even margins, olivaceous grey in the centre, followed by a pale olivaceous grey ring and greenish black periphery; iron-grey reverse; cultures sterile. Notes -Pseudocercospora abacopteridicola was only known from the type specimen, collected on Abacopteris urophylla (Thelypteridaceae) from Singapore (Yen & Lim 1980, Braun et al. 2013). The specimen collected in Brazil was found on a distantly related host -Adiathum sp. (Pteridaceae). However, as morphology and biometric data are indistinguishable, instead of describing the fungus from Brazil as new, we prefer to place it in Ps. abacopteridicola until DNA of the fungus from Singapore becomes available for a molecular comparison. Guatimosim, R.W. Barreto & Crous, sp. nov. -MycoBank MB812813;Fig. 16 Etymology. Name refers to bracken -the common English name for species of Pteridium.
Culture characteristics -Colonies on MEA slow-growing, 30 mm diam after 20 d in the dark; raised with velvety aerial mycelium, pale greenish grey centrally, and mouse grey at periphery; olivaceous grey in reverse; cultures sterile. Notes -Phylogenetically, Ps. brackenicola clusters with Ps. purpurea and Ps. sordida as sister clade (Fig. 2), but differs from them by having the following number of variable sites for each locus: Ps. purpurea (7 bp for ITS, 9 bp for act, 24 bp for tef1) and Ps. sordida (8 bp for ITS, 14 bp for act, 33 bp for tef1). Morphologically, both species are clearly different from Ps. brackenicola by having larger conidiophores (20 -200 × 3.5-4.5 µm in Ps. purpurea and 20 -90 × 3.5 -5 µm in Ps. sordida) and larger conidia (20 -100 × 2 -4.5 µm in Ps. purpurea and 20 -165 × 3 -5.5 µm in Ps. sordida) (Chupp 1954, Guo & Hsieh 1995. Additionally, the hosts of Ps. purpurea and Ps. sordida are higher plant families in the Perseaceae and Bignoneaceae, respectively (Farr & Rossman 2015). Pseudocercospora brackenicola is similar to Ps. davallicola (described on Davallia fejeensis from Brazil) and to Ps. lonchitidis (described on Lonchitis hirsuta from Venezuela) (Braun et al. 2013). Molecular data are lacking for both species, but there are various morphological differences that distinguish them. Firstly, the conidia in Ps. davallicola can be formed in short chains (absent in Ps. brackenicola), and the conidiophores of Ps. davallicola are solitary, whereas on Ps. brackenicola they form fascicles emerging from stromata, through stomata (Braun et al. 2013). Secondly, Ps. lonchitidis has erumpent, well-developed stromata (loosely dense, emerging through the stoma in Ps. brackenicola), straight and thicker conidiophores, 3 -5 µm wide in Ps. davallicola (curved to sinuous, 2-3 µm wide in Ps. brackenicola), and conidiogenous loci are subdenticulate (inconspicuous in Ps. brackenicola) (Braun et al. 2013). This is the first record of a Pseudocercospora sp. on the genus Pteridium. Pseudocercospora brackenicola causes a damaging disease on its host (bracken), which is a highly noxious weed. Further investigations are required to determine its potential role as biological control agent. Notes -There are four species of Pseudocercospora known from Lygodium, namely Ps. lygodii (on L. japonicum from Taiwan), Ps. lygodiicola (on L. japonicum from China), Ps. lygodiigena and Ps. polypodiacearum (both on Lygodium sp. from India) (Braun et al. 2013). Species boundaries among these taxa are based on morphological and biometric characters, which could be considered as tentative, as the host and distribution range of these taxa are quite similar. Currently there are no records of ex-type cultures or DNA information on any of these taxa.

Pseudocercospora lygodiicola
The fungus isolated from L. volubile in Brazil has morphological and biometric data similar to P. lygodiicola, but until the latter has been epitypified, we decided to extend its host range, rather than propose a new name for the Brazilian collection. Phylogenetically, Ps. lygodiicola clusters in the same clade with three other species isolated from ferns, namely Ps. cyatheicola, Ps. rumohrae and Ps. thelypteridis (Fig. 2).    Guatimosim, R.W. Barreto & Crous, sp. nov. -MycoBank MB812814;Fig. 18 Etymology. Name refers to the state in Brazil from where the fungus was collected, Paraná.
Culture characteristics -Colonies on MEA slow-growing, 28 mm diam after 20 d in the dark; smooth with even margins, raised, aerial mycelium velvety, surface olivaceous grey, mixed with pale olivaceous grey; iron-grey in reverse; cultures sterile. Notes -Both morphs (sexual and asexual) were found occurring hypophyllously, on different fronds from the same host. Pseudocercospora paranaensis clusters in an isolated clade (Fig. 2), having Ps. basitruncata as sister clade. Besides, Ps. basitruncata is known to be an extremely variable species, some features remaining relatively constant such as the irregular annellations on the conidiogenous cells, and the conidial shape. Smaller conidia tend to be cylindrical, whereas larger conidia are tapered to more obtuse apices (Crous 1998). Pseudocercospora paranaensis does not have any annellations on its conidiogenous cells, which proliferate sympodially instead. Additionally, Ps. paranaensis differs from Ps. basitruncata by having significantly smaller conidiophores (7-11 µm in the former and 12 -60 μm in the latter) and longer conidia (79-99 µm in the former and 45 -70 μm in the latter). Finally, Ps. basitruncata is only known from an unrelated species of Eucalyptus (Hunter et al. 2011, Crous et al. 2013a. Two other species of Pseudocercospora have already been recorded on members of Cyatheaceae, namely Ps. cyatheae described on Cyathea sp. from Japan and Ps. cyatheicola on Cyathea australis from Australia (Braun et al. 2013). With regards to Ps. cyatheae, the only sequence available in GenBank for this species is of the ITS region. Pseudocercospora paranaensis differs from Ps. cyatheae in ITS and clusters in a separate and highly supported clade (data not shown). Nevertheless, morphological criteria alone clearly separate the two species. Pseudocercopora cyatheae, in contrast to Ps. paranaensis, has epiphyllous caespituli, its conidiogenous cells have a rim-like thickening at the scars, and it also has thicker, cylindrical to obclavate conidia (30-50 × 3.7-5.5 μm) with rounded bases (Nakashima et al. 2006). Pseudocercospora cyatheicola is different from Ps. paranaensis both phylogenetically -grouping in a different clade of the tree (Fig. 2) -and morphologically -having amphigenous stromata, larger conidiophores (30-70 × 2-3 μm), and percurrently proliferating conidiogenous cells . Guatimosim, R.W. Barreto & Crous, sp. nov. -MycoBank MB812815;Fig. 19 Etymology. Name refers to the host genus from which it was isolated, Serpocaulon.
Culture characteristics -Colonies on MEA slow-growing, 31 mm diam after 20 d in the dark; flat, aerial mycelium cottony, with water droplets at periphery, pale olivaceous-grey combined lavender grey areas centrally, greenish grey towards periphery; olivaceous black centrally and olivaceous grey at periphery in reverse; cultures sterile. Notes -Pseudocercospora serpocaulonicola clustered within a new clade, together with an isolate recorded on Eucalyptus grandis from South Africa (CBS 110998), Ps. cordiana, Ps. paraguayensis and Ps. schizolobii (Fig. 2), but differs from them by having the following variable sites for each locus: Pseudocercospora sp. (9 bp for ITS), Ps. cordiana (30 bp for ITS, 1 bp for act, 1 bp for tef1), Ps. paraguayensis (6 bp for ITS, 1 bp for act, 4 bp for tef1) and Ps. schizolobii (5 bp for ITS, 5 bp for act, 4 bp for tef1). Morphologically, it was not possible to compare the present collection to Pseudocerco spora sp. (CBS 110998), as the fungarium specimen was in poor condition, and neither conidiophores nor conidia were seen. Moreover, the cultures proved to be sterile. Two other Pseudocercospora species known on ferns (for which no DNA data are available in GenBank) have a similar morphology to Ps. serpocaulonicola. These are Ps. microsori on Microsorum pustulatum from Australia, and Ps. phyllitidis, which occurs on various ferns belonging to different families, and has a cosmo politan distribution (Shivas et al. 2010, Braun et al. 2013. Pseudocercospora microsori differs from Ps. serpocaulonicola by having welldeveloped stromata (20-60 µm wide), longer (30-65 × 3-5 µm), densely fasciculate (5 -30 stalks per fascicle), red-brown conidiophores, and moderately wide (2.5 -4 µm), curved to flexuous conidia (Shivas et al. 2010). On the other hand, Ps. phyllitidis is known to be an extremely variable species and probably is polyphyletic. However, one distinctive feature that remains relatively constant for specimens belonging to this species is the persistency of the conidia, which remain attached to the conidiogenous cells for a long time (Braun et al. 2013). This feature is absent in Ps. serpocaulonicola. Additionally, Ps. phyllitidis has immersed stromata (ill-formed and subcuticular in Ps. serpocaulonicola) and moderately wider conidiophores (1.5 -4 µm), compared to Ps. serpocaulonicola (2 -3.5 µm) (Braun et al. 2013). This is the first record of a fungus causing disease on S. tritseriale.  (Fig. 2). However, Ps. cyatheicola is different from Ps. thelypteridis by having erumpent and amphigenous stromata, longer and narrower conidiophores (30-70 × 2-3 µm), percurrently proliferating conidiogenous cells, and pale brown conidia . Pseudocercospora rumohrae differs from the new species by the absence of stromata, with conidiophores arising directly from the hyphae, as well as longer and narrower conidia (60 -120 × 3-3.5 μm) (Braun et al. 2013).

Pseudocercospora thelypteridis
Pseudocercospora thelypteridis is known from the type material on Thelypteris laxa from Taiwan and China, and on Nephrolepis sp. from Brunei (Braun et al. 2013). However, as the morphology and biometric data are quite similar, we chose not to introduce a novel species for the fungus found in Brazil. This is the first record of P. thelypteridis from Brazil. Guatimosim Culture characteristics -Colonies on MEA slow-growing, 10-23 mm diam after 20 d in the dark; smooth to folded or concentrically folded, raised, aerial mycelium cottony or velvety, mouse grey, pale olivaceous grey or lavender grey; purplish grey or iron-grey in reverse; cultures sterile. Notes -Sexual and asexual morphs of Ps. trichogena were found in the same region but on different hosts. However, based on DNA phylogenetic analyses, there is no doubt that they belong to the same species. Phylogenetically, Ps. trichogena clusters in a highly diverse clade, differing from all species within it (Fig. 2). Morphologically, Ps. trichogena is similar to three other species recorded on Thelypteridaceae, namely Ps. abacopteridicola on Abacopteris urophylla from Singapore, Ps. pteridophytophila on Cyclosorus acuminatus from Asia and Ps. thelypteridis on Nephrolepis sp. and Thelypteris laxa from Asia (Braun et al. 2013, Farr & Rossman 2015. Among those, Ps. pteridophytophila is the only species for which there is molecular data available in GenBank (Kirschner & Liu 2014), though the ITS region differs from Ps. trichogena by 8 bp. Additionally, Ps. pteridophytophila and Ps. thelypteris differ from Ps. trichogena by having well-developed stromata, arising from the stomata with narrower conidiophores, 2-5 µm and 2-3 µm, respectively (Hsieh & Goh 1990), while Ps. abacopteridicola has narrower and smaller conidia (30 -80 × 2 -3 µm) and conidiophores (5 -15 × 2.5 -3 µm) (Yen & Lim 1980). Pseudocercospora trichogena is the first species of Pseudocercospora with a trichomatose habit recorded on ferns.
Culture characteristics -Colonies on MEA slow-growing, 25 mm diam after 24 d; raised, crustose, with lobate, feathery margins and cottony aerial mycelium at periphery, lavender grey centrally, and lavender grey mixed with leaden grey at periphery; leaden black reverse. On OA flat, aerial mycelium sparse, olivaceous grey centrally, buff to rosy buff periphery; cinnamon reverse. On PDA raised, yeast-like, rosy buff centrally, buff at the periphery; buff reverse; cultures sterile. Notes -Based on morphology, X. diplazii is similar to X. elongata, but differs from the latter by having smaller asci (45-60 µm long in X. elongata) and smaller and narrower ascospores, not constricted at the septum (20 -25 × 4 -5 µm, constricted at the septum in X. elongata) (Crous et al. 2007b). Phylogenetically (Fig. 3) Notes -The genus Zasmidium, based on Z. cellare, comprises species with conspicuously thickened, darkened conidiogenous loci and hila, as typical of Stenella (Braun et al. 2013). However, Stenella has wide, flat conidial hila and scars, and clusters within Teratosphaeriaceae, while Zasmidium has planate and somewhat thickened and darkened conidial hila and scars, and clusters within Mycosphaerellaceae (Arzanlou et al. 2007, Braun et al. 2013).

Zasmidium sp.
Culture characteristics -Colonies on MEA slow-growing, 53 mm diam after 24 d; flat, with undulate, lobate, feathery margins, mycelium centrally immersed, and velvety aerial mycelium periphery, vinaceous buff centrally, pale mouse grey periphery; isabelline centrally and iron-grey periphery reverse. On OA and PDA lavender grey with iron-grey periphery; olivaceous grey reverse; cultures sterile. Notes -Fungarium specimens of this fungus were in poor condition and no conidia were seen. Isolation was performed by conidiophore transfer only. All attempts to promote sporulation in vitro proved to be unsuccessful. It appears that this taxon is a cryptic lineage closely related to Zasmidium australiensis, described on the same host, Blechnum serrulatum, from Australia (Mulder 1989, Braun et al. 2013. Presently, there are no sequences or known cultures available for Z. australiensis. Guatimosim, R.W. Barreto & Crous, sp. nov. -MycoBank MB812819; Fig. 23 Etymology. Name refers to the host genus from which it was isolated, Cyathea.

DISCUSSION
The present survey presents a phylogenetic overview of the cercosporoid taxa and related sexual morphs that were collected during a systematic survey of fern fungi from Brazil. Quaedvlieg et al. (2014) recently provided a phylogenetic overview of fungi clustering in the Teratosphaeriaceae. In the latter study, the authors focused on pathogens of Eucalyptus, which makes it interesting to compare to the Brazilian fern fungi, as this could provide an insight into the question if the fungi occurring on ferns are somehow related to those attacking distant related taxa, such as Eucalyptus, or if they evolved independently with their fern hosts.
Forty-four cercosporoid species are known causing frond spots of Pterydophyta worldwide: 13 Cercospora spp., two Passalora spp., 28 Pseudocercospora spp. and one Zasmidium sp. (Braun et al. 2013). Although no pathogenicity tests were done, all species described on the present study were found associated with frond spot symptoms, indicating their probable habit as pathogens. However, further studies are necessary to clarify the pathogenicity of these fungi on ferns.
Most Cercospora species are morphologically very similar to taxa occurring in the C. apii species complex (Braun et al. 2013). In the present study, we were able to identify one new Cercospora species, and demonstrate that the host range of C. coniogrammes is wider than previously known, including ferns belonging to two additional families. Plant hosts from Pteridophyta represent some of the oldest lineages of vascular plants (Smith et al. 2008). It is interesting to note that C. coniogrammes is on one hand pro ving to have a wider host range within the Pteridophyta and, on the other hand, found to be basal in the phylogeny of the genus Cercospora (Groenewald et al. 2013; Fig. 1).
As for Pseudocercospora, a long list of names have been published for which there are no DNA data and ex-type cultures available (Braun et al. 2013), complicating a better understand- ing of the taxonomy of the genus. Recollecting and epitypifying all these species is a challenging, but necessary task for mycologists dealing with cercosporoid fungi. Three examples of taxonomic decisions that are still pending even after the present study involve Ps. abacopteridicola, Ps. lygodiicola and Ps. thelypteridis. Although we suspect that these Brazilian collections may in fact represent novel species, this can only be confirmed after the recollection of fresh materials from the type localities (Singapore, China and Taiwan, respectively - Yen & Lim 1980, Braun et al. 2013, followed by epitypification and a phylogenetic comparison.
Historically, the taxonomy of cercosporoid fungi has been based upon morphological and ecological features, including conjectured host specificity (Chupp 1954, Deighton 1965, 1973, 1974, 1976, Pons & Sutton 1988, Braun 1993a, b, c, 1995, Crous & Braun 1996, Braun & Mel'nik 1997, Crous et al. 2000, Braun et al. 2013. It is now widely accepted that this was an inadequate basis for the taxonomy of this complex plethora of fungi. Two examples of potentially mistaken conclusions based on morphology, symptoms and host-association alone were provided by our results: 1. Zasmidium cyatheae (only sexual morph found) and Neoceratosperma cyatheae (only asexual morph found), cooccurred on the same frond spot, on the fern Cyathea delgadii. Without pure cultures and access to molecular data the mistaken conclusion would be that Z. cyatheae was the sexual morph of N. cyathea.

A similar situation occurred for Paramycosphaerella sticheri
and Clypeosphaerella sticheri. Both were found attacking two different species in the same host genus Sticherus causing similar disease symptoms. It is likely that many conjectured connections between asexual and sexual morphs have been mistakenly made for cercosporoids and other fungal groups. Efforts towards clarifying these connections with modern criteria should be continued in order to gene rate an appropriate and consolidated taxonomy of cercosporoids and other fungal groups (Taylor et al. 2000, Crous & Groenewald 2005, Crous et al. 2009f, 2015b).
In the past, mycologists have hypothesized that plant pathogenic fungi associated with primitive plants were also evolutionarily basal to the evolution of fungi. Thus, Savile (1971) proposed that primitive plant hosts, such as ferns, would have primitive rust genera. Later, phylogenetic studies involving rust species in different genera have proven this hypothesis wrong. For example, Hemileia and Maravalia -sister genera at the base of the Pucciniales phylogenetic tree (Wingfield et al. 2004, Aime 2006) -are pathogens of higher plant taxa, especially in the Rubiaceae and Asclepiadaceae, respectively.
As for the cercosporoid and mycosphaerella-like species documented here, there is some evidence that the fungal species associated with ferns are evolutionarily basal to the evolution of their relatives. In the Cercospora phylogeny (Fig. 1), C. coniogrammes (recorded only from ferns) is basal to the evolution of all other Cercospora species, whilst the same pattern is reproduced in the Pseudocercospora phylogeny (Fig. 2), where Ps. cyatheicola, Ps. lygodiicola, Ps. rumohrae and Ps. thelypteridis, all isolated from ferns, appear to be evolutionarily basal in the clade where they cluster; in the phylogeny of mycosphaerella-like taxa (Fig. 3), a basal position was observed for Phaeophleospora hymenocallidis, Ps. hymenocallidicola and Ps. pteridivora (all from ferns), appearing evolutionarily basal to all other species in the genus for which sequence data were available.
As more sequence data become available for cercosporoids associated with ferns, this preliminary evidence may become stronger and allow for an elucidation of further cercosporoid genealogies and, hence, should permit a better understanding of the co-evolutionary history of this fungal group and its association with host plants.
The present study has significantly expanded our knowledge of cercosporoid and mycosphaerella-like fungi associated with frond spots in Brazilian Pteridophyta. Previously, only one cercosporoid and one mycosphaerella-like species (Ps. davalliicola and 'Mycosphaerella' tocoyenae, re spectively) were known to be associated with diseases on ferns in Brazil (Farr & Rossman 2015, Mendes & Urben 2015. The present work has expanded this number significantly by adding one new genus (Clypeosphaerella) and 15 new species to this list. Here we also provide novel molecular information that may be useful to obtain a better understanding of the evolution of cercosporoid and mycosphaerella-like fungi. We also hope that further exploration of these cultures will contribute in the future to a more robust phylogeny of these fungi across various families of host plants, and help establishing a better understanding of their host specificity and evolution. The clear abundance of novel taxa collected on ferns in Brazil, also underlines the scientific value of host or host-group based surveys as a source of mycological novelties. Finally, our findings confirm that mycologists in the tropics have thus far given little attention to fungi occurring on plant hosts with apparent limited economic relevance, such as ferns. Fern fungi in Brazil and other tropical regions are likely to represent an important source of a highly diverse mycobiota that still awaits discovery.