Multi-locus phylogeny of the genus Curvularia and description of ten new species

Curvularia is a cosmopolitan genus that includes species associated with plants, animals and humans, several of which are of clinical significance. Some of these species are important pathogens of grasses, causing devastating diseases on cereal crops in the family Poaceae. In the present multi-locus study, ex-type and reference strains of Curvularia, as well as several strains deposited in the CBS culture collection of the Westerdijk Fungal Biodiversity Institute, were included. Based on ITS, GAPDH and TEF1 sequences, as well as phenotypic data, ten new species are described and illustrated: C. arcana, C. austriaca, C. canadensis, C. ellisii, C. pseudoclavata, C. pseudoellisii, C. pseudointermedia, C. pseudoprotuberata, C. siddiquii and C. tribuli. Moreover, the new combinations C. cactivora and C. patereae are proposed, and an epitype for C. oryzae-sativae is designated. In addition, illustrations and descriptions are provided for C. cactivora, C. ellisii, C. crassiseptata, C. neergaardii, C. oryzae, C. oryzae-sativae, C. protuberata and C. verruciformis. The description of C. pseudobrachyspora is emended, and its host and distribution records are updated.


Introduction
Curvularia is a genus with a worldwide distribution that includes pathogens or saprobes of a wide range of plant hosts. Species occur mainly on members of the family Poaceae and represent important pathogens of grass and staple crops, including rice, maize, wheat and sorghum. Other hosts are inordinately enlarged intermediate cells, which contributes to its characteristic curvature. The curvature of the conidia is the main difference to the similar genus Bipolaris, since in the latter the curvature, when present, is throughout the length of the conidium. In Bipolaris, conidia are usually also longer than in Curvularia (Sivanesan 1987;Marin-Felix et al. 2017a). However, both genera include species that exhibit intermediate conidial characters (Manamgoda et al. 2012), making sequence data essential for proper species delimitation. In this context, several studies based on the internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS), large subunit of the rDNA (LSU) and partial fragments of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the translation elongation factor 1-α (TEF1) genes have been performed, resulting in several species being transferred from one genus to another (Manamgoda et al. 2012Tan et al. 2014). The Drechslera asexual morph of Pyrenophora, as well as species of Exserohilum and Johnalcornia, is also similar to Bipolaris and Curvularia (Tan et al. 2014;Hernández-Restrepo et al. 2018;Marin-Felix et al. 2019). Some Exserohilum species can be easily distinguished from the other three genera by the production of conidia with distinctly protruding hila, but some species of Curvularia produce similar structures, leading to wrong identifications . Pyrenophora differs from the other graminicolous genera by its muriform septate ascospores, but the asexual morph is similar to Curvularia, also leading to incorrect identifications (Marin-Felix et al. 2019). Johnalcornia can be distinguished by forming the second conidial septum in the apical cell and producing distinctive conidia-like chlamydospores (Tan et al. 2014). Cochliobolus, which is known as the sexual morph of Curvularia and Bipolaris, is characterised by brown or black, globose ascomata, bitunicate, cylindrical asci and filiform or flagelliform, hyaline ascospores, which are loosely arranged into a helix or parallel (Manamgoda et al. 2012). The only difference is the presence of stromata in some Curvularia species, a feature not observed in Bipolaris (Manamgoda et al. 2012). Because the sexual morph is rarely found in nature and difficult to induce in culture, it is of limited value to distinguish Bipolaris and Curvularia . Species of both Curvularia and Bipolaris are therefore mainly differentiated based on their asexual morphs .
Curvularia species are difficult to identify based only on morphology since many species share similar characters with overlapping dimensions. In order to get a proper delimitation and identification, several phylogenetic studies using ITS, GAPDH and TEF1 have recently been published (Manamgoda et al. 2012Tan et al. 2014Tan et al. , 2018Marin-Felix et al. 2017a, b). In the revision of the genus carried out by Marin-Felix et al. (2017a), 74 species were accepted based on DNA sequence data. Subsequently, 31 novelties have been introduced (Hyde et al. 2017;Marin-Felix et al. 2017b;Tan et al. 2018;Dehdari et al. 2018;Heidari et al. 2018;Hernández-Restrepo et al. 2018;Liang et al. 2018;Mehrabi-Koushki et al. 2018;Tibpromma et al. 2018). Hitherto, 105 species are accepted in the genus based on DNA sequence data. Some of these species were described a long time ago, with descriptions and/or illustrations not being readily available. In that context, Manamgoda et al. (2015) revised the genus and provided updated data for ten species. One of the objectives of this study is therefore to further enlarge our knowledge on the diversity and taxonomy of Curvularia. Another problem remaining unresolved is the lack of molecular data for those species that have only been described based on morphology, causing many of them to be ignored by the scientific community. A second objective is thus to provide sequence data of the main markers used in molecular studies of Curvularia and to determine the taxonomic position of many of these previously described species.

Isolates and morphological analysis
Strains deposited in the Westerdijk Fungal Biodiversity Institute (CBS) collection identified as Curvularia or related genera, i.e. Bipolaris, Exserohilum, Johnalcornia and Pyrenophora, were examined (Table 1). These strains were cultured on potato dextrose agar (PDA), which is the medium used in the recent revisions of the genus, at 25°C under an alternating 12-h UV light/ dark regime to induce sporulation. When the cultures were mature, the fertile fungal structures were mounted and measured in lactic acid, with at least 30 measurements of each structure. The strains that did not produce reproductive structures on PDA were also cultured on 2% malt extract agar (MEA), oatmeal agar (OA) and synthetic nutrient-poor agar (SNA) (Crous et al. 2019) with pieces of sterile maize leaves in order to induce sporulation. Observations and photomicrographs were obtained with a Nikon SMZ1500 dissecting microscope, and with a Nikon eclipse Ni compound microscope, using a DS-Ri2 digital camera (Nikon, Tokyo, Japan) and NIS-Elements imaging software v. 4.20.
Culture descriptions were done by incubating each isolate on three PDA plates in the dark. After a week, colony diameters were measured, colony morphologies described and the colours rated using the colour chart of Rayner (1970). Taxonomic novelties and typifications were registered in MycoBank (www.MycoBank.org; Crous et al. 2004).

DNA isolation and amplification
Genomic DNA was extracted and purified directly from fungal colonies growing on MEA according to the UltraClean™

Phylogenetic study
The phylogenetic analysis based on ITS, GAPDH and TEF1 was carried out, including strains available in the CBS collection and sequences of ex-type and reference strains of Curvularia spp. available in GenBank (Table 1). Each locus was aligned separately using MAFFT v. 7 (Katoh and Standley 2013) and manually adjusted in MEGA v. 6.06 (Tamura et al. 2013). The maximum likelihood (ML) and Bayesian inference (BI) were performed for the combined dataset using RAxML as was described by Hernández-Restrepo et al. (2016). The sequences generated in this study were deposited in GenBank, and the alignments in TreeBASE (www.treebase.org, S23887).

Results
The lengths of the fragments of the three genes used in the combined dataset were 505 bp (ITS), 477 bp (GAPDH) and 892 bp (TEF1). The length of the final alignment was 1874 bp. The consensus tree obtained from the RAxML analysis of the combined dataset is shown in Fig. 1, which agreed with the topology of the Bayesian analysis. It includes RAxML bootstrap support (BS) and Bayesian posterior probability at the nodes. In the combined phylogenetic tree ( Fig. 1), species of Curvularia formed a supported clade (92% bs/1 pp) clearly separated from other graminicolous helminthosporioid genera included in the phylogenetic study, i.e. Bipolaris, Exserohilum, Johnalcornia and Pyrenophora.
Seventeen strains included in the phylogenetic study were located in ten independent branches distant from other species of Curvularia. The morphological study of these strains revealed enough differences to propose ten new species, i.e. C. arcana, C. austriaca, C. canadensis, C. ellisii, C. pseudoclavata, C. pseudoellisii, C. pseudointermedia, C. pseudoprotuberata, C. siddiquii and C. tribuli.
Interestingly, CBS 198.87, the ex-type strain of Drechslera patereae, was located in the main clade representing the genus Curvularia. Therefore, a new combination is proposed in the taxonomy section. Moreover, five different isolates identified as Bipolaris cactivora were located in a fully supported subclade (100% bs/ 1 pp) within the Curvularia clade. Based on sequence similarities and morphological data, the new combination C. cactivora is also proposed.
The ex-type isolate (CBS 193.62) of the currently invalid C. ellisii, which is validly redescribed below, and the isolate CBS 127083 (obtained from a sexual cross) were located in a well-supported clade (86% bs/1 pp).
Finally, seven strains, CBS 207.59, CBS 533.70, CBS 336.64, CBS 337.64, CBS 339.64, MFLUCC 10-0739 and HNWN001, were located in a clade (89% bs/-pp) together with CPC 28808, the ex-type strain of C. pseudobrachyspora. Strain CBS 533.70 was found to differ morphologically from the extype strain, and thus, the original description is herewith emended, and new host and distribution reports are included. MB832460 Etymology. Name refers to the mystery of the origin of the ex-type strain.
Regrettably, no data about the ex-type strain are available. Curvularia arcana is located in a basal clade that includes C. intermedia and C. pseudointermedia (Fig. 1). For morphological differences, see notes under C. pseudointermedia.

MB830047
Etymology. Named after the country where the material was collected, Canada.

Curvularia crassiseptata
Notes This specie s w as initiall y d escrib ed as Exserohilum inaequale, due to the production of conidia with protuberant hila similar to those found in this genus (Sivanesan 1984). Subsequently, it was transferred to Curvularia changing the epithet to C. crassiseptata since inaequale was pre-occupied by another species (Zhang et al. 2004). Recently, the position of this species in Curvularia was confirmed by Hernández-Restrepo et al. (2018). In the present study, we provide a morphological description and illustrations of the type material.
Curvularia neergaardii is related to the new species C. pseudoellisii and C. ellisii, both proposed in the present study (Fig. 1). For a morphological comparison, see notes of C. pseudoellisii.
Curvularia patereae is related to C. mebaldsii, C. tsudae and C. penniseti (Fig. 1). These species all produce 3distoseptate conidia, except for C. patereae, which is characterised by 2-6-distoseptate conidia. Curvularia penniseti can be easily distinguished by its larger conidia (29-42 μm vs up to 30 μm in the other three species) with an enlarged middle cell, which is not or slightly enlarged in the other species. The four species of this clade are mainly reported on members of the Poaceae (Farr and Rossman 2019).
Curvularia pseudoclavata is similar to C. clavata, both species producing ovoid to clavate, mostly 3-distoseptate conidia. The size of their asexual structures is also similar. The only difference observed is in the ornamentation of their conidia, being smooth-walled in C. clavata (Sivanesan 1987) and verruculose in C. pseudoclavata. However, both species are phylogenetically distant. Curvularia pseudoclavata was found in Denmark, while C. clavata has never been reported in Europe (Farr and Rossman 2019). Curvularia clavata has been reported on a wide host range, including Oryza sativa, from which C. pseudoclavata was isolated.
Curvularia pseudoprotuberata is represented by CBS 385.69 and CBS 550.69 (Fig. 1), both strains isolated from soil from the same location in Canada, Ontario. Curvularia protuberata has also been reported in Canada, apart from other locations in North America, South America, Africa and Europe, on a wide range of host plants included in different families (see notes under C. protuberata).
Curvularia siddiquii Y. Marín & Crous, sp. nov. Fig. 17. Etymology. Named after Dr. Salimuzzaman Siddiqui, who assisted Ahmad and Quraishi (1960)   Asexual morph on PDA Hyphae hyaline to subhyaline, branched, septate, anastomosing, thin-walled, 2-4 μm. Conidiophores arising in groups, mononematous, macronematous, septate, straight or flexuous, geniculate at upper part, cell size decreasing towards apex, unbranched, sometimes branched, cell walls thicker than those of vegetative hyphae, pale brown, rarely paler towards apex, not swollen at the base, up to 600 μm. Conidiogenous cells smooth-walled, terminal or intercalary, proliferating sympodially, pale brown, subcylindrical to swollen, 5-16(21) × 6.5-8.5 μm. Conidia smooth-walled, curved, rarely straight, middle cells disproportionately enlarged, navicular, rarely ellipsoidal, pale brown to brown, sometimes apical cells paler than middle cells being subhyaline, (1)2-3(4)-distoseptate, (21.5)24-40 × (15)17-23 μm; hila inconspicuous to slightly conspicuous, flat to slightly protuberant, darkened, slightly thickened, 2-4 μm wide. Chlamydospores globose to subglobose or cylindrical, brown, thick-walled, terminally and intercalary, 8.5-22.5 μm diam. Microconidiation and sexual morph not observed. Culture characteristics Ex-type strain CBS 196.62: Colonies on PDA reaching 64-71 mm diam in 1 week, apricot to chestnut, fasciculate, irregular form, margin fimbriate; reverse umber to chestnut. Strain CBS 142.78: Colonies on PDA attaining 52-62 mm diam in 1 week, olivaceous, mouse grey and white in the centre, cottony, raised, margin fimbriate; reverse olivaceous to fuscus olivaceous, margin buff. Notes Curvularia siddiquii was introduced by Ahmad and Quraishi (1960) to accommodate a fungus isolated from air in Pakistan. However, this species is invalid due to the lack of a Latin description. Subsequently, Ellis (1971) synonymised C. siddiquii with Drechslera papendorfii, which clusters in Curvularia (Marin-Felix et al. 2017a) where it was initially described (van der Aa 1967). This synonymy was not molecularly corroborated since no sequence data were available at that moment. In the phylogenetic tree generated here (Fig. 1), the ex-type strains of C. siddiquii (CBS 196.62) and C. papendorfii (CBS 308.67) formed two separate lineages, representing two distinct species from other Curvularia spp. Therefore, we resurrected the former taxon as a new species. Morphologically, C. siddiquii can be distinguished from C. papendorfii by the length of its conidiophores (up to 200 μm in C. papendorfii vs up to 600 μm in C. siddiquii) and the production of chlamydospores, which have not been observed in C. papendorfii.  (CBS 196.62 ex-type). a-c Conidiogenous cells and conidia; d-g conidia; h chlamydospores. Scale bars a-c, h = 10 μm; g 5 μm, g applies to d-g Curvularia papendorfii has been reported from soil and on various genera of Poaceae, causing leaf spots (Farr and Rossman 2019), while the type material of C. siddiquii was isolated from air, and the substrate of the second strain belonging to this species is unknown. Both species have been reported only in Asia and Africa, respectively.

MB830062
Etymology. Name refers to Tribulus, the host genus from which this fungus was collected.
Notes In our phylogenetic study, C. tribuli was located on an independent branch far from other species of the genus (Fig.  1). This is the first species of Curvularia reported on Tribulus terrestris (Zygophyllaceae).
Notes Curvularia verruciformis is morphologically similar to C. verrucosa and C. verruculosa since all three species produce verrucose conidia. However, they differ in the number of conidial septa being mostly 3-distoseptate in C. verruculosa and 4-distoseptate in C. verruciformis and C. verrucosa (Ellis 1966;Sivanesan 1992). The conidia of C. verrucosa and C. verruculosa are wider (14-18 and 12-17 μm, respectively) than in C. verruciformis due to the middle cell being more enlarged. Conidia in C. verruciformis tend to be more geniculate which makes the longitude of the conidia shorter than in C. verruculosa (20-27.5 μm in C. verruciformis vs 20-35 μm in C. verruculosa). Curvularia verrucosa also produces more geniculate conidia than C. verruculosa, but these are longer than in the other two species (27-40 μm). Phylogenetically, these species are very distant (Fig. 1).
The closest relative of C. verruciformis is C. uncinata (Fig.  1), but both species can be easily distinguished by the ornamentation of the conidia, being smooth-walled in C. uncinata and verrucose in C. verruciformis (Sivanesan 1987).