Phylogeny and taxonomy of the scab and spot anthracnose fungus Elsinoë (Myriangiales, Dothideomycetes)

Species of Elsinoë are phytopathogens causing scab and spot anthracnose on many plants, including some economically important crops such as avocado, citrus, grapevines, and ornamentals such as poinsettias, field crops and woody hosts. Disease symptoms are often easily recognisable, and referred to as signature-bearing diseases, for the cork-like appearance of older infected tissues with scab-like appearance. In some Elsinoë-host associations the resulting symptoms are better described as spot anthracnose. Additionally the infected plants may also show mild to severe distortions of infected organs. Isolation of Elsinoë in pure culture can be very challenging and examination of specimens collected in the field is often frustrating because of the lack of fertile structures. Current criteria for species recognition and host specificity in Elsinoë are unclear due to overlapping morphological characteristics, and the lack of molecular and pathogenicity data. In the present study we revised the taxonomy of Elsinoë based on DNA sequence and morphological data derived from 119 isolates, representing 67 host genera from 17 countries, including 64 ex-type cultures. Combined analyses of ITS, LSU, rpb2 and TEF1-α DNA sequence data were used to reconstruct the backbone phylogeny of the genus Elsinoë. Based on the single nomenclature for fungi, 26 new combinations are proposed in Elsinoë for species that were originally described in Sphaceloma. A total of 13 species are epitypified with notes on their taxonomy and phylogeny. A further eight new species are introduced, leading to a total of 75 Elsinoë species supported by molecular data in the present study. For the most part species of Elsinoë appear to be host specific, although the majority of the species treated are known only from a few isolates, and further collections and pathogenicity studies will be required to reconfirm this conclusion.


INTRODUCTION
All members of the genus Elsino€ e (Myriangiales, Ascomycota) are specialised plant parasites causing diseases on many plant hosts, including some economically important crops such as avocado, cassava, citrus, grapevines, ornamentals such as poinsettias, field crops and woody hosts. Many species cause "signature-bearing diseases" easily recognised for their symptom-marker cork-like necrotic tissues. These are often raised, exhibiting cracks, and hence are referred to as scabs. In other Elsino€ e-host associations the symptoms that result from infection are different and are often called anthracnose (such as in infected grapevines) (Barrus & Horsfall 1928, Jenkins 1947, examination of specimens collected in the field is often frustrating because of the lack of fertile structures. In addition, isolation of Elsino€ e in pure culture can be very challenging because of their slow growth and resulting cultures easily becoming overgrown by contaminants. Although many species of the scab fungus have been described under Elsino€ e or Sphaceloma, only a few cause important diseases (Holliday 1980). Economically important diseases include avocado scab caused by E. perseae, citrus scab caused by E. fawcettii and E. australis, bean scab caused by E. canavaliae and E. phaseoli, grape spot anthracnose caused by E. ampelina, and cassava superelongation caused by "Sphaceloma manihoticola" (Jenkins 1925, Shear 1929, Jenkins 1931, Bruner & Jenkins 1933, Bitancourt & Jenkins 1936a, b, Boedijn 1961, Tan et al. 1996 (Fig. 1). In many cases the main impact is on the appearance of the harvested product, and its market acceptability rather than on crop productivity (Swart et al. 2001). On the positive side, several Elsino€ e species cause devastating diseases on important agricultural and environmental weeds and are beneficial species in this regard. Some examples are the scab fungi attacking alligator weed (Alternanthera philoxeroides), giant sensitive plant (Mimosa diplotricha), beggar tick (Bidens pilosa) (Guatimosim et al. 2015), and wild poinsettia (Euphorbia heterophylla) (Barreto & Evans 1998, Nechet et al. 2004. The order Myriangiales has two accepted families, namely Elsinoaceae and Myriangiaceae, which represent a sister group to Dothideales, the type order of the Dothideomycetes (Li et al. 2011, Hyde et al. 2013, Dissanayake et al. 2014. They generally have crustose to pulvinate, irregular ascostromata, in which the scattered asci are irregularly arranged in individual locules. Ascospores are hyaline to brown, transversely septate or muriform, which are irregularly arranged and liberated only by the breakup of the stromatal layers above them (Kirk et al. 2008, Hyde et al. 2013. Asexual morphs of Elsinoaceae are acervular coelomycetous fungi with polyphialidic conidiogenous cells, such as the Sphaceloma asexual morph of Elsino€ e in the present study (Jenkins 1932b, Kirk et al. 2008. Since the Myriangiales was introduced by Starb€ ack (1899), its classification has undergone several changes. Frederick & Frederick (1947) placed four families in this order (Dothioraceae, Elsinoaceae, Myriangiaceae and Pseudosphaeriaceae). von Arx (1963) originally reduced the Myriangiales to include the Myriangiaceae and Saccardiaceae, but later circumscribed the order to include a single family, Myriangiaceae (von Arx & Müller 1975). Subsequent treatments by other workers again saw an increase in the number of families, with Barr (1979) originally recognising seven, and later five (Eriksson & Hawksworth 1986, Barr 1987). The first multigene phylogenetic treatment was published by Schoch et al. (2006), who placed two families (Elsinoaceae and Myriangiaceae) in Myriangiales, with sister groups being delineated in subsequent studies (Tsuneda et al. 2008, Schoch et al. 2009). Kirk et al. (2008) included three families (Cookellaceae, Elsinoaceae and Myriangiaceae), while Lumbsch & Huhndorf (2010) accepted only Elsinoaceae and Myriangiaceae, and treated Cookellaceae as incertae sedis in Dothideomycetes, a conclusion that was supported by Hyde et al. (2013).
The Elsinoaceae was proposed by Saccardo & Trotter (1913) after the invalid "Elsino€ een" was introduced by von Höhnel (1909) as a separate family from Myriangiaceae, because of habitat and morphological characters. Woronichin (1914) treated this family as a synonym of Plectodiscellaceae based on a single species Plectodiscella piri, which he found occurring on the leaves of apple and pear. Jenkins (1932a) regarded Elsino€ e as valid name, and Frederick & Frederick (1947) placed Elsinoaceae in the Myriangiales. However, von Arx & Müller (1975) placed Elsino€ e, the type genus of the Elsinoaceae, in the Myriangiaceae according to the immersed or erumpent, pulvinate or irregular ascostromata, and being restricted to foliar pathogens causing scab disease. Based on observations of their restricted hosts, Barr (1979Barr ( , 1987 and Eriksson (1981) suggested that Elsinoaceae and Myriangiaceae should be maintained as two separate families in the Myriangiales.
The genus Elsino€ e was introduced by Raciborski (1900) with descriptions of three species (E. antidesmae, E. canavaliae and E. menispermacearum). It is characterised by forming scab-like lesions with pseudoascostromata containing three to eight bitunicate asci inside each locule. Asci are saccate to globose with eight hyaline, oblong or fusiform, septate ascospores (Fig. 2). The asexual morph is the acervular coelomycetous Sphaceloma, which has polyphialidic conidiogenous cells and hyaline, ellipsoid, aseptate conidia. Jenkins (1932a) treated Plectodiscella as a synonym of Elsino€ e and proposed a connection between Sphaceloma and the sexual morph Elsino€ e, supported by later studies using molecular data (Swart et al. 2001). More than 140 species epithets named Elsino€ e and more than 160 epithets of Sphaceloma asexual species have been recorded in Index Fungorum and MycoBank, with an estimated 48 species of Elsino€ e and 52 species of Sphaceloma in Kirk et al. (2008). Morphological characteristics of Elsino€ e species are difficult to observe, as the sexual morph is uncommon in nature, and the frequently observed asexual Sphaceloma morph is usually morphologically conserved. Molecular tools have therefore become increasingly important in resolving the connections between different stages of the lifecycle, and the interpretation of morphological variation (Cheewangkoon et al. 2009). Swart et al. (2001) delineated six Elsino€ e species associated with scab disease of Proteaceae from Australia, California (USA), South Africa, and Zimbabwe, and proposed three new species supported by ITS rDNA sequence data. Similar studies were conducted to describe Elsino€ e species associated with other plant hosts (Summerell et al. 2006, Everett et al. 2011, Crous et al. 2015b. In their phylogeny of the genus, Jayawardena et al. (2014) included 12 Elsino€ e species based on multi-gene data available in GenBank at the time. Ex-type sequence data is, however, available for only a few species. The far majority of the Elsino€ e species described to date will therefore need to be recollected and epitypified. To facilitate species recognition in Elsino€ e therefore, a phylogenetic backbone would first have to be established. The objectives of the present study were (i) to clarify species boundaries among Elsino€ e isolates from various host genera distributed over 17 countries; (ii) to provide a multigene phylogeny for the genus Elsino€ e based on a large set of well-identified cultures deposited in the CBS culture collection, supplemented by freshly collected specimens; (iii) to link Elsino€ e names to their Sphaceloma asexual morphs; and (iv) to try and elucidate host specificity or the relationship between Elsino€ e species and their respective host plants.

Isolates
One hundred and nineteen Elsino€ e isolates from 67 host genera representing 17 countries, including 64 ex-type isolates were included in this study (Table 1). The majority of the isolates were obtained from the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands (CBS) ( Table 1), while freshly collected specimens were placed in the working collection of Pedro Crous (CPC) housed at the Westerdijk Institute. Freeze-dried isolates were revived in 2 mL malt/peptone (50 %/50 %) and subsequently transferred to 2 % malt extract agar (MEA; Crous et al. 2009), and incubated at 22°C under a natural day-night cycle. For fresh specimens, single-conidial isolates were obtained using techniques from Crous et al. (1991). Additionally, an effort was conducted by R.W. Barreto to recollect scab fungi described in the past in Brazil allowing for epitypification of taxa lacking pure cultures. Surveys were concentrated in the State of São Paulo (from which most taxa described by A. Bitancourt and A. E. Jenkins were collected) but also included other south-eastern and southern Brazilian states, mostly in 2010 but with ad hoc collections continuing in later years.

DNA isolation, amplification and sequencing
Genomic DNA was extracted using the Wizard ® Genomic DNA Purification Kit (Promega, Madison, WI, USA) following the manufacturer's instructions, from fungal mycelium growing on MEA. The ITS region was amplified with the primers ITS5 and ITS4 (White et al. 1990), the LSU region with the primers LR0R (Rehner & Samuels 1994) and LR5 (Vilgalys & Hester 1990), the rpb2 region with primers RPB2-5F2 (Sung et al. 2007) and fRPB2-7cR (Liu et al. 1999), and the TEF1-α gene with the primers elongation-1-F and elongation-1-R (Hyun et al. 2001(Hyun et al. , 2009. The PCR mixture for the all regions consisted of 1 μL genomic DNA, 3 mM MgCl 2 , 20 μM of each dNTP, 0.2 μM of each primer and 0.25 U BIOTAQ DNA polymerase (Bioline). Conditions for PCR of ITS and LSU genes constituted an initial denaturation step of 2 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 48°C and 1 min at 72°C, and a final denaturation step of 8 min at 72°C, while the TEF1-α gene was performed as described by

Phylogenetic analyses
DNA sequences generated by each primer combination were used to obtain consensus sequences using SeqMan v. 7.1.0 in the DNASTAR Lasergene core suite software (DNASTAR Inc., Madison, WI, USA). Sequences were aligned using MAFFT v. 6 (Katoh & Standley 2013) and edited manually using MEGA v. 6.0 (Tamura et al. 2013). A partition homogeneity test (PHT) with heuristic search and 1 000 homogeneity replicates was performed using PAUP v. 4.0b10 to test the discrepancy among the ITS, LSU, rpb2 and TEF1-α sequence datasets in reconstructing phylogenetic trees. A maximum parsimony (MP) analysis was performed using PAUP v. 4.0b10 with a heuristic search option of 1 000 random-addition sequences with a tree bisection and reconnection (TBR) branch swapping algorithm (Swofford 2003). The branches of zero length were collapsed and all equally most parsimonious trees were saved. Other parsimony scores such as tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC) were calculated (Swofford 2003).
MrModeltest v. 2.3 was used to estimate the best nucleotide substitution model settings for each gene (Posada & Crandall 1998). Bayesian inference (BI) was performed based on the optimised model for each individual DNA dataset from the results of the MrModeltest, using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003). Two MCMC chains were run from random trees for 1 000 M generations and stopped when average standard deviation of split frequencies fell below 0.01. Trees were saved each 1 000 generations. The first 25 % of trees were discarded as the burn-in phase of each analysis, and the posterior probabilities (BPP) were calculated to assess the remaining trees (Rannala & Yang 1996). The branch support from the MP analysis was evaluated with a bootstrapping (BS) method of 1 000 replicates (Hillis & Bull 1993). Myriangium hispanicum (CBS 347.33) was selected as outgroup in all analyses. Phylograms were viewed using FigTree v. 1.3.1 (Rambaut & Drummond 2010). Novel sequences generated in the current study were deposited in GenBank (Table 1) and the aligned matrices used for phylogenetic analyses were maintained in TreeBASE (www. treebase.org; accession number: S19904).

Morphology
Descriptions of the sexual morph are based on host material, while those of the asexual morph are based on sporulating cultures (Fig. 3). Colonies were subcultured onto MEA, oatmeal agar (OA), potato dextrose agar (PDA), synthetic nutrient-poor agar (SNA), and tap water agar (WA) ). Cultures were incubated at moderate temperatures (22°C) under a 12 h near-ultraviolet (NUV) light (360 nm), 12 h dark cycle for 3 wk to induce sporulation. Structures were mounted in clear lactic acid, and 50 measurements determined per structure, with extremes of conidial measurements given in parentheses. Colony diameters were measured and the colony colours described after 3 wk according to the colour charts of Rayner (1970). Microscopic photographs were captured using a Nikon Eclipse 80i microscope equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast (DIC) illumination and the Nikon software NIS-Elements D Package v. 3.00. Adobe Bridge CS v. 6 and Adobe Photoshop CS v. 5 were used for the manual editing. Nomenclatural novelties and descriptions were deposited in MycoBank (Crous et al. 2004).

Phylogenetic analyses
The final combined alignment contained 119 Elsino€ e ingroup strains with a total of 2 532 characters including gaps (617 characters for ITS, 744 for LSU, 751 for rpb2 and 422 for TEF1α), of which 1 624 characters are constant, 221 variable characters are parsimony-uninformative and 687 characters are variable and parsimony-informative. MP analyses generated one tree, which is presented in Fig. 4

Taxonomy
At the onset of this study, it was estimated that Elsino€ e contained approximately 48 species, and Sphaceloma 52 (Kirk et al. 2008). After phylogenetic analyses and morphological examination of 119 isolates, we now recognise 75 species (Table 1), of which eight are newly described, 13 are epitypified, and 26 species names are suggested as new combinations based on the single nomenclature initiative (Wingfield et al. 2012, Crous et al. 2015a). All strains proposed as new species and for epitypification based on the multi-gene phylogeny were studied morphologically. Type details and notes on the host range and geographic distribution of previously described species are also included. Notes: Elsino€ e abutilonis was described by (Bitancourt & Jenkins 1950) causing scab disease on leaves and branches of Abutilon striatum in São Paulo, Brazil. Information on the original description is limited to a symptom description with acervuli referred to as "invisible" and conidia as "not seen".  Notes: Elsino€ e anacardii was described on cashew in India by , 1970 as causing anthrancnose spots on leaves and also on young shoots and fleshy peduncles that coalesce with age turning into scabs. Symptoms include numerous greyish white leaf spots on the lower leaf surface, 0.5-2 mm diam. Acervuli dark reddish-brown, circular to oblong, intraepidermal, appearing subcuticular when erumpent, 19-31 × 26-67 μm. The cultural characteristics on PDA of this fungus are quite distinct from the usual appearance of Elsino€ e colonies, having cottony white aerial mycelium on the surface, and green mycelium on the reverse side of the plate. The fact that the isolates studied here originate from completely distinct host families, suggests that there could have been some confusion during the culturing and subsequent deposit of these cultures. This matter can only be resolved based on fresh collections, as it appears highly unlikely that the same species could cause disease on these diverse hosts. The ITS, rpb2 and TEF1-α regions fail to distinguish E. anacardii and E. semecarpi.
Culture characteristics: Cultures on MEA, slow-growing (9-12 mm diam after 23 d), raised, cerebriform, compressing and cracking the medium, some pilose aerial mycelium centrally and over other parts of the colony, gelatinous clumps and mucilaginous drops abundant centrally, cinnamon with paler whitish periphery; reverse umber with many cracks in medium visible; colonies composed of a combination of thin-walled hyaline hyphae and dark pseudoparenchyma with muriform chlamydospores; sporulation abundant. Notes: Isolate CPC 18544 was initially identified as "Sphaceloma asclepiadis", which is characterised by yellowish, fusiform conidia, 10-15 × 3-4 μm, based on the type material from Asclepias curassavica in Brazil (Bitancourt & Jenkins 1949). However, morphological examination of the freshly collected isolate (CPC 18544) indicated that it could be distinguished from "Sphaceloma asclepiadis" by having smaller, ovoid conidia, 4-6.5 × 2-3.5 μm.  Notes: This fungus was originally described from Citrus sinensis in Brazil, causing a disease known as sweet orange fruit scab, with globose to obclavate asci, and 2-4 celled ascospores, 12-20 × 4-8 μm (Bitancourt & Jenkins 1936a, b). It was also found to be similar to Elsino€ e fawcettii but differentiating morphological characters were found, including well-developed globose ascostromata, and longer ascospores as well as different host circumscriptions (Bitancourt & Jenkins 1936a Notes: Elsino€ e banksiicola is known to cause visible brown amphigenous spots on Banksia leaves, up to 8 mm diam, sometimes also occurring on stems of Banksia prionotes in Victoria, Australia. Conidia hyaline, aseptate, ellipsoid, (4-) 8-9(-10) × (2.5-)3-4 μm in vitro (Pascoe et al. 2007). Only one other species of Elsino€ e is known from Banksia, namely E. banksiae. The two species are easily distinguished based on their symptomatology, morphology and cultural characteristics. Notes: Elsino€ e barleriicola is known to cause leaf and stem spots of Barleria gibsonii in India. Acervuli are dark brown to brownish red, ellipsoid to pyriform, intraepidermal, erumpent, 10-24 × 30-62 μm. Colonies on PDA are deep red on the surface, and reddish brown in reverse (Wani & Thirumalachar 1970).  Notes: Elsino€ e bidentis is known to infect leaves and stems on Bidens pilosa, B. segetum and B. subalternans in Brazil (Guatimosim et al. 2015). Symptoms include dark, irregular or elongated lesions, 0.5-2 mm diam on leaves; numerous coalescing spots slightly protruding, 0.2-0.6 mm diam on stems. Acervuli dark, slightly protruding, 15-50 μm diam (Bitancourt & Jenkins 1950). The description provided in the original publication is rather incomplete and based on a seemingly sterile specimen. The fungus was recently recollected by Guatimosim et al. (2015) who provided a complete description, quoted below: "Lesions on leaves and stems: on leaves, mostly along secondary veins, hypophyllous, depressed, irregular, 0.4-2.2 mm diam, leading to disintegration and flecking of host tissue, pale grey in centre; on stems, typical scab symptoms with numerous rounded to irregular warts, with russet vinaceous brown halos, and vinaceous centre, slightly wrinkly. Depending on intensity, leading to distortions of growing stems that may become sinuous or twisted and accompanied by defoliation and die-back of organs above infected areas. Internal mycelium septate, branched in acute angles, 2-3 μm diam, with some enlarged rounded cells, hyaline, smooth, often producing chlamydospores. Acervuli almost indistinct, erumpent, localised over a hyaline pseudoparenchyma, formed by 2-3 layers of swollen, irregular cells, 30-100 μm diam. Conidiogenous cells ampulliform, with an acute apex, 7 μm, hyaline, smooth. Conidia subcylindrical, 3-5(-8) × 2-4 μm, hyaline, smooth. Notes: Elsino€ e brasiliensis is known to cause leaf spots, petiole and stem cankers or galls on Euphorbia hyssopifolia (= Chamaesyce hyssopifolia) in Brazil (Bitancourt & Jenkins 1940a). Symptoms include small spots scattered or located at the margin, with a narrow dark border; stem cankers are circular to ellipsoid, elevated with medium to dark brown margin, 4 × 2 mm. This fungus is characterised by globose asci, 17-21 μm diam,  containing eight hyaline, 3-septate ascospores, 12-14 × 5-7 μm (Bitancourt & Jenkins 1940a). The culture CPC 18528 was isolated from the same host in Brazil, and therefore we designate it here as ex-epitype. In culture: Colonies raised, ridged, sometimes cracking at folds, cerebriform, compressing and cracking the medium, aerial mycelium absent or sparse, floccose to downy, vinaceous grey centrally with ochreous sectors and purplish grey periphery, with mucilaginous drops; reverse dark purple to ochreous; colonies composed of thick-walled hyphae and yellowish to dark brown pseudoparenchyma; slow growing, 15 mm diam after 23 d; sporulation abundant.
The complex of species reported on Chamaesyce spp., Euphorbia spp., Manihot spp. and related euphorbiaceous genera have been investigated more closely by plant pathologists because of the relevance of the superelongation disease of cassava and the impact of scab on weedy hosts as well as the possibility of weedy and wild members of Euphorbiaceae serving as reservoirs for the disease on cassava (Zeigler & Lozano 1983, Barreto & Evans 1998, Alvarez & Molina 2000, Alvarez et al., 2003, Nechet et al. 2004). An organised attempt to clarify the identity of the fungus behind superelongation of cassava (Zeigler & Lozano 1983) based on examination of fresh and herbarium specimens, cultural features and cross inoculations of isolates obtained from various Euphorbiaceae led to the conclusion that variability and overlap of morphological and cultural characters did not allow for a clear separation of taxa in this complex. Additionally host specificity tended to vary between isolates from a single host and was also inadequate as a basis for species separation. Based on their results these authors proposed that the fungus attacking C. hyssopifolia (among other hosts) belonged to Elsino€ e brasiliensisa conclusion confirmed with the present multi-gene phylogenetic study (Fig. 4). These authors also accepted Sphaceloma poinsettiae as a separate taxon having Euphorbia heterophylla and Eu. pulcherrima as hosts and considered Sphaceloma krugii as its synonym. This is in disagreement with the present study. Here isolates from several of these hosts belonged to separate clades, showing that there are at least four independent species of Elsino€ e attacking Chamaesyce spp., Euphorbia spp., and Manihot spp. Notes: Elsino€ e caleae was described by Bitancourt & Jenkins (1941) causing "spots and anthracnose" on leaves and stems of Calea pinnatifida in Brazil. On leaves lesions were circular to slightly irregular, amphigenous, 1-2 mm diam; on petioles and stems lesions were small, and slightly elongated, 0.5 × 0.6-2 mm. The fungus was described as having globose to subpyriform asci (21-26 × 21-24 μm), and hyaline, 3-septate (sometimes with longitudinal septa) ascospores (13-17 × 6-8 μm). Notes: Elsino€ e centrolobii was described by Bitancourt & Jenkins (1949) causing lesions on leaves of Centrolobium robustum in Brazil. Symptoms include small, rounded or slightly irregular leaf spots, torn when larger, causing deformations, 0.1-1.2 mm diam. The fungus was characterised by globose to oblong asci, 22-26 μm diam, and hyaline, 3-septate ascospores (sometimes with a longitudinal septum), 12-15 × 4-6 μm (Bitancourt & Jenkins 1949). The LSU region fails to distinguish E. centrolobii, E. fici, E. jasminae, and E. randii.
Etymology: Named after the host genus from which it was collected, Citrus.
Culture characteristics: Colonies irregular, erumpent, folded, surface apricot, with smooth, irregular margins and sparse white aerial mycelium; 10-15 mm diam after 3 wk; sterile. Notes: The isolate CPC 18535 was originally identified as "Sphaceloma fawcettii". It is, however, genetically distinguished from ex-type strains of Elsino€ e fawcettii (CBS 139.25) and others (CBS 231.64,CBS 232.64,CBS 233.64), based on four sequenced loci. Morphologically, E. citricola is very similar to E. fawcettii, and the two species cannot be distinguished based on conidial size alone (5.5-9 × 2.5-4.5 vs. 5-10 × 2-5 μm) (Jenkins 1925 Notes: The original description of Elsino€ e diospyri by Bitancourt & Jenkins (1950) on Japanese persimmon (Diospyros kaki) in Brazil includes a description of leaf spots symptoms (but no reference to scaby lesions being formed) which are white in the middle, dark brown or black at the margin, 0.1-0.5 mm diam. The authors also described the sexual morph as characterised by globose asci, 28 μm diam, containing eight hyaline, 1-3-transversally septate ascospores, 8-10 × 4-5 μm. The asexual morph is described as sporodochial, but no conidia were observed. Attempts at recollecting fresh material of the fungus in São Paulo in the context of this work proved unsuccessful.
Etymology: Named after the host genus on which it occurs, Embelia.

Elsino€ e eucalyptorum
Etymology: Named after the host species from which it was collected, Jasminum malabaricum.
Notes: Elsino€ e ledi was formerly treated as Aulographum ledi on Ledum glandulosum in the USA (Peck 1911). Zeller & Deremiah (1931) examined some older materials and treated it as Elsino€ e ledi based on its hyaline, subsphaeroid asci scattered in the entostroma with ellipsoid to fusoid, mostly 3-septate ascospores, 12-17.7 × 5-6.5 μm, which are similar to those of Elsino€ e ampelina. A.E. Jenkins examined the Oregon materials studied here and identified them as E. ledi according to Peck's type (Zeller & Deremiah 1931). We therefore designate this collection (CBS 167.33) as epitype, because it agrees well with the original records, having the same host and location. Colonies irregular, erumpent, folded; surface cinnamon, with white aerial mycelium and smooth, sinuate margins; 8-12 mm diam after 3 wk; sterile. Notes: Elsino€ e lepagei was described causing scab on leaves and branches of Manilkara zapota in Brazil (Bitancourt & Jenkins 1941). Symptoms include grey-brown, irregular, prominent, welldefined leaf spots, 0.5-2 mm diam and smaller (0.5-1 mm diam) lesions on stems. This fungus is characterised by globose asci, 16-22 × 12-21 μm, containing eight hyaline, 3-septate ascospores, 10-16 × 4-7 μm (Bitancourt & Jenkins 1941). In culture on PDA (original description): "Colonies slow-growing, compact, convoluted, downy aerial mycelium centrally, viscid marginal area, cinnamon buff." Elsino€ e leucospermi L. Swart & Crous, Mycologia 93: 370. 2001. Fig. 19 Notes: Elsino€ e leucospermi is known to cause leaf spots of Leucospermum and Leucadendron in Australia, South Africa, USA and Zimbabwe, and also could infect stems of Serruria (Swart et al. 2001). In Australia, scab disease symptoms have been observed on many genera of Proteaceae, including Banksia, Leucadendron, Mimetes, Protea and Serruria (Forsberg 1993, Pascoe et al. 1995, Crous et al. 2013. Elsino€ e is thought to be distributed via asymptomatic nursery material and this has probably occurred on an international scale. Asci ovoid to subglobose 16-28 × 13-18 μm. Ascospores hyaline, broadly ellipsoid with rounded to obtuse ends, 1-4 transverse, 1-2 vertical septa (oblique septa rare), constricted at the median septum, (10-)12-14(-19) × 4-5 μm. Conidiomata acervular, foliicolous but primarily caulicolous, raised, coalescing at maturity, composed of medium brown textura angularis, up to 200 μm diam and 1 mm long. Conidiophores subcylindrical, pale brown, verruculose, 0-2-septate, 20-30 × 3-6 μm. Conidiogenous cells polyphialidic, with 1-2 integrated loci, pale brown, verruculose, ampulliform to doliiform, 10-15 × 3-4 μm. Conidia hyaline, granular, aseptate, ellipsoid, with obtuse apex, constricted at the base to a subtruncate hilum, (2-)5-7(-8) × (1-) 2.5-3 μm in vivo, 5-7 × 2-3 μm in vitro (Swart et al. 2001). Notes: Elsino€ e lippiae is known to infect Phyla lanceolata (previous Lippia lanceolata) in the USA. Symptoms include numerous spots on leaves and stems that are scattered or aggregated, centres depressed, buff-coloured, with purple margins (Baines & Cummins 1939).  Notes: Elsino€ e mangiferae was described by  as the etiological agent of "wart disease" (= scab) of mango (Mangifera foetida) in Chile, Cuba and the USA. Symptoms were described in detail in the original description )in brief: small leaf spots, greyish pink, rounded to oval, slight elevated, 0.5-15 mm diam; on shoots spots rounded to oval, closely grouped to form larger patches of crusts (scabs), 1-1.5 mm diam. This fungus is characterised by dark reddish brown, ellipsoid to lenticular acervuli, 15-30 × 23-52 μm, and small, hyaline, spherical to ellipsoid conidia, "0-1-septate, frequently in chains, 6-29 × 2-4 μm"; ascostromata pulvinate, 80-160 μm diam and 30-48 μm thick, asci globose, 10-15 μm diam, ascospores 3septate (sometimes with one longitudinal septum), 10-13 × 4-6 μm; "microconidia" abundant. In culture (on PDA, according to the original description): Colonies raised, thinly convoluted, mostly glabrous, white to grey to pinkish downy aerial mycelium centrally, humid periphery. The situation regarding E. mangiferae was later complicated by the publication of Alcorn et al. (1999). While studying mango scab in Australia these authors examined samples from Australia and re-examined the type material and concluded that a species of Denticularia was involved in both Australian specimens and type material which fitted clearly into the description of the morphology for the asexual morph of the scab fungus included in Bitancourt & Jenkins' publication. It had erect and well developed conidiophores and long conidia and was recognised as a hyphomycetous morph and not close to any "Sphaceloma morph". Alcorn et al. (1999) proceeded to proposing a new combination Denticularia mangiferae for the fungus causing mango scab. Nevertheless, although obtaining pure cultures of the scab fungus from Australia and fulfilling Koch's postulates doubts remain as to which fungus were they actually handling during the inoculation studies. Their culture description is equivalent to that of most Elsino€ e spp. and they did not manage to produce typical Denticularia conidiophores or conidia in culture. The phylogenetic analysis of the type culture leaves no doubt as to the scab fungus on mango correctly belonging to Elsino€ e. It is reasonable to conjecture that the Denticularia found on scabbed tissues is either a saprobe or a mycoparasite that is regularly associated with E. mangiferae and led to  producing a mistaken description incorporating the information on Denticularia in their holomorph description. Later, while dealing with the complex again Alcorn et al. (1999) interpreted correctly the asexual morph present on the specimens as belonging to Denticularia but left aside the evidence provided by the description of the sexual morph in  and mistakenly proposed the new combination Denticularia mangiferae. One final puzzle is why there was successful completion of Koch's postulates by Alcorn et al. (1999) with their Denticularia isolate. A possible explanation for that might be that, while attempting to isolate Denticularia they have inadvertently obtained a pure culture of E. mangiferae to which Denticularia was associated and were then capable of reproducing the scab disease when it was used as inoculum. If that proves right, then the Denticularia on mango scab still needs to be named. It is interesting to note that during a survey for fungal pathogens to be used for weed biocontrol performed by RWB in Brazil (state of Rio de Janeiro) (Barreto & Evans 1995) a severe scab disease was found on Mimosa diplotricha (a major pantropical weed) and the dominating fungus found associated to the symptoms was also identified as a Denticularia and, at the time, mistakenly interpreted as the etiological agent of the diseasein fact caused by Elsino€ e mimosae.
Notes: This fungus causes a severe disease on M. diplotricha in South America on habitats ranging from the Ecuadorian Amazon to the highlands of the state of Rio de Janeiro (Brazil). It remains restricted to the neotropics and has clear potential for use as a classical biological control agent against its host (giant sensitive plant), which is a major pantropical weed. The LSU region fails to distinguish E. arachidis, E. bidentis, E. euphorbiae, E. genipae, E. krugii, E. mimosae, E. poinsettiae, E. sesseae, and E. fawcettii strain CBS 139.25. The rpb2 region failed to distinguish E. genipae, and E. mimosae. Notes: Elsino€ e oleae is known to cause leaf spots of Olea europaea in Italy. Symptoms include prominent, circular, oval or irregular spots that become linear, rugulose, 0.2-2 × 0.1-0.5 mm. This fungus is characterised by hyaline, ovoid asci, 25-30 × 10-14 μm, containing eight hyaline, fusiform, 3-septate ascospores, 12-15 × 3-4 μm. The asexual morph of this fungus has hyaline, ellipsoid, ovoid to subglobose conidia, 2-3.5 × 3-6 μm (Ciccarone & Graniti 1959). Notes: Occurring on stems of Othonna quinquedentata in South Africa. Symptoms include circular to subcircular lesions, pale grey-brown with dark red-brown borders, 1-10 mm diam. In culture on SNA: Conidia hyaline, guttulate, smooth, aseptate, ellipsoidal to subcylindrical, apex obtuse, base bluntly rounded to truncate, (5-)6-7 × (2.5-)3(-4) μm in vitro (Crous et al. 2015b). Because not all genes were successfully amplified, E. othonnae was omitted from the final multigene alignment (Table 1). ( Notes: Elsino€ e perseae was originally reported to infect leaves and fruits of Persea americana in the USA, causing scab disease. This fungus is characterised by hyaline, ovoid to oblongellipsoid, biguttulate conidia, 5-8 × 3-4 μm (Jenkins 1934). Following its initial description, it was broadly recorded in the warmer regions of the American continent (nt.ars-grin.gov/ fungaldatabases/) but also in Africa, America, Asia and New Zealand (Hartill 1991, Everett et al. 2011. It causes one of the most important diseases of avocado (Piccinini et al. 2005). Notes: Elsino€ e phaseoli is known to cause scab of beans (Phaseolus lunatus) in Cuba (Bruner & Jenkins 1933). It has subsequently been reported on Phaseolus and Vigna in Africa, America and Brazil (Lasca 1978, Phillips 1994. Symptoms include circular lesions on the leaves, occurring on the upper surface of the leaf, 2-3 mm diam; lesions on the stems elongated, white to grey, slightly sunken on green pods, turning redbrown, slightly raised, 2-3 mm diam. This fungus is characterised by ovoid to subglobose asci, 14-22 × 21-27 μm, and hyaline, oblong to ellipsoid ascospores, 10-15 × 4-5 μm, with 2-3 septa (Bruner & Jenkins 1933, Phillips 1994. The conidia are oblong to ellipsoid, 6 × 2 μm (Phillips 1994). Notes: Elsino€ e piri is known to cause apple and pear spot and anthracnose and is economically important in some organic orchards, but is rarely observed in orchards with a conventional fungicide regime (Scheper et al. 2013). It has a worldwide distribution and has been often recorded incorrectly spelled as "Elsino€ e pyri" (Jenkins 1932a). Symptoms include whitish grey leaf spots with brown margins, having visible dark brown ascostromata in the centre of the spots. Spots on fruits can vary in colour from white to pale yellow brown, to brown in the centre and surrounded by a dark red margin. This fungus is characterised by hyaline conidia, aseptate, 4-6 × 2.5-4 μm, which may be present on acervuli on leaves and fruits (Woronichin 1914, Jenkins 1932a, Scheper et al. 2013. The LSU region fails to distinguish E. fici-caricae, E. mattiroloanum, E. piri, and E. sicula. The TEF1-α region fails to distinguish E. piri, and E. sicula. Notes: Elsino€ e pitangae is known to cause lesions (referred to as anthracnose in the original description) on leaves and branches of Eugenia unifloraa native Brazilian myrtaceous fruit crop   (pitangueira)in São Paulo, Brazil. Symptoms include small spots, round, scattered, amphigenous, depressed, and perforated, occasionally in the centre with protruding edges, 0.2-2 mm diam. This fungus is characterised by globose to ovoid asci, 18-21 × 14-18 μm, and hyaline, 1-3-septate ascospores, 14-20 × 4-6 μm (Bitancourt & Jenkins 1940b). Cultures were briefly described by Bitancourt & Jenkins (1940b) on PDA as: slow-growing, compact, convoluted, black to dull purplish black with some short downy, whitish aerial mycelium. Notes: Elsino€ e poinsettiae was originally described from infected leaves and branches of Euphorbia pulcherrima var. plenissima in Florida, USA (Jenkins & Rühle 1942). Scab disease of this host was subsequently found in Brazil, Guatemala, Jamaica and Puerto Rico (Rubin 1961, Wehlburg 1968. Elsino€ e poinsettiae may cause economic losses in ornamental poinsettia nurseries. The name S. poinsettiae has been mistakenly used for the fungus causing scab on wild poinsettia (E. heterophylla) which was studied in detail as a potential biocontrol agent to be used against this major weed (Nechet et al. 2004). The phylogenetic analysis has shown that all isolates attacking E. heterophylla in fact belong to E. krugii.

Elsino€ e perseae
Conversely, E. pulcherrima appeared as a host of E. krugii, as revealed by the results of the molecular identification of isolate CPC 18537, a surprising result since the host-range evaluations of Nechet et al. (2004) of isolates of Sphaceloma obtained from E. heterophylla (then seemingly mistakenly identified as S. poinsettiae) involving 37 plant species, including E. pulcherrima among several members of the Euphorbiaceaeresulted only in the infection of E. heterophylla. It is known that there is variation in host range between different populations within species of Elsino€ e infecting Euphorbiaceae. In order to better clarify the situation for Elsino€ e attacking euphorbiaceous hosts a novel round of studies involving cross inoculations of a range of isolates from different hosts in the family following the lead of Zeigler & Lozano (1983), but supported by the results of the present molecular evaluation of these taxa and utilising modern tools for population studies is needed. Notes: Elsino€ e pongamiae was originally described as "Sphaceloma pongamiae" on Pongamia glabrae in India (Wani & Thirumalachar 1970). Symptoms include small, numerous spots on shoots and pods, often coalescing, forming greyish white crusts (= scab); numerous spots on leaves scattered, less often closely grouped with chalky-white to greyish pink margin, 1-2 mm diam. This fungus is characterised by spherical to ovoid, aseptate conidia, 1.5 × 1.5 μm (Wani & Thirumalachar 1970). The exepitype strain deposited at CBS (CBS 402.63) was isolated by the same collector and from the same host genus in India. In culture: Colonies circular, raised colonies, surface white to rosy buff, with smooth, sinuate margins and white aerial mycelium; 15-18 mm diam after 3 wk; sterile. The ITS and TEF1-α regions fail to distinguish E. embeliae and E. pongamiae.   Notes: Elsino€ e populi was originally described from infected leaves of Populus nigra in Europe, causing scab disease (Jenkins 1932a). This fungus is characterised by hyaline, globose to ovoid conidia, 4-5 × 3 μm (Saccardo 1878, Jenkins 1932a Notes: Occurring on leaves and petioles of Protea spp. in South Africa. It was described by (Swart et al. 2001) as follows: Symptoms include circular, raised leaf spots, white-grey with visible black ascostromata on both side of leaves. Ascospores hyaline to olivaceous, broadly ellipsoid with rounded ends, with 3-5 transverse, and 1-3 vertical septa; oblique septa sometimes present; mostly slightly constricted at the median septum, (14-) 16-17(-20) × (5-)6-7 μm. Conidia hyaline, granular, aseptate, ellipsoid, with obtuse apex, and rounded to subtruncate base, (5-) 6-7(-8) × 2-3(-4) μm. In culture: Colonies irregular, erumpent, folded, with sinuate, smooth margins, rose to red; aerial mycelium sparse, whitish; with diffuse red pigment in the medium. Because not all genes were successfully amplified, E. proteae was omitted from the final multigene alignment (Table 1). Notes: Elsino€ e protearum is known to cause leaf spots on Protea sp. in Australia and Zimbabwe (Swart et al. 2001). Ziehrl et al. (2000) also demonstrated that Elsino€ e spp. from South African Proteaceae could infect Australian proteaceous genera such as Banksia and Dryandra, but not Telopea, Grevillea or Hakea. Symptoms include circular, reddish leaf spots, erumpent with reddish sporodochia on the necrotic tissue, 5-15 mm diam. Conidia hyaline, aseptate, ellipsoid, with obtuse apex, constricted at the base to a subtruncate locus, (3.5-)5-6(-7) × (1.5-) 2-2.5 μm in vivo. In culture: Colonies irregular, erumpent, folded with sinuate, smooth margins, blood red, with diffuse red pigment (Swart et al. 2001).
Etymology: Named after the host genus from which it was collected, Salix sp.

DISCUSSION
The order Myriangiales has two families, Elsinoaceae and Myriangiaceae, that accommodate two and 10 genera, respectively (Dissanayake et al. 2014  SSU, LSU, rpb2, and TEF1-α sequences available . The same situation appeared to be the case in the Elsinoaceae, which was reassessed by Jayawardena et al. (2014) who excluded eight genera (Beelia, Butleria, Hemimyriangium, Hyalotheles, Micularia, Saccardinula, Stephanotheca and Xenodium) and included only Elsino€ e and Molleriella in this family. Prior to the present study, the available molecular dataset for Elsino€ e (asexual morph Sphaceloma) proved to be rather sparse (Swart et al. 2001, Summerell et al. 2006, Crous et al. 2015b. Different authors have discussed the relationship between Elsinoaceae and Myriangiaceae, but based on morphological features and phylogenetic analyses, the two families were accepted as distinct within the order Myriangiales (Schoch et al. 2006, Lumbsch & Huhndorf 2007, 2010, Boehm et al. 2009, Hyde et al. 2013).
In accordance with the "One Fungus = One Name" concept, a single name for polymorphic genera has generally followed the rule of choosing the oldest or the most commonly used name with the most species epithets (Hawksworth 2011, Taylor 2011, Rossman et al. 2015. In the case of Elsino€ e, the younger sexual name Elsino€ e Racib. (1900) was chosen for protection over that of the older asexual name, Sphaceloma de Bary (1874) (Wijayawardene et al. 2012, Rossman et al. 2015. Therefore, many names in Sphaceloma need to be formally recognised in Elsino€ e, with a first set of 26 Sphaceloma species being relocated to Elsino€ e in the present study. As is the case with many phytopathogenic genera of Dothideomycetes, the most common phylogenetic problem related to Elsino€ e taxonomy is that many species (e.g. E. ampelina, E. australe, E. fawcettii and E. perseae) are based on old specimens without accompanying sequence data (Jenkins 1925, 1932a, b, Bitancourt & Jenkins 1936a. In fact, until 2014 there were only 12 strains available in GenBank for multigene phylogenetic studies (ITS, LSU, TEF1-α) ). In the first taxonomic phylogenetic study of Elsino€ e, Swart et al. (2001) used ITS sequence data to evaluate the phylogenetic significance of six species from Proteaceae. According to the Dictionary of Fungi, Kirk et al. (2008) recognised about 50 species in Elsino€ e, although more than 140 species have been described to date (see Index Fungorum and MycoBank). A significant result of the present study was thus to extend the number of genes used in Elsino€ e phylogenetic studies, as well as the number of species subjected to DNA analyses.
The taxa investigated in the current study represent the largest collection of Elsino€ e and Sphaceloma strains ever subjected to DNA sequence analysis. A total of 73 single species lineages from 119 Elsino€ e strains were recognised based on ITS, LSU, rpb2 and TEF1-α sequence data, including eight new species, 13 epitypifications and 26 new combinations (Table 1). During the course of this study it was observed that, although the ITS is a useful locus for distinguishing most species of Elsino€ e (resolving 61 / 74 (82.4 %) of the species included in the phylogenetic tree), the rpb2 and TEF1-α regions performed much better at species resolution (resolving 65 / 71 (91.5 %) and 64/73 (87.7 %) of the species included in the phylogenetic tree, respectively). Specifically, the rpb2 and TEF1-α regions could distinguish the quarantine pathogen E. australis from its closest neighbours. The LSU region was able to distinguish only 51 of the 73 (69.9 %) species included in the phylogenetic tree. The total number of species counted for each of LSU, rpb2 and TEF1-α is slightly lower compared to ITS as species for which the respective gene sequence was missing was excluded from the count. This is of interest for disease diagnosis and quarantine services. The Elsino€ e species treated were sampled from various plants distributed over 17 countries in different continents including Africa, Asia, Australia, Europe, Latin America and North America. In spite of the limited number of strains per species, the vast majority of Elsino€ e species studied here appear to be host-specific (Fig. 1).
Phylogenetic studies based on type materials are hampered by the lack of authentic cultures, and thus epitypification from fresh collections is required to create a stable and workable taxonomy. There are several excellent studies on a number of Sphaceloma and Elsino€ e species associated with plant diseases in Brazil, India and the USA (Jenkins 1932a, b, Bitancourt & Jenkins 1936a, Narasimhan et al. 1969a, b, Wani & Thirumalachar 1969a, whereas very few species have any available cultures or DNA data, and thus cannot be included in recent studies of Elsino€ e. Epitypification of these taxa is urgently required (Cannon et al. 2012). Here we designated 13 epitypes based on specimens and cultures deposited at CBS, but no DNA data are presently available for the type species, E. canavaliae from Canavalia gladiata in Java, Indonesia.
With regard to host associations, species of Elsino€ e seem to have narrow host ranges, mostly limited to a single host species. Of the 73 species subjected to multi-gene analyses, only four were found to occur on more than one host. These include E. leucospermi (from Leucospermum and Leucadendron; Proteaceae), E. anacardii [from Anacardium, (Anacardiaceae) Annona (Annonaceae) and Rosa (Rosaceae)], E. violae [from Symphoricarpos (Caprifoliaceae) and Viola (Violaceae)] and E. piri (from Malus and Pyrus; Rosaceae). These unexpectedly broad host-range species need to be recollected and critically reexamined: E. anacardii in particular, for having hosts in broadly separate plant families. Each of the other 69 species included in this study is known to occur on only one host species or genus. An unexpected result is that we have no obvious distinct geographic distributions according to the phylogenetic tree obtained, which delineated two main subclades (Fig. 4). One subclade (MP/BPP = 89/1) contains 57 species, including the important phytopathogens E. ampelina (on grapevines) and E. fawcettii (on citrus). Another subclade (MP/BPP = 97/1) contains 14 species, including E. australis (on citrus).
It is clear that the leading mycologists dealing with Elsino€ e in the mid 20 th century (A. Bitancourt and A.E. Jenkins) have considered scab symptoms as a major character for recognising the presence of fungi belonging to Elsino€ e/Sphaceloma. To the point of having proposed new species based on disease symptoms alone, with no sporulation present on the specimens. Examples are S. allamandae and S. psidii (Bitancourt & Jenkins 1949), among others. In most instances, nevertheless, when no conidia or ascospores were found, identification had the support of successful isolations resulting in cultures having the appearance commonly found for fungi in this genusslow growing, raised, often cerebriform or corrugated, dark red, orange or brown colonies. In cases where not even cultures were obtained such species should be regarded as doubtful until fertile specimens and pure cultures are obtained since scab symptoms on plants may arise because of other fungal agents such as Venturia inaequalis (apple scab), bacteria such as Streptomyces sacabies (potato scab), or arthropod attack.
It is necessary to acknowledge the important legacy of Agesilau A. Bitancourt (Instituto Biol ogico de São Paulo -Brazil) and Anna E. Jenkins (USDA -USA) (Fig. 33) who have worked and published independently and in cooperation from the 1930s PHYLOGENY AND TAXONOMY OF GENUS ELSINO€ e www.studiesinmycology.org to the 1960s on the Elsinoaceae, and managed to collect and describe a significant proportion of the known species of Elsino€ e/ Sphaceloma. This wasn't a small feat considering the challenges involved in the collection, observation and isolation of Elsinoaceae. Many of the isolates included in the present publication were originally obtained by Bitancourt and Jenkins who cared for depositing their isolates in the CBS, and hence allowed for this work to be accomplished now. The fact that they remained viable after up to 80 yr in preservation underlines the sturdiness of Elsino€ e. Isolation of these fungi in pure culture has played an important role for confirmation of a suspected scab or spot anthracnose disease as having a Elsino€ e etiology. It now becomes even more important for the taxonomy of the genus, given the body of DNA sequences available following the present study. Not many publications provide descriptions of procedures for isolating these difficult microfungi, but Bitancourt & Jenkins (1939a) described a "routine procedure" which would be worthy of consideration, particularly, regarding the large number of species successfully isolated by them. Unfortunately, their "routine procedure" was based on single ascospores as starting point. As these are notoriously difficult to find, this method can hardly qualify as "routine".
The procedure for isolation of Elsino€ e spp. (used by R.W. Barreto) we advise others to use is as follows: a) always start from fresh material (herbarium material has never proven successful); b) under the dissecting microscope select a piece of scab or anthracnose tissue free of saprobes or mycoparasites; c) rub the scab lesion vigorously with cotton wool soaked with 96 % ethanol; d) allow to dry; e) with a sterile sharp scalpel peel the surface of lesion with a shallow tangential cut; f) flame the scalpel blade; g) stab the medium in the plate to cool it down, and make the point of the blade humid and sticky; h) drive the point of the blade into the inner tissue that appeared where the epidermis was pealed and remove a very small fragment of infected tissue; i) transfer it to different demarcated points on a plate containing a routine medium for fungi (MEA, PDA, or MEA with antibiotics); j) prepare several plates (success rate is low); k) follow culture development on plates closely as Elsino€ e colonies are slow-growing and any contaminants may overgrow the colonies; l) choose dense, pseudostromatic, slow growing colonies arising from the fragments (often honey, orange, reddish or brown, with diffuse pigment in agar) and transfer them to fresh plates or tubes. An alternative method (used by P.W. Crous) is to simply scrape the sterile tissue surface with a scalpel, and make dilution plates (on MEA with antibiotics) of the conidiomata/ascostromata, and later pick up typical Elsino€ e colonies that become visible after a 2-7 d (viewed with light from below on a dissecting microscope).
In future studies of Elsino€ e, fresh specimens should be collected to help clarify the species concepts of taxa presently still lacking types linked to multigene DNA data. It is frequently difficult to observe typical sexual structures in many of these taxa represented by old specimens, and strains soon become sterile in culture. Phylogenetically, the relationship between the two main subclades and the position of the type species Elsino€ e canavaliae, also awaits to be clarified. This epitypification, supplemented by DNA data of related genera such as Molleriella and Myriangium, will significantly improve our knowledge of the evolutionary relationships within the order.