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Persoonia 39, 2017: 74–90 ISSN (Online) 1878-9080 www.ingentaconnect.com/content/nhn/pimj https://doi.org/10.3767/persoonia.2017.39.04RESEARCH ARTICLE INTRODUCTION Phyllachorales is an order of biotrophic, obligate plant parasitic fungi in the class Sordariomycetes, i.e., inoperculate pyreno- mycetes. About 1 226 species are currently accepted in the order (Kirk et al. 2008), although 160 000 species have been estimated to occur worldwide (Cannon 1997). Phyllachorales are highly diverse in the tropics, relatively common in disturbed and natural vegetation, and likewise found in open and forested areas (Piepenbring et al. 2011). Species of Phyllachorales are leaf- or steam-inhabiting micro- fungi with shiny black stromata, which gave them the common name ‘tropical tar spot fungi’. They are morphologically char- acterised by: deep black stromata of various shapes (except in species of Polystigma which have brightly coloured stromata); pseudostroma inside the host tissue and usually beneath an epi- dermal clypeus; perithecia usually strongly melanised that may be superficial, erumpent or immersed in the host tissue (Fig. 1a–f); thin-walled paraphyses which frequently deliquesce; unitunicate asci of cylindrical to clavate shape, with an ascus crown and an inconspicuous apical ring not staining blue in iodine; and globose to filiform ascospores, which in most spe- cies are hyaline and 1-celled, with only a few genera including species with brown or septate ascospores (Parbery 1967, Cannon 1991, 1997). Although it has been difficult to connect asexual and sexual morphs in the order due to their obligately parasitic condition, some species of Phyllachorales have been linked to the asexual genus Linochora (Von Höhnel 1910), which may be inconspicuous and spermatial in function (Parbery & Langdon 1963, Parbery 1996). Due to their biotrophic nutrition mode, high host specificity is assumed and species concepts are based partly on the identity and systematic position of the corresponding host plants. Hence to identify species of Phyllachorales, it is necessary to identify the host plant. Traditionally, new species have been described on the basis of new host records at generic level. However, examination of species of Phyllachorales on the host families Poaceae and Fabaceae demonstrated that tropical tar spot spe- cies are not restricted to a single host genus, but may occur on species belonging to a group of closely related genera (Parbery 1978, Cannon 1991, 1997). Species delimitation based mainly on host identity may therefore lead to over-splitting of species (Cannon 1997). Phyllachorales are associated with diverse host plants, and most of the species are linked to angiosperms, with a few ex- ceptions including the lichenicolous Lichenochora species, the marine algicolous genus Phycomelaina, and some species on ferns and gymnosperms. Within the angiosperms, the following families are preferentially parasitized: Arecaceae, Fabaceae, Phylogeny of the order Phyllachorales (Ascomycota, Sordariomycetes): among and within order relationships based on five molecular loci M. Mardones1,2, T. Trampe-Jaschik1, S. Oster1, M. Elliott3, H. Urbina4, I. Schmitt1,5, M. Piepenbring1 1 Institute of Ecology, Evolution and Diversity, Faculty of Biosciences, Goethe University Frankfurt am Main, Biologicum, Max-von-Laue-Str. 13, 60439 Frankfurt am Main, Germany; corresponding author e-mail: melissamardones@gmail.com. 2 Escuela de Biología, Universidad de Costa Rica, San Pedro, 11501 San José, Costa Rica. 3 Department of Plant Pathology, University of Florida – IFAS, Fort Lauderdale Research and Education Center, Davie, FL-33314, USA. 4 Department of Botany and Plant Pathology, Purdue University, West La- fayette, IN-47907, USA. 5 Senckenberg Biodiversity and Climate Research Centre (BiK-F), Sencken- berganlage 25, 60325 Frankfurt am Main, Germany. Key words ancestral state reconstruction plant parasitic tar spot fungi Telimenaceae Abstract The order Phyllachorales (Pezizomycotina, Ascomycota) is a group of biotrophic, obligate plant parasitic fungi with a tropical distribution and high host specificity. Traditionally two families are recognised within this order: Phyllachoraceae and Phaeochoraceae, based mostly on morphological and host characteristics. Currently, the position of the order within the class Sordariomycetes is inconclusive, as well as the monophyly of the order, and its internal phylogenetic structure. Here we present a phylogeny of the order Phyllachorales based on sequence data of 29 species with a broad host range resulting from a wide geographical sampling. We inferred Maximum Likelihood and Bayesian phylogenies from data of five DNA regions: nrLSU rDNA, nrSSU rDNA, ITS rDNA, and the protein coding genes RPB2, and TEF1. We found that the order Phyllachorales is monophyletic and related to members of the subclass Sordariomycetidae within Sordariomycetes. Within the order, members of the family Phaeochoraceae form a monophyletic group, and the family Phyllachoraceae is split into two lineages. Maximum Likelihood ancestral state reconstructions indicate that the ancestor of Phyllachorales had a monocotyledonous host plant, immersed perithecia, and a black stroma. Alternative states of these characters evolved multiple times independently within the order. Based on our results we redefine the family Phyllachoraceae and propose the new family Telimenaceae with Telimena erythrinae as type species, resulting in three families in the order. Species of Telimena spp. occur in several monocotyledonous and eudicotyledonous host plants except Poaceae, and generally have enlarged black pseudostroma around the perithecia, a character not present in species of Phyllachoraceae. Article info Received: 19 September 2016; Accepted: 1 March 2017; Published: 20 June 2017. 75M. Mardones et al.: Phylogeny of Phyllachorales Fig. 1 Perithecia of species of Phyllachorales in different positions in the mesophyll of the leaves. a. Phyllachora graminis (isotype CUP3536) with immersed perithecia and few pseudostroma; b. Coccodiella miconiae (ppMP1342) with superficial perithecia; c. Camarotella costaricensis (MM-21) with erumpent perithecium; d. Polystigma pusillum (MM-113) with brightly coloured stroma; e. Serenomyces phoenicis (F59049) with subcuticular perithecium and without a clypeus; f. Telimena bicincta (epitype MM-133) with immersed perithecia and strongly developed pseudostroma. — Scale bars = 100 µm. Lauraceae, Melastomataceae, Moraceae, Myrtaceae, and Poa- ceae (Cannon 1997). Monographs for phyllachoraceous spe- cies are available for Arecaceae (Hyde & Cannon 1999), Faba- ceae (Cannon 1991), and Poaceae (Orton 1944, Parbery 1967). Additional host families studied include Asclepiadaceae (Pearce et al. 1999), Erythroxylaceae (Cannon & Evans 1999), Proteaceae (Pearce et al. 2001), and Rosaceae (Cannon 1996). The genus Phyllachora was introduced on a herbarium label in Fuckels exsiccate series ‘Fungi Rhenani’ with a single species, P. agrostis (Fuckel 1867 in Cannon 1991), currently accepted as Scirrhia agrostis in Dothideales (Eriksson 1967). Later the genus Phyllachora was lectotypified with Phyllachora graminis as generic type (Clements & Shear 1931), and the genus name in the sense of Fuckel (1870) was conserved to allow continued use in its currently accepted circumscription. The order Phyl- lachorales was formally described by Barr (1983). However, throughout the history of the group, several authors have placed phyllachoraceous fungi into various families and orders, stressing different morphological and ecological characteristics: Diaporthales (Cannon 1988), Dothideales (Saccardo 1876, Theissen & Sydow 1915), Polystigmatales or Polystigmata- ceae (Von Arx & Müller 1954, Eriksson 1982, Hawksworth et al. 1983), Sphaeriales (Nannfeldt 1932, Luttrell 1951, Müller & Von Arx 1962, 1973), and Xylariales (Barr 1983). For a detailed description of the taxonomical history of the order see Cannon (1991) and Pearce & Hyde (2006). The order Phyllachorales comprises the families Phyllachorace- ae and Phaeochoraceae. The family Phyllachoraceae was erected by Theissen & Sydow (1915) and is by far the largest family within the order with almost 1 200 described species (Kirk et al. 2008). The number of genera varies between 51 (Kirk et al. 2008) and 73 (www.indexfungorum.org). Many of 76 Persoonia – Volume 39, 2017 these genera, however, have less than ten species and 27 are monotypic. Phyllachora is the largest genus with 994 species; and Coccodiella, Lichenochora, Ophiodothella, Polystigma, and Trabutia are also large genera with 22, 40, 36, 24, and 35 species, respectively (www.indexfungorum.org). The family Phaeochoraceae is a small group with 19 accepted species in the genera Cocoicola, Phaeochora, Phaeochoropsis, and Serenomyces and is known to occur only associated with spe- cies of Arecaceae (Hyde et al. 1997). Phaeochoraceae were provisionally assigned to Phyllachorales since the family was erected (Hyde et al. 1997), mainly due to their unusual stromatic characteristics. However, no molecular studies are available to clarify the family’s phylogenetic placement. Molecular phylogenetic studies including members of the Phyl- lachorales are infrequent, mainly because it is difficult to obtain cultures of these biotrophic fungi. The existing molecular stud- ies indicate, without support and with a limited sampling, that the order might be related to Sordariales or Boliniales (Winka & Eriksson 2000, Wanderlei-Silva et al. 2003, Inderbitzin et al. 2004, Trampe 2010). A recent large-scale phylogenetic study confirms the position of Phyllachorales in the subclass Sordariomycetidae with high support (Maharachchikumbura et al. 2015). However, in this phylogeny based on four loci, Phyllachorales are represented only by three taxa and the single locus nrSSU. The phylogeny within the order Phyllachorales is still almost unresolved and its monophyly has not yet been resolved. It is known that the Glomerella /Colletotrichum complex that was placed within Phyllachorales in the past, belongs to the Glomer- ellales (Wanderlei-Silva et al. 2003, Réblová et al. 2011) fungi. Some studies suggested that Phyllachorales are a polyphyletic assemblage, since several taxa had to be excluded from this order: Ophiodothella and Sphaerodothis were transferred to Xylariales and Hypocreales, respectively (Wanderlei-Silva et al. 2003); Plectosphaera eucalypti to Xylariales (Summerell et al. 2006), and Polystigma amygdalinum to subclass Xylariomy- cetidae (Habibi et al. 2015). These studies also suggested that among the studied taxa, Phyllachora and Coccodiella are the only genera forming a monophyletic clade, closely related to Sordariales, and considered as true Phyllachorales. Due to the limited availability of molecular phylogenetic data, information is lacking concerning the evolution of morphological traits and the co-evolution trails with the hosts. The aims of this study are: 1 to confirm the phylogenetic position of Phyllachorales within Sordariomycetidae; 2 to determine the monophyly of Phyllachorales; 3 to define monophyletic clades within the order for the de- limitation of families; and 4 to reconstruct the evolution of morphological and ecological characteristics to assess their value as systematic criteria. To achieve these objectives, we inferred the first comprehensive multilocus phylogeny for the order Phyllachorales. MATERIALS AND METHODS Taxon sampling Fresh specimens representing the two recognised families of Phyllachorales were collected mainly in Costa Rica during 2012–2015 and Western Panama during 2007–2015 (Trampe 2010). Additional specimens were collected from Benin, Ecua- dor, Germany, Thailand, and the USA. A total of 48 collections of tropical tar spot fungi, representing 29 species and six genera were sequenced. Specimens collected in the context of the present study were deposited in the following herbaria: FR, M, UCHI, USJ, and HUTPL. Extraction, amplification, and sequencing of DNA DNA was isolated directly from hymenia of fresh, recently col- lected material or from dry specimens except for the species belonging to family Phaeochoraceae, which were available as cultures previously isolated by Elliott & Des Jardin (2014). To extract non-melanised cells with high quality DNA, for each stroma, the clypeus was cut off in half to exposed the hymenia of 1–10 perithecia (diam c. 0.2–0.5 mm) that were removed and placed into 1.5 mL sterilised microtubes containing Cetyl- trimethyl ammonium bromide (2 % CTAB). The isolation of genomic DNA from fresh material was performed with 600 μL of extraction buffer (2 % CTAB; 100 mM Tris-HCl, pH 8; 1.4 M NaCl, and 20 mM EDTA) and the DNA was extracted using phenol-chloroform : isoamyl alcohol (24 : 1). For dry material the E.Z.N.A® Forensic DNA Extraction Kit (VWR-Omega, USA) was used following the manufacturer’s instructions with a few modifications. The material was homogenized for 5–10 min using a Retsch Mixer Mill MM301 with STL buffer and 2.5 mm Zirconia beads. Isolated DNA was resuspended in sterile wa- ter and stored at -20 °C. DNA concentration was checked by electrophoresis in 0.8 % agarose and by the spectrophotometer NanoDrop 2000c (Thermo Fisher Scientific, USA). Several attempts were made to extract DNA from older herbarium specimens (type specimens) but they were unsuccessful. For that reason, the macro- and micro-morphology of extracted phyllachoraceous specimens were rigorously compared with the respective type specimen when it was possible. Five partial nuclear gene regions (three ribosomal loci and two protein-coding genes) were amplified and sequenced: one fragment of the large subunit nuclear ribosomal DNA (nrLSU) with primers NL1 and NL2 (O’Donnell 1993), one fragment of the small subunit nuclear ribosomal DNA (nrSSU) with prim- ers NS1 and NS4 (White et al. 1990), the complete internal transcribed spacer region of ribosomal DNA (ITS1-5.8S-ITS2) with primers ITS5 and ITS4 (White et al. 1990), one fragment of the second largest subunit of RNA polymerase II (RPB2) with primers fRPB2-5f and fRPB2-7cr (Liu et al. 1999), and one frag- ment of the translation elongation factor 1 (TEF1) with primers EF1-983f (Carbone & Kohn 1999) and EF1-2218r (Rehner & Buckley 2005). PCR reactions were performed on a PEQSTAR 2X GRADIENT Thermal Cycler (PEQLAB, Erlangen, Germany) using VWR Taq DNA polymerase (VWR-Omega, USA). The reactions followed this protocol: Each 50 μL PCR mixture included 10 μL of 5× buffer, 3 μL (25 mM) of magnesium chlo- ride (MgCl2), 0.8 μL (20 mM) of dNTP mix, 1 μL (10 mM) of each primer, 0.4 μL (5U/μL) of Taq Polymerase, 1 μL of template DNA, and 37.8 μL of sterile distilled water. For RPB2 and TEF1 4 μL of each primer and 4 μL of template DNA were used, and the amount of water decreased accordingly. Conditions of the PCR for nrLSU, nrSSU, and ITS were as follows: DNA denatura- tion 94 °C for 4 min; 35 cycles of DNA denaturation 94 °C for 30 s, primer annealing 55 °C for 30 s and TAQ extension 72 °C for 45 s, and a final TAQ extension 72 °C for 5 min, followed by storage at 8 °C. Thermal cycling parameters of the RPB2 and TEF1 genes were performed as described by Liu et al. (1999) and Rehner & Buckley (2005), respectively. PCR-products were checked on 1.5 % agarose electrophoresis gels stained with ethidium bromide. Amplified PCR products were purified with the Cycle Pure Kit (VWR-Omega, USA). The sequencing in both directions was performed with the same PCR primers with an Applied Biosystems 3730 DNA Analyzer in the BiK-F Laboratory Centre, Frankfurt am Main, Germany. Sequence alignment and model determination Alignments for each gene were made by MAFFT v. 7.164b (Katoh & Standley 2013) using the L-INS-i algorithm, and ad- justed manually using Mega v. 6.06 (Tamura et al. 2013). The 77M. Mardones et al.: Phylogeny of Phyllachorales program Gblocks v. 0.91b (Talavera & Castresana 2007) was used to remove poorly aligned positions and divergent regions from the DNA alignment using the parameters for a less strin- gent selection. Among the acquired sequences, often multiple sequences were recovered from a single stroma for a given genetic locus. To distinguish DNA of contaminants from phyllachoraceous DNA, all sequences were subjected to BLAST searches to verify the identity, and preliminary phylogenetic analyses were also performed including common plant parasitic fungi in Pezizo- mycotina. To test the level of congruence among loci, the Congruence Among Distance Matrices test (CADM, Legendre & Lapointe 2004) was performed using patristic distance matrices to test the null hypothesis of complete incongruence among loci. This analysis has been shown to have an accurate type-I error rate (Campbell et al. 2011). We assembled two datasets for phylogenetic analyses: a three- locus concatenated alignment (nrSSU, nrLSU, RPB2) including 88 specimens representing the three recognized subclasses of the Sordariomycetes following the classification of Zhang et al. (2006). The purpose of this analysis was to infer the posi- tion of Phyllachorales within Sordariomycetes, and to test the monophyly of the order. Sequences of further representative species of orders in Sordariomycetes were downloaded from GenBank (Table 1) mostly from studies published by Spatafora et al. (2006) and Zhang et al. (2006). Leotia lubrica was selected as outgroup taxon based on Zhang et al. (2006), and missing data were shown as gaps. The second dataset is a four-locus concatenated alignment (nrSSU, ITS, RPB2, TEF1) including 51 specimens representing members of the order Phyllochorales. The alignment contained mostly sequences generated in this study plus all sequences of the Phyllachorales available from GenBank. The purpose of Species Order Source GenBank Accession Numbers Reference nrLSU nrSSU RPB2 Aniptodera chesapeakensis Microascales ATCC 32818 U46882 U46870 DQ470896 Spatafora et al. (2006) Balansia henningsiana Hypocreales AEG96-27a AY489715 AY489683 DQ522413 Spatafora et al. (2006) Bionectria ochroleuca Hypocreales AFTOL-ID 187 DQ862027 DQ862044 DQ862013 Zhang et al. (2006) Bombardia bombarda Sordariales SMH 3391 DQ470970 DQ471021 DQ470923 Spatafora et al. (2006) Buergenerula spartinae Magnaporthales ATCC 22848 DQ341492 DQ341471 – Thongkantha et al. (2009) Calosphaeria pulchella Calosphaeriales CBS 115999 AY761075 AY761071 GU180661 Réblová & Seifert (2004) Camarops amorpha Boliniales SMH1450 AY780054 – AY780156 Miller & Huhndorf (2005) Camarops microspora Boliniales CBS 649.92 AY083821 DQ471036 DQ470937 Spatafora et al. (2006) Camarops tubulina Boliniales SMH4614 AY346266 – AY780157 Miller & Huhndorf (2005) Camarops ustulinoides Boliniales DEH 2164 DQ470941 DQ470989 DQ470882 Spatafora et al. (2006) Cercophora coprophila Sordariales SMH3794 AY780058 – AY780162 Miller & Huhndorf (2005) Chaetosphaerella phaeostroma Coronophorales SMH4257 AY695264 – FJ968940 Huhndorf et al. (2004) Chrysoporthe cubensis Diaporthales CBS 101281 AF408338 DQ862047 DQ862016 Zhang et al. (2006) Cordyceps cardinalis Hypocreales OSC 93610 AY184963 AY184974 EF469106 Sung et al. (2007) Cryphonectria parasitica Diaporthales ATCC 38755 NG027589 DQ862048 DQ862017 Zhang et al. (2006) Cryptosporella hypodermia Diaporthales CBS 171.69 DQ862028 DQ862049 DQ862018 Zhang et al. (2006) Diaporthe eres Diaporthales CBS 109767 AF408350 DQ471015 DQ470919 Spatafora et al. (2006) Diatrype disciformis Xylariales CBS 197.49 DQ470964 DQ471012 DQ470915 Zhang et al. (2006) Eutypa lata Xylariales CBS 208.87 DQ836903 DQ836896 DQ836889 Zhang et al. (2006) Falcocladium multivesiculatum Falcocladiales CBS 120386 JF831932 JF831928 – Jones et al. (2014) Falcocladium sphaeropedunculatum Falcocladiales CBS 111292 JF831933 JF831929 – Jones et al. (2014) Gelasinospora tetrasperma Sordariales CBS 178.33 DQ470980 DQ471032 DQ470932 Spatafora et al. (2006) Glomerella cingulata Glomerellales CBS 114054 AF543786 AF543762 DQ522441 Farr et al. (2006) Gnomonia gnomon Diaporthales CBS 199.53 AF408361 DQ471019 DQ470922 Spatafora et al. (2006) Graphium penicillioides Microascales CBS 506.86 AF027384 DQ471038 DQ470938 Spatafora et al. (2006) Halosphaeria appendiculata Microascales CBS 197.60 U46885 U46872 – Zhang et al. (2006) Hypocrea lutea Hypocreales ATCC 208838 AF543791 AF543768 DQ522446 Spatafora et al. (2007) Kylindria peruamazonensis Glomerellales CBS 838.91 GU180638 GU180609 GU180656 Réblová et al. (2011) Lasiosphaeria ovina Sordariales CBS958.72 AY587946 AY083799 AY600286 Miller & Huhndorf (2004) Melanospora tiffanii Melanosporales ATCC15515 AY015630 AY015619 AY015637 Zhang & Blackwell (2002) Melanospora zamiae Melanosporales ATCC 12340 AY046579 AY046578 AY046580 Zhang & Blackwell (2002) Microascus trigonosporus Microascales CBS 218.31 DQ470958 DQ471006 DQ470908 Spatafora et al. (2006) Monilochaetes infuscans Glomerellales CBS 869.96 GU180639 GU180620 GU180657 O'Connell et al. (2012) Ophioceras dolichostomum Magnaporthales CBS 114926 JX134689 JX134663 – Luo & Zhang (2013) Ophioceras leptosporum Magnaporthales CBS 894.70 JX134690 JX134664 – Luo & Zhang (2013) Ophiocordyceps irangiensis Hypocreales OSC 128578 DQ518770 DQ522556 DQ522445 Spatafora et al. (2007) Ophiodothella vaccinii Phyllachorales ATCC 36333 – U78777 – Wanderlei-Silva et al. (2003) Ophiostoma piliferum Ophiostomatales CBS 158.74 DQ470955 DQ471003 DQ470905 Spatafora et al. (2006) Ophiostoma stenoceras Ophiostomatales CBS 139.51 DQ836904 DQ836897 DQ836891 Zhang et al. (2006) Papulosa amerospora Cordanales JK 5547F DQ470950 DQ470998 DQ470901 Spatafora et al. (2006) Polystigma amygdalinum Phyllachorales EA-1 KM111540 KM111539 – Habibi et al. (2015) Pseudohalonectria lignicola Magnaporthales M95 JX134691 JX134665 – Luo & Zhang (2013) Roumegueriella rufula Hypocreales GJS 91-164 EF469082 EF469129 EF469116 Sung et al. (2007) Sordaria fimicola Sordariales CBS 15.5973 AY545728 AY545724 AY780194 Zhang et al. (2006) Sordaria macrospora Sordariales AFTOL-ID 393 AY346301 AY641007 AY641074 Huhndorf et al. (2004) Sphaerodothis acrocomiae Phyllachorales – – U76340 – Wanderlei-Silva et al. (2003) Sphaerostilbella berkeleyana Hypocreales CBS 102308 U00756 AF543770 DQ522465 Spatafora et al. (2007) Togninia vibratilis Togniniales CBS 117115 DQ649065 – HQ878611 Réblová & Mostert (2007) Tolypocladium capitatum Hypocreales OSC 71233 AY489721 AY489689 DQ522421 Spatafora et al. (2007) Tolypocladium japonicum Hypocreales OSC 110991 DQ518761 DQ522547 DQ522428 Spatafora et al. (2007) Valsa ambiens Diaporthales AR 3516 AF362564 DQ862056 DQ862025 Zhang et al. (2006) Verticillium dahliae Glomerellales ATCC 16535 DQ470945 AY489705 DQ522468 Spatafora et al. (2006) Xylaria acuta Xylariales ATCC 56487 AY544676 AY544719 DQ247797 Zhang et al. (2006) Table 1 Sequences downloaded from GenBank (in alphabetical order) used in this study. 78 Persoonia – Volume 39, 2017 this analysis was to infer the phylogenetic relationships within Phyllachorales. Camarops ustulinoides and Camarops micro- spora (Boliniales) were used as outgroup taxa, because some of our previous analyses (unpubl. data) have shown Boliniales as the sister group of Phyllachorales. The taxa of Phyllachorales used in both analyses are listed in Table 2 together with their location, host plant, and GenBank accession numbers. The alignments were deposited in TreeBASE (http://purl.org/phylo/ treebase/phylows/study/TB2:S19724). PartitionFinder v. 1.1.1 (Lanfear et al. 2012) following Akaike Information Criterion (AIC) was used to select the best-fit model of evolution for each gene fragment separately for Bayesian and Maximum Likelihood (ML) analyses. Data were partitioned by gene and by codon position in the case of the protein- coding sequences. For the first dataset, a GTR+G model was applied to nrLSU, TrN+G model to nrSSU, and TIM+I+G to RPB2. For the second dataset, a TrNef+G model was applied to ITS, TrN+G model to nrSSU, SYM+G model to TEF1, and TVMef+G to RPB2. Phylogenetic tree inference Phylogenetic analyses for each dataset were conducted ap- plying Maximum Likelihood (ML) and Bayesian methods. The ML analyses were performed in RAxML (Stamatakis 2014) implemented in raxmlGUI v. 0.9b2 (Silvestro & Michalak 2012). One thousand non-parametric bootstrap iterations were used with the available models of generalized time reversible (GTR- GAMMA model) and a discrete gamma distribution (Stamata- kis et al. 2008). Bayesian analyses were performed with the program MrBayes v. 3.2.6 (Ronquist et al. 2012) on XSEDE (Miller et al. 2010) in the CIPRES Science Gateway web portal (http://www.phylo.org/sub_sections/portal/). Two parallel runs with eight chains of Metropolis-coupled Markov chain Monte Carlo iterations were performed. Analyses were run for 100 Species Locality Voucher Host Host family GenBank Accession Numbers nrLSU nrSSU ITS RPB2 TEF1 Camarotella costaricensis Panama MM-149 Acrocomia aculeata Arecaceae KX430484 KX451863 KX451913 KX451954 KX451982 Panama MM-21 Acrocomia aculeata Arecaceae KX430490 KX451851 KX451900 KX451963 KX451988 Camarotella sp. Panama MM-27 Unknown Arecaceae KX430492 KX451852 KX451901 – – Coccodiella melastomatum Venezuela CMU78543 Miconia sp. Melastomataceae – U78543 – – – Coccodiella miconiae Panama ppMP1342 Miconia sp. Melastomataceae KX430506 KX451871 – – – Coccodiella miconiicola Panama TH571 Ossaea micrantha Melastomataceae KX430512 KX451880 – – – Coccodiella sp. Ecuador MM-165 Unknown Melastomataceae KX430488 KX451865 KX451917 KX451957 KX451986 Coccodiella toledoi Venezuela Unknown Miconia sp. Melastomataceae – U78544 – – – Cocoicola californica USA F59034 Washingtonia robusta Arecaceae KX430468 KX451866 KX451918 KX451958 KX451995 USA F59038 Washingtonia robusta Arecaceae KX430469 KX451867 KX451919 KX451959 KX451996 Phyllachora graminis Unknown Unknown Unknown Poaceae – – AF257111 – – Canada DAOM240981 Unknown Poaceae – – HQ317550 – – Germany RoKi3084 Arrhenatherum elatius Poaceae – KX451872 – – – Germany MM-166 Hordelymus europaeus Poaceae – KX451869 KX451920 KX451962 KX452001 Panama TH544 Dichanthelium viscidellum Poaceae KX430508 KX451873 – – – Sweden UME31349 Unknown Poaceae – – AF064051 – – Phyllachora maydis USA BPI893231 Zea mays Poaceae – – KU184459 – – Phyllachora sp. 1 Thailand MM-130 Unknown Poaceae – KX451883 – KX451949 KX451976 Phyllachora sp. 2 Thailand MM-128 Bamboo Poaceae – KX451859 KX451908 KX451964 KX451973 Phyllachora sp. 2 Thailand MM-129 Bamboo Poaceae – KX451860 KX451909 KX451948 KX451974 Phyllachora sp. 3 Costa Rica MM-135 Chusquea longifolia Poaceae – KX451885 – KX451951 KX451978 Phyllachora sp. 3 Costa Rica MM-78 Chusquea sp. Poaceae – KX451853 – KX451942 KX451990 Phyllachora sp. 3 Costa Rica MM-98 Chusquea longifolia Poaceae KX430502 KX451856 – KX451945 KX451994 Phyllachora sp. 3 Costa Rica MM-134 Chusquea longifolia Poaceae KX430479 KX451884 – KX451968 – Phyllachora sp. 3 Ecuador SO-07 Chusquea sp. Poaceae – KX451890 – – KX452009 Phyllachora sp. 4 Benin RMB1061 Panicum maximum Poaceae – KX451870 KX451921 – KX452002 Polystigma pusillum Costa Rica MM-113 Andira inermis Fabaceae KX430474 KX451858 KX451907 KX451947 KX451972 Costa Rica MM-147 Andira inermis Fabaceae – KX451862 – – KX451981 Panama MM-19 Andira inermis Fabaceae KX430489 KX451850 KX451899 KX451941 KX451987 Polystigma sp. Ecuador MM-163 Paspalum sp. Poaceae KX430487 KX451864 KX451916 – KX451985 Serenomyces phoenicis USA PLM314 Phoenix canariensis Arecaceae – KX451868 KX451928 KX451960 KX451997 USA PLM315 Phoenix canariensis Arecaceae KX430505 KX451886 – KX451961 KX451998 Telimena aequatoriensis Ecuador SO-05 Monnina hirta Polygalaceae – KX451889 – – KX452008 Telimena bicincta Costa Rica MM-133 Picramnia antidesma Picramniaceae KX430478 KX451861 KX451910 KX451950 KX451977 Costa Rica MM-108 Picramnia antidesma Picramniaceae – KX451857 KX451906 KX451946 KX451971 Telimena canafistulae Panama MM-13 Cassia fistula Fabaceae KX430477 KX451849 KX451898 – KX451975 Telimena engleri Ecuador MM-153 Anthurium sp. Araceae – KX451888 KX451914 KX451955 KX451983 Ecuador MM-159 Anthurium sp. Araceae – – KX451915 KX451956 KX451984 Panama TH551 Anthurium concinnatum Araceae KX430511 KX451875 KX451895 KX451939 KX451969 Ecuador SO-09 Anthurium cf. triphyllum Araceae – – KX451934 – KX452010 Telimena leeae Panama TH549 Cissus trianae Vitaceae KX430509 KX451874 – – – Telimena picramniae Panama MM-05 Picramnia sp. Picramniaceae KX430470 KX451848 KX451896 KX451940 KX451970 Telimena sp. 1 Panama MM-143 Eugenia acapulcensis Myrtaceae – KX451887 KX451911 KX451952 KX451979 Telimena sp. 1 Panama MM-144 Eugenia acapulcensis Myrtaceae – – KX451912 KX451953 KX451980 Telimena sp. 2 Costa Rica MM-92 Eugenia sp. Myrtaceae KX430501 KX451855 KX451905 KX451944 KX451993 Telimena sp. 3 Costa Rica MM-88 Symplocos panamensis Symplocaceae KX430499 KX451854 KX451904 KX451943 KX451991 Telimena sp. 4 Costa Rica MM-47 Rinorea sp. Violaceae – – KX451902 – KX451989 Telimena sp. 5 Ecuador SO-14 Wettinia sp. Arecaceae – KX451892 KX451936 – – Telimena sp. 5 Ecuador SO-21 Wettinia sp. Arecaceae – KX451893 KX451937 – KX452012 Telimena sp. 5 Ecuador SO-22 Unknown Arecaceae – KX451894 KX451938 – KX452013 Telimena ulei Ecuador SO-12 Dioscorea meridensis Dioscoreaceae – KX451891 KX451935 – KX452011 Panama TH574 Dioscorea urophylla Dioscoreaceae – KX451877 – – – Telimena zanthoxylicola Panama TH550 Zanthoxylum scheryi Rutiaceae KX430510 KX451879 – – – Table 2 Taxa of Phyllachorales used in this study. Sequences in bold were isolated/sequenced in the present study. 79M. Mardones et al.: Phylogeny of Phyllachorales million generations, with trees sampled every 1 000th genera- tion. Burn-ins were determined by checking the likelihood trace plots in Tracer v. 1.6 (Rambaut et al. 2014) and subsequently discarded. Tracer and the online version of AWTY (Nylander et al. 2008) were used to test convergence; no indication of lack of convergence was detected. Bayesian posterior prob- abilities (BPP) ≥ 95 % and Bootstrap values (BS) ≥ 70 % were considered to be significant. Ancestral state reconstruction of morphological and ecological characteristics An ancestral state reconstruction of four morphological and ecological characteristics used to delimit genera in Phyl- lachorales was performed with the Likelihood Ancestral States method of Mesquite v. 2.74 with an asymmetrical two-parameter model for binary data and a Mk1 model for multistate data (Maddison & Maddison 2015). The likelihood decision threshold value was set to 2. The Bayesian consensus tree based on the four-locus dataset was used for this reconstruction. The analysis was restricted to members of the Phyllachorales. The character- istics considered were: monocotyledonous or eudicotyledonous host plant, position of the perithecia in the leaves (completely immersed, erumpent, subcuticular, or superficial), the presence or absence of clypeus, and the colour of the stroma (black or brightly coloured). Other characteristics such as the family of the host plant, the presence or absence of ascospore septa, ascospore colour (hyaline or brown), and the anamorphic state also were considered, but they did not yield conclusive results because they lacked variation or data were missing for numerous taxa. Observations were taken from the respective specimen and from published literature. The outgroups were coded as uncertain. The character matrix used for this analysis is provided in Appendix 1. RESULTS Sequences and alignments produced in this study We generated a total of 156 sequences from 27 species of Phyllachorales: 23 sequences of nrLSU, 40 of nrSSU, 31 of ITS, 26 of RPB2, and 36 of TEF1. Congruence among loci For the three-locus dataset, CADM results showed no significant incongruence among loci, thus allowing concatenation of the three loci. The null hypothesis of complete incongruence among loci was rejected (W = 0.75; p < 0.0001). For the four-locus dataset, the null hypothesis of complete incongruence among loci was also rejected (W = 0.48; p < 0.0001), and the four loci were concatenated. Initially, we had planned to compile a five-locus dataset, also including nrLSU, however, the null hypothesis of complete incongruence among loci was accepted for nrLSU (W = 0.37; p > 0.05), and thus we did not consider nrLSU in the concatenated dataset. Phyllachorales within Sordariomycetes The separately aligned datasets for each marker consisted of 76 sequences/790 base pairs for nrLSU, 77/908 for nrSSU, and 61/939 for RPB2. The three-locus dataset consisted of 88 specimens representing 72 species in 16 orders of Sordario- mycetes. The final alignment was 2 637 base pairs in length. No conflicts were detected among the phylogenies produced by ML and Bayesian analyses; therefore we present only the ML tree for this dataset (Fig. 2). Support values for nodes were consistently higher in Bayesian analyses than in ML analyses. The phylogenies inferred from individual genes (data not shown) and the three-locus phylogeny (Fig. 2) showed the Sordariomy- cetes as a robust monophyletic clade comprising three well- supported subclasses, Hypocreomycetidae, Xylariomycetidae, and Sordariomycetidae, with Phyllachorales grouping within Sordariomycetidae. The Phyllachorales appeared as a mono- phyletic, but moderately to weakly supported clade (0.94/77), including taxa of the two families Phaeochoraceae and Phylla- choraceae with the Boliniales as a sister group (0.97/74). Three phyllachoraceous taxa fell outside the Phyllachorales clade. Polystigma amygdalinum and Ophiodothella vaccinii were located with weak support within the Xylariomycetidae. The single sequence representing Sphaerodothis acrocomiae formed a weakly supported clade together with taxa of Hypo- creales (Fig. 2). This sequence may stem from a hypocrealen hyperparasite. It was not possible, however, to test possible incongruence regarding these taxa since they are represented by sequences downloaded from GenBank and two of them were represented by only one of the markers (nrSSU). Phylogenetic relationships within the Phyllachorales The four-locus dataset included 53 sequences representing 29 species of Phyllachorales. The dataset was supplemented with additional sequences from GenBank (two SSU and four ITS sequences). The alignment consisted of 2 728 total characters. The topology of the tree identified by Bayesian analysis was almost identical to the one obtained by the ML analyses, there- fore we present the Bayesian tree for this dataset (Fig. 3). In the Bayesian tree, the 51 sequences of Phyllachorales clustered into one major clade with high support (100/1.0). Within the Phyllachorales, three clades (I–III) can be identified. Clade I, which received weak support in the ML analysis (0.98/55), was divided into three subclades: the well-supported subclade one containing members of the genera Camarotella on Arecaceae and Coccodiella on Melastomataceae; subclade two containing the type species Phyllachora graminis, other species of Phyllachora, and one species of Polystigma, all of them growing on Poaceae; and subclade three including other graminicolous species of Phyllachora on Chusquea spp. and Polystigma pusillum on Fabaceae. Clade II (1.0/100) is a monophyletic, strongly supported group restricted to species growing on Arecaceae, i.e., mem- bers of the genera Cocoicola and Serenomyces in the family Phaeochoraceae. Clade III is also strongly supported (1.0/99) and included species of tar spot fungi with immersed perithecia and infect- ing species belonging to many different plant families, but not Poaceae. Two subclades can be distinguished, one containing members growing on several monocotyledonous and eudi- cotyledonous host families and a second group with species growing on Dioscoreaceae. The internal relationships within Clade III were mostly unresolved, with low posterior probability and bootstrap values. Clades II and III may be sister groups but this relationship was not strongly supported (0.44/42). Our results indicated that the family Phyllachoraceae and the genus Phyllachora are polyphyletic being represented in two distinct clades, called Clades I and III here. The genus Polystigma is polyphyletic, with the three species treated in this study included in three groups, one in Xylariomycetidae and the other two within two different lineages within Clade I. There were several examples of different species from the same host species, genus, or family forming supported clades, for example two different species growing on Picramnia spp. (Picramniaceae), several species on Eugenia spp. (Myrta- ceae) or Chusquea spp. (Poaceae), and Coccodiella spp. on Melastomataceae. 80 Persoonia – Volume 39, 2017 Fig. 2 Phylogenetic position of Phyllachorales within Sordariomycetes. This is a Maximum Likelihood phylogeny based on three nuclear markers (nrLSU, nrSSU, RPB2). Support values are ML bootstrap values based on 1 000 replicates, and posterior probabilities from a Bayesian analysis. Nodes receiving ML bp > 70 %, or Bayesian PP > 0.94 are considered as strongly supported and are indicated by thickened branches; see TreeBASE files for individual support. Phyllachoraceous taxa which fall outside the order are indicated in red. Abbreviations: S = subclass Sordariomycetidae; H = subclass Hypocreomycetidae; X = subclass Xylariomycetidae. Hypocrea lutea ATCC208838 Bombardia bombarda SMH3391 Polystigma sp. MM-163 Phyllachora graminis TH544 Phyllachora sp.3 MM-134 Tolypocladium capitatum OSC71233 Telimena picramniae MM-05 Falcocladium sphaeropedunculatum CBS 111292 Ophiocordyceps irangiensis OSC128578 Cryphonectria parasitica ATCC38755 Camarops microspora CBS 649.92 Bionectria ochroleuca AFTOL-ID 187 Cryptosporella hypodermia CBS 171.69 Diaporthe eres CBS 109767 Sordaria macrospora AFTOL-ID 393 Togninia vibratilis CBS 117115 Camarops ustulinoides DEH2164 Lasiosphaeria ovina CBS 958.72 Telimena engleri TH551 Falcocladium multivesiculatum CBS 120386 Melanospora tiffanii ATCC15515 Telimena leeae TH549 Tolypocladium japonicum OSC110991 Cocoicola californica F59038 Telimena sp. 2 MM-92 Xylaria acuta ATCC56487 Coccodiella sp. MM-165 Leotia lubrica OSC100001 (outgroup) Phyllachora sp. 3 MM-98 Roumegueriella rufula GJS91-164 Polystigma pusillum MM-19 Graphium penicillioides CBS 506.86 Serenomyces phoenicis PLM315 Eutypa lata CBS 208.87 Coccodiella miconiae ppMP1342 Sphaerodothis acrocomiae U76340 Balansia henningsiana AEG96-27a Gelasinospora tetrasperma CBS 178.33 Kylindria peruamazonensis CBS 838.91 Diatrype disciformis CBS 197.49 Pseudohalonectria lignicola M95 Gnomonia gnomon CBS 199.53 Camarotella sp. MM-27 Papulosa amerospora JK5547F Melanospora zamiae ATCC12340 Chrysoporthe cubensis CBS 101281 Cercophora coprophila SMH3794 Telimena canafistulae MM-13 Valsa ambiens AR3516 Chaetosphaerella phaeostroma SMH4257 Telimena zanthoxylicola TH550 Camarotella costaricensis MM-149 Camarops amorpha SMH1450 Sphaerostilbella berkeleyana CBS 102308 Cocoicola californica F59034 Telimena bicincta MM-133 Microascus trigonosporus CBS 218.31 Halosphaeria appendiculata CBS 197.60 Camarotella costaricensis MM-21 Cordyceps cardinalis OSC93610 Ophioceras leptosporum CBS849.70 Camarops tubulina SMH4614 Ophioceras dolichostomum CBS 114926 Calosphaeria pulchella CBS 115999 Polystigma amygdalinum EA-1 Telimena sp. 3 MM-88 Ophiostoma stenoceras CBS 139.51 Phyllachora sp. 3 MM-78 Serenomyces phoenicis PLM314 Sordaria fimicola AFTOL-ID 216 Coccodiella miconiicola TH571 Ophiodothella vaccinii ATCC36333 Glomerella cingulata CBS 114054 Polystigma pusillum MM-113 Monilochaetes infuscans CBS 869.96 Ophiostoma piliferum CBS 158.74 Buergenerula spartinae ATCC22848 Verticillium dahliae ATCC16535 Aniptodera chesapeakensis ATCC32818 0.1 52/0.81 Boliniales S o rd a ri o m y c e te s Phaeochoraceae Phyllachoraceae sensu lato X H S P h y ll a c h o ra le s 81M. Mardones et al.: Phylogeny of Phyllachorales Ancestral state reconstruction of ecological and morphological characteristics The evolution of one ecological and three morphological characteristics was reconstructed by employing the Bayes- ian tree sampling of the four-locus dataset (Fig. 4a–d). The analysis of the host relationships suggested that the ancestor of Phyllachorales was growing most likely on a monocotyle- donous plant (Proportional Likelihood (PL) 0.9966, Fig. 4a). The ancestor of each clade most probably was also growing on a monocotyledonous plant (Clade I, PL = 0.9981; Clade II, PL = 0.9960; Clade III, PL = 0.9685). The position of the peri- thecia in the leaves varies from immersed in the mesophyll to superficial (Fig. 1a–f). This reconstruction suggested that for species of Phyllachorales the ancestral state were perithecia completely immersed in the mesophyll (PL = 0.9988, Fig. 4b), while erumpent or superficial perithecia apparently evolved at least once each within Clade I. The presence of subcuticular perithecia was supported as the ancestral state in Clade II (PL = 0.9928). The ancestor of Phyllachorales was predicted to have had a clypeus (PL = 0.9989) while the lack of a clypeus was predicted as the ancestral state (PL = 0.9933) for species within Clade II (Fig. 4c). A black colour of stromata was well supported as the ancestral state (PL = 0.9999) for Phyllacho- rales, and bright coloured stromata apparently evolved at least twice in Clade I (Fig. 4d). TAXONOMY Based on the phylogenetic relationships revealed by this study, as well as the ecological and morphological characteristics of the species grouped in the observed clades within the Phylla- chorales, three families were recognised: the Phaeochoraceae were accepted as previously described (Hyde et al. 1997), the Phyllachoraceae and Phyllachora need to be emended, while the Telimenaceae are newly described here. The genus Telimena is emended to accommodate established species of Phyllachora that belong to the family Telimenaceae. Fig. 3 Phylogenetic relationships within the order Phyllachorales. This is a Bayesian analyses based on four nuclear markers (nrSSU, ITS, RPB2, TEF1). Support values are posterior probabilities from a Bayesian analysis and ML bootstrap values based on 1 000 replicates. Nodes receiving Bayesian PP > 0.94, or ML bp > 70 % are considered as strongly supported and are indicated by thickened branches. Hosts are indicated in blue text. Camarotella sp. MM-27 Arecaceae Telimena bicincta MM-108 Picramnia antidesma Telimena sp.5 SO-14 Wettinia sp. Polystigma pusillum MM-113 Andira inermis Telimena engleri SO-09 Anthurium cf. triphyllum Telimena zanthoxylicola TH-550 Zanthoxylum scheryi Phyllachora sp.3 MM-78 Chusquea sp. Telimena engleri TH-551 Anthurium concinnatum Coccodiella miconiae ppMP1342 Miconia sp. Phyllachora graminis UME31349 Poaceae Phyllachora maydis BPI893231 Zea maydis Telimena canafistulae MM-13 Cassia fistula Phyllachora sp.3 MM-135 Chusquea longifolia Telimena engleri MM-159 Anthurium sp. Telimena sp.5 SO-22 Arecaceae Telimena sp.1 MM-144 Eugenia acapulcensis Telimena ulei TH-574 Dioscorea urophylla Polystigma pusillum MM-19 Andira inermis Telimena sp.4 MM-47 Rinorea sp. Telimena picramniae MM-05 Picramnia sp. Telimena leeae TH-549 Cissus trianae Telimena ulei SO-12 Dioscorea meridensis Coccodiella melastomatum CMU78543 Miconia sp. Serenomyces phoenicis PLM314 Phoenix canariensis Serenomyces phoenicis PLM315 Phoenix canariensis Telimena sp.3 MM-88 Symplocos panamensis Telimena sp.2 MM-92 Eugenia sp. Telimena aequatoriensis SO-05 Monnina hirta Camarops ustulinoides DEH 2164 Phyllachora sp.1 MM-130 Poaceae Phyllachora sp. 2 MM-128 Poaceae Phyllachora graminis MM-166 Hordelymus europaeus Cocoicola californica F59034 Washingtonia robusta Coccodiella sp. MM-165 Melastomataceae Telimena sp.1 MM-143 Eugenia acapulcensis Polystigma pusillum MM-147 Andira inermis Phyllachora sp.3 SO-07 Chusquea sp. Phyllachora graminis DAOM240981 Poaceae Camarops microspora CBS 649.92 Polystigma sp. MM-163 Paspalum sp. Coccodiella toledoi U78544 Miconia sp. Cocoicola californica F59038 Washingtonia robusta Camarotella costaricensis MM-21 Acrocomia aculeata Phyllachora graminis AF257111 Poaceae Telimena sp.5 SO-21 Wettinia sp. Phyllachora sp.2 MM-129 Poaceae Telimena bicincta MM-133 Picramnia antidesma Phyllachora graminis RoKi3084 Arrhenatherum elatius Coccodiella miconiicola TH-571 Ossaea micrantha Camarotella costaricensis MM-149 Acrocomia aculeata Telimena engleri MM-153 Anthurium sp. Phyllachora sp.4 RMB1061 Panicum maximum Phyllachora sp.3 MM-98 Chusquea longifolia 1.0/100 1.0/88 1.0/100 0.99/95 0.97/85 0.47/28 1.0/100 0.98/55 0.99/95 0.87/43 0.99/92 0.42/46 1.0/100 1.0/87 0.57/45 1.0/99 1.0/100 1.0/100 0.95/85 1.0/100 1.0/100 1.0/100 0.44/42 1.0/99 1.0/99 1.0/99 1.0/100 0.97/61 0.99/100 0.51/28 0.94/96 1.0/100 1.0/100 0.47/24 0.37/24 0.01 P h y lla c h o ra c e a e C la d e I P h a e o c h o - ra c e a e C la d e II T e lim e n a c e a e C la d e III 82 Persoonia – Volume 39, 2017 Camarops ustulinoides Camarops microspora Camarotella costaricensis Camarotella costaricensis Camarotella sp. Coccodiella melastomatum Coccodiella sp. Coccodiella toledoi Coccodiella miconiicola Coccodiella miconiae Phyllachora graminis Phyllachora graminis Phyllachora graminis Phyllachora graminis Phyllachora maydis Phyllachora graminis Polystigma sp. Phyllachora sp.1 Phyllachora sp.4 Phyllachora sp.2 Phyllachora sp.2 Phyllachora sp.3 Phyllachora sp.3 Phyllachora sp.3 Phyllachora sp.3 Polystigma pusillum Polystigma pusillum Polystigma pusillum Cocoicola californica Cocoicola californica Serenomyces phoenicis Serenomyces phoenicis Telimena canafistulae Telimena sp.4 Telimena leeae Telimena zanthoxylicola Telimena engleri Telimena engleri Telimena engleri Telimena engleri Telimena sp.6 Telimena sp.6 Telimena sp.6 Telimena sp.5 Telimena aequatoriensis Telimena picramniae Telimena bicincta Telimena bicincta Telimena sp.2 Telimena sp.2 Telimena sp.3 Telimena ulei Telimena ulei *Clypeus Absent Present * * * * * * * * * * * * * * c Camarops ustulinoides Camarops microspora Camarotella costaricensis Camarotella costaricensis Camarotella sp. Coccodiella melastomatum Coccodiella sp. Coccodiella toledoi Coccodiella miconiicola Coccodiella miconiae Phyllachora graminis Phyllachora graminis Phyllachora graminis Phyllachora graminis Phyllachora maydis Phyllachora graminis Polystigma sp. Phyllachora sp.1 Phyllachora sp.4 Phyllachora sp.2 Phyllachora sp.2 Phyllachora sp.3 Phyllachora sp.3 Phyllachora sp.3 Phyllachora sp.3 Polystigma pusillum Polystigma pusillum Polystigma pusillum Cocoicola californica Cocoicola californica Serenomyces phoenicis Serenomyces phoenicis Telimena canafistulae Telimena sp.4 Telimena leeae Telimena zanthoxylicola Telimena engleri Telimena engleri Telimena engleri Telimena engleri Telimena sp.6 Telimena sp.6 Telimena sp.6 Telimena sp.5 Telimena aequatoriensis Telimena picramniae Telimena bicincta Telimena bicincta Telimena sp.2 Telimena sp.2 Telimena sp.3 Telimena ulei Telimena ulei * Host Plant Monocots Eudicots * * * * * * * * * * * * * * * a Camarops ustulinoides Camarops microspora Camarotella costaricensis Camarotella costaricensis Camarotella sp. Coccodiella melastomatum Coccodiella sp. Coccodiella toledoi Coccodiella miconiicola Coccodiella miconiae Phyllachora graminis Phyllachora graminis Phyllachora graminis Phyllachora graminis Phyllachora maydis Phyllachora graminis Polystigma sp. Phyllachora sp.1 Phyllachora sp.4 Phyllachora sp.2 Phyllachora sp.2 Phyllachora sp.3 Phyllachora sp.3 Phyllachora sp.3 Phyllachora sp.3 Polystigma pusillum Polystigma pusillum Polystigma pusillum Cocoicola californica Cocoicola californica Serenomyces phoenicis Serenomyces phoenicis Telimena canafistulae Telimena sp.4 Telimena leeae Telimena zanthoxylicola Telimena engleri Telimena engleri Telimena engleri Telimena engleri Telimena sp.6 Telimena sp.6 Telimena sp.6 Telimena sp.5 Telimena aequatoriensis Telimena picramniae Telimena bicincta Telimena bicincta Telimena sp.2 Telimena sp.2 Telimena sp.3 Telimena ulei Telimena ulei Position of the perithecia Immersed Subcuticular Errumpent Superficial * * * * * * * * * * * * * * * * * b Camarops ustulinoides Camarops microspora Camarotella costaricensis Camarotella costaricensis Camarotella sp. Coccodiella melastomatum Coccodiella sp. Coccodiella toledoi Coccodiella miconiicola Coccodiella miconiae Phyllachora graminis Phyllachora graminis Phyllachora graminis Phyllachora graminis Phyllachora maydis Phyllachora graminis Polystigma sp. Phyllachora sp.1 Phyllachora sp.4 Phyllachora sp.2 Phyllachora sp.2 Phyllachora sp.3 Phyllachora sp.3 Phyllachora sp.3 Phyllachora sp.3 Polystigma pusillum Polystigma pusillum Polystigma pusillum Cocoicola californica Cocoicola californica Serenomyces phoenicis Serenomyces phoenicis Telimena canafistulae Telimena sp.4 Telimena leeae Telimena zanthoxylicola Telimena engleri Telimena engleri Telimena engleri Telimena engleri Telimena sp.6 Telimena sp.6 Telimena sp.6 Telimena sp.5 Telimena aequatoriensis Telimena picramniae Telimena bicincta Telimena bicincta Telimena sp.2 Telimena sp.2 Telimena sp.3 Telimena ulei Telimena ulei * Colour of the stromata Black Bright * * * * * * * * * * ** * * * * * d 1 2 3 1 1 1 2 2 2 33 3 Fig. 4 Ancestral character state reconstruction in the Phyllachorales based on the Bayesian tree. All analyses are based on maximum likelihood reconstruction with asymmetrical two-parameter (a, c, d) or Mk1 (b) models. The characteristics considered were: a. Host plant (possible states are monocotyledonous or eudicotyledonous host plant); b. position of the perithecia in the leaves (immersed, erumpent, subcuticular, or superficial); c. presence or absence of clypeus; d. colour of the stroma (black or brightly coloured). Relative likelihood probabilities for each character state are represented with a pie chart at the nodes. Squares denote supported nodes for which posterior probabilities and bootstrap values are presented in Fig. 3. Coloured asterisks near pies indicate that the corresponding state is judged best according to the threshold. 83M. Mardones et al.: Phylogeny of Phyllachorales Phaeochoraceae K.D. Hyde et al. Species of Phaeochoraceae present black stromata, usually significantly raising the substratum surface, perithecia im- mersed, clustered forming a single cavity, embedded in pseudo- stromata and not covered by a clypeus as in other species of Phyllachorales. Ascospores are typically thick-walled, oliva- ceous to brownish, aseptate and usually with a delicate striate ornamentation. They are biotrophic or saprotrophic on palms. For a more detailed description of this family see Hyde et al. (1997). Genera included in this family: Cocoicola, Phaeochora, Phaeo- choropsis, Serenomyces. Phyllachoraceae Theiss. & P. Syd., Ann. Mycol. 13, 3/4: 168. 1915. emend. Mardones, Trampe & M. Piepenbr. Stroma of various shapes, covered by a cuticular or epidermal shiny black clypeus, sometimes bright coloured, development around the ostioles of perithecia. Ascomata perithecioid, am- phigenous, epiphyllous or hyphophyllous, uni- to multiloculate, sometimes confluent, frequently surrounded by a bright yellow to reddish discolouration zone, and when superficial with an hy- postroma anchoring the ascomata with the host tissue. Pseudo- stroma sparse or absent. Perithecia superficial, erumpent or immersed in the host tissue, pyriform, globose, lenticular, or deformed by vascular bundles, with a periphysate ostiole, with a hyaline to pigmented peridium composed of textura intricata. Paraphyses hyaline, thin-walled, slightly longer than the asci, septate, often dissolving during maturation. Asci unitunicate, clavate or cylindrical, usually 8-spored, rarely 4-spored, apical ring normally not turning blue in iodine reagent (J-), sometimes with an ascus crown. Ascospores usually hyaline, rarely pale brown, globose to filiform, mostly cylindrical, thin and smooth- walled, mostly aseptate, sometimes surrounded by gel. Struc- tures supposed to be spermogonia infrequently found, pycnidial, spermatiogenous cells cylindrical, tapering towards the tip, proliferating percurrently, producing filiform, hyaline, aseptate scolecospores that are probably spermatial in function. Mostly biotrophic, growing mainly on members of Poaceae, but also associated with other families. Generic type of the family. Phyllachora Nitschke ex Fuckel, Jahrb. Nassauischen Vereins Naturk. 23–24: 216. 1870 (1869– 1870). Phyllachora Nitschke ex Fuckel., Jahrb. Nassauischen Vereins Naturk. 23–24: 216 (1870) emend. Mardones, Trampe & M. Piepenbr. Etymology. Name probably referring to the leaf habitat (gr. phyllas: leaf, chora: location, position). Type species. Phyllachora graminis (Pers.) Nitschke. In Fuckel, Jahrb. Nassauischen Vereins Naturk. 23–24: 216. 1870 (1869–1870). Infection spot variable in outline, often roundish, black, shiny. Clypeus mostly epidermal. Pseudostroma absent or sparse. Perithecia immersed in the host tissue. Periphyses present. Paraphyses filiform, septate, hyaline, often deliquescent. Asci cylindrical to clavate, with or without apical ring that does not stain blue in iodine, mostly 8-spored. Ascospores mostly hyaline, aseptate, smooth, mostly without gelatinous sheaths. Spermogonia acervulate or pycnidial, variable in shape, often associated with ascomata. Spermatiogenous cells cylindrical, tapering toward the apex, proliferation percurrent. Spermatia filiform, curved, hyaline. Telimenaceae Mardones, Trampe & M. Piepenbr., fam. nov. — MycoBank MB818222 Stroma of various shapes, covered by a cuticular or epidermal shiny blackened clypeus, which may have limited development around the ostiole or extensively above the ascomata and in some cases below the ascomata. Ascomata perithecioid, amphigenous, epiphyllous or hyphophyllous, uni- to multilocu- late, sometimes confluent, frequently surrounded by a bright yellow to reddish discolouration zone. Pseudostroma strongly developed, interfusing and conspicuously expanding into the host tissue. Perithecia subcuticular, epidermal, subepidermal or immersed in the host tissue, pyriform, globose, lenticular, or deformed by vascular bundles, with a periphysate ostiole, with a hyaline to pigmented peridium composed of textura intricata. Paraphyses hyaline, thin-walled, slightly longer than the asci, septate, often dissolving during maturation. Asci unitunicate, clavate or cylindrical, usually 8-spored, rarely 4-spored, apical ring normally not turning blue in iodine reagent (J-). Ascospores usually hyaline, rarely pale brown, globose to filiform, mostly cylindrical, thin and smooth-walled, aseptate to 3-septate, sometimes surrounded by gel. Spermogonia infrequently found, pycnidial, spermatiogenous cells cylindrical, tapering towards the tip, proliferating percurrently, developing filiform, hyaline, aseptate scolecospores, probably spermatial in function. Mostly biotrophic, growing on several monocotyledoneous and dicotyledonous families, except Poaceae. Type genus. Telimena Racib., Parasit. Alg. Pilze Java’s (Jakarta) 1: 18. 1900. Telimena Racib., Parasit. Alg. Pilze Java’s (Jakarta) 1: 18. 1900. emend. Mardones, Trampe & M. Piepenbr. Etymology. The name of the genus refers to the name of a Polish hero in literary works of A. Mickievicz. Type species. Telimena erythrinae Racib., Parasit. Alg. Pilze Java’s (Ja- karta) 1: 18. 1900. Java, Merapi, on Erythrina variegata L. (as E. lithosperma Miq.), s.d., Raciborski s.n. (type IMI302320!). Infection spots dark, on living or dead leaves. Ascomata soli- tary to aggregated, subcuticular, epidermal, subepidermal or immersed in the host tissue, amphigenous, epiphyllous or hypo- phyllous, clypeate, uni- to multilocular, ostiolate. Clypeus sub- cuticular or epidermal. Pseudostroma strongly developed. Hama- thecium with periphyses in the ostiole and evanescent para- physes. Asci unitunicate, cylindrical to broadly ellipsoidal, with iodine-negative apical ring, 8-spored. Ascospores hyaline to pale brown when mature, globose to filiform, straight to curved, smooth, 0–3-septate. Additional specimens examined. Telimena bicincta. Costa RiCa, San Jose, on Picramnia antidesma, 10 Mar. 1890, A. Tonduz 2183 (type BR- 76016-65). Telimena ecastophylli (as ‘T. caudata’). ECuadoR, on Ptero- carpus amazonum (as P. ulei), 24 Feb. 1938, H. Sydow s.n. (IMI307885); on Pterocarpus sp., 22 Feb. 1938, H. Sydow s.n. (IMI346458). vEnEzuEla, Puerto La Cruz, El Limón, Pterocarpus rohrii, 22 Jan. 1928, H. Sydow s.n. (IMI18828). Telimena graminella. PhiliPPinEs, Luzon, on Paspalum sp., Sept. 1913, M. Ramos in Flora of the Philippines 8224 (type IMI18829). Telimena haraeana (as ‘T. arundinariae’). JaPan, Nagato Prov., Shimonoseki, on Pleio- blastus simonii, 5 May 1955, Katumoto s.n. (IMI63381). Telimena rhoina (as ‘Homostegia rhoina’). usa, California, San Diego, on Rhus integrifolia, Mar. 1895, K. Brandegee No. 15 (type NY00830409). Notes — The genus Telimena was originally described by Raciborski (1900) for phyllachora-like species with 3-septate ascospores. Currently, 14 species have been described within this genus. Its type species, T. erythrinae, is a parasite of the dicotyledonous plant host Erythrina variegata (Fabaceae). In the past, Telimena have been related with genera Telimenopsis (currently a synonym), Telimenella and Telimenochora (Petrak 84 Persoonia – Volume 39, 2017 1931, Müller 1975, Barr 1977). Morphological characteristics of the ascopores, i.e., shape, number of septa and position of the septa, have been used to separate these genera from each other. For a detailed description of the former and their main differences see Sivanesan (1987). Examination of specimens of Telimena spp. (cited above, includ- ing type material) showed that stromata of Telimena spp. are similar to those of Phyllachora spp. with aseptate ascospores. Photos of sections of ascomata of Ph. graminis and T. bicincta are provided for comparison (Fig 1a, f). These sections show immersed perithecia in the mesophyll of the leaf, surrounded by pseudostroma. The stromatic development in species of Telimena seems rather variable, like in Phyllachora spp., with reduced stromatic development in species occurring in grasses and abundant pseudostroma in the remaining species. Our observations show that ascospores of Phyllachora spp. some- times are septate when mature, and we repeatedly observed aseptate ascospores as well as ascospores with one, two or three septa in the same specimen. The following taxa are combined into Telimena based on their phylogenetic position as shown by data presented herein. In addition, we propose a recently collected specimen of Telimena bicincta as epitype. Telimena aequatoriensis (Theiss. & Syd.) Mardones, Trampe & M. Piepenbr., comb. nov. — MycoBank MB818223 Basionym. Phyllachora aequatoriensis Theiss. & Syd., Ann. Mycol. 13, 5/6: 521. 1915. Synonym. Phyllachora dendritica Rehm, Hedwigia 31: 305. 1892. ECua- doR, Quito, Río Machangara, on Monnina sp., 10 Apr. 1892, G.V. Lagerheim in Rehm 1072 Ex. Herb. Sydow (type S F9301). Telimena bicincta (E. Bommer & M. Rousseau) Theiss. & Syd., Ann. Mycol. 13, 5/6: 601. 1915 Basionym. Montagnella bicincta E. Bommer & M. Rousseau, Bull. Soc. Roy. Bot. Belgique 35: 163. 1896. Costa RiCa, San Jose, on Picramnia anti- desma, 10 Mar. 1890, A. Tonduz 2183 (holotype BR-76016-65!). Epitype (MycoBank MBT375061, designated here): Costa RiCa, San José, San Pedro Montes de Oca, Campus Universidad de Costa Rica, N9°56'17" W84°2'59", on Picramnia antidesma, 19 Jan. 2015, Mardones MM-133 (epitype USJ 108929; isoepitype M). Telimena canafistulae (F. Stevens & Dalbey) Mardones, Trampe & M. Piepenbr., comb. nov. — MycoBank MB818224 Basionym. Phyllachora canafistulae F. Stevens & Dalbey, Bot. Gaz. 68: 55. 1919. PuERto RiCo, Mayaguez, on Cassia fistula, 14 June 1915, F.L. Stevens 7022 (holotype ILL00011456!; isotypes BPI 636604, BPI 636617, BPI 844649!, K, MAPR, NY 00986162; fide Cannon 1991). Synonym. Phyllachora azuanensis Petr. & Cif., Ann. Mycol. 30, 3/4: 235. 1932. dominiCan REPubliC, Azua, on Barbieria pinnata, 25 Ago. 1929, E.L. Ek- man 3565 in Herb. Ciferri (holotype BPI 636400 n.v.; isotypes NY 00986150, S F49803 n.v.). Telimena engleri (Speg.) Mardones, Trampe & M. Piepenbr., comb. nov. — MycoBank MB818225 Basionym. Phyllachora engleri Speg., Anales Soc. Ci. Argent. 19, 2: 96. 1885. PaRaguay, Barrancas de San Antonio, on Spathicarpa lanceolata, Jan. 1882, B. Balansa 3746 (holotype LPS130!). Synonyms. Botryosphaeria anthuriicola Massee, Bull. Misc. Inform. Kew: 185. 1899. Costa RiCa, Cartago, on Anthurium gracile, s.d., Donnell Smith 6813 (type K(M) 190501 n.v.; isotype BPI 797076 n.v.). Dothidella bifrons Starbäck, Bih. Kongl. Svenska Vetensk.-Akad. Handl., Afd. 3 25, no. 1: 46. 1899. PaRaguay, Concepcion, on Araceae, 17 Sept. 1893, G.A. Malme s.n. (holotype S F9201 n.v.). Phyllachora anthurii (E. Bommer & M. Rousseau) Speg., Bol. Acad. Nac. Ci. Córdoba 23, 3-4: 567. 1919. (1918). Dothidea anthurii E. Bommer & M. Rousseau, Bull. Soc. Roy. Bot. Belgique 35: 163. 1896. Phyllachora dioscoreae Rehm, Hedwigia 36, 6: 370. 1897. bRazil, Brasilien, on leaves of Dioscoreaceae, s.d., E. Ule 217 in Herb. Berol. Ex Herb. Rehm (syntype S F218511 n.v.). Phyllachora engleri f. anthurii Speg., Anales Soc. Ci. Argent. 26, 1: 37. 1888. PaRaguay, on Anthurium sp., Sept. 1883, B. Balansa 4082-4106 (type LPS 130!). Phyllachora engleri var. anthurii (Speg.) Pat., Bull. Herb. Boissier 3: 71. 1895. Phyllachora philodendri Pat. (as ‘philodendronis’), Bull. Soc. Mycol. France 8, 3: 134. 1892. ECuadoR, on Philodendron sp., Jan. 1892, Lagerheim s.n. (type FH n.v.). Phyllachora phylloplaca Chardón, Mycologia 32, 2: 197. 1940. bRazil, Vicosa, on Diclidanthera laurifolia, 22 Apr. 1933, Muller 491 (type CUP- MG-000491 n.v.). Additional specimens examined. Dothidea phylloplaca (as ‘phyllopla- cus’). guyana, prope Cayennam, on unidentified leaves, s.d., Leprieur 1152 (type PC 96772!). Phyllachora ipirangae. bRazil, Sao Paulo, Ipiranga, Villa Marianna, on Eugenia sp., 23 Aug. 1906, A. Usteri s.n. (type S F8918!). Sphaeria phylloplaca (as ‘phylloplacus’). suRinam, on unidentified leaves, 1827, Weigelt s.n. (isotypes HBG 6597, PC 96768). Notes — Currently, P. engleri is treated as a synonym of Phyllachora phylloplaca in Index Fungorum. Montagne (1855) published Dothidea phylloplaca and gave Sphaeria phylloplaca as synonym. Later, Saccardo (1883) cited Montagne’s descrip- tion of D. phylloplaca recombining the species to P. phylloplaca. Theissen & Sydow (1915) cited Sphaeria phylloplaca and P. ipirangae, as synonyms of P. phylloplaca on Eugenia sp. (Myrtaceae), and considered as distinct from P. engleri. Howev- er, this species is currently accepted as illegitimate (superfluous name). Re-examination of the type material of D. phylloplaca and S. phylloplaca confirmed that both specimens are growing on dicotyledonous plant hosts. Furthermore, D. phylloplaca is not accepted as a synonym of S. phylloplaca as described by Montagne (1855), because leaf material differs significantly in habitus and texture. Telimena leeae (Koord.) Mardones, Trampe & M. Piepenbr., comb. nov. — MycoBank MB818226 Basionym. Phyllachora leeae Koord., Verh. Kon. Akad. Wetensch., Afd. Natuurk., sect. 2, 13, 4: 182. 1907. Java, Gombong, on Leea rubra, 18 Mar. 1905, Kooders s.n. (holotype S F49896!). Telimena picramniae (Syd. & P. Syd.) Mardones, Trampe & M. Piepenbr., comb. nov. — MycoBank MB818227 Basionym. Dothidella picramniae Syd. & P. Syd., Ann. Mycol. 11, 3: 266. 1913. Costa RiCa, San Jose, on Picramnia bonplandiana (as P. antidesma), 10 Nov. 1912, Ad. Tonduz s.n. (isotype CUP Syd. F.exot.ex.0134 n.v.). Synonyms. Endodothella picramniae (Syd. & P. Syd.) Syd., in Theissen & Sydow, Ann. Mycol. 13, 5/6: 590. 1915. Phyllachora picramniae (Syd. & P. Syd.) Petr., Ann. Mycol. 38, 2/4: 259. 1940. Phyllachora picramniae F. Stevens, Illinois Biol. Monogr. (Urbana) 11, 2: 190. 1927. Costa RiCa, Aserri, on Picramnia bonplandiana (as P. antidesma), 26 June 1923, F.L. Stevens 119 (holotype ILL00005686!; isotypes BPI 639002!, CUP-014698, MICH 14837; paratypes CUP-014697, CUP- 014699, ILL00005686, ILL00005687). Telimena ulei (G. Winter) Mardones, Trampe & M. Piepenbr., comb. nov. — MycoBank MB818228 Basionym. Phyllachora ulei G. Winter, Grevillea 15, no. 75: 90. 1887. bRazil, Sao Francisco, on unknown plant, Aug. 1884, Ule 143 (holotype S F8887!). Telimena zanthoxylicola (Seaver) Mardones, Trampe & M. Piepenbr., comb. nov. — MycoBank MB818229 Basionym. Phyllachora zanthoxylicola Seaver, Mycologia 20, 4: 225. 1928. JamaiCa, on Zanthoxylum insularis, s.d., E.G. Britton 443 (holotype NY 01089448!). 85M. Mardones et al.: Phylogeny of Phyllachorales Synonym. Telimenopsis fagarae Speer, Trans. Brit. Mycol. Soc. 75, 3: 504. 1981 (1980). ECuadoR, Galapagos Islands, Insula Santa Cruz, on Zan- thoxylum fagara, 16 Oct. 1976, Gard & For.Nobis, s.n. (holotype IMI 245878 n.v., fide Speer 1980). DISCUSSION Phyllachorales within Sordariomycetes Our findings support the placement of Phyllachorales within the subclass Sordariomycetidae in the class Sordariomycetes, as suggested by previous molecular studies (Winka & Eriksson 2000, Wanderlei-Silva et al. 2003). The extended taxon sam- pling and the use of three markers (nrLSU, nrSSU, and RPB2) allow us to strongly corroborate these findings. This study confirms that the Phyllachorales and Boliniales are closely related orders, disproving that the sister group of Phyl- lachorales may be the Diaporthales (Cannon 1988). Although the order Boliniales mostly comprises saprotrophic fungi, spe- cies of both orders have perithecia immersed in stromata and unitunicate asci with an inamyloid apical ring (Untereiner et al. 2013). Also, in some species of Boliniales, the black stromata are described as clypeate (Sivanesan 1975, Réblová 1997). The Phyllachorales are supported as monophyletic based on the three-gene tree with moderate support. This is the first analysis that demonstrates the monophyly of the order includ- ing members of both families currently accepted in the order. The reason for the lack of a strong support for the monophyly of the order seems to be the sister group relationship among the three major clades. The three-locus dataset showed a close relationship between Clades I and III, and a separate Clade II while the four-locus dataset showed Clade II to be more closely related to Clade III. Species excluded from the Phyllachorales are Polystigma amygdalinum, Ophiodothella vaccinii, and Sphaerodothis acrocomiae. The reasons for the previous inclusion of these species in Phyllachorales were their biotrophic condition, immersed perithecia, and presence of stromatic tissue. Other molecular studies also supported these exclusions (Wanderlei-Silva et al. 2003, Habibi et al. 2015). Phylogenetic relationships within the Phyllachorales: clade-based assessment Our current study presents the up to now largest analysis of the Phyllachorales, with four gene regions from 29 species, yielding the most reliable phylogenetic analyses of Phyllachorales so far. Based on this analysis, three distinct monophyletic clades can be distinguished within the Phyllachorales. Clade I includes the type species of the order, P. graminis, to- gether with other tar spot fungi on Poaceae having immersed stromata, Po. pusillum on Fabaceae, Camarotella spp. on palms, and Coccodiella spp. on Melastomataceae. Species on Poaceae are grouped in two subclades, one containing P. graminis, P. maydis, one species of Polystigma sp., and two species on bamboo from Thailand. The other subclade contains Phyllachora spp. growing on Chusquea spp. In our study, the ML analysis provided no support for this clade. However, due to the limited taxon sampling included in this phylogeny, it seems too early to further subdivide this clade. A better-sampled phylogenetic study of these species as well as more species on Chusquea spp., and other grasses are needed to resolve the systematics of these species. Within the clade, sequences of P. graminis show a high level of variation, so apparently P. graminis is an assemblage of cryptic species. The position of Po. pusillum remains uncertain. In the three-locus dataset, sequences of Po. pusillum formed a separate clade outside Clade III without support (0.81/52). The same occurred with the analysis restricted to the nrLSU marker. This situation was the main cause of incongruent results that did not allow us to concatenate the five-locus dataset. The other subclade in Clade I comprises Camarotella spp. with strongly erumpent and flattened stromata and Coccodiella spp. with superficial stromata. These two genera seem to be mono- phyletic and closely related. Hyde & Cannon (1999) suggested that the species of Oxodeora and Coccostromopsis, also with typically erumpent stromata, are probably closely related to Coccodiella and Camarotella. However, no molecular data is available so far to corroborate this relationship. These results indicate that, surprisingly, tar spot fungi on Poaceae with im- mersed perithecia are more closely related to Camarotella spp. and Coccodiella spp. with perithecia in erumpent stromata, than to species with rather similar immersed perithecia growing on other monocotyledonous and dicotyledonous host plants in Clade III. Polystigma spp., which are leaf parasites with brightly coloured stromata, also are polyphyletic in Clade I (Cannon 1991). Three species of Polystigma were included in our analyses: Po. amyg- dalinum on Prunus dulcis, Po. pusillum on Andira inermis and Polystigma sp. on Paspalum sp. Several authors already sug- gested that the genus Polystigma is polymorphic, containing at least five well-defined assemblages of species (Cannon 1996, 1997, Pearce & Hyde 2006, Habibi et al. 2015). According to Cannon (1996) and his examination of the type species Poly- stigma rubrus on Prunus domestica, members of Polystigma should be restricted to species growing on Euro-Asiatic species of Rosaceae (Prunus spp.). However, the species of this group included in our analyses, Po. amygdalinum, was not grouped among phyllachoraceous fungi but with Trichosphaeriales and Xylariales in the Xylariomycetidae (Fig. 2), as previously report- ed by Habibi et al. (2015). This exclusion from Phyllachorales is morphologically supported by the presence of sympodial proliferation of conidia rather than percurrent proliferation typi- cal in species of Phyllachorales, and also by the accumulation of starch in the stromata of Polystigma spp., which is unusual in species of Phyllachorales. The other two species included in our analyses, Po. pusillum and Polystigma sp., were both placed in Clade I but not as closely related species. Poly- stigma pusillum has been related to the genus Physalospora (Hyponectriaceae) mainly due to the fact that microscopic fea- tures of the two genera are largely similar (Cannon 1991). As we mentioned before, our results confirm its placement within Phyllachorales, although its phylogenetic placement within the order is still uncertain. Another Polystigma sp. on Poaceae was grouped together with species of P. graminis. These results suggest that brightly coloured stromata evolved several times in the Phyllachorales. There are several Phyllachora spp. and Stigmatula spp. with poorly developed blackened tissue, so it is possible that species with brightly coloured stromata might be species of Phyllachora with reduced melanin pigmentation (Cannon 1996). Clade II includes species growing on Arecaceae, which are characterised by the lack of a clypeus and a more developed pseudostroma, as well as, by saccate evanescent asci and ascospores with appendages or weak striations (Hyde et al. 1997). Species of two genera were included in the analyses, Serenomyces and Cocoicola, which formed two different sub- clades. Serenomyces spp. can be distinguished by the pres- ence of individual ascomata with distinct necks, while Cocoicola spp. present multi-ostiolate ascomata without necks (Hyde & Cannon 1999). Clade III includes species of Phyllachorales growing on plants of numerous families of eudicots and monocots, except the family Poaceae. All the species belonging to this clade have immersed perithecia and hyaline ascospores, mostly without 86 Persoonia – Volume 39, 2017 septa. Only one species, Telimena bicincta on Picramnia spp., shows 3-septate ascospores but it is closely related to other species with aseptate ascospores. The number of septa in the ascospores has been used to separate Telimena spp. from species of other genera, but our results show that the presence or absence of septa in the spores is not always systematically informative in the present context. Taxonomic implications According to the current classification, Clades I and III include members of the accepted family Phyllachoraceae and Clade II corresponds to the family Phaeochoraceae. The two tradition- ally accepted families in Phyllachorales can be distinguished morphologically by the lack of a clypeus and a more developed pseudostroma in species of Phaeochoraceae, which is in con- trast to mostly immersed perithecia beneath a clypeus typical for species of Phyllachoraceae; as well as by the presence of olivaceous to brownish ascospores with striate ornamentation in Phaeochoraceae instead of smooth and hyaline ascospores typical of Phyllachoraceae. Species of Phyllachora in the traditional sense are present in Clades I and III, so the genus needs to be redefined. Therefore, we revise the taxonomy of Phyllachora and the Phyllachoraceae to be consistent with the multi-gene phylogeny and the host relationships. The type species, P. graminis, forms part of a strongly supported clade of grass-associated species. This group is well studied (Orton 1944, Parbery 1967), morphologi- cally homogenous, and should be considered Phyllachora s.str. The family Phyllachoraceae s.str. is emended and includes Phyllachora spp. that possess immersed perithecia and occur on Poaceae, and the erumpent perithecia genera Camarotella and Coccodiella. The suggested placement of Po. pusillum within Phyllachoraceae could not be confirmed. Further Phyllachora species on other monocotyledonous and eudicotyledonous hosts form the strongly supported Clade III. The emendation of the family Phyllachoraceae and the genus Phyllachora require the recognition of a new family and at least one genus to accommodate the species clustering in Clade III. We propose to transfer these species to the genus Teli- mena (Raciborski 1900), based on the phylogenetic position of T. bicincta, which is the oldest name available for species in- cluded in Clade III, and based on the type species of Telimena, T. erythrinae, which does not occur on a species of Poaceae but on Erythrina variegata (Fabaceae). Telimena was described for species with Phyllachora type stromata and 3-septate as- cospores, instead of aseptate ascospores in typical Phyllachora spp. As mentioned above, molecular results indicate that the number of septa of the ascospore is not a reliable character to delimit genera in the present context; therefore we include spe- cies with septate as well as aseptate ascospores in the same genus. The examination of the type specimen of T. erythrinae and specimens of other Telimena spp. showed that apart from the septation of the ascospores, stromatic characteristics of Telimena spp. are similar to those of species of Phyllachora not growing on Poaceae, as has been pointed out by several authors (Raciborski 1900, Von Höhnel 1911, Müller 1975, Barr 1977, Sivanesan 1987). The fact that Telimena comprises spe- cies with ascomata similar to those of Phyllachora spp. means that the generic concept of Telimena can be easily emended to include species considered Phyllachora spp. up to now. We assume that further molecular sequence data of the remaining Phyllachora spp. not occurring in Poaceae will not belong to Phyllachora s.str. but to Clade III due to their eudicotylenous host. We propose the new family Telimenaceae for taxa be- longing to Clade III. Species that are proposed here as new combinations into the genus Telimena correspond to species collected and sequenced by us. All of them have been compared with the corresponding type specimen and exhibited the same morphological character- istics. As no molecular sequence data could be obtained from the type material and most sequenced specimens were not col- lected at type localities, we mostly refrained from epitypification following recommendations by Hyde & Zhang (2008) as well as Zhang et al. (2013). Therefore, we decided to designate an epitype only for T. bicincta, which was obtained from the same location and host species as the type of this species. Our findings strongly support the separation of Clades I and III from Clade II based on morphological, molecular, and host data, but it is difficult to identify morphological synapomorphies to separate Clade I from Clade III. Nevertheless, a careful re-ex- amination of morphological characteristics of species classified in the families Phyllachoraceae s.str. (Clade I) and Telimena- ceae (Clade III) revealed characteristics that allow distinguish- ing species of the two families morphologically: Stromata of tar spot fungi classified in the new family Telimenaceae are located either in subcuticular, epidermal, subepidermal, or immersed position, whereas stromata of species in Phyllachoraceae are either completely immersed in host tissue in the case of Phyl- lachora spp. on grasses or erumpent to superficial in species of the other genera. In Phyllachora spp. on grasses examined by Parbery & Langdon (1964) the pseudostroma was absent; they did not document any stromatic tissue apart from the clypeus. On the contrary, in Telimena spp. on dicotyledonous plants, pseudostroma often is strongly developed, interfuses between adjacent stromata, and conspicuously expands into the host tissue. Evolution of parasite-host relationship Members of the order Phyllachorales mainly infect monocoty- ledonous or dicotyledonous plant hosts. Our analyses show that the ancestor of Phyllachorales may have grown on mono- cotyledonous hosts. All three families recognized in this study include monocotyledonous plants as hosts. These results strongly suggest intimate relationships between Phyllachorales and monocotyledonous plants in the early evolution of the order. In general, long-term evolutionary dynamics or coevolution between hosts and their symbionts (parasitic or mutualistic relationships) operates in parallel by co-speciation or through speciation by host shifts. Co-speciation usually involves the speciation of a symbiont at the same time as another species, while host-shift speciation can occur when the symbiont moves to a new host on which the symbiont’s immediate ancestor did not occur, and gives rise to new host-symbiont combinations (De Vienne et al. 2013). Based on the host expansion strategy, two groups can be dis- tinguished in the Phyllachorales, the family Phaeochoraceae which apparently did not expand its host range outside Are- caceae, and the families Telimenaceae and Phyllachoraceae, which expanded their host ranges to rather distant hosts. The facts that the phylogeny of Phyllachorales is not consistent with the phylogeny of their host plants, that a high number of distant host families are infected by phyllachoraceous fungi, and that several terminal groups of Phyllachorales species concentrate on hosts belonging to the same family, suggest that several host-shift speciation events followed by co-speciation might ex- plain host-parasite patterns in Phyllachoraceae and Telimena- ceae. We speculate that phyllachoraceous fungi first infected monocots, radiated on monocotyledonous hosts, and later expanded their host range to other dicotyledonous plant fami- lies by host jumps. For instance, in Clade I (Phyllachoraceae), 87M. Mardones et al.: Phylogeny of Phyllachorales Phyllachora species are restricted to grasses but the family also includes the genera Camarotella and Coccodiella with species growing on other hosts. In the genus Coccodiella, C. arundi- nariae (not included in our analyses) is the only species which occurs on the monocotyledonous family Poaceae, specifically on bamboos in Far East Asia, but most of the species of the genus occur on the dicotyledonous family Melastomataceae. We hypothesize that the ancestor of Coccodiella on Poaceae probably infected a melastomataceous plant and expanded its host range within this family. Evolution of morphological characteristics The position of the perithecia varies from completely immersed in the mesophyll of the leaf as in Phyllachora s.lat. spp. to com- pletely superficial as in Coccodiella spp. (Fig. 1a–f). Several other genera have been erected based on this characteristic, i.e., Camarotella spp. with erumpent perithecia, Trabutia spp. with subcuticular perithecia or Catacauma spp. with perithecia inserted between the clypeus and the epidermis. However, the location of the perithecia in the leaf has been suggested to be greatly influenced by the consistency of the host tissue (Parbery & Langdon 1964, Cannon 1991), and therefore not very reli- able as a taxonomical criterion. The reconstruction presented here suggests that the ancestral state for Phyllachorales was completely immersed perithecia, which apparently was lost in the family Phaeochoraceae and evolved to erumpent or superficial perithecia in some members of Phyllachoraceae. In the genus Telimena the position of perithecia varies from immersed to subepidermal. Based on our results, when the perithecia are superficial or erumpent, as in the genera Coc- codiella and Camarotella, this characteristic is reliable to define genera, while subepidermal and subcuticular positions of perithecia might be dependent on the texture and anatomy of the host tissue. Our data also indicate that phyllachoraceous fungi growing on palms form a distinctive group, probably due to the very particular anatomical characteristics of palms. In these fungi, the expansion of the stromata and the shape of the perithecia are affected by leaves with closely spaced, strongly lignified, parallel vascular bundles, typical of Arecaceae. Our phylogeny shows that palm-inhabiting species have evolved independently at least three times within Phyllachorales, as Arecaceae is the only host family occurring in all three fungal families. According to Hyde & Cannon (1999), there are three different types of stro- mata in phyllachoraceous fungi on palms, one elongated and erumpent as in Camarotella spp. (Clade I, Phyllachoraceae), another one inserted between the outermost layers of the host tissue, as in family Phaeochoraceae (Clade II), and a few spe- cies having immersed perithecia, as in Telimenaceae (Clade III), which are confined to species of palms with less lignified leaf tissue. Our results do not confirm that the ancestor of Phyl- lachorales was growing on a palm. Therefore, more research is needed to elucidate the role of palm-inhabiting species in the evolutionary history of Phyllachorales. Most species within Phyllachorales produce black stromata caused by dense fungal cells with melanin deposits, the only exception being Polystigma spp. Our analyses suggest that the presence of black stromata is the ancestral state in Phyl- lachorales, and that brightly coloured stromata evolved at least twice in the order. The clypeus is a shield of black fungal cells located above the perithecia, which is restricted to the epidermal cells of the host and is thought primarily to protect the developing tissues from damage caused by UV radiation or to absorb heat from the sun promoting growth (Durrell & Shields 1960, Sherwood 1981). Species of Phyllachorales share the presence of a clypeus as a synapomorphy, which apparently was lost only once in the Phaeochoraceae, thus, the clypeus is thought to represent an evolutionarily stable characteristic in the order. Conclusions and future work This study demonstrates the monophyly of Phyllachorales and its placement in Sordariomycetidae with Boliniales as a sister group. Although several genera, which possibly do not belong to Phyllachorales, were not represented in our dataset, it is clear that there is a core group representing this order. The phylogenetic relationships within the order are partially eluci- dated. The placement of Phaeochoraceae within Phyllachorales is confirmed, however, more sampling in the three families is necessary to better assess their internal relationships. Our data also supports the split of the family Phyllachoraceae and the genus Phyllachora, and the establishment of the additional family Telimenaceae and the genus Telimena. With monophyly demonstrated, efforts should be made to find characteristics that help to distinguish between families. In this study, apart from the molecular and ecological distinctions, few morphologi- cal synapomorphies were detected to differentiate the families Telimenaceae and Phyllachoraceae. Potentially informative characteristics that should be evaluated are the ascus wall and the morphology of the ascus apex. Additional morphological and ultrastructural work by electron microscopy will contribute to our understanding of the asci. Other valuable characteristics might be the asexual/spermatogonial states of Phyllachorales and the morphology of the haustoria. Also, additional molecular mark- ers are necessary for a profound phylogenetic study of some specific clades. Although our study contains a comprehensive dataset, it is still not possible to clearly circumscribe the family Phyllachoraceae and to elucidate the monophyly of the genera within Phyllachorales. DNA should be generated for species of Phyllachora on grasses and for several other genera that have been erected historically based on single characteristics of ascospores, i.e., Apiosphaeria, Ophiodothella, Sphaerodothis, Stigmochora, Telimenochora. Obtaining fresh specimens of these fungi to place them within a molecular framework should be an important objective of future studies, to prove the validity of the characteristics used to delimit them. Further molecular analyses including sequences of more species, from new loca- tions and host plants, will contribute to an understanding of the evolution of host ranges. We predict that this re-evaluation will produce the reassessment of several genera. The results of ancestral state reconstruction analyses rely on the phylogeny of the present analysis that includes only a small fraction of species known for the corresponding systematic relationships. Therefore, the ancestral state reconstructions are not certain and the true number of state changes is probably underestimated. A broader sampling will probably reveal further state changes specially regarding subsequent host jumps and also concerning the position of the perithecia in the leaves in species belonging to Phyllachoraceae and Telimenaceae. Acknowledgements We thank Orlando Cáceres, Julieta Carranza, Darío Cruz, Tina Hofmann, and Carlos Rojas for their help during the fieldwork, José Macia-Vicente for fruitful discussion on an early state of the manuscript on molecular phylogeny, Ralph Mangelsdorff for valuable advice on nomen- clature and Federico Albertazzi (CIBCM, UCR) for facilities for DNA extrac- tion in Costa Rica. Special thanks to Ralph Mangelsdorff, Carlos Morales, Rafael Rincón, and Héctor Zumbado for their help with the identification of host plants. 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