Resolving the phylogenetic and taxonomic status of dark-spored teleomorph genera in the Botryosphaeriaceae

Artur Alves

The ribosomal DNA from the Zygomycete Mucor miehei has been characterised. The complete rDNA unit was cloned by heterologous PCR using primers whose sequence matched conserved regions of the rDNA from related fungal species. The sequence of the overlapping PCR products revealed the existence of a repeated unit of 9574 bp. The genes encoding the different rRNA species were identified by their homology to the corresponding sequences from other fungi. We estimate that the rDNA unit is present in the genome of M. miehei in about 100 copies. This estimation was made by comparing the intensity of its hybridisation signal in a Southern blot with that of the mmp gene coding for aspartyl protease, which was assumed to be contained in single copy. The size and structure of the M. miehei rDNA unit was similar to that of other fungi. The genes encoding the 25S, 18S and 5.8S RNAs are closely linked within the repeated unit which also contains the 5S gene. This latter gene appears to be transcribed in the opposite direction. The 25S, 18S and 5.8S genes showed 70–80% homology to the corresponding genes from other fungi, whereas the degree of homology for the 5S gene was much lower. The highest homology (about 80%) corresponded to the few available sequences from other Mucor species. Homology to genes from other Zygomycota was no higher than that observed for genes from the Ascomycota or Basidiomycota fungi.

Sequences of the RNase P RNA gene were investigated for the phylogenetic analysis of the genus Saccharomonospora. Aligned nucleotide sequences, determined from the PCR-amplified RNase P RNA gene of representative strains of the genus Saccharomonospora, displayed 94.2 +/- 1.3% interspecific variances. The intraspecific similarity value was 99.7-100% in all species tested. Saccharomonospora azurea K161T and 'Saccharomonospora caesia' K76T displayed identical RNase P RNA gene sequences in the region that was determined and Saccharomonospora sp. K180 showed sequences distinct from validly described species with a similarity value of 94.6 +/- 1.0%. The phylogenetic trees constructed by aligning the sequences either within the genus Saccharomonospora or with other Gram-positive bacteria were similar to the ones derived using sequences of the 16S rDNA gene. Advantageous features of this gene for application as a molecular phyletic marker are discussed.

Persoonia 21, 2008: 29 – 55 www.persoonia.org RESEARCH ARTICLE doi:10.3767/003158508X340742 Resolving the phylogenetic and taxonomic status of dark-spored teleomorph genera in the Botryosphaeriaceae A.J.L. Phillips 1, A. Alves 2, S.R. Pennycook 3, P.R. Johnston 3, A. Ramaley 4, A. Akulov 5, P.W. Crous 6 Key words Barriopsis Diplodia Dothiorella EF1-α ITS Lasiodiplodia LSU Neodeightonia Phaeobotryon Phaeobotryosphaeria phylogeny Spencermartinsia Sphaeropsis SSU Abstract Species in the Botryosphaeriaceae are common plant pathogens and saprobes found on a variety of mainly woody hosts. Teleomorphs typically have hyaline, aseptate ascospores. However, some have been reported with brown ascospores and their taxonomic status is uncertain. A multi-gene approach (SSU, ITS, LSU, EF1-α and β-tubulin) was used to resolve the correct phylogenetic position of the dark-spored ‘Botryosphaeria’ teleomorphs and related asexual species. Neodeightonia and Phaeobotryon are reinstated for species with brown ascospores that are either 1-septate (Neodeightonia) or 2-septate (Phaeobotryon). Phaeobotryosphaeria is reinstated for species with brown, aseptate ascospores that bear an apiculus at either end. The status of Sphaeropsis is clariﬁed and shown to be the anamorph of Phaeobotryosphaeria. Two new genera, namely Barriopsis for species having brown, aseptate ascospores without apiculi and Spencermartinsia for species having brown, 1-septate ascospores with an apiculus at either end are introduced. Species of Dothiorella have brown, 1-septate ascospores and differ from Spencermartinsia in the absence of apiculi. These six genera can also be distinguished from one another based on morphological characters of their anamorphs. Although previously placed in the Botryosphaeriaceae, Dothidotthia, was shown to belong in the Pleosporales, and the new family Dothidotthiaceae is introduced to accommodate it. Article info Received: 29 April 2008; Accepted: 4 July 2008; Published: 16 July 2008. InTRoduCTIon The genus Botryosphaeria based on the type species, B. do thidea, typically has ascospores that are hyaline and aseptate, although they can become brown and septate with age (Saccardo 1877, von Arx & Müller 1954, 1975, Denman et al. 2000). Because some species of Botryosphaeria have ascospores that become brown with age, von Arx & Müller (1954) placed Dothidea visci with brown ascospores in Botryosphaeria as B. visci. Later, von Arx & Müller (1975) also placed the darkspored Neodeightonia subglobosa in Botryosphaeria. Since this is the type species of Neodeightonia (1970), this genus was reduced to synonymy with Botryosphaeria (1863). In recognising these synonymies, von Arx & Müller (1954, 1975) broadened the concept of Botryosphaeria to include species with brown ascospores. At least 18 anamorph genera have been associated with Botryo sphaeria. Denman et al. (2000) recognised only two of these, namely Fusicoccum and Diplodia. However, in view of the range of morphologies found in Botryosphaeria anamorphs, the 1 2 3 4 5 6 Centro de Recursos Microbiológicos, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829516, Caparica, Portugal; corresponding author e-mail: alp@fct.unl.pt. CESAM, Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal. Landcare Research, Private Bag 92170, Auckland Mail Centre, Auckland 1142, New Zealand. 7 Animas Place, Durango, CO 81301, USA. Department of Mycology and Plant Resistance, V.N. Karasin National University of Kharkov, Svobody sq. 4, Kharkov 61077, Ukraine. CBS Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. proposal by Denman et al. (2000) is probably too conservative. Although Denman et al. (2000) suggested that Lasiodiplodia could be a synonym of Diplodia, authors of recent papers accept these as distinct genera (Pavlic et al. 2004, Burgess et al. 2006, Damm et al. 2007, Alves et al. 2008). Phillips et al. (2005) resurrected the genus Dothiorella for species with 1-septate conidia that darken at an early stage of development, and teleomorphs that have brown, 1-septate ascospores. Phylogenetically (ITS+EF1-α) these species fell within the broad morphological concept of Botryosphaeria (Phillips et al. 2005) as recognised by von Arx & Müller (1954, 1975). For these reasons, Phillips et al. (2005) described the teleomorphs of Dothiorella as two new species of Botryosphaeria with brown, 1-septate ascospores. Subsequently, Luque et al. (2005) described another dark-spored Botryosphaeria, namely B. viticola, with a Dothiorella anamorph. Crous et al. (2006) referred to the clade with Dothiorella anamorphs as Dothidot thia because of the strong resemblance of the teleomorphs to that genus. However, in a morphological study of Dothidotthia aspera from diverse hosts, Ramaley (2005) showed that the anamorph is a hyphomycete, Thyrostroma negundinis, and that this species and possibly D. symphoricarpi, type of Dothi dotthia, are unrelated to the Botryosphaeriaceae (Schoch et al. 2006). Although the teleomorphs of Botryosphaeria tend to be morphologically conserved, the anamorphs display a wide range of morphologies. Based on the morphological diversity of the anamorphs linked to species of Botryosphaeria, Crous et al. (2006) suggest that these taxa represent more than a single genus. By including teleomorphs with brown ascospores in © 2008 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 30 Table 1 Isolates studied in this paper. Accession number 1 Host Locality Barriopsis fusca Bimuria novaezelandiae Botryosphaeria corticis CBS 174.26 CBS 107.79 CBS119047 ATCC 22927 CBS 115476 CBS 110302 CBS 418.64 CBS 675.92 CBS 147.52 AFTOL 54 AFTOL 1591 AFTOL 1599 IMI 373225 CBS 910.73 CBS 112549 CBS 112546 CBS 242.51 CBS 168.87 CBS 261.85 CBS 188.87 CBS 112553 CBS 230.30 CBS 393.84 CBS 109727 CBS 109725 CBS 109943 CBS 116470 CBS 116472 CBS 113423 CBS 109944 CBS 112555 CBS 119049 AFTOL 274 CPC 12928 CPC 12930 CPC 12932 CPC 12933 CPC 12929 AFTOL 1359 CBS 115041 CBS 113188 IMI 63581b CBS 115038 CAA 005 CAP 187 JL 599 AFTOL 1360 CBS 111645 AFTOL 1305 CBS 110492 CBS 118741 CBS 115812 CBS 116355 CBS 356.59 CBS 494.78 Citrus sp. soil Vaccinium corymbosum Vaccinium sp. Prunus sp. Vitis vinifera Tsuga heterophylla Medicago sativa Coffea robusta Zea mays man, skin, foot dung of rabbit Unknown Acer pseudoplatanus Quercus suber Quercus ilex Unknown Cupressus sempervirens Cupressus sempervirens Juglans regia Vitis vinifera Phoenix dactylifera Pinus nigra Pinus radiata Pinus patula Pinus patula Prunus africana Prunus africana Pinus greggii Pinus greggii Vitis vinifera Vitis sp. Unknown Fendlera rupicola Euonymus alatus Acer negundis Acer negundis Symphoricarpos rotundifolia Daphne cannabina Quercus ilex Quercus suber Ulmus sp. Malus pumila Pistacia vera Prunus dulcis Corylus avellana Rubus sp. Parthenocissus quinquefolia Salix sp. Unknown Santalum album Syzygium cordatum Syzygium cordatum Theobroma cacao Cassava-field soil Cuba New Zealand USA USA Switzerland Portugal Canada USA Zaire Unknown Netherlands Netherlands USA Germany Portugal Spain Italy Israel Israel France Portugal USA Netherlands South Africa South Africa Indonesia Ethiopia Ethiopia Mexico Mexico Portugal Italy Unknown USA USA USA USA USA India Spain Spain United Kingdom Netherlands USA Portugal Spain Unknown USA USA Unknown Australia South Africa South Africa Sri Lanka Colombia Botryosphaeria dothidea ‘Botryosphaeria’ tsugae Byssothecium circinans Capnodium coffeae Cochliobolus heterostrophus Davidiella tassiana Delitschia winteri Didymella cucurbitacearum Diplodia acerina Diplodia corticola Diplodia coryli Diplodia cupressi Diplodia juglandis Diplodia mutila Diplodia pinea A Diplodia pinea C Diplodia rosulata Diplodia scrobiculata Diplodia seriata Dothidea sambuci Dothidotthia sp. Dothidotthia sp. Dothidotthia sp. Dothidotthia sp. Dothidotthia symphoricarpi Dothiora cannabinae Dothiorella iberica Dothiorella sarmentorum Dothiorella sp. Dothiorella sp. Dothiorella sp. Elsinoë veneta Guignardia bidwelli Hysteropatella clavispora Lasiodiplodia crassispora Lasiodiplodia gonubiensis Lasiodiplodia parva GenBank2 SSU LSU ITS EF1-α β-tubulin EU673182 AY016338 EU673175 EU673176 EU673173 EU673174 EU673208 AY016339 DQ247808 AY544727 DQ678022 DQ678026 AY293779 EU673160 EU673206 EU673207 EU673162 EU673209 EU673210 EU673161 EU673213 EU673214 EU673219 EU673220 EU673222 EU673221 EU673211 EU673212 EU673217 EU673218 EU673215 EU673216 AY544722 EU673225 EU673226 EU673227 EU673228 EU673224 DQ479933 EU673155 EU673156 EU673158 EU673159 EU673157 EU673163 EU673164 DQ678007 EU673223 DQ678006 EU673189 EU673190 EU673193 EU673194 EU673200 EU673201 DQ377857 AY016356 EU673244 EU673245 AY928047 EU673243 DQ377867 AY016357 DQ247800 AY544645 DQ678074 DQ678077 AY293792 EU673234 AY928051 EU673262 EU673235 EU673263 EU673264 DQ377891 AY928049 EU673265 DQ377893 EU673269 EU673270 EU673271 DQ377896 DQ377897 EU673267 EU673268 AY928050 EU673266 AY544681 EU673272 EU673274 EU673275 EU673276 EU673273 DQ470984 AY928053 EU673230 AY928052 DQ377860 EU673231 EU673232 EU673233 DQ678060 DQ377876 AY541493 EU673251 DQ377901 DQ377902 EU673252 EU673257 EU673258 EU673330 – DQ299245 DQ299247 AY236949 AY259092 DQ458888 – – – – – – EU673315 AY259100 AY259090 EU673317 DQ458893 DQ458894 EU673316 AY259093 DQ458886 DQ458895 DQ458897 DQ458896 DQ458898 EU430265 EU430266 DQ458900 DQ458899 AY259094 DQ458889 – – – – – – – AY573202 AY573198 AY573212 AY573206 EU673312 EU673313 EU673314 – – – EF622086 DQ103550 DQ458892 AY639594 EF622082 EF622084 EU673296 – EU017539 EU673291 AY236898 AY573218 DQ458873 – – – – – – EU673282 AY573227 EU673310 EU673284 DQ458878 DQ458879 EU673283 AY573219 DQ458869 DQ458880 DQ458882 DQ458881 DQ458883 EU430267 EU430268 DQ458885 DQ458884 AY573220 DQ458874 – – – – – – – AY573222 EU673278 AY573235 AY573223 EU673279 EU673280 EU673281 – – – EF622066 EU673303 DQ458877 DQ103567 EF622062 EF622064 EU673109 – EU673107 EU673108 AY236927 EU673106 DQ458855 – – – – – – EU673139 DQ458853 EU673117 EU673105 DQ458861 DQ458862 EU673119 DQ458850 DQ458849 DQ458863 DQ458865 DQ458864 DQ458866 EU673132 EU673131 DQ458868 DQ458867 DQ458856 DQ458857 – – – – – – – EU673096 EU673097 EU673102 EU673101 EU673098 EU673100 EU673099 – – – EU673134 EU673133 DQ458860 EU673126 EU673113 EU673114 Persoonia – Volume 21, 2008 Species 1 Suriname Costa Rica Queensland USA New Guinea USA Venezuela Algeria Unknown Australia Switzerland USA Switzerland Unknown Netherlands Argentina USA Spain Spain United Kingdom Portugal Portugal Australia Australia New Zealand Portugal USA Hawaii Hawaii Hawaii Hawaii Hawaii Hawaii New Zealand New Zealand South Africa Luxembourg Germany Ukraine Ukraine Unknown Unknown Brazil Netherlands Unknown Venezuela Venezuela Unknown Spain Spain South Africa France New Zealand New Zealand New Zealand New Zealand USA EU673198 EU673199 EU673191 EU673195 EU673196 EU673197 EU673192 DQ384068 DQ470993 DQ677994 DQ678017 DQ678013 DQ813513 AF164370 DQ471017 AY016347 EU673203 EU673204 EU673205 EU673202 EU673148 EU673149 EU673153 EU673154 EU673151 EU673150 EU673152 EU673183 EU673184 EU673185 EU673186 EU673187 EU673188 EU673180 EU673181 EU673179 EU673177 EU673178 – – AY544725 DQ678014 AY016349 DQ678027 AY544726 EU673146 EU673147 AY545724 EU673165 EU673166 EU673167 EU673168 EU673171 EU673172 EU673169 EU673170 AY544692 EU673255 EU673256 DQ377903 AY928054 EU673253 EU673254 DQ377904 DQ384106 DQ470946 DQ678044 DQ678069 DQ678065 DQ813509 DQ678086 DQ470968 AY016365 EU673259 EU673260 EU673261 DQ377866 AY928043 EU673229 DQ377920 DQ377921 AY928045 AY928046 AY928044 DQ377898 EU673248 DQ377899 EU673249 DQ377900 EU673250 EU673246 EU673247 DQ377894 DQ377870 DQ377868 – – AY544684 DQ678066 AY016366 DQ678078 AY544686 DQ377931 DQ377932 AY545728 DQ377873 EU673236 EU673237 EU673238 EU673241 EU673242 EU673239 EU673240 AY544648 EF622081 EF622077 DQ103553 DQ458890 AY640255 DQ458891 DQ103547 – – – – – – – – – EU673338 EU673339 EU673340 EU673337 AY259091 EU673311 AY615185 AY615186 AY236943 AY259098 – EU673331 EU673332 EU673333 EU673334 EU673335 EU673336 EU673328 EU673329 AY343379 EU673324 EU673325 EU673326 EU673327 – – – – – AY693974 DQ436935 – AY905554 AY905555 EU673318 EU673319 EU673322 EU673323 EU673320 EU673321 – EF622060 EF622057 EU673304 DQ458875 AY640258 DQ458876 EU673305 – – – – – – – – – EU673307 EU673308 EU673309 EU673306 AY573217 EU673277 DQ093221 DQ093220 AY236888 AY573221 – EU673297 EU673298 EU673299 EU673300 EU673301 EU673302 EU673294 EU673295 AY343340 EU673292 EU673293 – – – – – – – AY693975 DQ436936 – AY905559 AY905562 EU673285 EU673286 EU673289 EU673290 EU673287 EU673288 – EU673112 EU673111 EU673136 DQ458858 EU673110 DQ458859 EU673129 – – – – – – – – – EU673138 EU673115 EU673116 EU673137 DQ458848 EU673092 AY615172 AY615173 AY236917 EU673095 – EU673125 EU673121 EU673124 EU673120 EU673123 EU673122 EU673140 EU673141 EU673130 EU673127 EU673128 – – – – – – – EU673094 EU673093 – EU673104 EU673103 EU673118 EU673135 EU673144 EU673145 EU673142 EU673143 – Acronyms of culture collections: AFTOL – Assembling the Fungal Tree of Life; CAA – A. Alves, Universidade de Aveiro, Portugal; CAP – Alan J.L. Phillips, Universidade Nova de Lisboa, Portugal; CBS – Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CMW – M.J. Wingfield, FABI, University of Pretoria, South Africa; IMI – CABI Bioscience, Egham, U.K.; JL – J. Luque, IRTA, Spain. Sequence numbers in italics were retrieved from GenBank. All others were obtained in the present study. 31 2 Citrus aurantium Gmelina arborea Eucalyptus grandis Unknown Fruit along coral reef coast Vitis vinifera Acacia mangium sandy desert soil Unknown Medicago rugosa Prunus spinosa Platanus occidentalis Phragmites australis Opuntia sp. Quercus robur Chrysomphalus aonidium Phoenix dactylifera Phoenix canariensis Phoenix dactylifera keratomycosis in eye Vitis vinifera Vitis vinifera Mangifera indica Mangifera indica Pinus nigra Vitis vinifera Ribes sp. Sophora chrysophylla Sophora chrysophylla Sophora chrysophylla Sophora chrysophylla Sophora chrysophylla Sophora chrysophylla Citrus sinensis Citrus sinensis Vitis vinifera Viscum album Viscum album Viscum album Viscum album Unknown Unknown man, hair Betula verrucosa Unknown Eucalyptus hybrid Eucalyptus hybrid Unknown Vitis vinifera Vitis vinifera Medicago sativa Poniciana gilliesii Citrus sinensis Citrus sinensis Citrus sinensis Citrus sinensis downed rotting wood A.J.L. Phillips et al.: Dark-spored teleomorph genera in Botryosphaeriaceae CBS 447.62 CBS 116459 Lasiodiplodia rubropurpurea CBS 118740 Lasiodiplodia theobromae CBS 124.13 CBS 164.96 CAA 006 Lasiodiplodia venezuelensis CBS 118739 Lepidosphaeria nicotiae CBS 559.71 Leptosphaeria maculans AFTOL 277 Lewia eureka AFTOL 267 Lophiostoma crenatum AFTOL 1581 Massaria platani AFTOL 1574 Massarina arundinacea CBS 619.86 Montagnula opulenta AFTOL 1734 Mycosphaerella punctiformis AFTOL 942 Myriangium duriaei CBS 260.36 Neodeightonia phoenicum CBS 169.34 CBS 123168 CBS 122528 Neodeightonia subglobosa CBS 448.91 Neofusicoccum luteum CBS 110299 CBS 110497 Neofusicoccum mangiferum CBS 118531 CBS 118532 Neofusicoccum parvum CMW 9081 CBS 110301 Neofusicoccum ribis CBS115475 Phaeobotryon mamane CPC 12264 CPC 12440 CPC 12442 CPC 12443 CPC 12444 CPC 12445 Phaeobotryosphaeria citrigena ICMP 16812 ICMP 16818 Phaeobotryosphaeria porosa CBS 110496 Phaeobotryosphaeria visci CBS 100163 CBS 186.97 CBS 122526 CBS 122527 Phaeosphaeria avenaria AFTOL 280 Phoma herbarum AFTOL 1575 Piedraia hortae CBS 480.64 Pleomassaria siparia AFTOL 1600 Preussia terricola AFTOL 282 Pseudofusicoccum stromaticum CBS 117448 CBS 117449 Sordaria fimicola – Spencermartinsia vitícola CBS 117009 CBS 117006 Spencermartinsia sp. (as Diplodia medicaginis) CBS 500.72 Spencermartinsia sp. (as Diplodia spegazziniana) CBS 302.75 Spencermartinsia sp. ICMP 16827 Spencermartinsia sp. ICMP 16828 Spencermartinsia sp. ICMP 16819 Spencermartinsia sp. ICMP 16824 Xylaria hypoxylon AFTOL 51 Lasiodiplodia pseudotheobromae 32 Botryosphaeria, Phillips et al. (2005) broadened the concept of the genus even further. Through a study of partial sequences of the LSU gene, Crous et al. (2006) showed that Botryosphaeria s.l. is composed of 10 phylogenetic lineages that correspond to different anamorph genera. To avoid the unnecessary introduction of new generic names, they opted to use existing anamorph generic names for most of the lineages, and restricted the use of Botryosphaeria to B. dothidea and B. corticis. In their phylogeny, a large clade consisting of Diplodia and Lasiodiplodia species was largely unresolved. Within this clade are species known to have hyaline ascospores, e.g. B. corticola, B. stevensii, and others reported to have dark ascospores, e.g. B. subglobosa and B. visci. The aim of the present study was to use a multigene approach to determine the correct taxonomy and phylogeny of the darkspored Botryosphaeria-like teleomorphs and their associated anamorphs and to resolve the phylogenetic position of the genus Dothidotthia. MATERIALS And METHodS DNA isolation, PCR amplification and sequencing Genomic DNA was extracted from mycelium following the method of Alves et al. (2004). PCR reactions were carried out with Taq polymerase, nucleotides and buffers supplied by MBI Fermentas (Vilnius, Lithuania) and PCR reaction mixtures were prepared according to Alves et al. (2004), with the addition of 5 % DMSO to improve the ampliﬁcation of some difﬁcult DNA templates. All primers used were synthesised by MWG Biotech AG (Ebersberg, Germany). A portion of the nuclear ribosomal SSU gene was ampliﬁed with primers NS1 and NS4 (White et al. 1990). The ampliﬁcation conditions were as follows: initial denaturation of 5 min at 95 °C, followed by 35 cycles of 45 s at 94 °C, 45 s at 48 °C and 90 s at 72 °C, and a ﬁnal extension period of 10 min at 72 °C. The nucleotide sequence of the SSU region was determined using the above primers along with the internal sequencing primers NS2 and NS3 (White et al. 1990). Part of the nuclear rRNA cluster comprising the ITS region plus the D1/D2 variable domains of the ribosomal LSU gene was ampliﬁed using the primers ITS1 (White et al. 1990) and NL4 (O’Donnell 1993) as described by Alves et al. (2005). Nucleotide sequences of the ITS and D1/D2 regions were determined as described previously (Alves et al. 2004, 2005) using the primers ITS4 (White et al. 1990) and NL1 (O’Donnell 1993) as internal sequencing primers. The primers EF1-688F (Alves et al. 2008) and EF1-986R (Carbone & Kohn 1999) and Bt2a and Bt2b (Glass & Donaldson 1995) were used to amplify and sequence part of the translation elongation factor 1-alpha (EF1-α) gene and part of the β-tubulin gene, respectively. Ampliﬁcation and nucleotide sequencing of the EF1-α and β-tubulin genes was performed as described previously (Alves et al. 2006, 2008). The ampliﬁed PCR fragments were puriﬁed with the JETQUICK PCR Puriﬁcation Spin Kit (GENOMED, Löhne, Germany). Both strands of the PCR products were sequenced according to the procedures described previously (Alves et al. 2004), while Persoonia – Volume 21, 2008 some were sequenced by STAB Vida Lda (Portugal). The nucleotide sequences were read and edited with FinchTV 1.4.0 (Geospiza Inc. http://www.geospiza.com/ﬁnchtv). All sequences were checked manually and nucleotide arrangements at ambiguous positions were clariﬁed using both primer direction sequences. Phylogenetic analyses Sequences were aligned with ClustalX v. 1.83 (Thompson et al. 1997), using the following parameters: pairwise alignment parameters (gap opening = 10, gap extension = 0.1) and multiple alignment parameters (gap opening = 10, gap extension = 0.2, transition weight = 0.5, delay divergent sequences = 25 %). Alignments were checked and manual adjustments were made where necessary. Phylogenetic information contained in indels (gaps) was incorporated into the phylogenetic analyses using simple indel coding as implemented by GapCoder (Young & Healy 2003). Phylogenetic analyses of sequence data were done using PAUP v. 4.0b10 (Swofford 2003) for Maximum-parsimony (MP) analyses and Mr Bayes v. 3.0b4 (Ronquist & Huelsenbeck 2003) for Bayesian analyses. Trees were visualised with TreeView (Page 1996). Maximum-parsimony analyses were performed using the heuristic search option with 1 000 random taxa addition and tree bisection and reconnection (TBR) as the branch-swapping algorithm. All characters were unordered and of equal weight and gaps were treated as missing data. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the most parsimonious trees was evaluated from 1 000 bootstrap replications (Hillis & Bull 1993). Other measures used were consistency index (CI), retention index (RI) and homoplasy index (HI). Bayesian analyses employing a Markov Chain Monte Carlo method were performed. The general time-reversible model of evolution (Rodriguez et al. 1990), including estimation of invariable sites and assuming a discrete gamma distribution with six rate categories (GTR+Γ+G) was used. Four MCMC chains were run simultaneously, starting from random trees for 1 000 000 generations. Trees were sampled every 100th generation for a total of 10 000 trees. The ﬁrst 1 000 trees were discarded as the burn-in phase of each analysis. Posterior probabilities (Rannala & Yang 1996) were determined from a majority-rule consensus tree generated with the remaining 9 000 trees. This analysis was repeated three times starting from different random trees to ensure trees from the same tree space were sampled during each analysis. In this study we assessed the possibility of combining the individual datasets by comparing highly supported clades among trees generated from the different datasets to detect conflict. High support typically refers to bootstrap support values ≥ 70 % and Bayesian posterior probabilities ≥ 95 % (Alfaro et al. 2003). If no conflict exists between the highly supported clades in trees generated from these different datasets, it is likely that the genes share similar phylogenetic histories and phylogenetic resolution and support could ultimately be increased by combining the datasets (Miller & Huhndorf 2004). 33 A.J.L. Phillips et al.: Dark-spored teleomorph genera in Botryosphaeriaceae coded alignment gaps. Indels were coded separately and added to the end of the alignment as characters 1682 –1742. Of the 1742 characters, 1203 were constant, while 168 were variable and parsimony uninformative. Maximum parsimony analysis of the remaining 371 parsimony informative characters resulted in a single tree with TL = 1443, CI = 0.5038, RI = 0.7056 and HI = 0.4962. The overall topology of the 50 % majority-rule consensus tree of 10 000 trees sampled during the Bayesian analysis was similar to the MP tree. The MP tree is presented in Fig. 1 with bootstrap support above the branches. The Bayesian tree is available in TreeBASE (SN 3881). RESuLTS Phylogenetic analyses Partial nucleotide sequences of the SSU ribosomal DNA (1134 bp), the ITS region (500–600 bp), the D1/D2 variable domains of the LSU ribosomal DNA (614 bp), β-tubulin (approx. 400 bp) and EF1-α genes (approx. 300 bp) were determined for several isolates. The other sequences used in the analyses were retrieved from GenBank (Table 1). Sequences of the ﬁve genes were aligned and analysed separately by MP and Bayesian analyses, and the resulting trees were compared. No conflicts were detected between single gene phylogenies indicating that the datasets could be combined. New sequences were deposited in GenBank (Table 1) and the alignments in TreeBASE (SN 3881). Dothidotthia sp. CBS119688 58 61 Dothidotthia sp. CBS119691 99 Combined SSU and LSU rDNA sequences of Dothidotthia symphoricarpi isolates were aligned with a set of sequences retrieved from GenBank (Table 1) representing several orders in the Dothideomycetes, as well as two Sordariomycetes sequences that were selected as outgroup taxa (Sordaria fimicola and Xylaria hypoxylon). The combined SSU+LSU alignment consisted of 38 taxa and contained 1742 characters including 89 72 Dothidotthia symphoricarpi CBS119687 Didymella cucurbitacearum 97 Leptosphaeria maculans 81 Massaria platani 55 Delitschiaceae Botryosphaeria dothidea 99 Diplodia mutila Botryosphaeriaceae Hysteropatella clavispora Capnodium coffeae 100 Hysteriales Davidiellaceae Capnodiaceae Mycosphaerella punctiformis 88 100 Hysteriaceae Davidiella tassiana 81 87 Botryosphaeriales Lasiodiplodia theobromae Guignardia bidwelli 67 Lophiostomataceae Testudinaceae Dothiorella sarmentorum 57 99 95 Teichosporaceae Neofusicoccum ribis 78 100 Melanommataceae Pleomassariaceae Sporormiaceae Lophiostoma crenatum 99 Montagnulaceae Pleomassaria siparia Byssothecium circinans Lepidosphaeria nicotiae Massarinaceae Bimuria novae zelandiae Preussia terricola Delitschia winteri 66 Montagnula opulenta 82 95 Massariaceae Massarina arundinacea 98 Pleosporaceae Pleosporales Cochliobolus heterostrophus 97 72 Phaeosphaeriaceae Lewia eureka 100 77 59 Leptosphaeriaceae Phaeosphaeria avenaria 56 87 family incertae sedis Phoma herbarum 98 98 Dothidotthiaceae Dothidotthia sp. CBS119690 Dothidotthia sp. CBS 19686 Piedraia hortae Elsinoë veneta Mycosphaerellaceae Piedraiaceae Elsinoaceae Myriangium duriaei Dothidea sambuci Dothideaceae Dothiora cannabinae Dothioraceae Capnodiales Myriangiaceae Myriangiales Dothideales Xylaria hypoxylon 10 Sordaria fimicola Fig. 1 Single most parsimonious tree resulting from maximum parsimony analysis of combined SSU and LSU sequence data for 38 taxa. MP bootstrap values are given at the nodes. 34 Persoonia – Volume 21, 2008 Fig. 2 One of eight most parsimonious trees resulting from maximum parsimony analysis of combined SSU, LSU, ITS, β-tubulin and EF1-α sequence data for 76 isolates in the Botryosphaeriaceae. MP bootstrap values are given at the nodes. D. pinea C CBS109725 D. pinea C CBS109943 D. pinea A CBS393.84 83 D. pinea A CBS109727 D. scrobiculata CBS109944 100 100 D. scrobiculata CBS113423 D. seriata CBS112555 99 D. seriata CBS119049 65 100 D. mutila CBS112553 Diplodia 100 D. mutila CBS230.30 D. rosulata CBS116470 61 100 D. rosulata CBS116472 98 100 D. cupressi CBS168.87 1 D. cupressi CBS261.85 80 “B.” tsugae CBS418.64 91 100 D. corticola CBS112549 D. corticola CBS112546 N. phoenicum CBS123168 100 N. phoenicum CBS122528 100 Neodeightonia N. phoenicum CBS169.34 2 N. subglobosa CBS448.91 L. theobromae CBS124.13 100 L. theobromae CAA006 99 73 L. theobromae CBS164.96 100 L. pseudotheobromae CBS447.62 100 L. pseudotheobromae CBS116459 L. parva CBS356.59 87 97 L. parva CBS494.78 100 L. gonubiensis CBS115812 56 L. gonubiensis CBS116355 100 81 L. rubropurpurea CBS118740 3 L. venezuelensis CBS118739 100 L. crassispora CBS110492 L. crassispora CBS118741 P. mamane CPC12442 P. mamane CPC12443 86 P. mamane CPC12440 Phaeobotryon 100 P. mamane CPC12445 4 P. mamane CPC12444 87 P. mamane CPC12264 Physalospora fusca CBS174.26 5 Barriopsis 100 100 P. visci CBS100163 77 P. visci CBS186.97 100 P. porosa CBS110496 Phaeobotryosphaeria 6 100 P. citrigena ICMP16812 P. citrigena ICMP16818 100 B. dothidea CBS115476 100 B. dothidea CBS110302 Botryosphaeria 100 B. corticis CBS119047 B. corticis ATCC22927 73 D. iberica CBS115041 60 D. iberica CBS113188 Dothiorella sp. CAA005 D. sarmentorum CBS115038 D. acerina CBS910.73 Dothiorella D. sarmentorum IMI63581b 96 D. coryli CBS242.51 D. juglandis CBS188.87 100 Dothiorella sp. JL599 Dothiorella sp. CAP187 98 Spencermartinsia sp. ICMP16827 98 Spencermartinsia sp. ICMP16828 78 Spencermartinsia sp. CBS500.72 Spencermartinsia 100 S. viticola CBS117006 9 S. viticola CBS117009 61 100 Spencermartinsia sp. CBS302.75 100 ICMP16819 10 Incertae sedis ICMP16824 100 N. parvum CMW9081 70 N. parvum CBS110301 100 N. mangiferum CBS118531 Neofusicoccum N. mangiferum CBS118532 100 N. luteum CBS110299 N. luteum CBS110497 99 Lasiodiplodia 66 100 75 7 62 100 99 100 8 100 100 11 Pseudofusicoccum stromaticum CBS117449 Pseudofusicoccum stromaticum CBS117448 10 100 35 A.J.L. Phillips et al.: Dark-spored teleomorph genera in Botryosphaeriaceae Within the ingroup taxa six well-supported clades could be identiﬁed, which correspond to known orders belonging to the Dothideomycetes, namely the Dothideales, Myriangiales, Capnodiales, Hysteriales, Botryosphaeriales and Pleosporales. The isolates identiﬁed as D. symphoricarpi formed a distinct and well-supported subclade (MP bootstrap = 89 %, posterior probability = 1.00) within the Pleosporales clade. The D. sym phoricarpi clade was clearly separated from all families included in the analyses. In both MP and Bayesian analyses the isolates grouped close to Didymella cucurbitacearum and Phoma her barum (family incertae sedis; de Gruyter et al. in prep.). The object of the multigene dataset (SSU, LSU, ITS, β-tubulin and EF1-α) analyses was to determine the phylogenetic relationships of the species with brown ascospores. Therefore Pseudofusicoccum stromaticum was used as outgroup because it lies basal to the Botryosphaeriaceae (Crous et al. 2006). The alignment of 76 isolates consisted of 3470 characters including alignment gaps. Indels were coded separately and added to the end of the alignment as characters 3255–3470. In the analyses, alignment gaps were treated as missing data. The combined dataset contained 3470 characters, of which 83 were variable and parsimony-uninformative and 2555 were constant. Maximum parsimony analysis of the remaining 832 parsimony-informative characters resulted in eight equal, most parsimonious trees (TL = 1966 steps, CI = 0.6175, RI = 0.9103, RC = 0.5621, HI = 0.3825). The 50 % majority-rule consensus tree of 10 000 trees sampled during the Bayesian analysis had an overall topology similar to the MP trees. One of the MP trees is shown in Fig. 2 with bootstrap support at the branches. The Bayesian tree is available in TreeBASE (SN 3881). In both analyses 11 clades were identiﬁed within the ingroup. For convenience these clades are numbered 1–11 in Fig. 2. All of the clades received high bootstrap (87–100 %) and posterior probabilities (0.99 –1.00) support. TAXonoMY Position of Dothidotthia Until now the genus Dothidotthia has been regarded as a member of the Botryosphaeriaceae (Barr 1987). One species of Dothidotthia, D. aspera, was recently shown to have a hyphomycetous anamorph in Thyrostroma (Ramaley 2005), quite unlike the pycnidial anamorphs of members of the Botryosphaeriaceae. The type species of Dothidotthia, D. sym phoricarpi, was included in an analysis of the Dothideomycetes (Fig. 1). These data show that Dothidotthia belongs in the Pleosporales, outside any of the known families, and thus a new family in the Pleosporales is introduced. Dothidotthiaceae Crous & A.J.L. Phillips, fam. nov. — MycoBank MB511706 Ascomata aggregata, erumpescentia, globosa, atrobrunnea. Pseudoparaphyses hyalinus, septatis. Asci octisporis, bitunicati, clavati. Ascosporae brunneae, uniseptatae, ellipsoidae. Typus. Dothidotthia Höhn. Anamorph. Thyrostroma. Ascomata in gregarious clusters, rarely solitary, erumpent, globose, dark brown; wall consisting of 3–6 layers of dark brown textura angularis; basal region giving rise to dark brown, thickwalled hyphae, that extend from the bottom of the ascomata into the substrate. Pseudoparaphyses hyaline, septate, branched in upper part above asci. Asci 8-spored, bitunicate, sessile, clavate, straight to curved. Ascospores brown, ellipsoid, transversely 1-septate. Anamorph hyphomycetous, Thyrostroma. Dothidotthia Höhn., Ber. Deutsch. Bot. Ges. 36: 312. 1918 Type species. Dothidotthia symphoricarpi (Rehm) Höhn. Dothidotthia symphoricarpi (Rehm) Höhn., Ber. Deutsch. Bot. Ges. 36: 312. 1918. — Fig. 3–5 Basionym. Pseudotthia symphoricarpi Rehm, Ann. Mycol. 11: 169. 1913. ฀ ≡ Dibotryon symphoricarpi (Rehm) Petr., Ann. Mycol. 25: 301. 1927. ฀ ≡ Gibbera symphoricarpi (Rehm) Arx, Acta Bot. Neerl. 3: 85. 1954. Anamorph. Thyrostroma negundinis (Berk. & M.A. Curtis) A.W. Ramaley, Mycotaxon 94: 131. (2005) 2006. Basionym. Coryneum negundinis Berk. & M.A. Curtis, Grevillea 2: 153. 1874. For additional synonyms see Ramaley (2005). Ascomata pseudothecial, in gregarious clusters, rarely solitary, erumpent, up to 550 µm diam and 500 µm high; apex somewhat papillate to depressed; wall consisting of 3–6 layers of dark brown textura angularis, 20–80 µm wide, giving rise to dark brown, thick-walled hyphae, 4–6 µm wide, that extend from the bottom of the ascomata into the substratum; reduced to short lateral projections (10 –15 µm long) elsewhere on the outer ascomatal wall. Pseudoparaphyses hyaline, septate, 2–3 µm wide, generally not constricted at septa, and branched in upper part above asci. Asci 8-spored, bitunicate, sessile, clavate, 70–120 × 15–22 µm, straight to curved. Ascospores uniformly pale brown, (20 –)22–23(– 26) × (8 –)9–10(–11) µm, ellipsoid, tapering towards subacutely rounded ends, medianly 1-septate, prominently constricted at septum, widest just above septum, smooth. Specimens examined. Dothidotthia symphoricarpi. USA, North Dakota, on branches of Symphoricarpos occidentalis, holotype of D. symphoricarpi herb. NY; Colorado, San Juan Co, c. 0.5 mile up Engineer Mountain Trail from turnoff at mile 52.5, Hwy 550, dead twigs of Symphoricarpos rotundifolius, 24 June 2004, A.W. Ramaley 0410, epitype designated here as BPI 871823, culture ex-epitype CPC 12929 = CBS 119687. Notes — Barr (1989) introduced the combination Dothi dotthia aspera, but incorrectly listed D. symphoricarpi as synonym. Dothidotthia aspera (Fig. 6) has ascospores that are ellipsoidal with rounded ends, constricted at the medium septum, widest just above the septum, medium brown, smooth to ﬁnely verruculose, (20 –)32 – 35(– 37) × (12 –)13 –14(–15) µm. Ascospores of D. symphoricarpi are much smaller, namely (20 –)22–23(– 26) × (8 –)9–10(–11) µm, ellipsoid with rounded ends, constricted at the median septum, widest above septum, ﬁnely verruculose, pale brown, and not medium brown as in D. aspera. Ramaley (2005) collected several specimens in this complex, one of which, BPI 871823, is selected to serve as epitype of D. symphoricarpi. Given the new circumscription of 36 Persoonia – Volume 21, 2008 a d b c f e i g h j Fig. 3 Pseudotthia symphoricarpi holotype NY. a. Ascomata; b. immature asci; c. asci and pseudoparaphyses; d. mature ascus with pale brown 1-septate ascospores; e. brown 1-septate ascospores; f. base of sessile ascus; g. brown 1-septate ascospores; h–j. conidia of Thyrostroma anamorph in association with ascomata. — Scale bars: a = 500 µm; b–h = 10 µm. 37 A.J.L. Phillips et al.: Dark-spored teleomorph genera in Botryosphaeriaceae a c b d e Fig. 4 Dothidotthia symphoricarpi epitype BPI 871823. a. Longitudinal section through an ascoma; b. detail of a section through the ascoma wall; c, d. asci with pale brown, 1-septate ascospores; e. pale brown, 1-septate ascospores. — Scale bars = 10 µm. this species, the other specimens treated by Ramaley (2005) appear to represent D. aspera, which is morphologically and phylogenetically distinct from both D. symphoricarpi based on the larger ascospores. Dothidotthia aspera (Ellis & Everh.) M.E. Barr, Mycotaxon 34: 519. 1989 — Fig. 6 Basionym. Amphisphaeria aspera Ellis & Everh., Bull. Torrey Bot. Club 27: 52. 1900. Ascomata pseudothecial, gregarious in groups, rarely solitary, erumpent, up to 600 µm diam, 500 µm high; apex rounded, short papillate to depressed; wall consisting of 3–6 layers of dark brown textura angularis, 20–80 µm wide, giving rise to dark brown, thick-walled hyphae, 4–6 µm wide, that extend from the bottom of the ascomata into the substratum; reduced to short lateral projections elsewhere on the outer ascomatal wall. Pseudoparaphyses hyaline, septate, 2–3 µm wide, not constricted at the septa, branched in the upper parts. Asci 8-spored, bitunicate, sessile, clavate, 65–140 × 10–23 µm. Ascospores medium brown, ellipsoidal with rounded ends, 1-septate, constricted at the median septum, smooth to ﬁnely verruculose, (20 –)32–35(– 37) × (12–)13–14(–15) µm. Specimens examined. Dothidotthia aspera. USA, Colorado, E. Bethel 517, holotype of Amphisphaeria aspera herb. NY. – Dothidotthia spp. USA, Colorado, Durango, 7 Animas Place, dead twigs of Euonymus alatus, 29 June 2004, A.W. Ramaley 0411, BPI 871820, culture CPC 12930 = CBS 119688; Colorado, Durango, between Animas Place and Animas River, dead twigs of Acer negundo, 8 July 2004, A.W. Ramaley 0414, BPI 871819, anamorph culture CPC 12933 = CBS 119691, teleomorph CPC 12932 = CBS 119690; Colorado, La Plata Co, c. 1.75 mile up Carbon Junction Trail, dead twigs of Fendlera rupicola, 11 May 2004, A.W. Ramaley 0403, BPI 871821, culture CPC 12928 = CBS 119686. Taxonomy of species with brown ascospores in the Botryosphaeriaceae The multigene phylogeny revealed 11 clades within the dataset of isolates studied (Fig. 2). Valid generic names are available and currently in use for clade 1 (Diplodia), clade 3 (Lasiodiplodia), 38 Persoonia – Volume 21, 2008 a b c d Fig. 5 Thyrostroma sp. CBS 119691, anamorph of Dothidotthia aspera. a–d. Conidia and conidiophores. — Scale bars = 10 µm. 39 A.J.L. Phillips et al.: Dark-spored teleomorph genera in Botryosphaeriaceae a b d e c f g Fig. 6 Amphisphaeria aspera holotype NY. a. Ascomata; b, c. asci with ascospores; d. pseudoparaphyses and ascospores; e. ascospores; f. ascospore and two conidia; g. conidia. — Scale bars: a = 500 µm ; b –g = 10 µm. clade 7 (Botryosphaeria), clade 8 (Dothiorella) and clade 11 (Neofusicoccum). Neodeightonia and Phaeobotryon are reinstated for clades 2 and 4, respectively. Phaeobotryosphaeria is reinstated for clade 6 and shown to be the teleomorph of Sphaeropsis, the status of which is clariﬁed. No generic names are available for the remaining clades and for this reason Bar riopsis and Spencermartinsia are introduced for clades 5 and 9, respectively. The status of clade 10 remains unresolved. Barriopsis A.J.L. Phillips, A. Alves & Crous, gen. nov. — MycoBank MB511712 Ascomata pseudothecia, brunnea vel nigra. Pseudoparaphyses hyalinae, septatae. Asci clavati, stipitati, octospori, bitunicati cum loculo apicali bene evoluto. Ascosporae ellipsoides, unicellulares, ovoidea, brunnea. Type species. Barriopsis fusca A.J.L. Phillips, A. Alves & Crous. Etymology. Named in honour of Margaret E. Barr, who dedicated a large part of her career to resolving the taxonomy of the Dothideomycetes. Ascomata pseudothecial, scattered or clustered, brown to black, wall composed of several layers of textura angularis, ostiole central. Pseudoparaphyses hyaline, smooth, multiseptate, constricted at septa. Asci bitunicate, clavate, stipitate, thick-walled with thick endotunica and well-developed apical chamber. Ascospores aseptate, ellipsoid to ovoid, brown when mature, without terminal apiculi. Note — The absence of apiculi differentiate this genus from Sphaeropsis and Phaeobotryosphaeria. The aseptate, brown ascospores without apiculi are unique in the Botryosphaeriaceae. Barriopsis fusca (N.E. Stevens) A.J.L. Phillips, A. Alves & Crous, comb. nov. — MycoBank MB511713; Fig. 7 Basionym. Physalospora fusca N.E. Stevens, Mycologia 18: 210. 1926. = Phaeobotryosphaeria fusca Petr., Sydowia 6: 317. 1952. = Botryosphaeria disrupta (Berk. & Curtis) Arx & E. Müll., Beitr. Kryptogamenfl. Schweiz 2, 1: 37. 1954. Ascomata scattered, immersed, brown to black, separate or aggregated, wall composed of textura angularis, uniloculate, ostiole single, central. Pseudoparaphyses hyaline, smooth, 3–4.5 µm wide, multiseptate with septa 14–18 µm apart. Asci bitunicate, clavate, 8-spored, stipitate, thick-walled with thick endotunica and well-developed apical chamber, 125 –180 × 30–36 µm. Ascospores biseriate, aseptate, ellipsoid to oval, 40 Persoonia – Volume 21, 2008 a c b d Fig. 7 Barriopsis fusca BPI 599052. a. Immature asci; b. mature asci with brown ascospores; c. ascus with ascospores; d. ascospores. — Scale bars: a, b = 20 µm; c, d = 10 µm. straight or slightly curved, apex and base obtuse, without terminal apiculi, wall externally smooth, internally ﬁnely verruculose, brown, widest in the middle, (30 –)31– 36.5(– 38.5) × (15.5–)16–18.5(–21) μm, 95 % conﬁdence limits = 32.6–33.4 _ × 17.0 –17.5 µm (χ฀± S.D. = 33.0 ± 1.5 × 17.2 ± 1.0 µm, L/W ratio = 1.9 ± 0.15). Specimens examined. CUBA, Herradura, on twigs of Citrus sp., 15 Jan. 1925, N.E. Stevens, holotype BPI 599052, culture ex-type CBS 174.26. – USA, Florida, Orlando, on twigs of Citrus sp., 20 Feb. 1923, C.L. Shear, BPI 599054. Notes — The ex-type culture could not be induced to sporulate, no doubt because it has been in culture for more than 80 years. According to Stevens (1926) the anamorph is lasiodiplodia-like and he described it as follows: “conidia initially hyaline, aseptate and thick-walled becoming dark brown and septate with irregular longitudinal striations, (20 –)23–25(– 28) × (11–)12 –13(–16) µm”. Stevens (1926) placed this species in Physalospora, but he was obviously hesitant to do so, judging from his statement “To place in the genus Physalospora a fungus with coloured ascospores is of course to do violence to the ideas of that genus”. On account of the bitunicate asci and brown ascospores of this species, Physalospora is clearly unsuitable. Petrak & Deighton (1952) transferred this species to Phaeobotryosphaeria as Phaeobotryosphaeria fusca, presumably on account of its dark ascospores. We examined the type species of Phaeobotryosphaeria (P. yerbae) and found it to have terminal apiculi on the ascospores. Therefore, Phaeo botryosphaeria would also appear to be unsuitable. For this reason we propose the new genus Barriopsis for this fungus. The brown, aseptate ascospores without terminal apiculi are characteristic of this new genus. 41 A.J.L. Phillips et al.: Dark-spored teleomorph genera in Botryosphaeriaceae b a d e f c g j h i k l m n Fig. 8 Dothiorella iberica. a–g: LISE 94944; h–n: CBS 115041. a. Vertical section through an ascoma; b. ascus with brown, 1-septate ascospores; c. immature asci and one ascus with four ascospores; d. details of ascoma wall; e. pseudoparaphyses; f. ascospores; g. ascospore; h. young conidiogenous cells; i. conidiogenous cells with developing conidia; j, k conidia viewed at two different levels of focus to show internally verruculose wall; l, m. conidia; n. germinating conidia. — Scale bars: a = 50 µm; b –n: 10 µm. 42 Persoonia – Volume 21, 2008 b a d c e Fig. 9 Neodeightonia subglobosa CBS 448.91. a. Globose conidiomata; b, c. conidiogenous cells; d. hyaline conidia; e. mature brown conidia. — Scale bars: a = 250 µm; b–e = 10 µm. b a e f d c g h Fig. 10 Neodeightonia phoenicum CBS 122528. a. Conidiogenous layer; b–e. conidiogenous cells; f. hyaline, aseptate conidia; g, h. brown, 1-septate conidia with longitudinal striations. — Scale bars = 10 µm. 43 A.J.L. Phillips et al.: Dark-spored teleomorph genera in Botryosphaeriaceae Dothiorella Sacc., Michelia 2: 5. 1880 Type species. Dothiorella pyrenophora Sacc. Dothiorella pyrenophora Sacc., Michelia 2: 5. 1880 Notes — The genus Dothiorella has been the source of much confusion in the past and the name has been used in more than one sense. Dothiorella has been used for anamorphs with hyaline, aseptate conidia similar to those normally associated with Fusicoccum and Neofusicoccum. Presumably this confusion started when Petrak (1922) transferred F. aesculi to Dothiorella, citing the species as the conidial state of B. be rengeriana (Sutton 1980). In later years, Dothiorella has been used for fusicoccum-like anamorphs with multiloculate conidiomata (Grossenbacher & Duggar 1911, Barr 1987, Rayachhetry et al. 1996). Sivanesan (1984) confused matters further by placing Dothiorella pyrenophora in synonymy with Dothichiza sorbi, which has small, hyaline, aseptate conidia and is the anamorph of Dothiora pyrenophora (Fr.) Fr. However, he was referring to Dothiorella pyrenophora Sacc. (1884), which is a later homonym of Dothiorella pyrenophora Sacc. (1880) (Sutton 1977). The taxonomic history of Dothiorella has been explained by Sutton (1977) and Crous & Palm (1999), and is illustrated by Crous & Palm (1999). Dothiorella was reduced to synonymy under Diplodia by Crous & Palm (1999), who used a broad morphological concept for Diplodia. Phillips et al. (2005) re-examined the type of Dothiorella pyrenophora Sacc. (K 54912) and stated that it differed from Diplodia by having conidia that are brown and 1-septate early in their development, while they are still attached to the conidiogenous cells. In Diplodia conidial darkening and septation takes place after discharge from the pycnidia. Crous et al. (2006) re-examined the types of both Diplodia and Dothio rella and conﬁrmed these morphological differences. Teleomorphs of Dothiorella have pigmented, septate ascospores. Dothiorella sarmentorum (Fr.) A.J.L. Phillips, A. Alves & J. Luque, Mycologia 97: 522. 2005 Basionym. Sphaeria sarmentorum Fr., Kongl. Vetensk. Acad. Handl., n.s. 39: 107. 1818. ≡ Diplodia sarmentorum (Fr.) Fr., Summa Veg. Scand. 2: 417. 1849. = Diplodia pruni Fuckel, Jahrb. Nassauischen Vereins Naturk. 23–24: 169. (1869 –1870) 1870. Teleomorph. Botryosphaeria sarmentorum A.J.L. Phillips, A. Alves & J. Luque, Mycologia 97: 522. 2005. Specimens examined. ENGLAND, Warwickshire, on Ulmus sp., Aug. 1956, E.A. Ellis, holotype of Otthia spiraeae, IMI 63581b, culture ex-holotype IMI 63581b. – SWEDEN, Lund, Botanical Garden, on Menispermum canadense, 1818, E.M. Fries (holotype of Sphaeria sarmentorum) Scleromyc. Suec. 18, isotype K(M) 104852. Dothiorella iberica A.J.L. Phillips, J. Luque & A. Alves, Mycologia 97: 524. 2005 — Fig. 8 Teleomorph. Botryosphaeria iberica A.J.L. Phillips, J. Luque & A. Alves, Mycologia 97: 524. 2005. Specimens examined. SPAIN, Aragon, Tarazona, on dead twigs of Quercus ilex, 18 Dec. 2002, J. Luque, holotype of B. iberica LISE 94944, culture extype CBS 115041, LISE 94942 = CBS 115035. Notes — This species is similar to Dothiorella sarmentorum but can be distinguished on characteristics of the asci, ascospores and conidia. Thus, in D. iberica the asci are shorter and more clavate, the ascospores characteristically taper towards the base, and on average the conidia are slightly longer. Within the Dothiorella clade there is a subclade consisting of Diplodia coryli (CBS 242.51) and Diplodia juglandis (CBS 188.87). However, neither of these two isolates is authentic, and neither could be induced to sporulate. Thus, their identity and distinction from other species could not be determined. Two other isolates (CAP187 from Prunus dulcis in Portugal, and JL599 from Corylus avellana in Spain) identiﬁed as Dothiorella spp. formed two further clades. However, these clades are represented by single isolates and were not considered any further. Nevertheless, it is clear that Dothiorella is genetically diverse and further collections will undoubtedly yield more species. Neodeightonia C. Booth, Mycol. Pap. 119: 17. (1969) 1970. Type species. Neodeightonia subglobosa C. Booth. Neodeightonia subglobosa C. Booth, Mycol. Pap. 119: 19. (1969) 1970. Anamorph. Sphaeropsis subglobosa Cooke, Grevillea 7: 95. 1879, as ‘subglobosum’. Specimens examined. SIERRA LEONE, Njala (Kori), on dead culms of Bambusa arundinacea, 17 Aug. 1954, F.C. Deighton, holotype IMI 57769 (c). – UNKNOWN LOCATION, human, keratomycosis of eye, Aug. 1991, Kirkness, CBS 448.91. Note — We examined the type specimen of Neodeightonia subglobosa and found only immature asci with hyaline ascospores. However, Punithalingam (1969) clearly described and illustrated the ascospores as brown and 1-septate. Von Arx & Müller (1954) transferred N. subglobosa to Botryosphaeria, and because this is the type species of the genus, they reduced Neodeightonia to synonymy under Botryosphaeria. However, morphologically (based on the dark, 1-septate ascospores) and phylogenetically, this genus is distinguishable from Botryosphaeria, and the genus is therefore reinstated here. Punithalingam (1969) referred to a germ slit in the conidia. Crous et al. (2006) suggested that this is in fact a striation on the conidial wall, and that more than one could occur per conidium, a feature conﬁrmed in the present study (Fig. 9). Such striate walls suggest an afﬁnity to Lasiodiplodia. Nevertheless, Neodeightonia can be distinguished from Lasiodiplodia by the absence of pycnidial paraphyses. Thus, conidial striations distinguish Neodeightonia from Diplodia, and the absence of pycnidial paraphyses distinguishes it from Lasiodiplodia. Neodeightonia phoenicum A.J.L. Phillips & Crous, sp. nov. — MycoBank MB511708; Fig. 10 Conidiomata brunnea vel nigra, in contextu hospitis inclusa, multilocularia, globosa. Cellulae conidiogenae holoblasticae, hyalinae, cylindricae, percurrenter cum 1–2 proliferationibus proliﬁcentes, vel in plano eodem periclinaliter incrassatae. Conidia (14.5 –)17– 21(–24) × (9 –)10–12.5(–14) µm ovoidea vel ellipsoidea, apicibus rotundato, in fundo rotundato, parietibus crassis, primaria hyalinae, cum maturitate brunnea, longitudinaliter striata et unum septa formantia. 44 Persoonia – Volume 21, 2008 Anamorph. Macrophoma phoenicum Sacc., Annuario Reale Ist. Bot. Roma 4: 195. 1890. ฀ ≡ Diplodia phoenicum (Sacc.) H.S. Fawc. & Klotz, Bull. Calif. Agric. Exp. Station 522: 8. 1932. ฀ ≡ Strionemadiplodia phoenicum (Sacc.) Zambett., Bull. Trimestriel Soc. Mycol. France 70: 235. (1954) 1955. Conidiomata formed on pine needles in culture pycnidial, multiloculate, dark brown to black, immersed in the host, becoming erumpent when mature. Conidiogenous cells hyaline, smooth, cylindrical, swollen at base, holoblastic, proliferating percurrently to form one or two annellations, or proliferating at same level giving rise to periclinal thickenings. Conidia ovoid to ellipsoid, apex and base broadly rounded, widest in middle to upper third, thick-walled, initially hyaline and aseptate, becoming dark brown and 1-septate some time after discharge from pycnidia, with melanin deposits on inner surface of wall arranged longitudinally giving a striate appearance to conidia, (14.5–)17– 21(–24) × (9–)10–12.5(–14) μm, 95 % conﬁdence _ limits = 18.6 –19.5 × 11.2 –11.8 µm (χ฀± S.D. = 19.1 ± 1.7 × 11.5 ± 1.1 µm, l/w ratio = 1.7 ± 0.2). Specimens examined. SPAIN, Catalonia, Tarragona, Salou, on Phoenix sp., F. Garcia, holotype CBS H-20108, culture ex-type CBS 122528; Catalonia, Barcelona, Vilanova i la Geltrú, on Phoenix canariensis, 17 May 2004, M. Rojo, CBS 123168. – USA, California, on Phoenix dactylifera, Mar. 1934, H.S. Fawcett, CBS 169.34. a d b Notes — Zambettakis (1955) placed this species in Strione madiplodia (based on the striate conidia). However, the teleomorphic genus Neodeightonia is available for this species. The absence of pycnidial paraphyses distinguishes Neodeightonia from Lasiodiplodia, while the striate conidia distinguish it from Diplodia. Although Punithalingam (1969) reported that the teleomorph of N. subglobosa forms in culture, our isolates of N. phoenicum failed to do so, even after long periods of incubation (> 3 mo). Phaeobotryon Theiss. & Syd., Ann. Mycol. 13: 664. 1915 Type species. Phaeobotryon cercidis (Cooke) Theiss. & Syd. Phaeobotryon cercidis (Cooke) Theiss. & Syd., Ann. Mycol. 13: 664. 1915. — Fig. 11 Basionym. Dothidea cercidis Cooke, Grevillea 13: 66. 1885, as ‘Dothidea (Bagnisiella)’. ฀ ≡ Bagnisiella cercidis (Cooke) Berl. & Voglino, Add. Syll. Fung. 1–4: 223. 1886. ฀ ≡ Auerswaldia cercidis (Cooke) Theiss. & Syd., Ann. Mycol. 12: 270. 1914. Specimen examined. USA, Carolina, on bark of Cercis canadensis, ex Herb. MC Cooke No 795, K134204. c e f g h Fig. 11 Bagnisiella cercidis K 134204. a. Ascomata; b. immature ascus; c. asci with immature ascospores; d. hyaline ascospores; e–g. hyaline, aseptate ascospores with terminal apiculi (arrows); h. broken, brown, 2-septate ascospore. — Scale bars: a = 400 µm; b–h = 10 µm. 45 A.J.L. Phillips et al.: Dark-spored teleomorph genera in Botryosphaeriaceae a b c Fig. 12 Phaeobotryon quercicola Fungi Rehnani 534 in G. a. Vertical section through an ascoma; b. immature ascus; c. mature, 2-septate brown ascospores. — Scale bars: a = 100 µm; b, c = 10 µm. Notes — In the original description of Dothidea cercidis, ascospores were reported as hyaline. However, Theissen & Sydow (1914) observed them to become brown with age, 32–38 × 12–13 µm. Subsequently Theissen & Sydow (1915) introduced the genus Phaeobotryon Theiss. & Syd. to accommodate this species. The asci are clavate, bitunicate, approx. 150 –170 × 30–35 µm. The pseudoparaphyses are branched, septate, constricted at septa, anastomosing, 4 – 5 µm wide. The ascospores are ellipsoidal, initially hyaline, becoming pale brown, turning brown at maturity, 2-septate (cells equal in length), with a characteristic punctiform outgrowth (conical apiculus) at each end of the spore, (27–)30–35(– 38) × (8 –) 12–14(–15) µm. The latter features, namely 2-septate, brown ascospores with a conical apiculus at each end, are considered characteristic for the genus. Phaeobotryon quercicola (A.J.L. Phillips) Crous & A.J.L. Phillips, comb. nov. — MycoBank MB511711; Fig. 12 Basionym. Botryosphaeria quercicola A.J.L. Phillips, Mycologia 97: 526. 2005 (based on Otthia quercus Fuckel, Jahrb. Nassauischen Vereins Naturk., 23–24: 170. (1869 –1870) 1870. Notes — As illustrated by Phillips et al. (2005), Phaeobotryon quercicola has brown, 2-septate ascospores with a conical apiculus at each end, thus suggesting that it would be better accommodated in Phaeobotryon than Botryosphaeria. 10 – 23 µm apart, constricted at septa, 3 – 4 µm wide. Asci bitunicate, 8-spored, stipitate, thick-walled with thick endotunica and well-developed apical chamber, 120 –150(– 200) × 25–30 µm, with biseriate ascospores. Ascospores ellipsoid to ovate, (30 –)37– 40(– 45) × (11–)13 –15(–16) µm, 2-septate, with 3 cells of equal length, not constricted at septa, ﬁnely verruculose, widest in middle with conical apiculus at one or both ends. Spermatogonia morphologically similar to conidiomata, also formed in culture. Spermatia hyaline, rod-shaped with rounded ends, 3–5 × 2 µm. Conidiomata pycnidial, ostiolate, separate or aggregated, globose, black, immersed to erumpent, unilocular, up to 350 µm diam; wall consisting of 4–6 layers of brown textura angularis. Conidiogenous cells cylindrical to doliiform, hyaline, smooth, proliferating percurrently near apex, 10–14 × 4–8 µm. Conidia ellipsoid to oblong or subcylindrical or obovoid, brown, smooth to ﬁnely verruculose, moderately thickwalled, granular, guttulate, ends rounded, 1(– 2)-septate, base with inconspicuous scar, slightly flattened, (30 –)35–38(– 43) × (12 –)14–15(–16) µm. Specimen examined. HAWAII, Manna Koa Park, Saddle Road, on stems of Sophora chrysophylla, July 2005, W. Gams, holotype CBS H-20109, culture ex-type–CPC 12440 = CBS 122980. Phaeobotryosphaeria Speg., Ann. Inst. Rech. Agron. 17, 10: 120. 1908. Type species. Phaeobotryosphaeria yerbae Speg. Phaeobotryon mamane Crous & A.J.L. Phillips, sp. nov. — MycoBank MB506581; Fig. 13 Phaeobotryon cercidis similis sed ascosporae majoribus, (30–)37–40(– 45) × (11–)13 –15(–16) µm. Anamorph. Dothiorella-like, but with up to two transverse septa. Etymology. Named for its host, Sophora chrysophylla, which is known as ‘mamane’ in Hawaii. Ascomata pseudothecial, dark brown to black, stromatic, globose, aggregated in botryose clusters or separate, immersed, becoming erumpent, ostiolate, up to 350 µm diam; wall consisting of 4 – 6 cell layers of dark brown textura angularis. Pseudoparaphyses hyaline, smooth, multiseptate, with septa Anamorph. Sphaeropsis Sacc., Michelia 2: 105. 1880, nom. cons. Ascomata pseudothecial, brown to black, unilocular, thickwalled. Pseudoparaphyses hyaline, septate. Asci bitunicate, 8-spored, thick-walled with thick endotunica and well-developed apical chamber. Ascospores brown, aseptate with small apiculus at either end. Conidiomata pycnidial, eustromatic, immersed to erumpent, thick-walled, wall composed of several layers of dark brown textura angularis. Ostiole single, central, papillate. Paraphyses hyaline, aseptate, thin-walled. Conidiogenous cells hyaline, discrete, proliferating internally to form periclinal thickenings. Conidia oval, oblong or clavate, straight, aseptate, moderately thick-walled. 46 Persoonia – Volume 21, 2008 a d b e c g f i j l m h k n o Fig. 13 Phaeobotryon mamane. a–h: CBS H-20109; i –o: CBS 122980. a–c. Asci and ascospores; d–h. ascospores with terminal apiculi (arrows); i. conidiomata forming on pine needle; j –m. conidiogenous cells with developing conidia; n. aseptate and 1-septate conidia; o. 2-septate conidium. — Scale bars: a–h, j –o = 10 µm; i = 350 µm. 47 A.J.L. Phillips et al.: Dark-spored teleomorph genera in Botryosphaeriaceae Phaeobotryosphaeria yerbae Speg., Ann. Inst. Rech. Agron. 17, 10: 120. 1908 — Fig. 14 Anamorph. Not reported but presumably a Sphaeropsis species. Ascomata pseudothecial, brown to black, multiloculate, immersed, becoming erumpent, ostiolate, papillate, up to 500 µm diam, wall composed of several layers of dark brown textura angularis. Pseudoparaphyses hyaline, smooth, 4–6 µm wide, multiseptate, with septa 10–18(– 22) µm apart, constricted at septa. Asci bitunicate, clavate, 8-spored, ascospores biseriate in the ascus, stipitate, thick-walled with thick endotunica and well-developed apical chamber, 120 –150 × 25–30 µm. Ascospores dark brown when mature, ovoid, (32–)34–42(–48) × (14–)16–18(–20) µm, aseptate, externally smooth, internally a ﬁnely verruculose, widest in middle with a hyaline apiculus at either end. Specimen examined. ARGENTINA, Misiones, Campo das Cuias, y San Pedro, on branches of Ilex paraguayensis, Feb. 1907, C. Spegazzini, holotype LPS 2926. Phaeobotryosphaeria visci (Kalchbr.) A.J.L. Phillips & Crous comb. nov. — MycoBank MB512100; Fig. 15 Basionym. Dothidea visci Kalchbr., Hedwigia 8: 117. 1869. ≡ Phaeobotryon visci (Kalchbr.) Höhn., Sber. Akad. Wiss. Wien, Math.naturw. kl., Abt I 128: 591. 1919. ≡ Botryosphaeria visci (Kalchbr.) Arx & E. Müll., Beitr. Kryptogamenfl. Schweiz, Band II, Heft I: 41. 1954. Anamorph. Sphaeropsis visci (Fr.) Sacc., Michelia 2: 105. 1880. For synonyms see Sutton (1980). b e c d f Fig. 14 Phaeobotryosphaeria yerbae LPS 2926. a. Immature (left) and mature (right) asci; b. mature ascus with brown, aseptate ascospores; c. septate pseudoparaphyses; d. dark brown, aseptate ascospores; e, f. dark brown, aseptate ascospores with apiculi. — Scale bars = 10 µm. 48 Persoonia – Volume 21, 2008 b a c g j e d f h i k l Fig. 15 Phaeobotryosphaeria visci. a–f: CWU (MYC) AS 2271; g–l: CBS 122527. a. Immature asci; b. mature ascus with brown, aseptate ascospores; c–f. brown, aseptate ascospores with apiculi (arrows); g. conidioma formed in culture on a pine needle; h, i. conidia forming on conidiogenous cell between paraphyses (arrows); j. developing conidia; k. paraphyses; l. conidia. — Scale bars: a, b = 20 µm; c –f, h –l = 10 µm; g = 50 µm. 49 A.J.L. Phillips et al.: Dark-spored teleomorph genera in Botryosphaeriaceae b a f d h g e c i j Fig. 16 Phaeobotryosphaeria citrigena. a–g: PDD 89238; h–j: ICMP 16812. a–c. Asci with brown ascospores; d. pseudoparaphyses; e–g. brown, aseptate ascospores with apiculi (arrows); h. conidium developing on a conidiogenous cell; i. hyaline, aseptate conidia; j. conidiomatal paraphyses. — Scale bars: a = 50 µm; b–d = 20 µm; e–j = 10 µm. 50 Persoonia – Volume 21, 2008 Ascomata pseudothecial, brown to black, uni- or multiloculate, separate, immersed, ostiolate, up to 500 µm diam, wall composed of several layers of dark brown textura angularis. Pseudoparaphyses hyaline, smooth, 4–6 µm wide, multiseptate, with septa 11–19(–26) µm apart, constricted at septa. Asci bitunicate, 8-spored, ascospores biseriate in the ascus, stipitate, thick-walled with thick endotunica and well-developed apical chamber, 180–230 × 35–50 µm. Ascospores pale-brown when mature, ovoid, (27.5–)31–37.5(–38.5) × (14.5–)15–19(–19.5) μm, aseptate, externally smooth, internally ﬁnely verruculose, widest in middle with an apiculus at either end. Conidiomata immersed to erumpent and superﬁcial, unilocular, up to 300 μm wide, wall composed of dark brown textura angularis. Paraphy ses hyaline, aseptate, up to 40 µm long and 4 µm wide with a bulbous tip 5 µm diam. Conidiogenous cells hyaline, discrete proliferating internally to form periclinal thickenings, (4–)8.5–11 × 4–6.5 µm. Conidia (27–)29–33(–50) × (14.5–)15.5–19(–22) µm, oval, apex obtuse, base obtuse or truncate, moderately thick-walled, initially hyaline, becoming brown, externally smooth, internally ﬁnely verruculose. ‘Svjatie Gory’, Donetsk district, on branches of Viscum album, 10 Mar. 2007, Á. Akulov, CWU (MYC) AS 2271, cultures CBS 122526, CBS 122527. Note — Until now the connection between Phaeobotryo sphaeria and its anamorph has not been proven. On the specimen examined here there is a Botryosphaeria-like ascomycete with brown ascospores. Single ascospore isolations from this specimen resulted in cultures of S. visci, thus proving the connection between the two states. Features that distinguish this teleomorph from others with brown ascospores in the Botryosphaeriaceae are the aseptate ascospores with an apiculus at either end. Phaeobotryosphaeria citrigena A.J.L. Phillips, P.R. Johnst. & Pennycook, sp. nov. — MycoBank MB511714; Fig. 16 Phaeobotryosphaeria visci similis sed ascosporae rufus-brunneae, et conidiae minoribus, (27–)28–33(– 34) × (14.5–)15–18.5(–19) µm. Anamorph. Sphaeropsis sp. Etymology. Named for its association with Citrus. Specimens examined. GERMANY, Klein Ziethen, near Angermünde, on fallen twigs of Viscum album, 22 July 1996, T. Graefenhan, CBS 186.97. – LUXEMBOURG, Weilenbach, near Echternach, on fallen twigs of Viscum album, 14 June 1997, H.A. van der Aa, CBS 100163. – UKRAINE, National Nature Park a Ascomata pseudothecial, brown to black, separate or aggregated, immersed, becoming erumpent, ostiolate, wall composed of several layers of dark brown textura angularis. Pseudopara b c e d f g h Fig. 17 Phaeobotryosphaeria porosa CBS 110496. a. Pycnidium with elongated neck; b. conidium developing between paraphyses; c. paraphyses; d. conidia and conidiogenous cells; e, f. immature conidium at two different levels of focus to show pores in the conidium wall; g, h. mature conidium at two different levels of focus to show verruculose inner surface of the wall. — Scale bars: a = 500 µm, b–h = 10 µm. A.J.L. Phillips et al.: Dark-spored teleomorph genera in Botryosphaeriaceae physes hyaline, smooth, 4–6 µm wide, multiseptate, with septa 11–26 µm apart; constricted at septa. Asci bitunicate, 8-spored, stipitate, thick-walled with thick endotunica and well-developed apical chamber, 180 – 230 × 35 – 43(– 50) µm, with biseriate ascospores. Ascospores reddish brown when mature, ellipsoid to ovoid with both ends rounded, (27.5 –)29 – 37.5(– 38.5) × (14.5–)15.5–18(–19.5) µm, with an apiculus at either end, aseptate, externally smooth, internally ﬁnely verruculose, widest in middle to upper third. Conidiomata immersed to erumpent and superﬁcial, unilocular, up to 500 μm wide, wall composed of several layers of dark brown textura angularis. Paraphyses hyaline, aseptate, up to 25 µm long and 3–3.5 µm wide. Co nidiogenous cells hyaline, discrete, proliferating internally to form periclinal thickenings, 8–11 × 4–6.5 µm. Conidia (27–) 28–33(– 34) × (14.5–)15–18.5(–19) µm, oval, apex obtuse, base obtuse or truncate, moderately thick-walled, initially hyaline, becoming brown, externally smooth, internally ﬁnely verruculose, aseptate. Specimens examined. NEW ZEALAND, Northland, Kerikeri, Davies Orchard (#2), Inlet Road, on recently dead bark-covered twigs of Citrus sinensis, 6 Sept. 2006, S.R. Pennycook, P.R. Johnston & B.C. Paulus, holotype PDD 89238, culture ex-type ICMP 16812; Northland, Kerikeri, Davies Orchard (#3), Inlet Road, on recently dead bark-covered twigs of Citrus sinensis, 6 Sept. 2006, S.R. Pennycook, P.R. Johnston & B.C. Paulus, PDD 89239, culture ICMP 16818. Notes — Conidia of P. citrigena remained hyaline for long periods and only rarely did we observe dark conidia. Conidial dimensions of this species are similar to S. visci, but its ascospores are reddish brown in contrast to the pale brown ones of S. visci. Phaeobotryosphaeria porosa (Van Niekerk & Crous) Crous & A.J.L. Phillips, comb. nov. — MycoBank MB511715; Fig. 17 Basionym. Diplodia porosum Van Niekerk & Crous, Mycologia 96: 790. 2004. Specimen examined. SOUTH AFRICA, Western Cape Province, Stellenbosch, on shoots of Vitis vinifera, 2002, J.M. van Niekerk, holotype CBS H-12039, culture ex-type CBS 110496. Notes — Van Niekerk et al. (2004) did not mention pycnidial paraphyses, but these were clearly seen when these isolates were re-examined (Fig. 17). This species is unique within the Botryosphaeriaceae because of its large, thick-walled conidia with large pores (1 µm wide) that are clearly visible by light microscopy. However, the pitted walls, although unique and distinctive, should be regarded as informative at the species level in the same way that this character was regarded in the original description. Spencermartinsia A.J.L. Phillips, A. Alves & Crous, gen. nov. — MycoBank MB511762. Ascomata pseudothecia, ostiolati. Asci bitunicati, octo-spori, clavati, stipitati, pseudoparaphysibus multis ﬁliformibus, septatis, latis interspersi. Ascosporae biseriati, uniseptati cum terminali apiculi. Conidiomata stromatiformia. Cellulae conidiogenae holoblasticase, proliferatione percurrenti, ut videtur annellationibus, vel inplano eodem periclinaliter incrassate. Conidia brunnea, uniseptata. 51 Type species. Spencermartinsia viticola (A.J.L. Phillips & J. Luque) A.J.L. Phillips, A. Alves & Crous. Etymology. Named in honour of Prof. dr Isabel Spencer-Martins, founder of the Centro de Recursos Microbiológicos in Portugal. Ascomata pseudothecial, ostiolate. Pseudoparaphyses thinwalled, hyaline, septate, constricted at septa. Asci bitunicate, 8-spored, clavate, stipitate, developing amongst thin-walled, septate pseudoparaphyses, with biseriate ascospores. Asco spores hyaline when young, brown when mature, uniseptate with an apiculus at each end. Conidiomata stromatic. Conidio genous cells lining inner surface of conidiomata, holoblastic, proliferating internally producing periclinal thickenings, or proliferating percurrently to form annellations. Conidia brown, 1-septate. Note — Spencermartinsia differs from Dothiorella in having 2-celled ascospores with an apiculus at either end of the ascospores. Spencermartinsia viticola (A.J.L. Phillips & J. Luque) A.J.L. Phillips, A. Alves & Crous, comb. nov. — MycoBank MB511763; Fig. 18 Basionym. Botryosphaeria viticola A.J.L. Phillips & J. Luque, Mycologia 97: 1116. (2005) 2006. Anamorph. Dothiorella viticola A.J.L. Phillips & J. Luque, Mycologia 97: 1116. (2005) 2006. Specimens examined. SPAIN, Catalonia, Vimbodí, near the Monastery of Poblet, on pruned canes of Vitis vinifera cv. Garnatxa Negra, 12 Aug. 2004, J. Luque & S. Martos, holotype LISE 95177, culture ex-type CBS 117009; ditto, 28 May 2003, J. Luque & Mateu, LISE 95178, culture CBS 117006. Notes — The ex-type isolate of Spencermartinsia viticola (CBS 117009) clustered with an isolate previously identiﬁed as Diplodia spegazziniana (CBS 302.75). The latter isolate is misidentiﬁed and is not representative of this species. An additional isolate originally identiﬁed by Luque et al. (2005) as B. viticola (CBS 117006), exhibited some differences in culture morphology from the ex-type strain and other strains (Luque et al. 2005). For example, the reverse side of cultures of CBS 117006 became red-brown after 3–5 d on PDA at 25 °C with a progressive darkening of the pigment after 6–10 d. Furthermore, there were some differences in ITS and EF1-α sequences between CBS 117006 and CBS 117009 (one substitution and one deletion in ITS and nine substitutions in EF1-α). Although these morphological and phylogenetic differences may reflect species differences, no name was applied to CBS 117006 because only one isolate was available for study. Also contained within Spencermartinsia was a single isolate of ‘Diplodia’ medicaginis (CBS 500.72), which formed a unique clade. Again, only a single isolate was available, the name of which is unresolved. Isolates ICMP 16827 and ICMP 16828 from Citrus sinensis in New Zealand formed another subclade, and thus would be regarded as a distinct phylogenetic species. However, neither of the isolates could be induced to sporulate, and no morphological data are available. Therefore, no names will be applied until their morphology can be determined. Isolates ICMP 16819 and ICMP 16824, also from Citrus sinensis in New Zealand, formed a sister clade to Spencer martinsia that was supported by a high MP bootstrap value 52 Persoonia – Volume 21, 2008 a b e c f i d g h j k Fig. 18 Spencermartinsia viticola. a–h: LISE95177; i –k: CBS 117009. a. Ascomata erumpent through host bark; b. ascoma cut through horizontally showing the white contents with dark spots corresponding to asci with ascospores; c. vertical section through an ascoma; d. septate pseudoparaphyses; e. clavate ascus containing eight biseriate, dark brown, 1-septate ascospores; f. ascospores; g. ascospores with small rounded apiculi (arrows); h. conidiomata partially erumpent through the host bark; i. conidiogenous cells; j. conidiogenous cells with annellations. The cell on the right has a dark brown, 1-septate conidium attached; k. conidia. — Scale bars: a, h = 500 µm; b = 250 µm; c = 50 µm; d–g, i –k = 10 µm. A.J.L. Phillips et al.: Dark-spored teleomorph genera in Botryosphaeriaceae (100 %). Neither of these two isolates has been induced to sporulate. In view of the lack of morphological data, the status of these two isolates remains uncertain. dISCuSSIon In this paper the phylogenetic position and taxonomy of species of Botryosphaeriaceae with brown ascospores were studied. The taxonomic position of Dothidotthia was resolved, and within the Botryosphaeriaceae we recognise a number of genera with brown ascospores. For some of these genera we reinstate old names, while others are described as new. In keeping with the proposal to use a single name for pleomorphic fungi (Rossman & Samuels 2005) we propose a single generic name for each clade. For example, since Dothidotthia was shown to fall in the Pleosporales, this name is no longer available for the teleomorph of Dothiorella, and therefore we propose that the anamorph genus name, Dothiorella, be used for both the anamorph and the teleomorph of this clade. Within the Botryosphaeriaceae, species with brown ascospores are found in three separate lineages, which lead to at least six genera. These lineages are dispersed randomly in different branches of the phylogenetic tree. Considering that brown ascospores are a common feature in other families in the Dothideomycetes (von Arx & Müller 1954, 1975), it is possible that this character has been retained in these lineages from the ancestors of the family, rather than being a character that has evolved at different times. Other morphological features that were used to differentiate genera were the presence or absence of apiculi on ascospores, septation of ascospores, striations on conidia, and the presence or absence of paraphyses in conidiomata. It is interesting to note that striations are strongly developed in Lasiodiplodia, weaker in Neodeightonia and absent from Diplodia. Furthermore, conidiomatal paraphyses are found in Lasiodiplodia but are absent from Neodeightonia and Diplodia. Two of the lineages with brown ascospores lie within a clade that was previously regarded to contain species with Lasiodiplodia and Diplodia anamorphs (Crous et al. 2006). In their phylogenetic study (Crous et al. 2006), based on LSU sequences, this clade could not be resolved. In the present study it could be resolved only by combining sequences of two protein coding genes with sequences of three ribosomal genes. In this way this clade was resolved into six genera, four of which have dark ascospores. One lineage (clade 2, Fig. 2) clustered between Diplodia and Lasiodiplodia. The name Neodeightonia already exists for this genus and it is reinstated in this paper. This genus was introduced by Booth (in Punithalingam 1969) for a single species, namely N. subglobosa. Von Arx & Müller (1975) transferred this species to Botryosphaeria within their broad concept of the genus. When Crous et al. (2006) reassessed Botryosphaeria, reducing it to B. corticis and B. dothidea, their isolate of B. subglobosa resided in an unresolved clade consisting of Diplodia, Lasiodiplodia and Tiarosporella. From the data presented here it is clear that Neodeightonia subglobosa, type of Neodeightonia, is phylogenetically and morphologically 53 distinct from other genera in the Botryosphaeriaceae. Diplodia phoenicum was shown to be another species in this genus. Rather than introduce a new anamorph genus to accommodate Diplodia phoenicum we followed the procedures suggested by Rossman & Samuels (2005) and used the teleomorph genus name for this species. Conidia of both N. subglobosa and N. phoenicum have striations on the conidial wall similar to those seen in Lasiodiplodia, albeit somewhat less distinct. However, anamorphs of Neodeightonia do not have paraphyses, which are typical of Lasiodiplodia, and the striate conidial wall distinguishes Neodeightonia from Diplodia. A second lineage, basal to Diplodia, Lasiodiplodia and Neo deightonia, was resolved into three clades (clades 4–6) that could be distinguished from one another on the morphology of the teleomorphs, especially septation of the ascospores and the presence or absence of ascospore apiculi. These genera can also be differentiated on morphology of the anamorphs. Phaeobotryon is available for one of these clades, Phaeobot ryosphaeria for another, but as far as we could tell, no suitable names are available for the third one, and Barriopsis is introduced to accommodate Physalospora fusca, which has aseptate ascospores without apiculi. Von Arx & Müller (1954, 1975) placed Phaeobotryon in synonymy with Botryosphaeria. However, as determined here, Phaeobotryon is morphologically and phylogenetically distinct from all the other genera we studied. For this reason we have reinstated the generic name Phaeobotryon for isolate CBS 122980, and for other isolates in the same clade. The 1–2-septate ascospores of these fungi with an apiculus at either end correspond with Bagnisiella cer cidis K134204, which is the basionym of Phaeobotryon cercidis and type species of the genus Phaeobotryon. Ascospores of the isolates from Sophora chrysophylla are larger than P. cer cidis and for this reason these isolates were described as a new species. Although Von Arx & Müller (1954) considered Phaeobotryosphaeria a synonym of Botryosphaeria, in this study we show that it is morphologically and phylogenetically distinct from the other two genera in this clade and the name is reinstated for species with brown, aseptate ascospores with terminal apiculi. The anamorph of Phaeobotryosphaeria was shown to correspond to Sphaeropsis. Although we have adopted to follow the system of one name for one genus, it is important to clarify some of the controversy surrounding the genus Sphaeropsis. This genus has been the subject of considerable debate, much of which has revolved around the question of a suitable genus name for the pine pathogen sometimes referred to as Sphaeropsis sap inea. The main point of debate has been whether this species should revert to its older name of Diplodia pinea or whether it should remain in Sphaeropsis. From the literature it seems that this species has been regarded as typical of the genus Sphaeropsis, both morphologically and phylogenetically. For example, the phylogenetic studies of Jacobs & Rehner (1998) and Denman et al. (2000) placed Sphaeropsis sapinea in the Diplodia clade, which prompted Denman et al. (2000) to suggest that Sphaeropsis is a synonym of Diplodia. This decision was also supported by subsequent studies (Zhou & Stanosz 2001, Alves et al. 2004). When Sutton (1980) stated that percurrently proliferating conidiogenous cells are a feature of Sphaeropsis 54 that are not found in Diplodia it is not clear if he was referring to S. visci or S. pinea. Nevertheless, Denman et al. (2000) referred to percurrent proliferations in Diplodia, further conﬁrming their suggestion that Sphaeropsis is a synonym of Diplodia. Phillips (2002) and Alves et al. (2004) conﬁrmed that this type of conidiogenesis occurs in Diplodia mutila. However, it is important to point out that when Saccardo (1880) established Sphaeropsis for species of Diplodia with dark conidia, he cited S. visci as the type species. We examined a number of strains isolated from Viscum album that correlate in all ways with the original description of S. visci and could ﬁnd only internal proliferation of the conidiogenous cells, resulting in periclinal thickenings and typical phialides (sensu Sutton 1980). Moreover, this was the only type of conidiogenesis that we could detect in the other species that we consider to belong in Sphaeropsis (D. poro sum and S. citrigena). As we illustrate here, the anamorphs of Sphaeropsis are morphologically (pycnidial paraphyses) and phylogenetically distinct from Diplodia. Thus, as revealed by the phylogeny presented here, Sphaeropsis, typiﬁed by S. visci, is a valid and distinct genus. Moreover, the pine pathogen often referred to as S. sapinea resides in Diplodia. The other species in Phaeobotryosphaeria deserve some mention. Phaeobotryosphaeria porosa is distinct in the large pits in the conidial wall. When this species was described from grapevines in South Africa (van Niekerk et al. 2004) it was placed in Diplodia, although the authors suggested that its unique conidial morphology might necessitate a new genus. At that time Sphaeropsis was not clearly deﬁned and indeed had been suggested as being a synonym of Diplodia. Despite the unique character of conidial pits, D. porosum has features that place it within the morphological concept of Phaeobotryosphaeria. These features include relatively large, thick-walled conidia, phialidic conidiogenous cells with periclinal thickenings, and pycnidial paraphyses. Phylogenetically (Fig. 2) it also falls within Phaeobotryosphaeria. Thus, it seems that conidial pits are of taxonomic signiﬁcance at species level only, in the same way as they were regarded when this species was ﬁrst described by van Niekerk et al. (2004). Finally, a third species is described in Phaeobotryosphaeria, namely P. citrigena from dead citrus twigs in New Zealand. The third lineage (clades 8–10) is sister to Neofusicoccum, and the name Dothiorella has been used for the anamorphs of these species. This lineage was resolved into at least two, possibly three genera. Clades 8 and 9 could be distinguished from one another on the morphology of their ascospores. No teleomorph is yet known for clade 10. Dothiorella is already available for clade 8, and a new genus Spencermartinsia is introduced for clade 9. Dothiorella is based on D. pyrenophora, but no cultures are available for this species. When Phillips et al. (2005) reinstated Dothiorella, they determined that D. sarmentorum corresponded in all ways with the concept for this genus. A clade sister to Dothiorella was composed of two subclades (clades 9 and 10). It is not entirely clear if these two clades represent two genera or a single genus. Spencermartinsia viticola was considered to be a species of Dothiorella by Luque et al. (2005), who pointed out that some morphological aspects of the anamorph (colony morphology) differentiated this species from others in Dothiorella. A more detailed examination of Persoonia – Volume 21, 2008 this species revealed that the ascospores bear an apiculus at either end. This feature, together with the phylogenetic difference indicates that this clade represents another genus closely related to Dothiorella, and for which we introduce the name Spencermartinsia. The distinct apiculi differentiate this genus from Dothiorella, and for this reason we propose it as a new genus. This clade (9) is phylogenetically diverse and appears to be composed of several species. The type species (S. viticola) is represented in Fig. 2 by the ex-type culture of Do. viticola (CBS 1187009). Another isolate with this name (CBS 117006) resides in a separate clade, and thus probably represents another species. Since we have only a single example of this species we decline at this stage to apply a species name to it. Similarly, CBS 500.72 (D. medicaginis) is another distinct species represented by a single isolate, which we also decline to name. The two isolates from Citrus (ICMP 16827 and ICMP 16828) did not sporulate in culture during the course of this work and thus cannot be fully characterised. Nevertheless, they too represent a third species in Spencermartinsia. The conidia from which these isolates were grown match closely those illustrated by Gure et al. (2005) from an isolate from Podocarpus falcatus seeds, which these authors referred to Dothiorella. We are continuing to study these isolates with the aim of applying species epithets. Isolates ICMP 16819 and ICMP 16824 form a further clade (clade 10). These isolates were grown from 1-septate, dark brown, striate conidia collected from twigs of Citrus. The conidia become pigmented and septate while still attached to the conidiogenous cell, a characteristic of Dothiorella and Spencermartinsia. This fungus failed to sporulate in culture, and has yet to be linked to a teleomorph. For this reason we were unable to determine if these two isolates form a distinct genus and such a decision will have to wait until more isolates in this clade have been studied. Acknowledgements This work was ﬁnanced by the European Regional Development Fund and Fundação para a Ciência e a Tecnologia (FCT) under project POCI/AGR/56140/2004. A. Alves was supported by grant No. SFRH/BPD/24509/2005 from FCT, and a visit to CBS by a SYNTHESIS grant (No. NL-TAF-1876). A. Phillips was supported by grant No. SFRH/ BCC/15810/2006 from FCT. J. 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