6. Molecular Studies and Phylogeny

MOLECULAR STUDIES AND PHYLOGENY 14 6. Molecular studies and phylogeny Previous molecular studies employing rDNA ITS sequence data (CROUS et al. 2001) have shown cladosporium-like taxa to cluster adjacent to the main monophyletic Myco- sphaerella clade, suggesting a position apart of the latter genus. Comprehensive ITS (ITS- 1, 5.8S, ITS-2) and 18S rRNA sequence analyses carried out by BRAUN et al. (2003) provided further evidence for the separation of Cladosporium s. str. In the latter paper, results of molecular examinations published by other authors were summarised and phylograms of cladosporium-like fungi were discussed in detail. Human-pathogenic cladophialophora-like hyphomycetes (Herpotrichiellaceae), Sorocybe resinae (Fr.) Fr. (Amorphotheca resinae Parbery) (Amorphothecaceae), Alternaria malorum (Rühle) U. Braun, Crous & Dugan (Cladosporium malorum Rühle) (Pleosporaceae) and cladosporioid Venturia anamorphs (Fusicladium) (Venturiaceae) formed separate monophyletic clades and could be excluded from Cladosporium s. str. Within a big clade formed by members of the Mycosphaerellaceae, true Cladosporium species were shown to represent a sister clade to Mycosphaerella with cercosporoid anamorphs. The new teleomorph genus Davidiella was proposed to accommodate the teleomorphs of Cladosporium formerly placed in Mycosphaerella s. lat. It could be demonstrated that relatively minor differences in the conidiogenous loci and conidial hila support the different phylogenetic affinities. SEIFERT et al. (2004) used morphological characters, ecological features and DNA sequence data to characterise Cladosporium staurophorum taxonomically and phylogenetically and assigned it to the new genus Devriesia. Together with three additional heatresistant species, C. staurophorum formed a monophyletic group, with marginal position in the Mycosphaerellaceae, but clearly distinct from Cladosporium s. str. The new hyphomycetous genus Metulocladosporiella, an additional segregate of Cladosporium s. lat., has recently been introduced to accommodate Cladosporium musae, the causal agent of Cladosporium speckle disease of banana (CROUS et al. 2005). DNA sequence data derived from the ITS and LSU gene regions of C. musae isolates showed that this species is part of a large group of hyphomycetes in the Chaetothyriales with dematiaceous blastoconidia in acropetal chains. Cladosporium adianticola R.F. Castañeda, a foliicolous hyphomycete known from leaf litter in Cuba, proved to be also a member of this clade and closely related to C. musae. WIRSEL et al. (2002) and PARK et al. (2004) carried out phylogenetic studies within Cladosporium s. str. WIRSEL et al. (2002) analysed ITS data of strains isolated from common reed in Germany, compared them with sequences from GenBank and cultures from the CBS (Utrecht, the Netherlands), and distinguished three species, viz., Cladosporium herbarum, C. oxysporum and Cladosporium sp. Beside ITS sequences, they generated two additional phylogenies, viz., analyses based on the differentiation of the fungi by their capacity to metabolize different carbon sources and a second approach, using actin gene sequences, in which they discovered a highly variable intron sequence. Species phylogenies based on this protein-encoding gene exhibited higher resolution compared with the ITS tree leading to further differentiation in terminal branches. Furthermore, it could be shown that all strains with smooth conidial surfaces clustered together, as did all isolates with rough-walled conidia, thus reflecting a possible division among plantassociated Cladosporia based on conidial ornamentation. However, due to the limited dataset, including only few Cladosporium species, a final conclusion could not be drawn. In the study carried out by PARK et al. (2004), the sequences of the D1/D2 regions of the LSU rDNA genes and the ITS regions of the rDNA were employed in order to establish molecular standards for the demarcation of the common airborne species C. herbarum, C. cladosporioides and C. sphaerospermum. MOLECULAR STUDIES AND PHYLOGENY 15 The two neighbour-joining analyses of ITS datasets of the Cladosporium s. lat. complex presented within this work have been carried out at the CBS and kindly provided by Ewald Groenewald. The first phylogram (Fig. 1) contains isolates from five main groups [Pleosporaceae, Herpotrichiellaceae, Mycosphaerellaceae, Venturiaceae and the species complex of Cladosporium paeoniae Pass. / C. chlorocephalum (Fresen.) E.W. Mason & M.B. Ellis]. The Pleosporaceae form a well-supported clade (100 % bootstrap support) containing isolates of species of the genera Alternaria Nees and Lewia M.E. Barr & E.G. Simmons. The Herpotrichiellaceae are separated into two groups, the first consists of a clade supported by a bootstrap support value of 100 % comprising species of Cladophialophora and Phialophora Medlar, and the second well-supported clade contains strains of Cladosporium adianticola (99 % bootstrap support), Metulocladosporiella musicola Crous, Schroers & Groenewald (91 % bootstrap support) and Metuloclado- sporiella musae (E.W. Mason) Crous, Schroers, Groenewald, U. Braun & K. Schub. (99 % bootstrap support). Within the Mycosphaerellaceae cluster, a strongly supported Davidiella clade (100 %) containing Cladosporium anamorphs is formed. A clade for the Venturiaceae is also well supported containing species of Venturia as well as isolates of Fusicladium and Pseudocladosporium hachijoensis (Matsush.) U. Braun. Cladosporium paeoniae and C. chlorocephalum cluster closely together with a bootstrap support value of 100 %, but grouped outside of the Davidiella/Cladosporium clade (100 % bootstrap support). ITS sequences similar to those of the latter species could not be obtained by <a href="/goto?url=http://www.ncbi.nlm.nih.gov/BLAST/) (pers" target="_blank">BLAST-searches (www.ncbi.nlm.nih.gov/BLAST/) (pers. comm. with Ewald Groene- </a>wald), so the grouping with ‘Trimmatostroma salinum’ is only due to the limited dataset selected here. The second neighbour-joining tree contains isolates of the Mycosphaerellaceae and Amorphothecaceae. The Amorphothecaceae clade is well-supported with a bootstrap support value of 100 % and consists of Amorphotheca resinae and ‘Cladosporium’ breviramosum. The Mycosphaerellaceae clade consists of isolates of Mycosphaerella, a strongly supported clade (100 %) of Devriesia containing Cladospo- rium staurophorum and a big monophyletic clade of Davidiella comprising numerous Cladosporium species. The phylograms derived in the present study reflect and support the results of previous phylogenic studies published by BRAUN et al. (2003), SEIFERT et al. (2004) and CROUS et al. (2005) in which several clades (genera, families) with cladosporium-like taxa are clearly delineated from Cladosporium s. str. Furthermore, it could be demonstrated that C. paeoniae and C. chlorocephalum form a well-supported group outside of the Davidiella / Cladosporium clade. This separate position could be confirmed by means of light and scanning electron microscopy showing the conidiogenous loci and hila to be different from those of Cladosporium s. str. in being non-coronate (see Fig. 113 and Pl. 34, Figs B, C, E, F). Although both species are morphologically and ecologically quite distinct, ITS sequence data of both species proved to be completely identical, which led to the conclusion that the latter species represent two stages of the same fungus (synanamorphs). However, the taxonomic affinity of both species is not yet clear. Further critical studies are required, in which additional isolates and other genes are included. The independent and separate phylogenetic position of the genus Cladosporium in relation to Mycosphaerella, confirmed by morphotaxonomic and molecular studies, has been well established in literature. Isolates of Davidiella and Cladosporium s. str. consistently cluster together in ITS and LSU analyses, providing support for the monophyly of the anamorphic genus Cladosporium and the teleomorphic genus Davidiella (BRAUN et al. 2003). Within the big Davidiella/Cladosporium clade presented here (Fig. 2) three small subclades can be recognised, the first consisting of isolates of Davidiella tassiana (De Not.) Crous & U. Braun and its anamorph Cladosporium herbarum [incl. var. macrocarpum (Preuss) M.H.M. Ho & Dugan], the second containing two isolates of C. cladosporioides, and the MOLECULAR STUDIES AND PHYLOGENY 16 third comprising isolates of C. sphaerospermum. Isolates derived from new collections of several species made during the course of the present studies, e.g., C. phyllogenum and C. phyllophilum McAlpine (= C. exoasci Lindau) cluster within this clade confirming their correct placement in Cladosporium. C. oncobae also belongs in Cladosporium s. str. Since the two cultures isolated by Frank Hill cluster at two different places within the monophyletic clade, these cultures have to be checked to clarify their identities since the inclusion of a contamination has to be taken into consideration. There are similar problems with several isolates of ‘C. cladosporioides’. However, it must clearly be stated that ITS data are often not sufficient to assess phylogenetic relationships, above all on subgeneric and species level. UNTEREINER (2000) stated that the lack of resolution in phylogenies inferred from rDNA sequence data, particularly at the level of species and genus, argues strongly in favour of the use of combined data sets including sequences from other regions of the nuclear ribosomal repeat or sequences from multiple loci. Analyses based on a single locus are often not sufficient to differentiate phenotypically clearly distinct species, as recently demonstrated by SAMSON et al. (2004) for species of Penicillium Link subgen. Penicillium analysed on the base of ß-tubulin sequences. Sequence data from the internal transcribed spacers (ITS 1, ITS 2) within the genus Cladosporium are very uniform showing little variation resulting in a barely resolved clade, so that final conclusions at subgeneric and species rank are not yet possible. Multilocal analyses of the genome, based on a larger number of isolates from different geographical regions are necessary to redefine species borders within Cladosporium (WIRSEL et al. 2002). MOLECULAR STUDIES AND PHYLOGENY 17 Phomopsis vaccinii Alternaria conjuncta AF392988 Alternaria infectoria Y17066 100 Lewia ethzedia AF392987 Lewia infectoria CPC 4271AF397248 Alternaria malorum CPC 4572 100 100 Alternaria malorum CPC 4571 Alternaria malorum var. polymorpha CPC 4570 ‘Cladosporium’ adianticola CBS 735.87 74 100 Metulocladosporiella musae CBS 161.74 100 Metulocladosporiella musae CBS 113863 Metulocladosporiella musicola CBS 113861 Metulocladosporiella musicola CBS 113860 100 100 Metulocladosporiella musicola CBS 113865 Metulocladosporiella musicola IMI327290 96 100 Metulocladosporiella musicola CBS 113862 Metulocladosporiella musicola CBS 113864 Cladophialophora bantiana AF131079 Cladophialophora bantiana AF397182 Capronia semiimmersa MUCL 39979 Phialophora americana U31838 100 <ul style="display: flex;"><li style="flex:1">100 </li><li style="flex:1">90 </li></ul>100 Phialophora verrucosa AF397136 Phialophora verrucosa MUCL 15537 80 99 Rhinocladiella compacta AF397133 Cladophialophora minourae CBS 556.83 Cladophialophora carrionii CBS 160.54 Cladophialophora carrionii CBS 260.83 100 Cladosporium sphaerospermum CPC 3686 Davidiella tassiana ATCC 11281 Davidiella tassiana ATCC 201094 100 100 Cladosporium cladosporioides CPC 5100 Cladosporium cladosporioides CPC 3683 94 100 ‘Cladosporium paeoniae’ CPC 11383 100 ‘Cladosporium chlorocephalum’ CPC 11969 100 Trimmatostroma salinum AJ238672 100 Trimmatostroma salinum AJ238675 Trimmatostroma salinum AJ238676 Venturia pyrina AF065844 Venturia cerasi AF065847 100 Pseudocladosporium hachijoense CPC 5391 Venturia inaequalis AF065837 Venturia inaequalis AF531078 Fusicladium convolvulorum CPC 3884 Fusicladium effusum AY251085 100 100 0.1 substitutions per site Fusicladium effusum AF065850 Fusicladium effusum AF065851 Fusicladium effusum AY251084 89 Fig. 1: Phylogram of neighbour joining tree obtained from ITS sequencing data using F84 substitution model. Bootstrap support values are shown at nodes. The GenBank sequence Phomopsis vaccinii AF317578 was used as outgroup. The subclade including species of Davidiella and Cladosporium is marked by coloration. MOLECULAR STUDIES AND PHYLOGENY 18 Phomopsis vaccinii Amorphotheca resinae ATCC 200942 Cladosporium breviramosum ATCC 64696 Cladosporium breviramosum ATCC 76215 Hormoconis resinae CBS 184.54 100 96 64 Amorphotheca resinae CPC 3692 Hormoconis resinae CBS 183.54 Mycosphaerella punctiformis CBS 113265 Passalora arachidicola AF297224 88 Passalora bellynckii AF222831 Passalora dissiliens AF222835 Passalora vaginae AF222832 99 Mycovellosiella vaginae AF222832 Passalora henningsii AF284389 Pseudocercospora kibouchiae Hill 1061 Mycosphaerella aurantia AY509744 Passalora fulva AF393701 89 90 73 80 100 Passalora fulva AY251069 Devriesia thermodurans S108 1 Devriesia shelburnense S54 1 <ul style="display: flex;"><li style="flex:1">63 </li><li style="flex:1">100 </li></ul>Devriesia acadiense S101 1 54 99 100 Devriesia staurophorum CBS 374.81b Devriesia staurophorum CBS 375.81 Cladosporium staurophorum ATCC 200934 Mycosphaerella tassiana CPC 11600 Mycosphaerella tassiana CPC 11603 Mycosphaerella tassiana CPC 11601 Mycosphaerella tassiana CPC 11602 Cladosporium herbarum CBS 813.71 Mycosphaerella tassiana CPC 11604 Cladosporium macrocarpum CBS 175.62 Cladosporium sp. on Corylus CPC 11330 Cladosporium colocasiae CPC 4323 Cladosporium cladosporioides CBS 100 89 84 Cladosporium cucumerinum CBS 172.54 Cladosporium vignae CBS 121.25 Cladosporium sp. on Lycopodium CBS 114078 Cladosporium cladosporioides f. pisicola CBS 145.35 Cladosporium sp. 2 on Oncoba CPC 11664 Cladosporium cucumerinum CBS 173.54 100 Cladosporium cladosporioides CPC 3682 Cladosporium cladosporioides CPC 3683 Cladosporium tenuissimum ATCC 38027 Cladosporium uredinicola CPC 5390 70 Cladosporium phyllophilum CPC 11324 Cladosporium phyllogenum CPC 11327 Cladosporium phyllophilum CPC 11333 Cladosporium sp. 1 on Oncoba CPC 11663 AY361988 Cladosporium sphaerospermum ATCC 12092 0.1 substitutions per site 100 AY361958 Cladosporium sphaerospermum ATCC 11289 AJ244228 Cladosporium sphaerospermum CBS 122.47 Fig. 2: Phylogram of neighbour joining tree obtained from ITS sequencing data using F84 substitution model. Bootstrap support values are shown at nodes. The GenBank sequence Phomopsis vaccinii AF317578 was used as outgroup. The subclade including species of Davidiella and Cladosporium is marked by coloration.

6. Molecular Studies and Phylogeny

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