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Biodiversity in the Cladosporium herbarum complex (Davidiellaceae, Capnodiales), with standardisation of methods for Cladosporium taxonomy and diagnostics

The Cladosporium herbarum complex comprises five species for which Davidiella teleomorphs are known. Cladosporium herbarum s. str. (D. tassiana), C. macrocarpum (D. macrocarpa) and C. bruhnei (D. allicina) are distinguishable by having conidia of different width, and by teleomorph characters. Davidiella variabile is introduced as teleomorph of C. variabile, a homothallic species occurring on Spinacia, and D. macrospora is known to be the teleomorph of C. iridis on Iris spp. The C. herbarum complex combines low molecular distance with a high degree of clonal or inbreeding diversity. Entities differ from each other by multilocus sequence data and by phenetic differences, and thus can be interpreted to represent individual taxa. Isolates of the C. herbarum complex that were formerly associated with opportunistic human infections, cluster with C. bruhnei. Several species are newly described from hypersaline water, namely C. ramotenellum, C. tenellum, C. subinflatum, and C. herbaroides. Cladosporium pseudiridis collected from Iris sp. in New Zealand, is also a member of this species complex and shown to be distinct from C. iridis that occurs on this host elsewhere in the world. A further new species from New Zealand is C. sinuosum on Fuchsia excorticata. Cladosporium antarcticum is newly described from a lichen, Caloplaca regalis, collected in Antarctica, and C. subtilissimum from grape berries in the U.S.A., while the new combination C. ossifragi, the oldest valid name of the Cladosporium known from Narthecium in Europe, is proposed. Standard protocols and media are herewith proposed to facilitate future morphological examination of Cladosporium spp. in culture, and neotypes or epitypes are proposed for all species treated.


InTroducTIon
Cladosporium herbarum (Pers. : Fr.) Link, type species of the genus Cladosporium Link, is one of the most common environmental fungi to be isolated worldwide. It abundantly occurs on fading or dead leaves of herbaceous and woody plants, as secondary invader on necrotic leaf spots, and has frequently been isolated from air (Samson et al. 2000), soil (Domsch et al. 1980), foodstuffs, paints, textiles, humans (de Hoog et al. 2000 and numerous other substrates. It is also known to occur on old carpophores of mushrooms and other fungi (Heuchert et al. 2005) and to be a common endophyte (Riesen & Sieber 1985, Brown et al. 1998, El-Morsy 2000, especially in temperate regions. Under favourable climatic conditions C. herbarum also germinates and grows as an epiphyte on the surface of green, healthy leaves (Schubert 2005).
Persoon (1794) introduced C. herbarum as Dematium herbarum Pers., which was later reclassified by Link (1809) as Acladium herbarum (Pers.) Link. In 1816, Link included C. herbarum together with three additional species in his newly described genus Cladosporium. Clements & Shear (1931) proposed C. herbarum as lectotype species of the latter genus, a decision followed by de Vries (1952) and Hughes (1958). Several authors provided detailed treatments of C. herbarum (de Vries 1952, Ellis 1971, Domsch et al. 1980, Prasil & de Hoog 1988, and there are literally thousands of records of this species in the literature. McKemy & Morgan-Jones (1991) and Ho et al. (1999) examined C. herbarum in culture and published detailed descriptions of its features in vitro.
Cladosporium macrocarpum Preuss, a second component within the herbarum complex, has hitherto been known and treated as an allied, but morphologically distinct species on the basis of its wider and somewhat larger, frequently 2-3-septate, more regularly verrucose conidia, shorter conidial chains and more pronounced prolongations of the conidiophores. Dugan & Roberts (1994) carried out examinations of morphological and reproductive aspects of both species, and in so doing demonstrated a morphological continuum between C. macrocarpum and C. herbarum, concluding that the name herbarum should have preference. Therefore, Ho et al. (1999) introduced the new combination C. herbarum var. macrocarpum (Preuss) M.H.M. Ho & Dugan. Although transitional forms have been discussed to occur between the two species, several authors still prefer to retain C. macrocarpum as a separate species.
In an attempt to elucidate the species within the C. herbarum complex, therefore, a multilocus DNA sequence typing approach was used, employing five genes, namely the internal transcribed spacers of the rDNA genes (ITS), actin, calmodulin, translation elongation factor 1-α, and histone H3. These data were supplemented with morphological examinations under standardised conditions, using light and scanning electron microscopy, as well as cultural characteristics and growth studies.

Isolates
Isolates included in this study were obtained from the culture collection of the Centraalbureau voor Schimmelcultures (CBS), Utrecht, Netherlands, or were freshly isolated from a range of different substrates. Single-conidial and ascospore isolates were obtained using the techniques as explained in Crous (1998) for species of Mycosphaerella Johanson and its anamorphs. Isolates were inoculated onto 2 % potato-dextrose agar (PDA), synthetic nutrient-poor agar (SNA), 2 % malt extract agar (MEA) and oatmeal agar (OA) (Gams et al. 2007), and incubated under continuous near-ultraviolet light at 25 °C to promote sporulation. All cultures obtained in this study are maintained in the culture collection of the CBS (Table 1). Nomenclatural novelties and descriptions were deposited in MycoBank (www.MycoBank.org).

DNA isolation, amplification and sequence analysis
Fungal colonies were established on agar plates, and genomic DNA was isolated as described in Gams et al. (2007). Partial gene sequences were determined as described by Crous et al. (2006) for actin (ACT), calmodulin (CAL), translation elongation factor 1alpha (EF), histone H3 (HIS) and part (ITS) of the nuclear rDNA operon spanning the 3' end of the 18S rRNA gene (SSU), the first internal transcribed spacer, the 5.8S rRNA gene, the second internal transcribed spacer and the 5' end of the 28S rRNA gene (LSU). The nucleotide sequences were generated using both PCR primers to ensure good quality sequences over the entire length of the amplicon. Subsequent sequence alignment and phylogenetic analysis followed the methods of Crous et al. (2006). Gaps longer than 10 bases were coded as single events for the phylogenetic analyses; the remaining gaps were treated as new character states. Sequence data were deposited in GenBank (Table 1) and the alignment and tree in TreeBASE (www.treebase.org).

data analysis
The number of entities in the dataset of 79 strains was inferred with Structure v. 2.2 software (Pritchard et al. 2000, Falush et al. 2003 using an UPGMA tree of data of the ACT gene compared with CAL, EF and HIS with the exclusion of the nearly invariant ITS region. For this analysis group indications were derived from a tree produced with MrAic (Nylander 2004). The length of the burn-in period was set to 1 000 000, number of MCMC repeats after burn-in 10 000, with admixture ancestry and allele frequencies correlated models, assuming that all groups diverged from a recent ancestral population and that allele frequencies are due to drift. Uniform prior for ALPHA was set to 1.0 (default) and allele frequencies with λ set to 1.0 (default). The numbers of MCMC repetitions after burn-in were set as 10 000 and 100 000. The number of clusters (K) in Structure was assumed from 5 to 7. Population differentiation F ST (index: θ) was calculated with 1-6 runs using the same software. The null hypothesis for this analysis is no population differentiation. When observed theta (θ) is significantly different from those of random data sets (p < 0.05), population differentiation is considered.
Association of multilocus genotypes was screened with the multilocus option in bionuMericS v. 4.5. To test for reproductive mode in each population, the standardised index of association (I S A ; Haubold et al. 1998) was calculated with StArt2 software (Jolley et al. 2001). The null hypothesis for this analysis is complete panmixia. The values of I S A were compared between observed and randomised datasets. The hypothesis would be rejected when p < 0.05. Mean genetic diversity (H) and diversities of individual loci were calculated with liAn v. 3.5 (Haubold & Hudson 2000). Degrees of recombination or horizontal gene transfer were also visualised using SplitStree v. 4.8 software (Huson & Bryant 2006). Split decomposition was carried out with default settings, i.e., character transformation using uncorrected (observed, "P") distances, splits transformation using "equal angle", and optimise boxes iteration set to 2.

Morphology
As the present study represents the first in a series dealing with Cladosporium spp. and their Davidiella Crous & U. Braun teleomorphs in culture, a specific, standardised protocol was established by which all species complexes will be treated in future. Morphology of the anamorph: Microscopic observations were made from colonies cultivated for 7 d under continuous near-ultraviolet light at 25 °C on SNA. Preparations were mounted in Shear's solution (Gams et al. 2007). To study conidial development and branching patterns, squares of transparent adhesive tape (Titan Ultra Clear Tape, Conglom Inc., Toronto, Canada) were placed on conidiophores growing in the zone between the colony margin and 2 cm inwards, and mounted between two drops of Shear's solution under a glass coverslip. Different types of conidia are formed by Cladosporium species for which different terms need to be adopted. ramoconidia are conidia with usually more than one (mostly 2 or 3) conidial hilum, which typically accumulate at the tip of these conidia. Conidiogenous cells with more than one conidiogenous locus are first formed as apical parts of conidiophores. Such apical Cladosporium herbarum SpecieS coMplex parts of conidiophores are called ramoconidia if they secede at a septum from the conidiophore (Kirk et al. 2001). The septum at which the ramoconidium secedes often appears to be somewhat refractive or darkened. Ramoconidia are characterised by having a truncate, undifferentiated base (thus they lack a differentiated, coronate basal hilum formed in the context of conidiogenesis) and they can be very long, aseptate to sometimes multi-septate. Although they were formed initially as part of the conidiophore, they function as propagules. Only few of the species known until now have the ability to form true ramoconidia. secondary ramoconidia also have more than one distal conidial hilum but they always derive from a conidiogenous locus of an earlier formed cell, which can be either a conidiogenous cell or a ramoconidium. Secondary ramoconidia are often shorter but somewhat wider than ramoconidia; they are often septate, and typically have a narrowed base with a coronate hilum (Fig. 1). Conidia in Cladosporium are cells with a coronate basal hilum, which is formed in the context of conidiogenesis and with either a single (when formed as intercalary units in unbranched parts of chains) or without any distal conidial hilum (when formed at the tip of conidial chains). For the first, the term "intercalary conidium" and for the latter, "small terminal conidium" is used. Intercalary conidia typically are larger and more pigmented and have a more differentiated surface ornamentation than the small terminal conidia. In older literature true ramoconidia were often cited as "ramoconidia s. str.", whereas secondary ramoconidia have been referred to as "ramoconidia s. lat." Morphology of the teleomorph: Teleomorphs were induced by inoculating plates of 2 % tap water agar onto which autoclaved stem pieces of Urtica dioica (European stinging nettle) were placed. Inoculated plates were incubated on the laboratory bench for 7 d, after that period they were further incubated at 10 °C in the dark for 1-2 mo to stimulate teleomorph development. Wherever possible, 30 measurements (× 1 000 magnification) were made of conidia and ascospores, with the extremes of spore measurements given in parentheses. Cultural characteristics: Colonies were cultivated on PDA, MEA and OA plates for 14 d at 25 °C in the dark, after which the surface and reverse colours were rated using the charts of Rayner (1970). Linear growth was determined on MEA, PDA and OA plates by inoculating three plates per isolate for each medium, and incubating them for 14 d at 25 ºC, after that period colony diameters were determined.

Low-temperature scanning electron microscopy
Isolates of Cladosporium spp. were grown on SNA with 30 g agar/L for 3-4 d at room temperature under black light. Relevant parts of the small colonies with conidiophores and conidia were selected under a binocular, excised with a surgical blade as small agar (3 × 3 mm) blocks, and transferred to a copper cup for snap-freezing in nitrogen slush. Agar blocks were glued to the copper surface with frozen tissue medium (KP-Cryoblock, Klinipath, Duiven, Netherlands) mixed with 1 part colloidal graphite (Agar Scientific, Stansted, U.K.). Samples were examined in a JEOL 5600LV scanning electron microscope (JEOL, Tokyo, Japan) equipped with an Oxford CT1500 Cryostation for cryo-electron microscopy (cryoSEM). Electron micrographs were acquired from uncoated frozen samples, or after sputter-coating by means of a gold/ palladium target for 3 times during 30 s (Fig. 2). Micrographs of uncoated samples were taken at an acceleration voltage of 3 kV, and consisted out of 30 averaged fast scans (SCAN 2 mode), and at 5 kV in case of the coated samples (PHOTO mode).

Phylogeny and differentiation
The manually adjusted alignment contained 80 sequences (including the outgroup sequence) and the five loci were represented by a total of 1 516 characters including alignment gaps which were used in the analysis. Of the 1 516 characters, 369 were parsimonyinformative, 259 were variable and parsimony-uninformative, and 888 were constant.
Forty equally most parsimonious trees (TL = 1 933 steps; CI = 0.569; RI = 0.786; RC = 0.447), one of which is shown in Fig.  3, were obtained from the parsimony analysis of the combined genes. Neighbour-joining analysis using three substitution models (uncorrected "p", Kimura 2-parameter and HKY85) on the sequence data yielded trees with identical topologies. These differed from the tree presented in Fig. 3 with regard to the placement of C. macrocarpum strain CPC 12054 which was placed as a sister branch to the C. bruhnei Linder clade in the distance analyses (results not shown) because it shares an identical CAL sequence. All cryptic species consisting of multiple strains are clustering in well-supported clades with bootstrap support values ranging from 71 % (C. herbarum) to 100 % [e.g. C. ramotenellum K. Schub., Fig. 2. Terms used to describe conidium wall ornamentation under the cryo-electron microscope. Adapted from David (1997 Schub.]. The intraspecific variation in the C. bruhnei clade is due to genetic variation present in the sequence data of all loci except for ITS, those in the C. macrocarpum clade in all loci except for ITS and ACT, and those in the C. herbarum clade in all loci except for ITS and CAL (data not shown). However, none of the variation for these species could be linked to host specificity or morphological differences. In general, ITS data did not provide any resolution within the C. herbarum complex, whereas EF data provided species clades with very little intraspecific variation and ACT, CAL and HIS revealed increasing intraspecific variation (ACT the least and HIS the most).
The mean genetic diversity (H) of the entire data set excluding the nearly invariant ITS region was 0.9307, with little difference between genes (ACT = 0.9257, CAL = 0.9289, EF = 0.9322, HIS = 0.9361). The loci showed different numbers of alleles (ACT: 22,CAL: 16,EF: 21,HIS: 20,ITS: 6). Differentiation of entities when calculated with Structure software using the admixture/correlated model showed highest value with K = 6. At this value F ST varied between 0.1362 and 0.3381. Linkage disequilibrium calculated using the standardised index of association (I S A ) for the entire dataset (observed variance V o = 0.5602, expected variance V e = 0.2576) was 0.3914 (P = 0.0001), consistent with a small amount of recombination that did not destroy the linkage between alleles. Only few groups appeared to be separated for all alleles; degrees of gene flow are indicated in Fig. 4. SplitStree software produced unresolved star-shaped structures for all genes, without any sign of reticulation (Fig. 5).

Generic concept of the teleomorph
The introduction of the teleomorph genus Davidiella was mainly based on phylogenetic studies within the Mycosphaerellaceae (Braun et al. 2003), where it could be demonstrated that "Mycosphaerella" species with Cladosporium anamorphs formed a sister clade to Mycosphaerella (Crous et al. , 2001. Braun et al. (2003)

Cladosporium herbarum SpecieS coMplex
Cultural characteristics: Colonies on PDA reaching 22-32 mm diam after 14 d at 25 ºC, olivaceous-grey to iron-grey, sometimes whitish, smoke-grey to pale olivaceous due to abundant aerial mycelium covering almost the whole colony, with age collapsing becoming olivaceous-grey, occasionally zonate, velvety to floccose, margin narrow, entire edge, white, glabrous to somewhat feathery, aerial mycelium sparse to abundant, white, fluffy, growth regular, flat to low convex, sometimes forming few exudates in the colony centre, sporulating. Colonies on MEA reaching 21-32 mm diam after 14 d at 25 ºC, grey-olivaceous, olivaceous-grey to dull green or irongrey, sometimes whitish to pale smoke-grey due to abundant aerial mycelium, olivaceous-grey to iron-grey reverse, velvety, margin narrow, entire edge to slightly undulate, white, radially furrowed, glabrous to slightly feathery, aerial mycelium sparse to abundant, mainly in the centre, white, fluffy, growth convex to raised, radially furrowed, distinctly wrinkled in the colony centre, without prominent exudates, sporulating. Colonies on OA reaching 20-32 mm diam after 14 d at 25 ºC, smoke-grey, grey-olivaceous to olivaceous-grey, greenish black or iron-grey reverse, margin narrow, entire edge, colourless to white, glabrous, aerial mycelium sparse to abundant, dark smoke-grey, diffuse, high, later collapsed, felty, growth flat, without prominent exudates, sporulation profuse.  Notes: Cladosporium bruhnei proved to be an additional component of the herbarum complex. The species resembles C. herbarum s. str. as already stated by Linder (1947), but possesses consistently narrower conidia, usually 2.5-5 µm wide, and the conidiophores often form only a single apical swelling. The species was described by Bruhne (l.c.) as Hormodendrum hordei from Germany but type material could not be located. Linder (1947) examined No. 1481a-5 (Canada, N. Quebec, Sugluk, on Elymus arenarius var. villosus, 31 Jul. 1936, E. Meyer), presumably in the National Museum, and stated that this specimen agreed well with the description and illustration given by Bruhne (l.c.). Although the species occurs on numerous substrates and is widely distributed, it has not yet been recognised as a distinct species since it has probably been interpreted as a narrow variant of C. herbarum. Based on morphology and DNA sequence data, the CBS strain CBS 177.71 chosen by Prasil & de Hoog (1988) as representative living strain of C. herbarum, rather clusters together with isolates of C. bruhnei. The strain CBS 813.71 is an albino mutant of the latter species as it does not appear to contain colour pigment. Furthermore, all isolates from humans treated until now as C. herbarum proved to be conspecific with the narrow-spored C. bruhnei.
Cultural characteristics: Colonies on PDA reaching 19-37 mm diam after 14 d at 25 ºC, grey-olivaceous to olivaceous-grey, whitish to smoke-grey or pale olivaceous-grey due to abundant aerial mycelium, velvety, reverse olivaceous-grey or iron-grey, margin almost colourless, regular, entire edge, glabrous to feathery, aerial mycelium abundant mainly in the colony centre, dense, felty, woolly, sometimes becoming somewhat reddish brown, fawn coloured, growth regular, flat to low convex with an elevated colony centre, sometimes forming few large prominent exudates, sporulation profuse. Colonies on MEA reaching 17-37 mm diam after 14 d at 25 ºC, smoke-grey to pale olivaceous-grey towards margin, olivaceous-grey to iron-grey reverse, velvety, margin white, entire edge to slightly undulate, aerial mycelium abundant, dense, fluffy to felty, growth low convex or raised, radially furrowed, folded and wrinkled in the colony centre, without prominent exudates but sporulating. Colonies on OA reaching 12-28 mm diam after 14 d at 25 ºC, olivaceous-grey to iron-grey, due to abundant aerial mycelium pale olivaceous-grey, olivaceous-grey reverse, margin narrow, more or less undulate, white, aerial mycelium white, loose to dense, high, fluffy to felty, covering large parts of the colony, growth flat to low convex, without prominent exudates, sporulating. Substrate and distribution: On fading and decaying plant material, on living leaves (phylloplane fungus), as secondary invader, as an endophyte, isolated from air, soil, foodstuffs, paints, textiles and numerous other materials; cosmopolitan.
Notes: De Vries (1952) incorrectly selected a specimen of Link's herbarium at herb. B as lectotype. Prasil & de Hoog (1988) discussed this typification and designated one of Persoon's original specimens as lectotype in which C. herbarum could be recognised. The latter material, which is in poor condition, could be re-examined within the course of these investigations and showed conidia agreeing with the current species concept of C. herbarum being (6-)9.5-14.5(-21) × (5-) 6-7(-8) µm. Since the identity of the strain CBS 177.71 chosen by Prasil & de Hoog (1988) as representative living strain of C. herbarum could not be corroborated, an epitype with a living ex-epitype culture is designated. The holotype specimen of D. tassiana (RO) is morphologically similar to that observed on the epitype of C. herbarum, having ascospores which are (17-)21-23(-25) × (6-)7-8(-8.5) µm, turning brown and verruculose in asci with age. However, no hamathecial remnants were observed in ascomata in vivo.
The connection to the teleomorph D. tassiana could be confirmed, which is in agreement with the findings of von Arx (1950) and Barr (1958). Ascospore isolates formed the typical C. herbarum anamorph in culture, and these anamorph cultures developed some immature fruiting bodies within the agar. When inoculated onto water agar plates with nettle stems, numerous ascomata with viable ascospores were formed in culture. For additional synonyms see Dugan et al. (2004).   Mycelium branched, 2-8 µm wide, septate, not constricted at the septa, hyaline to pale brown, smooth, walls slightly thickened, sometimes guttulate. Conidiophores very long, usually terminally arising from ascending hyphae, erect to subdecumbent, slightly to distinctly flexuous, geniculate-sinuous, usually several times, subnodulose due to geniculate, sympodial proliferation forming swollen lateral shoulders, unbranched, rarely branched, up to 720 µm long, 6-11 µm wide, swellings 8-11(-14) µm wide, pluriseptate, often very regularly septate, not constricted at the septa, pale to medium olivaceous-brown, somewhat paler towards the apex, smooth to minutely verruculose, walls only slightly thickened. Conidiogenous cells integrated, terminal as well as intercalary, cylindrical-oblong, 15-55 µm long, proliferation percurrent to sympodial, usually with a single geniculation forming laterally swollen shoulders often below a septum, conidiogenous loci confined to swellings, usually one locus per swelling, rarely two, protuberant, (2-)2.5-4 µm diam, somewhat thickened and darkened-refractive. Conidia solitary, sometimes in short, unbranched chains, straight to curved, young conidia pyriform to subcylindrical, connection between conidiophore and conidium being rather broad, subhyaline to pale olivaceous-brown, walls slightly thickened, then enlarging and becoming more thick-walled, cylindrical-oblong, soleiform with age, both ends rounded, usually with a slightly to distinctly bulbous base, visible from a very early stage, but broadest part often towards the apex not at the base, (18-) 30-75(-87) × (7-)10-16(-18) µm [av. ± SD, 53.3 (± 17.8) × 12.6 (± 2.2) µm], (0-)2-6(-7)-septate, usually not constricted at the septa, rarely slightly constricted, septa often becoming sinuous with age, pale to medium olivaceous-brown, sometimes darker, verrucose to echinulate, walls thickened, especially in older conidia, up to 1 µm thick, hila protuberant, often stalk-like or conically prolonged, up to 2 µm long, (2-)2.5-3.5(-4) µm diam, with age becoming more sessile, sometimes just visible as a thickened plate just below the outer wall layer, especially in distal scars of branched conidia, periclinal rim often distinctly visible, hila somewhat thickened and darkened-refractive; microcyclic conidiogenesis not observed. Fig. 19. Cladosporium herbarum (CPC 11600). A. Overview of hyphal growth and conidiophore formation of a colony on SNA. Conidiophores are often formed on very wide (approx. 10 μm), septate hyphae that often grow near the agar surface. B. A more detailed view on colony organisation reveals the ornamented conidia. Note the septum near the conidiophore (arrow). C. Detail of spore ornamentation and hila on a secondary ramoconidium (arrow). Ornamentation is visible during early stages of spore formation (arrow). D. Structure of the conidiophore, illustrating the complex morphology of the spore-forming apparatus. In addition, secondary ramoconidia, conidia, and a hilum on the conidium are visible. E. Complex structure of the spore-forming apparatus. F. Details of secondary ramoconidia with complex scar-pattern on the right cell. G. Details of a secondary ramoconidium giving rise to conidia. Note the lack of ornamentation at the location of spore formation. Scale bars: A = 50 µm, B, F = 10 µm, C-E, G = 5 µm.
Cultural characteristics: Colonies on PDA reaching 30-43 mm in diam after 14 d at 25 ºC, dark dull green to olivaceous-grey, olivaceous-grey, dark olivaceous-to iron-grey reverse, pulvinate, velvety, sometimes somewhat zonate, paler zones towards the margin, margin regular, entire edge, almost colourless to white, glabrous to feathery, aerial mycelium sparse to more abundant in the colony centre or covering large areas of the colony, hairy, fluffy or felty, whitish to smoke-grey, sometimes becoming reddish, livid red to vinaceous, growth flat, regular, sometimes forming few prominent exudates, exudates sometimes slightly reddish, sporulation profuse with two kinds of conidiophores, low and high. Colonies on MEA reaching 31-50 mm in diam after 14 d at 25 ºC, grey-olivaceous to olivaceous-grey or iron-grey, sometimes pale olivaceous-grey to whitish due to abundant aerial mycelium, olivaceous-grey or iron-grey reverse, velvety or powdery, margin narrow, entire edge, colourless to white, glabrous, aerial mycelium sparse to abundant, hairy or felty, growth regular, flat to low convex, radially furrowed, without prominent exudates, sporulation profuse. Colonies on OA reaching 29-40 mm in diam after 14 d at 25 ºC, grey-olivaceous, olivaceous-grey to dark smoke-grey, olivaceousblack or iron grey reverse, margin entire edge, narrow, colourless or white, glabrous, aerial mycelium sparse, mainly in the colony centre, felty, white to smoke-grey or grey-olivaceous, felty, growth flat, regular, without exudates, sporulating. Substrate and distribution: Decaying plant material, human, hypersaline water, water; widespread.

Cladosporium graminum, described by Persoon
A characteristic difference between ascomata of C. macrocarpum in comparison to those of C. herbarum, are the smaller, globose pseudothecia, asci with longer stalks, prominence of pseudoparaphyses, and rather inconspicuous luminar ascospore inclusions.
Notes: Type material of Napicladium ossifragi is not preserved in Rostrup's herbarium (on Narthecium ossifragum, Faeroe Islands, Viderö, Viderejde and Österö, Svinaa, sine dato, leg. Ostenfeld & Harz). However, other authentic collections seen and examined by Rostrup are deposited at CP. Lind (1913) re-examined these samples, synonymised N. ossifragi with H. magnusianum and correctly introduced the combination H. ossifragi. Nevertheless, the correct oldest name for this fungus has been ignored by most authors. David (1997), who clearly stated that N. ossifragi is the earliest name for this species, preferred to use the name C. magnusianum because the typification of Rostrup's name was still uncertain. Despite the lacking type material, there is no doubt about the correct identity of N. ossifragi since authentic material of this species, examined by and deposited in Rostrup's herbarium (CP), is preserved. Therefore, there is no reason to reject the oldest valid name for this species. The original collection of C. magnusianum cited by Jaap (1902) (on leaves of Narthecium ossifragum, Denmark, Tǿnder, Rǿmǿ, peatbog by Twismark, Jul.-Aug. 1901, Jaap), but not designated as type, is not preserved (David 1997). It is neither deposited at B, HBG nor S. However, in the protologue Jaap (1902) also referred to material of this species found near Hamburg, which is, hence, syntype material available for lectotypification. Mycelium sparingly branched, 2-7 µm wide, septate, not constricted at the septa, subhyaline to pale brown, smooth or almost so, walls somewhat thickened, guttulate or protoplasm appearing granular, sometimes enveloped by a slime coat. Conidiophores arising mostly terminally from ascending hyphae, sometimes also laterally from plagiotropous hyphae, erect, more or less straight, broadly cylindrical-oblong, once or several times slightly to distinctly geniculate-sinuous, forming more or less pronounced lateral shoulders, nodulose, unbranched, 100-320(-500) × 7-11 µm, swellings 10-14 µm wide, becoming narrower and paler towards the apex, septate, not constricted at the septa, septa mainly basal, apical cell often very long, pale to medium olivaceous-brown, subhyaline at the apex, smooth or almost so, sometimes minutely verruculose, walls usually distinctly thickened, sometimes even two-layered, up to 1(-2) µm thick, protoplasm granular, often clearly contrasting from the outer wall. Conidiogenous cells integrated, terminal and intercalary, cylindrical-oblong, slightly to distinctly geniculatesinuous, nodulose with conidiogenous loci confined to swellings or lateral shoulders, 30-110 µm long, proliferation percurrent to sympodial, with a single or three, sometimes up to five geniculations per cell, usually only a single locus per swelling, protuberant, very prominent, short cylindrical, peg-like, clearly composed of a dome and surrounding rim, dome often higher than the periclinal rim, broad, somewhat paler than rim, conically narrowed, (2-)2.5-4 µm wide, up to 2 µm high, thickened and darkened-refractive.  Notes: Cladosporium pseudiridis closely resembles C. iridis, a common and widespread species causing leaf spots on numerous Iris spp. and a few additional hosts of the host family Iridaceae, but the latter species is easily distinguishable by having longer and narrower, more frequently septate conidia, (18-)30-75(-87) × (7-)10-16(-18) µm, (0-)2-6(-7)-septate.
Cultural characteristics: Colonies on PDA attaining 20 mm diam after 14 d at 25 ºC, pale olivaceous-grey due to abundant aerial mycelium, olivaceous-grey towards margins, iron-grey to olivaceous-black reverse, margin regular, entire edge, aerial mycelium abundant, cottony, dense, high, growth regular, low convex, radially furrowed in the centre, growing deep into the agar, with age numerous small to large prominent exudates, sporulation sparse. Colonies on MEA attaining 16 mm diam after 14 d at 25 ºC, white to pale smoke-grey, fawn reverse, velvety, margin undulate, glabrous, aerial mycelium abundant, dense, high, fluffy, growth raised with elevated colony centre, laterally furrowed, without prominent exudates. Colonies on OA attaining 18 mm diam after 14 d at 25 ºC, olivaceous, white to pale olivaceous-grey in the centre due to abundant aerial mycelium, olivaceous-grey reverse, margin white, entire edge, glabrous, aerial mycelium loose to dense, high, fluffy to felty, growth flat to low convex, regular, without prominent exudates, sporulating. Notes: This new species is well characterised by its slightly to distinctly geniculate-sinuous, often zigzag-like conidiophores and its conidia formed solitary or rarely in short unbranched chains and is therefore morphologically not comparable with any of the species described until now. Most Cladosporium species with conidia usually formed solitary or in short unbranched chains have previously been treated as species of the genus Heterosporium Klotzsch ex Cooke, now considered to be synonymous with Cladosporium. All of them, including the newly introduced C. arthropodii K. Schub. & C.F. Hill from New Zealand, which also belongs to this species complex , possess very large and wide, often pluriseptate conidia quite distinct from those of C. sinuosum (David 1997). Cladosporium alopecuri (Ellis & Everh.) U. Braun, known from the U.S.A. on Alopecurus geniculatus is also quite different by having larger and wider conidia, 20-40 × 7-13(-15) µm, and wider conidiogenous loci and conidial hila, 3.5-5 µm diam (Braun 2000).
Cladosporium herbarum is superficially similar but the conidiophores of the latter species are sometimes only slightly geniculate-sinuous but never zigzag-like and the verruculose to verrucose conidia are frequently formed in unbranched or branched chains.  Etymology: Refers to its nodulose conidiophores.
Cultural characteristics: Colonies on PDA attaining 24 mm diam after 14 d at 25 °C, grey-olivaceous to olivaceous, olivaceousgrey, iron-grey or olivaceous-black reverse, velvety, margin regular, entire edge, white or pale greenish olivaceous, glabrous to feathery, aerial mycelium sparse, only few areas with abundant Cladosporium herbarum SpecieS coMplex mycelium, diffuse, growth regular, flat or with a raised and wrinkled colony centre, radially furrowed, effuse, usually without prominent exudates, with age several exudates formed, sporulation profuse, colonies consisting of two kinds of conidiophores, short and a few longer ones. Colonies on MEA reaching 25 mm diam after 14 d at 25 °C, greenish olivaceous to grey-olivaceous in the centre, olivaceous-grey to iron-grey reverse, velvety, margin entire edge, crenate or umbonate, narrow, pale greenish olivaceous, sometimes radially furrowed, aerial mycelium absent or sparse, growth low convex with distinctly wrinkled colony centre, without prominent exudates, abundantly sporulating. Colonies on OA attaining 25 mm diam after 14 d at 25 °C, dark grey-olivaceous to olivaceous due to profuse sporulation, iron-grey reverse, sometimes releasing some olivaceous-buff pigments into the agar, velvety, margin regular, entire edge or crenate, narrow, colourless or white, glabrous or feathery, aerial mycelium sparse, growth flat with slightly raised colony centre, prominent exudates lacking, sporulation profuse. Notes: Cladosporium cladosporioides is morphologically comparable with the new species but deviates in having usually smooth conidiophores and conidia, with the conidia being mainly aseptate. C. subtilissimum is represented by three isolates of different origins  and substrates. Besides these strains, several additional isolates listed under excluded strains are morphologically indistinguishable from C. subtilissimum in culture, but genetically different, clustering in various subclades. They are indicated as Cladosporium sp. in the tree (Fig. 3).
Notes: Cladosporium subtilissimum and C. cladosporioides are morphologically comparable with the new species C. tenellum, but C. cladosporioides deviates in having usually smooth conidiophores and conidia with only few conidiogenous loci and conidial hila crowded at the apex and somewhat wider conidiophores, 3-5(-6) µm; and in C. subtilissimum the small terminal conidia are not globose but rather narrowly obovoid to limoniform, the conidiogenous loci and conidial hila are somewhat wider, (0.5-)0. 8-2(-2.2) µm, and at the apices of conidiophores and conidia only few scars are formed.
Cladosporium variabile (Cooke) G.A. de Vries, Contr. Knowl. Genus Cladosporium: 85. 1952 (CPC 12753). A. Survey of hyphae that grow on the agar surface. Some of the fungal cells have a swollen appearance and could develop into a "foot cell" that gives rise to a conidiophore. B. A number of aerial hyphae obstruct the swollen, large structures on the agar surface, which give rise to conidiophores. Some of them appear ornamented. C. A series of conidia formed on a conidiophore (bottom of the micrograph). D. Detail of the ornamented conidia. The ornamentations are isolated and dispersed. Note also the ornamentation-free scar zone and the hilum of the left cell. E. Two conidia behind an aerial hypha. F. Two conidiophores forming secondary ramoconidia. Note the bulbous shape of the spore-forming apparatus. This micrograph is from an uncoated sample. Scale bars: A-C, F = 10 µm, D = 2 µm, E = 5 µm. Ascomata pseudothecial, black, superficial, situated on a small stroma, globose, up to 250 µm diam, with 1-3 ostiolate necks; ostioles periphysate, with apical periphysoids present; wall consisting of 3-6 layers of dark brown textura angularis, textura epidermoidea in surface view. Asci fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoid, straight to slightly curved, 8-spored, 70-95 × 18-28 µm; with pseudoparenchymatal cells of the hamathecium persistent. Ascospores tri-to multiseriate, overlapping, hyaline, with irregular lumina, thick-walled, straight to slightly curved, fusoid-ellipsoidal with obtuse ends, widest near the middle of the apical cell, medianly 1-septate, not to slightly constricted at the septum, at times developing a second septum in each cell, several ascospores with persistent, irregular mucoid sheath, (22-)26-30(-35) × (7-)7.5-8(-9) µm.
This leaf-spotting fungus superficially resembles C. macrocarpum, but besides its pathogenicity to Spinacia, C. variabile differs from the latter species in having distinctly larger and more frequently septate conidia on the natural host, forming twisted and spirally coiled aerial mycelium in culture and in having lower growth rates in culture (29 mm after 14 d on PDA versus 38 mm on average in C. macrocarpum). Furthermore, the conidial septa of C. variabile are often distinctly darkened, become sinuous with age and the apex and base of the conidia often appear to be distinctly darkened. A Davidiella teleomorph has not previously been reported for this species.

The cladosporioides complex
This species complex will be treated in an additional paper in this series, dealing with the epitypification of this common and widespread species, and with numerous isolates identified and deposited as C. cladosporioides.

dIscussIon
In the present study, a multilocus genealogy supported by light and SEM microscopy, and cultural characteristics was used to redefine species borders within Cladosporium, especially within the C. herbarum complex. Most of the diagnostic features used for species delimitation on host material (Heuchert et al. 2005, Schubert 2005), proved to be applicable in culture. However, morphological features were often more pronounced in vivo than in vitro. For instance, conidiophore arrangement is not applicable to cultures, conidiophore and conidium widths were often narrower in culture than on the natural host, and macro-as well as microconidiophores were often observed in culture, but not on host material. All species belonging to the C. herbarum complex are characterised by possessing conidia which are ornamentated, the ornamentation ranging from minutely verruculose to verrucose, echinulate or spiny whereas in the C. sphaerospermum complex species with both smooth-walled as well as ornamented conidia are included (Zalar et al. 2007). The surface ornamentation varies based on the length of surface protuberances and in the density of ornamentation. Furthermore, the conidia are mainly catenate, formed in unbranched or branched chains. However, species previously referred to the genus Heterosporium, which usually produce solitary conidia or unbranched chains of two or three conidia at the most on the natural host, also belong to this species complex (e.g., C. iridis). In vitro these chains can become longer and may even be branched. The conidiophores formed in culture are mostly macro-but may also be micronematous, sometimes forming different types of conidia that vary in shape and size from each other. Most of the species possess nodulose conidiophores with the conidiogenesis confined to the usually lateral swellings. However, this phenetic trend is not consistently expressed in all of the species belonging to the C. herbarum complex. The various Cladosporium species within the C. herbarum complex were observed to have subtle differences in their phenotype which were visible via cryo-electron microscopy (cryoSEM), and are discussed below. Fungal colonies: CryoSEM provides the opportunity to study the organisation of the fungal colony at relatively low magnifications. Cladosporium tenellum proved to be the only fungus able to form aerial hyphal strands under the conditions studied. Cladosporium variabile formed abundant aerial hyphae, but in C. spinulosum these were sparse, and only conidiophores were observed on the agar surface. Three-day-old colonies of C. subinflatum formed numerous, long aerial hyphae, and no conidiophores could be discerned under the binocular. After 11 d the aerial hyphae seemed to have disappeared, giving rise to conidiophores. Cladosporium antarcticum, C. variabile and C. ramotenellum showed very large, swollen (> 10 μm) cells which gave rise to conidiophores. With C. variabile possible earlier stages of these cells were visible (Fig. 48), which gave rise to conidiophores. More than one conidiophore could be formed on such a structure (C. variabile and C. ramotenellum). Cladosporium herbarum has very wide hyphae on the agar surface, which gave rise to conidiophores as lateral branches. These wide hyphae were observed to anastomose, which may provide a firm interconnected supporting mycelium for these conidiophores. In C. herbaroides these wide hyphae could also be discerned, but conidiophore formation was less obvious. Similarily, C. tenellum has wide, parallel hyphae that gave rise to conidiophores.
These observations reveal fungal structures in Cladosporium that have not previously been reported on, and that raise intriguing biological questions. For instance, why are hyphal strands observed in some species (C. tenellum), and not in others, and what happens to the aerial hyphae during incubation in some species such as C. subinflatum? Furthermore, these preliminary results suggest that CryoSEM provide additional features that can be used to distinguish the different species in the C. herbarum complex. Fine details of morphological stuctures: CryoSEM provides the opportunity to study fine details of the conidiophore, (ramo)conidia and scars. Samples can be studied at magnification up to × 8 000, revealing details at a refinement far above what is possible under the light microscope (LM) (Fig. 2). However, the LM micrographs provide information about the different compartments of ramoconidia, as well as the thickness and pigmentation of the cell wall of different structures. With other words, the different techniques are complementary, and both reveal fungal details that build up the picture that defines a fungal species.
Conidiophores can vary with respect to their width and the length. Cladosporium ramotenellum, C. antarcticum and C. variabile have tapered conidiophores formed on large globoid "foot cells". The conidiophore itself can be branched. Cladosporium spinulosum has conidiophores that rise from the agar surface, but can have a common point of origin. These conidiophores are not tapered, but parallel and slender. The conidiophores of C. bruhnei and C. herbaroides are rather long, and can appear as aerial hyphae.
An important feature of the conidiophore is the location were the conidia are formed. Conidiophore ends can be simple and tubular, or rounded to more complex, several times geniculate, with several scars. Conidiophore ends become more elaborate over time.
Cladosporium spinulosum and C. tenellum have nearly tubular conidiophore ends, with often very closely aggregated scars. The conidiophore ends of C. subinflatum are also near tubular with a hint of bulbousness. Cladosporium subtilissimum is similar, but with somewhat more elevated scars that look denticulate. Cladosporium variabile has nodulose, somewhat swollen apices with often sessile, almost inconspicuous scars. In the case of C. macrocarpum, these structures are also nodulose to nodose and somewhat bent, with only slightly protuberant loci. Cladosporium ramotenellum has tubular conidiophore ends with pronounced scars. Cladosporium antarcticum has very characteristic, tapered ends, and widely dispersed (5 μm) scars. More complex conidiophore ends are more irregular in shape, and have scars dispersed over a longer distance, such as observed in C. bruhnei, C. herbaroides, and C. herbarum.
Secondary ramoconidia are usually the first conidia formed on a conidiophore. They are often multicellular, and have one basal cladosporioid hilum, and more at the apex. Few Cladosporium species additionally form true ramoconidia representing apical parts of the conidiophore which secede at a septum resulting in an undifferentiated non-coronate base and function as conidia. Ramification of conidial chains is realised through these conidia. They can occur in up to three stages, which results in elaborated spore structures. The basal secondary ramoconidium is invariably the largest, and cell size decreases through a series of additional secondary ramoconidia, intercalary conidia, and small, terminal conidia. The elongation of secondary ramoconidia varies among the different species. Cladosporium macrocarpum has broadly ellipsoid to cylindrical secondary ramoconidia usually with broadly rounded ends, like C. variabile, while C. spinulosum has secondary ramoconidia that can often hardly be discerned from the conidia that are formed at later stages. The conidia of the other species roughly fall between these species. The most notable structures on these conidia are their ornamentation, scar pattern and morphology. Cladosporium spinulosum forms numerous globose to subsphaerical spores with digitate, non-tapered surface ornamentation, which is unique for all the species discussed here. In his study on Cladosporium wall ornamentation, David (1997) recognised three classes of echinulate surfaces (aculeate, spinulose, digitate), and five classes of verrucose surfaces (muricate, granulate, colliculate, pustulate and pedicellate) (Fig. 2). The ornamentation particles vary in shape, width, height and density. The most strongly ornamented conidia of the species examined by SEM are formed by C. ossifragi, with the ornamentation both large (up to 0.5 μm wide) and high, and can be regarded as densely muricately ornamented. Strong ornamentation is also seen in C. herbaroides, which is mostly granulate. Cladosporium tenellum (with muricate, granulate and colliculate tendencies) and C. bruhnei (mostly granulate with some muricate projections) have relatively large ornamentation structures with slightly more space between the units than the other two species. Cladosporium antarcticum, C. ramotenellum, C. variabile and C. subtilissimum exhibit rather large granulate ornamentations that have a more irregular and variable shape. Cladosporium subinflatum shows the widest dispersed structures of the series, being muricate. In contrast, C. macrocarpum has a very neat and regular pattern of muricate ornamentation. The area of formation of new spores on conidia is invariably not ornamented, and hila all have the typical Cladosporium morphology with a central dome and a ring-like structure around it. Branching patterns: Spores usually show a "line of weakness" between them where the coronate scars form. It seems that scars at both sides of the line of weakness have the central dome structure, which appears to play a major role in the effective mechanism Cladosporium employs for spore dispersal, with the dome actively pushing the conidia apart. This mechanism is also illustrated in David (1997, fig. 2E). Indeed, conidia of Cladosporium are very easily dislodged; even snap freezing or the electrical forces inside the SEM often result in dislodgement of the spores in a powdery "wave". It is no surprise, therefore, that Cladosporium conidia are to be found in most air samples. In Cladosporium, conidia are mostly formed in chains, with the size invariably decreasing from the base to the apex of the row. Upon formation each conidium is separated from the conidiophore, or previously formed conidium, and hence from its nutrients. The basal ramoconidium or secondary ramoconidia have the nutrients and metabolic power to produce Cladosporium herbarum SpecieS coMplex a number of additional secondary ramoconidia that in turn could produce a chain of intercalary conidia, and finally, some small, single-celled, terminal conidia. Further research is still necessary to determine if specific branching patterns can be linked to different species.
A surprising finding from the present study is the huge diversity in species and genotypes that exist in nature, be it in the indoor environment, on fruit surfaces, or in extreme ecological niches such as salterns, etc. It is clear that detailed studies would be required to find and characterise other species of Cladosporium and obtain a better understanding of their host ranges and ecology. A further surprise lay in the fact that several of these species are capable of sexual reproduction, and readily form Davidiella teleomorphs in culture. The Davidiella states induced here were all from homothallic species. Further attention now needs to be given to elucidating teleomorphs from other species which, as in Mycosphaerella , Ware et al. 2007) could be heterothallic, and experiencing clandestine sex.
Despite the occurrence of many different genotypes in variable genes, the degree of diversity in the entire data set was low. For the majority of the species ITS was almost invariant, with only six genotypes in the entire dataset. This suggests a very recent evolution. The standardised index of association (I S A ) was high (0.3914), indicating an overabundance of clonality and / or inbreeding, the latter possibly matching with observed homothallism of Davidiella teleomorphs. Clonality was visualised with SplitStree software, where star-shaped representations without any sign of reticulation were obtained for all genes, though at different branch lengths (Fig. 5). With Structure software an optimal subdivision was achieved at six putative groups. Some of them were distinctly separated, yielding a theta (θ) around 0.14, but in most cases there was considerable overlap in representation of motifs, with θ at significantly higher values. Results are difficult to interpret due to the small size of the data set compared to the number of predicted groups, and due to unknown but probably large sampling effects. With optimal subdivision of the 79 strains at a hypothesised value of K = 6 ( Fig. 4), still a large degree of inter-group similarity was noted, as was the case at any other level of K. This was particularly obvious when data from the most variable genes (EF and ACT) are superimposed (Fig. 4). The ACT groups are further subdivided by EF data, but in many cases the same EF motif (indicated with arrows) was encountered in different (multilocus) species, for example in C. antarcticum, C. spinulosum, Davidiella sp., and the various clusters comprising Cladosporium strains which are phenotypically almost indistinguishable but genetically distinct from C. subtilissimum. A similar situation was found with the distribution of EF genotypes (indicated with doughnuts) in C. herbarum and C. macrocarpum. Nevertheless, the data set showed significant structuring, partly correlating with geography, e.g. the EF-determined cluster of C. bruhnei that contained isolates from different sources in The Netherlands. Differences may be over-accentuated by known sampling effects, particularly in C. herbarum and C. macrocarpum, where single-spore isolates from a single collection are included.
Taken together the data suggest a recent, preponderantly clonal evolution, combined with limited natural selection at a low level of evolutionary pressure. As a result, many genotypes produced by hot spots in the genes analysed have survived, leading to nearly random variation in the data set. Many combinations of motifs that possibly could emerge have maintained in the course of time due to the absence of recombination. This indicates that the observed structure is that of populations within a single species, and consequently a distinction of clonal "species" could be redundant. This conclusion is underlined by the fact that a single source in a single location can be colonised by various genotypes, such as grapes in the U.S.A. containing three different, closely related genotypes. However, the phenomenon of co-inhabitation by different Mycosphaerella species on the same lesion of Eucalyptus has been described before (Crous 1998, Crous et al. 2004 and it is therefore not surprising that different genotypes occurring close together are also observed for the related genus Cladosporium. There is no obvious ecological difference between genotypes, and hence isolates seem to have equal fitness. However, in general we noticed a remarkable concordance of genetic and phenetic characters. The morphological study was done prior to sequencing, and nearly all morphotypes clustered in separate molecular entities. There are some exceptions, such as with C. antarcticum with striking morphology that was almost identical on the molecular level to Cladosporium spp. that resemble C. subtilissimum and would normally have been interpreted to be a mutant. Conversely, nearly all genetically distinguishable groups proved to be morphologically different, with the exception of members of the C. subtilissimum s. lat. complex (indicated as Cladosporium sp. in Fig. 3 and Table 1). The possibility remains that the found genetic parameters correlate with phenetic markers other than morphology, such as virulence, toxins or antifungal susceptibilities. For this reason we introduce the established entities here as formal species. They can be diagnosed by ACT sequencing or by phenetic characters provided in the key. For simple routine purposes, however, they can be seen and treated as the "C. herbarum complex", based on their close phylogenetic relationships.

AcKnoWledGeMenTs
Several colleagues from different countries provided material and valuable cultures without which this work would not have been possible. In this regard, we thank particularly Alina G. Greslebin (CIEFAP, Esquel, Chubut, Argentina), Frank Dugan and Lindsey DuToit (Washington State Univ., U.S.A.), and Annette Ramaley (Colorado, U.S.A.). Konstanze Schubert was financially supported by a Synthesys grant (No. 2559) which is gratefully acknowledged. We thank Marjan Vermaas for preparing the photographic plates, Rianne van Dijken for the growth studies, and Arien van Iperen for maintaining the cultures studied. Bert Gerrits van den Ende is acknowledged for assisting with the molecular data analyses.