Alignments and phylogenetic analyses

Sequences were assembled and edited using SeqMan II software (DNAStar, Inc., Madison, Wis.). Manual alignments were performed using MacClade v. 4.08 (Maddison & Maddison 2003). Ambiguous regions (sensu Lutzoni et al. 2000) and introns were delimited manually and excluded from the alignments. Congruence was tested using a 70 % reciprocal bootstrap criterion (Mason-Gamer & Kellogg 1996, Reeb et al. 2004). Final phylogenetic analyses of the two-gene and one-gene datasets were performed using Stamatakis's “randomised axelerated (sic) maximum likelihood for high performance computing” (RAxML VI-HPC, Stamatakis et al. 2005, 2008) on the Cipres Web Portal (http://www.phylo.org/sub_sections/portal/). For the two-gene analysis, the maximum likelihood search followed a “GTRMIX” model of molecular evolution applied to two partitions, nucLSU and nucSSU. The same model was applied to the one-gene analysis without partition. Support values were obtained in RAxML with bootstrap analyses of 500 pseudoreplicates. The trees are labeled with the updated scientific names.

Phylogenetic inference

As shown in Fig. 1, the species of Acremonium mostly fall into three groups, namely the Hypocreales, the Plectosphaerellaceae, and the Sordariales. The bulk of species fall into the Hypocreales. Nine of the named Acremonium species in this analysis belong to the Plectosphaerellaceae. The Sordariales are represented in Fig. 1 only by Acremonium alabamense, the only named Acremonium species in Acremonium section Chaetomioidea. Outside these groups Acremonium atrogriseum, represented by numerous conspecific isolates, belongs to the family Cephalothecaceae (Fig. 1C), along with Albertiniella polyporicola and Cephalotheca sulfurea; this family is sister to the Coniochaetales. Another Acremonium species, A. thermophilum, falls into the Cephalothecaceae clade grouping with Albertiniella polyporicola. An isolate provisionally identified as Acremonium alternatum, CBS 109043 is a member of the Cephalothecaceae. The complex status of A. alternatum is discussed below.

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Fig. 1.

A–C. The phylogenetic position of Acremonium and related fungi within the Sordariomycetes, as seen in combined analysis of the large and small subunits of the nuclear ribosomal RNA gene (LSU + SSU) analysed by maximum likelihood via RAxML VI-HPC following a GTRMIX model applied to two partitions. 100 % bootstrap values are indicated by a black dot on the relevant internode.

The Acremonium species in the Hypocreales form an array of poorly to well distinguished clades, most of which do not correspond to previously recognised genera or suprageneric taxa. Included within the Hypocreales in the Sarocladium clade labeled the “strictum-clade” is the well known soil fungus long known as Acremonium strictum (Fig. 1A). The soil fungus and human opportunistic pathogen traditionally called A. kiliense is also included as is the maize corn endophyte known as A. zeae. The corresponding clade in Fig. 2C based on LSU reveals that this group of fungi includes the rice pathogen Sarocladium oryzae as a saltatory morphological apomorph. No teleomorphs are known to be associated with this group. This clade consists of fungi forming conidia in mucoid heads; it is closely related to a clade of species forming catenulate conidia, namely the Acremonium bacillisporum clade including A. bacillisporum, A. glaucum, A. implicatum pro parte, and, in a separate subclade, A. ochraceum (Figs ​(Figs1A, 1A, ​,2C).2C). The “bacillisporum-clade” and “strictum-clade” grouped together in an overall Sarocladium clade (Figs ​(Figs1, 1, ​,2).2). Two catenulate-conidial isolates labeled A. alternatum are also loosely associated with the A. bacillisporum clade in Fig. 2C. In Fig. 1A, one isolate CBS 406.66 is connected to the Sarocladium and A. breve/curvulum clades with a 96 % bootstrap value.

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Fig. 2.

A–E. The phylogenetic position of Acremonium and related fungi within the Hypocreales, as seen in nucLSU analysed by maximum likelihood via RAxML VI-HPC following a GTRMIX model applied to a single partition. 100 % bootstrap values are indicated by a black dot on the relevant internode.

Another major hypocrealean Acremonium clade in Fig. 1A contains A. breve, A. radiatum, A. gamsii and, more distantly, with 96 % bootstrap support, A. curvulum. In Fig. 2C where phylogenetic signal is lower, A. curvulum loses its tight association with A. breve and its relatives and appears in unsupported juxtaposition with the genus Trichothecium and the corresponding teleomorph genus, Leucosphaerina. The clade containing Trichothecium roseum and Leucosphaerina indica (see taxonomic comments below) also contains two anamorph species that were long placed in different genera based on conidiogenesis, namely, Acremonium crotocinigenum and Spicellum roseum, here recombined into Trichothecium.

The next clade in Fig. 1A is a loosely structured assemblage consisting of members of Acremonium subgenus Gliomastix, some of which are delineated below as members of a phylogenetically delineated genus Gliomastix, plus the teleomorphic genera Bionectria, linked to the well known penicillate hyphomycete anamorph genus Clonostachys (Schroers 2001), Hydropisphaera, and Roumegueriella. As Fig. 2B shows in more detail, the type species of the genus Gliomastix, originally named Gliomastix chartarum but currently called G. murorum, is in a relatively well supported clade (92 % bootstrap support) along with G. masseei, G. polychroma, and G. roseogrisa, three other species with melanised conidia that were placed in Acremonium subg. Gliomastix by Gams (1971). Related to Gliomastix are two clades of non-melanised Acremonium species placed in A. subg. Gliomastix, the A. persicinum clade, and A. pteridii clade. Smaller clades containing species in A. subg. Gliomastix such as A. biseptum, A. cerealis, A. luzulae, and A. rutilum (= A. roseum) are included in the large Gliomastix/Bionectria clade, which has 78 % bootstrap support. This clade includes additional teleomorphic fungi such as Heleococcum, Hydropisphaera, Nectriopsis, Ochronectria, Selinia, and Stephanonectria, along with the anamorph Sesquicillium microsporum.

The Gliomastix/Bionectria clade, the sclerotigenum/Geosmithia clade, and other members of the Bionectriaceae form a weakly supported clade with 74 % bootstrap value as shown in Fig. 1A. Included in the sclerotigenum/Geosmithia clade is the penicillate anamorph genus Geosmithia sensu stricto and the ex-type isolates of Acremonium pinkertoniae and A. sclerotigenum as well as the cephalosporin-producer Acremonium chrysogenum and its close relative, the thermophilic A. flavum. It also includes non-type isolates identified as A. blochii and A. egyptiacum. In the LSU-tree (Fig. 2A) the sclerotigenum/Geosmithia clade includes an extensive group of Acremonium species and cleistothecial Bionectriaceae with Acremonium-like anamorphs, namely, Emericellopsis, Hapsidospora, Mycoarachis, and Nigrosabulum. Among the anamorphic species in this group are most of the phylogenetically disparate isolates identified as the type species of Acremonium, A. alternatum. One of these, CBS 407.66, is designated below as epitype of A. alternatum. Prominent subclades include the Acremonium sclerotigenum clade containing the ex-type isolates of A. sclerotigenum and A. sordidulum.

Another major, well supported bionectriaceous subclade associated with the sclerotigenum clade is the Emericellopsis clade (Fig. 2A). It includes the type species of the synnematal hyphomycete genus Stilbella, S. fimetaria (Seifert 1985) as well as the type of Stanjemonium (Gams et al. 1998) and the marine Acremonium tubakii sensu stricto and A. fuci (Zuccaro et al. 2004). Stilbella fimetaria is closely related to the ex-type isolate of Acremonium salmoneum isolated from dung, also a typical habitat for S. fimetaria (Seifert 1985). An adjacent weakly supported clade includes Hapsidospora, Mycoarachis, and Nigrosabulum, and the two Acremonium species named for yellow pigmentation, A. chrysogenum and A. flavum. Although associated with A. chrysogenum and A. flavum in Fig. 1B, A. pinkertoniae and A. borodinense form a distinct clade in Fig. 2A along with an isolate included in the polyphyletic A. blochii (CBS 993.69), plus the cleistothecial teleomorphs Bulbithecium hyalosporum and Leucosphaerina arxii, both of which have unnamed Acremonium anamorphs. In Fig. 2A, the A. chrysogenum subclade appears to be distinct from the other clades containing A. sclerotigenum, Emericellopsis, and Geosmithia. The other clades within the overall sclerotigenum/Geosmithia clade include the A. fusidioides clade containing several acremonia forming similar conidial chains (A. cavaraeanum, A. fusidioides, A. hansfordii, A. hennebertii, one of the isolates labeled A. alternatum). A small A. brachypenium clade associated with the A. sclerotigenum clade includes A. brachypenium plus the ex-type strain of Cephalosporium purpurascens placed by Gams (1971) in A. persicinum. There is also an isolate of the polyphyletic, untypified species A. potronii. Basal to these clades is another small clade that links an entomogenous isolate identified as Verticillium insectorum with two isolates from human sources identified as A. blochii; these conidial chain-forming isolates are sister to an isolate of the chain-forming entomogenous species Acremonium zeylanicum. The “A. blochii” isolate CBS 427.93, linked with a 99 % bootstrap value to A. pinkertoniae in Fig. 1B, is one of the two isolates associated with Acremonium zeylanicum in Fig. 2A.

Adjacent and loosely linked to the bionectriaceous clades in Fig. 2B is a small clade in Fig. 2C containing the ex-type isolate of Acremonium incrustatum plus an isolate labeled A. potronii, and a sequence attributed to Linkosia fusiformis, although this sequence most likely represents a contaminant.

Below the sclerotigenum/Geosmithia clade in Fig. 1A and above the Hypocreaceae in Fig. 2E fall clades representing the Clavicipitaceae sensu lato. These clades include the families Clavicipitaceae sensu stricto, Ophiocordycipitaceae, and Cordycipitaceae. Although many species in this group of three families have Acremonium-like anamorphic states, only two described Acremonium species are associated here. In Fig. 2E A. camptosporum sits basally in a clade adjacent to the Clavicipitaceae and is close to the poorly understood teleomorphic species Clypeosphaeria phillyreae, assuming the latter is correctly associated with the sequence attributed to it. Simplicillium obclavatum, originally described as Acremonium obclavatum, provides the only other clavicipitaceous species in Fig. 2 representing a named species of Acremonium.

Below the Clavicipitaceaceae in Fig. 1B is a clade of ambiguous affinities containing Acremonium guillematii, A. minutisporum and A. vitellinum. This group also appears as two to three unaffiliated clades in Fig. 2C. An insignificant branch in Fig. 1B subtends Acremonium exiguum, A. psammosporum, and an isolate identified as A. potronii. In Fig. 2D, just A. exiguum and the A. potronii entity remain associated while Acremonium psammosporum segregates into a basal hypocrealean clade of its own in Fig. 2E.

The Nectriaceae is represented by Nectria cinnabarina in Fig. 1B along with the ex-type isolate of the tropical opportunistic pathogen of humans, Acremonium recifei. Fig. 2D shows A. recifei subtending multiple taxa with three non-type isolates splitting off as a separate clade. These clades have approximately the same status in the Nectriaceae as the genus Nalanthamala, including N. diospyri, the former Acremonium diospyri. Another nectriaceous Acremonium in Fig. 2D is A. tsugae, which is closely related to Cylindrocarpon cylindroides. The broad morphotaxonomic concept of Acremonium berkeleyanum is polyphyletic consisting of isolates placed in the nectriaceous genus Cosmospora (Fig. 2D). Acremonium berkeleyanum sensu lato is represented in Fig. 2D by the newly recombined Cosmospora species, C. lavitskiae and C. khandalensis based on the ex-type isolates of Gliomastix lavitskiae and Cephalosporium khandalense (Gräfenhan et al. 2011). Another purported synonym of A. berkeleyanum, a Cadophora isolate received as A. butyri CBS 301.38, falls outside the Hypocreales (Fig. 1C).

Basally in the Hypocreales in Fig. 1B, Acremonium roseolum appears in loose association with Stachybotrys species. In Fig. 2D, it appears in a clade along with the teleomorph Scopinella solani and three Acremonium inflatum isolates, including CBS 403.70, an atypical, catenate-conidial isolate identified at CBS as A. atrogriseum. Nearby but statistically unlinked clades include Stachybotrys and allied fungi such as Peethambara spirostriata and Didymostilbe echinofibrosa (Castlebury et al. 2004).

Acremonium nigrosclerotium represents an isolated Acremonium near the families Hypocreaceae and Niessliaceae (Fig. 1B). In Fig. 2D, A. nigrosclerotium is intercalated among two genotypes ascribed to N. exilis, and loosely associated (77 % bootstrap) with Acremonium pseudozeylanicum and the type culture of Cephalosporium ballagii, currently in synonymy with Acremonium charticola (Gams 1971).

A distant outlier is Acremonium lichenicola at the bottom of Fig. 1C. This isolate, CBS 425.66, chosen to represent this species in lieu of ex-type material, blasts as a pezizalean fungus with affinities to another hyaline, phialidic fungus, Phialophora alba.

A number of genera in addition to Acremonium were investigated for possible affinity with Acremonium clades as shown in Fig. 2. The sporodochial genus Sarcopodium was investigated and found to split into two groups (Fig. 2C, D). One isolate identified as S. circinatum grouped with Sarcopodium circinosetiferum and S. vanillae in a widely separated clade along with Lanatonectria teleomorphs and a sequence identified as Pseudonectria rousseliana (Fig. 2C). This clade appeared in LSU sequencing to be independently situated within the Hypocreales. Acremonium rhabdosporum appeared as a statistically unsupported, possible distant relative. The other two isolates of S. circinatum formed a clade near Myrothecium in the Stachybotrys/Peethambara clade (Fig. 2D). Also appearing in this clade was Parasarcopodium ceratocaryi, a monotypic genus recently described by Mel'nik et al. (2004).

Phylogenetic analysis compared to the morphological treatment of Acremonium

Gams (1971, 1975) divided Acremonium into three major sections, Simplex, a name later updated as the type section Acremonium, Gliomastix, and Nectrioidea. Of these sections, only Gliomastix withstands phylogenetic scrutiny as a unit, albeit a loosely associated one.

The type section Acremonium contained four widely phylogenetically scattered major clades (Fig. 2), specifically the A. sclerotigenum clade, Sarocladium clade, A. curvulum clade, and A. breve clade. As seen best in Fig. 1, the Sarocladium clade and the A. breve and A. curvulum clades comprise a distinct group that falls within the Hypocreales but outside any currently recognised family. Acremonium sclerotigenum falls into a distinct clade within the Bionectriaceae that also contains Emericellopsis and Geosmithia. This clade also includes about half the investigated CBS isolates identified as the type species of Acremonium, A. alternatum, including CBS 407.66 as well as some isolates such as CBS 223.70 revealed as morphological variants of A. sclerotigenum. Despite the substantial phylogenetic distance between A. sclerotigenum and A. strictum, relatively glabrous, cylindrical-conidial isolates of A. sclerotigenum not producing sclerotia on special media (lupine stem agar according to Gams, 1971, later replaced at CBS by nettle stem agar) are essentially micromorphologically indistinguishable from A. strictum. Table 1 shows CBS 287.70 O as an A. sclerotigenum isolate identified in CBS as A. strictum; ITS sequencing studies of additional strains (data not shown) have found two more such isolates, CBS 319.70 D and CBS 474.67.

The convergence among isolates of phylogenetically remote species is remarkable. An unknown proportion of the literature on A. strictum is based on studies of A. sclerotigenum. For example, in a study influential in medical mycology, Novicki et al. (2003) labeled ITS-sequenced isolates of A. sclerotigenum in GenBank as “Acremonium strictum genogroup II.” The complexity of A. sclerotigenum, not its earliest valid name, goes beyond the scope of this paper. Perdomo et al. (2010) have recently investigated the diversity of medically important isolates within this species.

Besides the four clades mentioned above, Acremonium sect. Acremonium species also make up the non-synnematal anamorphs of the Emericellopsis clade, most of the A. fusidioides clade, and most of the small A. camptosporum, A. exiguum, A. minutisporum, A. pinkertoniae, and A. pseudozeylanicum clades. Gams (1975) accommodated A. byssoides, now known to belong in Simplicillium lanosoniveum (Zare & Gams 2001), in Acremonium sect. Acremonium, while commenting that it was suggestive of Verticillium sect. Prostrata, later recognised as Simplicillium (Zare & Gams 2001). He withheld A. byssoides from Verticillium because the colony margin was relatively flat and slightly fasciculate, rather than cottony. To some extent Acremonium sect. Acremonium was based on keying out all the relatively flat or fasciculate Acremonium-like species together provided that they lacked the dark conidia or chondroid hyphae of Gliomastix.

Acremonium sect. Nectrioidea as delineated by Gams (1971) included many Nectria sensu lato anamorphs. Some of these species are now placed in the genus Cosmospora by Gräfenhan et al. (2011). These include members of the A. berkeleyanum complex as well as A. arxii and A. cymosum. Acremonium falciforme in A. sect. Nectrioidea had already been recognised as a member of the Fusarium solani complex (Summerbell & Schroers 2002) and A. diospyri had been transferred into Nalanthamala along with other nectriaceous species (Schroers et al. 2005). Acremonium tsugae appears to be a microconidial Cylindrocarpon species. The Acremonium recifei complex still remains as an undisposed major group of nectriaceous Acremonium species originally included in A. sect. Nectrioidea. The placement of A. sect. Nectrioidea species A. alcalophilum, A. brunnescens, A. furcatum, A. nepalense, A. restrictum, and A. stromaticum in the Plectosphaerellaceae has already been shown by Zare et al. (2007). Acremonium apii also has been shown to belong to this family as a synonym of Verticillium alboatrum, and its ex-type strain, CBS 130.51, was used as the representative isolate of that species by Zare et al. (2007).

Other anomalous elements of A. sect. Nectrioidea include A. crotocinigenum in the Trichothecium clade, A. radiatum in the phylogenetically isolated A. breve clade, A. biseptum in the A. cerealis clade near Gliomastix, A. salmoneum in the Emericellopsis clade near Stilbella fimetaria, A. chrysogenum in a bionectriaceous clade containing cleistothecial teleomorphs such as Nigrosabulum, A. rutilum in a clade otherwise containing isolates identified as A. persicinum, and a non-type A. hyalinulum isolate in another clade peripheral to Gliomastix. When Sarocladium zeae as A. zeae in A. sect. Nectrioidea was compared to the phylogenetically related S. kiliense as A. kiliense in A. sect. Acremonium by Gams (1971, p. 16), he noted that the latter species may sometimes also be strongly branched and thus resemble the former. The exigencies of dichotomous morphological keying tended to sort closely related species into widely separated Sections of the genus.

The main heterogeneous element included in Gams' (1971) original concept of sect. Gliomastix was the “Striatisporum series.” These were later distinguished as the separate genus Sagenomella (Gams 1978). Both Sagenomella and the recently described genus Phialosimplex are members of the Eurotiales (Sigler et al. 2010). Another anomalous element in sect. Gliomastix, Acremonium atrogriseum, is here removed to the Cephalothecaceae.

Other species included by Gams (1971, 1975) in A. sect. Gliomastix that can now be seen to be separated from the Gliomastix/Bionectria clade include “Cephalosporium purpurascens,” synonymised by Gams (1971) with A. persicinum as well as A. brachypenium, A. hennebertii, A. incrustatum, and A. inflatum. Species outside the Gliomastix/Bionectria clade that have well developed chondroid hyphae include A. hennebertii and A. incrustatum.

Acremonium atrogriseum and Acremonium cf. alternatum CBs 109043 in the Cephalothecaceae: a study in comparative morphology vs. phylogeny

Acremonium atrogriseum and an isolate identified as Acremonium cf. alternatum CBS 109043 belong in the Cephalothecaceae (Fig. 1C). This isolate is a white coloured acremonioid fungus forming fusoid conidia in long chains. It also forms small, dark structures that may be aborted ascomatal initials. Sequencing of the ITS region (data not shown) reveals it to be a representative of Phialemonium obovatum. It is identical in all bases but one to the ITS sequence of ex-type strain CBS 279.76 (AB278187) and in all but two bases to another isolate of this species, CBS 116.74. Phialemonium obovatum was described as having conidia in slimy heads (Gams & McGinnis 1983). CBS 109043 shows that either mucoid heads or chains may be formed in this species, as in Acremonium persicinum, A. sclerotigenum, and Gliomastix murorum. Gams (1971) mentions an isolate of Sarocladium bacillisporum that tends to produce mucoid heads. Colonies producing conidia in chains often have a different look from their head-forming conspecifics; the mass colour of the chains may give the colony colours not found in the species descriptions, such as the chalk white colour of CBS 109043 in contrast to the normally pale greenish brown of P. obovatum or the greenish grey of A. sclerotigenum isolate 223.70, in contrast to the normal pale salmon pink of non-catenate A. sclerotigenum.

Existing morphological keys and descriptions not just in Acremonium but in all the acremonioid fungi need to be cautiously and skeptically interpreted. At the very least, identifications for publication should be tested by sequencing. We hope that the LSU sequences in this paper will provide the foundation for a phylogenetically sound approach to the systematics and ecology of acremonioid fungi.