The Colletotrichum gigasporum species complex

In a preliminary analysis, 21 Colletotrichum strains with large conidia preserved in the CBS culture collection clustered with a recently described species, C. gigasporum, forming a clade distinct from other currently known Colletotrichum species complexes. Multi-locus phylogenetic analyses (ITS, ACT, TUB2, CHS-1, GAPDH) as well as each of the single-locus analyses resolved seven distinct species, one of them being C. gigasporum. Colletotrichum gigasporum and its close allies thus constitute a previously unknown species complex with shared morphological features. Five of the seven species accepted in the C. gigasporum species complex are described here as novel species, namely C. arxii, C. magnisporum, C. pseudomajus, C. radicis and C. vietnamense. A species represented by a single sterile strain, namely CBS 159.50, was not described as novel species, and is treated as Colletotrichum sp. CBS 159.50. Furthermore, C. thailandicum is reduced to synonymy with C. gigasporum.

The most distinctive morphological feature of C. gigasporum is the long straight conidia (up to 32 μm long, av. length 26 μm). Rakotoniriana et al. (2013) discussed the morphological differences between C. gigasporum and other species that produce large conidia, e.g. C. crassipes, C. echinatum, C. macrosporum, C. taiwanense and C. vinosum. Based on phylogenetic analyses of ITS and TUB2 sequence data, they showed C. gigasporum to belong to a distinct clade, distant from other currently accepted Colletotrichum species.
Numerous Colletotrichum isolates detected in a blastn search on GenBank have similar ITS sequences to that of the extype strain of C. gigasporum, e.g. isolates from Coffea arabica in Vietnam (Nguyen et al. 2010), Hibiscus rosa-sinensis in Thailand (Noireung et al. 2012), Magnolia liliifera in Thailand (Promputtha et al. 2007), Taxus chinensis var. mairei in China (Wu et al. 2013) and Theobroma cacao, Trichilia tuberculata and Virola surinamensis in Panama (Rojas et al. 2010). In our preliminary ITS analysis, 21 isolates retrieved from the CBS collection clustered with C. gigasporum, but showed considerable genetic variability, suggesting further species belonging to a previously unreported species complex.
The objectives of this study are to clarify the genetic and taxonomic relationships of Colletotrichum strains from various hosts and geographic areas thought to be closely related to C. gigasporum, and to describe the new species from this complex.

Isolates
Colletotrichum isolates with large conidia were obtained from the culture collection of the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands (CBS). All descriptions are based on ex-type cultures. Features of other strains are added if deviant. Cultures of additional isolates used for morphological and phylogenetic analyses are maintained in the CBS culture collection (Table 1).

Morphological analysis
To enhance sporulation, 5-mm-diam plugs from the margin of actively growing cultures were transferred to the centre of 9-cm-diam Petri dishes containing synthetic nutrient-poor agar medium (SNA) (Nirenberg 1976) amended with autoclaved filter paper and double-autoclaved stems of Anthriscus sylvestris placed onto the agar surface. Strains were also studied after growth on oatmeal agar (OA). Cultures were incubated for 10 d at 20 °C under near UV light with a 12 h photoperiod. Measurements and photographs of characteristic structures were made according to methods described by Liu et al. (2012). Appressoria on hyphae were observed on the reverse side of colonies grown on SNA plates. Microscopic preparations were made in clear lactic acid, with 30 measurements per structure, and observed with a Nikon Eclipse 80i microscope using differential interference contrast (DIC) illumination. Colony characters and pigment production on SNA and OA incubated at 20 °C were noted after 10 d. Colony colours were scored according to Rayner (1970). Growth rates were measured after 7 and 10 d.
The DNA sequences obtained from forward and reverse primers were used to obtain consensus sequences using MEGA v. 5.1 (Tamura et al. 2011), and subsequent alignments were generated using MAFFT v. 6 (Katoh & Toh 2010), and manually edited using MEGA v. 5.1.
Sequences of the 21 Colletotrichum strains studied in this paper as well as sequences of 50 reference strains (Table 1) downloaded from GenBank (www.ncbi.nlm.nih.gov/genbank/) and NIAS GenBank (www.gene.affrc.go.jp /about_en.php) were included in individual alignments and eight single gene phylogenies were generated using a distance-based method. The ITS alignment included a further 22 sequences that were found in blastn searches in GenBank in addition to those in Table 1. Distance matrixes of the aligned sequences were calculated using the Kimura 2-parameter model (Kimura 1980), and analysed with the Neighbour-joining (NJ) algorithm (Saitou & Nei 1987) using MEGA v. 5.1, excluding positions with gaps. The reliability of the inferred trees was estimated by bootstrap analyses with 1 000 replicates.
A maximum parsimony analysis was performed on the multilocus alignment including five of the eight loci (ACT, CHS-1, GAPDH, ITS, TUB2) of a total of 71 strains (Table 1) using PAUP v. 4.0b10 (Swofford 2002). Ambiguously aligned regions were excluded from all analyses. Unweighted parsimony (UP) analysis was performed. Trees were inferred using the heuristic search option with TBR branch swapping and 1 000 random sequence additions. Maxtrees were unlimited, branches of zero length were collapsed and all multiple parsimonious trees were saved. Clade stability was assessed in a bootstrap analysis with 1 000 replicates, each with 10 replicates of random stepwise addition of taxa. A second phylogenetic analysis of the concatenated alignment using a Markov Chain Monte Carlo (MCMC) algorithm was done to generate trees with Bayesian posterior probabilities in MrBayes v. 3.2.1 (Ronquist & Huelsenbeck 2003). Nucleotide substitution models were determined using MrModeltest v. 2.3 (Nylander 2004) for each gene region and included in the analyses. Two analyses of four MCMC chains were run from random trees for 10 000 000 generations and sampled every 1 000 generations. The first 25 % of trees were discarded as the burn-in phase of each analysis and posterior probabilities determined from the remaining trees. Monilochaetes infuscans strain CBS 869.96 was used as outgroup in all analyses. Sequences derived in this study were lodged in GenBank, the multi-locus alignment and tree in TreeBASE (http://www.treebase.org/treebase-web/search/ studySearch.html) (S15175), and taxonomic novelties in Myco-Bank (www.MycoBank.org; Crous et al. 2004a).

Phylogeny
The eight NJ trees derived from the single gene sequence alignments (ACT, CAL, CHS-1, GAPDH, GS, HIS3, ITS, TUB2) confirmed that the 21 CBS isolates and the ex-type and other strains of C. gigasporum constituted a monophyletic lineage, distant from other known major clades of the genus Colletotrichum recognised by Cannon et al. (2012). The NJ trees are not shown in this study except for the phylogeny based on ITS data ( Fig. 1). Isolates studied in this paper (marked with red squares) are separated into seven subclades, which could also be confirmed with the other seven single gene phylogenies.  Fig. 2, where the 21 strains studied belong to a major clade consisting of seven subclades. More than half of the strains clustered in the largest subclade (C. gigasporum) with a high bootstrap support and Bayesian posterior probability value (100/1.00). The Bayesian tree confirmed the tree topology of the trees obtained with maximum parsimony.

Fig. 1
Neighbour-joining tree of ITS sequences from 21 isolates generated in this study and 43 isolates from other studies, retrieved from GenBank. The tree was constructed using MEGA v.5.1 software. The Kimura-2-parameter method was used. Bootstrap support values (1 000 replicates) above 50 % are shown at the nodes. Ex-type cultures are emphasised in bold, and include the taxonomic name as originally described. Our isolates are marked with a red square, and the strain number is followed by host and country of origin. Stars indicate reported pathogens, triangles indicate reported endophytes, GenBank accessions are followed by taxonomic name as originally identified, strain number, host and country of origin. The tree is rooted with Monilochaetes infuscans.
Closest match in a blastn search with the ITS sequence of strain CBS 132511 (with 99 % identity, 8 bp differences) was an endophytic isolate (DQ780412) from Magnolia liliifera probably in Thailand (Promputtha et al. 2007) and an endophytic isolate (FJ205460) from an orchid in Taiwan  Notes -Colletotrichum gigasporum is characterised by large conidia ((22-)25 -29(-32) × (6 -)7-9 μm). Phylogenetic analyses by Rakotoniriana et al. (2013) based on the ITS and TUB2 sequences placed it in a distinct clade far from the cur-rently accepted Colletotrichum species. Another species with large conidia (27-30 × 9-10 μm), C. thailandicum, was described from diseased Alocasia sp. and Hibiscus rosa-sinensis from Thailand (Noireung et al. 2012). Colletotrichum thailandicum is morphologically similar to C. gigasporum; the ITS and β-tubulin sequences of both fungi are identical or near-identical (differed in two nucleotide position in β-tubulin). In addition, phylogenetic analyses of single locus data, including ITS (Fig.  1), and multi-locus data (Fig. 2), show that the ex-type strains of the two species cluster together in one strongly supported clade. Since C. gigasporum was published online earlier (8 August 2012) than C. thailandicum (September 2012), we regard C. thailandicum as a synonym of C. gigasporum.
Strain CBS 109355, isolated from a phaeohyphomycotic cyst from a Brazilian man, was originally identified as C. crassipes, mainly based on morphology of the appressoria with crenate or deeply lobed margins and its size of conidia ). In addition, strains CBS 159.75 and IMI 302450, which were deposited as C. crassipes in the CBS and IMI culture collections, were compared morphologically with CBS 109355 by Castro et al. (2001). However, strains CBS 159.75 and CBS 109355 were reidentified as C. gigasporum in the present study (Fig. 2). Hitherto, the taxonomic status of C. crassipes as well as the genetic relationship between C. gigasporum and C. crassipes remain unclear due to the lack of an ex-type culture and DNA sequence data. Thus, an epitype is needed to stabilise the nomenclature of C. crassipes.
In addition to being a disease-causing agent of humans, C. gigasporum is also associated with Musa sp. (Fig. 1, 2), the anthracnose of which is commonly considered to be caused by C. musae that belongs to the C. gloeosporioides species complex (Weir et al. 2012). However, C. gigasporum is phylogenetically distinct from C. musae, and its conidia are significantly larger than those of C. musae. Additional Colletotrichum species associated with Musa spp. include C. cavendishii, C. liukiuensis and C. paxtonii. Colletotrichum gigasporum differs from C. liukiuensis (Sawada 1959), a species on leaves of M. liukiuensis in Taiwan, and C. cavendishii (Petrak 1925), a species on living leaves of M. cavendishii by producing larger conidia (20.5-25.5 × 6-9 μm vs 12-14 × 4.8-5.5 μm and 10-19 × 4.5-7 μm, respectively). Colletotrichum paxtonii, a species associated with banana in St. Lucia, belongs to the C. acutatum complex (Johnston & Jones 1997, Damm et al. 2012a) and is therefore not closely related to C. gigasporum.
Our 5-locus phylogram shows that several strains from diverse countries and hosts cluster with C. gigasporum (syn. C. thailandicum). Based on our blastn search in GenBank, the results of which are included in the ITS phylogeny, 22 additional ITS sequences from GenBank cluster with the ex-type strain of C. gigasporum, including sequences derived from strains isolated from plants as endophytes or pathogens and even strains that were isolated from human tissue (Fig. 1). This is in accordance with the conjecture that ecologically C. gigasporum can occur as either endophyte or pathogen (Rakotoniriana et al. 2013). The isolates from which most of these GenBank sequences were generated had been previously identified as C. crassipes, C. gloeosporioides, C. incarnatum, C. orbiculare or C. taiwanense (sexual morph Glomerella septospora) (Fig. 1).
The ascospores and conidia of C. gigasporum resemble those of C. taiwanense with respect to their size. However, C. gigasporum produces aseptate conidia and 0-1-septate ascospores (Rakotoniriana et al. 2013), while the conidia of C. taiwanense may become 1-5-septate with age and ascospores are mostly 3-septate and may become up to 6-or 8-septate when old (Sivanesan & Hsieh 1993). Colletotrichum taiwanense, originally described from Styrax formosanus in Taiwan, is currently poorly characterised using molecular methods (Hyde et al. 2009,  Cannon et al. 2012). Unfortunately, a subculture from the extype isolate of C. taiwanense (IMI 353024) is contaminated; the original strain could not be recovered. Several plant pathogenic strains from various hosts (none of them from Styrax) that were previously identified as C. taiwanense were reidentified as C. gigasporum based on the ITS-rDNA phylogram in this study (Fig. 1). Colletotrichum gigasporum differs from C. incarnatum (Zimmermann 1901), a species first described from Coffea liberica in Java, by producing larger conidia (20.5 -25.5 × 6-9 μm vs 14 -19 × 5 μm).
Some strains from Mora excelsa in Guyana had been previously identified as C. orbiculare (Lu et al. 2004) and grouped with C. gigasporum in our ITS tree. However, C. orbiculare was recently redefined and shown to belong to a different species complex together with C. lindemuthianum (Damm et al. 2013).
Although the ITS-rDNA phylogram revealed that C. gigasporum strains formed two subclades (Fig. 1), the bootstrap values are too low to support two distinct species, which could also be verified by the multi-locus phylogram (Fig. 2).
The closest matches in a blastn search in GenBank with the ITS sequence of strain CBS 398.84 were with 100 % identity EF672323 from the endophytic isolate VegaE4-36 from Coffea arabica from Hawaii, USA (Vega et al. 2010), EU686812 from an endophytic isolate from Rhipidocladum racemiflorum from Panama (Higgins et al. 2011), as well as KF436311 from the endophytic isolate TK780 from a tropical woody plant from Panama (Higginbotham et al. 2013). The closest match with the TUB2 sequence (with 96 % identity, 16 bp differences) was isolate MUCL 41702 from Orchis in Singapore (FN599826; Rakotoniriana & Munaut unpubl. data).
The name C. camelliae, although not listed by Hyde et al. (2009) and Cannon et al. (2012), is widely used for the causal agent of the brown blight disease of tea (Sosa de Castro et al. 2001, Muraleedharan & Baby 2007. However, the status of C. camelliae and its taxonomic relationship with G. cingulata f. sp. camelliae remain unresolved (Weir et al. 2012). There are 11 ITS sequences of Colletotrichum sp. from tea in GenBank (EF063686, FJ515007, EU732732, FJ216456, HQ832797, JQ809665, HQ832801, AB548281, AB218993, GQ916544, HE655519), of which sequence HQ832801 associated strain nested within the C. boninense complex in the ITS phylo genetic tree, while the others belong to several clades within the C. gloeoporioides complex (data not shown). Appropriate fresh collections associated with brown blight symptoms of tea from Sri Lanka are needed for epitypification to clarify the phylogenetic relationships of this taxon. Colletotrichum pseudomajus can be distinguished from C. camelliae by its significantly larger conidia (22-30.5 × 6.5 -9.5 μm vs 15 -17 × 4 -5 μm).
Colletotrichum pseudomajus is morphologically similar to G. major except for the presence of paraphyses and the shape of its ascospores. Paraphyses were reported to be absent in G. major, but thin-walled, hyaline and septate paraphyses are present in C. pseudomajus; ascospores of G. major are ellipsoid, not allantoid, with obtuse or subacute tips (Tunstall 1935), while those of C. pseudomajus are lunate, with more or less acute tips (Fig. 5). Currently, the phylogenetic position of G. major is unresolved due to the lack of an ex-type isolate. Thus, an epitype is needed to stabilise the nomenclature of G. major and to clarify the relationship between C. pseudomajus and G. major.
The closest matches in a blastn search with the ITS sequence of CBS 571.88 with 100 % identity were JX009424, the sequence generated from the same isolate by Weir et al. (2012), and JQ809667 from the endophytic isolate JD08-18 from Camellia sinensis in China (Fang et al. 2013), as well as JN418782 from the endophytic isolate E10202g from Otoba parvifolia in Ecuador (Barba et al. unpubl. data). Closest match with the TUB2 sequence (with 93 % identity, 32 bp differences) was isolate MUCL 41702 from Orchis in Singapore (FN599826; Rakotoniriana & Munaut unpubl. data). The blastn search with the GAPDH sequence of CBS 571.88 showed similarity with JN050231 (85 % identity, 34 bp differences) from isolate BCC 38879 from Hibiscus rosa-sinensis in Thailand (Noireung et al. 2012) which is here referred to C. gigasporum, and JX009422 (99 % identity, 1 bp difference), a sequence generated from the same isolate. The only base difference in the end of the sequence was due to sequencing error by Weir et al. (2012).
Colletotrichum radicis F. Liu,L. Cai,Crous & Damm,Fig. 6 Etymology. Referring to the host organ, a plant root, from which it was isolated.
Culture characteristics -Colonies on OA flat with entire margin, aerial mycelium lacking; colonial diam 64-71 mm in 7 d, > 90 mm in 10 d. Colonies on SNA flat with entire margin, aerial mycelium lacking, medium hyaline, buff around Anthriscus stem; colonial diam 64 -75 mm in 7 d, > 90 mm in 10 d.
Specimen examined. Costa riCa, La Selva, host plant unknown (isolated from a plant root), unknown collection date and collector (isolated by G. Weber in Mar. 1993) (holotype CBS H-21494, culture ex-type CBS 529.93).
Another species known to occur on Coffea sp. from Vietnam in this complex is C. gigasporum (CBS 125476 and CBS 125475), which can be distinguished from C. vietnamense by each of the eight genes used in this study, including ITS (Fig. 1).
The closest matches with the ITS sequence of CBS 125478 were FJ968584 (with 100 % identity), a sequence generated from the same isolate by Nguyen et al. (2010), and EF672327 (with 100 % identity) from the endophytic isolate PR61F2, also from Coffea arabica , but from coffee berries in Puerto Rico, a country in Central America (Vega et al. unpubl. data). Closest match with the TUB2 sequence was KC293665 (with 96 % identity, 20 bp differences) from isolate gnqczg15 from China (Huang et al. unpubl. data).

dISCuSSIon
Many of the strains included in the present study were deposited in the CBS culture collection as C. crassipes (Speg.) Arx. However, C. crassipes is a species with uncertain taxonomic status. There is significant confusion regarding its morphology in the literature. Spegazzini (1878) originally described this fungus as Gloeosporium crassipes from Vitis vinifera from Conegliano, Italy with conidia measuring 20 -30 × 7-8 μm. Subsequently, von Arx (1957) combined Gloeosporium crassipes in Colletotrichum as C. crassipes along with 17 synonyms. The conidial size of C. crassipes was reported as 22 -31 × 6 -8 μm, broadly matching the original description; and the appressoria as irregular, usually lobed, measuring 8 -12 μm (von Arx 1957). Sutton (1980) presented a different morphological concept of C. crassipes, which was characterised by conidia measuring 10-15 × 4.5-6.5 μm, long clavate or circular appressoria with crenate or deeply divided edges, 10.5 -14 × 7-9.5 μm, and reduced another two names to synonymy with it. However, when Sutton summarised an accepted taxa list of Colletotrichum species, C. crassipes was characterised with conidia again with a different size (14 -28 × 5 -7 μm), and he suspected that this species may consist of a number of separate taxa (Sutton 1992). Moreover, several isolates identified as C. crassipes that have sequences lodged in GenBank actually belong to C. gloeosporioides s.lat. (Weir et al. 2012). Recollecting and epitypification of this taxon is required to stabilise the phylogenetic position of C. crassipes.
Although morphological features are not stable and change under different growth conditions and with repeated subculturing, species of the C. gigasporum species complex form larger conidia than most of the other species in the genus Colletotrichum, which provides a valuable character for species complex level diagnosis. Conidia of two other species with large conidia, C. euphorbiae and C. sansevieriae, differ in shape; they are slightly clavate with a round apex tapering to a truncate to slightly acute base (Nakamura et al. 2006, Crous et al. 2013). These two species do not belong to the C. gigasporum complex.
While single gene data, especially ITS data, are usually not sufficient for species recognition in most of the Colletotrichum species complexes or groups (Cannon et al. 2012) and multilocus phylogenies are therefore now routinely used as the primary basis on which to describe new Colletotrichum species , b, Weir et al. 2012, Liu et al. 2013a, species of the C. gigasporum species complex can be easily distinguished from each other using the individual gene data included in this study (Fig. 1).
Colletotrichum gigasporum appears to have a wide host range and geographic distribution. Isolates treated in this paper and those deposited in GenBank originate mainly from Africa (East Africa, Madagascar), Central and South America (Brazil, Chile, Columbia, Ecuador, Guyana, Mexico, Panama), Asia (China, India, Japan, Korea, Thailand, Vietnam) and New Zealand (Fig. 1). Besides, this species is associated with various host plants as pathogens and endophytes, from air and stored grain, indicating that it is not host-specific and apparently has different life styles. This character is not unique to C. gigasporum, many other Colletotrichum species have been reported as both pathogens and endophytes, e.g. C. boninense, C. karstii and C. liriopes (Yang et al. 2011, Damm et al. 2012b, Tao et al. 2013. For instance, C. boninense causes diseases of Crinum asiaticum var. sinicum and Solanum lycopersicum, and is also an endophyte of Bletilla ochracea and Dacrycarpus dacrydioides (Damm et al. 2012b, Tao et al. 2013. The relationship between plant endophytic and pathogenic isolates of the same Colletotrichum species needs more research, as some endophytes may be latent pathogens (Lu et al. 2004).