The trimmed and edited sequences of each marker were as follows: ITS1, 140 nucleotides; 5.8S, 117 nucleotides and ITS2, 164 nucleotides. MAFFT analysis (http://mafft.cbrc.jp/alignment/server/ (Katoh 2005, 2008) revealed no evidence of chimeric sequences present in the ITS data set and indicated good quality ITS sequences with no stochastic or artifactual nucleotide data.

RNA sequences of the ITS1 and ITS2 markers were aligned using the align and fold approach (Figure 1). The consensus minimum free energy (MFE) structures for the ITS1 marker according to species are illustrated in Figure 2. The delta G required for formation of the secondary structures was on average -55.1 kcal/mol. The GC content of the ITS1 sequences was on average 56.7%. Within the entire data set, there were 24 polymorphic sites, 19 singleton sites and 12 indel sites with two indel haplotypes. The indel haplotype diversity was calculated to be 0.054. Nucleotide diversity (Pi) was calculated as 0.02296 ± 0.00794. Total number of mutations (Eta) was 27. When only type and ex-type sequences were considered, there were five polymorphic sites and five mutations. A comparison of predicted structures for each sequence revealed deviations from the consensus secondary structure of the ITS1 region of C. gloeosporioides sensu stricto (Figure 2). Overall, four ITS1 ribotypes were proposed based on variations in type sequences: Ribotype 1 – C. gloeosporioides sensu stricto, Ribotype 2: C. asianum, Ribotype 3: C. fructicola, Ribotype 4: C. siamense and C. tropicale (Figure 2).

The consensus secondary structure of ITS1 marker of C. gloeosporioides sensu stricto consisted of a long, double helix; at its central part, the double helix contained one large, internal loop in addition to other asymmetrical internal loops. There were three non-canonical G-U base pairings and a number of base substitutions. Non-canonical G–U pairing presents certain degeneracy in base-pairing which may provide structural flexibility and can be allowed within rRNA secondary structures without resulting in significant structural changes (Mullineux and Hausner 2009). Comparisons of consensus structures suggest that insertions/deletions that impact upon helix length or base changes that occur in loops or bulges do not necessarily affect the formation of mature functional rRNA and these regions may be susceptible to such changes (Bridge et al., 2008).

There is no consensus structure for this marker across all eukaryotes, however, the GGCRY-RYGYC motif was found in the ITS1 sequence alignment and has been identified as similar to the inverted repeats found in ascomycetes and to the stem of helix 1C in some angiosperms (Liu and Schardl 1994).

There was one taxonomic motif unique to C. gloeosporioides sensu stricto that characterized the second internal loop in the predicted structure which was identified as “UACA” (Figure 3). All other taxa had “UAUA” except for isolates PAW-Cg110, PAW-Cg122, PAW-Cg6 and PAW-Cg7 which had “UAUG”. There was also a unique taxonomic motif in the secondary structure predicted for C. asianum identified as “CACU” but exists as “CCCU” in all other taxa. C. fructicola had differences in the internal loop and terminal loops T2 and T3. C. siamense and C. tropicale shared more structural similarities than with any of other species. C. asianum had a unique structure that appeared to be taxon-specific.

As a result of the number of polymorphisms in sequences of PAW-Cg110, PAW-Cg122, PAW-Cg6 and PAW-Cg7, the predicted consensus secondary structure was distinct from all other taxa and a fifth ribotype was proposed. Among eukaryotes, there is an apparent variability in the number of helices and structural details that occur in the ITS1 transcript, for example, four helices were identified in Chlorobionta (Coleman et al. 1998; Gottschling et al. 2001) and Saccharomyces cerevisiae (van Nues et al., 1994), but seven helices were proposed for Digenea (von der Schulenburg et al., 1999). Since there is no consensus structure for this ITS marker as there is for the ITS2 marker, it is difficult to determine whether these variants are normally distributed or may be representative of a separate species within the C. gloeosporioides sensu lato complex but which was not considered in this study.

There were no polymorphic sites within the aligned 5.8S sequences and the entire 117 nt region was conserved with 0.0000 entropy (Hx) value. The delta G required for formation of the secondary structure of the 5.8S gene was -15.2 kcal/mol, and the GC content was 39.3%.

Conserved motifs for the 5.8S gene are not widely reported or described in fungi. However, at least three motifs of the 5.8S gene are conserved among angiosperms: Motif I: 5′-CGAUGAAGAACGUAGC-3′ (Harpke and Peterson 2008); Motif II: 5′-GAAUUGCAGAAUCC-3′ (Jobes and Thien, 1997); Motif III: (5′-UUUGAACGCA-3′) (Harpke and Peterson, 2008). All three motifs were identified in the 5.8S aligned sequences with the exception of a single base substitution (U > C) in Motif II: 5′-GAAUUGCAGAAUUC-3′ (Figure 4). In the 5.8S DNA sequence, Motif II contains an EcoRI restriction site (GAA/TTC) which is conserved in fungi and may serve to discriminate between cognate Motif II sequences in fungi and angiosperms (Jobes and Thien 1997). The presence of all three motifs in each sequence of the 5.8S alignment indicated that no pseudogenes were included in the data set. A search of the Rfam database (http://rfam.xfam.org/; Burge at al., 2012) resulted in a sequence match (bit score =172.3, error value =1.1e-47) for 5.8S rRNA-RF00002 Accession given in the Rfam database. The 5.8S rRNA plays a critical role in ribosome movement and in protein translation and as such, displays a high degree of pan-eukaryotic conservation (Abou-Elela and Nazar 1997).

The ITS2 sequence alignment contained 27 polymorphic sites, 24 singleton sites and four indel sites in the aligned sequences of the data set with four indel haplotypes. The indel haplotype diversity was calculated to be 0.593. Nucleotide diversity (Pi) was calculated as 0.01064 ± 0.00717, which indicates a lower level of diversity to that of the ITS1 marker. The total number of mutations (Eta) was 28. When type sequences were considered, there were two polymorphic sites and two mutations. These polymorphisms ultimately gave rise to two ribotypes (Figure 5); one belonging to all taxa except for C. asianum and C. siamense species (Ribotype 1) and the other appeared exclusive to C. asianum and C. siamense species (Ribotype 2 and 3 respectively). The delta G required for formation of the secondary structures ranged from -58.59 and -52.80 (for Ribotypes 2 and 3, respectively) to -52.93 kcal/mol (for Ribotype 1). The GC content of these sequences averaged 56.17% which is very similar to that of the ITS1 marker.

Homology modelling provided high quality models and recovered a consensus secondary structure for the two ribotypes (Figure 5) which consisted of four helices radiating from a central loop. Four helices were identified and were designated I, II, III and IV. The consensus secondary structure of this region has been described as having four helices of which helix III may be the longest, and contains an “UGGU” motif 5’ to the apex. Sequence variations of this helix, such as “UGGGU”, “UGG”, or “GGU”, have been described in addition to the existence of a U-U mismatch in the second helix which is conserved in the vast majority of eukaryotes (Schultz et al. 2005; Coleman 2007). For each ribotype, percentages of helix transfer were 100% for any of the four helices. C. asianum and C. siamense had 87.14% similarity match for helix IV when ribotypes 1 and 2 (a) and (b) were compared. The ITS2 database motif description was a “U-U” mismatch (helix II, left, at 395–409 nt) “U-U” mismatch (helix II, right) with AAA between helices II and III (at 429–443 nt), “UGGU” on helix III at the 5′ end. The GC content of the ITS2 was 56.19% which was similar to that of the ITS1 marker and indicated that no pseudogenes were present in the data set.

The consensus structure of the ITS2 region was mapped for all type and query sequences (Figure 6). There were several unique taxonomic motifs identified in the secondary structures predicted for C. gloeosporioides sensu stricto especially at terminal loop 2. Similarly for C. asianum and C. siamense, there were distinct base changes, differences in internal loop number, structure and position which appeared to be taxon-specific. Slippage of RNA polymerase during transcription may result in production of mononucleotide repeats (“UUUU”) in the RNA sequences (Levinson and Gutman 1987; Hillis and Dixon 1991). These inadvertent errors in transcription may lead to an increase in the number of detected ITS2 ribotypes.

In this study, the observed variability of the ITS1 marker was higher than that of ITS2 which is in-keeping with the findings of Freire et al. (2012) and Nilsson et al. (2008). Although several conserved nucleotide sequence motifs have been identified in 5.8S and ITS2 sequences (Liu and Schardl 1994; Mai and Coleman 1997), it is the retention of functionally conserved secondary structures that enable the ITS array to play a critical role in the production of mature rRNA molecules (van Nues et al. 1994, 1995; Joseph et al. 1999; Michot et al. 1999; Venema and Tollervey 1999). It is apparent from the level of variation in the nucleotide sequence and predicted secondary structures of the ITS1 and ITS2 markers that different selective pressures may be acting at each markers. Within the ITS array, some regions are under evolutionary constraints at the level of the nucleotide sequence (Liu and Schardl 1994; Mai and Coleman 1997), while others are under positive selection at the level of the secondary structure with the emergence of concomitant compensatory base changes to preserve this structure (van Nues et al. 1994, 1995; Joseph et al. 1999).

Studies have shown that within the C. gloeosporioides sensu lato species complex, ITS sequences are capable of resolving approximately 50% of the accepted species (Weir et al. 2012). This reflects the low number of base changes in the ITS region across the C. gloeosporioides sensu lato species complex. Member species are often distinguished by only one or two base changes. In some cases, the variation in the ITS sequence is insufficient to distinguish among certain members of this species complex. There was only one other study by Bridge et al. (2008) in which the structure types of the ITS1 marker for C. gloeosporioides were compared. At the time of that study, many of the now-known epitype member species of this complex were not used and it is, therefore, difficult to determine the reliability of the predicted structures and precisely to which intra-specific level the identified structures belonged.

Type sequences of C. gloeosporioides sensu stricto, C. asianum, C. fructicola, C. siamense and C. tropicale (Additional file 1: Table S1) were mined from GenBank. These species within the C. gloeosporioides species complex have been commonly identified as the causal agents of anthracnose of tropical fruit (Phoulivong et al. 2010). In selecting the sequences from holotype and epitype specimens for analyses, there were three important considerations, (i) the type sequences were selected based on the study by Cannon et al. (2012), and were included to provide sufficient range of sequence diversity, (ii) the species used are well- characterized and recognized at the phylogenetic and morphological levels, and (iii) the selected species have been analyzed in previous independent studies.

To obtain test sequences, papaya fruit displaying symptoms of anthracnose were collected during the period 2011 to 2013 (Additional file 1: Table S2). DNA was extracted from pure single spore cultures of Colletotrichum sp. using the E.Z.N.A. fungal DNA extraction kit® according to the manufacturer’s instructions (Omega bio-tek Ltd., USA). The entire ITS region (496 bp) was amplified using the universal primer pair ITS4/5 (White et al. 1990) and sequenced with independent base call verification (Amplicon Express, WA, USA). Representative sequences were submitted to GenBank (KM117226 to KM117228). A total of 56 sequences were used in the final data set for generating consensus secondary structures for ITS1, 5.8S and ITS2 markers: 30 type sequences obtained from holotype and epitype specimens and 26 query sequences belonging to the C. gloeosporioides sensu lato complex. Other fungal sequences were also mined from GenBank (HQ238968, JF780523, EU480703, HQ238962, EF543854) and were used as out-groups to assist in defining of helical domains of the three markers based on mfold alignments in the UNAFold webserver (http://mfold.rna.albany.edu/) (Zuker 2003; Markham and Zuker 2008).

Errors can occur during PCR amplification when two different DNA templates may be present. The resulting amplicon may be chimeric, that is, a mosaic of these original sequences (Jumpponen 2011). Such chimeric sequences may be misinterpreted as novel which can artificially inflate estimates of diversity and interfere with phylogenetic inference and species discrimination if undetected (Hugenholtz and Huber 2003). ITS sequences were checked for possible chimeras using the UNITE PlutoF Chimera checker (Nilsson et al. 2010; Edgar et al. 2011) and the Chimera Test developed in the Fungal Metagenomics Project at the University of Alaska (https://biotech.inbre.alaska.edu/fungal_portal/?program=chimera_test).

Alignments were carried out using the online version of the sequence alignment program MAFFT version 6 ((http://mafft.cbrc.jp/alignment/server/ (Katoh et al. 2005; Katoh and Toh 2008). This sequence analysis will also aid in determining if the ITS sequences were composed of stochastic, artifactual nucleotide data. The start and end point of each marker were first defined for each species using the pipeline available on the ITS2 database website (http://its2.bioapps.biozentrum.uni-wuerzburg.de/). Ultimately, three sets of sequence alignments were generated: ITS1, 5.8S and ITS2 as separate data sets.

The sequence length of the ITS1 and ITS2 region for a given species can be variable, however, the two markers should have similar GC content if they are authentic sequences under functional and selective constraints and not pseudogenes (Harpke and Peterson 2008; Mullineaux and Hausner 2009). The GC content of the ITS1, 5.8S and ITS2 sequences was determined using BioEdit version 7.2.0 software. DNASP version 5.10 (Rozas et al. 2003; Librado and Rozas 2009) was used to determine the nucleotide diversity (Pi), polymorphic and singleton sites, indel sites and indel haplotypes among the ITS1, 5.8S and ITS2 sequences.

Ribosomal secondary structure and motif detection was determined for the ITS2 marker. Secondary structure predictions of rRNA sequences are sensitive to single base changes which in turn, can affect hydrogen base-pairing especially along the stem aspect of a stem-loop secondary structure (Matthews et al. 2005). Consequently for this study, the ITS sequences and their electropherograms were manually reviewed and evaluated for signal quality and accurate nucleotide assignment in order to prevent user-induced errors in structural predictions. Because the core folding pattern of the ITS2 sequence is already known, this presents an external criterion or reference to check for the correctness of the predicted structures (Schultz and Wolf 2009). For the ITS2 consensus secondary structure prediction, the ITS2 database pipeline (http://its2.bioapps.biozentrum.uni-wuerzburg.de/) (Koetschan et al. 2010; Merget et al. 2012; Koetschan et al. 2012) was used. Consensus secondary structures for the ITS1 and 5.8S markers were determined using LocaRNA-P simultaneous RNA alignment and folding option of the Freiburg RNA Tools pipeline (http://rna.informatik.uni-freiburg.de:8080/LocARNA/Input.jsp) (Will et al. 2007, Will et al. 2012 and Smith et al. 2010), and the RNA folding form option of the mfold webserver using default conditions for temperature (37°C) and ionic conditions (http://mfold.rna.albany.edu/) (Zuker 2003; Markham and Zuker 2008). Consensus secondary structures for ITS1, 5.8S and ITS2 markers as radial view structures were re-drawn and annotated for publication purposes using VARNA 3.9 (Darty et al. 2009).