Annals of Microbiology (2018) 68:763–772 https://doi.org/10.1007/s13213-018-1381-8 ORIGINAL ARTICLE Secondary structure prediction of ITS rRNA region and molecular phylogeny: an integrated approach for the precise speciation of Muscodor species Neha Kapoor1,2 & Lokesh Gambhir3 & Sanjai Saxena1 Received: 23 March 2018 /Accepted: 21 September 2018 /Published online: 29 September 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018 Abstract Muscodor is a non-sporulating, volatile organic compounds producing endophytic fungi that has been extensively explored as a bio-fumigant and bio-preservative. Novel species of this genus have been mainly identified using ITS sequences. However, the ITS hyper-variability hinders the creation of reproducible alignments and stable phylogenetic trees. Conserved structural data of the ITS region represents as a vital auxiliary information for accurate speciation of fungi. In the present study, secondary structural data of ITS1, 5.8S, and ITS2 region of all Muscodor species were generated using LocaRNAweb server. The predicted secondary structural data displayed greater variability in ITS1 region in comparison to ITS2. The structural data of all sequences exhibited characteristic conserved features of eukaryotic rRNA. Evolutionary conserved motifs were found among all 5.8S and ITS2 sequences. Profile neighbor joining (PNJ) tree based on combined sequence-structural information of ITS region was generated in ProfDists. The PNJ tree resolved into four major groups whereby M. fengyangenesis and M. albus species formed monophyletic clades. However, three M. albus species along with other Muscodor species emerged as sister branches to the existing clades, thereby, improving the precision of phylogenetic analysis for identification of novel species of Muscodor genus. Hence, the results indicated that structural analysis along with primary sequence information can provide new insights for precise identification of Muscodor species. Keywords Endophytic fungi . rRNA . Muscodor species . ITS . Secondary structure Introduction been isolated and reported from tropical and subtropical flora in Australia, Central/South America, China, Thailand, and The genus Muscodor was established with the discovery of India (Strobel 2015). The unique attribute of this genus is Muscodor albus, a sterile endophytic fungus which was first the release of a characteristic mixture of volatile organic com- isolated from Cinnamomum zeylanicum in a botanical garden pounds, which have been exploited as volatile antimicrobial of Honduras. Since then, many members of this genus have agents, potential fuels, as well as biofumigants (Hutchings et al. 2017). Based on the morphological and molecular char- acteristics and the profile of these volatile organic compounds, Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-018-1381-8) contains supplementary 19 species have been added to this genus up to now (Saxena material, which is available to authorized users. et al. 2015). The major constraint in identification and speciation of * Sanjai Saxena Muscodor genus is its non-sporulating nature. There are mul- [email protected]; [email protected] tiple strategies to accurately identify novel Muscodor species among which the most commonly used is ITS rRNA sequence 1 Department of Biotechnology, Thapar Institute of Engineering and analysis (Suwannarach et al. 2013). ITS region is a highly Technology, Patiala, Punjab 147004, India polymorphic multigene family. However, the polymorphism 2 Present address: Department of Microbiology, Uttaranchal (P.G) is not uniform across the ITS cassette due to the presence of College of Biomedical Sciences and Hospital, Dehradun, Uttarakhand 248001, India highly conserved 5.8S region between ITS1 and ITS2 of the nuclear rRNA cistron. Hyper-variable regions ITS1 and ITS2 3 Department of Biotechnology, Shri Guru Ram Rai University, Dehradun, Uttrakhand 248001, India have been used as a primary choice of molecular identification 764 Ann Microbiol (2018) 68:763–772 as well as formal fungal barcode. Hence, reliability of these M. fengyangensis species complex. As it has been found that sequences is of extreme importance (Nilsson et al. 2012; the substantial portion of nucleotide sequences in publicly Schoch et al. 2012). However, the hyper variability of ITS1 available databases are chimeric, the sequence under the pres- region as compared to ITS2 region complicates the generation ent study was therefore checked for the purity using UNITE of reproducible sequence alignments and reconstruction of PlutoF chimera checker (Nilsson et al. 2010; Edgar et al. 2011) stable and robust phylogenetic trees for accurate speciation of fungi. Multi-locus-based taxonomy is commonly adopted Sequence assembly and phylogenetic tree for precise speciation of various fungi (Donnell et al. 2012; construction Marques et al. 2013). However, this strategy is limited by the lack of sequence information of Muscodor species (Yuan et al. An intensive phylogenetic analysis of all the retrieved se- 2011). Thus, there is a need for delineating an alternative quences based on ITS1, 5.8S, ITS2, and all of them together combinatorial strategy incorporating additional parameter was conducted in MEGA 5.2 (Tamura et al. 2011). The se- along with sequence alignment to construct a phylogenetic quences were aligned using ClustalW in MEGA 5.2, and evo- tree for accurate speciation. lutionary relationship was inferred by employing neighbor Though ITS1 and ITS2 sequences display greater se- joining (Saitou and Nei 1987) and maximum likelihood meth- quence variation among different species of the same ge- od. Bootstrap analysis (1000 bootstrap) was conducted to infer nus, they still exhibited significant levels of structurally the consensus tree (Felsenstein 1985) for the representation of conserved regions (Hausner and Wang 2005). ITS1 and phylogenetic diversity and evolutionary relationship. ITS2 conserved secondary structures have been deduced for a wide variety of eukaryotic groups including fungi Secondary structure prediction and motif detection (Barik et al. 2011; Koetschan et al. 2014), dinoflagellates (Thornhill and Lord 2010), and nematodes (Ma et al. 2008) Secondary structures of ITS1, 5.8S, and ITS2 marker were for gaining insights to illustrate phylogenetic relationships generated using LocaRNA-P simultaneous RNA alignment at different taxonomic levels (Schultz and Wolf 2009). and folding option of the Freiburg RNA Tools web server Hence, an integrated approach utilizing primary sequence (http://rna.informatik.uni-freiburg.de:8080/LocARNA/Input. data and secondary structure to generate reproducible jsp)(Smithetal.2010;Willetal.2012)andRNAfoldweb alignments and stable phylogenetic tree appears to be an server ([email protected]) hosted by Institute of Theoretical amenable method of delineating species identification and Chemistry, University of Vienna (Hofacker 2004). The three enhancing phylogenetic resolution (Seibel et al. 2006; conserved motifs M1, M2, and M3 among eukaryotes were Wolf et al. 2008;Letschetal.2010; Koetschan et al. also predicted. The minimum free energy (MFE) method 2014). For this reason, in the present study, we have uti- utilizing dynamic programming algorithm and partition lized the cumulative sequence-structure data of ITS region function algorithm was adopted to predict the secondary (ITS1 and ITS2) from rRNA for the precise speciation of structures as it provides the lowest free energy secondary Muscodor species. Secondary structures of ITS region structure that indicates high occurrence likelihood. Apart were predicted and compared. Further, the secondary struc- from the MFE, centroid structures (i.e., the structure with ture data and the sequence alignment were employed to minimal base-pair distance to all structures in the thermo- construct a phylogenetic tree. dynamic ensemble) and positional entropy (entropy of base given by the probability of forming the pair) were used to compare and validate the MFE-generated structures Material and methods (Mathews et al. 2004;Gruberetal.2008). Retrieval of datasets Sequence-structure assembly, alignment, and phylogenetic tree construction Type sequences of Muscodor species reported till date were retrieved from GenBank database at NCBI server on 14th ThesequenceandstructuredataofallMuscodor species February, 2016. The criteria behind selecting holotype lies were synchronously aligned using ClustalW (Larkin et al. within the fact that the type sequences exhibit enough se- 2007) to generate a multiple alignment of sequence- quence diversity and all the species were characterized at mor- structure data in 4SALE v 1.7 (Seibel et al. 2006;Seibel phological and molecular levels. Final data set comprised 36 et al. 2008). Further, phylogenetic relationship among all sequences for phylogenetic reconstruction and secondary Muscodor sp. was inferred by profile neighbor joining as structure prediction for ITS1, 5.8S, and ITS2 regions, out of implemented in ProfDistS 0.9.9 (Friedrich et al. 2005;Wolf which 19 were type sequences belonging to holotype speci- et al. 2008) by the use of sequence structure specific gen- mens, 12 sequences to M. albus, and 5 sequences were from eral time reversible (GTR) model of substitution and 1000 Ann Microbiol (2018) 68:763–772 765 Table 1 Showing ΔG required for the formation of secondary structure bootstrap replicates. The visualization of the phylogenetic