Secondary Structure Prediction of ITS Rrna Region and Molecular Phylogeny: an Integrated Approach for the Precise Speciation of Muscodor Species

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 of ITS1 and ITS2 marker region of different Muscodor species tree was carried out in FigTree v1.4.2 (Rambaut 2007). S.No Species name Minimum free energy (kcal/mol)

ITS1 ITS2 Results 1 M. albus − 64.74 − 53.49 2 M. roseus − 74.60 − 51.20 Sequence analysis and construction of phylogenetic 3 M. crispans − 74.60 − 51.70 tree 4 M. yucatanensis − 63.40 − 45.70 5 M. vitigenus − 69.90 − 59.20 The sequence length of each marker region ranges from 6 M. satura − 57.00 − 54.30 158 to 250 nucleotides for ITS1, 157 nucleotides for 5.8S 7 M. cinnanomi − 98.60 − 60.10 and 125–216 nucleotides for ITS2 region. The sequences 8 M. equiseti − 75.40 − 54.80 were of good quality and devoid of any artifacts in the 9 M. musae − 81.10 − 67.10 nucleotide data. The precise boundaries of ITS1 and ITS2 10 M. suthepensis − 73.10 − 52.80 RNA structure in M. albus (12 strains) and − − 11 M. fengyangenesis 91.55 45.96 M. fengyangensis (5 strains) were deduced by STAR − − 12 M. kashayum 59.70 46.40 (structure-based alignment reliability) profile plots − − 13 M. darjeelingensis 60.20 46.40 (Supplementary file, S1). The phylogenetic tree using pri- − − 14 M. tigerii 59.90 46.40 mary sequence data of ITS region was constructed using − − 15 M. strobelii 48.70 38.50 neighbor joining and maximum likelihood method (data − − 16 M. ghoomensis 54.00 44.40 presented in Supplementary file S2). However, using ITS − − 17 M. indica 44.80 42.60 region, these methods were not able to resolve different 18 M. hevae − 82.80 − 50.10 species of Muscodor genus with significant bootstrap sup- 19 M. oryzae − 101.20 − 59.30 port. Therefore, incorporating secondary structures along with sequence similarities to obtain better resolving ΔG for 5.8S rRNA is − 44.20 kcal/mol Muscodor speciation became an amenable hypothesis.

Fig. 1 The predicted minimum free energy (MFE) secondary structures of ITS1 region from seven different Muscodor species a Muscodor albus (Consensus structure), b M. yucatanensis, c M. satura, d M. roseus, e M. suthepensis, f M. oryzae, g M. crispans. All the species formed common three helix configuration core structure except M. crispans which was folded in two helix arrangement 766 Ann Microbiol (2018) 68:763–772

Fig. 2 The predicted minimum free energy (MFE) secondary structures of ITS1 region from seven different Muscodor species a M. kashayum, b M. tigerii, c M. darjeelingensis, d M. strobelii, e M. musae, g M. equiseti

Prediction of secondary structure 101.20 kcal/mol and − 38.50 to − 67.10 kcal/mol for ITS1 and ITS2 regions, respectively (Table 1). In the eu- The ΔG required for the formation of secondary structure karyotic taxa examined so far, the folding pattern of ITS1 of different Muscodor species ranges from − 44.80 to − secondary structure is variable from one taxonomic group

Fig. 3 The predicted minimum free energy (MFE) secondary structures of ITS1 region from seven different Muscodor species a Muscodor vitigenus, b M. ghoomensis, c M. heveae, d M. indica, e M. fengyangensis Ann Microbiol (2018) 68:763–772 767 to another, but the region is characterized by the formation 43.94%. The predicted secondary structure comprised a cen- of a main central loop along with multiple double-stranded tral internal loop from which four helices emerged (Fig. 4). All helices (Lalev and Nazar 1998;Campbelletal.2005; the three motifs viz. M1, M3 (Harpke and Paterson 2008), and Mullineux and Hausner 2009). However, the MFE folding M2 (Jobes and Thien 1997)reportedincaseofangiosperms pattern of ITS2 region is characterized by the formation of were identified in the 5.8S region of all Muscodor species as conserved core structure as a central bulge with four (base substitutions are highlighted in bold) M3: (5′- double-stranded helices radiating from it. These helices UUUGAACGCA-3′), M1 (5′-CGAUGAAGAACGCAGC- are designated as I-IVof which helices II and III were most 3′), and M2 (5′-GAAUUGCAGAAUUC-3′). There was a sin- conserved (Coleman 2007; Hunter et al. 2007;Kocotand gle base substitution in motif M1 (C > U) and M2 (U > C). Santos 2009; Glass et al. 2013). Structure prediction of ITS2 region Structure prediction of ITS1 region The analysis of MFE secondary structures based on ITS2 re- The MFE secondary structures based on ITS1 region of all gion exhibited diverse folding pattern among different Muscodor species exhibited a consistent folding pattern Muscodor species. All the Muscodor species except consisting of central loop with variable number of helices M. fengyangenesis, M. tigerii, M. kashayum, M. indica, as reported in case of various other eukaryotes M. strobelii,andM. darjeelingensis are known to contain U- (Gottschling et al. 2001; Gottschling and Plotner 2004; U, A-G mismatch bulges and highly conserved eukaryotic Beiggi and Piercey-Normore 2007; Thornhill and Lord 2010). Formation of three helix folding pattern with helix II being the longest has been reported and explored for speciation of anaerobic fungi (Koetschan et al. 2014). ITS1-based secondary structures of all Muscodor species formed big central loop while six species viz. M. albus, M. roseus, M. yucatanensis, M. satura, M. suthepensis, and M. oryzae folded in a common three helix configuration. The helices were designated as I, II, and III, of which helix II was the longest with other structural motifs such as unpaired, mismatched nucleotides in small internal loops, bulges, and hairpin loop (Fig. 1a–f). M. crispans exhibited two helices formation from the central loop with helix I being the longest in comparison to above-mentioned Muscodor species (Fig. 1g). Further, M. kashayum, M. tigerii,andM. darjeelingensis also folded into three- helix core structure of different nucleotide length and vary- ing presence of bulges and internal loops (Fig. 2a–c). Interestingly, M. strobelii, M. musae, M. cinnanomi,and M. equiseti formed five helices of which M. musae, M. cinnanomi,andM. equiseti formed two major (designated as I-II) and three minor helices (designated a- c). Within minor helices, helix ‘b’ of M. musae, M. cinnanomi and helix ‘a’ of M. equiseti included unpaired and mismatched nucleotides (Fig. 2d–g). Further, five Muscodor species viz. M. vitigenus, M. ghoomensis, M. heveae, M. indica,and M. fengyangenesis folded into four helix configuration, of which M. ghoomensis revealed the formation of unpaired helix in between the helix II and III (Fig. 3).

Structure prediction of 5.8S region

The 5.8S region spanning 157 nucleotides was highly con- Fig. 4 The predicted consensus secondary structure of conserved 5.8S Δ region of Muscodor species. The three conserved motifs (M1, m2, and served. The G required for the formation of secondary struc- M3) were detected and highlighted in yellow color for M3, red for m1, ture was − 44.20 kcal/mol and GC content was estimated to be and blue for M2 768 Ann Microbiol (2018) 68:763–772

Fig. 5 The predicted minimum free energy (MFE) secondary structures of ITS2 region from seven different Muscodor species a Muscodor albus (consensus structure), b M. roseus, c M. oryzae, d M. heveae, e M. equiseti, f M. vitigenus, g M. satura. Conserved motif 5′- UGGU-3′ and U-U mismatch was detected

motif 5′-UGGU-3′ at the apex of helix II (Schultz et al. 2005; 5′ apex of the helix has been shown to differ slightly as 5′- Wolf et al. 2005;Coleman2007). However, in case of UAGU-3′, which may occur due to single-base substitution in M. fengyangenesis, highly conserved motif UGGU near the some eukaryotes (Schultz et al. 2005). The consensus

Fig. 6 The predicted minimum free energy (MFE) secondary structures of ITS2 region from six different Muscodor species a M. suthepensis, b M. cinnanomi, c M. yucatanensis, d M. crispans, e M. fengyangensis (consensus structure), f M. musae. Conserved motif 5′-UGGU-3′ and U-U mismatch was detected Ann Microbiol (2018) 68:763–772 769 secondary structure of M. albus, M. roseus, M. oryzae,and M. darjeelingensis, M. tigerii, M. indica,andM. strobelii M. heveae folded to form central loop containing three helices produced a long double helix differing in all species due to (designated I-III) radiating outwards. Helix III of M. albus branching, presence, and absence of additional bulges and formed A-G mismatch bulge and a small hairpin loop at the internal loops (Fig. 7). apex while M. heveae formed two unpaired loop nucleotides (Fig. 5a–d). Further, secondary structures of M. equiseti, Phylogenetic tree reconstruction M. vitigenus,andM. satura also folded in three helix configuration with additional three minor helices (designated a-c) The PNJ tree based on the combined sequence-structure emerging from the central core (Fig. 5e–g). dataset of 19 different species of Muscodor genus resolved However, M. suthepensis, M. cinnanomi, the M. fengyangenesis species as distinct monophyletic group M. yucatanensis, M. crispans,andM. fengyangenesis (group III). Twelve strains of M. albus did not cluster into a folded to form a large central loop containing four helices single group (group I). However, Muscodor sp. OM-01 while M. musae folded into five-helix configuration (Accession no. KF229762) clustered in group II along with (Fig. 6). The helix III of M. equiseti, M. vitigenus, M. vitigenus, M.equiseti, and M. yucatanensis when using M. yucatanensis, M. fengyangenesis, M. musae, combined sequence and structure information for phylogenet- M. satura,andhelixIVincaseofM. crispans was homol- ic study. Further, M. albus GP-100 (Accession no. ogous, longest, and contained a small internal ring of un- AY555731), M. albus (Accession no. AY244642), and paired nucleotides, non-canonical A-C, U-C mismatch M. albus 9-6 (Accession no. HM034857) emerged as a sister bulges and AUUA (M. equiseti, M. vitigenus,and clade from group III and group IV, respectively. Only M. satura), AAUC (M. musae and M. crispans), ACAU M. crispans and M. suthepensis were clustered significantly (M. yucatanensis), UAAUUUCUU (M. fengyangenesis) with high bootstrap support value in group IV while all other sequence near the apex. M. ghoomensis, M. kashayum, species viz. M. ghoomensis, M. indica, M. kashayum,

Fig. 7 The predicted minimum free energy (MFE) secondary structures of ITS2 region from six different Muscodor species a Muscodor ghoomensis, b M. kashayum, c M. darjeelingensis, d M. tigerii, e M. indica, f M. strobelii 770 Ann Microbiol (2018) 68:763–772

M. darjeelingensis, M. strobelii,andM. heveae were emerging differ at intergenic as well as intragenic levels. Earlier stud- out as sister clade (Fig. 8). iesonITS1regionofanaerobicfungi(Phylum Neocallimastigomycota) were shown to form common three helix structure (Koetschan et al. 2014). In the present Discussion study, nine Muscodor species formed common three helix configured structure with a common central loop. Further, Molecular phylogeny plays a crucial role in the identifica- M. kashayum, M. darjeelingensis,andM. tigerii ITS1 re- tion of Muscodor genus which is a group of non- gion sequence folded three helix configuration but showed sporulating endophytic fungi. The sequence analysis of demarcation due to lack of sequence conservation in com- ITS rRNA region has been the method of choice for phy- mon core structure. On the other hand, ITS2 folding pat- logenetic and taxonomic placement studies of fungi tern of these three species shared similarities with minor (Rampersad 2014). However, sequence variability of differences in outer bulges of unpaired sequences. Thus, ITS1 region across species of the same genus is a con- theITS1regionofMuscodor species was observed to be straint in constructing reproducible and robust phylogentic significantlyvariableascomparedtoITS2regionatstruc- relationship. Interestingly, in the present study, it was ob- tural level. These findings are in accordance with Freire served that the phyogenetic trees based on individual re- et al. (2012) in which the authors explored the structural gions of ITS1, 5.8S, ITS2, and combined ITS1–5.8S-ITS2 features of ITS1 and ITS2 markers of the obligate cassette using neighbor joining method were not able to biotrophic fungus, Phakopsora pachyrhizi. resolve the species in the respective groups. Similar speci- The 5.8S region was observed to be highly conserved ation of different Muscodor species was observed in max- and motif analysis confirmed the presence of conserved imum likelihood-based tree of ITS region, thus eliminating eukaryotic motifs (M1, M2, and M3) among all the method biasing. Hence, it was hypothesized that inte- Muscodor species but with a single base substitution in grating secondary structure data along with primary se- MotifM1(C>U)andM3(U>C).Similarsinglebase quence, a better speciation of Muscodor species could have substitution in motif M3 (U > C) was found in 5.8S region been achieved. The proposed secondary structure models of Colletotrichum gloeosporiodes sensu lato species com- of ITS1 and ITS2 marker regions of Muscodor species plex (Rampersad 2014). P. pachyrhizi displayed three

Fig. 8 Profile neighbor joining (PNJ) tree of Muscodor species based on ITS sequence and structure data. The tree was generated by using general time reversible model of evolution with 1000 bootstrap replicates Ann Microbiol (2018) 68:763–772 771 substitutions in motif M1 (C > U, A > G, U > C) and single Funding information This study was financially supported by the substitution (U > C) in M3 (Freire et al. 2012). The ITS2 Department of Biotechnology (DBT) (National Biodiversity Development Board), Government of India, through project no. BT/ region mapping of all Muscodor species except PR/10083/NDB/52/95/2007 (Principal Investigator: Prof. Sanjai Saxena). M. fengyangenesis consisted of pyrimidine-pyrimidine (U-U) mismatch and highly conserved 5′-UGGU-3′ motif of eukaryotes. The secondary structures of ITS2 in References Muscodor species displayed conserved features as expect- ed for eukaryotes in ITS2 region. 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