Sequence comparison of the ribosomal DNA internal transcribed spacers (ITSs) and 5.8S ribosomal gene and their secondary structures in 17 Pteriomorphian bivalves
K Ashokan, M Pillai, S Angadi, D Mundaganur
5.8srrna gene, internaltranscribed spacer, phylogentic analysis, pteriomorphian bivalves, secondary structure
K Ashokan, M Pillai, S Angadi, D Mundaganur. Sequence comparison of the ribosomal DNA internal transcribed spacers (ITSs) and 5.8S ribosomal gene and their secondary structures in 17 Pteriomorphian bivalves. The Internet Journal of Genomics and Proteomics. 2008 Volume 5 Number 1.
The internal transcribed spacer (ITSs) of nuclear ribosomal DNA (rDNA) is one of the most extensively sequenced markers, and the region is a component of an rDNA cistron, which consists of 18S, ITS1, 5.8S, ITS2 and 28s. ITSs, exists in several hundred copies in most eukaryotes. They are located in one or several loci and distributed in one or several chromosomes. The nuclear rDNA copies within a genome can be highly homogenous because of concerted evolution of intra and inter chromosomal loci. ITS1 and ITS2 are non coding regions located in rDNA between 18s and rRNA gene respectively (Insune
The transcripts folding structure of the ITS1 and ITS2 provide some signals that guide the ribosomal coding regions where they are processed into small 5.8S and large ribosomal RNA (Vander Sande
The class bivalves are one of the most important members of most marine and fresh water ecosystems. It includes 6 subclasses. The subclass pteriomorpha includes entirely marine forms. Bivalves are one of the abundant and diverse groups of marine fauna. The phylogeny of mollusca, including bivalves is controversial subject (Sigwat
Materials and methods
ITS1, ITS2 and 5.8S sequences of the 17 pteriomorphian bivalve species belongs to diverse geographical locations that are deposited in Genbank were investigated. The accession numbers, species scientific name, common name and geographical locations are listed in Table1
Sequence analysis and phylogenetic tree construction
Multiple sequence alignment was performed by using MegaAlign of DNA star package using Clustal W method. The trees were produced by Neighbor-joining (NJ) and maximum parsimony (MP) methods using MEGA (Molecular Evolutionary Genetics Analysis, Version.4) (Tamura
those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura K, Nei M & Kumar S 2004) and are in the units of the number of base substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). Phylogenetic analyses were conducted in MEGA4 (Tamura
The evolutionary history was also inferred using the Maximum Parsimony method (Eck & Dayhoff 1966). The MP tree was obtained using the Close-Neighbor-Interchange algorithm (Eck & Dayhoff 1966) with search level 2 (Eck & Dayhoff 1966, Nei & Kumar 2000) in which the initial trees were obtained with the random addition of sequences (10 replicates). Phylogenetic analyses were conducted in MEGA4 (Tamura
Estimates of Evolutionary Divergence between Sequences.
The number of base substitutions per site from analysis between sequences is shown. All results are based on the pair wise analysis of 17 sequences. Analyses were conducted using the Maximum Composite Likelihood method in MEGA4
The motif was identified by using SeSiMCMC (Sequence Similarities by Markov Chain Monte-Carlo) algorithm, which finds DNA motifs of
Secondary structure prediction
The RNA secondary structure for ITSs was predicted by using RNADRAW online software (Christoffersen
The ribosomal DNA ITSs, 5.8S rRNA gene and rDNA ITS2+5.8S rRNA gene regions are analyzed for its sequence length, GC contents, AT contents, Motifs and secondary structural parameters.
Sequence analysis of first internal transcribed spacer
The length of ITS1 sequence ranged from 21bp to 475bp and GC contents from 25.84% to 50.00 % in 17 species
Among Mimichlamy species the genetic distance ranged from 1.751%-9.009 %. Among Perna species the genetic distance ranged from 0.055%-10.494 %. The other species shows genetic distance variably lower than these genera.
We find relatively conserved motifs in ITS1 region across the 17 species of pteriomorphian bivalve species, the consensus sequence (39bp) of these motifs is depicted in Fig 3.The motifs show 11 polymorphic sites, 7 of which were transition and 4 of which were transversion.
Phylogenetic analysis was conducted with neighbor-joining and maximum parsimony method. All obtained tree showed the same topology and differed only in their supportive values for certain branch (Fig 2).
Sequence analysis of the second internal transcribed spacer
The length of ITS2 sequence ranged form 233bp to 504bp and GC contents from 28.91% to 51.69% (Table 2). The length variation for ITS2 was less variable in all selected species. The contents of GC in ITS2 sequence were higher than AT in all the species studied. Sacostrea kegaki had the longest ITS2 sequence (504bp) with 48.10% GC contents,
The length of 5.8S rRNA gene was 127bp to 167bp (Table 2).The GC content ranged from 32.73% to 46.67% depending on species. The sequence divergence ranged from 0.000% to 19.319% across 17 species analyzed (Table 4).
Phylogenetic analysis with ITS2 contain 5.8S rRNA gene sequence
In contrast to ITS2, the construction of phylogeny using ITS1 was not applicable because of some disadvantages, such as ITS1 length variation, the presence of tandem repeated sequence, and large number of indels. ITS1 sequence analysis using neighbor-joining and maximum-parsimony method gives poor resolution. Therefore ITS1 was not used for phylogentic analysis.
Secondary structure analysis of ITSs and ITS2 + 5.8S RrRNA gene
Secondary structure of the remaining species is highly variant. Three common motifs, having sequesnce
The present study provided information about the nucleotide sequence of ITS1 and ITS2 regions, complete sequence of 5.8S ribosomal DNA gene in 17 species of pteriomorphian bivalves. Their characteristics and variation were demonstrated through sequencing. The length of ITS1 in the family veneridae were longer that in 4 Pectinade scallop with size 209bp to 276bp for ITS1 and 270bp to 294bp for ITS2 (Insua et al., 2003) respectively. The length ranges in ITS1 and ITS2 reported in the present study was one of the largest observed in bivalves .The size of ITS is species- dependent and difference could be significant among species. The largest ITS1, such as that in Ladybird beetle
Extensive sequence variation and obvious length polymorphism were in both ITS1 and ITS2 regions in all the three family, pectinidae, mytilinidae and ostriodae similar to crustaceae Eriocheir formoca (Chen et al., 2002) and other bivalves (Ding et al., 2004). In the present study, the construction of topology in pteriomorphian bivalve species using ITS1 sequence information is not applicable because of the higher length variation, the presence of number of repeated sequences and large number of indels in ITS1. But, it is better in phylogenetic analysis at lower taxonomic levels. In contrast, the interspecific ITS2 were shorter than that of ITS1, providing advantage and convenience in designing primer and sequencing, the study of genetic structure in all the three families of pteriomorphian bivalves. In the present study, three relatively conserved motifs in ITS2 were found in the all the species analyzed. This indicates that these motifs might be involved in certain nucleotide acid-related functions, such as in rRNA processing (Insua et al., 2003). This study also reports two dinucleotide microsatallite (AC) and (GC) in ITS1 and ITS2 regions. These microsatallites can be used as good markers in future studies. Presence of dinucliotide and trinucleotide microsatallites were reported in ITSs sequences in Lasigona (King et al. 1999, Chen, 2006). Inter individual divergences in ITS2 and ITS2 + 5.8S regions were detected in Perna picta, Ostrea circumpicta (0.01%) and Semipallium fulvicostat, Mimichlamys nobilis, Mimichlamys sp.NT-2006,
The secondary structure prediction was performed in both ITS1, ITS2 and ITS2 contain partial 5.8S rRNA gene to find out the conserverdness of the sequence in various species of pteriomorphian bivalve species (Table 3). The stems (double stranded paired region) stabilize RNA secondary structure and the number of stems present in each ITS1, ITS2 and ITS2 + 5.8S rRNA is given in Table 2. ITS2 RNA structure form species in ostreidae and spondylidae family shows highest negative free energy ranging -162 kcal to -222.7 k cal. This is followed by mytilidae species and pectinidae species ranging from -128 kcal to -141.01 kcal, indicating the divergence of this family occurred at different periods, with greater stability in ostraeidae RNA might indicate it evolve before the evolution of pectinidae and mytilidae. Both ostreidae and pectinidae evolved in Ordovician period, but mytilidae evolved in Devonian period (Schneider, 2001). To substantiate the early evolution of ostreidae require further study in molecular level. The same trend in negative free energy was found in ITS1 also, but the variation is higher than ITS2.Visual comparison shows that this is related to the trend in the cladogram given in Fig 2 .This convergence at secondary structural level among species from different geographical isolates may be due to the evolutionary pressure on ITSs to maintain the RNA secondary structure involved in post transcriptional processing of rRNA (Shinohara et al., 1999). Secondary structures predictions for ITS1 and ITS2 regions show that their domain, base pair to form a core region central to several stem features inferring the conserverdness is more important for the proper rRNA folding pattern (Wesson et al., 1992). Fig 5 shows the distribution of loops among different isolates. The segments of ITS2 having score >50 are further probed carefully for target site to asses the likelihood of un-paired segments. Interestingly, the observed phylognetic trend was identical with respect to the target accessibility sites for the 17 isolates. The order of preference is, inter loops; bulge loops multiple loops, hairpin loops and exterior loops in all the species analyzed, except in Mimichlamys senatoria, Decatopecten radula, Ostrea circumpicta and Crassostrea crenulifera where multiple loops are second to interior loops. These results suggest that the differences and conserverdness observed between ITSs of different species are not ‘natural’ and are not simple accumulated random nucleotide changes, but bare a significant functional load. In the previous study of 3 related mosquito genus (aedes, psorophora and haemogogu) (Wesson et al., 1992) it was found that intra spacer variable region appear to co-evolve and that ITS2 variation is constrained to some extent by its secondary structure. Further studies on yeast (Vander Sanade et al., 1992) have demonstrated that the ITS2 is central for the correct and efficient processing after the removal of ITS2 from its RNA precursor is dispersed through the entire ITS2 region and indel that affect secondary structure differentially alters rRNA processing. Critical changes in the rRNA folding pattern brought about by sequence evolution in the ITS spacer regions may thus have an important influence on the kinetics of precursor RNA formation, and ultimately on the efficient functioning of the rDNA cluster.
The spacer regions, ITS1 and ITS2of rDNA are widely and routinely used in analysis of species relationships by using a phylogentic recon structure method in various organisms. It was successfully applied in analyzing of phylogenetic relationship among the Biomphaaria species and among pearl oysters and the conclusions from phylogenetic tree was well in agreement with those from analysis based on morphological systematics and other molecular techniques, such as polymer chain reaction and restriction fragment length polymorphism analysis (He et al., 2005, Vidgal et al., 2000). Our study demonstrated that ITS1 provide week phylogentic relationship among species from three families (Pectinidae, mytilidae and ostreidae). In this study the tree obtained by sequence analysis revealed that member of pectinidae (Amusium pleuronectes, Mimichlamys nobilis) has close relationship with mytiloidae (Perna picta, Perna canaliculus, Aulacomya atra maoriana) but pectinidae species Mimichalmys senatoria, Spondylus varius shows more relationship to ostreidae. Phylogentic analysis of ITS2 sequence through both methods (Neighbor-joining and Maximum parsimony) generated forms with similar topology that were very concurrent with the morphological taxonomy proposed previously. Therefore ITS2 sequence characteristics and secondary structure prediction are efficient tools in reconstruction of evolutionary relationship among these organisms, and they can be applied in establishment of species relationship, or reevaluation of the traditional taxonomy.
The present study shows two contrasting aspects of ITSs regions i.e. the general trend of variability among the species as well as the conservedness between few species. Surprisingly the species displaying the conservedness belong to different geographical locations with diverse ecological conditions. Our study implies that ITSs region though have less selective pressure than the ribosomal regions but still evolve slower than the intergenic spacers, indicating that some selective pressure do exist on them, probably from the constraint to maintain the RNA secondary structure required for post-transcriptional processing and are more species specific than geographically influenced. Several common structural folds were shared among the selected pteriomorphian bivalve species for maintaining functional equivalents. Identifying the homologous regions and reconstructing their evolution increase the traits available for the phylogenetic analysis. The present studies indicate that the class pectinidae shows more closeness to mytiloidae than to ostreidae with monophyletic relationship among the species. It also shows that ITS2 is more powerful, than ITS1, tool with their secondary structural analysis to reconstruct phylogenetic tree.
The authors thanks to PVP College principal Dr.AShok.V.Babar to provide us broadband internet facility and various soft wares required for the present study and valuable advise to complete the work in time.