7. MOLECULAR IDENTIFICATION OF POLYCHAETES BASED. ON 18S rrna GENE

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1 7. MOLECULAR IDENTIFICATION OF POLYCHAETES BASED 7.1. Introduction ON 18S rrna GENE Molecular tools have revolutionized the exploration of biodiversity, especially in organisms for which traditional taxonomy is difficult (Tang et al., 2012). The small subunit (SSU) 18S rrna gene is one of the most frequently used genes in phylogenetic studies and an important marker for random target PCR in environmental biodiversity screening. 18S rdna is a nuclear ribosomal gene, commonly referred to as the nuclear small ribosomal subunit (SSU), ~ nucleotides in length. This gene is part of a tandem repeated element in the nuclear genome that also includes the 28S, 5.8S, and the internal transcribed spacer regions (ITS) (Hillis and Dixon, 1991). In general, 18S rdna gene sequences are easy to access due to highly conserved flanking regions for the use of universal primers (Hillis and Dixon, 1991). Their repetitive arrangement within the genome provides excessive amounts of template DNA for PCR, even in smallest organisms. The 18S rrna gene is part of the ribosomal functional core and is exposed to similar selective forces in all living beings (Moore and Steitz, 2002). Thus, when the first large-scale phylogenetic studies of the animal kingdom based on 18S rrna sequences were published - the gene was renowned as the prime candidate for reconstructing the metazoan tree of life (Field et al., 1988). In fact, 18S rdna sequences later provided evidence for the splitting of Ecdysozoa and Lophotrochozoa (Aguinaldo et al., 1997; Halanych, 2004), thus contributing to the most recent revolutionary change in our understanding of metazoan relationships. Methodological innovation 103

2 within the last years came from the incorporation of secondary structure into phylogenetic analyses. In particular RNA specific substitution models considering paired sites in rrna genes have been shown to outperform standard DNA models (Telford et al., 2005; Reumont et al., 2009; Tsagkogeorga et al., 2009; Jow et al., 2002; Dohrmann et al., 2008; Mallatt et al., 2010). In the traditional classification polychaete annelids have been classified into over 80 families (Fauchald, 1977). The phylogenetic relationships of these polychaete taxa are matter of debates in the papers on annelid morphology (Meyer and Bartolomaeus, 1996; Purschke, 1997; Rouse and Fauchald, 1997; 1998; Westheide, 1997; Bartolomaeus, 1998; Hausen and Bartolomaeus, 1998; Westheide et al., 1999; Hausen, 2001). Rouse and Fauchald (1997) suggest that the Polychaeta comprise two major clades, the Scolecida and the Palpata. The highest ranked taxa within the Palpata are the Canalipalpata (comprising the remaining so-called Sedentaria ) and the Aciculata (formerly termed Errantia). This view was challenged by Bartolomaeus, (1998) and Hausen, (2001), who interrogated the monophyly of these taxa. Whereas the above-mentioned hypotheses on polychaete systematics have all been obtained by analysing morphological data, many attempts have been made to unravel polychaete relationships using molecular data (McHugh, 1997; Kojima, 1998; Brown et al., 1999; McHugh, 2000; Halanych et al., 2001; Rota et al., 2001; Struck et al., 2002; Bleidorn et al., 2003a). While none of these analyses can be regarded as a major breakthrough in polychaete systematics, some of them have shown that traditional polychaete families are often well supported (Struck et al., 2002; Bleidorn et al., 2003). Increased taxon sampling, gives the possibility to test the hypotheses on polychaete systematics developed through the cladistic analysis by Rouse and Fauchald (1997). 104

3 Studies in molecular data and particularly sequences of the 18S rrna gene are the most frequently used to analyse annelid phylogeny (McHugh, 2000; 2005; Bleidorn et al., 2003a). However, the phylogeny within the Polychaeta has not been resolved by these approaches. Neither of the above mentioned suggestions can be supported nor be rejected by the molecular data at hand today (McHugh, 2000; 2005). In this chapter it was aimed to estimate the relationship between the polychaete taxa based on 18S rrna gene sequence Materials and methods Samples The DNA samples used in the previous chapter on COI gene was taken for the study of 18S rrna gene also. Briefly, polychaetes samples were collected from Vellar estuary, Tamil Nadu, India and DNA was extracted by the standardized method (chapter 1) PCR amplification of 18S rrna gene The small subunit gene was amplified using primers obtained from previous literature (Medlin et al., 1988) showed in Table 7.1. Table 7.1. Primer and sequence details for 18S rrna gene Name of the primer 18SA 18SB Sequences of the primer 5 -AACCTGGTTGATCCTGCCAGT-3 5 -TGATCCTTCCGCAGGTTCACCT-3 105

4 Polymerase chain reaction conditions Mastermix components Distilled water - 18 µl 10x Mgcl2 Buffer µl dntp (each 2.5mM) - 2 µl Taq DNA polymerase (3 U/ µl) µl Primer mix - 1 µl Template DNA - 1 µl Polymerase chain reactions were performed in the following temperature and timing condition programmed in TechGene TM, thermal cycler. Initial denaturation at 94 ºC for 5 min. Number of cycles 35 Denaturation at 94 ºC for 30 sec. Annealing at 50 ºC for 60 sec. Extension at 72 ºC for 60 sec. Final extension at 72 ºC for 10 min. The amplified products were checked on 1.5% Agarose gel electrophoresis. The molecular weight was also checked using molecular weight marker (100 bp ladder). 106

5 Sequencing The purified DNA products were sent to Macrogen, Inc. (Seoul, Korea) for sequencing. The DNA sequence analyzer, 3730xl DNA analyzer with BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems, USA) was used Sequence editing and characterization The obtained sequences were edited based on the electropherogram peak clarities in MEGA 5.0 software. Sequences with noisy peaks were excluded from the analysis BLAST search The amplified sequences belonging to 18S rrna gene were confirmed by percent similarity in the NCBI s BLASTn program. Higher percentage similarity (95-100%) against the reference sequence was used to confirm the identity of the species Genbank submission All the sequences were submitted to the NCBI s Genbank through BankIt according to NCBI s procedure with required information Estimation of genetic distance Overall genetic distance between family, within family and genetic distance between genus and within genus were estimated using 1060 positions of 18S rrna gene. The analysis involved 26 nucleotide sequences. All positions containing gaps and missing data were eliminated. Analyses were conducted using the Kimura 2-parameter model (K2P) (Kimura, 1980). 107

6 Phylogenetic analysis Nucleotide sequence of 14 taxa from the present study along with 12 reference sequence from Genbank was incorporated for the phylogenetic study analysis. There were a total of 1060 positions were considered in the final dataset. Maximum likelihood (ML) statistical method: Phylogeny Test Test of Phylogeny - Bootstrap method No. of Bootstrap Replications Substitution Model Substitutions Type - Nucleotide Model/Method - Kimura 2-parameter model Rates and Patterns Rates among Sites - Uniform rates Data Subset Gaps/Missing Data Treatment - Complete deletion Tree Inference Options ML Heuristic Method - Nearest-Neighbor-Interchange (NNI) Initial Tree for ML Initial tree automatically selected 108

7.3. Results 7.3.1. PCR amplification of 18S rrna gene 18S rrna region of ribosomal gene was successfully amplified using universal primers in 14 out of 15 species. The Fig. 7.1 shows the amplified fragments of 18S rrna gene.

7 7.3. Results PCR amplification of 18S rrna gene 18S rrna region of ribosomal gene was successfully amplified using universal primers in 14 out of 15 species. The Fig. 7.1 shows the amplified fragments of 18S rrna gene. 900 bp Figure 7.1. Agarose gel electrophoresis of amplified 18S rrna gene (Lane no. 1-7 Amplified 18S rrna gene; M 100 bp ladder) From the gel analysis, the single highly intense band without smearing strongly indicated the amplification of 18S rrna gene. The base pair length was predicted as approximately 900 bp by comparing with standard molecular marker (100 bp ladder) Sequencing The purified DNA products were sent to Macrogen, Inc. (Seoul, Korea) for sequencing. The DNA sequence analyzer, 3730xl DNA analyzer with BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems, Foster City, CA, USA) was used for sequence analysis. 109

8 Sequence characterization and alignment DNA sequencing results reveals a 669 to 1060 bp fragment of the 18S rrna at both 5 and 3 ends. All sequences were aligned by using CLUSTAL W (Thompson et al., 1994) under default settings and subsequently manually edited by using BioEdit (Hall, 1999). Gap positions and regions that could not be aligned unambiguously were excluded from the analysis. The edited nucleotide sequence of the partial 18S rrna gene was then used as a query in the public available BLAST database (Basic Local Alignment Search Tool) search ( The alignment shows the high homology of 95 to 99% identity with more than 90% query coverage with previously published COI sequences in the NCBI s nucleotide database. Genbank submission NCBI s BankIt tool was used for sequence submissions and all the sequences were assigned the accession numbers ranging from KF to KF and KF (Table 7.2) Nucleotide content variations Nucleotide compositions of all the 14 identified polychaete taxon were studied and the percent composition is presented in Table 7.3. The fours nucleotides i.e. A, T, G and C were divided into two groups namely A+T and C+G. The maximum A+T content (52.30%) was recorded in Perinereis camiguinoides and minimum (43.80%) in Naineris quadricuspida. Similarly, the maximum C+G value of 56.20% was observed in Naineris quadricuspida and least content of 47.7% was estimated in Perinereis camiguinoides. The mean A+T content among the 14 taxon of polychaete was found to be 48.9 ± 2.7, whereas the mean C+G content was 51.1 ±

9 Table 7.2. List showing the polychaetes species used in the present study along with S. No. reference taxon with accession numbers Species Family Source Genbank Accession number 1 Marphysa sanguinea Eunicidae P KF Marphysa sanguinea Eunicidae G GQ Lumbrineris latreilli Lumbrineridae P KF Lumbrineris latreilli Lumbrineridae G AF Lumbrineris inflata Lumbrineridae P KF Lumbrineris inflata Lumbrineridae G AY Glycera alba Glyceridae P KF Glycera alba Glyceridae G HQ Glycera capitata Glyceridae P KF Glycera capitata Glyceridae G GQ Naineris quadricuspida Orbiniidae P KF Naineris quadricuspida Orbiniidae G AY Scoloplos armiger Orbiniidae P KF Scoloplos armiger Orbiniidae G AY Laonice cirrata Spionidae P KF Perinereis camiguinoides Nereididae P KF Platynereis dumerilii Nereididae P KF Platynereis dumerilii Nereididae G AY Namalycastis abiuma Nereididae P KF Namalycastis abiuma Nereididae G HQ Namalycastis jaya Nereididae P KF Namalycastis jaya Nereididae G JX Capitella capitata Capitellidae P KF Capitella capitata Capitellidae G U Ceratocephale loveni Onuphidae P KF Ceratocephale loveni Onuphidae G DQ *P: Present study; G: Genbank 111

10 Genetic distance between family and within family A total of eight polychaete families were recorded from Vellar estuary. Table 7.4 shows the K2P genetic distance between family (below diagonal) and within family (bold). Lower level of variation in genetic distance was observed among all the studied polychaete based on 18S rrna gene. The maximum K2P distance (1.602) was found between Orbiniidae and Capitellidae family. Whereas, the minimum K2P distance of was observed between Lubrineridae and Glyceridae. However, the maximum K2P distance within family was observed in Nereididae (0.188) and the minimum value of was recorded within Capitellidae and Glyceridae family Genetic distance between genus Genetic distance was measured by K2P parameter between genus and the result are shown in Table 7.5. The results revealed considerable variations among genus. The maximum K2P distance (1.620) was found between Scoloplos and Capitella genus. Whereas the minimum K2P distance (0.026) was observed between Platynereis and Laonice. However, the maximum K2P distance within genus was observed in Perinereis (0.043) whereas the minimum K2P distance of was evident in the two genus namely Capitella and Glycera. The intra genus K2P distance was observed as in five genera namely Ceratocephale, Laonice, Marphysa, Naineris, and Platynereis. 112

11 Table 7.3. Percentage nucleotide composition of 18S rrna gene sequences of Polychaetes S. No. Species Nucleotide composition %A + T % C + G 1 Marphysa sanguinea Lumbrineris latreilli Lumbrineris inflata Glycera alba Glycera capitata Naineris quadricuspida Scoloplos armiger Laonice cirrata Perinereis camiguinoides Platynereis dumerilii Namalycastis abiuma Namalycastis jaya Capitella capitata Ceratocephale loveni Diopatra neapolitana - - Mean ± SD 48.9 ± ±

12 Table 7.4. K2P genetic distance between family (below diagonal) and within family (bold) based on 18S rrna gene sequence analysis Family Capitellidae Nereididae Glyceridae Spionidae Lubrinneridae Eunicidae Capitellidae Capitellidae Nereididae Glyceridae Spionidae Lubrinneridae Eunicidae Orbiniidae

13 Table 7.5. K2P genetic distance between genus (below diagonal) and within genus (bold) based on 18S rrna gene sequence analysis Genus Capitella Ceratocephale Glycera Laonice Lumbrineris Marphysa Naineris Namalycastis Perinereis Platynereis Scoloplos Capitella Ceratocephale Glycera Laonice Lumbrineris Marphysa Naineris Namalycastis Perinereis Platynereis Scoloplos

14 Mean K2P genetic variation From the figure 7.2. it is quite evident that the mean K2P genetic variation was maximum (0.5360) within family followed by within genus (0.4235). The least mean variation can be observed amid intra genus (0.0006) polychaete individuals. Figure 7.2. Mean K2P genetic variation in inter family, intra family, inter genus and intra genus in Polychaetes based on 18S rrna gene sequence data Phylogenetic relationship The alignment of 26 sequences produced a data set of 1060 positions, in which 704 sites were parsimony informative, 217 positions were constant and 133 sites were parsimony uninformative. 116

15 The evolutionary history was inferred by using the maximum likelihood method based on the Kimura 2-parameter model. Initial tree(s) for the heuristic search were obtained automatically, when the number of common sites was < 100 or less than one fourth of the total number of sites, the maximum parsimony method was used. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. The maximum likelihood tree clearly demonstrates the efficacy of 18S rrna gene in discriminating polychaete individuals both at genus and species level (Fig. 7.3). The perspicacity can be ascertained by strong bootstrap support of over 99%. The polychaete species used in the present study is highlighted by blue colour pyramid (Fig. 7.3). All the polychaete families formed a distinct clade except the Perinereis (due to non-availability of reference taxon). Topologically two major clades were formed; one major clade comprising 13 species belonging to six families (Nereididae, Gyceridae, Lumbrineridae, Eunicidae, Orbiniidae) and the next clade residing only Capitellidae (one species). From the ML tree it can be also visualized that the clade entailing Nereididae, Orbiniidae, Eunicidae, Capitellidae, Lumbrineridae and Glyceridae family showed perfect distinction between the genus within the family by strong bootstrap value support of 99 and

16 Figure 7.3. Maximum Likelihood tree of studied Polychete species based on 18S rrna gene sequence data 118

17 7.4. Discussion A key limitation inherent to most biodiversity studies is the arduous task of identifying organisms in the wild. Reliance upon good taxonomy and species identification goes beyond bio- diversity surveys because it has wide implications for a number of ecological and evolutionary disciplines (Yoccoz, 2012). Molecular data have proved to be an important tool for solving problems on the relationships at lower taxonomic The analysis of nuclear rdna, which encodes rrna gene, is commonly used in assessing phylogenetic relationships among taxa (Hwang and Kim, 1999; Manylov et al., 2003). In this study, the most conservative portions of the 18S rrna gene were successfully used to investigate the phylogenetic relationships of polychaetes of Vellar estuary. Nucleotides sequence ranging from 669 to 1060 bp were successfully amplified and sequenced representing the conservative portions of the 18S rrna molecule. The 18S rdna sequences were adenine and thymine G+C rich (51.1%) compared to mean A+T content (48.9%). High GC content shows higher stability of the organism (Vinogradov, 2003). In the case of COI gene in the previous chapter A+T was the dominating one (59.0%). Most utilization of DNA sequence-based identification uses distance measures to make inference as to species designation, while classical taxonomy uses character-based delineation. Accordingly, changing to character based identifications may prove to be more compatible with classical taxonomy (DeSalle et al., 2005). Similar, to COI gene, lower level of variation was observed within and between polychaete genus and family respectively based on 18S rdna sequence data. Two main reasons could be attributed for this observation. Firstly, 119

18 the low sample size and same geographical region. Second, the low resolution may be due to a rapid radiation of the Annelida (Brown et al., 1999; McHugh, 2000; Rota et al., 2001) which is generally ascribed to an attrition of information during time (Abouheif et al., 1998). Compared to the diversity in polychaetes, sequences of only a few polychaete taxa are available and in most cases only a single representative stands for a larger taxon (Kojima, 1998; McHugh, 2000). It therefore does not surprise that even those taxa that can be supported as monophyla on the basis of morphological data do not form a single clade in molecular analyses. A strong conviction is that the low number of polychaete species analyzed causes the low resolution and knowledge about whether an increased taxon sampling increases the resolution is essential, so that molecular data can be used to substantiate the monophy of certain polychaeta taxa. ML analysis once again revealed the monophyly of polychaete taxon. Monophyletic clades were formed among Nereididae, Glyceridae, Spionidae, Onuphidae, Capitellidae, Eunicidae and Orbiniidae family. Similar interpretations were made by Rota et al. (2001); Struck et al. (2002) and Bleidorn et al. (2003) based on 18S rdna sequences. A pair of taxa is generally known as sisters, for example in clade I represented by Nereididae family shows Platynereis dumerilli and Laonice cirrata as sister taxon. Similarly, Naineris quadricuspida and Scoloplos armiger belonging to Orbiniidae could be considered as sister species. However, individuals in remaining six clades could also be considered as a sister taxon with their respective clade. This is due to the sharing of a common ancestor at the node that joins them together, undoubtedly evident in the Fig.7.3. Interestingly, capitellidae family which clustered together with Orbiniidae based 120

19 on COI gene doesn t showed similar clustering based on 18S rdna sequences. It formed a separate clade indicating the difference in the perseverance of phylogenetic divergence among genes. This could be also attributed to inclusion of fewer number of polychaete taxon for phylogenetic tree construction. The problems which are connected to the attempt to unravel the phylogenetic relationships are reflected in the analysis using molecular data. Whereas most polychaete families represent well supported monophyla, the relationships to one another remain obscure - a result which was also observed in former investigations (Rota et al., 2001; Struck et al., 2002; Bleidorn et al., 2003; McHugh, 2005). However, the consistency among different delimitation metrics was much lower for 18S than for COI gene (Tang et al., 2012). Recently Tang et al. (2012) also concluded that taxonomic entities estimated from analyses using 18S rdna are not reliable proxies for diversity and very likely underestimate true species richness. Only a fixed threshold of 99.5% based on 18S rdna (approaching the rate of potential PCR errors) can produce DNA estimates comparable to morphological estimates, but these are known to be underestimates because of the prevalence of cryptic species. The use of 18S rdna might be appropriate to compare levels of relative diversity at higher taxonomic scales, but species-level patterns, such as ecological distributions and geographical turnover, should not be inferred from these data. Furthermore, the degree of lumping per morphospecies estimated from 18S rdna varies among different taxonomic groups and thus diversity might be very different depending on the composition of taxonomic groups. 121

20 Thus, the 18S-rDNA seems to be a use-full molecular marker within the polychaetes to test the monophyly of a family. Also, increasing the taxon sampling of 18S rrna gene sequences of different polychaete groups could be a promising methodological solution to the problem of reconstructing relationships which resulted from rapid radiation, yet there is no guarantee for its success. 122

6. DNA BARCODING AND PHYLOGENETIC ANALYSIS OF POLYCHAETES

6. DNA BARCODING AND PHYLOGENETIC ANALYSIS OF 6.1. Introduction POLYCHAETES Appropriate knowledge about the diversity of life at different hierarchical levels, including physiological, ecological, biogeographical,