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Comparative Phylogenetic Study of Four Genes of Mitochondrial Genome in Tenpounder Fishes (Order: Elopiformes)

Vellaichamy RAMANADEVI, Muthusamy THANGARAJ*

Annamalai University, Centre of Advanced Study in Marine Biology, 608 502 Parangipettai, Tamil Nadu, India; [email protected] (*corresponding author)

Abstract

The Elopiformes represent a group of seven known species of fishes found in marine and estuarine ecosystem and many of them are endemic to East Asia. To date published morphological and molecular phylogenetic hypothesis of Tenpounder fishes are part congruent and there are some areas of significant disagreement with respect to species relationships. The present study analyzed the sequence data from four genes (Cytb, CO1, 16S and 12S rRNA) of mitochondrial genome for the attempt to estimate the relationships among five species such as Elops saurus, E. affinis, E. smithi, E. machnata and E. hawaiensis and to assess the phylogenetic utility of these markers.

The Kimura 2- parameter (K2P) genetic distance, average nucleotide frequencies, nucleotide substitution patterns and phylogenetic trees were reconstructed using neighbour joining (NJ) method. The interspecies K2P genetic distance was 0.0158 and intraspecies distance was 0.0042 based on the barcoding gene, CO1 sequence data. Whereas the interspecies K2P genetic distance was 0.6140 and intraspecies genetic distance was 0.0020 based on the Cytb data. The four mitochondrial marker genes used in this study showed different type of cluster and we could not confirm the relationship between the five Elops species. This is due to the independent mutation rate of each mtDNA genes. However, this problem can be overcome by analysing in parallel other gene markers.

Keywords: Elops, genetic distance, mtDNA genes, phylogeny, Tenpounders

Introduction

The ladyfishes or tenpounders (genus Elops) are widely distributed in tropical-subtropical, marine and coastal waters (Mcbride et al., 2010). Six species of Elops are rec- ognized worldwide (Eschmeyer and Fong, 2008), but the taxonomy of the group is poorly known and some authors recognize fewer species (Nelson, 2006). Taxonomic un- certainty of Elops is exemplified by the ladyfish, E. sau- rus, currently recognized as the only species of Elops in the western Atlantic Ocean (Mcbride et al., 2010). Smith (1989) also noted that E. saurus and E. smithi had largely allopatric distributions. Recent work has failed to support the phenotypic hypothesis for this two species (Mcbride et al., 2010). Mcbride et al. (2010) examined specimens of E. saurus and Elops sp. using common morphological, meristic characters and Cytb data that have been used to distinguish six species of Elops worldwide (E. saurus, E.

affinis, E. lacerta, E. senegalensis, E. machnata and E. ha- waiensis). Many taxonomic studies of Elops examined less than 20 specimens per species and for some species even single specimen also used. Hence, there may be even more species of Elops awaiting discovery (Mcbride et al., 2010).

Phylogenetically, Obermiller and Pfeiler (2003) identify E. saurus and E. smithi based on 12S and 16S rRNA gene sequence. So far E. senegalensis and E. lacerta are not yet entered into the GenBank database and there is no phylo-

genetic analysis work has been made using molecular tools.

Based on the study of Mcbride et al. (2010) E. saurus and E. smithi showed ecological specialization in tropical and subtropical habitats may be a foundation for speciation.

Recently three independent research groups have published results from molecular phylogenetic stud- ies of the elopomorphs using mitochondrial ribosomal DNA sequences (Filleul and Lavoue, 2001; Obermiller and Pfeiler, 2003; Wang et al., 2003). Wang et al. (2003) analyzed the elopomorph interrelationships based on the complete 12S rRNA gene sequences (1073 bp) from 42 teleosts including the Elopomorpha (34 spp.). Forey et al.

(1996) employed a total-evidence approach in analyzing elopomorph relationships, combining partial nucleotide sequences of the mitochondrial 12S rRNA (345 bp), 16S rRNA (535 bp), and nuclear 18S rRNA (1870 bp) genes and morphological data (56 characters) from 13 species, thereafter subjecting the combined data to MP analysis, and suggested that the saccopharyngiforms are deeply nested within the anguilliforms. Obermiller and Pfeiler (2003) also analyzed mtDNA sequences (754 bp) in seg- ments of the 12S and 16S rRNA genes from 45 species including 33 elopomorphs, nine osteoglossomorphs, and three clupeomorphs and did not support the monophyly of Elopomorpha. Inoue et al. (2004) investigated Elopo- morpha monophyly and interrelationships at the ordinal level using complete mitochondrial genomic data from 33 Received 14 May 2013; accepted 17 July 2013

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Sequence analysis

The CO1 gene partial sequences of Elops machnata were unambiguously edited using BioEdit sequence edi- tor, aligned using CLUSTAL-W and checked manually.

Haplotype definitions have been submitted to the NCBI GenBank (Acc. No. KF006255, KF006256, KF006257).

To give more support to the present data, CO1, Cytb, 16S rRNA and 12S rRNA sequences of other available species were retrieved from GenBank and the details are given in Tab. 1. Nucleotide diversity, genetic variation, nucle- otide composition and pairwise evolutionary distance among haplotypes were determined by Kimura 2-Param- eter method (Kimura, 1980) using the software program MEGA 3.1 (Kumar et al., 2004). The neighbour-joining (NJ) trees for CO1, Cytb, 16S rRNA and 12S rRNA were constructed and to verify the robustness of the internal nodes of these trees, bootstrap analysis was carried out us- ing 1000 pseudoreplications.

Results and discussion

A total of 58 sequences were from five Elops species were included and analysed in this study. Simplicity and un-ambiguity were observed among the sequences and no introns, deletions or stop codons were observed any of the CO1 and Cytb sequences. The CO1 sequence analy- sis revealed that the average nucleotide frequency was A

= 24.16 ± 0.78%, T = 26.74 ± 0.17%, G = 18.96 ± 0.60, C = 30.14 ± 1.72 (Fig. 1). The interspecies transition and transversion was 86.23 and 13.78 respectively (Fig. 2). Ta- jima’s statistics for E. machnata and E. hawaiensis were not significantly (p<0.01) different among individuals. Kimu- ra 2 Parameter (K2P) genetic distance in four Elops spe- cies is given in Tab. 2 (below diagonal). The K2P genetic distance was high (0.023) between E. hawaiensis and E.

affinis. Very low K2P distance (0.003) was exhibited be- tween E. hawaiensis and E. machnata. The Cytb sequence analysis among the four species showed that the average nucleotide frequency was A = 25.19 ± 0.65%, T = 26.54

± 0.12%, G = 18.24 ± 0.53, C = 30.03 ± 0.90 (Fig. 1).

The interspecies transition and transversion was 57.01 and 42.99 respectively and the R value was 1.28 (Fig. 2) based on the Cytb data. Tajima’s statistics for Cytb sequence in E. smithi was not significantly (p<0.01) different among individuals (Tab. 4). The K2P genetic distance based on the Cytb data among the Elops species is given in Tab. 3.

The K2P genetic distance was high (1.231) between E.

saurus and E. affinis and the distance was low (0.010) be- tween E. hawaiensis and E. smithi.

The 16S rRNA gene sequence analysis showed the av- erage nucleotide frequency was A = 31.63 ± 0.82%, T = 13.99 ± 0.42%, G = 23.39 ± 0.83, C = 30.99 ± 1.20 (Fig.

1). The interspecies transition and transversion was 52.96 and 37.04 respectively and the R value was 1.70 (Fig. 2).

The K2P genetic distance based on 16S rRNA gene data of Elops species is given in Tab. 4. The high (0.048) K2P species represent the major teleostean and elopomorph

lineages. Mitogenomic data strongly supported the order Elopiformes occupied the most basal position in the elo- pomorph phylogeny, with the Albuliformes and a clade comprising the Anguilliformes and the Saccopharyngi- formes forming a sister group.

Mitochondrial DNA provides a potential tool for studying population and phylogenetic analysis and the different genes of mitochondrial genome are used for phy- logeny analysis at different levels of taxa, family, species and individual’s level. Hence, an attempt has been made to report the phylogenetic analysis based on the updated nu- cleotide sequence data from GenBank for the four regions of the mitochondrial genome (Cytb, CO1, 16S rRNA and 12S rRNA) to assess the pattern of species relation- ship and also to examine the rates and types of nucleotide substitutions among the Tenpounder fish species.

Materials and methods Sample collection

Forty specimens of Elops machnata were collected from Vellar estuary (Lat 11°29' N; Lon 79°46' E) southeast coast of India. Immediately after the collection, the specimens were kept in the icebox and the fishes were identified up to the species level using the FAO fish identification sheets (Thomson, 1984). The voucher specimens are maintained in Marine Biotechnology Laboratory, CAS in Marine Bi- ology, Annamalai University. The fin-clips were preserved in 95% ethanol and stored at 4°C until used.

DNA isolation

The DNA was isolated by standard Proteinase-K/Phe- nol-Chloroform-ethanol method (Sambrook et al., 1989) and the concentration of isolated DNA was estimated us- ing a UV spectrophotometer. The DNA was diluted in TAE buffer to a final concentration of 100 ng⁄μL.

Gene amplification and sequencing

The CO1 gene was amplified in a 50 μL volume with 5 μL of 10X Taq polymerase buffer, 2 μL of MgCl2 (50 mM), 0.25 μL of each dNTP (0.05 mM), 0.5 μL of each primer (0.01 mM), 0.6 U of Taq polymerase and 5 μL of genomic DNA. The primers used for the amplification of the CO1 gene were FishF1-5' TCAACCAACCACAAAGACAT- TGGCAC 3' and FishR1-5' TAGACTTC TGGGTG- GCCAAAGAATCA 3' (Ward et al., 2005). The thermal regime consisted of an initial step of 2 min at 95°C fol- lowed by 35 cycles of 40 s at 94°C, 45 s at 54°C and 1 min at 72°C followed in turn by final extension of 10 min at 72°C.The PCR products were visualized on 1.5% agarose gels, and the most intense products were selected for se- quencing. The cleaned up PCR product was sequenced by a commercial sequencing facility (Ramachandra Innovis, Chennai, India).

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genetic distance was observed between E. saurus and E.

hawaiensis and low (0.024) among E. saurus and E. affinis.

The 12S rRNA gene sequence analysis result showed the average nucleotide frequency was A = 28.81 ± 0.43%, T

= 21.19 ± 0.31%, G = 26.44 ± 0.32, C = 23.56 ± 0.70 (Fig. 1). The transition and transversion among the species was 37.85 and 62.15 respectively and the R value was 0.68 (Fig. 2). The K2P genetic distance based on 12S rRNA gene sequence data of the fish species is given in Tab. 2 (above diagonal). The K2P genetic distance was high (0.035) between E. saurus and E. affinis and low (0.004) genetic distance was observed between E. hawaiensis and E. machnata.

The neighbour-joining method was actually employed in this study to get a solid phylogenetic information by the dendrogram. All the 58 sequences of the tenpounder fishes were subjected in the phylogenetic analysis. The neighbour joining trees by K2P model for the four mito- chondrial genes were created to provide a graphical rep- resentation of the patterning of divergence of five Elops species. The neighbour joining phylogenetic tree based on CO1 gene sequences is given in Fig. 3. As per the NJ tree, two distinct clades as two sub-trees within the same genus were recognized with high bootstrap value. Among the two sub-trees, one has an independent assemblage of E. hawaiensis and E. machnata with 98% bootstrap value.

Another clade representing the other two species such as Tab. 1. Elops species and their mitochondrial genes with accession number and reference

Sl. No Species Accession Number

CO1 Cyt b 12S rRNA 16S rRNA

1. Elops machnata JF493412a - AF417340c -

2. Elops machnata JF493410a - - -

3. Elops machnata JF493413a - - -

4. Elops machnata JF493411a - -

5. Elops machnata KF006255b - - -

6. Elops machnata KF006256b - - -

7. Elops machnata KF006257b - - -

8. Elops saurus GU702393d AP004807f AP004807f KC146866a

9. Elops saurus GU702337d NC_005803f - KC146864a

10. Elops saurus GU702338d - - KC146867a

11. Elops saurus JQ365344d - - KC146865a

12. Elops saurus JN025309e - - KC146863a

13. Elops saurus GU224782a - - AF455766g

14. Elops saurus GU224783a - - -

15. Elops saurus GU225201a - - -

16. Elops saurus GU225600a - - -

17. Elops saurus GU225200a - - -

18. Elops hawaiensis EF609347h HQ616667k - X99175l

19. Elops hawaiensis EF607367i HQ157200k - -

20. Elops hawaiensis EF607365i HQ616666k - -

21. Elops hawaiensis EF607366i HQ157201k - -

22. Elops hawaiensis EF607364i AB051070f - -

23. Elops hawaiensis EU595109a NC_005798f NC_005798f -

24. Elops hawaiensis JF952722j - X99176l -

25. Elops affinis GU440309a DQ082913a AF454710g AF455765g

26. Elops affinis EU403078a - - AY836585a

27. Elops smithi - GQ183893m - -

28. Elops smithi - GQ183889m - -

29. Elops smithi - GQ183887m - -

30. Elops smithi - GQ183885m - -

31. Elops smithi - GQ183883m - -

32. Elops smithi - GQ183890m - -

33. Elops smithi - GQ183888m - -

34. Elops smithi - GQ183886m - -

35. Elops smithi - GQ183884m - -

aUnpublished; bPresent study; cWang et al.,2003; dRosso et al., 2012; eApril et al., 2011; fInoue et al., 2004; gObermiller and Pfeiler, 2003; hWard and Holmes, 2007;

iZhang, 2011; jZhang and Hanner, 2011; kKwun and Kim, 2011; lForey et al., 1996; mMcBride et al., 2010

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bootstrap value. The second clad representing E. affinis with bootstrap value of 59% and the third branch was E.

hawaiensis. Fig. 6 shows the neighbour joining tree based on 12S rRNA gene sequences. In this tree, one large clus- ter has an assemblage of E. hawaiensis, E. saurus and E.

machnata. Another small clade representing the deviated species, E. affinis as like Cytb NJ tree.

Species identification and phylogenetic relationship based on traditional methods and molecular methods are mostly concordant (Ward et al., 2005). The efficiency of species identification by molecular methods is judged by E. saurus and E. affinis. Fig. 4 shows the neighbour joining

phylogenetic tree based on Cytb gene sequences. In this tree, two distinct clusters are raised with high bootstrap value. Among the two clusters, one large has an assemblage of E. hawaiensis, E. saurus and E. smithi with 54% boot- strap value. Another clade representing the other one spe- cies, E. affinis.

The neighbour joining phylogenetic tree based on 16S rRNA gene sequences is given in Fig. 5. As per this tree, three distinct clades as sub-trees within the Elops genus were recognized with high bootstrap value. Among the three sub-trees, one has the cluster of E. saurus with 84%

Fig. 1. Percentage of nucleotide composition in four mitochon- drial genes of Elops species

Fig. 2. The nucleotide substitution patterns of four mitochon- drial genes based on the Kimura (1980) 2-parameter model (Ts-Transition; Tv-Transversion; R-estimated Transition/

Transversion bias)

Fig. 3. Neighbour joining tree of Elops species based on CO1 gene sequences

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may cause the inability of CO1 barcodes. In such cases, a secondary independent molecular marker is required to solidify or confirm identification if applicable (Smith et al., 2007). In this study five Elops species were found ge- netically distinct from each other based on four mtDNA gene sequences which demonstrates simplicity and unam- biguity. Morphologically very similar species like E. affinis and E. saurus form sister clade by all the four gene based NJ trees. Whereas, E. machnata and E. hawaiensis form an independent sister clade in NJ tree of CO1 gene. Because of the data deficient in GenBank we could not clearly resolve E. smithi from all other species. The observed ge- netic divergence from CO1 gene is sufficient to differenti- ate individuals of different Elops species. In this study the level of intra-species variation was low which may be due to low number of haplotype identified in the sample with limited numbers collected for this study. Similarly, Lakra et al. (2011) reported very low intra-specific genetic diver- gence for scombroid fishes and Ward et al. (2005) showed in many marine teleost species. Peris et al. (2009) also re- ported very low interspecies genetic distance for Indian the levels of intraspecific homogeneity and interspecific

heterogenenity displayed by the intended method (Hall- den et al., 1994; Lievens et al., 2001). Mitochondrial CO1 gene, as an attractive “species barcode”, its high efficiency in species identification has been reported in Australia ma- rine fishes (Ward et al., 2005), Canadian freshwater fishes (Hubert et al., 2008), ornamental fishes in the market of North America (Steinke et al., 2009b) and marine fishes of Japan (Zhang and Hanner, 2011). Due to the high ef- ficiency in species identification, some ichthyologists ad- vocate the inclusion of a DNA barcode in the formal de- scription of species (Victor, 2007; Astarloa et al., 2008).

Somehow, it deserves attention to recent speciation, in- trogressive hybridization, and taxonomic splitting, which

Tab. 2. K2P genetic distance between Elops species based on CO1 gene sequences (below diagonal), based on 12S rRNA gene sequences (above diagonal)

Species Elops machnata Elops saurus Elops hawaiensis Elops affinis

Elops machnata ***** 0.009 0.004 0.026

Elops saurus 0.014 ***** 0.013 0.035

Elops hawaiensis 0.003 0.014 ***** 0.030

Elops affinis 0.020 0.021 0.023 *****

Tab. 3. K2P genetic distance between Elops species based on Cytb gene sequences

Species Elops smithi Elops saurus Elops hawaiensis Elops affinis

Elops smithi *****

Elops saurus 0.013 *****

Elops hawaiensis 0.010 0.013 *****

Elops affinis 1. 210 1.231 1.207 *****

Tab. 4. K2P genetic distance between Elops species based on 16S rRNA gene sequences

Species Elops saurus Elops hawaiensis Elops affinis Elops saurus *****

Elops hawaiensis 0.048 *****

Elops affinis 0.024 0.038 *****

Fig. 4. Neighbour joining tree of Elops species based on Cytb gene sequences

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the mean GC content in CO1 was 49.10% and in Cytb it was 48.27% among the five Elops species.

The genetic distance between Elops sp. and E. saurus, which occur sympatrically in Florida (Smith, 1989), was 0.021, similar to that found between the allopatric E. sau- rus and E. hawaiensis (0.024) (Obermiller et al., 2003) which inhabit different ocean basins. The present study revealed that the K2P genetic distance between the al- lopatric E. saurus and E. hawaiensis was 0.014 based on CO1 data and it was 0.013 based on Cyt b and 12S rRNA gene sequence data respectively.

It is unfeasible to build the phylogeny of Elopiforme fishes only based on mitochondrial DNA fragments alone.

The disadvantage of 16S rDNA sequences is the lack of discrimination power among closely related species. How- ever, this problem can be overcome by analysing in par- allel other gene markers. Polyphyly/paraphyly in the NJ tree probably results from “bad taxonomy” when named species fail to identify the genetic limits of separate evo- lutionary entities, particularly for perplexing taxa involv- ing cryptic species (Nice and Shapiro, 2001). All the four mitochondrial marker genes used in this study showed different type of cluster and we could not confirm the re- carangid fishes. For many marine fishes, there is a lack of

phylogeographic structure among populations (Palumbi, 1994; Hellberg et al., 2002). Zhang (2011) reported that, individuals from long distance localities, some intraspecific genetic variations reduced to zero within families Carangi- dae, Sciaenidae, and Mullidae. However, some pairwise K2P distances exceeded 1.00% within the coastal species such as Acentrogobius caninus, Scomber japonicus, Terapon jarbua, Upeneus sulphureus, Elops hawaiensis, Gymnotho- rax pseudothyrsoideus, and Dendrophysa russelii. Based on the CO1 data, the present study also confirmed that the interspecies K2P genetic variation is more than 1.00%. It implied that biological mechanisms were responsible for the fluctuation of intraspecific genetic divergences in ma- rine fishes.

Ward et al. (2005) reported an overall higher GC con- tent in fishes based on complete MtDNA genome ranging from 38.4-43.2% and in CO1 alone it was 42.2-47.1%, which reflects the 3rd base variation. Peris et al. (2009) also reported considerable variation was exhibited in carangids in the 3rd base position. Ravitchandirane et al. (2012) showed the mean GC content was 36.8 - 42.6% among the nine Nemipterus species. In this study it has been observed

Fig. 5. Neighbour joining tree of Elops species based on 16S rRNA gene sequences

Fig. 6. Neighbour joining tree of Elops species based on 12S rRNA gene sequences Tab. 5. Tajima’s Neutrality Test for CO1 and Cytb genes in Elops species

Gene Species M S Ps Θ π D

CO1

Elops machnata 7 4 0.006885 0.002810 0.002295 -0.876418

Elops saurus 10 16 0.027586 0.009751 0.011916 1.027479

Elops hawaiensis 7 3 0.005803 0.002368 0.001658 -1.358415

Elops affinis 4 0 0.000000 0.00000E+000 0.000000 n/c

Cytb

Elops smithi 9 13 0.027660 0.010177 0.007092 -1.446159

Elops saurus 3 0 0.000000 0.00000E+000 0.000000 n/c

Elops hawaiensis 5 577 0.973019 0.426140 0.634963 3.190463

Elops affinis 3 0 0.000000 0.00000E+000 0.000000 n/c

M = number of sequences, S = Number of segregating sites, ps = S/m, Θ = ps/a1, π = nucleotide diversity, and D = Tajima test statistic

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dependent mutation rate of each mtDNA genes. Different nucleotide positions and genes within mtDNA are known to evolve at heterogeneous rates within a lineage (Brown et al., 1982; Gillespie, 1986; Moritz et al., 1987), and par- ticular mtDNA genes (such as cytochrome oxidase) also show rate differences as high as fivefold across lineages (Brown and Simpson, 1982; Crozier et al., 1989). As per Zhang (2011), if we cannot set a threshold of the genetic variation in species delimitation, we find ourselves sunk in the dilemma facing new or cryptic species. On the one hand, the morphological taxonomy cannot give a definite identification. On the other hand, we cannot claim that it may be a new species based on molecular analysis with- out the species delimitation (Zhang, 2011). An assumed threshold is helpful to expedite discovery of new species and biodiversity, especially in dealing with little-studied biota, although a single, uniform threshold for species de- limitation seems arbitrary because the rates of molecular evolution vary widely within and among lineages (Zuck- erkandl and Pauling, 1965; Will and Rubinoff, 2004; De- Salle et al., 2005).

Conclusions

The study has successfully assessed the utility of the four genes (Cytb, CO1, 16S and 12S rRNA) of mitochondrial genome to estimate the relationships among five Elops spe- cies such as Elops saurus, E. affinis, E. smithi, E. machnata and E. hawaiensis. The four mitochondrial marker genes used in this study showed different type of cluster and we could not confirm the relationship between the five Elops species. This is due to the independent mutation rate of each mtDNA genes. However, this problem can be over- come by analysing in parallel by other gene markers also.

Further studies involving all the Elopiformes in the world and also by increasing the sample size in future studies will clarify the issue.

Acknowledgements

The authors would like to thank The Director, CAS in Marine Biology and The Authorities of Annamalai Uni- versity for encouragement and facilities.

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