DNA barcoding of the Bryde’s Whale Balaenoptera edeni Anderson (Cetacea: Balaenopteridae) washed ashore along Kerala coast, India

A. Bijukumar ¹, S.S. Jijith ², U. Suresh Kumar ³ & S. George ⁴

¹ Department of Aquatic Biology and Fisheries, University of Kerala, Thiruvananthapuram, Kerala 695581, India

^2,3 Regional Facility for DNA Fingerprinting, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala 695014, India

⁴ Chemical Biology Group, Rajiv Gandhi Centre for Biotechnology,Thiruvananthapuram, Kerala 695014, India

Email: ¹ abiju@rediffmail.com (corresponding author), ² jijithss@gmail.com, ³ sureshkumar@rgcb.res.in, ⁴ sgeorge@rgcb.res.in

Date of publication (online): 26 March 2012

Date of publication (print): 26 March 2012

ISSN 0974-7907 (online) | 0974-7893 (print)

Editor:E. Vivekanandan

Manuscript details:

Ms # o2859

Received 01 July 2011

Final received 16 November 2011

Finally accepted 02 March 2012

Citation: Bijukumar, A., S.S. Jijith, U.S. Kumar& S. George (2012). DNA barcoding of the Bryde’s Whale Balaenoptera edeni Anderson (Cetacea:Balaenopteridae) washed ashore along Kerala coast, India. Journal of Threatened Taxa4(3): 2436–2443.

Copyright: © A. Bijukumar, S.S. Jijith, U. Suresh Kumar & S. George 2012. Creative Commons Attribution 3.0 UnportedLicense. JoTT allows unrestricted use of this article in any medium for non-profit purposes, reproduction and distribution by providing adequate credit to the authors and the source of publication.

Author Details and Contribution:

Dr. A. Bijukumar is currently working as Associate Professor and Head of the Department of Aquatic Biology and Fisheries, University of Kerala. His fields of research include taxonomy and biodiversity informatics. Earlier he worked as Scientific Officer of State Committee on Science, Technology and Environment and Member Secretary-in-Charge and Principal Scientific Officer of Kerala State Biodiversity Board. Initiated major works on marine biodiversity informatics for Kerala and DNA barcoding of marine mammals and molluscs. In this paper, sampling, photography, morphological taxonomy and the paper preparation was done by him.

U. Suresh Kumar, holdsMPhil degree in bioinformatics and is currently working as the DNA examiner of the Regional Facility for DNA Fingerprinting at Rajiv Gandhi Centre for Biotechnology (RGCB), Trivandrum. His major research interests are DNA barcoding and DNA fingerprinting. Currently conducting training programmes on molecular markers, human DNA fingerprinting and DNA barcoding. Sequence analysis of the work was done by him in the work.

S.S.Jijith possesses Masters in Biotechnology and is currently working as a project fellow in Regional facility for DNA fingerprinting at RGCB. His current project addresses the development of a reference DNA barcodingdatabase of selected mammals of Kerala Forest. For this work DNA isolation and amplification was done by him.

Dr. S. George is currently working as a Scientist in the chemical biology laboratory of Rajiv Gandhi Centre for Biotechnology, Trivandrum. His main area of research is centered around amphibians of Western Ghats with particular interest in DNA barcoding and bioprospecting. At present he is the Principal Investigator of DNA barcoding projects on amphibians, mammals and molluscs of India. In this paper he has contributed towards the writing of molecular taxonomy and interpretation of data.

Acknowledgements: AB thank Kerala State Council for Science, Technology and Environment for financial support of the work and friends in the print media who frequently inform us stranding of whales. We thank Dr. Radhakrishna Pillai, Director, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram for the support. We appreciate the field support extended by the students Sirajudheen, Ravinesh, Rajesh, Smirthi Raj, VarunRaj and Soosan.

Abstract: Three whales washed ashore along Kerala coast of southwest India were identified as Bryde’s Whale Balaenoptera edeni Anderson based on sequencing of mitochondrial cytochrome c oxidase subunit 1 and cytochromeb genes. The results of mtDNA sequencing in the present study confirm the presence of B. edeni species of ‘Bryde’s Whale complex’ in the coastal waters of India.

Keywords: Balaenoptera, Bryde’s Whale complex, cytochromeb, cytochrome c oxidase subunit 1, morphometry, mitochondrial DNA.

For figures, images, tables -- click here

Introduction

Though an integral component of marine ecosystems, marine mammals, particularly whales, are given little attention by conservation biologists and taxonomists in India. Baleen whales are included in the suborder Mysticeti (Ceteacea:Balaenopteriidae) and are characterisedby the presence of a filtering structure in the mouth called Baleen or Whalebone, flippers representing the forelimbs, a tail with horizontal flukes and nasal openings (blowholes) on top of the head (Jefferson et al. 1993). In Indian coastal waters this suborder includes the Blue Whale Balaenoptera musculus, Fin Whale B. physalus, Sei Whale B. borealis, Bryde’sWhale B. edeni, Mink Whale B. acutorostrata and the Humpback Whale Megaptera novaeangliae (Kumaran 2002; Sathasivam2004; Jayasankar & Anoop2010).

Stranding of marine mammals occurs frequently in India, yet precise identification is not done in many cases due to lack of local taxonomic expertise and poor condition of specimens (George et al. 2011). Since all cetaceans are important from the conservation point of view, precise documenting of their presence would provide valuable information regarding the distribution and migratory nature of different species in the seas around India. Of late, DNA barcoding or sequencing of mitochondrial genes, particularly cytochromec oxidase subunit 1 (cox1) (Amaral et al. 2007; George et al. 2011) and cytochrome b (cytb) (Ross et al. 2003; Dalebout et al. 2004; Herath2007; Sholl et al. 2008; Jayasankar et al. 2007, 2008; Viricel & Rosel2011), has been used to successfully identify cetaceans.

Three whales were stranded at Kollam (Thanni Beach; 08⁰49’44.4”N & 76⁰33.3’14.3”E; 24 May 2011; Image 1),Anchuthengu (MuthalapozhiBeach; 08⁰40’23”N & 76⁰45’23”E; 04 June 2011; Image 2) and Poonthura (CheriyathuraBeach; 08⁰26’36.21”N & 76⁰56’32.70”E; 10 June 2011; Image 3) along southern Kerala. The precise identity of the specimens at Anchuthenguand Poonthura could not be made since the specimens were putrefied. The whale stranded at Kollam measured 960cm (total length) and was identified as Bryde’s Whale Balaenoptera edeni, Anderson, based on morphological features and morphometry (Table 1). The Bryde’s Whale can be distinguished from other baleen whales by the presence of three conspicuous ridges on the snout, 40–70 throat pleats extending to the navel and a tall and falcate dorsal fin that generally rises abruptly out of the back (Jefferson et al. 1993).

Tissue samples were collected from all the whales to confirm identification by the sequencing of two mitochondrial genes, cox1 and cyt b. The samples in absolute ethanol were processed for the extraction of DNA using QIAGEN DNeasy Blood and Tissue Kit (cat No.69506) and cox1 and cyt-b genes were amplified using universal primers [cox 1: Forward primer- 5’-GGTCAACAAATCATAAAGATATTGG-3’, Reverse primer-5’-TAAACTTCAGGGTGACCAAAAAATCA-3’, Tm value :45–51 ⁰C (Folmer et al. 1994); cyt b: Forward primer- 5’-TACCATGAGGACAAATATCATTCTG-3’, Reverse primer-5’-CCTCCTAGTTTGTTAGGGATTGATCG-3’, Tm value: 46⁰C (Verma & Singh 2003)] in a 25µl reaction volume with QIAGEN Taq PCR master mix kit in GenAmp PCR System 9700 (Applied Biosystems). The following thermal cycling conditions were used for amplifications: 95⁰C for 5 min, followed by 10 cycles of 95⁰C for 30s, 45⁰C for 40s, 72⁰C for 90s, followed by 30 cycles of 95⁰C for 30s, 51⁰C for 40s, 72⁰C for 90s, and a final extension step at 72⁰C for 5 min (for cox 1) and 95⁰C for 5 min, followed by 40 cycles of 95⁰C for 30s, 46⁰C for 30 s, 72⁰C for 30s, and a final extension step at 72⁰C for 7 min (for cytb).

All the PCR products were visualized on 1% agarosegels and the most intense products were selected for sequencing. Sequencing was performed directly using the corresponding PCR primers and products were labelledusing the BigDye Terminator V.3.1 Cycle sequencing Kit (Applied Biosystems, Inc.) and sequenced using an ABI 3730 capillary sequencer following manufacturer’s instructions. Sequence similarity search was done to identify the species of the tissue, with all entries in the DNA sequence database GenBank using Basic Local Alignment Search Tool (BLAST, Altschul et al. 1990). Twenty-six cytb sequences and 28 cox1 sequences were used for the phylogenetic analysis and after final alignment the lengths were 400bp for cytb and 513bp for cox1. Phylogeneticposition of the query sequences was determined using the maximum likelihood and maximum parsimony methods using MEGA Ver. 5 (Tamura et al. 2007; Kumar et al. 2008) and the branch support was evaluated using 1000 bootstrap replicates (Felsenstein 1985) (Figs. 1–4). The best fitnuclear substitution model was selected as HKV+I for cytband HKY+G for cox1 using model test, implemented in MEGA Ver. 5.

The BLAST search of cox1 and cytb showed 99.8% sequence identity with Bryde’s Whale Balaenoptera edeni. The phylogenetictrees obtained with maximum likelihood and maximum parsimony were very similar by clustering all the three stranded whales with other B. edeni sequences except Acc. No.X75583 (cytb) of the GenBank, which was confirmed as B. brydei after BLAST search. The GenBank accession numbers of the cox1 and cytb sequence data generated in the study is given in Table 2.

Bryde’s Whales are the least known of the large baleen whales and are reported from warm temperate, subtropical, and tropical oceans between 40⁰N and 40⁰S (Kato 2002). In India presence of this species has been reported only through occasional stranding data (Table 3) and behaviour, seasonal occurrence and abundance in our coastal waters remains to be documented.

Balaenoptera edeni was first described by Anderson (1879) from a stranded specimen in Burma and was named Eden’s Whale, after Sir Ashley Eden, the British High Commissioner to Burma at the time. In 1912, Olsen described a new species of mysticete whale from South Africa, and named this new species Balaenoptera brydei after Johan Bryde, the Norwegian consul to South Africa, who set up the first whaling station in Durban (Olsen 1913). Balaenoptera edeni and B. brydei were subsequently synonymised based on skeletal comparisons (Junge 1950) and B. edeni was used as the scientific name and Bryde’sWhale as the common name. This synonymisation was not accepted by many taxonomists and molecular analysis of mtDNA from all nominal species of ‘Bryde’s whale complex’ has separated brydei from edeni and resulted in a third species called B. omurai described from specimens collected mostly in tropical waters of the western Pacific and eastern Indian oceans (Wada et al. 2003). The studies by Wada et al. (2003) demonstrated that B. edeni forms a sister taxon to B. brydei (Sasaki et al. 2006).

Although the recent findings outlined above support that B .edeni and B. brydei may be separate species, and that genetic differentiation is high among different oceanic regions, further molecular studies are required to identify which populations of Bryde’s Whales belong to each species, and consensus on a type specimen for brydei is required. Eden’s Whale and Bryde’s Whale may be used as the common name of B.edeni and B. brydei respectively as suggested by Wada et al.(2003) and George et al. (2011). The results of mt DNA sequencing in the present study confirms the presence of B. edeni species of ‘Bryde’s Whale complex’ in the coastal waters of India.

According to the recent International Union for Conservation of Nature (IUCN) assessment, Bryde’s whale taxonomy is unresolved and they are classified as ‘Data Deficient’ (Reilly et al. 2008). They are currently listed in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and in Appendix II of the Convention on the Conservation of Migratory Species of Wild Animals (CMS), under the United Nations.

Marine mammal strandings may be attributed to natural or anthropogenic factors and the stranding data can provide insight on spatial distribution, seasonal movements, and mortality factors pertaining to marine mammal populations (Woodhouse 1991). A deep injury was noticed on the back of the whale washed ashore Thanni beach which could be due to a ship collision. Vessel collisions are considered an important source of mortality for Bryde’s Whale in New Zealand waters (Stockin et al. 2008). In many cases the causes of death in stranded marine mammals are not properly investigated, and detailed necropsy studies and post-mortem examination would help in evaluating the impact of anthropogenic interactions.

References

Altschul, S.F., W. Gish, W. Miller, E.W. Myers & D.J. Lipman (1990). Basic local alignment search tool. Journal of Molecular Biology 215(3): 403–410

Amaral, A.R., M. Sequeira & M.M. Coelho (2007). A first approach to the usefulness of cytochrome c oxidase barcodes in the identification of closely related delphinid cetacean species. Marine and Freshwater Research 58: 505–510.

Anderson, J. (1879). Anatomical and zoological researches comprising an account of the zoological results of the two expeditions to Western Yunnan in 1868 and 1875; and a monograph of the two cetacean genera, Platanistaand Orcaella. London, B. Quaritch 551–564.

Dalebout, M.L., C.S. Baker, J.G. Mead, V.G. Cockcroft & T.K. Yamada (2004). A comprehensive and validated molecular taxonomy of beaked whales, family Ziphiidae.Journal of Heredity 95: 459–473.

Felsenstein, J. (1985). Confidence limits on phylogenies, an approach using bootstrap. Evolution 39: 783–791.

Folmer, O., M. Black, W. Hoeh, R. Lutz& R. Vrijenhoek (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.

George, S., K. Meenakshi& A. Bijukumar (2011). Molecular taxonomy of marine mammals stranded along Kerala coast, India. Current Science 100: 117–120.

Herath, D.R. (2007). Identification of a stranded whale by mitochondrial DNA analysis - www.DNA-surveillance program in action. Asian Fisheries Science 20: 319–324.

Jayasankar, P. & B. Anoop (2010). Identification of Marine Mammals of India. Narendra Publishing House, Delhi, 124pp+10pl.

Jayasankar, P., B. Anoop, R. Peter, V.V. Afsal & M. Rajagopalan (2007). Species of a whale and an unknown fish sample identified using molecular taxonomy. Indian Journal of Fisheries 54: 339–343.

Jayasankar, P., B. Anoop, E. Vivekanandan, M. Rajagopalan, K.M.M. Yousuf, P. Reynold, P.K. Krishnakumar, P.L. Kumaran, V.V. Afsal & A.K. Anoop (2008). Molecular identification of delphinidsand finless porpoise (Cetacea) from the Arabian Sea and Bay of Bengal. Zootaxa 1853: 57–67.

Jefferson, T.A., S. Leatherwood & M.A. Webber (1993). FAO Species Identification Guide. Marine Mammals of the World. FAO, Rome, 320pp.

Junge, G.C.A. (1950). On a specimen of the rare fin whale, Balaenoptera edeni Anderson, stranded on Pulu Sugi near Singapore. Zoologische Verhandelingen 9: 1–26.

Kumaran, P.L. (2002). Marine mammal research in India - a review and critique of the methods. Current Science 83: 1210–1220.

Kumar, S., J. Dudley, M. Nei & K. Tamura (2008). MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinformatics 9: 299–306.

Mohan, R.S.L. (1992). Observations on the whales Balaenoptera edeni, B. musculus and Megaptera novaeangliae washed ashore along the Indian coast with a note on their osteology. Journal of the Marine Biological Association of India 34: 253–255.

Olsen, O. (1913). On the external characteristics and biology of Bryde’s Whale (Balaenoptera brydei) a new rorqualfrom the coast of South Africa. Proceedings of the Zoological Society of London, 1073–1090pp.

Reilly, S.B., J.L. Bannister, P.B. Best, M. Brown, R.L. Brownell Jr., D.S. Butterworth, P.J. Clapham, J. Cooke, G.P. Donovan, J. Urbán & A.N. Zerbini(2008). Balaenoptera edeni. In: IUCN 2010. IUCN Red List of Threatened Species. Version 2010.4. <www.iucnredlist.org>. Downloaded on 31 May 2011.

Ross, H.A., G.M. Lento, M.L. Dalebout, M. Goode, G. Ewing, P. Mclaren, A.G. Rodrigo, S. Lavery & C.S. Baker (2003). DNA Surveillance: Web-based molecular identification of whales, dolphins and porpoises. Journal of Heredity 94: 111–114.

Sasaki, T., M. Nikaido, S. Wada, T.K. Yamada, Y. Cao, M. Hasegawa & N. Okada (2006). Balaenoptera omurai is a newly discovered baleen whale that represents an ancient evolutionary lineage. Molecular Phylogenetics and Evolution 41: 40–52.

Sathasivam, K. (2002). Two whale records from Tamil Nadu, Journal of the Bombay Natural History Society 99: 289–290.

Sathasivam, K. (2004). Marine Mammals of India. WWF-India and Universities Press (India) Pvt. Ltd., Hyderabad, 180pp.

Sholl, T.G.C., F.F. Nascimento, O. Leoncini, C.R. Bonvicino & S. Siciliano(2008). Taxonomic identification of dolphin love charms commercialized in the Amazonian region through the analysis of cytochrome b DNA. Journal of the Marine Biological Association of the United Kingdom 88: 1207–1210.

Stockin, K.A., N. Wiseman, A. Hartman, N. Moffat & W.D. Roe (2008). Use of radiography to determine age class and assist with the post-mortem diagnostics of a Bryde’s Whale (Balaenoptera brydei). New Zealand Journal of Marine and Freshwater Research 42: 307–313.

Verma, S. K. & L. Singh (2003). Novel universal primers establish identity of enormous number of animal species for forensic application. Molecular Ecology Notes 3: 28–31.

Viricel A. & P.E. Rosel (2012). Evaluating the utility of cox1 for cetacean species identification. Marine Mammal Science 28(1): 37–62.

Wada, S., M. Oishi & T.K. Yamada (2003). A newly discovered species of living baleen whale. Nature 426: 278–181.

Woodhouse, C.D. (1991). Marine mammal beachingsas indicators of population events, pp. 111–116. In: Reynolds, J. E. & D. K. Odell (eds.). Marine mammal strandings in the United States: Proceedings of The Second Marine Mammal Stranding Workshop; 3–5 Dec. 1987, Miami. FL,U.S. Department of Commerce, NOAA Technical Report NMFS 98, 157pp.