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

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


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 characterised by 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's Whale B. edeni, Mink Whale B. acutorostrata and the Humpback Whale Megaptera novaeangliae (Kumaran 2002;Sathasivam 2004;Jayasankar & Anoop 2010).
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 cytochrome c oxidase subunit 1 (cox1) (Amaral et al. 2007;George et al. 2011) and cytochrome b (cyt b) (Ross et al. 2003;Dalebout et al. 2004;Herath 2007;Sholl et al. 2008;Jayasankar et al. 2007Jayasankar et al. , 2008;;Viricel & Rosel 2011), has been used to successfully identify cetaceans.
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'-GGTCA ACAAATCATAAAGATATTGG-3', Reverse primer-5'-TAAACTTCAGGGTGACCAAAAAATCA-3', Tm value : 45-51 0 C (Folmer et al. 1994)  The following thermal cycling conditions were used for amplifications: 95 0 C for 5 min, followed by 10 cycles of 95 0 C for 30s, 45 0 C for 40s, 72 0 C for 90s, followed by 30 cycles of 95 0 C for 30s, 51 0 C for 40s, 72 0 C for 90s, and a final extension step at 72 0 C for 5 min (for cox 1) and 95 0 C for 5 min, followed by 40 cycles of 95 0 C for 30s, 46 0 C for 30 s, 72 0 C for 30s, and a final extension step at 72 0 C for 7 min (for cytb).
All the PCR products were visualized on 1% agarose gels and the most intense products were selected for sequencing.Sequencing was performed directly using the corresponding PCR primers and products were labelled using 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).Twentysix 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.Phylogenetic position 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 fit nuclear substitution model was selected as HKV+I for cytb and 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 phylogenetic trees 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 40 0 N and 40 0 S (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's Whale 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 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 postmortem examination would help in evaluating the impact of anthropogenic interactions.
Height of dorsal fin (fin tip to base) 96 10 Fluke span 282 11 Width of flukes (distance from nearest point on anterior border of fluke notch) 88

Figure 1 .
Figure 1.Maximum Parsimony phylogram using cytb partial sequences of the samples compared with other reference sequences of Balaenoptera spp. in GenBank.The numbers on the tree branches indicate bootstrap values

Figure 2 .
Figure 2. Maximum Parsimony phylogram using cox1 partial sequences of the samples compared with other reference sequences of Balaenoptera spp. in GenBank.The numbers on the tree branches indicate bootstrap values

Figure 3 .
Figure 3. Maximum Likelihood phylogram using cytb partial sequences of the samples compared with other reference sequences of Balaenoptera spp. in GenBank.The numbers on the tree branches indicate bootstrap values

Figure 4 .
Figure 4. Maximum Likelihood phylogram using cox 1 partial sequences of the samples compared with other reference sequences of Balaenoptera spp. in GenBank.The numbers on the tree branches indicate bootstrap values

Table 1 . Morphometry of Bryde's Whale Balaenoptera edeni Anderson washed ashore at Thanni Beach, Kerala volume
with QIAGEN Taq PCR master mix kit in GenAmp PCR System 9700 (Applied Biosystems).