Index References

Cetacea (whales, dolphin, porpoises) are important reference model organisms used in many evolutionary studies. The order Cetacea traditionally consists of three suborders: the extinct Archaeoceti, Odontoceti (toothed whales) and Mysticeti (baleen whales) (Arnason et al., 2004). Generally, it has been shown that, in the evolution of cetaceans from terrestrial to fully marine life, these organisms highly specialized their morphological changes as well as the acquisition of echolocating ability. For this reason, comprehensive molecular phylogenetic analyses, including the mitochondrial cytochrome b gene (Arnason and Gullberg, 1996), the milk casein gene (Gatesy et al., 1996) and short and long interspersed repetitive elements (SINEs and LINEs) (Nikaido et al., 1999), have been of great interest to evolutionary biologists. Although the phylogenetic analysis of the origin of whales has been extensively studied over the past decade, genomic markers associated with the repetitive sequences relating to the phylogeny of the whale have not been well explored.

In our previous paper, we reported a highly conserved repetitive sequence in the upstream region of the false killer whale (Pseudorca crassidens) PRNP gene (Kim et al., 2008). The repetitive unit is composed of a consensus sequence of 1,869 bp, displays 97% sequence similarity, and is repeated 19.2 times. Comprehensive bioinformatic analysis has disclosed the possibility that this could be a new potential whale-specific genomic marker, and could thus be used to trace the origin of this species and its genomic relationships among those closely related during the evolutionary process.

To validate the quality and the phylogenic relationship, we obtained tissue samples from the Cetacean Research Institute at the National Fisheries Research and Development Institute in Korea. The whale species examined in this study were as follows: Delphinus delphis (Short-beaked common dolphin), Lagenodelphis hosei (Fraser’s dolphin), Tursiops truncatus (Bottlenose dolphin), Lagenorhynchus obliquidens (Pacific white-sided dolphin), Grampus griseus (Risso’s dolphin), Pseudorca crassidens (False killer whale), Orcinus orca (Killer whale), Neophocaena phocaenoides (Finless porpoise), Phocoena phocoena (Harbor porpoise), Mesoplodon stejnegeri (Stejneger’s beaked whale), Berardius bairdii (Baird’s beaked whale), Ziphius cavirostris (Cuvier’s beaked whale), Physeter catodon (Sperm whale), Balaenoptera omurai (Omura’s baleen whale), Balaenoptera acutorostrata (Minke whale), and Megaptera novaeangliae (Humpback whale). To validate the quality and the phylogenic relationship of these samples, genomic DNA from tissues were extracted and purified by Blin and Stafford’s method (Blin and Stafford, 1976). For taxonomic characterization of these tissue samples, we first designed specific primers to 16S rDNA based on the consensus sequences of whale mitochondria DNA using the NCBI database: mt16s-5' GCAGCCAYCAATWRAGAAAGCGTTMMAGCTC, mt16s-3' GAACAARTGATTATGCTACCTTTGCAC. Polymerase chain reaction (PCR) was typically performed in 50 μl reactions using genomic DNA, and the products were sequenced using an ABI 3730XL DNA analyzer (Applied Biosystems, USA). The sequence data have been deposited in DDBJ/EMBL/GenBank under the accession numbers from AB481388 to AB481403. A rooted phylogenetic tree using DNA sequence data was constructed from evolutionary distance matrices by a neighbor-joining algorithm using the maximum composite likelihood method of the MEGA4 program (Tamura et al., 2007). As a result, they were classified into two orders (Odontoceti and Mysticeti) and five family groups (Fig. 1a): Delphinidae (7), Phocoenidae (2), Ziphiidae (3), Physeteridae (1) and Balaenopteridae (3).

View Details

Fig. 1 (a) A phylogenetic tree based on 16S rDNAs. The multiple sequence alignment used to construct the tree was generated using CLUSTALW 2.0 (Larkin et al., 2007). The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). Each node was tested using the bootstrap approach by taking 1000 replications and a random seeding of 64238 to ascertain the reliability of nodes. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 711 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007). The numbers indicated are in percentages against each node. The branch lengths are drawn to scale indicated. The abbreviations and accession numbers for the sequences are the following: Short-beaked common dolphin, AB481388; Bottlenose dolphin, AB481402; Fraser’s dolphin, AB481403; Risso’s dolphin, AB481395; False killer whale, AB481400; Pacific white-sided dolphin, AB481390; Killer whale, AB481399; Finless porpoise, AB481391; Harbor porpoise, AB481401; Cuvier’s beaked whale, AB481398; Baird’s beaked whale, AB481396; Stejneger’s beaked whale, AB481397; Sperm whale, AB481393; Minke whale, AB481389; Omura’s baleen whale, AB481392; Humpback whale, AB481394. Red filled circles represent results in “c” and “d” experiments. (b) Consensus sequence (1,869 bp) of the repetitive unit. PCR primer positions are indicated with red arrows and characters and the direction of the arrows indicate sense/antisense orientation. (c) PCR amplification from 16 whale species. (d) Southern blot analysis from 16 whale species. The genomic probe was hybridized to BamHI-digested genomic DNA of the targeted whales. The control used here is the QGB1-059P03 BAC clone (GenBank Accession number AP009416).

To investigate whether the 1,869 bp repetitive unit plays a role as a genomic marker, we designed PCR primers, whs1-CTACTGGGTAAGTATGGCAGT and whs2-CGGCCTCAAGGTACGCAGTTC, and performed PCR amplification with the unique primer designed in the repetitive sequence unit using genomic DNA from 16 whale species (Fig. 1b). The PCR products were then separated by electrophoresis on a 1.5% agarose gel. As a result, not only did amplification occur in seven of 16 species, but these PCR products were also approximately the same size (Fig. 1c). Interestingly, all seven species that were amplified belonged to the Delphinidae family phylogenetic classification. As such, this result represents that the repetitive sequence has specificity in the Delphinidae family. In addition, DNA blotting was performed by standard protocols to validate our PCR results (Sambrook and Russell, 2001). Similar to our PCR results, strong hybridization signals were also observed in only seven species of Delphinidae, which were approximately the same in length as the control (Fig. 1d).

Pioneering efforts concerning repetitive sequences in cetaceans have shown the existence of a highly repetitive component in the Delphinidae family, which was estimated to be approximately 1,580 bp in size by southern hybridization (Widegren et al., 1985). A subsequent study revealed the repetitive unit’s acute sequences and differences in length, which showed specificity among subfamilies of the Delphinidae family (Gretarsdottir and Arnason, 1992). In addition, this suggests that the repetitive units had likely been inserted into the Delphinids after the split of the family Delphinidae and Phocoenidae during the evolutionary process, and that long repetitive sequences could be useful markers for understanding this process in the whale lineage from a common ancestor, especially given the divergence of the dolphin lineage.

Conclusively, our study has revealed that the novel 1,869 bp repetitive sequence has both sensitivity and specificity in the members of the Delphinidae family. This result suggests that the repetitive sequence is a unique DNA component, which most likely was inserted into Delphinids after the split of the family Delphinidae and Phocoenidae during the evolutionary process, similar to the 1,580 bp element identified in the work of Widegren et al. (Widegren et al., 1985).

This study was supported by grant 2007-04269 from the Ministry of Education, Science and Technology and grant KGM1230812 from the Korea Research Institute of Bioscience and Biotechnology (KRIBB).