Taxonomy

The taxonomy of species in Witness for the Whales follows Rice (1998), with the following exceptions: for the Balaenidae, four species are recognised following Rosenbaum et al. (2000); for the Balaenopteridae, Antarctic, North Pacific, North Atlantic and dwarf minke whales are recognised as distinct taxa (the latter three are recognised as subspecies of Balaenoptera acutorostrata by Rice, 1998); for the Ziphiidae, 21 species are recognised following Dalebout et al. (2002); and for the Platanistidae, two species are recognised following Hamilton et al. (2001). Link to schematic representation of cetacean phylogeny

We are aware of the confused taxonomy of the sei whale-Bryde's whale complex of species which likely comprises at least three unique taxa. We are also aware of the problems associated with the apparent lack of genetic differentiation among some members of the Stenella-Tursiops- Delphinus complex of species (as discussed in Dizon et al. 2000). The programme includes a caution message for test sequences identified as derived from this subfamily, Delphininae.

Data sources and DNA Extraction

Sample sources and validation of species-identity of reference specimens

The cetacean reference sequences in this database were derived from dead, stranded animals, victims of fisheries bycatch, skeletal material from museum specimens or from biopsy samples collected from living animals in the field. For some species, reference sequences were obtained from the international genetic database, Genbank, as generated from studies by other known species specialists. In general, a sequence was included in the reference databases only if the specimen from which it was obtained was examined by an expert in cetacean morphology, and diagnostic skeletal material or photographic records were collected (Dizon et al. 2000). However, not all sequences in the current version (Vs. 3.1) of Witness for Whales are fully validated.

DNA extraction

For sequences generated in our home laboratory at the Univesity of Auckland, total genomic DNA was isolated from tissue samples using proteinase K digestion, following standard methods (Sambrook et al. 1989), as modified by C. S. Baker et al. (1994). For museum specimens represented by teeth or bone, DNA was extracted using the silica method (Boom et al. 1990; Hoss and Paabo 1993), as modified by Matisoo-Smith et al. (1997). All work with museum tooth and bone samples was conducted in a designated 'ancient'DNA laboratory in which no previous work on modern cetacean tissue had been conducted. Sufficient material for DNA extraction was obtained from these specimens using methods described in Pichler and Baker (2000) and Pichler et al. (2001).

PCR amplification

• Fresh tissue samples: mtDNA control region
  For specimens represented by fresh tissue samples, a 500 base pair (bp) fragment of the 5'end of the mitochondrial (mt) DNA control region (D-loop) was amplified, using the primers, M13-Dlp1.5-L and Dlp5-H (e.g., Dalebout et al. 1998; Dalebout 2002). In some cases, a longer 800 bp fragment of this locus was amplified using the primers, M13-Dlp1.5-L and Dlp8G-H (e.g., Lento et al. 1998; Pichler et al. 2001). Note that the Genbank control region sequences from other studies included in the reference database may have been generated using slightly different primers.
• Freshtissue samples: mtDNA cytochrome b
  For the Ziphiidae (beaked whales), a 424 bp fragment of the 5'end of the cytochrome b gene was also amplified, using the 'universal'primers, GLUDG-L and CB2-H (Palumbi 1996). Polymerase-chain reaction (PCR) amplification of sequences from both loci followed standard protocols (Palumbi 1996).
• Historical tooth Kishino and Hasegawa 1989). The parameter values used are: * the transition/transversion ratio (Ts/Tv) = 2, and * the equilibrium nucleotide frequencies are:~ freq(A) = 0.30751~ freq(C) = 0.22645~ freq(G) = 0.13893~ freq(T) = 0.32711~ as calculated empirically across all reference sequences ===Building the Phylogenetic Tree=== A phylogenetic tree is build to include the members of the reference set of sequences chosen by the user and the sequence that the user has submitted. The tree is built using the Neighbor-Joining (NJ) algorithm (Saitou and Nei 1987) and rooted using an outgroup appropriate for each data set. ===Maximum Likelihood Analysis=== The reference alignment, and the associated phylogenetic tree, are considered to be prior knowledge about the relationships among the reference organisms. Potentially the query sequence can be joined to that tree on any branch. We seek the connection point that has the highest statistical likelihood, thereby giving the maximum likelihood estimate of the relationship between the query and reference sequences. The maximum likelihood connection point is represented in the output by a dashed branch. For a particular connection point the determined likelihood score is the maximum likelihood estimate under the associated topology (that is, all the branch lengths are reoptimised for each connection point). The Shimodaira-Hasegawa (SH) test is used for assessing a confidence limit on the connection point with the highest expected likelihood. The expected likelihood of a connection point is the expectation of likelihood under the true process of evolution (as a random variable). The SH test calculates such a confindence limit by simulating replicate datasets under an approximation of the least configurable configuration (LFC) in which is that all connection points have equivalent expected likelihoods, and comparing the observed differences in likelihood with the expected distribution of likelihoods under the LFC. The utilised implementation of the SH test simulates 1000 non-parameteric bootstraps, and uses the RELL (Shimodaira and Hasegawa 1999) approximation. Branches that represent connection points within the confidence limit are colour red. A critical value of ?= 0.05 is used (95%confidence limit). ==References== #Arnason, U., Gullberg, A., and Widegren, B. 1991. The complete nucleotide sequence of the Mitochondrial DNA of the fin whale, Balaenoptera physalus. Journal of Molecular Evolution 33, 556-568. #Baker, C. S., Slade, R. W., Bannister, J. L., Abernethy et al. 1994. Hierarchical structure of mitochondrial DNA gene flow among humpback whales Megaptera novaeangliae, world-wide. Molecular Ecology 3, 313-327. #Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheim-van Dillen, P. M. E. and van der Noorda, J. 1990. Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28, 495-503. #Dalebout, M. L. 2002. Species Identity, genetic diversity and molecular systematic relationships among the Ziphiidae (beaked whales). PhD thesis, School of Biological Sciences, University of Auckland, Auckland, New Zealand. #Dalebout, M. L., J. G. Mead, C. S. Baker, A. N. Baker, and A. L. van Helden. 2002. A new species of beaked whale Mesoplodon perrini sp. n. (Cetacea: Ziphiidae) discovered through phylogenetic analyses of mitochondrial DNA sequences. Marine Mammal Science 18, 577-608. #Dalebout, M. L., G. J. B. Ross, C. S. Baker, R. C. Anderson, P. B. Best, V. G. Cockcroft, H. L. Hinsz, V. Peddemors, and R. L. Pitman. 2003. Appearance, distribution and genetic distinctiveness of Longmans beaked whale, Indopacetus pacificus. Marine Mammal Science 19, in press.

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