Material and Methods

Live animals of Spirula were caught near Fuerteventura (Canary Islands, Spain, Fig. 7.2). They were preserved in 95-100% ethanol. A small tissue sample was taken from the arm tip of each animal. DNA was isolated from these samples following the Chelex method (Walsh et al., 1991) modified by Söller et al. (2000). Chelex supernatant was purified with the DNeasy Kit (Quiagen, Hilden).

A fragment of mitochondrial ribosomal 16S RNA gene (16S) and the cytochrome oxidase III gene (COIII) were used as target sequences. The DNA of mt16S rDNA was amplified by PCR using universal primers 16Sar and 16Sbr (Simon et al., 1991). For the COIII fragment, primers from Barriga Sosa et al. (1995) and Warnke

On the species status of Spirula spirula

On the species status of Spirula spirula

Fig. 7.2 Map of localities of Spirula sample (arrows show sample location, map modified after Scotese, 20)01).

(1999) were used. These primers were designed for obtaining the DNA of COIII from various Octopus species. A polymerase chain reaction (PCR) was performed in 50 jl reaction volumes containing 10 mM Tris-HCL, pH 8.8; 25 mM KCL; 2 mM MgSO4; 0.2 mM each of dATP, dCTP, dGTP, dTTP; 0.2 jiM of both forward and reverse primers; 0.5 U Taq polymerase (Pharmacia Biotech, Freiburg i. Br.), 110 jl DNA solution (purified Chelex supernatant).

The fragments were sequenced and arranged together with three further Spirula sequences from the EMBL database [EMBL Acc-Nr.: X79574, Bonnaud et al. (1994), fragment of mt16S DNA; EMBL Acc-Nr.: X97957, Bonnaud et al. (1996), fragment of mtCOIII; EMBL Acc-Nr.: AY293659, Nishiguchi et al. (2004), fragment of mt16S DNA)] in a multiple sequences alignment. The Spirula specimens used by Bonnaud et al. (1994, 1996) were recovered from New Caledonia. For the animal mentioned by Nishiguchi et al. (2004), the Atlantic Ocean was given as the collecting site. The sequences of this study have been deposited in the EMBL database (accession numbers follow: AJ966784 Spirula spirula isolate 1, fragment of mt16S DNA; AJ966785, Spirula spirula isolate 2, fragment of mt16S DNA; AJ966786, Spirula spirula isolate 2, fragment of mtCOIII; AJ966787, Spirula spirula isolate 1, fragment of mtCOIII). The sequences of the animal 1, 3, and 4 were identical. Because of this, just the sequence of animals 1 and 2 were deposited in the database.

Sequence divergences were examined using the PAUP* program (Swofford, Smithonian Institution, Washington, DC). Additionally, the nucleotide sequences of COIII were translated into the amino acid sequence using the invertebrate mito-chondrial code. The putative phylogenetic relationships were calculated applying both distance- and character-based analyses of the data. The trees were rooted with Octopus vulgaris (EMBL Acc-Nr.: AJ012121, Söller et al., 2000) as an outgroup representative.

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