Phylogeny and Jaw Ontogeny of Beloniform Fishes1

Integrative and Comparative Biology, Nov 2004 by Lovejoy, Nathan R, Iranpour, Mahmood, Collette, Bruce B

Here, we present an extension and test of Lovejoy's (2000) findings. We have expanded the matrix by adding 14 ingroup taxa to the previous 39, which significantly improves taxonomic coverage of hemiramphids and flyingfishes. We have also added a novel source of character data, by sequencing a 1 kilobase fragment of the nuclear gene, Recombination Activating Gene 2, or RAG2. Our analysis is based on the nuclear RAG2 and Tmo-4C4 genes, and the mitochondrial cytochrome-b and 16S rRNA genes. We use the resultant phylogenetic hypothesis to explore the evolution of jaw ontogeny.

METHODS

Fishes were collected in the field by ourselves or colleagues. Gill tissue was either frozen immediately in liquid nitrogen or preserved in 95-100% ethanol or buffer of 20% DMSO, 0.25 M EDTA at pH 8, saturated with NaCl (Seutin et al., 1991). Tissue preserved in buffer and stored at room temperature reliably yields amplifiable DNA (after storage for up to four years). Voucher specimens were preserved in 10% buffered formalin, transferred to 70% ethanol or 5055% isopropanol and deposited in museum collections (catalogue numbers for voucher specimens are listed with sequences in GenBank).

Samples represent all beloniform families, and with the addition of new taxa, 30 of 39 beloniform genera are represented in the study: ten of ten needlefish genera, three of four saury genera, nine of thirteen halfbeak genera, seven of seven flyingfish genera, and one of four ricefish genera. Whenever possible, sequences were collected from two individuals of each species, providing a total of 104 terminal taxa for analysis, representing 54 different beloniform species.

Data collection

Both mitochondrial and nuclear genes were used for analysis. However, rather than sequencing a single complete mitochondrial gene, smaller segments of two separate genes, cytochrome b (cyt b) and 16S rRNA (16S), were examined. This decision was based on the expectation that sampling a range of genes, with different rates and patterns of molecular evolution, would provide phylogenetic information that spanned a broader range of taxonomic divergence. The nuclear gene, Tmo-4C4 (Tmo) is an anonymous, putative protein-coding locus identified and used for phylogeny by Streelman and Karl (1997). It provided resolution of families and genera within labroids, and was thus expected to provide useful information for deeper parts of the beloniform tree. Recombination Activating Gene 2 (RAG2) is a nuclear gene that appears to evolve slightly faster than Tmo-4C4 (Lovejoy and Collette, 2001), and has proven useful for species-level phylogenetic analyses (Sullivan et al., 2000; Lovejoy and Collette, 2001).

For each sample, approximately 25 mg of tissue was rinsed briefly in water, then DNA purified using Qiagen's spin-column tissue kit. Cells were lysed at 55° in 20 µl of Proteinase K (20 mg/ml) for three to six hours. Lysate was bound to the spin column membrane, and washed twice by centrifugation. DNA was then eluted by centrifugation twice with 200 µl of low salt buffer.