Molecular systematics of cytochrome oxidase I and 16S from Neochlamisus leaf beetles and the importance of sampling

If a gene tree is to be judiciously used for inferring the histories of clo sely related taxa, (1) its topology must be sufficiently resolved and robus t that noteworthy phylogenetic patterns can be confidently documented, and (2) sampling of species, populations, and pertinent biological variation mu st be sufficiently broad that otherwise misleading sources of genetic varia tion can be detected. These principles are illustrated by the complex gene tree of Neochlamisus leaf beetles that I reconstructed using 90,000 bp of c ytochrome oxidase I (COI) and 168 mitochondrial DNA (mtDNA) sequences from over 100 specimens. Cytochrome oxidase I haplotypes varied up to 25.1% with in Neochlamisus and up to 11.1% within the gibbosus species group, while ex hibiting very low A+T bias for insect mtDNA (63%), low transition saturatio n, and conservative patterns of amino acid variation. 16S exhibited lower s equence divergences and greater A+T bias and transition saturation than COI , and substitutions were more constrained in stems than in loops. Compariso ns with an earlier study of Ophraella leaf beetles highlighted conservative and labile elements of molecular evolution across genes and taxa. Cytochro me oxidase I parsimony and neighbor-joining analyses strongly supported a r obust mtDNA genealogy that revealed the monophyly of Neochlamisus and of th e gibbosus species group. Phylogeographic relationships suggested that the eastern U.S. gibbosus group derives from southwestern velutinus group ances tors. Haplotypes from individual velutinus group species clustered monophyl etically, as expected. However, haplotypes from each of several gibbosus gr oup taxa were polyphyletically distributed, appearing in divergent parts of the tree. 16S provided a less-resolved gibbosus group topology that was co ngruent with the COI tree and corroborated patterns of mitochondrial polyph yly. By subsampling haplotypes corresponding to particular species, populat ions, and ecological variants of gibbosus group taxa, I demonstrate that re covered topologies and genetic distances vary egregiously according to samp ling regime. This study thus documents the potentially dire consequences of inadequate sampling when inferring the evolutionary history of closely rel ated and mitochondrially polyphyletic taxa.