The "lava lizards" (Microlophus) are distributed throughout the Galápagos Archipelago, and consist of radiations derived from two independent colonizations. The "Eastern Radiation" includes M. bivittatus and M. habeli endemic to San Cristobal and Marchena Islands. The "Western Radiation" includes five to seven historically recognized species distributed across almost the entire Archipelago. We combine dense geographic sampling and multilocus sequence data to estimate a phylogenetic hypothesis for the Western Radiation, to delimit species boundaries in this radiation, and to estimate a time frame for colonization events.Our phylogenetic hypothesis rejects two earlier topologies for the Western Radiation and paraphyly of M. albemarlensis, while providing strong support for single colonizations on each island. The colonization history implied by our phylogeny is consistent with general expectations of an east-to-west route predicted by the putative age of island groups, and prevailing ocean currents in the Archipelago. Additionally, combined evidence suggests that M. indefatigabilis from Santa Fe should be recognized as a full species. Finally, molecular divergence estimates suggest that the two colonization events likely occurred on the oldest existing islands, and the Western Radiation represents a recent radiation that, in most cases, has produced species that are considerably younger than the islands they inhabit. K E Y W O R D S :Galápagos, lizards, mitochondrial DNA, molecular timing of colonization, nuclear DNA, oceanic islands, phylogeny.Oceanic islands have been model systems in evolutionary studies for well over a century (Emerson 2002;Whittaker et al. 2008), and the Galápagos Archipelago, located about 960 km west from the coast of Ecuador, has figured prominently among them. Galápagos is one of the most recent oceanic island formations (Christie et al. 1992), and a consensus biogeographic history (reviewed in Grehan 2001) has favored an over-water colonization model for the origin of its many endemic radiations. Most of these studies have emphasized early models of Galápagos colonization events that were initially constrained to a 4 to 5 million year time frame set by the estimated ages of the oldest current islands (Cox 1983). The subsequent discovery of underwater seamounts representing former Galápagos islands to the east of the current archipelago on the east-shifting Nazca Plate, extended the 1 6 0 6

We use a multigene data set (the mitochondrial locus and nine nuclear gene regions) to test phylogenetic relationships in the South American "lava lizards" (genus Microlophus) and describe a strategy for aligning noncoding sequences that accounts for differences in tempo and class of mutational events. We focus on seven nuclear introns that vary in size and frequency of multibase length mutations (i.e., indels) and present a manual alignment strategy that incorporates insertions and deletions (indels) for each intron. Our method is based on mechanistic explanations of intron evolution that does not require a guide tree. We also use a progressive alignment algorithm (Probabilistic Alignment Kit; PRANK) and distinguishes insertions from deletions and avoids the "gapcost" conundrum. We describe an approach to selecting a guide tree purged of ambiguously aligned regions and use this to refine PRANK performance. We show that although manual alignment is successful in finding repeat motifs and the most obvious indels, some regions can only be subjectively aligned, and there are limits to the size and complexity of a data matrix for which this approach can be taken. PRANK alignments identified more parsimony-informative indels while simultaneously increasing nucleotide identity in conserved sequence blocks flanking the indel regions. When comparing manual and PRANK with two widely used methods (CLUSTAL, MUSCLE) for the alignment of the most length-variable intron, only PRANK recovered a tree congruent at deeper nodes with the combined data tree inferred from all nuclear gene regions. We take this concordance as an objective function of alignment quality and present a strongly supported phylogenetic hypothesis for Microlophus relationships. From this hypothesis we show that (1) a coded indel data partition derived from the PRANK alignment contributed significantly to nodal support and (2) the indel data set permitted detection of significant conflict between mitochondrial and nuclear data partitions, which we hypothesize arose from secondary contact of distantly related taxa, followed by hybridization and mtDNA introgression.