Unraveling the evolutionary radiation of the thoracican barnacles using molecular and morphological evidence: A comparison of several divergence time estimation approaches

Abstract

The Thoracica includes the ordinary barnacles found along the sea shore and is the most diverse and well-studied superorder of Cirripedia. However, although the literature abounds with scenarios explaining the evolution of these barnacles, very few studies have attempted to test these hypotheses in a phylogenetic context. The few attempts at phylogenetic analyses have suffered from a lack of phylogenetic signal and small numbers of taxa. We collected DNA sequences from the nuclear 18S, 28S, and histone H3 genes and the mitochondrial 12S and 16S genes (4,871 bp total) and data for 37 adult and 53 larval morphological characters from 43 taxa representing all the extant thoracican suborders (except the monospecific Brachylepadomorpha). Four Rhizocephala (highly modified parasitic barnacles) taxa and a Rhizocephala + Acrothoracica (burrowing barnacles) hypothetical ancestor were used as the outgroup for the molecular and morphological analyses, respectively. We analyzed these data separately and combined using maximum likelihood (ML) under "hill- climbing" and genetic algorithm heuristic searches, maximum parsimony procedures, and Bayesian inference coupled with Markov chain Monte Carlo techniques under mixed and homogeneous models of nucleotide substitution. The resulting phylogenetic trees answered key questions in barnacle evolution. The four-plated Iblomorpha were shown as the most primitive thoracican, and the plateless Heteralepadomorpha were placed as the sister group of the Lepadomorpha. These relationships suggest for the first time in an invertebrate that exoskeleton biomineralization may have evolved from phosphatic to calcitic. Sessilia (nonpedunculate) barnacles were depicted as monophyletic and appear to have evolved from a stalked (pedunculate) multiplated (5+) scalpelloidlike ancestor rather than a five-plated lepadomorphan ancestor. The Balanomorpha (symmetric sessile barnacles) appear to have the following relationship: (Chthamaloidea(Coronuloidea(Tetraclitoidea, Balanoidea))). Thoracican divergence times were estimated under ML-based local clock, Bayesian, and penalized likelihood approaches using an 18S data set and three calibration points: Heteralepadomorpha = 530 million years ago (MYA), Scalpellomorpha = 340 MYA, and Verrucomorpha = 120 MYA. Estimated dates varied considerably within and between approaches depending on the calibration point. Highly parameterized local clock models that assume independent rates (r ≥ 15) for confamilial or congeneric species generated the most congruent estimates among calibrations and agreed more closely with the barnacle fossil record. Reasonable estimates were also obtained under the Bayesian procedure of Kishino et al. (2001, Mol. Biol. Evol. 18:352-361) but using multiple calibrations. Most of the dates estimated under the Bayesian procedure of Aris-Brosou and Yang (2002, Syst. Biol. 51:703-714) and the penalized likelihood method using single and/or multiple calibrations were inconsistent among calibrations and did not fit the fossil record.