Incorporating molecular phylogenetics with larval morphology while mitigating the effects of substitution saturation on phylogeny estimation: A new hypothesis of relationships for the flatfish family Pleuronectidae (Percomorpha: Pleuronectiformes).

The family Pleuronectidae contains 23 genera and 60 species of primarily dextral flatfishes that maintain a broad distribution, inhabiting all oceans of the Northern Hemisphere. Adults are asymmetrical fishes that are mostly benthic; however, pre-transformation larvae are symmetrical, swimming upright in the water column. A previous phylogenetic analysis, utilizing characters of adult morphology, recovered four major lineages of Pleuronectidae. Evidence from external larval morphology, however, indicates an alternative hypothesis. To resolve intergeneric relationships, DNA sequences (totaling 2391 bp) from the nuclear recombination activating gene 2 (RAG2), and the mitochondrial genes, NADH dehydrogenase subunit 1 (ND1) and 16S, for 22 pleuronectid genera, were analyzed using Bayesian and Maximum Likelihood methods. The markers were partitioned and analyzed for substitution saturation and only the third codon position of ND1 (ND1-3) was found to have reached saturation. Two datasets, one with the saturated data and one without, were analyzed and compared to assess the effects of the saturated data on topology and clade support. Change-point analysis was carried out on saturated transitions and was used to determine the first point where the saturation curve began behaving non-linearly. The level 1 change point was then treated as a threshold pairwise genetic distance. Those clades whose members all had a pairwise distance less than this value were treated as inferred from unsaturated data. Ancestral state reconstruction, utilizing parsimony with unordered states, was carried out on the molecular phylogenies and was used to determine the distribution of character states of larval morphology within the family. The presence of postanal ventral melanophores, distal finfold pigment, preopercular and otic spines, and the pattern of melanophores in the median finfolds of larvae maintain strong phylogenetic signals in the context of these new molecular phylogenies. These findings reaffirm the usefulness of larval characters in systematic ichthyology, especially as an additional line of evidence when hypotheses of relationships based on DNA sequences and adult morphology disagree.