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We present the results of a study that implements a recently developed phylogenetic algorithm that combines fixed-states nucleotide optimization with breakpoint analysis to identify and examine the evolution of a mitochondrial intergenic spacer between the tRNAVal and 16S rRNA loci in a clade of fairy basslets (Serranidae: Anthiinae). The results of the analysis indicate that this spacer evolved once and that it may be increasing in size through evolutionary time. The resulting molecular hypothesis corroborates much of the previous morphological phylogenetic work.