Evolution of the actin gene family in testate lobose amoebae (Arcellinida) is characterized by two distinct clades of paralogs and recent independent expansions.

The evolution of actin gene families is characterized by independent expansions and contractions across the eukaryotic tree of life. Here, we assess diversity of actin gene sequences within three lineages of the genus Arcella, a free-living testate (shelled) amoeba in the Arcellinida. We established four clonal lines of two morphospecies, Arcella hemisphaerica and A. vulgaris, and assessed their phylogenetic relationship within the "Amoebozoa" using small subunit ribosomal DNA (SSU-rDNA) genealogy. We determined that the two lines of A. hemisphaerica are identical in SSU-rDNA, while the two A. vulgaris are independent genetic lineages. Furthermore, we characterized multiple actin gene copies from all lineages. Analyses of the resulting sequences reveal numerous diverse actin genes, which differ mostly by synonymous substitutions. We estimate that the actin gene family contains 40-50 paralogous members in each lineage. None of the three independent lineages share the same paralog with another, and divergence between actins reaches 29% in contrast to just 2% in SSU-rDNA. Analyses of effective number of codons (ENC), compositional bias, recombination signatures, and genetic diversity in the context of a gene tree indicate that there are two groups of actins evolving with distinct patterns of molecular evolution. Within these groups, there have been multiple independent expansions of actin genes within each lineage. Together, these data suggest that the two groups are located in different regions of the Arcella genome. Furthermore, we compare the Arcella actin gene family with the relatively well-described gene family in the slime mold Dictyostelium discoideum and other members of the Amoebozoa clade. Overall patterns of molecular evolution are similar in Arcella and Dictyostelium. However, the separation of genes in two distinct groups coupled with recent expansion is characteristic of Arcella and might reflect an unusual pattern of gene family evolution in the lobose testate amoebae. We provide a model to account for both the existence of two distinct groups and the pattern of recent independent expansion leading to a large number of actins in each lineage.