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Sea cucumbers (Holothuroidea) are a diverse clade of echinoderms found from intertidal waters to the bottom of the deepest oceanic trenches. Their reduced skeletons and limited number of phylogenetically informative traits have long obfuscated morphological classifications. Sanger-sequenced molecular datasets have also failed to constrain the position of major lineages. Noteworthy, topological uncertainty has hindered a resolution for Neoholothuriida, a highly diverse clade of Permo-Triassic age. We perform the first phylogenomic analysis of Holothuroidea, combining existing datasets with 13 novel transcriptomes. Using a highly curated dataset of 1100 orthologues, our efforts recapitulate previous results, struggling to resolve interrelationships among neoholothuriid clades. Three approaches to phylogenetic reconstruction (concatenation under both site-homogeneous and site-heterogeneous models, and coalescent-aware inference) result in alternative resolutions, all of which are recovered with strong support and across a range of datasets filtered for phylogenetic usefulness. We explore this intriguing result using gene-wise log-likelihood scores and attempt to correlate these with a large set of gene properties. While presenting novel ways of exploring and visualizing support for alternative trees, we are unable to discover significant predictors of topological preference, and our efforts fail to favour one topology. Neoholothuriid genomes seem to retain an amalgam of signals derived from multiple phylogenetic histories.

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino acid substitutions in the H1-H2 extracellular loop of Na+,K+-ATPase (NKA). Previous work revealed that hystricognath rodents (e.g., chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino acid insertions in the H1-H2 loop, but their functional significance was not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance in homologs of each of the two species that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla NKA unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse NKA, the amino acid insertion and substitution Q111R both contribute to an augmented CTS resistance without compromising ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific mutational effects on CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.