Convergent adaptation to the same environment by multiple lineages frequently involves rapid evolutionary change at the same genes, implicating these genes as important for environmental adaptation. Such adaptive molecular changes may yield either change or loss of function; loss of protein function can eliminate newly deleterious proteins or reduce energy necessary for protein production. We previously found a striking case of recurrent pseudogenization of the Paraoxonase 1 (Pon1) gene among aquatic mammal lineages – Pon1 became a pseudogene at least six times independently in aquatic and semiaquatic mammals with clear genetic lesions, such as stop codons and frameshifts. Here, we assess the landscape and pace of pseudogenization by studying Pon1 sequences, expression levels, and blood plasma activity across four major aquatic and semiaquatic mammal lineages: pinnipeds, cetaceans, otters, and beavers. We also observed in beavers and pinnipeds an unexpected reduction in expression of Pon3, a paralog with similar expression patterns but different substrate preferences. Ultimately, in all lineages with aquatic/semiaquatic members, we find that preceding any coding level pseudogenization events in Pon1, there is a drastic decrease in expression, followed by relaxed selection, thus allowing for accumulation of disrupting mutations. The recurrent and rapid loss of Pon1 even suggests that it is adaptive in aquatic mammals. Accordingly, we examined diving and dietary traits across pinniped species as potential driving forces of Pon1 loss. We found that loss is best correlated with diving activity and is likely the result of selective pressures associated with hypoxia and hypoxia-induced inflammation.

Expression patterns using transcriptomic data were performed for RNA-seq libraries created from museum liver samples, as well as liver RNA-seq libraries available on the Short Read Archive (SRA). For museum samples, the extracted RNA for 4 samples was sent to Novogene for library preparation (polyA enrichment) and sequencing via NovaSeq PE150. An additional 13 individuals had RNA-seq libraries made using Swift Biosciences RNA library prep kit, before being sent to Novogene for sequencing – these libraries were trimmed for adapters and quality filtered via BBduk in the BBtools package. Data downloaded from the SRA included transcriptomic data derived exclusively from liver for other pinniped species, as well as other carnivores, cetaceans, ungulates, rodents and chiropterans. Each library was mapped using Salmon against the closest-related reference available – for pinnipeds, they were mapped to all 6 pinniped references available. When possible, multiple individuals were used for the same species to assess within-species expression variation. Regardless of origin, expression levels of PON1, PON2, PON3, and endogenous controls were assessed in all transcriptomes. There were a total of 43 different transcriptome references used, thus each transcript was renamed according to the closest BLAST hit from the human transcriptome (GCF_000001405.39_GRCh38.p13_rna), and then isoforms for each gene collapsed into one per gene.

Included in this repository are...

• Raw TPM count files from SALMON for Carnivores, Rodents, Ungulates, Cetaceans, and Chiropterans.
• Scripts and files associated with the PGLS
• Trees associated with CODEML
• PON3 sequence alignments