Gene expression, evolution, and the genetics of electrosensing in the smalltooth sawfish, Pristis pectinata

Abstract

Sawfishes (Pristidae) are large, highly threatened rays named for their tooth-studded rostrum, which is used for prey sensing and capture. Of all five species, the smalltooth sawfish, Pristis pectinata, has experienced the greatest decline in range, currently found in only ~20% of its historic range. To better understand the genetic underpinnings of these taxonomically and morphologically unique animals, we collected transcriptomic data from several tissue types, mapped them to the recently completed reference genome, and contrasted the patterns observed with comparable data from other elasmobranchs. Evidence of positive selection was detected in 79 genes in P. pectinata, several of which are involved in growth factor/receptor tyrosine kinase signaling and body symmetry and may be related to the unique morphology of sawfishes. Changes in these genes may impact cellular responses to environmental conditions such as temperature, dissolved oxygen, and salinity. Data acquired also allow for examination of the molecular components of P. pectinata electrosensory systems, which are highly developed in sawfishes and have likely been influential in their evolutionary success.

Keywords: conservation; electrosensing; evolution; sawfish; transcriptome.

FIGURE 1

Establishment of a near‐complete gene set for the smalltooth sawfish, Pristis pectinata. (a) Log length distribution of transcripts in assembled transcriptome with total number of transcripts. (b) Outcome of Benchmarking Universal Single Copy Ortholog (BUSCO) analysis to assess completeness of assembled transcriptome. (c) WeGO (Ye et al., 2018) gene ontology analysis of P. pectinata and little skate, Leucoraja erinacea, transcriptomes. Genes are classified according to biological process, molecular function, or cellular component and plotted by percent of genes related to meaningful biological activities. (d) Venn diagram (Hulsen et al., 2008) showing overlap of the number of transcripts provided with the public genome (GCA_009764475.1, green), the Cufflinks (purple) assisted annotation of the public genome, and transcripts in the de novo assembled transcriptome (pink). (e) BUSCO assessment for all unique gene content from combined transcriptome and genome resources, and annotations associated with GCA_009764475.1.

FIGURE 2

Smalltooth sawfish, Pristis pectinata, genes under positive selection. (a) Phylogeny of taxa used in positive selection analysis with approximate divergence times shown as millions of years ago at each node and number of transcripts in each transcriptome noted at each terminal branch. Species included: Latimeria menadoensis, Callorhinchus milii, Scyliorhinus retifer, Leucoraja erinacea, and P. pectinata. (b) Results of k‐means clustering analysis of genes with a percentage of sites under selection using omega value, percent of sites, change in grand average of hydropathy value relative to little skate, L. erinacea. (c) Enriched gene ontology (GO) terms related to biological processes from genes which were grouped into Cluster 1 by principal components analysis plotted by ‐log(p‐value). (d–h) Changes in hydrophobicity and polarity of protein sequence relative to L. erinacea. Sequence alignment gaps are shown by shaded regions. Functional protein domains retrieved from InterproScan and literature are shown as colored boxes below each plot. (i) Sequence alignment of PCR‐amplified region of interest from HoxA5 between Pristis sawfishes and L. erinacea with codons of interest colored by nucleotide. (j) Alignment of region of interest from amplification of CCDC103. Alignments are colored by nucleotide.

FIGURE 3

Smalltooth sawfish, Pristis pectinata, electroreception machinery. (a) FPKM normalized expression of voltage‐gated calcium channel α and β subunits in rostral skin. (b) Multiple sequence alignment of Cacnα1D depicting missing 91‐residue segment in P. tinata ion channel domain relative to other elasmobranchs. Species included: Callorhinchus milii (C. mil), Leucoraja erinacea (L. eri), Amblyraja radiata (A. rad), Scyliorhinus retifer (S. ret), P. pectinata (P. pec), Rhinchodon typus (R. typ), and Chiloscyllium plagiosum (C. pla). Alignments are colored by amino acid chemistry. (c) Phylogenetic construction of potassium channels and subunits with bootstrap values noted (left). Sequences were aligned with ClustalW and tree was constructed by RAxML with 1000 bootstraps. Shaker channel sequences are highlighted in red, while BK channels are highlighted in light blue. Annotated protein domains of each sequence obtained using Interpro Scan (right). cyto, cytoplasm; extracell, extracellular; ion pore, ion channel pore; sig pep, signal peptide; and TM, transmembrane. (d) Normalized expression of potassium channels potentially involved electrosensing in rostral skin. Kcnm‐transcripts correspond to BK channel subunits and Kv are voltage‐gated Shaker channels. Novel β refers to the uncharacterized BK subunit found in transcriptome data. (e) GGSashimi plot of alternatively spliced BK channel transcripts in all P. pectinata tissues. Transcriptome expression confirms that the BK channel transcript with highest expression in the rostral skin retains exon 21 (exon 29 in L. erinacea).