Homospermidine synthase evolution and the origin(s) of pyrrolizidine alkaloids in Apocynaceae

Premise: Enzymes encoded by paralogous genes producing identical specialized metabolites in distantly related plant lineages are strong evidence of parallel phenotypic evolution. Inference of phenotypic homology for metabolites produced by orthologous genes is less straightforward, since orthologs may be recruited in parallel into novel pathways. In prior research on pyrrolizidine alkaloids (PAs), specialized metabolites of Apocynaceae, the evolution of homospermidine synthase (HSS), an enzyme of PA biosynthesis, was reconstructed and a single origin of PAs inferred because HSS enzymes of all known PA-producing Apocynaceae species are orthologous and descended from an ancestral enzyme with the motif (VXXXD) of an optimized HSS. The
Methods: We increased sampling, tested the effect of amino acid motif on HSS function, revisited motif evolution, and tested for selection to infer evolution of HSS function and its correlation with phenotype.
Results: Some evidence supports a single ori..., Taxon sampling forDHS/HSS sequencing
One hundred seventy (170) accessions of 159 Apocynaceae species (Appendix S1: Table S2), including 10 known PA-producing species were sampled (Appendix S1: Table S2). The outgroup was Gelsemium sempervirens (Gelsemiaceae) (Antonelli et al., 2021). Previously published Apocynaceae DHS and HSS sequences from 25 species were also included (Livshultz et al., 2018).
Calotropis genome query
DHS- and HSS-like genes were extracted from the Calotropis gigantea genome (Hoopes et al., 2018) via tblastx searches with exon sequences of Parsonsia alboflavescens DHS and HSS (MG817648.1, MG817649.1) in Geneious Prime v.2020.0.3 (https://www.geneious.com).
DNA extraction, library preparation, targeted enrichment, sequencing
DNA extraction, library preparation, targeted enrichment, and paired-end sequencing were previously described by Straub et al. (2020). Probes were designed based on the Apocynaceae DHS and HSS sequences published by Livshultz et al. (2018), manual...,  
, # Data from: Homospermidine synthase evolution and the origin(s) of pyrrolizidine alkaloids in Apocynaceae

[https://doi.org/10.5061/dryad.1c59zw3z2](https://doi.org/10.5061/dryad.1c59zw3z2)

## Description of the data and file structure

These datasets and results files are part of a project that studies the evolution of a gene paralog in to test hypotheses about the evolution of pyrrolizidine alkaloid defense in Apocynaceae, adding both taxa sampling and sequence to previous study on a similar topic. The alignment are iterations (explained below) of alignments of the deoxyhypusine synthase (dhs) gene and its putatively neofunctionalized homospermidine synthase (hss) gene. The latter gene is the first gene in the pyrrolizidine alkaloid biosynthesis pathway and it interestingly exists in plant species, including many in Apocynaceae, that do not produce pyrrolizidine alkaloids. There is an amino acid motif that differs between DHS and HSS, and has been connected to HSS function. It was h...,