Phylogenetics of Apocynaceae and the evolution of angiosperm alkaloids
Authors:   , Smith Chelsea Raymond , Livshultz Tatyana , Potapova Marina 
Published: July 2022

Reconstructing the evolution of plant adaptations to insect herbivores requires a layered approach that integrates phenotype and genotype. Given that plant defenses experience positive selection when they first evolve, and selection on phenotype feeds through to selection on genotype, patterns of selection on genes involved in a specialized metabolic pathway can place the origin of a metabolite even when ancestral state reconstruction of the phenotype is ambiguous. Pyrrolizidine alkaloids (PAs), defensive specialized metabolites, are rare in Apocynaceae, with scattered occurrence in only a few lineages. Prior research on the evolution of homospermidine synthase (hss), the only characterized gene of PA biosynthesis in Apocynaceae, inferred a single origin of PAs followed by multiple losses based on patterns of gene duplication, loss, and amino acid motif evolution. All Apocynaceae hss-like genes evolved from the ubiquitous deoxyhypusine synthase (dhs) via a single duplication in the MRCA of all known PA-producing species. Furthermore, the characteristic HSS motif (VXXXD) was reconstructed as evolving from the characteristic DHS motif (IXXXN) in this ancestor. Reversions to the DHS-like IXXXN motif and evidence of pseudogenization in the Asclepiadoideae hss-like clade were inferred as evidence of PA biosynthesis loss. With an expanded dataset (142 taxa, 275 sequences, 70% larger), I revisited HSS motif evolution in Apocynaceae and tested for selection on hss-like sequences to infer the number of times a highly functional HSS evolved. In addition, my collaborator, Elisabeth Kaltenegger (University of Kiel), tested the effect of amino acid motif on the function of HSS from PA-producing Parsonsia alboflavescens (Dennst.) Mabb. via mutagenesis and in vitro enzymatic assays. In contradiction to the prediction of selection for a functionally optimized HSS in the MRCA of all PA-producing Apocynaceae followed by loss of function in many independent lineages, the ancestral HSS, now reconstructed with an intermediate IXXXD motif, did not experience positive selection, and there was no evidence of significant relaxation of selection in the hss-like sequence clade relative to the dhs clade as would be predicted by pervasive loss of function. Instead, our data supports multiple independent recruitments of the ancestral hss-like locus to a functionally optimized HSS: the canonical HSS motif (VXXXD) evolved multiple times independently; one of the branches where the VXXXD motif evolved experienced significant positive selection; a branch site test shows that the first position of the motif, amino acid position 269 (I or V), experienced significant positive selection; and the hss-like clade experienced significant intensification of positive selection relative to the dhs-like clade. All mutagenized HSS enzymes produced abundant homospermidine, regardless of amino acid motif; however, the D273N mutation restored a minute amount of DHS-like function. Therefore, we infer the reconstructed IXXXD ancestral HSS in Apocynaceae had lost the ancestral DHS-like function and could produce homospermidine. Further conclusions, however, about the evolution of PA biosynthesis in Apocynaceae will require the identification of additional genes in the PA biosynthetic pathway and ancestral state reconstruction of the PA phenotype on a well-resolved and supported phylogeny. Ancient rapid radiations have impeded the resolution of several nodes in the Apocynaceae phylogeny that are key to reconstructing phenotypic evolution and biogeographic history in the family. Plastome-based phylogenetic analyses had produced resolved topologies of presumed rapid radiations in Apocynaceae. The first nuclear phylogenomic analysis of Apocynaceae, based on the Angiosperm353 probe set, supported several nodes that were incongruent with the plastome topology, implicating lineage sorting and/or chloroplast capture. Furthermore, the nuclear phylogenomic analysis did not support the positions of several key taxa, including the tribe Rhabdadenieae and subfamily Periplocoideae in the APSA clade. Here, taxonomic sampling (175 species, 5-fold larger), with a focus on APSA clade lineages, and nuclear gene sequencing (837 genes, 2-fold larger), using a family-specific probe set, were both expanded and used to construct species trees with summary coalescent and concatenation analyses. After accounting for long branch attraction, both concatenated and coalescent trees reconstructed the same APSA clade topology. After the successive divergence of Wrightieae, Nerieae, and Malouetieae, are two sister clades: 1) a New World and Asian clade and 2) an Old World clade. In the New World and Asian clade, Rhabdadenieae is fully supported as sister to Apocyneae and MOE (Mesechiteae, Odontadenieae, Echiteae). In the Old World clade, Periplocoideae diverges first, followed by Baisseeae, then Secamonoideae and Asclepiadoideae. Increased taxon and sequence sampling resolved the APSA clade topology with strong support, suggesting a similar strategy may be successful in other parts of Apocynaceae phylogeny. Identical plant specialized metabolites are often produced by distantly related taxa. The homology of these metabolites can be studied by reconstructing the evolution of genes involved in specialized metabolic pathways: orthology or paralogy of pathway genes supports homology or convergence/parallelism, respectively. Tropane alkaloids (TAs), like PAs, are putatively defensive specialized metabolites with scattered occurrence across angiosperms. Despite copious pharmacological research on the closely related TA-producing families Solanaceae and Convolvulaceae, and the distantly related Erythroxylaceae, homology of the genes involved in the TA biosynthetic pathways in these families has never been investigated. Here, 430 genomes from 375 angiosperm species, including TA-producing families and non-TA-producing families, were searched for homologs of the first three genes in the TA biosynthetic pathway: ornithine decarboxylase (odc), a gene of primary metabolism, and putrescine methyltransferase (pmt) and methylputrescine oxidase (mpo), both genes of specialized metabolisms paralogous to genes of primary metabolism. Gene trees were constructed to investigate patterns of duplication and identify candidate pmt and mpo loci within the spermidine synthase (spds) and diamine oxidase (dao) gene families, respectively, based on orthology with functionally characterized pmt and mpo genes. These genome searches revealed that odc has been lost in multiple angiosperm families and is not required to produce putrescine, the polyamine precursor for TA biosynthesis. The duplication event that led to the evolution of functionally characterized pmt genes of Solanaceae and Convolvulaceae occurred in their most recent common ancestor. In contrast, the duplication event that led to the evolution of the only functionally-characterized mpo was a Solanaceae-specific duplication. Therefore, despite the phenotypic reconstruction of a TA-producing most recent common ancestor of Solanaceae and Convolvulaceae, it is ambiguous whether the TA biosynthetic pathway evolved in this ancestor or independently in each of these two families. Candidate pmt sequences could not be identified outside Solanaceae and Convolvulaceae nor candidate mpo outside of Solanaceae. Analyses of spds and dao homologs to identify functional pmt and mpo in TA producing taxa are top priority to enable reconstruction of the evolution of TA biosynthesis across angiosperms.