APPLICATION ARTICLE Enabling evolutionary studies at multiple scales in Apocynaceae through Hyb-Seq Shannon C. K. Straub1,4 , Julien Boutte1, Mark Fishbein2 , and Tatyana Livshultz3 Manuscript received 1 July 2020; revision accepted 12 September PREMISE: Apocynaceae is the 10th largest flowering plant family and a focus for study of 2020. plant–insect interactions, especially as mediated by secondary metabolites. However, it has 1 Department of Biology, Hobart and William Smith Colleges, 300 few genomic resources relative to its size. Target capture sequencing is a powerful approach Pulteney Street, Geneva, New York 14456, USA for genome reduction that facilitates studies requiring data from the nuclear genome in non- 2 Department of Plant Biology, Ecology, and Evolution, Oklahoma model taxa, such as Apocynaceae. State University, 301 Physical Sciences, Stillwater, Oklahoma 74078, USA METHODS: Transcriptomes were used to design probes for targeted sequencing of putatively 3 Department of Biodiversity, Earth, and Environmental Sciences single-copy nuclear genes across Apocynaceae. The sequences obtained were used to assess and the Academy of Natural Sciences, Drexel University, 1900 the success of the probe design, the intrageneric and intraspecific variation in the targeted Benjamin Franklin Parkway, Philadelphia, Pennsylvania 19103, USA genes, and the utility of the genes for inferring phylogeny. 4Author for correspondence: [email protected] RESULTS: From 853 candidate nuclear genes, 835 were consistently recovered in single Citation: Straub, S. C. K., J. Boutte, M. Fishbein, and T. Livshultz. copy and were variable enough for phylogenomics. The inferred gene trees were useful for 2020. Enabling evolutionary studies at multiple scales in coalescent-based species tree analysis, which showed all subfamilies of Apocynaceae as Apocynaceae through Hyb-Seq. Applications in Plant Sciences 8(11): e11400. monophyletic, while also resolving relationships among species within the genus Apocynum. Intraspecific comparison of Elytropus chilensis individuals revealed numerous single- doi:10.1002/aps3.11400 nucleotide polymorphisms with potential for use in population-level studies. DISCUSSION: Community use of this Hyb-Seq probe set will facilitate and promote progress in the study of Apocynaceae across scales from population genomics to phylogenomics. KEY WORDS Apocynaceae; dogbane; genome reduction; Hyb-Seq; low-copy nuclear genes; milkweed; phylogenomics; targeted sequencing. Target capture sequencing of nuclear genes is one approach that fa- et al., 2017; Peng et al., 2017; Villaverde et al., 2018; de la Harpe cilitates large-scale phylogenomic studies of non-model organisms et al., 2019; Jones et al., 2019). (Lemmon and Lemmon, 2013; Dodsworth et al., 2019). In plants, As sequencing of DNA and RNA has become easier and cheaper, this approach has clarified evolutionary relationships in multiple several large-scale projects have sequenced, assembled, and made lineages (e.g., Mandel et al., 2015; Fisher et al., 2016; Heyduk et al., available to the research community numerous plant transcrip- 2016; Léveillé-Bourret et al., 2018; Boutte et al., 2019; Couvreur tomes (e.g., Phytometasyn, https://bioin forma tics.tugraz.at/phyto et al., 2019; Herrando-Moraira et al., 2019; Bagley et al., 2020). metas yn/; Medicinal Plant Genomics Resource, http://medic inalp Obtaining data from many nuclear genes facilitates the use of lantg enomi cs.msu.edu; 1000 Plants, www.onekp.com). Now that species tree methods based on the multispecies coalescent model transcriptomes from multiple species within the same plant fam- (Mirarab and Warnow, 2015; Edwards et al., 2016) and provides ily are publicly available, tools (e.g., MarkerMiner; Chamala et al., data sets of the size required to address problems that have been 2015) that can take advantage of this wealth of information about unsolvable using traditional molecular systematics approaches nuclear genes in the design of targeted sequencing probes can be (e.g., Léveillé-Bourret et al., 2018; Herrando-Moraira et al., 2019), easily utilized. The MarkerMiner pipeline conducts a reciprocal or even whole plastome data (e.g., Straub et al., 2014). This method BLAST search between each transcriptome and a reference pro- allows sufficient flexibility to design probes in conserved regions teome to identify putative orthologs, which are then filtered based that flank more variable intron and intergenic regions, providing on known single-copy and low-copy genes in angiosperms (DeSmet sequence data that are useful for comparative analysis of distantly et al., 2013). Putative orthologs from multiple input transcriptomes related species, among closely related species within a genus, or are then clustered by reference protein identifiers and aligned. The even within a single species (Weitemier et al., 2014, 2019; Crowl alignments are useful for targeted enrichment probe design and Applications in Plant Sciences 2020 8(11): e11400; http://www.wileyonlinelibrary.com/journal/AppsPlantSci © 2020 Straub et al. Applications in Plant Sciences published by Wiley Periodicals LLC on behalf of Botanical Society of America. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. 1 of 9 Applications in Plant Sciences 2020 8(11): e11400 Straub et al.—Apocynaceae nuclear gene probes • 2 of 9 other downstream applications (Chamala et al., 2015). This ap- MarkerMiner version 1.2 (Chamala et al., 2015) was employed proach has been successful in aiding development of targeted se- to identify putatively single-copy genes using the Vitis vinifera L. quencing probes in multiple plant lineages (Villaverde et al., 2018; proteome reference and a minimum similarity of 85%. The de- Morais et al., 2019; Jantzen et al., 2020). fault minimum transcript length of 900 bp was used because it The milkweed and dogbane family, Apocynaceae, has publicly approximates the setting used by Weitemier et al. (2014) for target available transcriptomes from several species. With ca. 5300 spe- capture sequencing probe design in Asclepias using the Hyb-Seq cies, Apocynaceae is the 10th largest family of flowering plants pipeline. Intron/exon boundaries were determined by comparison and has a worldwide distribution (Endress et al., 2018). Its mem- with the V. vinifera genome. This work was completed utilizing the bers are widely known for their production of secondary metab- High Performance Computing Center facilities of Oklahoma State olites that function in defense against herbivores, some of which University at Stillwater. have evolved mechanisms to detoxify and sequester these com- MarkerMiner output alignments were submitted to MYcroarray pounds (Malcolm and Brower, 1989). Some metabolites, such (now Daicel Arbor Biosciences, Ann Arbor, Michigan, USA) for as vinblastine and vincristine, are used as human medicines the design and synthesis of myBaits biotinylated RNA probes of (Cragg and Newman, 2005). Pollination biology and floral evo- 120 nucleotides with 2× tiling. Probes with multiple BLAST hits to lution have been another focal area of study due to the presence the A. syriaca nuclear genome assembly (Weitemier et al., 2019) or of complex derived floral structures, such as pollinia, gynostegia, those containing repetitive sequence were excluded from the final and coronas (Endress, 1994; Endress and Bruyns, 2000; Fishbein, probe set of 48,974 probes. An additional 2707 probes for targeting 2001; Fishbein et al., 2018). Complete resolution of the backbone two nuclear genes, paralogs dhs and hss, were present in the probe of the phylogeny of Apocynaceae has proven to be difficult using set used for enrichment that generated data for a separate project traditional molecular systematics methods, a supermatrix ap- investigating pyrrolizidine alkaloid biosynthesis (Livshultz et al., proach, and even plastome data sets (Livshultz, 2010; Fishbein 2018). These two genes are not considered further in the reported et al., 2018). However, understanding the evolution of second- results of this paper. ary metabolite biosynthetic pathways and other traits, such as The genes targeted across Apocynaceae were compared with floral structure and pollen aggregation, in these plants requires the set of genes targeted in Asclepias by Weitemier et al. (2014) a well-resolved phylogeny, which could be inferred based on in- and the universal angiosperm probe set (Johnson et al., 2019) formation from hundreds of nuclear genes using a target capture using reciprocal BLASTN (E-value threshold of 10−6, mini- sequencing approach. mum percentage of identity of 80%; Altschul et al., 1990) and Previously, Weitemier et al. (2014) used the Hyb-Seq pipe- Python custom scripts (Python version 2.7.12; Python Software line to identify loci for probe design for targeted sequencing in Foundation, 2016). one genus of Apocynaceae, Asclepias L. They identified 768 sin- gle-copy genes and demonstrated their utility in two subtribes Taxon sampling, library preparation, and targeted sequencing of Asclepiadeae (Asclepiadoideae), and this set has subsequently been utilized to successfully resolve relationships among Asclepias Probes were tested for target enrichment success in 15 species species (Boutte et al., 2019). However, Weitemier et al. (2014) also of Apocynaceae spanning the diversity of the family and