The following article appeared in Molecular Phylogenetics and Evolution 117: 111-123 (2017); and may be found at: https://doi.org/10.1016/j.ympev.2017.05.014 This is an open access article under the Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license https://creativecommons.org/licenses/by-nc-nd/4.0/ Molecular Phylogenetics and Evolution 117 (2017) 111–123 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Recalcitrant deep and shallow nodes in Aristolochia (Aristolochiaceae) illuminated using anchored hybrid enrichment ⇑ Stefan Wanke a, , Carolina Granados Mendoza b,c,1, Sebastian Müller a,1, Anna Paizanni Guillén d,e, Christoph Neinhuis a, Alan R. Lemmon f, Emily Moriarty Lemmon g, Marie-Stéphanie Samain e a Technische Universität Dresden, Institut für Botanik, Zellescher Weg 20b, 01062 Dresden, Germany b CONACYT División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica A.C., Camino a la Presa de San José 2055, Lomas 4a. sección, C.P. 78216 San Luis Potosí, San Luis Potosí, Mexico c Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado Postal 70-367, 04510 Coyoacán, Distrito Federal, Mexico d Universidad Michoacana de San Nicolás de Hidalgo, 58066 Morelia, Michoacán, Mexico e Instituto de Ecología, A.C., Centro Regional del Bajío, 61600 Pátzcuaro, Michoacán, Mexico f Department of Scientific Computing, Florida State University, Dirac Science Library, Tallahassee, FL 32306-4102, USA g Department of Biological Science, Florida State University, 319 Stadium Drive, PO Box 3064295, Tallahassee, FL, 32306-4295, USA article info abstract Article history: Recalcitrant relationships are characterized by very short internodes that can be found among shallow Received 29 September 2016 and deep phylogenetic scales all over the tree of life. Adding large amounts of presumably informative Revised 12 May 2017 sequences, while decreasing systematic error, has been suggested as a possible approach to increase phy- Accepted 15 May 2017 logenetic resolution. The development of enrichment strategies, coupled with next generation sequenc- Available online 20 May 2017 ing, resulted in a cost-effective way to facilitate the reconstruction of recalcitrant relationships. By applying the anchored hybrid enrichment (AHE) genome partitioning strategy to Aristolochia using an Keywords: universal angiosperm probe set, we obtained 231–233 out of 517 single or low copy nuclear loci origi- Enrichment strategy nally contained in the enrichment kit, resulting in a total alignment length of 154,756 bp to Next generation sequencing Phylogenomics 160,150 bp. Since Aristolochia (Piperales; magnoliids) is distantly related to any angiosperm species Polyploidy whose genome has been used for the plant AHE probe design (Amborella trichopoda being the closest), Short internodes it serves as a proof of universality for this probe set. Aristolochia comprises approximately 500 species Recent diversification grouped in several clades (OTUs), whose relationships to each other are partially unknown. Previous phy- Universal nuclear probe set logenetic studies have shown that these lineages branched deep in time and in quick succession, seen as short-deep internodes. Short-shallow internodes are also characteristic of some Aristolochia lineages such as Aristolochia subsection Pentandrae, a clade of presumably recent diversification. This subsection is here included to test the performance of AHE at species level. Filtering and subsampling loci using the phylo- genetic informativeness method resolves several recalcitrant phylogenetic relationships within Aristolochia. By assuming different ploidy levels during bioinformatics processing of raw data, first hints are obtained that polyploidization contributed to the evolution of Aristolochia. Phylogenetic results are discussed in the light of current systematics and morphology. Ó 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction plant lineages all over the tree of life. For many years, resolving such nodes in phylogenies has been recognized as most challeng- Hundreds of loci are potentially needed to resolve recalcitrant ing (e.g. Richardson et al., 2004). An ongoing discussion surrounds phylogenetic relationships (Leaché and Rannala, 2010; Wickett the question of increasing taxonomic sampling versus increment- et al., 2014; Prum et al., 2015), which are very short internodes that ing character sampling for previously poorly resolved phylogenies are often present at shallow scales, as well as among deep level (e.g. Goldman, 1998; Geuten et al., 2007; Leaché and Rannala, nodes. These so-called short branched clades are found in many 2010; Townsend and López-Giráldez, 2010; San Mauro et al., 2012). Sequencing costs per DNA base pair are continuously decreas- ⇑ Corresponding author. ing and at the same time accuracy of raw data is increasing (Cai E-mail address: [email protected] (S. Wanke). et al., 2012; Lemmon and Lemmon, 2013). Despite reduced 1 Authors contributed equally to this study. http://dx.doi.org/10.1016/j.ympev.2017.05.014 1055-7903/Ó 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 112 S. Wanke et al. / Molecular Phylogenetics and Evolution 117 (2017) 111–123 sequencing costs, whole genome sequencing is still very expensive Table 1 if applied to a broad sampling and, in most cases, far beyond nec- List of recently published studies using enrichment strategies in plants. essary, as only a fraction of the genome is potentially needed Study Studied Taxon Taxon # of recovered (Ruane et al., 2015). Furthermore, it requires extensive down- level loci stream (post-sequencing) processing with many custom scripts, Fragoso-Martínez et al. Salvia (Lamiaceae) Genus 448 nuclear whereas only a small amount of the obtained data may be well sui- (2017) loci ted for phylogenetic questions (Carstens et al., 2012; Lemmon and Heyduk et al. (2016) Sabal (Arecaceae) Genus 133 nuclear loci Lemmon, 2013). Aiming at more cost-efficient results, only pre- Mandel et al. (2015) Asteraceae Family 795 nuclear sumably informative loci can be sequenced. As such, it is avoided loci to produce a large amount of potentially uninformative molecular Mitchell et al. (2017) Protea (Proteaceae) Genus 498 nuclear data that might demand high computational effort or present the loci difficulty of discerning between orthologs and paralogs when Sass et al. (2016) Zingiberales Order 308 nuclear loci genes are multi-copy. Furthermore, the acquired data can be sub- Schmickl et al. (2015) Oxalis (Oxalidaceae) Genus 727 nuclear sampled to increase the phylogenetic information while simulta- loci neously decreasing systematic error (Lemmon and Lemmon, Sousa et al. (2014) Medicago (Fabaceae) Genus 50 nuclear loci 2013). Consequently, the era of next generation sequencing used Stephens et al. (2015a) Sarracenia Genus 199 nuclear (Sarraceniaceae) loci in phylogenomics contains a new challenge: applying new tech- Stephens et al. (2015b) Helianthus Genus 170 nuclear niques to select and sequence only those loci that are informative (Asteraceae) loci and necessary for answering the particular phylogenetic question. Syring et al. (2016) Pinus albicaulis Species 4452 nuclear One recently developed technique is ‘‘Anchored Hybrid Enrich- (Pinaceae) loci ment” (AHE). In this approach selected loci are enriched with the help of enrichment probes, oligonucleotide sequences of ca. 60– 120 bp that hybridize to moderately conserved target regions and deep internodes where lineages split early in quick succession, (compared to ultraconserved elements), followed by sequencing and second, shallow level nodes at the species level (Townsend and of the enriched target regions, and more variable flanking regions, Leuenberger, 2011). In order to be informative for a specific diver- on a high-throughput sequencing platform (Lemmon and Lemmon, gence event in a phylogenetic tree, a character must suffer a 2013). Lemmon et al. (2012) designed a vertebrate-wide enrich- change in state in that specific time frame and remain unchanged ment probe set and obtained a fully resolved and well-supported along the complete branch length. Short and deep internodes are species tree for vertebrates. More recently, this methodology was difficult to resolve for two reasons. Firstly, the probability that a applied to snakes (Pyron et al., 2014; Ruane et al., 2015; Pyron character state change occurs in a short time period (short intern- et al., 2016; Chen et al., 2017), lizards (Leaché et al., 2014; ode) is lower than after a long period (long internode). Secondly, a Brandley et al., 2015; Tucker et al., 2017; Domingos et al., 2017; character has a higher probability to subsequently change its state Manthey et al., 2016), frogs (Peloso et al., 2015), fish (Eytan et al., in deep branches than in shallow ones. Therefore, a phylogenetic 2015; Stout et al., 2016), birds (Prum et al., 2015), flies (Young signal derived from a character state change during a short and et al., 2016), and spiders (Hamilton et al., 2016). Although solid evi- deep internode is likely to be obscured by phylogenetic noise dence exists from diverse animal lineages, a probe set for angios- caused by subsequent