Botany SUPERMATRIX ANALYSES AND MOLECULAR CLOCK ROOTING OF FABALES: EXPLORING THE EFFECTS OF OUTGROUP CHOICE AND LONG BRANCH ATTRACTION ON TOPOLOGY Journal: Botany Manuscript ID cjb-2019-0109.R2 Manuscript Type: Article Date Submitted by the 16-Nov-2019 Author: Complete List of Authors: Aygoren Uluer, Deniz; Ahi Evran Universitesi, Cicekdagi Vocational College, Department of Plant and Animal Production; Forest, Félix;Draft Royal Botanic Gardens Kew, Hawkins, Julie; University of Reading, School of Biological Sciences, Lyle Building Keyword: Fabales, long branch attraction, molecular clock rooting, rapid radiation Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/botany-pubs Page 1 of 45 Botany SUPERMATRIX ANALYSES AND MOLECULAR CLOCK ROOTING OF FABALES: EXPLORING THE EFFECTS OF OUTGROUP CHOICE AND LONG BRANCH ATTRACTION ON TOPOLOGY Deniz Aygoren Uluer1,4, Félix Forest2, Julie A. Hawkins3 1, 4 Ahi Evran University, Cicekdagi Vocational College, Department of Plant and Animal Production, Boyalik Mahallesi, Stadyum Caddesi, Turan Sok. No:18. 40700 Cicekdagi, Kirsehir, Turkey, [email protected] 2 Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, United Kingdom, [email protected] 3 School of Biological Sciences, Lyle Building, University of Reading, Whiteknights, Reading, Berkshire, RG6 6BX, United Kingdom, [email protected] Draft 4Author for correspondence: Deniz Aygoren Uluer, Ahi Evran University, Cicekdagi Vocational College, Department of Plant and Animal Production, Boyalık Mahallesi, Stadyum Caddesi, Turan Sok. No:18 40700 Cicekdagi, Kirşehir, Turkey, email: [email protected], Work phone: +903862805500, Fax: +903862805528. https://mc06.manuscriptcentral.com/botany-pubs Botany Page 2 of 45 ABSTRACT Fabales is a cosmopolitan angiosperm order which consists of four families, Leguminosae (Fabaceae), Polygalaceae, Surianaceae and Quillajaceae. Despite the great interest in this group, a convincing phylogeny of the order is still not available. Therefore, the aim of the current study is to explicitly test for possible LBA problems within Fabales for the first time and determine whether low tree stemminess and unequal branch lengths could worsen this problem. Supermatrix analysis of Fabales was carried out using previously published plastid matK, trnL, rbcL and newly sequenced nuclear sqd1 regions for 678 taxa in total, including 43 outgroup taxa from families of Fabidae. We employed additional analyses, such as simulations, network analyses, sampling different outgroup taxa (random or real), removing fast evolving sites and fast evolving taxa and molecular clock rooting, to identify both long branch attraction (LBA) and/or rooting problems. These analyses clearly show that the Fabales phylogeny hasDraft been influenced by the sampling of outgroup taxa, but not LBA. However, network analyses show that even though it is weak, there is a consistent phylogenetic signal among the rapidly radiated Fabales families, which can be traced by further analyses. While, molecular clock rooting analysis yielded a (Leguminosae(Polygalaceae(Surianaceae+Quillajaceae))) topology with strong support for the first time here, supermatrix analyses yielded a ((Leguminosae+Polygalaceae)(Surianaceae+Quillajaceae)) with low-moderate support. Keywords — Fabales, long branch attraction, molecular clock rooting, rapid radiation. https://mc06.manuscriptcentral.com/botany-pubs Page 3 of 45 Botany INTRODUCTION Fabales is divided into four families which are very diverse morphologically and molecularly, Leguminosae, Polygalaceae, Surianaceae and Quillajaceae (Bello et al. 2009; APG IV 2016). Molecular studies and fossil evidence suggest an ancient-rapid radiation for Fabales (e.g., Crane et al. 1990; Zi- Chen et al. 2004; Lavin et al. 2005; Pigg et al. 2008; Bello et al. 2009). The monophyly of the order is strongly supported by several studies (e.g., Bello et al. 2009; Bello et al. 2012; APG IV 2016; Koenen et al. 2019), but the overall phylogenetic relationships across the order and position of the root remain controversial, changing from one study to another; a situation common in higher level phylogenetic studies of ancient, rapid radiations. (Bello et al. 2009). This unresolved phylogenetic problem for Fabales also hinder further evolutionary questions such as estimating diversification rates (e.g., Smith et al. 2011; Koenen et al. 2013). In almost every phylogenetic study of Fabales to date, different rootings and different phylogeneticDraft relationships have been found (e.g., Crayn et al. 1995; Doyle et al. 2000; Savolainen et al. 2000; Soltis et al. 2000; Kajita et al. 2001; Persson 2001; Wojciechowski et al. 2004; Lavin et al. 2005; Forest et al. 2007; Bruneau et al. 2008; Bello et al. 2009; Soltis et al. 2011; Bello et al. 2012; Sun et al. 2016; Koenen et al. 2019); however, none of these studies has focussed on the reason for this incongruence and only a few have employed broad enough taxon sampling with suitable outgroup taxa. Ancient rapid radiations have been one of the hardest problems for phylogenetic studies to resolve, due to short internal branches, which show a limited time span between speciation events and have a weak phylogenetic signal compared with long external branches; in addition to other problems, such as high extinction rates before or after a rapid radiation, the evolutionary rate heterogeneity of branches and long branch attraction (LBA) artefacts even closely related outgroup sequences are sampled (Felsenstein 1978; Wägele 1999; Fishbein et al. 2001; Rokas and Carroll 2006; Shavit et al. 2007; Whitfield and Lockhart 2007; Murdock 2008; Jian et al. 2008; Whitfield and Kjer 2008; Kodandaramaiah et al. 2010; Philippe et al. 2011; Rothfels et al. 2012). Ancient-rapid radiations have https://mc06.manuscriptcentral.com/botany-pubs Botany Page 4 of 45 been described as having low tree stemminess sensu Smith (1994), bush-like trees sensu Rokas and Carrol (2006), broom-and-handle sensu Crisp et al. (2004) or starburst phylogenies sensu Albertson et al. (1999). These types of problematic phylogenies have been reported for many angiosperm clades such as Mesangiospermae (Zeng et al. 2014), eudicots (Moore et al. 2010; Soltis et al. 2011), Brassicaceae (Huang et al. 2015), Fabales (Bello et al. 2009; Bello et al. 2012) and early-diverging Leguminosae (Azani et al. 2017). In the worst cases, rapid radiations may be represented as hard or near-hard polytomies, such as when the genes used have limited phylogenetic signal allowing the inference of relationships, especially for the internal branches (Braby et al. 2005; Whitfield and Kjer 2008; Kodandaramaiah et al. 2010). However, only a few hard polytomy cases have been reported until now (e.g., Kodandaramaiah et al. 2010), and Fabales is not one of them (Bello et al. 2009). Gene tree incongruence has been reported as more serious for these short internal branches (Salichos and Rokas 2013; Sun et al. 2015).Draft Additionally, in these cases, phylogenetic results may be very sensitive to the specific genes used, and to the phylogenetic method and outgroup taxon choice (Roberts et al. 2009; Kirchberger et al. 2014; Borowiec et al. 2019), because outgroup taxa may recover an incorrect or random root (Smith 1994). Even if it is often trivialised, rooting is one of the hardest steps in phylogenetic reconstruction (Boykin et al. 2010). Due to the high levels of homoplasy, particularly in molecular data, finding the “correct-rooted tree” is always less probable than finding the “correct-unrooted tree” (Sourdis and Krimbas 1987; Smith 1994; Graham et al. 2002). In the case of rapid radiations, this situation becomes even more severe (Smith 1994; Shavit et al. 2007; Sterli 2010) as the combination of low tree stemminess (i.e., rapid radiations) and high levels of homoplasy reduces the chance of finding the correct root (Smith 1994), as in the case of Fabales. On the other hand, LBA was first described by Felsenstein (1978) as the spurious attraction of unrelated-long-branched taxa, and therefore homoplasy. LBA has been attributed to poor taxon sampling due to the extinction or unavailability of extant taxa, the Maximum Parsimony method, https://mc06.manuscriptcentral.com/botany-pubs Page 5 of 45 Botany distant outgroup taxa and outgroup taxa, which have very different base compositions from ingroup taxa (Wheeler 1990; Shavit et al. 2007; Dabert et al. 2010; Kodandaramaiah et al. 2010; Li et al. 2012; Grant 2019). Over time, both empirical and simulated data and analyses have confirmed the widespread distribution of this phenomenon. LBA can also be an important problem, particularly for rapid radiations, because even closely related outgroup taxa can introduce long branches to the phylogeny, compared with the short internal branches of the ingroup (Lyons-Weiler et al. 1998; Moreira and Philippe 2000; Wägele and Mayer 2007). Furthermore, ancient radiations may be more prone to LBA as a result of branch length heterogeneity, base homoplasy and compositional bias, even when the model-based methods, such as maximum likelihood (ML), are employed (Wheeler 1990; Foster and Hickey 1999; Fishbein et al. 2001; Gribaldo and Philippe 2002). For these reasons, any study aiming to resolve a particularly difficult phylogeny question needs to be mindful of the influence of rooting, outgroupDraft effect and LBA as sources of systematic error. Therefore, the aim of the current study is to explicitly test