bioRxiv preprint doi: https://doi.org/10.1101/2020.04.01.021071; this version posted April 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 1 New approaches to species delimitation and population structure of corals: two case studies 2 using ultraconserved elements and exons 3 4 Running Title: New genomic approaches for coral species delimitation 5 6 7 Katie L. Erickson1#, Alicia Pentico1#, Andrea M. Quattrini1,2*, Catherine S. McFadden1 8 9 1 Department of Biology, Harvey Mudd College, Claremont CA 91711 USA 10 2 Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian 11 Institution, Washington DC 20560 USA 12 13 #equal contributions 14 *corresponding author [email protected] 15 16 17 18 Draft 19 20 21 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.01.021071; this version posted April 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 24 Abstract 25 As coral populations decline worldwide in the face of ongoing environmental change, 26 documenting their distribution, diversity and conservation status is now more imperative than 27 ever. Accurate delimitation and identification of species is a critical first step. This task, 28 however, is not trivial as morphological variation and slowly evolving molecular markers 29 confound species identification. New approaches to species delimitation in corals are needed to 30 overcome these challenges. Here, we test whether target enrichment of ultraconserved elements 31 (UCEs) and exons can be used for delimiting species boundaries and population structure within 32 species of corals by focusing on two octocoral genera, Alcyonium and Sinularia, as exemplary 33 case studies. We designed an updated bait set (29,363 baits) to target-capture 3,040 UCE and 34 exon loci, recovering a mean of 1,910 ± 168 SD per sample with a mean length of 1,055 ± 208 35 bp. Similar numbers of loci were recovered from Sinularia (1,946 ± 227 SD) and Alcyonium 36 (1,863 ± 177 SD). Species-level phylogenies were highly supported for both genera. Clustering 37 methods based on filtered SNPs delimited species and populations that are congruent with 38 previous allozyme, DNA barcoding, reproductive and ecological data for Alcyonium, and offered 39 further evidence of hybridization among species. For Sinularia, results were congruent with 40 those obtained from a previous study using Restriction Site Associated DNA Sequencing. Both 41 case studies demonstrate the utilityDraft of target-enrichment of UCEs and exons to address a wide 42 range of evolutionary and taxonomic questions across deep to shallow time scales in corals. 43 44 Keywords: cnidaria, population genomics, target enrichment, UCE, Octocorallia 45 46 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.01.021071; this version posted April 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 47 Introduction 48 Corals are arguably one of the most ecologically important groups of metazoans on earth. 49 By their ability to produce large colonies by asexual propagation and CaCO3 precipitation, they 50 create massive biogenic structures, thereby engineering entire reef-based ecosystems in both 51 shallow and deep waters (Riegl & Dodge, 2019; Roberts, Wheeler, & Freiwald, 2006). These 52 ecological communities are among those most imperiled by climate change, with dire predictions 53 suggesting that they may not survive the next century (Carpenter et al., 2008; Pandolfi, Connolly, 54 Marshall, & Cohen, 2011; Hughes, Bellwood, Connolly, Cornell, & Karlson, 2014). As coral 55 populations and species are in decline, it is important to establish baselines of abundance, 56 distribution, and diversity, all of which rely on accurately delimiting and identifying species. 57 Unfortunately, our current lack of understanding of basic taxonomy and species boundaries in 58 many groups of corals prohibits us from better understanding their roles in ecosystems, and 59 ultimately, from effectively managing their habitats. 60 Traditionally, morphological traits such as colony growth form and skeletal structure 61 have been used to distinguish corals at all taxonomic levels (Fabricius & Alderslade, 2001; 62 Veron, 2000). At the species level, however, morphological differences can be subtle, and both 63 phenotypic variation and plasticity of growth form often confound assessment of species 64 boundaries (e.g., Forsman, Barshis,Draft Hunter, & Toonen, 2009; Marti-Puig et al., 2014; McFadden 65 et al., 2017; Paz-García, Hellberg, García-de-León, & Balart, 2015). The advent of integrated 66 taxonomic approaches that utilize molecular characters to inform taxonomy has greatly improved 67 our understanding of species boundaries in numerous coral taxa, and led to the recognition of 68 homoplasy in many morphological characters previously considered to be diagnostic of species 69 and higher taxa (Arrigoni et al., 2014; Benayahu, Ofwegen, & McFadden, 2018; Budd & 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.01.021071; this version posted April 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 70 Stolarski, 2009; McFadden et al., 2017). The relative invariance of the mitochondrial genome in 71 corals and other anthozoan cnidarians (e.g., sea anemones) has, however, hindered the 72 application of integrated approaches to species delimitation in this ecologically important group. 73 As a result of their slow rates of mitochondrial gene evolution coupled with a lack of 74 alternative marker development, both DNA barcoding efforts and studies of phylogeography in 75 most corals lag far behind other groups. The universal COI barcode does not reliably distinguish 76 species within the anthozoan cnidarians (Huang, Meier, Todd, & Chou, 2008; McFadden et al., 77 2011; Shearer & Coffroth, 2008). No alternative, universally agreed upon single-locus barcode 78 has been proposed or widely used for either the scleractinian corals or related sea anemones, and 79 a multilocus barcode that has been used for the octocorals (mtMutS + COI + 28S rDNA) has 80 recognized limitations (McFadden et al., 2011; McFadden, Brown, Brayton, Hunt, & Ofwegen, 81 2014; Quattrini et al., 2019). Relatively few studies have developed alternative markers such as 82 nuclear gene regions (Ladner & Palumbi, 2012; Prada et al. 2014) or microsatellites (e.g., 83 Baums, Boulay, Polato, & Hellberg, 2012; Gutiérrez-Rodríguez & Lasker, 2004; LeGoff, Pybus, 84 & Rogers, 2004; Quattrini, Baums, Shank, Morrison, & Cordes, 2015) for use in species 85 delimitation or phylogeographic studies. As a result, understanding of population structure, 86 phylogeographic patterns, and the processes driving evolutionary diversification in corals and 87 other anthozoans lags far behind theDraft progress that has been made in other ecologically important 88 marine phyla (Cowman, Parravicini, Kulbicki, & Floeters, 2017; Crandall et al., 2019; Hodge & 89 Bellwood, 2016). 90 The recent development of genomic sequencing methods holds considerable promise for 91 resolution of species boundaries and phylogeography in corals and other anthozoans. Several 92 studies have now demonstrated that Restriction Site Associated DNA sequencing (RAD-Seq) 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.01.021071; this version posted April 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 93 effectively delimits species when conventionally-used barcodes have failed (Forsman et al., 94 2017; Herrera & Shank, 2016; Johnston et al., 2017; Pante et al., 2015; Quattrini et al., 2019). 95 However, few studies have yet documented the effectiveness of RAD-Seq for population-level 96 work in anthozoans (Leydet, Grupstra, Coma, Ribes, & Hellberg, 2018; Bracco, Liu, Galaska, 97 Quattrini, & Herrera, 2019). There are also a few inherent disadvantages to RAD-Seq relative to 98 sequence enrichment methods. These include a stark drop in orthologous loci that can be 99 extracted from distantly related taxa (Cairou, Duret, & Charlat, 2013; Viricel, Pante, Dabin, & 100 Simon-Bouhet, 2013), limiting the ability of RAD-Seq to determine deeper phylogenetic 101 relationships, and rendering this method impractical for combining datasets of distantly-related 102 taxa (Harvey, Smith, Glenn, Faircloth, & Brumfield, 2016). 103 Target capture enrichment of Ultraconserved Elements (UCEs) and exons could be a 104 welcome addition to the methods used for species delimitation and studies of phylogeography 105 and population structure in corals and other anthozoans. UCEs consist of highly conserved 106 regions shared across species, which