bioRxiv preprint doi: https://doi.org/10.1101/2020.01.07.896902; this version posted January 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Phylogenomics supported by geometric morphometrics reveals 2 delimitation of sexual species within the polyploid apomictic 3 Ranunculus auricomus complex (Ranunculaceae) 4 5 Karbstein, Kevin1,2,*, Tomasello, Salvatore1, Hodac, Ladislav1, Dunkel, Franz G.3, 6 Daubert, Mareike1 & Hörandl, Elvira1,* 7 8 Running Title 9 Species delimitation of sexual goldilocks 10 11 1 University of Goettingen, Albrecht-von-Haller Institute for Plant Sciences, Department of Systematics, 12 Biodiversity and Evolution of Plants (with Herbarium), Untere Karspuele 2, D-37073, Goettingen, 13 Germany 14 2 University of Goettingen, Georg-August University School of Science (GAUSS), Wilhelmsplatz 1, D- 15 37073, Goettingen, Germany 16 3 Am Saupurzel 1, D-97753 Karlstadt, Germany 17 * corresponding authors: email: [email protected], [email protected] 18 19 20 Acknowledgments 21 We thank the German Research Foundation for project funding (DFG, Ho4395/10-1) to E.H., within 22 the priority program ‘Taxon-Omics: New Approaches for Discovering and Naming Biodiversity’ (SPP 23 1991). We also acknowledge Jennifer Krüger, Birthe H. Barke and Julius Schmidt for technical help, 24 Claudia Pätzold for bioinformatic support, and Silvia Friedrich for curating the garden plants. We 25 thank the Botanical Museum, University of Oslo (O) for silica-gel material of R. pygmaeus. 26 27 Data Accessibility Statement 28 Code availability. Exemplary bash and R scripts used in analyses are deposited on the Dryad data 29 repository (). 30 Data availability. The authors declare that basic data supporting the findings are available within the 31 Supporting Information. Demultiplexed RADseq reads are stored on National Center for 32 Biotechnology Information Sequence Read Archive (SRA): BioProject ID XXX. Flow cytometric, 33 genetic and morphometric data are available on Dryad data repository (). 34 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.07.896902; this version posted January 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 35 Abstract 36 Species are the basic units of biodiversity and evolution. Nowadays, they are widely considered as 37 ancestor-descendant lineages. Their definition remains a persistent challenge for taxonomists due to 38 lineage evolutionary role and circumscription, i.e., persistence in time and space, ecological niche or a 39 shared phenotype of a lineage. Recognizing and delimiting species is particularly methodically 40 challenging in fast-evolving, evolutionary young species complexes often characterized by low genetic 41 divergence, hybrid origin, introgression and incomplete lineage sorting (ILS). Ranunculus auricomus 42 is a large Eurasian apomictic polyploid complex that probably has arisen from the hybridization of a 43 few sexual progenitor species. However, even delimitation and relationships of diploid sexual 44 progenitors have been unclearly ranging from two to twelve species. Here, we present an innovative 45 workflow combining phylogenomic methods based on 86,782 parameter-optimized RADseq loci and 46 target enrichment of 663 nuclear genes together with geometric morphometrics to delimit sexual 47 species in this evolutionary young complex (< 1 Mya). For the first time, we revealed a fully resolved 48 and well-supported maximum likelihood (ML) tree phylogeny congruent to neighbor-net network and 49 STRUCTURE results based on RADseq data. In a few clades, we found evidence of discordant 50 patterns indicated by quartet sampling (QS) and reticulation events in the neighbor-net network 51 probably caused by introgression and ILS. Together with coalescent-based species delimitation 52 approaches based on target enrichment data, we found five main genetic lineages, with an allopatric 53 distribution in Central and Southern Europe. A concatenated geometric morphometric data set 54 including basal and stem leaves, as well as receptacles, revealed the same five main clusters. We 55 accept those five morphologically differentiated, geographically isolated, genetic main lineages as 56 species: R. cassubicifolius s.l. (incl. R. carpaticola), R. flabellifolius, R. envalirensis s.l. (incl. R. 57 cebennensis), R. marsicus and R. notabilis s.l. (incl. R. austroslovenicus, R. calapius, R. 58 mediocompositus, R. peracris and R. subcarniolicus). Our comprehensive workflow combing 59 phylogenomic methods supported by geometric morphometrics proved to be successful in delimiting 60 closely related sexual taxa and applying an evolutionary species concept, which is also transferable to 61 other evolutionarily young species complexes. 62 63 Keywords 64 Europe, geometric morphometrics, RADseq, Ranunculus auricomus, sexual species, target enrichment 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.07.896902; this version posted January 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 65 Introduction 66 Species are the basic units of biodiversity and evolution, but their definition remains a persistent 67 challenge for taxonomists (Sukumaran & Knowles, 2017). Most modern authors consider species as 68 ancestor-descendant lineages (De Queiroz, 2007; Sukumaran & Knowles, 2017), a concept that is 69 applicable to both sexual and asexual lineages. Nevertheless, its rather challenging to define their 70 evolutionary role and circumscription, i.e. persistence in time and space, ecological niche or a shared 71 phenotype (Freudenstein & al., 2017; Hörandl, 2018). Hörandl (2018) proposed a species delimitation 72 workflow based on four main principles particularly addressing apomictic polyploid complexes: First, 73 separate and classify the obligate sexual progenitor species; second, merge sexual progenitors and 74 highly facultative apomictic lineages into one species; third, classify the main clusters of the 75 remaining facultative apomicts as species; and fourth, treat obligate apomictic lineages as 76 agamospecies. 77 However, recognizing and delimiting even the sexual progenitor species is methodically challenging 78 in fast-evolving, evolutionary young species complexes (e.g. Burgess & al., 2015). Commonly, they 79 are characterized by low genetic divergence, occasionally by hybrid origin, persistent gene flow, 80 introgression and incomplete lineage sorting (ILS). During speciation processes, ILS is rather the rule 81 than an exception causing incongruences in phylogenetic reconstructions (see e.g., Oliver, 2013; Pease 82 & al., 2018). The progenitor species can also be sexual polyploids. Natural hybridization and 83 polyploidy are important evolutionary processes and are regarded as key factors for diversification of 84 Angiosperms (Soltis & Soltis, 2000; Jiao & al., 2011; Alix & al., 2017). Reticulate evolution (e.g., 85 hybridization, allopolyploidy and ILS), however, results in the phenomenon that gene trees do not 86 match species trees (e.g., McBreen & Lockhart, 2006; Pirie & al., 2009). 87 The application of molecular markers has helped to reconstruct not only relationships of sexual 88 progenitor species but also hybrid origin, reticulate relationships and population genetic structure of 89 apomictic lineages within species complexes (Dobeš & al., 2004; Hörandl, 2004; Paun & al., 2006a, 90 2006b; Kirschner & al., 2015). Nevertheless, most complexes are evolutionarily young and many 91 originated or diversified in the context of Pleistocene glaciations. Therefore, genetic divergence 92 among taxa is usually quite low. Traditional genetic markers (for example internal transcribed spacer 93 (ITS) markers) often failed to reconstruct fully resolved phylogenies (e.g., Hörandl & al., 2009; Krak 94 & al., 2013; Kirschner & al., 2015). Population genetic markers like microsatellites or AFLPs have 95 been widely used, but are often not homologous among more divergent taxa (Freeland & al., 2011). 96 Hence, species-level relationships in apomictic complexes often remained unresolved. 97 The application of phylogenomic methods provides a magnitude more loci compared to traditional 98 genetic markers and can resolve relationships of taxa with less than few million (or even less than 99 100.000) years of divergence (Pellino & al., 2013; Tripp & al., 2017; Tomasello et al., in subm.). 100 Currently, two main approaches are used in species-level systematics: Target enrichment and 101 restriction site-associated DNA sequencing (RADseq; and the similar Genotyping-by-Sequencing = 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.07.896902; this version posted January 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 102 GBS method). Target enrichment (i.e., nuclear genes selected out of a reference genome) is most 103 useful for diverged species and genus-wide relationships (Folk & al., 2015; Schmickl & al., 2016; 104 Tomasello, 2018). RADseq has repeatedly proven its efficiency in resolving intraspecific 105 relationships, phylogenies of closely related species, but also within genera (relationships between less