Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2019 A phylogenomic analysis of the floral transcriptomes of sexually deceptive and rewarding European orchids, Ophrys and Gymnadenia Pineiro Fernandez, Laura ; Byers, Kelsey J R P ; Cai, Jing ; Sedeek, Khalid E M ; Kellenberger, Roman T ; Russo, Alessia ; Qi, Weihong ; Aquino Fournier, Catharine ; Schlüter, Philipp M Abstract: The orchids (Orchidaceae) constitute one of the largest and most diverse families of flowering plants. They have evolved a great variety of adaptations to achieve pollination by a diverse group of pollinators. Many orchids reward their pollinators, typically with nectar, but the family is also well- known for employing deceptive pollination strategies in which there is no reward for the pollinator, in the most extreme case by mimicking sexual signals of pollinators. In the European flora, two examples of these different pollination strategies are the sexually deceptive genus Ophrys and the rewarding genus Gymnadenia, which differ in their level of pollinator specialization; Ophrys is typically pollinated by pseudo-copulation of males of a single insect species, whilst Gymnadenia attracts a broad range of floral visitors. Here, we present and describe the annotated floral transcriptome of Ophrys iricolor, an Andrena- pollinated representative of the genus Ophrys that is widespread throughout the Aegean. Furthermore, we present additional floral transcriptomes of both sexually deceptive and rewarding orchids, specifi- cally the deceptive Ophrys insectifera, Ophrys aymoninii, and an updated floral transcriptome of Ophrys sphegodes, as well as the floral transcriptomes of the rewarding orchids Gymnadenia conopsea, Gym- nadenia densiflora, Gymnadenia odoratissima, and Gymnadenia rhellicani (syn. Nigritella rhellicani). Comparisons of these novel floral transcriptomes reveal few annotation differences between deceptive and rewarding orchids. Since together, these transcriptomes provide a representative sample of the genus-wide taxonomic diversity within Ophrys and Gymnadenia (Orchidoideae: Orchidinae), we employ a phyloge- nomic approach to address open questions of phylogenetic relationships within the genera. Specifically, this includes the controversial placement of O. insectifera within the Ophrys phylogeny and the place- ment of “Nigritella”-type morphologies within the phylogeny of Gymnadenia. Whereas in Gymnadenia, several conflicting topologies are supported by a similar number of gene trees, a majority ofOphrysgene topologies clearly supports a placement of O. insectifera as sister to a clade containing O. sphegodes. DOI: https://doi.org/10.3389/fpls.2019.01553 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-182304 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Pineiro Fernandez, Laura; Byers, Kelsey J R P; Cai, Jing; Sedeek, Khalid E M; Kellenberger, Roman T; Russo, Alessia; Qi, Weihong; Aquino Fournier, Catharine; Schlüter, Philipp M (2019). A phylogenomic analysis of the floral transcriptomes of sexually deceptive and rewarding European orchids, Ophrys and Gymnadenia. Frontiers in Plant Science, 10:1553. DOI: https://doi.org/10.3389/fpls.2019.01553 2 ORIGINAL RESEARCH published: 29 November 2019 doi: 10.3389/fpls.2019.01553 A Phylogenomic Analysis of the Floral Transcriptomes of Sexually Deceptive and Rewarding European Orchids, Ophrys and Gymnadenia Laura Piñeiro Fernández 1,2*, Kelsey J. R .P. Byers 2,3, Jing Cai 2,4, Khalid E. M. Sedeek 2,5,6, Roman T. Kellenberger 2,7, Alessia Russo 1,2,8, Weihong Qi 9, Catharine Aquino Fournier 9 and Philipp M. Schlüter 1* 1 Institute of Botany, University of Hohenheim, Stuttgart, Germany, 2 Department of Systematic and Evolutionary Botany, University of Zurich, Zurich, Switzerland, 3 Department of Zoology, University of Cambridge, Cambridge, United Kingdom, 4 Center for Ecological and Environmental Sciences, Northwestern Polytechnical University, Xi’an, China, 5 Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia, 6 Agricultural Genetic Engineering Research Institute (AGERI), Agriculture Research Centre, Edited by: Giza, Egypt, 7 Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom, 8 Department of Plant Jen-Tsung Chen, and Microbial Biology, University of Zurich, Zurich, Switzerland, 9 Functional Genomics Centre Zurich, Zurich, Switzerland National University of Kaohsiung, Taiwan The orchids (Orchidaceae) constitute one of the largest and most diverse families of Reviewed by: fl Ashley N. Egan, owering plants. They have evolved a great variety of adaptations to achieve pollination by Aarhus University, Denmark a diverse group of pollinators. Many orchids reward their pollinators, typically with nectar, Alejandra Vázquez-Lobo, but the family is also well-known for employing deceptive pollination strategies in which Universidad Autónoma del Estado de Morelos, Mexico there is no reward for the pollinator, in the most extreme case by mimicking sexual signals *Correspondence: of pollinators. In the European flora, two examples of these different pollination strategies Laura Piñeiro Fernández are the sexually deceptive genus Ophrys and the rewarding genus Gymnadenia, which [email protected] Philipp M. Schlüter differ in their level of pollinator specialization; Ophrys is typically pollinated by pseudo- [email protected] copulation of males of a single insect species, whilst Gymnadenia attracts a broad range of floral visitors. Here, we present and describe the annotated floral transcriptome of Specialty section: Ophrys iricolor,anAndrena-pollinated representative of the genus Ophrys that is This article was submitted to Plant Systematics and Evolution, widespread throughout the Aegean. Furthermore, we present additional floral a section of the journal transcriptomes of both sexually deceptive and rewarding orchids, specifically the Frontiers in Plant Science deceptive Ophrys insectifera, Ophrys aymoninii, and an updated floral transcriptome of Received: 20 July 2019 fl Accepted: 07 November 2019 Ophrys sphegodes, as well as the oral transcriptomes of the rewarding orchids Published: 29 November 2019 Gymnadenia conopsea, Gymnadenia densiflora, Gymnadenia odoratissima,and Citation: Gymnadenia rhellicani (syn. Nigritella rhellicani). Comparisons of these novel floral Piñeiro Fernández L, Byers KJRP, transcriptomes reveal few annotation differences between deceptive and rewarding Cai J, Sedeek KEM, Kellenberger RT, Russo A, Qi W, Aquino Fournier C and orchids. Since together, these transcriptomes provide a representative sample of the Schlüter PM (2019) A Phylogenomic genus-wide taxonomic diversity within Ophrys and Gymnadenia (Orchidoideae: Analysis of the Floral Transcriptomes of Sexually Deceptive and Orchidinae), we employ a phylogenomic approach to address open questions of Rewarding European Orchids, phylogenetic relationships within the genera. Specifically, this includes the controversial Ophrys and Gymnadenia. placement of O. insectifera within the Ophrys phylogeny and the placement of “Nigritella”- Front. Plant Sci. 10:1553. doi: 10.3389/fpls.2019.01553 type morphologies within the phylogeny of Gymnadenia. Whereas in Gymnadenia, several Frontiers in Plant Science | www.frontiersin.org 1 November 2019 | Volume 10 | Article 1553 Fernández et al. Phylogenomics of European Orchids conflicting topologies are supported by a similar number of gene trees, a majority of Ophrys gene topologies clearly supports a placement of O. insectifera as sister to a clade containing O. sphegodes. Keywords: phylogenomics, orchids, Ophrys, Gymnadenia, transcriptome, pollination strategy INTRODUCTION and facilitates reproductive isolation between orchid species (Xu et al., 2011; Xu et al., 2012; Paulus, 2018). Ophrys is a recently Orchidaceae and Asteraceae constitute the largest families of diverged genus (crown age estimated ca. 5 Ma) with ancestral wasp flowering plants. Over 800 orchid genera and 25,000 species have pollination (Breitkopf et al., 2015), but extant species are been described, with an average rate of 500 species and 13 genera commonly pollinated by solitary bees, e.g. Eucera or Andrena described per year (Cribb et al., 2003; Chase et al., 2015). Orchids (Paulus and Gack, 1990; Gaskett, 2011). Successful floral isolation have colonized a great variety of geographical ranges, from and species divergence in the genus may easily be achieved by Scandinavia to Tierra del Fuego (Antonelli et al., 2009; shifts between similar pollinators, where small changes in genes Domínguez and Bahamonde, 2013), although the vast majority involved in the pheromone profiles can lead to attraction of new, of species occur in tropical and neotropical areas (Dressler, related pollinators (Schlüter et al., 2011; Sedeek et al., 2014; 1993). The key to their success has variously been Schlüter, 2018). For instance, after two independent shifts to hypothesized to reside in their epiphytic habitat (for tropical (mostly) Andrena solitary bee pollination (Breitkopf et al., orchids) or in their high level of pollinator specialization 2015), two parallel adaptive radiations have taken place (Gravendeel et al., 2004; Cozzolino and Widmer,