Received: 19 February 2020 | Revised: 9 March 2020 | Accepted: 30 March 2020 DOI: 10.1002/ece3.6312 ORIGINAL RESEARCH Divergent selection on flowering phenology but not on floral morphology between two closely related orchids Elodie Chapurlat1 | Iris Le Roncé1,2 | Jon Ågren1 | Nina Sletvold1 1Plant Ecology and Evolution, Department of Ecology and Genetics, Evolutionary Abstract Biology Centre, Uppsala University, Uppsala, Closely related species often differ in traits that influence reproductive success, sug- Sweden gesting that divergent selection on such traits contribute to the maintenance of spe- 2Master BioSciences, École Normale Supérieure de Lyon, Université Claude cies boundaries. Gymnadenia conopsea ss. and Gymnadenia densiflora are two closely Bernard Lyon 1, Université de Lyon, Lyon, related, perennial orchid species that differ in (a) floral traits important for pollination, France including flowering phenology, floral display, and spur length, and (b) dominant pol- Correspondence linators. If plant–pollinator interactions contribute to the maintenance of trait differ- Nina Sletvold, Plant Ecology and Evolution, Department of Ecology and Genetics, ences between these two taxa, we expect current divergent selection on flowering Evolutionary Biology Centre, Uppsala phenology and floral morphology between the two species. We quantified pheno- University, Norbyvägen 18 D, SE-752 36 Uppsala, Sweden. typic selection via female fitness in one year on flowering start, three floral display Email: [email protected] traits (plant height, number of flowers, and corolla size) and spur length, in six popula- Funding information tions of G. conopsea s.s. and in four populations of G. densiflora. There was indication Extensus Foundation; Swedish Research of divergent selection on flowering start in the expected direction, with selection Council; Swedish Research Council Formas for earlier flowering in two populations of the early-flowering G. conopsea s.s. and for later flowering in one population of the late-flowering G. densiflora. No divergent selection on floral morphology was detected, and there was no significant stabilizing selection on any trait in the two species. The results suggest ongoing adaptive differ- entiation of flowering phenology, strengthening this premating reproductive barrier between the two species. Synthesis: This study is among the first to test whether di- vergent selection on floral traits contribute to the maintenance of species differences between closely related plants. Phenological isolation confers a substantial potential for reproductive isolation, and divergent selection on flowering time can thus greatly influence reproductive isolation and adaptive differentiation. KEYWORDS divergent selection, flowering phenology, Gymnadenia, phenological isolation, plant–pollinator interactions, reproductive barriers, species divergence This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd Ecology and Evolution. 2020;10:5737–5747. www.ecolevol.org | 5737 5738 | CHAPURLAT ET al. 1 | INTRODUCTION flower morphology, but also exhibit partly overlapping quantitative variation in these traits in the wild (Jersáková et al., 2010; Stark, In angiosperms, flowering time and flower morphology critically in- Michalski, Babik, Winterfeld, & Durka, 2011). Second, both orchids fluence mating patterns because of their effects on pollen transfer. depend on pollinators for successful fruit set, and significant pol- Timing of flowering determines which pollinators can visit the flow- linator-mediated selection on flowering phenology, floral display, ers (Elzinga et al., 2007) and the shape, color, scent, and size of flow- and spur length has been documented in G. conopsea s.s. (Chapurlat, ers and inflorescences are important traits for attracting pollinators Ågren, & Sletvold, 2015; Sletvold & Ågren, 2010; Sletvold, and/or for the efficiency of pollination (Ida & Kudo, 2010; Jersáková, Trunschke, Wimmergren, & Ågren, 2012). Third, the pollinator com- Jürgens, Šmilauer, & Johnson, 2012; Raguso, 2008; Trunschke, munities partly differ between the two species, and on Öland, G. Sletvold, & Ågren, 2019). Differentiation in floral traits between conopsea s.s. is mainly visited by nocturnal pollinators, while G. den- taxa may thus play an important role in reducing interspecific pol- siflora is mainly visited by diurnal pollinators with shorter proboscis len transfer and contribute to reproductive isolation, either through than the nocturnal ones (Chapurlat, Anderson, Ågren, Friberg, & phenological isolation (premating barrier caused by differences in Sletvold, 2018; this study). Fourth, genetic studies suggest inter- flowering time; e.g., Kudo, 2006; Nuismer & Cunningham, 2005; specific gene flow and introgression between the species, where Stiles, 1975) or floral isolation (premating barrier caused by dif- introgression is associated with reduced fitness (Gustafsson & ferences in morphological, visual or olfactory traits; e.g., Fulton & Lönn, 2003; Lönn, Alexandersson, & Gustafsson, 2006). Our ob- Hodges, 1999; Maad & Nilsson, 2004; Nilsson, 1983; Sun, Schlüter, jective is to test for divergent selection on flowering phenology Gross, & Schiestl, 2015). If floral trait differences between closely and floral morphology between the two Gymnadenia species. On related taxa are maintained by selection, we should expect current Öland, G. conopsea s.s. flowers earlier than G. densiflora, produces divergent selection on these traits. shorter inflorescences with fewer flowers and longer spurs, and is Adaptive divergence occurs when selection drives the evolution pollinated by species with longer proboscis (see below). If trait dif- of traits toward different optima in different populations or species. ferences are adaptive, we expect optimal flowering to be earlier, Depending on the current trait distributions in relation to these re- optimal flower production and plant height to be lower and opti- spective optima, divergent selection can be linear in different direc- mal spur length to be longer in G. conopsea s.s. than in G. densiflora. tions (e.g., Hall & Willis, 2006) or stabilizing with different optima Given sufficient trait variation, this should be evident as directional (e.g., Benkman, 2003). Divergent selection on flowering phenology selection of opposite sign, or stabilizing selection with different op- has been documented between lowland and montane populations of tima in the two species. Mimulus guttatus (Hall & Willis, 2006), between lowland and alpine populations of Arabidopsis lyrata (Sandring, RiihimäKi, Savolainen, & Ågren, 2007), and between diploid and tetraploid Heuchera grossula- 2 | MATERIALS AND METHODS riifolia (Nuismer & Cunningham, 2005). Divergent selection on floral morphology has been detected in several studies, including traits 2.1 | Study species that influence the efficiency of pollen transfer such as tube or spur length (Gómez, Perfectti, Bosch, & Camacho, 2009; Rymer, Johnson, Gymnadenia conopsea (L.) s.l. is a terrestrial orchid distributed across & Savolainen, 2010), and traits that influence the attraction of polli- Eurasia (Hultén & Fries, 1986). The tuberous, nonclonal, and long- nators such as corolla size (Campbell, 2003; Gómez et al., 2009) and lived perennial plant prefers calcareous soils in grazed or mown number of inflorescences (Sandring et al., 2007). Most of these stud- meadows and margins of marshes and fens (Øien & Moen, 2002). ies provide examples of divergent selection within species, and only The Gymnadenia conopsea (L.) s.l. complex is highly variable with a few studies have tested whether floral differentiation between regard to morphology, scent production, flowering phenology, and species is maintained by divergent selection. While there was diver- habitat (Gustafsson & Lönn, 2003; Jersáková et al., 2010; Soliva & gent selection on corolla size between two Ipomopsis species visited Widmer, 1999; Stark et al., 2011). The most recent classification by hummingbirds and hawkmoths (Campbell, 2003), this was not the based on genetic data recognizes two taxa within the G. conopsea case between two Lobelia species specialized on hummingbirds and (L.) s.l. complex: G. conopsea (L.) R.Br. s.s. and G. densiflora A. Dietr bumblebees, respectively (Johnston, 1991). To elucidate which traits (Bateman et al., 2003; Stark et al., 2011). These two taxa were previ- contribute to the maintenance of species boundaries, it is necessary ously considered subspecies based on morphological similarity, but to study selection on floral traits that are differentiated between they do not even have a sister-species relationship as phylogenetic closely related taxa. analyses of the genus have shown that G. odoratissima is the sister In this study, we quantify phenotypic selection on flowering species of G. conopsea s.s. (Bateman et al., 2003; Brandrud, Paun, phenology, three floral display traits and spur length in the closely Lorenz, Baar, & Hedrén, 2019; Sun et al., 2015). Gymnadenia odor- related orchids Gymnadenia conopsea s.s. and Gymnadenia densi- atissima differs from the other taxa in color, floral scent, and mor- flora on the island of Öland, southern Sweden. These two species phology and was thus not previously included in the G. conopsea (L.) constitute an excellent