International Journal of Molecular Sciences Article How Are the Flower Structure and Nectar Composition of the Generalistic Orchid Neottia ovata Adapted to a Wide Range of Pollinators? Emilia Brzosko *, Andrzej Bajguz * , Magdalena Chmur, Justyna Burzy ´ nska, Edyta Jermakowicz, Pawel Mirski and Piotr Zieli ´ nski   Citation: Brzosko, E.; Bajguz, A.; Chmur, M.; Burzy´ nska, J.; Jermakowicz, E.; Mirski, P.; Zieli´ nski, P. How Are the Flower Structure and Nectar Composition of the Generalistic Orchid Neottia ovata Adapted to a Wide Range of Pollinators?. Int. J. Mol. Sci. 2021, 22, 2214. https://doi.org/10.3390/ ijms22042214 Academic Editor: Jen-Tsung Chen Received: 29 December 2020 Accepted: 19 February 2021 Published: 23 February 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Faculty of Biology, University of Bialystok, Ciolkowskiego 1J, 15-245 Bialystok, Poland; m.chmur@uwb.edu.pl (M.C.); j.burzynska@uwb.edu.pl (J.B.); edytabot@uwb.edu.pl (E.J.); p.mirski@uwb.edu.pl (P.M.);p.zielinski@uwb.edu.pl (P.Z.) * Correspondence: emilka@uwb.edu.pl (E.B.); abajguz@uwb.edu.pl (A.B.); Tel.: +48-85-738-8424 (E.B.); +48-85-738-8361 (A.B.) Abstract: Plant-pollinator interactions significantly influence reproductive success (RS) and drive the evolution of pollination syndromes. In the context of RS, mainly the role of flower morphology is touched. The importance of nectar properties is less studied, despite its significance in pollination effectiveness. Therefore, the aim of this study was to test selection on flower morphology and nectar chemistry in the generalistic orchid Neottia ovata. In 2019–2020, we measured three floral displays and six flower traits, pollinaria removal (PR), female reproductive success (FRS), and determined the soil properties. The sugars and amino acids (AAs) were analyzed using the HPLC method. Data were analyzed using multiple statistical methods (boxplots, ternary plot, one-way ANOVA, Kruskal-Wallis test, and PCA). Variation of flower structure and nectar chemistry and their weak correlation with RS confirms the generalistic character of N. ovata. In particular populations, different traits were under selection. PR was high and similar in all populations in both years, while FRS was lower and varied among populations. Nectar was dominated by glucose, fructose, and included 28 AAs (Ala and Glu have the highest content). Sugars and AAs influenced mainly FRS. Among soil parameters, carbon and carbon:nitrogen ratio seems to be the most important in shaping flower structure and nectar chemistry. Keywords: amino acids; female reproductive success; pollinaria removal; natural selection; orchids; plant-pollinator interactions; sugars 1. Introduction Plants dependent on animals in the pollination process evolved different strategies to attract pollinators, thereby increasing reproductive success. The main parts of these strate- gies are flower traits (the size, shape, color, scent, and nectar) adapted to a given pollinator or their whole group. Pollinator-mediated selection on floral traits is well documented, and adaptation of plants to the most effective pollinators drives the evolution of pollination syndromes [1]. The flagship example of the unusual diversity of flowers and equally differentiated pollination mechanisms is Orchidaceae, which is one of the biggest families among flowering plants [2]. About one-third of its representatives deceive pollinators through sexual or food deception [2–4]. Other groups of orchids reward pollinators in a different way, producing oils, nectar, resin, wax, and fragrances [5,6]. Among rewards offered by orchids, nectar is the most effective [2,7,8]. Fruiting in nectariferous orchids is significantly higher than in nectarless [2,8]. Although nectariferous orchids constitute a large part of the family, and the role of nectar in highly effective pollination is indisputable, information on its chemical composition in Orchidaceae is very scarce. Moreover, many data derive from studies using less sensitive methods in comparison to those applied Int. J. Mol. Sci. 2021, 22, 2214. https://doi.org/10.3390/ijms22042214 https://www.mdpi.com/journal/ijms