RESEARCH ARTICLE AMERICAN JOURNAL OF BOTANY Associations between sex-organ deployment and morph bias in related heterostylous taxa with diff erent stylar polymorphisms 1 Victoria Ferrero 2,3,8 , Spencer C. H. Barrett 4 , Danny Rojas 5,6 , Juan Arroyo 7 , and Luis Navarro2 PREMISE OF THE STUDY: Populations of heterostylous species are characterized by two or three fl oral morphs with reciprocal positioning of stigmas and anthers. Theoretical models predict equal morph frequencies (isoplethy) when disassortative mating is prevalent in populations, but biased morph ratios may occur when variation in the expression of heterostyly causes deviations from intermorph mating. METHODS: We explore the role of sex-organ deployment in governing morph ratios in two closely related genera of Boraginaceae, exhibiting striking varia- tion in fl oral traits associated with the heterostylous syndrome. We sampled 66 populations of six species of Glandora and 39 populations of three species of Lithodora across their distributional range in the Mediterranean. In each population we estimated morph ratios and measured several fl oral traits. We used phylogenetically corrected and noncorrected regressions to test the hypothesis that diff erences in sex-organ reciprocity and herkogamy are associ- ated with deviations from isoplethy. KEY RESULTS: Biased morph ratios occurred in 24% of populations, particularly in Lithodora . Populations biased for the long-styled morph (L-morph) were more frequent than the short-styled morph (S-morph). Distylous species were less likely to exhibit biased ratios than species with stigma-height dimor- phism. In Lithodora fruticosa , a species lacking reciprocity, decreased herkogamy in the S-morph was associated with increasing L-morph bias, perhaps resulting from self-interference. CONCLUSION: Striking variation in the expression of heterostyly in Glandora and Lithodora is associated with biased morph ratios, which probably result from pollinator-mediated mating asymmetries within populations. KEY WORDS disassortative mating; distyly; fl oral morph ratios; Glandora ; heterostyly; Lithodora ; Mediterranean plants; stigma-height dimorphism Heterostyly is a genetic polymorphism in which populations are ( Darwin, 1877 ; Barrett, 1992 ). Th e genetic control of the polymor- composed of two (distyly) or three (tristyly) fl oral morphs that dif- phism in distylous plants usually involves a single Mendelian dial- fer in the reciprocal placement of stigmas and anthers within fl owers lelic locus in which the long-styled morph is of genotype ss and the short-styled morph is of genotype Ss , although in several species the dominance relations at the S -locus are reversed (Lewis and 1 Manuscript received 28 September 2016; revision accepted 29 November 2016. 2 Department of Plant Biology and Soil Sciences, Faculty of Biology, University of Vigo, As Jones, 1992 ). Th e fl oral morphs are maintained in populations by Lagoas-Marcosende 36200 Vigo, Spain; negative frequency-dependent selection resulting from intermorph 3 CFE, Centre for Functional Ecology and Department of Life Sciences, University of (disassortative) mating. With this genetic system and disassortative Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal; mating, a 1:1 morph ratio (isoplethy; Finney, 1953 ) is expected in 4 Department of Ecology and Evolutionary Biology, University of Toronto, 25 Willcocks equilibrium populations. Street, Toronto, Ontario, Canada M5S 3B2; 5 Department of Biology and Centre for Environmental and Marine Studies, University of Th e classic textbook depiction of heterostyly as a balanced poly- Aveiro, 3810-193 Aveiro, Portugal; morphism (e.g., Roughgarden, 1979 ; Silvertown and Charlesworth, 6 Department of Ecology and Evolution, Stony Brook University, 650 Life Sciences Building 2009 ; Charlesworth and Charlesworth, 2010 ) is largely based on Stony Brook, New York 11794 USA; and knowledge of distyly in Primula and emphasizes how the reciprocal 7 Departamento de Biología Vegetal y Ecología, Universidad de Sevilla, Apartado 1095 41080 Sevilla, Spain positioning of sex organs (reciprocal herkogamy) associated with a 8 Author for correspondence (e-mail: [email protected]) self and intramorph incompatibility system (heteromorphic incom- doi:10.3732/ajb.1600345 patibility) promotes outcrossing in populations. Th e heterostylous 50 • AMERICAN JOURNAL OF BOTANY 104 (1): 50 – 61 , 2017; http://www.amjbot.org/ © 2017 Botanical Society of America JANUARY 2017 , VOLUME 104 • FERRERO ET AL. —MORPH RATIOS IN SPECIES WITH DIFFERENT STYLAR POLYMORPHISMS • 51 syndrome has evolved on numerous occasions in unrelated animal- siring opportunities through illegitimate pollen transfer should se- pollinated families of fl owering plants ( Ganders, 1979 ; Lloyd and lect for greater sex-organ reciprocity and greater herkogamy. In Webb, 1992a ; Barrett et al., 2000 ), and is perhaps the most well- contrast, in species in which intramorph mating is permitted, because studied discrete fl oral polymorphism. of the absence of heteromorphic incompatibility, pollen wastage Investigations of heterostyly have broadened since Darwin’s does not occur and we might expect less stringent selection for re- seminal work on Primula and Lythrum ( Darwin, 1877 ) to include ciprocal herkogamy and perhaps smaller herkogamy distances. many more families. It is now evident that reciprocal herkogamy Recent investigations of the fl oral biology of several heteromor- may vary considerably in expression and can be associated with phic genera have revealed an unusual combination of reproductive diverse compatibility systems, while still functioning to promote features not evident in previously studied heterostylous taxa. In varying degrees of disassortative pollen transfer (Barrett and several species of Anchusa , Lithodora, Glandora (Boraginaceae), Richards, 1990 ; Dulberger, 1992 ; Lloyd and Webb, 1992a , b ; Barrett and Narcissus (Amaryllidaceae) the style morphs are self-incom- and Cruzan, 1994 ; Ferrero et al., 2012 ; Zhou et al., 2015 ). Moreover, patible but intramorph compatible—a pattern inconsistent with although the frequencies of style morphs in populations are gov- heteromorphic incompatibility and indicative that they possess dis- erned by the aggregate patterns of mating in preceding generations, tinct self-recognition systems ( Anchusa — Dulberger, 1970 ; Philipp a variety of stochastic and deterministic processes can result in morph and Schou, 1981; Schou and Philipp, 1984 ; Lithodora and Glan- ratios that deviate signifi cantly from equality. Founder events and dora — Ferrero et al., 2011a , 2012 ; Narcissus — Dulberger, 1964 ; genetic drift commonly result in biased morph ratios (anisoplethy), Barrett et al., 1997; Arroyo et al., 2002; Baker et al., 2000b ; Simón- especially in species in which features of the life history (e.g., Porcar et al., 2015b ). In Anchusa ( Schou and Philipp, 1983 ) and clonality and episodic sexual recruitment) slow progress to the iso- Narcissus ( Dulberger, 1964 ; Sage et al., 1999 ; Santos-Gally et al., plethic equilibrium ( Ornduff , 1972; Morgan and Barrett, 1988 ; 2015 ; Simón-Porcar et al., 2015b ) self-rejection involves late-acting Eckert and Barrett, 1995). Although less commonly documented, ovarian incompatibility. Signifi cantly, sex-organ reciprocity is less morph-specifi c diff erences in reproductive fi tness can also cause well developed in these taxa despite clear polymorphism for style biased morph ratios in heterostylous populations (Barrett et al., length. Anther heights are usually less well diff erentiated between 1983 ; 2004 ; Brys et al., 2008a ; Weber et al., 2013 ). Th us, determin- the style morphs, and where this occurs in dimorphic taxa, the poly- ing the causes of anisoplethic morph ratios in heterostylous popu- morphism is referred to as stigma-height dimorphism, with popula- lations is a complex problem that usually commences with a study tions composed of L- and S-morphs (reviewed in Barrett et al., 2000 ). of the reproductive correlates of morph-ratio variation, an approach Th eoretical models of the evolution of distyly include stigma- we use here. height dimorphism as an intermediate stage in the transition from Haldane (1936) fi rst recognized that distylous populations stylar monomorphism to distyly ( Charlesworth and Charlesworth, should proceed more rapidly than tristylous populations to an iso- 1979 ; Lloyd and Webb, 1992b ). Comparative evidence in Narcissus plethic equilibrium. He pointed out that in the absence of “illegiti- and Lithodora involving phylogenetic reconstructions of the evolu- mate unions” (self and intramorph mating) in distylous populations, tionary history of stylar polymorphisms generally support these the frequencies of the L- and S-morphs should be fully restored to models (Graham and Barrett, 2004 ; Pérez-Barrales et al., 2006 ; equality in one generation aft er any particular perturbation ( Haldane, Ferrero et al., 2009). However, stigma-height dimorphism is clearly 1936 , p. 396). Th is inference assumed a tight association between a stable polymorphism in each of these taxa because it is reported the stamen and style polymorphism and heteromorphic incompat- from a signifi cant number of species. Moreover, at least in Narcis- ibility. However, not all species with stylar polymorphism possess sus , there is experimental evidence that despite incomplete sex- strong heteromorphic incompatibility, and in species in which self organ reciprocity and intramorph compatibility,