Sex ratio variation in gynodioecious species of Echium endemic to the Canary Islands

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1 211 NOTE / NOTE Sex ratio variation in gynodioecious species of Echium endemic to the Canary Islands Marcel E. Dorken Abstract: Species of Echium from the Canary Islands represent an adaptive radiation of plants involving evolutionary changes in habitat and life form. Alongside these changes, evolutionary transitions of the sexual system have occurred: approximately one quarter of Echium species from the Canary Islands are dimorphic for gender, paralleling other adaptive radiations of plants on oceanic islands, such as Schiedea in Hawaii. Here, I report on the variation in the frequency of female plants among eight gynodioecious populations representing four species of Echium on the island of Tenerife, and provide preliminary estimates of the seed fertility of females and hermaphrodites, which is an important determinant of the sex ratio for gynodioecious species. Across populations, female frequencies ranged between 6% and 54%, a much wider range than had previously been reported for gynodioecious species of Echium on the Canary Islands. Substantial frequencies of female plants occurred, even though no differences in the seed fertility of females and hermaphrodites were detected. Key words: Canary Islands, Echium, island radiation, female frequency, gynodioecy, sex ratio. Résumé : Les espèces d Echium des îles Canaries représentent une radiation adaptative de plantes impliquant des changements évolutifs dans l occupation de l habitat et dans la forme biologique. En plus de ces modifications, on observe des transitions évolutives du système sexuel; environ le quart des Echium des îles Canaries sont dimorphes quant au genre, ce qui correspond à la radiation des espèces sur d autres îles, comme les Schiedea en Hawaii. L auteur rapporte ici la variation des fréquences femelles au sein de huit populations gynodioïques représentant quatre espèces sur l île de Ténériffe, et fournissant les mesures préliminaires de la fertilité des plantes femelles et hermaphrodites par leur fonction femelle, un important facteur déterminant pour le rapport des sexes chez les espèces gynodioïques. La fréquence des femelles varie entre 6 % et 54 %, un écart beaucoup plus large que ce qui a déjà été rapporté pour les Echium des îles Canaries. On observe une fréquence femelle substantielle, même si aucune différence n a pu être observée dans la fertilité des graines des plantes femelles et hermaphrodites. Mots-clés :Îles Canaries, Echium, radiation insulaire, fréquence femelle, gynodiécie, ratio des sexes. [Traduit par la Rédaction] Introduction Oceanic islands have a higher than average incidence of gender dimorphism (Baker 1967; Sakai and Weller 1999), an association that has been explained by the advantages of outcrossing to newly established lineages on islands (Baker 1967; Thomson and Barrett 1981). Transitions to unisexuality have been particularly well studied in the Hawaiian genera Bidens and Schiedea (Helenurm and Ganders 1985; Sun and Ganders 1986, 1988; Weller et al. 1998; Sakai et al. 2006). For Schiedea, separate sexes appear to have evolved in at least two lineages representing approximately one third of the species in the genus (Sakai et al. 2006). Shifts from mesic or wet habitats to dry and windy habitats have been implicated in the evolution of separate sexes in this group Received 15 July Published on the NRC Research Press Web site at botany.nrc.ca on 9 February M.E. Dorken. Biology Department, Trent University, 1600 West Bank Drive, Peterborough, ON K9J 7B8, Canada ( marceldorken@trentu.ca). (Sakai et al. 2006). A key inference from studies of Schiedea is that shifts in habitat, and associated changes in plant morphology and life history, might have led to novel selection pressures that favoured the evolution of gender dimorphism (Rankin et al. 2002). The genus Echium from the Macaronesian islands (the Canary, Madeira, and Cape Verde archipelagos) has several features that parallel Hawaiian Schiedea. For Echium, the colonization of the islands has been followed by expansion into habitats that range between semi-desert to more mesic conditions (Bramwell 1975; García-Maroto et al. 2009). Coinciding with these habitat shifts, changes in life form have also occurred, with short-lived annuals and monocarpic herbs, and long-lived woody shrubs represented in the group (Bramwell 1975; Böhle et al. 1996; García-Maroto et al. 2009). There are 28 species of Echium in Macaronesia, which diverged from a single herbaceous ancestor (Böhle et al. 1996; García-Maroto et al. 2009) during the Miocene (Mansion et al. 2009). As in Schiedea, a high proportion of these species are dimorphic for gender, with seven species Botany 88: (2010) doi: /b09-106

2 212 Botany Vol. 88, 2010 described as gynodioecious (the co-occurrence of females and hermaphrodites; Bramwell 1972). Sexual systems other than hermaphroditism and gynodioecy have not been described for Macaronesian Echium. The evolution of gynodioecy involves the invasion and spread of male-sterile phenotypes (i.e., female plants) that are governed by the expression of genes that occur in the cytoplasmic and (or) nuclear genomes (Lewis 1941; Budar et al. 2003; Bailey and Delph 2007a; Delph et al. 2007). Under nuclear inheritance of male sterility, females are required to have at least twice the fertility through their female function as hermaphrodites, which transmit genes to the next generation via both seeds and pollen (i.e., via both their female and male functions; Lewis 1941; Charlesworth and Charlesworth 1978). Although some gynodioecious species possess nuclear transmission of male-sterility (e.g., Fragaria virginiana; Valleau 1923; Ahmadi and Bringhurst 1991), in the majority of cases a combination of cytoplasmic and nuclear genes regulate male-sterility (Delph et al. 2007). Here, maternally inherited cytoplasmic male sterility (CMS) is balanced by nuclear restorers of fertility, converting plants with CMS into hermaphrodites. Although the evolutionary dynamics of female frequencies can be complicated by the fitness effects of genes causing male sterility (and of the nuclear restorers of male fertility; Bailey et al. 2003; Delph et al. 2007), under this mode of inheritance, females can be maintained even if they possess a slight advantage in seed production (Lewis 1941; Lloyd 1974). Because of the different modes of transmission of maternally vs. biparentally inherited genes, the frequencies of females maintained under nuclear vs. cytonuclear inheritance of gynodioecy can differ widely. Under nuclear inheritance, the maximum equilibrium frequency of females is 50%, a value that can be exceeded under cytonuclear inheritance (Lewis 1941; Lloyd 1974; Delph et al. 2007). Under both modes of inheritance, however, the relative seed fertility of females and hermaphrodites plays a role in regulating the frequency of females in populations (Lloyd 1974, 1976; Bailey and Delph 2007b). Here, I report sex ratios from eight populations of woody Echium from the island of Tenerife (Canary Islands) and present preliminary data on the seed fertility of females vs. hermaphrodites to explore the factors regulating female frequencies in this group. Materials and methods Study species and locality information Three of the four species of Echium included in this study have highly restricted geographic ranges, occurring only on the island of Tenerife and only in a small number of localities on that island (E. giganteum, E. leucophaeum, and E. virescens; Bramwell and Bramwell 2001). The fourth species, E. aculeatum, is more widespread, occurring on three of the seven main islands (Bramwell and Bramwell 2001; García-Maroto et al. 2009). Locality information for each of these species examined is presented in Table 1 (more detailed locality information is available upon request). All four species are woody shrubs with a candelabra architecture that grow up to 2.5 m in height (Bramwell and Bramwell 2001). Cymes are grouped together forming aggregate inflorescences at the tips of erect branches, and flowers open simultaneously across multiple inflorescences per plant. Sex ratios for six species of gynodioecious Echium (E. aculeatum, E. brevirame, E. giganteum, E. leucophaeum, E. virescens, E. webbii; all of which occur in the Canary Islands) were described by Bramwell (1972), who noted some variability in the expression of male sterility among plants. In particular, he described four classes of flower types: (a) hermaphrodite flowers, in which all stamens develop normally and produce fertile pollen; (b) intermediate flowers, in which a subset of stamens appear to be normally developed; (c) pistillate flowers with rudimentary stamens, in which degenerate filaments and non-dehiscent, sterile anthers occur; (d) pistillate flowers without stamens. Type (a) characterizes hermaphroditic plants. Type (b) was uncommon, and, on plants in which these flowers were detected, represented only a small proportion of flowers (e.g., in the population of E. leucophaeum at the Mirador de Aguaide intermediate flowers represented fewer than 1% of open flowers on plants expressing this phenotype, and occurring on fewer than 5% of the plants in the sample). Plants with type-(b) flowers produced mostly type-(c) flowers, and because evaluating the gender of plants, rather than of individual flowers, was the goal of this study, plants that produced a mixture of (b)- and (c)-type flowers were classified as females. Type-(d) flowers were not observed in this study and were reported by Bramwell (1972) to occur on only a subset of plants in one population of E. strictum. As noted by Bramwell (1972), and as observed in this study, type-(c) flowers represent the vast majority of flowers on plants expressing any degree of male sterility. For the six species considered by Bramwell (1972), sex ratios varied between 17% and 30%. Bramwell s (1972) reports of sex ratios did not include the sample size and because sex ratios are highly sensitive to sample size, one of the goals of this study was to report sex ratios from entire populations, or from samples of at least 100 plants. Accordingly, in April 2007, eight populations of Echium representing four species were evaluated for variation in female frequencies. At each site, if the population comprised fewer than 100 flowering plants, the sex of all flowering plants was recorded. At sites with more than 100 plants, at least 100 plants, all occurring within a contiguous area were included in the sample. At two sites, components of fertility were evaluated. At Chinamada, I evaluated the daily display size of females and hermaphrodites by counting the number of open flowers (E. leucophaeum) on one randomly chosen compound inflorescence (i.e., all of the cymes grouped at the terminus of a branch), as well as the total number of compound inflorescences per plant. These two values were multipled to yield estimates of the daily display size for the first 19 plants (8 females and 11 hermaphrodites). Plants were sampled by taking measurements from all plants encountered along a transect through a contiguous portion of the population. For the population of E. giganteum at San Marcos, I estimated seed set by counting the total number of flowers produced on the 20 basal-most cymes on a randomly chosen compound inflorescence and the total number of nutlets produced by these flowers. The gynoecium of the flowers of the species studied here consists of four uni-ovulate seg-

3 Dorken 213 Table 1. Variation in female frequencies (sample size is indicated in parentheses) for four species of Echium sampled at eight localities. Species Female frequencies reported by Bramwell (1972) Locality Female frequency (N) E. leucophaeum 0.17 (1) Chinamada 0.38 (101) Chamorga 0.23 (186) E. giganteum (3) San Marcos 0.12 (50) E. aculeatum 0.20 (2) Tabaibapass 0.06 (100) Santiago del Teide I 0.22 (136) Santiago del Teide II 0.15 (136) El Carrizal Alto 0.59 (259) E. virescens 0.19 (1) Güímar 0.29 (62) Note: For comparison, the range of female frequencies reported by Bramwell (1972) is given for each species, with the number of sites sampled in that paper indicated in parentheses. ments, each of which may form a nutlet. Thus, seed set can be estimated as the number of nutlets produced per flower divided by four. Seed set was estimated in this way for the 34 plants (6 females, 28 hermaphrodites) occurring in the largest contiguous portion of the population. Results There was substantial variation in sex ratios among species and among populations within species, with female frequencies ranging between 0.06 and 0.59 (Table 1). These two extreme values came from different populations of the same species (E. aculeatum). There were no significant differences in daily floral display size between females and hermaphrodites of E. leucophaeum at Chinamada (Fig. 1a; one-way analysis of variance: F [1,17] = 0.76, P > 0.30). The seed set of E. giganteum was low at San Marcos, averaging 13% across all plants sampled, and again, no difference between females and hermaphrodites was detected (Fig. 1b; one-way analysis of variance: F [1,33] = 1.15, P > 0.25). Discussion Females were observed in all species reported as gynodioecious by Bramwell (1972) and their frequencies varied substantially among populations. In one population, female frequencies reached 50%, the maximum that can be obtained at equilibrium under nuclear gynodioecy (Lewis 1941). In addition, the variability of female frequencies among populations found here contrasts with the pattern reported by Bramwell (1972). In particular, there was a substantial difference between the extremes of the values found here ( ) compared with the results of that study ( ). Below, I discuss some of the features of gynodioeicous populations that affect variation in female frequencies and highlight issues that require further study. Under cytonuclear gynodioecy, the genes regulating malesterility (and its restoration) are subject to changes in the intensity of selection (Bailey and Delph 2007b), as well as from frequency-dependent selection (McCauley and Brock 1998; Dufay et al. 2009). As a result, sex ratios can be expected to vary substantially over time (e.g., Belhassen et al. 1989; Bailey and Delph 2007b; Barr 2008), and between populations (Bailey and Delph 2007a; Barr 2008). Although the relative seed fertility of females and hermaphrodites is an important determinant of female frequencies under maternal inheritance of male sterility, cyclical changes in the sex ratio are an expectation arising from models of the dynamics of gynodioecious populations (Gouyon et al. 1991; Bailey and Delph 2007b). As a result, a direct correspondence between relative seed fertilities and the female frequencies is not a general prediction for populations with this mode of inheritance. If the frequency of females in Echium on the Canary Islands is regulated by the joint action of nuclear and cytoplasmic factors, as it appears to be for Echium vulgare (Klinkhamer et al. 1994), and for the majority of gynodioecious species (Budar et al. 2003; Delph et al. 2007), then differences in female frequencies between the populations sampled here might be the result of the substantial fluctuations in female frequencies over time noted for gynodioecious species with this mode of inheritance (e.g., Belhassen et al. 1989; Barr 2008). In species with nuclear gynodioecy, on the other hand, the frequency of females is closely associated with the relative seed fitness of females vs. hermaphrodites (Lloyd 1974, 1976; Charlesworth and Charlesworth 1978; Delph 1990; Eckhart 1992; Ashman 1999). With this mode of inheritance, hermaphrodites function increasingly as males as the female frequencies approach 50%, and their fitness through seed is effectively nil. In contrast, in populations in which females occur at low frequencies, the seed fitness of females should approach 2 that of hermaphrodites the minimum seed fitness advantage required for the maintenance of females under nuclear gynodioecy (Lewis 1941; Lloyd 1976). For example, for populations of Phacelia linearis in which the frequency of female plants ranges from 2% to 16%, females produce as many seeds as the hermaphrodites (Eckhart 1992). The frequency of female plants in some of the Tenerife populations studied here were substantially higher than those reported for Phacelia, even though there were no detectable differences in seed set between females and hermaphrodites for one of these populations [i.e., the San Marcos population of E. giganteum with 12% female plants, a frequency approaching the highest value reported by Eckhart (1992) for Phacelia]. It should be noted that seed set does not relate directly to total seed production, and interpreting a role for seed set assumes that the females and hermaphrodites in this population produced similar numbers of flowers (each of which produces four ovules). Clearly, further research on the magnitude of seed fertility differences across populations, particularly for those popula-

4 214 Botany Vol. 88, 2010 Fig. 1. Estimates of the number of open flowers per plant (a) and seed set (b) for females and hermaphrodites of two species of gynodioecious Echium (E. leucophaeum, and E. giganteum, respectively). Data shown are means ± 1 SE. and sample sizes are indicated below each estimate. tions with contrasting frequencies of females, would aid in the interpretation of the causes underlying variation in female frequencies across populations of Echium in the Canary Islands. Even if females and hermaphrodites produce similar quantities of seeds, the quality of those seeds might differ if hermaphrodites are self-compatible and their seed fertility is reduced because of inbreeding depression. Indeed, the candelabra morphology of the Echium species studied here is likely to make self-pollination by hermaphrodites a common occurrence. These plants simultaneously present large numbers of flowers and rates of geitonogamous pollen exchange could be substantial (e.g., Eckert 2000). Moreover, inbreeding depression in long-lived, woody plants often affects the earliest stages of development, including seed formation (Husband and Schemske 1996). If inbreeding depression affects the seed fertility of Echium species on the Canary Islands, it might be expected to reduce the seed fertility of hermaphrodites in comparison with females. However, the seed set of females of E. giganteum at the San Marcos site was similar to that of hermaphrodites. Further research is required to evaluate whether hermaphrodites are self compatible, and if so, the extent to which seeds are formed through self pollination and the magnitude of inbreeding depression in selfed progeny. Inbreeding depression could be ruled out if Macaronesian Echium were self incompatible. However, self-incompatibility is relatively uncommon for plants on isolated oceanic islands (Baker 1967; Barrett 1996; and see Schueller 2004), and there have been no conclusive examinations of the presence of self incompatibility in Echium from the Canary Islands. Moreover, the congeneric species, E. vulgare, has been shown to be self-compatible (Melser et al. 1997; Rademaker et al. 1999). If these plants are self compatible, much of the variation in female frequencies might be caused by differences in selfing rates among populations. Under nuclear gynodioecy, female frequencies would be regulated, in part, by the product of the selfing rate and the magnitude of inbreeding depression (Charlesworth and Charlesworth 1978; Sun and Ganders 1986). Under cytonuclear gynodioecy, the effect of selfing on female frequencies depends on the mode of inheritance of nuclear restorers of male fertility (Charlesworth and Laporte 1998; Emery and McCauley 2002), but even here, differences in selfing rates among populations would be expected to cause variation in female frequencies (Emery and McCauley 2002). While investigating sex ratios of E. aculeatum at El Carrizal Alto, I noticed that E. strictum, which occurs at that site and is another shrubby species of Echium, also has substantial frequencies of females [17% of a sample of 23 plants; and see Bramwell (1972)]. Together with four other species, all of the gyndioecious species of Canary Islands Echium for which sex ratios have been reported have been grouped into a single clade (García-Maroto et al. 2009). However, García- Maroto et al placed E. strictum in a separate lineage with a species not known to be gynodioecious, raising the possibility that gynodioecy has evolved more than once, or that there have been reversions from gynodioecy. Convergent evolution of sexual systems have been inferred for other radiations of island plants (e.g., Hawaiian Schiedea; Sakai et al. 2006), and further study of sexual systems in Macaronesian Echium, together with an explicit phylogenetic reconstruction of breeding system evolution in this group would help clarify the number of lineages in which separarate sexes have evolved since the colonization of Macaronesia. Acknowledgements I thank Alfredo Reyes-Betancourt and Arnoldo Santos- Guerra for helpful advice, Camille Barr for helpful comments on an earlier version of the manuscript, Femke de Man for assistance in the field, and the University of Oxford and St. Hugh s College for a Career Development Fellowship that funded this work.

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