Heredity (2004) 92, 459–465 & 2004 Nature Publishing Group All rights reserved 0018-067X/04 $25.00 www.nature.com/hdy Mating patterns and genetic diversity in the wild Daffodil Narcissus longispathus (Amaryllidaceae) SCH Barrett1, WW Cole1 and CM Herrera2 1Department of Botany, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2; 2Estacio´n Biolo´gica de Don˜ana, CSIC, Apartado 1056, E-41080, Sevilla, Spain Despite the importance of Narcissus to ornamental horticul- different from zero (f ¼ 0.001), indicating that few selfed ture, there have been no population genetic studies of wild offspring survive to maturity because of inbreeding depres- species, many of which have narrow distributions. Here, we sion. Species-wide estimates of genetic diversity for the measure selfing rates and levels of genetic diversity at six populations were Ps ¼ 0.38, Hes ¼ 0.119 and As ¼ 1.27 allozyme loci in six populations of Narcissus longispathus,a with significant genetic differentiation among populations self-compatible daffodil endemic to a few mountain ranges in y ¼ 0.15. The observed patterns of genetic differentiation southeastern Spain. The populations were distributed among among populations are likely influenced by the mating four distinct river valleys encompassing two main watersheds system, and a combination of local topography, watershed in the Sierra de Cazorla mountains. Selfing rates averaged affinities and gene flow. 0.37 (range 0.23–0.46), resulting in significant inbreeding Heredity (2004) 92, 459–465, advance online publication, coefficients for the progeny (f ¼ 0.324). In contrast, estimates 10 March 2004; doi:10.1038/sj.hdy.6800441 of inbreeding in parental genotypes were not significantly Keywords: allozymes; genetic diversity; inbreeding depression; Narcissus; selfing rates Introduction small population size generally act to reduce the frequency of pollinator visitation (A˚ gren, 1996), resulting Mating patterns in flowering plants are governed by in the potential for increased selfing and local inbreeding. complex interactions between reproductive traits and the Under these demographic conditions, the selection of ecology of populations (Lloyd, 1980; Holsinger, 1996; self-compatibility through the breakdown of self-incom- Barrett and Pannell, 1999). An important component of patibility may be favoured as a means of reproductive mating is the amount of self-fertilization that a popula- assurance (Lloyd, 1980; Piper et al, 1986). This is likely to tion experiences. This is because selfing has direct occur under low-density conditions if populations con- genetic consequences, including its effect on the intensity sistently receive insufficient pollinator service to maintain of inbreeding depression (Charlesworth and Charles- fertility (Kunin, 1992). Indeed, the reported association worth, 1987) and the partitioning of genetic diversity between self-compatibility and plant rarity may reflect, in within and among populations (Hamrick and Godt, part, the mating constraints that spatial isolation presents 1989). In animal-pollinated species, pollen dispersal and for animal-pollinated plants with restricted distributions levels of selfing may be influenced by the environmental (Kunin and Shmida, 1997, but see Karron, 1987). conditions during flowering. Species that bloom in early Narcissus longispathus Pugsley (Amaryllidaceae) is a spring when unfavourable weather conditions often bee-pollinated, perennial herb endemic to a few moun- prevail are susceptible to unpredictable pollinator tain ranges in southeastern Spain (Moreno Saiz and Sainz service. Probably as a response to uncertain pollination Ollero, 1992). It is related to the widely distributed, environments many early blooming herbs possess floral highly polymorphic, self-incompatible N. pseudonarcissus; traits, such as extended floral longevities and self- the most commonly cultivated trumpet daffodil of compatibility, which reduce the likelihood of reproduc- horticulture. In a study of the floral biology of N. tive failure (Schemske et al, 1978; Barrett and Helenurm, longispathus in the Sierra de Cazorla mountains, Herrera 1987; Herrera, 1995). However, self-compatible species (1995) demonstrated that N. longispathus is self-compa- with long floral lifespans may be vulnerable to unpre- tible producing equivalent amounts of seed following dictable pollinator service, which can potentially result in experimental self- and cross-pollinations. This contrasts increased self-fertilization. with other species in the genus most of which are The spatial distribution, size and abundance of moderately to strongly self-sterile (reviewed in Barrett populations might also be expected to influence mating et al, 1996; Sage et al, 1999). In the Sierra de Cazorla patterns in animal-pollinated plants. Spatial isolation and mountains, N. longispathus is represented by scattered populations confined to stream margins or poorly drained meadow fragments at 1000–1500 m. Flowering Correspondence: SCH Barrett, Department of Botany, University of occurs from late February to mid-April, a period Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2. E-mail: [email protected] characterized by cool, rainy weather that frequently Received 18 November 2002; accepted 27 January 2004; published limits the activity of the species’ main pollinator (Andrena online 10 March 2004 bicolor, Andrenidae). Despite low pollinator visitation, Genetics of wild daffodils SCH Barrett et al 460 most flowers of N. longispathus are pollinated. Using controlled crosses, Herrera (1995) demonstrated weak pollen limitation of fruit set, but no evidence that insufficient pollen delivery limits seed set per fruit. To what extent self-fertilization contributes towards the Sierra maintenance of female fertility in N. longispathus is not de Cazorla known. To address this issue, we investigated the mating CB system of this species. quivir There have been no studies of the population genetics of wild daffodil species to evaluate levels of diversity. As Guadal N. longispathus is a rare endemic closely related to several LA IRUELA cultivated species, we were interested in investigating the VAD ILLO Vt-2 Vc amounts of genetic diversity within and among popula- Vt-1 tions. Such information may be of value for future efforts for the conservation of gene pools of close relatives of Gualay cultivated daffodils. Populations of N. longispathus in the Sierra de Cazorla mountains are distributed among Gu-C several distinct watersheds and river valleys. Therefore, Gu-D NORTH we were also interested in examining how geographical isolation and watershed membership might influence their genetic relationships. 1 km Here, we address several questions concerning the mating system and population genetic structure of Guadalentín N. longispathus: (1) What rates of self-fertilization occur in populations and are these influenced by population size? (2) Is there evidence of selection against selfed Figure 1 Geographical distribution of six populations of N. longispathus sampled from the Sierra de Cazorla mountains, SE offspring during growth and reproduction due to Spain. Arrows indicate the direction of flow of water along river inbreeding depression? (3) What are the levels of genetic valleys. diversity in populations and does their spatial distribu- tion influence genetic relationships? These questions were addressed by sampling seed families from six populations in the Sierra de Cazorla mountains and Table 1 Populations of N. longispathus in the Sierra de Cazorla investigating polymorphism at allozyme loci. mountains, SE Spain, which were investigated in this study Code Population Population size, n Plants sampled Materials and methods CB Cuevas Bermejas 40 14 Gu-C Tornillos de Gualay-C 300 12 Sampling and electrophoresis Gu-D Tornillos de Gualay-D 300 15 Vc Valdecuevas 600 15 We randomly sampled open-pollinated capsules at Vt-1 Valdetrillos-1 250 15 maturity from six populations of N. longispathus during Vt-2 Valdetrillos-2 150 15 May–June 1990. The location of populations is mapped in Figure 1 and details of population size and sample sizes for families used in the genetic analyses are provided in Tables 1 and 3. Size of populations was graphy-paper wicks (Whatman 3 mm), and placed estimated as the number of spatially distinct clumps of directly onto 11–12% starch gels. Enzymes were resolved ramets. The six populations were distributed among four on two buffer systems: aspartate amino transferase, distinct river valleys encompassing two main water- alcohol dehydrogenase, adeline kinase (AK), fructose- sheds. The Cuevas Bermejas (CB) population is isolated bisphosphate aldolase, diaphorase, glutamate dehydro- from the remaining populations and is located on a genase, cytosol aminopeptidase, menadione reductase and separate watershed (Guadalquivir River). The remaining triose phosphate isomerase, on lithium borate (pH 8.3); five populations are distributed among three river and aconitase, acid phosphotase (ACP), creatine kinase, valleys within a single watershed (Guadalentı´n River). fumarate hydratase, glycerol-3-phosphare dehydrogenase, Most populations were small but populations Gu-C and isocitric dehydrogenase, malate dehydrogenase, per- Gu-D were two distinct nuclei, about 200 m apart, oxidase (PER), phosphoglucose dehydrogenase (6PGD), located on opposite banks of the Gualay River, and phosphoglucose isomerase, shikimic dehydrogenase on belonged to a large and more or less continuous histidine citrate (pH 6.5). Gels were stained