Evolution, 57(7), 2003, pp. 1520±1534 COMPONENTS OF REPRODUCTIVE ISOLATION BETWEEN THE MONKEYFLOWERS MIMULUS LEWISII AND M. CARDINALIS (PHRYMACEAE) JUSTIN RAMSEY,1,2,3 H. D. BRADSHAW,JR.,1,4 AND DOUGLAS W. SCHEMSKE1,5 1Biology Department, Box 355325, University of Washington, Seattle, Washington 98195 2E-mail: [email protected] 4E-mail: [email protected] Abstract. Evolutionists have long recognized the role of reproductive isolation in speciation, but the relative con- tributions of different reproductive barriers are poorly understood. We examined the nature of isolation between Mimulus lewisii and M. cardinalis, sister species of monkey¯owers. Studied reproductive barriers include: ecogeo- graphic isolation; pollinator isolation (pollinator ®delity in a natural mixed population); pollen competition (seed set and hybrid production from experimental interspeci®c, intraspeci®c, and mixed pollinations in the greenhouse); and relative hybrid ®tness (germination, survivorship, percent ¯owering, biomass, pollen viability, and seed mass in the greenhouse). Additionally, the rate of hybridization in nature was estimated from seed collections in a sympatric population. We found substantial reproductive barriers at multiple stages in the life history of M. lewisii and M. cardinalis. Using range maps constructed from herbarium collections, we estimated that the different ecogeographic distributions of the species result in 58.7% reproductive isolation. Mimulus lewisii and M. cardinalis are visited by different pollinators, and in a region of sympatry 97.6% of pollinator foraging bouts were speci®c to one species or the other. In the greenhouse, interspeci®c pollinations generated nearly 50% fewer seeds than intraspeci®c controls. Mixed pollinations of M. cardinalis ¯owers yielded .75% parentals even when only one-quarter of the pollen treatment consisted of M. cardinalis pollen. In contrast, both species had similar siring success on M. lewisii ¯owers. The observed 99.915% occurrence of parental M. lewisii and M. cardinalis in seeds collected from a sympatric population is nearly identical to that expected, based upon our ®eld observations of pollinator behavior and our laboratory experiments of pollen competition. F1 hybrids exhibited reduced germination rates, high survivorship and reproduction, and low pollen and ovule fertility. In aggregate, the studied reproductive barriers prevent, on average, 99.87% of gene ¯ow, with most reproductive isolation occurring prior to hybrid formation. Our results suggest that ecological factors resulting from adaptive divergence are the primary isolating barriers in this system. Additional studies of taxa at varying degrees of evolutionary divergence are needed to identify the relative importance of pre- and postzygotic isolating mechanisms in speciation. Key words. Ecological isolation, hybridization, Mimulus, pollen competition, pollinator isolation, reproductive iso- lation, speciation. Received August 16, 2001. Accepted January 27, 2003. Biologists disagree on the conditions that are necessary behavioral differences between species and postzygotic bar- and suf®cient to delimit related taxa as different species. It riers of hybrid inviability, hybrid sterility, and F2 breakdown has been suggested, for example, that species boundaries (Dobzhansky 1937; Mayr 1942). should be established by the existence of reproductive bar- A variety of reproductive barriers contribute to total iso- riers (biological species concept; Coyne et al. 1988), the na- lation in most taxa (Dobzhansky 1937; Mayr 1947, 1963; ture of phylogenetic relationships between taxa (phylogenetic Coyne 1992; Schluter 2001; Price and Bouvier 2002). Mayr species concept; Nixon and Wheeler 1990), or trait differ- (1947) speculated that ecological isolation, sexual differenc- ences that are consistent and easy to observe (taxonomic es, and low hybrid ®tness contribute to the isolation of many species concept; Cronquist 1978). In spite of these arguments, species pairs, yet studies of isolating mechanisms generally most evolutionists agree that reproductive isolation plays a target one or a few barriers to gene ¯ow without reference key role in the formation and maintenance of species in na- to other components of isolation. For example, intrinsic post- ture. Dobzhansky (1937) identi®ed a number of factors that zygotic barriers have been the subject of considerable atten- function to limit gene ¯ow between related taxa. In general, tion because of their ease of study in the laboratory, but it traits conferring reproductive isolation are thought to evolve is not known if these reproductive barriers evolve before or in allopatry by conventional processes of drift and selec- after speciation is complete (Schemske 2000). By contrast, tionÐtheir function in speciation is incidental. In some cases, ecogeographic isolation is rarely included as a component of however, prezygotic barriers may evolve speci®cally to pre- reproductive isolation, yet genetically based differences in vent the formation of un®t hybrids (reinforcement; Dob- habitat preference are well known (Clausen et al. 1940) and zhansky 1937; Noor 1997). Reproductive barriers are clas- may often reduce opportunities for hybrid formation. si®ed according to their timing in the life history, and include The relative contribution of pre- and postzygotic barriers prezygotic mechanisms such as ecogeographic, temporal, and is unknown, as is the degree to which diverse types of pre- zygotic barriers function to isolate species (Coyne and Orr 3 Present address: Department of Botany, University of Guelph, 1998; Schemske 2000). Here we estimate stage-speci®c and Guelph, Ontario, N1G 2W1 Canada; E-mail: [email protected]. cumulative contributions of different reproductive barriers 5 Present address: Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824, and W. K. Kellogg Bi- between Mimulus lewisii and M. cardinalis (Phrymaceae; ological Station 3700 E. Gull Lake Drive, Hickory Corners, Mich- Beardsley and Olmstead 2002), sister species of monkey- igan 49060-9516; E-mail: [email protected]. ¯owers (Beardsley et al. 2003). In sequential order of their 1520 q 2003 The Society for the Study of Evolution. All rights reserved. REPRODUCTIVE ISOLATION IN MIMULUS 1521 life-history stages, we calculated the degree of reproductive cent phylogenetic analyses indicate that the genus Mimulus isolation between M. lewisii and M. cardinalis caused by should be included in a new family, the Phrymaceae. This ecogeographic isolation, pollinator ®delity, pollen competi- family is named after the monotypic genus Phryma (from tion, and F1 hybrid ®tness (seed germination, seedling sur- eastern North America) and in addition to Mimulus includes vival, adult reproduction, and fertility). We then combined six genera (Leucocarpus, Hemichaena, Berendtiella, Glos- these stage-speci®c measures following the methods pro- sostigma, Peplidium and Elacholoma) that are in the same posed by Coyne and Orr (1989) to estimate total reproductive major clade as Mimulus (Beardsley and Olmstead 2002). The isolation and the relative contribution of the studied barriers traditional placement of M. cardinalis and M. lewisii in sec- to total isolation. tion Erythranthe is well supported by the molecular analyses. This approach provides a quantitative assessment of the current barriers to gene ¯ow between populations and thus Ecogeographic Isolation motivates studies of the genetic basis of the primary isolating We determined the elevational and geographic distribution barriers in these species (Schemske and Bradshaw 1999). In of M. lewisii and M. cardinalis in California using herbarium addition, the estimated total reproductive isolation between specimens. Elevation data were obtained from 104 M. lewisii taxa provides a direct test of Mayr's biological species con- and 100 M. cardinalis collections, and 57 M. lewisii and 132 cept (Mayr 1942). The biological species concept has been M. cardinalis specimens were used for examining two-di- widely criticized by botanists (Mishler and Donoghue 1982; mensional (latitude, longitude) spatial distributions. No du- Raven 1986), yet to our knowledge no study has evaluated plicate specimens (individuals of the same species collected the key criterion of total reproductive isolation as would be at the same site) were included. Collection information was required to assess whether the biological species concept can used to determine the elevation, latitude, and longitude of be empirically applied in natural populations. A test of the the sampled populations. We compared the average elevation biological species concept is of particular interest in M. lew- of the species using a Mann-Whitney U-test, and calculated isii and M. cardinalis because Hiesey et al. (1971, p. 24) the degree of overlap in the species' elevational range. considered these taxa as ``a single biological species'' based We performed computer simulations to estimate the degree on the ease with which fertile F hybrids can be produced in 1 of ecogeographic isolation between M. lewisii and M. car- the laboratory. dinalis. For each iteration of the simulation, 100,000 virtual quadrats were assigned randomly over the combined geo- MATERIALS AND METHODS graphic distribution of the species. Within each quadrat the Mimulus lewisii and M. cardinalis are rhizomatous peren- simulations determined whether one or more M. lewisii and nial herbs found in moist seep, stream, and river habitats in one or more