American Journal of Botany 97(3): 412–422. 2010.

G ENETIC FACTORS ASSOCIATED WITH MATING SYSTEM CAUSE A PARTIAL REPRODUCTIVE BARRIER BETWEEN TWO PARAPATRIC SPECIES OF () 1

Vanessa A. Koelling2 and Rodney Mauricio

Department of Genetics, University of , Athens, Georgia 30602-7223 USA

Reproductive barriers play a major role in the origin and maintenance of biodiversity by restricting gene fl ow between species. Although both pre- and postzygotic barriers often isolate species, prezygotic barriers are thought to contribute more to reproduc- tive isolation. We investigated possible reproductive barriers between Leavenworthia alabamica and L. crassa, parapatric species with high morphological and ecological similarity and the ability to hybridize. Using greenhouse and fi eld experiments, we tested for habitat isolation and genetic incompatibilities. From controlled crosses, we identifi ed unilateral incompatibility (a partial pr- ezygotic barrier associated with the self-incompatibility system), but no evidence of other genetic incompatibilities. We found a

small reduction in pollen viability of F 1 hybrids and early germination of F1 , F 2, and BC hybrids relative to L. alabamica and L. crassa in a common garden experiment, but the effect on fi tness was not tested. Field studies of hybrid pollen viability and germi- nation are needed to determine if they contribute to reproductive isolation. In a reciprocal transplant, we found no evidence of habitat isolation or reduced hybrid survival (from seedling to adult stage) or reproduction. These data suggest unilateral incompat- ibility partially reproductively isolates L. alabamica and L. crassa , but no other reproductive barriers could be detected.

Key words: Brassicaceae; genetic incompatibilities; hybrid fi tness; habitat isolation; Leavenworthia alabamica ; Leavenwor- thia crassa ; reproductive barriers; unilateral incompatibility.

Barriers to reproduction are key in the origin and mainte- cedar glade endemics that have been studied by evolutionary nance of biodiversity. Reproductive barriers limit gene fl ow biologists since the 1960s. These sister taxa look very similar between populations, allowing them to diverge into new spe- (Rollins, 1963), live in the same unique habitat type, overlap in cies or maintain species divergence upon secondary contact range but do not co-occur in the same sites, have the same chro- ( Dobzhansky, 1937 ; Mayr, 1942 ). Many types of reproductive mosome number ( Baldwin, 1945 ), are easily crossed in the barriers are possible (reviewed in Coyne and Orr, 2004 ), but greenhouse, and appear to have experienced the same adaptive they are generally classifi ed into two groups: those that occur pressures with respect to mating system ( Lloyd, 1965 ). Possible prior to fertilization (prezygotic) and those that occur after fer- hybrids have been found in the wild in areas of ecological dis- tilization (postzygotic). Some empirical studies have found turbance (Rollins, 1963), yet L. alabamica and L. crassa differ both pre- and postzygotic barriers isolate recently diverged spe- markedly in a diagnostic trait of this family: fruit morphology cies or ecotypes, but prezygotic barriers seem to be more im- (Appendix S1, see Supplemental Data with the online version portant to total reproductive isolation (e.g., Husband and Sabara, of this article; Rollins 1963, 1993 ). They can also be differenti- 2003; Ramsey et al., 2003; Kay, 2006; Martin and Willis, 2007; ated at neutral markers ( Beck et al., 2006 ). What, then, repro- Nosil, 2007; Lowry et al., 2008). However, studies of a diverse ductively isolates these species? array of species are needed to evaluate the generality of this One possible prezygotic barrier between these species is hab- fi nding. itat isolation, which develops when individuals are unable to To investigate the relative contributions of pre- and postzy- grow or reproduce in the same habitat due to adaptation to eco- gotic barriers to reproductive isolation, we focused our work on logical conditions (e.g., Clausen et al., 1940 ; Feder and Bush, Leavenworthia alabamica and L. crassa (Brassicaceae), parapatric 1989). Although the glade sites in which L. alabamica and L. crassa grow look superfi cially identical, these species may be adapted to different, cryptic glade habitats. To our knowledge, 1 Manuscript received 26 June 2009; revision accepted 27 December 2009. The authors thank J. Busch, E. Kuntz, J. Colvard, R. Baucom, J. Ross- no studies of habitat differentiation between these species have Ibarra, M. Poelchau, J. Sterling, T. Haselkorn, J. Estill, S. Scott, L. Mitchell, ever been conducted. M. Brown, J. Wolf, E. Hedgepeth, S. Brown, J. Sexton, and N. Bowman for Another possibility is that environment-independent or de- invaluable fi eld and greenhouse assistance; the Bramlett family and The pendent genetic incompatibilities are postzygotic barriers be- Nature Conservancy of for access to do fi eldwork; and G. tween these species. Environment-independent genetic Schneider for mapmaking assistance. They also thank R. Baucom, T. incompatibilities result from chromosomal (Rieseberg, 2001; Marriage, S. Bodbyl, J. Mojica, J. Kelly, and two anonymous reviewers for Brown et al., 2004 ), genic ( Dobzhansky, 1936 ; Muller, 1939 ), improvements to the manuscript. A Ruth L. Kirschstein National Research or cytonuclear (Michaelis, 1954; Levin, 2003) interactions, and Service Award from the National Institutes of Health to V.A.K. supported manifest as hybrid inviability or sterility when taxa are crossed this research. 2 Author for correspondence (e-mail: [email protected]); present ( Stebbins, 1958 ). Chromosomal or genic incompatibilities act address: Department of Ecology and Evolutionary Biology, University of irrespectively of the direction of the cross (i.e., the identity of Kansas, 1200 Sunnyside Ave., Lawrence, KS 66045 USA the mother or father). Cytonuclear incompatibilities, on the other hand, often result in unidirectional cross success (re- doi:10.3732/ajb.0900184 viewed in Grant, 1975 and Tiffi n et al., 2001 ). In addition,

American Journal of Botany 97(3): 412–422, 2010; http://www.amjbot.org/ © 2010 Botanical Society of America 412 March 2010] Koelling and Mauricio — Reproductive barriers in L EAVENWORTHIA 413 hybrid fi tness may be reduced only in particular environments constraints, all other were germinated in the greenhouse during the fall or due to genotype by environment interactions (i.e., environment- spring when greenhouse temperatures averaged 23 ° C. Plants were watered daily dependent genetic incompatibilities; e.g., Wang et al., 1997; and fertilized weekly with Peter ’ s 20-10-20 Peat Lite Special at ~250 ppm. Generation 0 consisted of nine L. alabamica and nine L. crassa plants (three Hatfi eld and Schluter, 1999 ). Rollins (1963) produced fi rst and populations from each species, three individuals from each population) crossed second-generation hybrids of L. alabamica and L. crassa in the to produce F 1 hybrids and intraspecifi c F 1’ s (Fig. 2). In Generation 0 (Year 1), greenhouse, but did not analyze their morphology or test their no between-population, within-species crosses were made, and crosses were fi tness in the fi eld, leaving open the question of whether genetic replicated four times, but in Generation 0 (Year 2), all possible crosses were incompatibilities are present. made, and crosses were replicated fi ve times. Generation 1 crosses consisted of In addition, within L. alabamica and L. crassa, some popula- 18 F 1 hybrids crossed to produce F2 hybrids, and 12 F 1 plants (six F 1 hybrids and six intraspecifi c F ’ s) crossed to produce BC hybrids. Because these are tions are self-incompatible (SI) and others are self-compatible 1 annual species, BCs could not be made with parent plants, so F1 ’ s derived from (SC) (Lloyd, 1965) allowing us to test for a postmating, prezy- intraspecifi c crosses were used. All possible Generation 1 crosses were made gotic barrier: unilateral incompatibility (UI). UI is a unidirec- and replicated fi ve times. tional crossing barrier found only in plants with genetic SI To test for unilateral incompatibility (UI), each used in crosses was systems ( Lewis and Crowe, 1958 ). UI occurs when a plant that is selfed to determine if it was SI or SC. Plants designated as SI were those that SI cannot successfully pollinate ovules of a SC plant; conversely, did not set seed (because the sporophytic incompatibility system can be “ leaky ” , we sometimes, but rarely, observed seeds in plants designated as SI; no plants the reciprocal cross is successful. UI has been documented be- designated as SI ever had more than 2 seeds on a plant). We had an approximate tween species and suggested as a reproductive barrier between idea of which plants in Generation 0 would be SI or SC based on their popula- them ( Harrison and Darby, 1955 ; Grun and Radlow, 1961 ). But tion of origin, but did not control the absolute number of SI and SC individuals its documented presence within some species (Martin, 1963; in the experiment. We also had no information about the genotypes of our Lloyd, 1968; Pandey, 1981), including Leavenworthia , has left plants at the S-locus, so some SI × SI crosses likely failed as a result of the SI the status of UI as a reproductive barrier between species unclear. reaction. Before crossing plants, we used a pair of fi ne forceps to open buds and re- We took the opportunity to revisit this question here. move all six undehisced anthers 1 d before anthesis to prevent self-fertilization. In the current study, our goal was to identify reproductive Crosses were made on the fi rst day of anthesis by brushing a pollen donor ’ s barriers between parapatric L. alabamica and L. crassa using a anther on the stigma of the pollen recipient until covered with pollen. Crosses combination of greenhouse and fi eld experiments. We con- were marked with tape wrapped around the fl ower pedicel. Fruits were allowed ducted crosses in the greenhouse to determine if environment- to mature, and then seeds per fruit counted. independent genetic incompatibilities were present between L. In Generation 0 (Year 1), pollen quality was determined visually (only well- developed and dehiscent anthers were used in crosses). For all subsequent gen- alabamica and L. crassa. Hybrids were then measured in a erations, we quantifi ed the percentage viable pollen using Alexander ’ s stain greenhouse common garden to investigate the possibility they (Kearns and Inouye, 1993), which stains viable pollen grains red and inviable would exhibit phenotypes that could reduce their fi tness rela- grains green. We brushed one anther per fl ower (three total) gently on a glass tive to the parent species. Finally, we used a reciprocal trans- slide to remove pollen. Stain (1 – 2 drops) was then placed on the slide and plant experiment to address the following questions: (1) Is heated a few seconds to fi x it. We sampled 300 pollen grains per slide. habitat isolation present between species? and (2) Do hybrids We also determined the mean number of ovules per plant (average of three fl owers). Pistils were placed between two glass slides, then gentle pressure was exhibit reduced fi tness in the fi eld? We then discuss these data applied with the top slide until the ovules were visible under a dissecting scope with regards to reproductive isolation between these species. as dark spots along each half of the ovary. Using this method, we counted ovules without dissecting them from the pistil. Leavenworthia alabamica pro- duced, on average, 12 ovules, whereas L. crassa produced eight (V. Koelling, unpublished data). MATERIALS AND METHODS We performed mixed-model ANOVAs in the program JMP 6.0 (SAS Insti- tute, 2005) to test for differential success of (1) hybrid v. within-species crosses, Study species— Leavenworthia alabamica and L. crassa are sister species (2) crosses mismatched at plastid and nuclear genomes, and (3) crosses differ- (Beck et al., 2006) with a haploid chromosome number of 11 (Baldwin, 1945) ing in SI status for each generation in each year. Maternal plant was included as that contain both self-incompatible (SI) and self-compatible (SC) populations a random effect. All other effects tested were fi xed, including year. Year was ( Rollins, 1963 ; Lloyd, 1965 , 1967 ). Compared to SI populations, SC popula- treated as a fi xed effect because plants were grown during different seasons tions have small fl owers, reduced stigma– anther distances, anthers oriented to between years. In all analyses, model fi tting was done using restricted maxi- dehisce toward the stigma, small pollen to ovule ratios, and little or no fl oral mum likelihood with unbounded variance. Degrees of freedom were calculated scent ( Lloyd, 1965 ). They are pollinated by generalist, solitary bee species using the Kenward– Roger method (Littell et al., 2006). A signifi cant maternal (Lloyd, 1965) and are winter annuals endemic to limestone glades of the plant effect was tested by whether the 95% confi dence interval of the variance Moulton and Valleys of Alabama ( Rollins, 1963 ). Limestone glades component contained zero. A Bonferroni correction was used to control for are characterized by thin soil over a limestone base ( Baskin et al., 1995 ). Seeds type I error. The response variable was the arcsine – square root transformation of L. alabamica and L. crassa germinate from September to mid- to late-Octo- of the mean proportion seed set per cross per plant (i.e., the mean number of ber, and individuals fl ower and fruit in the spring (March-early May). During seeds produced by each plant for each cross divided by the mean number of the winter and spring, glade soils are often saturated with water; Leavenworthia ovules produced by that plant). This accounted for the difference in ovule pro- may be adapted to grow in such anaerobic conditions (Baskin and Baskin, duction between the two species. 1976 ). Seeds are dormant through the hot, dry summer months ( Caudle and Baskin, 1968 ; Baskin and Baskin, 1971 ). Comparing fl oral morphology, fl owering time, and germination of hybrids and parent species— Greenhouse common garden— To determine if hybrids Testing for environment-independent genetic incompatibilities— Green- displayed phenotypes that could reduce their fi tness in the fi eld compared to L. house crosses— To assess the ability of L. alabamica and L. crassa to hybrid- alabamica and L. crassa , fl oral traits were measured on plants from each gen- ize, we crossed plants grown from seed collected from natural populations eration in each year ( Fig. 2 ). Three fl owers were measured per plant within its ( Table 1 ; Fig. 1 ) and then crossed progeny from those initial crosses. Each in- fi rst week of fl owering. The following numbers of each plant type were mea- dividual used in crosses had a different maternal parent. This experiment was sured: 18 L. alabamica , 18 L. crassa , 98 F1 ’ s, 152 F2 ’ s, and 63 BC hybrids. performed in 2004 (Year 1) and replicated in 2005 (Year 2). We measured petal width, petal length, corolla tube length, long and short Seeds were planted in Fafard (Syngenta Group, Agawam, Massachusetts, fi lament length, pistil length, ovule number, the percentage viable pollen, and USA) potting soil mixed with lime (approximately 128 g lime per bag), and anther orientation. Because fl owers of Leavenworthia have four long and two rosettes transplanted into 15 cm pots. Generation 0 (Year 1) was germinated in short stamens ( Rollins, 1993 ), we measured long and short fi laments to calcu- an 18 ° C growth chamber (14 h daylength). Due to slow germination and space late anther exsertion from the corolla (long or short fi lament length – corolla tube 414 American Journal of Botany [Vol. 97

Table 1. Populations of Leavenworthia alabamica and L. crassa used in this study. The name, number, self-incompatibility (SI) status, latitude, longitude, and use of each population are listed.

Species Population Pop. no. SI Status Latitude (N) Longitude (W) Used in

L. alabamica Isbell 1 SI 34.45725 − 87.75298 Reciprocal transplant Tuscumbia 2 SC 34.70833 − 87.83132 Crosses Hatton 3 SI 34.50993 − 87.44204 Reciprocal transplant Winchell 4 SI 34.47049 − 87.57300 Crosses Landersville 5 SI 34.44783 − 87.39458 Crosses CPCC 6 SC 34.36453 − 86.99656 Reciprocal transplant Prairie Grove Glades 7 SI 34.51778 − 87.50533 Reciprocal transplant and site L. crassa CR 203 8 SI 34.40858 − 87.14747 Crosses Quarry 9 SC 34.34869 − 86.98722 Crosses, Reciprocal transplant Bramlett 10 SC 34.35055 − 86.99978 Reciprocal transplant and site NCP 11 SI 34.37742 − 86.99978 Crosses, Reciprocal transplant

length) and anther– stigma distance (long or short fi lament length – pistil length). correlation matrix. Using the “ eigenvalues greater than one” rule (Quinn and Because short fi laments were always much shorter than pistils, such that short Keough, 2002), two principal components were rotated using the varimax anthers were never near the stigmas, short anther – stigma distance was excluded method to simplify the structure of the data. The data on pollen viability were from analyses. not included in the PCA due to missing values. All lengths were measured to the nearest 0.01 mm using an ocular ruler on a We used ANOVA to determine whether plant types differed in pollen via- dissecting microscope. At anthesis, fl owers were dissected with a pair of fi ne bility, the log-transformed number of days to germination, and the number of forceps, and fl ower parts were pressed between two glass slides. One petal and days to fi rst fl ower. We also checked whether germination and fl owering time one long and short fi lament were measured. See “ Greenhouse Crosses” for were correlated during each year. The correlations were 0.5757 and 0.2464 for ovule and pollen viability count methods. Anther orientation (introrse, turned years one and two respectively. Due to the fact that even moderately correlated toward or extrorse, turned away from the stigma) was scored visually prior to variables diminish the power of MANOVA (Tabachnick and Fidell, 2001), we fl ower dissection. We also recorded the date of germination and fi rst fl ower of analyzed germination and fl owering time separately. Because germination con- each plant. ditions differed between years, each year was analyzed separately. Mean com- Before analyzing fl oral trait differences between L. alabamica , L. crassa , parisons were made using Tukey ’ s honestly signifi cant difference (HSD) test. and hybrids, we determined trait correlations. We calculated Pearson ’ s correla- All analyses were done in JMP 6.0 ( SAS Institute, 2005 ). tion coeffi cient for all possible correlations between petal width, petal length, corolla tube length, pistil length, ovule number, anther exsertion, and anther- Testing for habitat isolation and environment-dependent genetic incom- stigma distance. As a categorical variable, anther orientation was not included patibilities— Reciprocal transplant— We conducted a reciprocal transplant us- in the correlation analysis. We found petal length was highly correlated with ing L. alabamica , L. crassa, and F1 , F2 , and BC hybrids to test for habitat petal width ( ρ = 0.8251, P < 0.0001) and pistil length ( ρ = 0.8373, P < 0.0001). isolation and to assess hybrid fi tness in the fi eld. Seeds were planted on 7 Octo- Due to these high correlations, petal length was not used in subsequent ber 2006 in a randomized block design ( Cochran and Cox, 1992 ), with two analyses. blocks per site. Germination began in the University of Georgia greenhouse We used principal components analysis (PCA) to determine if L. alabamica (see Greenhouse crosses for growth conditions) on 11 October 2006. Some and L. crassa fl owers differed from those of hybrids. To describe where indi- additional seed was planted on 27 October 2006 to replace seeds that did not vidual plants fi t in multidimensional space with respect to petal width, corolla germinate. Seeds came from two sources: (1) fi eld collections of four L. ala- tube length, pistil length, ovule number, anther exsertion, anther– stigma dis- bamica and three L. crassa populations, and (2) crosses (Table 1; see Green- tance, and anther orientation, principal components were calculated using a house crosses for a description of crosses). Plants were transplanted to two fi eld

Fig. 1. Location of Leavenworthia alabamica (black) and L. crassa (white) populations used in this study. Inset map shows their location within Ala- bama. Triangles denote the reciprocal transplant sites. Populations 2 and 6 are at either end of the range of L. alabamica , whereas 8 and 9 are at either end of L. crassa ’ s range. March 2010] Koelling and Mauricio — Reproductive barriers in L EAVENWORTHIA 415

Fig. 2. Crossing design. Leavenworthia alabamica and L. crassa (Generation 0) were grown in the greenhouse from fi eld-collected seed and crossed to produce intraspecifi c F1 ’ s and F1 hybrids (Generation 1). F1 hybrids were then crossed to produce F2 hybrids, and crossed to intraspecifi c F 1 plants to produce backcross (BC) hybrids (Generation 2). The experiment was replicated in 2 years. Number of individuals crossed is shown in parentheses below the plant type. The sample size (N ) of each cross type is shown for each generation. See Materials and Methods for details on crosses. sites on 5 and 6 November 2006. Reciprocal transplant sites were typical glades carbon to nitrogen ratio was also measured. We used t tests in JMP 6.0 (SAS centrally located within each species ’ range ( Fig. 1 ). Institute, 2005 ) to analyze differences between sites for each nutrient measure. Rosette diameter was measured on the day of planting so that each plant ’ s initial size was known. To reduce transplantation shock, we covered each block with a spun-bonded, polyester material and hand-watered the blocks. After 1 month, the polyester was removed, and plants were allowed to grow uncovered RESULTS for the remainder of the experiment. Plants were hand-watered at each site three times (twice in late March and once in early April). Plant mortality was mea- Environment-independent genetic incompatibilities— sured throughout the experiment but was very low. In mid-April, each senes- Crossing experiment— Because the crossing experiment was cent plant was collected before silique dehiscence. We counted the number of replicated in 2 years, in each analysis we tested for differences fl owers (from pedicels remaining on the plant), fruits, and seeds produced per between years and for an interaction between year and the other plant. We used PROC MIXED in the program SAS 9.1.3 ( SAS Institute, 2006 ) to main effects. Only signifi cant effects are reported in the text. perform a mixed model nested analysis of covariance to determine whether See Table 2 for all ANOVA results and Appendix S2 (see Sup- fl ower, fruit, and seed set was greater for each species in its home site relative plemental Data with the online version of this article) for all to its nonhome site and to compare hybrids and parents. Due to the differing untransformed means and sample sizes. maternal environments of plants derived from natural populations compared to To test for genic incompatibilities between L. alabamica and those from crosses, the two groups were analyzed separately. Effects tested in L. crassa , we compared the average seed set of hybrid vs. pure the analysis of fi eld-derived plants were site, block within site, species, species by site, and block by species within site. Effects tested in the analysis of cross- species crosses. Absence of genic incompatibility between L. derived plants were site, block within site, plant type, plant type by site, and alabamica and L. crassa would be consistent with equal seed block by plant type within site. Block within site and block by species/plant production following crosses within and between species. For type within site were random effects. All other effects tested were fi xed. Model Generation 0, we found no signifi cant difference in the propor- fi tting was done using restricted maximum likelihood with unbounded vari- tion of within-species and F1 hybrid seed set (online Appendix ance. The signifi cance of random effects was tested using a likelihood ratio test. S3). The same analysis was performed on Generation 1, where Degrees of freedom were calculated using the Kenward – Roger method ( Littell et al., 2006 ). Response variables were square root transformations of fl ower, a signifi cant year effect was detected (F 1, 835 = 203.597, P < fruit, and seed number. All analyses included plant size (initial rosette diame- 0.0001), but not a signifi cant effect of seed type (online Appen- ter) as a covariate. dix S3), or their interaction. These data indicate that L. ala- Finally, to investigate potential differences between the transplant sites in bamica , L. crassa , and F 1 plants produced pure species and essential plant nutrients, we obtained 10 soil samples from each transplant site hybrid seed equally well. and sent them to the Stable Isotope Laboratory at the University of Georgia for We tested for the presence of cytonuclear incompatibility be- element analysis. Mass spectrometry was used to analyze soil samples for the following elements (all measured as percentage by mass unless otherwise tween L. alabamica (A) and L. crassa (C) based on whether noted): calcium, phosphorus, potassium, magnesium, sodium, nitrogen (µ g cross success differed between individuals with plastid and nu- NO3 -N and NH4 -N per g dry soil, and total %N), and carbon (total %). The clear genomes from the same species (A × A, C × C) and those 416 American Journal of Botany [Vol. 97

Table 2. Tests for three types of genetic incompatibility: genic, 7.388, P < 0.0001). In addition, crosses between hybrids dif- cytonuclear, and unilateral. Results of mixed model ANOVAs on the fered signifi cantly between years (F 1, 835.6 = 616.114, P < arcsine-square root transformed mean proportion seed set of crosses of 0.0001), compatibility type (F 3,840.8 = 65.635, P < 0.0001; Fig. Leavenworthia alabamica , L. crassa, and hybrids with a Bonferroni- 3C ), and their interaction (F = 13.637, P < 0.0001). Con- corrected tablewide α = 0.0167. Maternal plant was included in each 3, 830.6 analysis as a random effect. trasting SC × SI to SI × SC crosses, we found a signifi cant dif- ference (t = 10.661, df = 1, P < 0.0001). Contrasts comparing Analysis Source of variation Num, dem dfF ratio P -value SC x SC to SC x SI and SI × SC to SI × SI crosses were not signifi cant, and the same result was true when each year was Genic incompatibility tested separately. In all analyses, SC × SC crosses set signifi - Generation 0 Year 1, 519 0.529 0.4675 Seed type 1, 519 0.333 0.5642 cantly more seed than SI × SI crosses. Fewer SI × SI crosses set Seed type × year 1, 519 2.126 0.1454 seed due to the operation of the SI system, which we could not Generation 1 Year 1, 835 203.597 < 0.0001 control because we did not know the S -alleles of each SI plant. Seed type 3, 667.9 0.384 0.7649 These data indicate UI occurs between species and hybrids, but Seed type × year 3, 828.1 1.671 0.1718 not within species, and may be a partial reproductive barrier Cytonuclear incompatibility between L. alabamica and L. crassa . Generation 0 Year 1, 516 0.536 0.4644 Cross type 3, 516 1.622 0.1832 Cross type × year 3, 73.3 1.866 0.1429 Floral morphology, fl owering time, and germination of Generation 1 Year 1, 832.6 782.944 < 0.0001 hybrids and parent species— Common garden— Of more than Cross type 3, 835 2.570 0.0531 300 hybrids, none had fl owers that were extreme in size, shape, Cross type × year 3, 631.7 3.087 0.0267 or placement of reproductive parts relative to L. alabamica and Unilateral incompatibility L. crassa . We obtained seven principal components, two of Within-species Year 1, 198.7 0.060 0.8065 which had eigenvalues greater than one and explained 61.5% of (Generation 0) the trait variance. We found L. alabamica and L. crassa did not Compatibility type 3, 187.9 17.548 < 0.0001 Compatibility 3, 202.1 0.752 0.5226 differ along the principal component axes (Fig. 4A), and the F1 , type × year F2 , and BC hybrids also fell within the same range of values Between-species Year 1, 302.2 1.190 0.2761 ( Fig. 4B ). (Generation 0) We found a signifi cant difference between plant types in pol- Compatibility type 3, 299.6 11.741 < 0.0001 len viability (F 4, 230 = 6.775, P < 0.0001), with F 1 hybrids hav- Compatibility 3, 306.4 7.388 < 0.0001 ing the lowest percentage viable pollen, but not differing type × year Generation 1 Year 1, 835.6 616.113 < 0.0001 signifi cantly from L. crassa (Table 3 ). We also found that par- Compatibility type 3, 840.8 65.635 < 0.0001 ents and hybrids differed signifi cantly in years 1 and 2 for both Compatibility 3, 830.6 13.637 < 0.0001 log days to germination (year 1: F 4, 153 = 61.965, P < 0.0001; type × year year 2: F 4, 178 = 26.269, P < 0.0001) and days to fi rst fl ower (year 1: F 4, 154 = 9.251, P < 0.0001; year 2: F 4, 182 = 7.256, P < 0.0001). Figure 5A and Table 4 show that hybrids germinated with plastid genomes from one species and a portion of the nu- more quickly than the parents in both years, although the differ- clear genome from the other (A × C, C × A). If no cytonuclear ence was smaller in year 2. In year 1, hybrids also fl owered incompatibility is present, then the average amount of seed set earlier than L. alabamica and L. crassa , but there was no clear from these cross types will not differ. In Generation 0, we found pattern in year 2 (Fig. 5B; Table 4). Thus, hybrids germinate, no signifi cant differences between cross types in seed set (on- but do not necessarily fl ower more quickly than their parents. line Appendix S4). A contrast comparing A × C to C × A was not signifi cant. For Generation 1, seed set differed signifi cantly Habitat isolation and environment-dependent genetic in- between years (F 1, 832.6 = 782.944, P < 0.0001), but we found no compatibilities— Reciprocal transplant — We found mean signifi cant difference between cross types (Appendix S4). A fl ower number differed signifi cantly between the species (F 1, contrast comparing A × C to C × A was not signifi cant when 2.577 = 58.5709, P = 0.0077; L. alabamica: 11.10 ± 0.50 fl owers; pooled across years or when years were tested separately. These L. crassa : 9.22 ± 0.47 fl owers) and blocks within sites ( χ 2 = data indicate that L. alabamica , L. crassa, and F 1 plants set seed 313.6, P < 0.0001), but not between sites, species by sites, or equally well if the cross was between matched (A × A, C × C) species by block within sites. We found no signifi cant differ- or opposed (A × C, C × A) plastid and nuclear genomes, and ence in fruit or seed set between sites, species, species by sites, thus there is no evidence of a cytonuclear incompatibility. or block by species within sites (see Fig. 6A for species by site In addition, we tested for UI in crosses within species, be- seed set; Table 5 ; online Appendix S5). In addition, blocks tween species, and between hybrids. For crosses within species, within sites differed signifi cantly in fruit and seed set, and there we found a signifi cant difference in seed set between compati- was a signifi cant effect of plant size on fl owers (F 1, 607.5 = bility types (F 3, 187.9 = 17.548, P < 0.0001; Fig. 3A ). Contrasts 509.4378, P < 0.0001), fruits ( F1, 606.3 = 293.1328, P < 0.0001), comparing SC × SI to SI × SC and SI × SC to SI × SI crosses and seeds (F 1, 606.4 = 283.2786, P < 0.0001) due to the tendency found no signifi cant difference, but SC × SC and SC × SI crosses of large plants to set more than small plants. These data indicate differed signifi cantly ( t = 2.859, df = 1, P = 0.0047). For crosses L. alabamica and L. crassa set equivalent amounts of fruit and between species, we found a signifi cant effect of compatibility seed irrespective of the site in which they grew, but fruit and type ( F3, 299.6 = 11.741, P < 0.0001; Fig. 3B). A contrast com- seed set differed within a site. However, within-site differences paring SC × SI to SI × SC crosses was signifi cant ( t = 5.204, were not species-specifi c. df = 1, P < 0.0001). Contrasts comparing SC × SC to SC × SI and We also tested the fi tness of over 250 hybrids in the fi eld, and SI × SC to SI × SI crosses were not signifi cant. There was also found no signifi cant difference between sites, plant type, or a signifi cant compatibility type by year interaction ( F 3, 306.4 = plant type by sites in fl ower, fruit, or seed set (see Fig. 6B for March 2010] Koelling and Mauricio — Reproductive barriers in L EAVENWORTHIA 417

plant type by site seed set; Table 5; online Appendix S5). For all response variables, random effects (blocks within sites, block by plant type within sites) were signifi cant. Again we saw a signifi cant effect of plant size on fl owers (F 1, 367.4 = 359.0542, P < 0.0001), fruits ( F 1, 367.4 = 229.1194, P < 0.0001), and seeds ( F1, 367.1 = 231.7802, P < 0.0001) because large plants set more than small plants. These data indicate L. alabamica , L. crassa , and hybrids had equal seed set in either site, but that there were differences within a site, some of which were due to differences among plant types. In addition, we found signifi cant differences between the two sites in nitrogen (as measured by NO 3-N; t = − 3.7538, df = 14.50, P = 0.0020; online Appendix S6), total carbon ( t = 4.3191, df = 15.61, P = 0.0006), calcium (t = − 4.1329, df = 12.86, P = 0.0012), potassium ( t = 3.7098, df = 14.85, P = 0.0021), and magnesium (t = 5.4132, df = 9.09, P = 0.0004). These data demonstrate that the transplant sites were not uni- form in nutrients important for plant growth and reproduction.

DISCUSSION

Biodiversity is generated and maintained via the evolution of reproductive barriers between populations or species. Prezy- gotic barriers, because they occur fi rst, are often found to signif- icantly limit gene fl ow ( Husband and Sabara, 2003 ; Ramsey et al., 2003 ; Kay, 2006 ; Martin and Willis, 2007 ; Nosil, 2007 ; Lowry et al., 2008 ). Our goal in this study was to identify pre- and postzygotic barriers between L. alabamica and L. crassa and determine their relative contribution to total reproductive isolation. L. alabamica and L. crassa are parapatric species with similar morphology and ecology, leading to the question: how are these species reproductively isolated? We tested for two possible prezygotic barriers: (1) habitat isolation and (2) unilateral incompatibility, as well as environment-independent and -dependent genetic incompatibilities (postzygotic barriers).

Environment-independent genetic incompatibilities— De- spite the large number of crosses we made between L. alabam- ica and L. crassa (Appendix S2; see online Supplemental Data), we did not fi nd a pattern of cross success indicative of environ- ment-independent genetic incompatibilities. Neither Generation 0 nor Generation 1 plants differed signifi cantly in their ability to produce within-species or hybrid seed (online Appendix S3). Nor was there a signifi cant difference in either generation’ s seed set when crosses with mismatched plastid and nuclear genomes (A × C and C × A) were compared to those with matched ge- nomes (A × A and C × C; Appendix S4). Populations used in Generation 0 were selected from throughout the species’ ranges ( Fig. 1 ), which should have maximized the opportunity for ge- netic incompatibilities to occur. Thus, we conclude no genetic incompatibilities are present between L. alabamica and L. crassa , a fi nding consistent with the results of Rollins (1963) . Fig. 3. Unilateral incompatibility (UI) in crosses of Leavenworthia . Given the presumed complexity of genetic incompatibilities SC × SC = self-compatible × self-compatible, SC × SI = self-compatible × and, consequently, the diffi culty in losing them once evolved self-incompatible, SI × SC = self-incompatible × self-compatible, and SI × SI = self-incompatible × self-incompatible. Means are shown with 95% confi dence intervals and signifi cant differences marked with an asterisk (*). Proportion seed set is the mean number of seeds set per cross per plant crosses. A contrast of SC × SI and SI × SC crosses showed signifi cant UI divided by the mean number of ovules produced by that plant. Crosses are (t = 5.204, df = 1, P < 0.0001). Sample sizes were as follows: SC × SC = 80, listed with maternal parent fi rst. (A) Within-species crosses showed no UI, SC × SI = 82, SI × SC = 82, SI × SI = 80. (C) F 1 hybrid crosses. A contrast but SC × SC crosses set signifi cantly more seed than SC × SI ( t = 2.859, df of SC × SI and SI × SC crosses also showed signifi cant UI (t = 10.661, = 1, P = 0.0047) or the other groups. Sample sizes were as follows: SC × df = 1, P < 0.0001). Sample sizes were as follows: SC × SC = 306, SC × SI = SC = 70, SC × SI = 44, SI × SC = 44, SI × SI = 58. (B) Between-species 181, SI × SC = 180, SI × SI = 185. See Table 2 for ANOVA results. 418 American Journal of Botany [Vol. 97

Fig. 4. Hybrid fl owers do not differ from those of Leavenworthia alabamica and L. crassa. Seven traits were examined using principal component analysis. Components 1 and 2 were obtained through varimax rotation of the fi rst two principal components. (A) L. alabamica ( N = 18) and L. crassa ( N = 18) do not differ in fl oral morphology. (B) F1 , F2 , and BC hybrids (N = 98, 152, and 63, respectively) do not differ from L. alabamica and L. crassa .

( Gould, 1970 ), their absence makes it unlikely that they were not properly categorized as SC × SI or SI × SC because he did ever present. not know the SI status of the individuals used. In contrast, we selfed all individuals and determined whether they were SI or Unilateral incompatibility— Due to the mating system poly- SC. Furthermore, Lloyd (1968) based his conclusions on cross morphism present in L. alabamica and L. crassa, and their close means only and a much smaller sample size than in our evolutionary relationship (Beck et al., 2006), we had a rare op- analysis. portunity to test if UI acts as a reproductive barrier in this sys- We found a clear pattern of UI between species ( Fig. 3B ) and tem. Previous work by Lloyd (1968) documented what he hybrids (Fig. 3C), but not within species (Fig. 3A). These data termed “ partial unilateral incompatibility ” within L. alabamica suggest UI functions as a postmating, prezygotic reproductive and L. crassa. However, Lloyd did not self the plants used in barrier partially isolating L. alabamica and L. crassa . crosses to determine the SI status of each individual; he used Since its discovery, UI has been posited as a reproductive prior estimates of greenhouse autogamy rates to categorize barrier between species (Harrison and Darby, 1955; Grun and populations as fully selfi ng, intermediate, or fully outcrossing. Radlow, 1961) and possibly a species-recognition system func- He then crossed individuals from each population type and tionally related to SI (Lewis and Crowe, 1958; Heslop-Harri- found that the “ SI × SC rule” did not hold for many of his son, 1982; Hiscock and Dickinson, 1993). Our results are crosses. It is possible, however, that some of his crosses were consistent with UI as a reproductive barrier between species, March 2010] Koelling and Mauricio — Reproductive barriers in L EAVENWORTHIA 419

Table 3. The amount of viable pollen produced by Leavenworthia Table 4. Germination and fl owering time differences between alabamica , L. crassa , and their hybrids. The sample size (N ) is shown Leavenworthia alabamica , L. crassa , and their hybrids. Means are for each plant type. Data are pooled across years. Means are shown shown with standard errors. The sample size ( N) is shown for each with standard errors and different letters denote differences between plant type in each year. See text for ANOVA results. means at α = 0.05. Plant type N Days to germination Days to fi rst fl ower Plant type N Percentage viable pollen Year 1 L. alabamica 9 0.985 ± 0.007 a L. alabamica 9 42.33 ± 0.83 108.00 ± 5.66 L. crassa 9 0.972 ± 0.012 a, b L. crassa 9 41.89 ± 0.73 115.58 ± 5.53 F 1 96 0.945 ± 0.005 b F1 40 27.90 ± 2.67 90.42 ± 3.66 F 2 79 0.966 ± 0.004 a F2 72 8.99 ± 0.47 91.36 ± 0.85 BC 42 0.965 ± 0.006 a BC 28 10.21 ± 0.89 90.17 ± 1.31 Year 2 L. alabamica 9 18.00 ± 1.14 86.89 ± 4.85 L. crassa although we suggest one caveat. There were fewer within-spe- 9 18.00 ± 0.96 95.55 ± 5.83 F 1 58 13.74 ± 0.60 99.45 ± 1.14 cies SC × SI and SI × SC crosses than between-species in our F2 76 11.08 ± 0.28 91.17 ± 1.07 experiment due to the fact that between-population crosses BC 31 10.16 ± 0.24 91.21 ± 1.93 were not performed in year 1 (Fig. 2), and only one population had both SI and SC individuals. Although the difference in sample size was not great (44 vs. 82; online Appendix S2) and our sample size was larger than that of Lloyd (1968), we cannot power to detect UI in our within-species analysis. If UI also entirely rule out the possibility that we did not have suffi cient occurs within species, it is possible the UI pattern results from the breakdown of SI in self-compatible plants. Further charac- terization of UI and its relationship to SI in these species is needed to determine whether UI is, in fact, a reproductive bar- rier as our results suggest.

Phenotypic differentiation of parent species and hy- brids— We compared L. alabamica and L. crassa to their hy- brids for a suite of fl oral traits, pollen viability, and the timing of germination and fl owering. Despite the fact that six parent popu- lations located across the geographic range of each species were used to produce hybrids (which should have maximized the op- portunity for differences between L. alabamica, L. crassa, and hybrids in fl oral phenotypes), we found no evidence that fl owers differed between the parents and hybrids in a way that would affect their fi tness (Fig. 4). However, there was a difference in pollen viability between L. alabamica and F1 hybrids (Table 3). Measurements of pollen viability in the fi eld are needed to deter- mine what impact there might be on plant fi tness. With respect to germination and fl owering time, hybrids ger- minated and fl owered more quickly than their parents in year 1 (Table 4; Fig. 5). In year 2, hybrids germinated more quickly than their parents, but the difference was approximately 1 week rather than 2– 4 (Table 4). Also in year 2, we found no clear pattern of difference between parents and hybrids in days to fi rst fl ower (Fig. 5B). Years likely differed due to the fact that in year 1, parents were germinated in an 18 ° C growth chamber, rather than under warm greenhouse conditions as all other plants, thereby slowing germination of parents in year 1 and infl ating the difference between hybrids and parents. Because we did not measure germination in the fi eld, we do not know the effect of early germination on fi tness of hybrids. In Leavenworthia, seeds are dormant during the summer months when limestone glades are extremely hot and dry (Caudle and Baskin, 1968; Baskin and Baskin, 1971). In the fall, when tem- peratures are cooler and substantial rainfall has occurred, seeds Fig. 5. Germination and fl owering time differences between Leaven- begin to germinate. Germination continues until temperatures worthia alabamica , L. crassa, and hybrids in 2 years. Means are shown begin to drop in October. Baskin and Baskin (1972) found that with 95% confi dence intervals. Years 1 and 2 are shown as white and gray seeds of L. stylosa germinating in July had reduced survival bars, respectively. Germination time is the number of days from planting to germination. (A) F , F , and BC hybrids germinated earlier than the parent compared to those germinating in mid- to late-September and 1 2 early October ( L. stylosa’ s normal germination season), but species in both years. (B) F1 , F 2 , and BC hybrids fl owered earlier than L. alabamica and L. crassa in year 1, but not in year 2. See text for ANOVA produced more fruits and seeds than the later-germinating results and Table 4 for means and sample sizes. plants. The least fi t plants in their study germinated after early 420 American Journal of Botany [Vol. 97

October. Hybrids in our study germinated about 1 month (year 1) However, we were unable to germinate plants in the fi eld or 1 week (year 2) earlier than individuals of L. alabamica and because exceedingly wet soil conditions and small seed size L. crassa, differences much smaller than between the early and prohibited reliable tracking of individual seeds. Thus, our ex- late germinators in the L. stylosa study. It is possible to imagine periment could not detect early selection on L. alabamica , scenarios in which early germination could reduce (no seed dor- L. crassa, or hybrids in the fi eld. Selection can be very strong at mancy) or increase (quicker response to favorable germination germination and emergence (e.g., Donohue et al., 2005), and conditions) fi tness of hybrids. Further experimentation is needed we may have missed the signature of habitat isolation at this to determine the fi tness impact of germination timing and whether stage. Given that hybrids germinated more rapidly than L. ala- it has any role in the reproductive isolation of these species. bamica and L. crassa in the greenhouse, our results for hybrid fi tness in the fi eld may also have been different if germination Habitat isolation and hybrid fi tness— We tested for habitat time had been measured or if we were able to germinate seeds isolation between L. alabamica and L. crassa , and for environ- in the fi eld. ment-dependent hybrid fi tness using a reciprocal transplant. The lack of evidence for habitat isolation between L. ala- We found no home-site advantage for either species of Leaven- bamica and L. crassa suggests they may not be adapted to cryp- worthia . Rather, L. alabamica and L. crassa produced fl owers, tic niches available within limestone glade habitat and are fruits, and seeds equally in each site (Fig. 6A). It is possible a therefore not reproductively isolated from one another due to an seed viability difference is present, but because collected seeds inability to survive or reproduce in the same habitat. This result were not germinated, we do not know their viability. However, is consistent with the fi ndings of Busch (2005) , in which a re- we feel this is unlikely given that seeds were of normal size, ciprocal transplant experiment between populations of L. ala- shape, and color relative to other seeds of these species (V. bamica found no local adaptation. Koelling, personal observation). Soil analyses showed our sites differed in available nutrients, We observed no reduction in the fi tness of hybrids in the providing an opportunity for edaphic selection. It is, however, fi eld, as was the case in the greenhouse. This fi nding is consis- possible that the measured differences in soil nutrient content tent with the many examples Arnold (2006) provides of fi t hy- are transitory or that our sites did not differ in factors important brids in nature. for delineating the species ’ respective niches ( Baskin and Baskin, 1988). Reciprocal transplants at other glade sites may reveal a pattern of habitat isolation not detected in this study. Many studies have documented habitat isolation between species or found adaptation to different ecological niches in closely related plant species (e.g., Cruzan and Arnold, 1993 ; Campbell, 2003 ; Husband and Sabara, 2003 ; Ramsey et al., 2003 ; Rieseberg et al., 2003 ; Kay, 2006 ; Martin and Willis, 2007 ). Local adaptation within and between populations from different parts of a species ’ range is also found in numerous studies (e.g., Clausen et al., 1940; Bradshaw, 1960; Schemske, 1984; Linhart and Grant, 1996). In other words, habitat isola- tion or local adaptation is usually the rule (Leimu and Fischer, 2008), not the exception (although there are exceptions, e.g., Galloway and Fenster, 1999 ; Baack and Stanton, 2005 ). Why we found no habitat isolation between L. alabamica and L. crassa is unknown. Perhaps limestone glades are relatively uni- form ecologically, and therefore, selection has not favored hab- itat isolation. A number of possible constraints ( Antonovics, 1976 ), including gene fl ow, may have prevented the develop- ment of habitat isolation between these species.

Reproductive isolation in Leavenworthia— In this study, we combined greenhouse and fi eld experiments to comprehen- sively address the nature of reproductive isolation between two parapatric species in the genus Leavenworthia , L. alabamica and L. crassa . From a large, replicated crossing experiment, we Fig. 6. No local adaptation or reduced hybrid fi tness in a reciprocal found no evidence for genic or cytonuclear incompatibilities transplant experiment. Means are shown with 95% confi dence intervals. and conclude that these reproductive barriers are unlikely to Legends show plant types in each analysis. (A) Leavenworthia alabamica play a role in the reproductive isolation of these species. We and L. crassa plants grown from fi eld-collected seed did not set more seeds did, however, fi nd evidence that unilateral incompatibility oc- in their home sites than in their nonhome sites. Sample sizes in the L. ala- curs in this system, appears functionally related to self-incom- bamica home site were as follows: L. alabamica = 173, L. crassa = 181. patibility, and may act as a partial reproductive barrier between Sample sizes in the L. crassa home site were as follows: L. alabamica = 139, L. alabamica and L. crassa. SC populations of L. alabamica L. crassa = 124. (B) F1 , F 2 , and BC hybrids did not differ from L. alabamica and L. crassa in seed set in either the L. alabamica or L. crassa site. Sample occur within the range of L. crassa (Fig. 1). If UI acts as a par- tial reproductive barrier in this system, then gene fl ow between sizes in the L. alabamica home site were as follows: F1 = 41, F 2 = 44, BC = 40, L. alabamica = 38, L. crassa = 40. Sample sizes in the L. crassa home site SI L. crassa and SC L. alabamica in this region should be were as follows: F 1 = 41, F 2 = 41, BC = 34, L. alabamica = 33, L. crassa = 33). largely in the direction of L. alabamica. Further experimenta- See Table 5 for ANOVA results and Appendix S5 for means. tion is needed to test this prediction. March 2010] Koelling and Mauricio — Reproductive barriers in L EAVENWORTHIA 421

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