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Reproductive Biology |
2Oklahoma Biological Survey and 3Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019 USA
Received for publication July 15, 2005. Accepted for publication December 23, 2005.
ABSTRACT
We examined the effect of reproductive and life history strategies on the amount and partitioning of genetic variation in three annual species of Nuttallanthus. The North American species N. canadensis, N. floridanus, and N. texanus have regional to widespread ranges that overlap in the southeastern USA, are characterized by homogeneous populations and high fecundity, and possess showy, fragrant flowers seemingly adapted for insect pollination and outbreeding. Field and greenhouse studies on plants from 25 populations indicated that reproductive strategies were similar among species and showed predominant self-fertilization via cleistogamy and self-pollination prior to anthesis in chasmogamous flowers. Species were reproductively isolated and demonstrated complete cross-incompatibility after experimental crosses and no evidence for hybridization in mixed populations. Genetic variation was assessed using starch gel electrophoresis to resolve 15 isozyme loci in 50 populations. Conspecific genetic identity (I) values were high (0.819–0.936), but interspecific comparisons indicated many qualitative allelic differences and correspondingly low I values (0.516–0.623). Low levels of polymorphism and observed heterozygosity within populations and the disproportionate amount of gene diversity distributed among populations were concordant with reproductive data. The pattern of genetic differentiation was most similar to that observed in species with a predominantly inbreeding mating system.
Key Words: cleistogamy • isozyme • Nuttallanthus • Plantaginaceae • reproductive biology
North American species of Nuttallanthus D.A. Sutton are widespread herbaceous annuals and produce showy, weakly personate, bluish, bilabiate, and spurred flowers that attract a variety of insect visitors. Variation among reproductive characters has been used to delimit species and infraspecific taxa. Purported intergradation of floral characters also has been used to propose hypotheses of interspecific hybridization (Pennell, 1920
, 1922
; Munz, 1926
). Because North American species of Nuttallanthus share most life history traits, the effect of differences in floral characters, breeding system, and reproductive isolation on the amount and pattern of genetic variation can be assessed effectively.
The genus Nuttallanthus (toadflax) was segregated from Linaria Miller by Sutton (1988)
based primarily on floral and seed characters including size of the abaxial lip of the corolla, development of the palate and occlusion of the tube, diameter of the nectar spur, and seed morphology. Nuttallanthus includes one South American species and three species of native North American annual or, rarely, biennial herbs (Sutton, 1988
): N. canadensis (L.) D.A. Sutton, N. floridanus (Chapman) D.A. Sutton, and N. texanus (Scheele) D.A. Sutton.Intergradation of floral characters among specimens of L. canadensis L. (= N. canadensis) and L. texana Scheele (= N. texanus) was considered "quite complete" by Pennell (1920)
and Munz (1926)
, who both recognized L. canadensis var. texana (Scheele) Pennell. Their submergence of L. texana influenced the floristic and taxonomic treatments of several workers (Rothmaler, 1954
; Cronquist et al., 1984
; Hitchcock and Cronquist, 1998
). Because of its unique floral and seed characters, N. floridanus has been treated consistently at the rank of species.
Observations of reproductive character variation resulted in infraspecific delimitation of plants possessing cleistogamous flowers and white corollas by Fernald (1936
, 1943
) as L. canadensis forma cleistogama Fernald and L. canadensis forma albina Fernald, respectively. Cleistogamy in L. canadensis has been observed by numerous botanists (e.g., Britton and Brown, 1898
) and investigated by Webster (1900)
and Hill (1909)
, who reported and illustrated its occurrence at the end of the growing season. In chasmogamous flowers, the narrow spur and the virtual absence of a palate that occludes the corolla tube suggested to Pennell (1935
, p. 302) that flowers of the American toadflaxes were "tending from pollination by bees to that by butterflies, although the nearly spurless L. floridana may cater rather to flies." Similar observations were made previously by Robertson (1888
, p. 228), who noted that L. canadensis was "visited by bees, but more often by butterflies."
The North American toadflaxes have comparable life history traits and vary mostly in reproductive features and geographic distribution. In common with the genus Linaria, Nuttallanthus has a chromosome base number of x = 6, with polyploidy (4x) indicated only in N. texana (Raven, 1963
). Nuttallanthus canadensis and N. texanus have the largest geographical ranges of any native species of New World Antirrhineae (Elisens, 1985
). Whereas N. canadensis is native throughout much of temperate North America and is naturalized in South America and in Europe, N. texanus is native to the southern USA and Mexico, may be native to temperate South America, and is naturalized in other temperate regions (Sutton, 1988
). Nuttallanthus floridanus is more narrowly distributed and occurs in the Atlantic and Gulf coastal plain in Alabama, Florida, Georgia, and Mississippi. Species ranges overlap in the southeastern USA, and although population sizes vary, all three species occur occasionally in mixed populations. All North American species commonly grow in well-drained, sandy soils of dunes and open woodlands as well as in fields and other disturbed areas.
Phylogenetic relationships are not resolved among the American toadflaxes or among Nuttallanthus and genera in tribe Antirrhineae (e.g., Hileman and Baum, 2003
; Vargas et al, 2004
). Although a close phylogenetic relationship is suggested with species of Linaria (Hileman and Baum, 2003
), there are numerous characters differentiating Nuttallanthus from Linaria (Sutton, 1988
). Compared to other native New World Antirrhineae, Nuttallanthus is not closely related phylogenetically and has many unique features in its seed morphology and anatomy (Elisens and Tomb, 1983
; Elisens, 1985
), floral structure (Rothmaler, 1943
), chromosome base number (x = 6), and pollen morphotype (Elisens, 1986
). Nuttallanthus canadensis also possesses unique tubular nuclear inclusions compared to Linaria (Speta, 1982
; Bigazzi, 1989
). Morphological data is generally concordant with molecular phylogenetic investigations (Ghebrehiwet et al., 2000
; Vargas et al., 2004
), which indicate the distinctness of the linareoid lineage in tribe Antirrhineae and to New World genera.
Prior to this investigation, no detailed investigation of the reproductive biology and genetic variation was undertaken on species of Nuttallanthus. Our investigation employed starch gel electrophoresis of soluble enzymes and breeding system and crossing experiments to elucidate the genetic variation and reproductive biology of the North American species of Nuttallanthus. The principal goals of this study were to determine the amount of genetic differentiation within and among populations and species in the genus, to test the hypothesis that there are three distinct species, and to describe the types of mating systems and degree of reproductive isolation exhibited by these species.
MATERIALS AND METHODS
Isozyme analysis
A total of 649 individuals from 50 populations representing three species of Nuttallanthus were examined for electrophoretic variation: 325 individuals from 22 populations of N. canadensis, 110 individuals from eight populations of N. floridanus, and 214 individuals from 20 populations of N. texanus (Appendix, Fig. 1). Voucher specimens of the populations sampled were deposited at the Bebb Herbarium of the University of Oklahoma (OKL). A maximum of 20% of the individuals in any population was sampled, although this limited sample sizes from small populations. Individuals of populations 42 and 43 consisted of plants grown from a bulk seed collection obtained in the field. For all other populations, plants were bagged individually and placed in plastic containers on ice for transport to the lab, where they were refrigerated at 4°C prior to protein extraction. Tissues from young, actively-growing leaves served as the enzyme source.
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. Leaf extracts were centrifuged and the supernatant was absorbed onto wicks of Whatman 17 MM chromatography paper, which were placed in 1.5 mL microcentrifuge tubes at –70°C for approximately 1 h. Samples were electrophoresed on 11% starch gels using two buffer systems (Soltis et al., 1983
) to resolve 15 loci for 10 enzyme systems: aspartate aminotransferase (AAT, EC 2.6.1.1), alcohol dehydrogenase (ADH, EC 1.1.1.1), 
-glycerophosphate dehydrogenase (GPD, EC 1.1.1.8), glucose 6-phosphate isomerase (GPI, EC 5.3.1.9), isocitrate dehydrogenase (IDH, EC 1.1.1.41), malate dehydrogenase (MDH, EC 1.1.1.37), 6-phosphogluconate dehydrogenase (6PGD, EC 1.1.1.43), phosphoglucomutase (PGM, EC 5.4.2.2), superoxide dismutase (SOD, EC 1.15.1.1), and triosephosphate isomerase (TPI, EC 5.3.1.1). System I consisted of an electrode buffer of 0.18 M Tris and 0.004 M EDTA titrated to pH 8.6 with boric acid and a gel buffer prepared from a 1 : 3 aqueous dilution of the electrode buffer; System I was used to resolve AAT, ADH, GPD, GPI, PGM, SOD, and TPI. The electrode buffer of System II was prepared from 0.065 M L-histidine free base titrated to pH 6.5 with citric acid monohydrate and a gel buffer was obtained from a 1 : 3 aqueous dilution of the electrode buffer; System II was used to resolve IDH, MDH, and 6PGD. Agarose-overlay and staining procedures used to detect enzyme activity followed the protocols of Soltis et al. (1983)
. The genetic bases of the enzyme banding patterns observed were inferred from their concordance with published data regarding the basic number of loci expected in the absence of gene duplication and patterns of enzyme expression (Gottlieb, 1982
; Crawford, 1990
). Isozyme and allozyme patterns were consistent with the known substructure and compartmentalization of the resolved enzymes. Loci were numbered and alleles were lettered beginning with the most anodal (fastest-migrating) form.
Allele and genotype frequencies were determined for each population and species. Table 1 lists the summary frequencies for species; genotype frequencies by locus and population are available from the senior author. The percentage of polymorphic loci, mean number of alleles per locus and per polymorphic locus, mean observed and expected heterozygosity, and the effective number of alleles per locus was calculated manually for each population and was averaged across all populations of each species. Species-level statistics were computed by treating all sampled individuals within each species as members of a single population (Hamrick and Godt, 1990
), which facilitates comparison of genetic diversity estimates within species. Spearman's rank-order correlation analysis was performed with SPSS, version 11.5.0 (SPSS, 2002
) to examine the association of population size with genetic diversity.
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); the proportion of genetic diversity among populations (GST) and Wright's (1951) estimate of gene flow (Nm) were calculated for each species. BIOSYS-1 ( Swofford and Selander, 1989
) and GENESTAT2 (Whitkus, 1988
) provided computational software. To further assess the level of genetic differentiation among populations, Wright's (1965) F statistics (FIS, FIT, and FST) were computed for each polymorphic locus and were averaged across loci within each species. The Weir and Cockerham (1984)
estimator of Wright's FST (
) was not calculated, although sampling intensity (50 populations, 649 individuals) suggests that differences between GST and would be slight. Wright's fixation indices (F) were calculated for each locus and population of the species; the statistical significance of observed deviations of heterozygote proportions from Hardy-Weinberg expectations was tested using a Chi square analysis (Wright, 1965
, 1978
; Nei, 1977
). NTSYSpc version 2.11c (Rohlf, 2002
) was used to calculate Nei's (1972)
genetic identity and distance, Roger's distance as modified by Wright (1978)
, and Cavalli-Sforza and Edwards (1967)
chord distance for all pairwise comparisons among populations and species. Dendrograms were generated using these coefficients with the Distance Wagner procedure (Farris, 1972
) and the unweighted pair-group method using arithmetical averages (UPGMA) as discussed in Sneath and Sokal (1973)
; cophenetic correlations were calculated for each dendrogram. NTSYSpc also was used to conduct nonmetric-multidimensional scaling (MDS) (Kruskal, 1964a
) of genetic identities among populations. Species-level gene diversity statistics and genetic distances and identities were determined using all loci; only polymorphic loci were included in calculations of genetic diversity within and among populations, because statistics excluding monomorphic loci better represent the partitioning of genetic variation within and among populations (Berg and Hamrick, 1997
). Spearman's rank-order correlation analysis was performed using SPSS version 11.5.0 (SPSS, 2002
) to assess the association of genetic distance and geographic distance (in kilometers) between populations.
Reproductive and crossing studies
A minimum of five individuals were propagated from seed collected from individuals in each of 25 populations used in reproductive experiments (Appendix). Because of inconsistent germination of untreated seed, germination treatments involved soaking seed in a solution of gibberellic acid (0.1 g/L) for 10 min, washing with distilled water, and incubating at room temperature on water-saturated filter paper in sealed petri dishes. Seedlings were transplanted to a soil-based potting mix and cultivated under pollinator-free conditions in a growth chamber set at 13.3 h of light at 28°C and 10.7 h of dark at 16°C. Breeding system and crossability studies involved tests for autogamy (observation of untreated cleistogamous and chasmogamous flowers), apomixis (emasculation of flowers followed by no hand pollination), self-compatibility (emasculation followed by geitonogamous hand pollination), and cross-compatibility (emasculation followed by intra- and interspecific hand pollination). Buds of manipulated flowers were emasculated with needle-point forceps prior to anthesis, because the anthers of cleistogamous and chasmogamous flowers usually dehisced prior to the opening of the perianth. Hand pollinations were accomplished 1 day following emasculation; forceps sterilized in 95% ethanol were used to transfer recently dehisced anther sacs to the receptive stigmas of emasculated flowers. Flowers used in reproductive experiments were not bagged. Because of the small size of the floral structures, all floral manipulations were performed utilizing a stereomicroscope at 12.5x.
Over 240 hand pollinations representing all possible directional interspecific crosses were made; a minimum of 30 and a maximum of 50 hand pollinations were made for each directional interspecific combination. In addition, 75 or 80 intraspecific hand pollinations were performed among plants from populations of each species, and 100 manual self-pollinations were performed per species. Seed production and germination rate was recorded for 100 capsules produced by both cleistogamous and chasmogamous flowers of each species and for all manipulated flowers that produced mature capsules and seeds. Germination was assessed for 25 seeds from each capsule using the treatment described.
Univariate statistical analyses were performed using SPSS for Windows, version 11.5.0 (SPSS, 2002
). Descriptive statistics of seed production and germination rate were calculated for each species; normality tests were performed for each treatment category. To compare treatment categories among populations and species, one-way analysis of variance was employed; post-hoc testing was performed using Fisher's least significant difference test.
RESULTS
Genetic variation
Fifteen loci coding for 10 enzymes were scored from populations of three species of Nuttallanthus: two for AAT, GPI, MDH, and TPI, and one each for ADH, GPD, IDH, 6PGD, PGM, and SOD (Table 1). Four loci were observed for MDH; MDH-4 was polymorphic for two alleles, MDH-3 was invariant, and MDH-1 represented a composite for two loci, because MDH-1 and MDH-2 overlapped extensively and loci and alleles could not be distinguished reliably. Because three banding patterns were observed, MDH-1 and MDH-2 were scored as a single polymorphic locus with patterns A, B, and C corresponding to allelic differences (cf. Small et al., 1999
). Additional isozymes were detected for AAT, ADH, IDH, MDH, 6PGD, and PGM, but were not scored because of low or inconsistent levels of activity or poor resolution. Additional enzyme systems detected but not scored were ME using buffer system I and ALD, GA3PD, ME, MNR, and SDH using buffer system II.
The number of isozymes detected was typical for diploid plant species, with the exception of MDH and PGM, which generally have three and two loci, respectively (Gottlieb, 1982
; Weeden and Wendel, 1989
). No differences in isozyme number among species of Nuttallanthus were observed. The number of allelomorphs observed at polymorphic loci ranged from two (AAT-1, MDH-4, SOD) to five (AAT-2).
Mean values for genetic identity (I) coefficients for pairwise comparisons of 50 populations within and among species are presented in Table 2. Average I values within species varied from 0.819 (N. texanus) to 0.936 (N. canadensis). The lower values for conspecific comparisons in N. texanus reflected the presence of two groups with different allozyme profiles (Table 1). Mean genetic identities were considerably lower between than within species, with interspecific I values ranging from 0.516 (N. canadensis x N. floridanus) to 0.623 (N. canadensis x N. texanus). Comparable patterns of genetic similarity were observed regardless of the coefficient employed. All pairwise comparisons among species of Nuttallanthus (average I = 0.580) were lower than the average identity value (I = 0.670) for plant congeners reported by Gottlieb (1981)
.
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Species-level genetic variation estimates based on allele presence and frequencies are summarized in Table 3. Among all populations, the mean number of alleles per locus (A) ranged from 1.00 to 1.64, the mean number of alleles per polymorphic locus (Ap) varied from 2.0 to 3.0, and the percentage of polymorphic loci (P) ranged from 0.0 to 57.1. The average proportion of heterozygous loci observed per individual (Ho) ranged from 0.0 to 0.054; the average proportion of heterozygous loci per individual expected for populations in Hardy–Weinburg equilibrium (He) ranged from 0.0 to 0.283. Among species, N. canadensis exhibited the highest mean values of A (1.25), P (23.7), and He (0.076), whereas N. floridanus populations showed the lowest levels of genetic variation for A (1.15), P (14.3), and He (0.043). Spearman's rank-order correlation analyses indicated positive and statistically significant associations between population (sample) size in Nuttallanthus and A (rs = 0.50, p < 0.001), P (rs = 0.52, p < 0.001), Ho (rs = 0.33, p = 0.019), and He (rs = 0.49, p < 0.001). The effective number of alleles per locus (Ae) within populations varied from 1.00 to 1.39. Ae is equal to the actual number of alleles only when all alleles exist in equal frequency; it provides a measure of allelic evenness. This value was lower than the mean number of alleles per locus (A) observed in all polymorphic populations, suggesting that a portion of the allelic diversity within populations was present in the form of low-frequency alleles (Nei, 1987
).
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for annuals (GST = 0.357), for species with regional distributions (GST = 0.216), and for species of temperate regions (GST = 0.246), but were comparable to those reported for selfing species (GST = 0.510). Wright's (1969)
estimate of gene flow (Nm = (1 – FST )/4FST) ranged from 0.110 in N. texanus to 0.331 in N. canadensis; Nm estimates of less than 1.0 suggest relatively little gene flow among populations (Slatkin and Barton, 1989
). Observed and expected (for randomly outcrossing species) frequencies of heterozygous loci in the three species of Nuttallanthus were calculated but not presented. Observed heterozygosity was lower than expected heterozygosity for all polymorphic loci; the ratios of observed to expected heterozygosities were consistent with data for species characterized by selfing breeding systems (Hamrick and Godt, 1990
). Wright's F statistics (FIS, FIT, and FST) for polymorphic loci within each species of Nuttallanthus are provided in Table 4. The average amount of genetic variation distributed among populations (FST) ranged from 0.430 in N. canadensis to 0.694 in N. texanus. For all three species, the average total inbreeding coefficient (FIT) appeared to be more greatly influenced by nonrandom mating within populations (FIS) than by differences in allele frequencies among populations (FST). Of the 144 single-locus fixation indices tested, 138 (96%) were both positive and significantly different from Hardy–Weinberg expectations (p < 0.05), indicating a deficiency of heterozygotes at polymorphic loci in these species. An excess of heterozygotes was observed only at a single locus (SOD) in a population of Nuttallanthus canadensis, but this excess was not significant.
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), chord distance (Cavalli-Sfoza and Edwards, 1967), and Nei's genetic identity and distance produced similar topologies. The UPGMA dendrogram based on Nei's genetic identity coefficients resulted in the highest cophenetic correlation (0.920) and is reproduced here (Fig. 2). The dendrogram clearly illustrates both geographic and taxonomic coherence; conspecific populations cluster together and, within individual species, populations tend to cluster with others from the same geographic region. Two discrete clusters of populations (groups 1 and 2) of N. texanus are apparent; one cluster is composed predominately of populations from the southeastern United States, California, Oklahoma, and central Texas, whereas populations from Arkansas, Louisiana, eastern Texas, and Oklahoma make up group 2. The two groups of populations of N. texanus have minor qualitative allelic dissimilarities among loci, but the groups have major frequency differences at four loci: AAT-2, GPD, PGM-1, and TPI-2 (Table 1).
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, pp. 9–44; Kruskal, 1964b
). Similar to the UPGMA dendrogram, the MDS plot illustrates the pronounced genetic differentiation among the three species of Nuttallanthus and distinguishes two groups of N. texanus.
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Reproductive and crossing studies
Seed production in three species of Nuttallanthus following controlled flower manipulations and hand pollinations is presented in Table 5. Individuals of all species of Nuttallanthus demonstrated autogamy and produced cleistogamous flowers both early and late in the life cycle in addition to chasmogamous flowers (Fig. 4A–C). In cleistogamous flowers (Fig. 4D), the limbs of the undeveloped corolla securely enclosed the male and female reproductive structures, and self-fertilization occurred in the flower bud. Capsules produced by cleistogamous flowers were smaller and produced significantly fewer seeds than did those of chasmogamous flowers. Pollen appeared to be required for successful fruit and seed development, because no emasculated, unpollinated flower produced mature capsules and seeds. Within each of the three species of Nuttallanthus, no significant difference in seed production was observed between individuals self-pollinated with intraindividual (geitonogamous) pollen or with intraspecific cross pollen. Capsules produced through artificial hand pollination yielded significantly fewer seeds than those from unmanipulated cleistogamous or chasmogamous flowers, which may have resulted from limited pollen transfer between hand-pollinated flowers.
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DISCUSSION
Genetic differentiation and systematic implications
The observed patterns of allozyme divergence among North American Nuttallanthus were consistent with the recognition of three distinct species as proposed by Sutton (1988)
: N. canadensis, N. floridanus, and N. texanus. Both qualitative and quantitative allelic differences among species were evident at many enzyme loci. Two species were characterized by marker alleles that were either fixed or present in high frequencies (>0.8) in all populations: 6PGD-2c and TPI-1d in N. canadensis, GPD-1b and PGM-1d in N. floridanus. Additionally, high frequency, species-specific alleles present in all but a few populations were observed in N. canadensis (SOD-1a) and N. texanus (TPI-1b). Over 40% of the total alleles (19 of 46) were unique to individual species, but summary frequencies for 11 of those not mentioned before were less than 0.1 and the remaining two were less than 0.4. Pairwise genetic identity (I) values between species (average 0.580) were lower than average genetic identities within species (0.888). Although a wide range of I values has been documented among congeneric species, interspecific I values in Nuttallanthus were lower than those reported for most angiosperm congeners (Crawford, 1983
) and the mean I value of 0.67 reported by Gottlieb (1981)
.
UPGMA clustering and MDS ordination analyses (Figs. 2, 3) as well as dendrograms (not illustrated) produced using Distance Wagner, Roger's, and chord distance coefficients resulted in similar patterns identifying three major groups of populations corresponding to species boundaries. The UPGMA dendrogram indicated a closer genetic relationship between N. canadensis and N. texanus than either species exhibited with N. floridanus. Although populations were sampled throughout most of the geographic range of N. floridanus, there were many regions where populations were not sampled for N. canadensis and N. texanus. Regional differentiation reflecting minor variation in allele frequencies was observed among conspecific populations of N. canadensis and N. floridanus, but two distinct subclusters of populations were present within N. texanus (groups 1 and 2 in Fig. 2). Divergence between these two groups of populations primarily reflected alternative highest frequency alleles at three loci (GPD-1, MDH-1, and PGM-1) and fixation of the allele TPI-2a among group 2 populations. Consequently, average conspecific I values in N. texanus (0.819) were considerably lower than those found in N. canadensis (0.936) and N. floridanus (0.909). The latter two species had conspecific genetic identities comparable to those within groups of N. texanus (0.878 in group 1, 0.928 in group 2). Genetic identities between groups 1 and 2 of N. texanus (0.737) were intermediate between average infraspecific (0.888) and interspecific (0.580) values observed among North American Nuttallanthus.
Despite compatibility between groups of N. texanus in experimental crosses, only low levels of gene exchange were indicated among natural populations. For example, the alleles AAT-2a (mid-frequency in group 2) and TPI-2a (fixed in group 2) occurred at low frequencies in neighboring group 1 populations 59 (Oklahoma) and 37 (Alabama). Similarly, the allele PGM-1a (high frequency in group 2) was detected only in a neighboring group 1 population (59) in Oklahoma. Sampled populations of groups 1 and 2 in N. texanus were allopatric except for one mixed population (58) in a disturbed agricultural field from central Oklahoma. The level of genetic divergence between groups and their general allopatry or parapatry reflects a pattern observed in many plant species (Crawford, 1989
; Rieseberg et al., 2004
) that may be indicative of incipient divergence (Levin, 2000
), although it is not known whether these differences reflect ecological, historical, or other life history factors. Based on limited geographical sampling and comparatively low levels of morphological (Crawford, 2003
) and genetic divergence, we do not recommend taxonomic recognition of groups within N. texanus.
The type and amount of genetic divergence between species indicate they have been isolated for a substantial period of time and have not exchanged genes in the recent past. The complete cross-incompatibility among species was concordant with the pattern of allozyme divergence indicating effective reproductive barriers between species. No individuals of suspected hybrid origin were observed in sympatric populations in Alabama, Florida, Georgia, and South Carolina using either morphological or allozymic criteria. In mixed populations of different species, many Lepidopteran and Hemipteran visitors were observed on flowers of plants of different species (Crawford, 2003
), but our data indicate little evidence for gene flow and hybridization. Although cross-compatibilities can vary among populations of some species (Niklas, 1997
) and are best considered plesiomorphic traits (Mishler and Theriot, 2000
), our results indicating complete reproductive isolation, numerous accumulated genetic differences, and unique morphological characters support hypotheses proposing specific rank among North American taxa in Nuttallanthus. Recognition of infraspecific taxa using cleistogamy as a character is clearly unjustified, because many (if not all) individuals of the three species of Nuttallanthus are capable of producing cleistogamous flowers.
Genetic variation and reproductive system
Greenhouse and field observations indicated that all species of Nuttallanthus had similar flowering phenologies and comparable reproductive strategies. Cleistogamous flowers were produced early and late in the life cycle; chasmogamous flowers were self-pollinated effectively before anthesis, but attracted numerous insect visitors after anthesis. Nectar amounts and composition are unknown, but nectar was not observed in flowers grown in greenhouses and growth chambers. Among conspecific populations, cleistogamy and self-pollination in chasmogamous flowers limited outcrossing resulting in high levels of homozygosity within populations, and the apportionment of genetic variation among rather than within populations. Species of Nuttallanthus have effective post-mating isolating barriers (Levin, 1978
), although the pollination biology and pre-mating barriers were not examined for this study. It is not known whether factors involving pollen–pistil interactions (prezygotic) or seed development (postzygotic) resulted in cross-incompatibility among species. Seed set was not observed in the greenhouse after controlled interspecific pollinations, and hybrids were not observed in natural populations including those with two or more species present.
Besides reproductive system, several other life history features are critical in influencing the observed pattern of genetic variation. Fecundity in Nuttallanthus is generally high with seed number averaging 
100 per fruit and numerous fruits produced per individual. Seed are densely packed in capsules and are small in size (length 0.4–0.7 mm; Crawford, 2003
) with angles or small ridges (Sutton, 1988
); dispersal may be mediated primarily by gravity or wind (Ridley, 1930
; van der Pijl, 1972
). Seed longevity is not known. Species of Nuttallanthus are herbaceous annuals (occasionally biennials) that share habitat preferences for well-drained sandy soils in early successional communities or in sites characterized by frequent disturbance. Although geographic ranges are best categorized as widespread (N. canadensis and N. texanus) or regional (N. floridanus), there are marked annual changes in population size (Crawford, 2003
). The majority of populations sampled appeared to be structurally homogeneous, except for two populations of N. texanus from south central Oklahoma; population 58 included group 1 and group 2 allozyme profiles, and no. 59 consisted of subpopulations confined to "soil islands" on a granitic outcrop.
Collectively, these life history attributes and our reproductive and genetic observations suggest that the amount and apportionment of genetic variation observed within and among populations of toadflaxes have been impacted most significantly by drift processes (Nei et al., 1975
) acting in concert with an annual life form, (primarily) selfing breeding system, high fecundity, spatially and temporally fluctuating population sizes, and founding events through seed dispersal. Several measures of genetic variation, diversity, and heterozygosity reported here match closely those summarized for other organisms with similar life history attributes (Hamrick and Godt, 1990
; Britten, 1996
; Schoen et al., 1996
). Particularly compelling are reproductive data indicating that populations of Nuttallanthus are self-compatible and self-pollinating and genetic data indicating most populations appear to be mostly self-fertilizing, which is supported by the low levels of heterozygosity and high levels of allelic fixation at most loci (Tables 2, 4). Field studies in two Oklahoma populations of N. texanus (P. Crawford, unpublished data) also supported a mode of reproduction characterized by predominant autogamy and facultative xenogamy. Experiments involving emasculated flowers that were either available for open pollination or excluded from pollinators usually failed to produce seed (P. Crawford, unpublished data) despite active floral visitation by Lepidopteran, Hymenopteran, and Hemipteran (but no Dipteran) insects. The primary apportionment of genetic diversity among rather than within populations (GST estimates from 42% in N. canadensis to 69% in N. texanus) and gene flow (Nm) estimates less than 0.35 (Table 3) also were consistent with predominant selfing (Gottlieb, 1981
; Loveless and Hamrick, 1984
) and limited gene exchange among populations (Wright, 1951
; Slatkin and Barton, 1989
), respectively. Autogamous seed set was significantly greater in chasmogamous flowers (Table 6) suggesting a cost in fecundity for cleistogamy.
Genetic and reproductive data in Nuttallanthus are generally concordant and demonstrate the utility of combining approaches when examining the effects of life history strategies on genetic variation among populations and species. Similar to most annuals, all three species are predominantly inbreeding and have gene diversity partitioned among populations, despite production of chasmogamous flowers that attract insects and appear to promote outcrossing. Whereas N. canadensis and N. texanus have similar life history traits, floral features, and amounts of gene variation, N. floridanus has a (smaller) regional geographic range, lower seed numbers per capsule, and highly reduced nectar spurs associated with the lowest levels of gene variation. Additional studies are required to determine whether the groups present in N. texanus represent incipient speciation, whether nectar production is functional or variable among species, and whether differences in floral biology affect insect visitation and pollination mechanism.
Taxon
State. County: Population code = Latitude, Longitude (N = no. of individuals examined, Collector no.).
Nuttallanthus canadensis (28 populations)
Alabama. Baldwin Co.: 1* = 30.414N, 87.598W (16, Crawford 358–373); 2 = 30.238N 87.882W (5, Crawford 686–690). Mobile Co.: 3 = 30.433N 88.144W (10, Crawford 324–333); 4 = 30.243N 88.078W (2, Crawford 649–650).
Delaware. Sussex Co.: 5* = 38.574N 75.056W (30, Crawford 211–240).
Florida. Bay Co.: 6*
= 30.204N 85.847W (25, Crawford 380–399). Calhoun Co.: 7 = 30.464N 85.045W (8, Crawford 853–860). Franklin Co.: 8* = 29.833N 84.876W (19, Crawford 406–415, 417–425); 9* = 29.909N 84.394W (13, Crawford 449–461); 10 = 29.853N 84.664W (3, Crawford 808–810). Lafayette Co.: 11* = 30.139N 83.290W (10, Crawford 493–502). Wakulla Co.: 12* = 30.136N 84.326W (10, Crawford 817–826). Walton Co.: 13 = 30.309N 86.102W (5, Crawford 705–709).
Georgia. Camden Co.: 14* = 30.759N 81.658W (20, Crawford 509–528). Candler Co.: 15* = 32.355N 81.989W (10, Crawford 596–605). Glynn Co.: 16* = 31.020N 81.435W (10, Crawford 532–541). Liberty Co.: 17* = 31.675N 81.414W (10, Crawford 545–554).
Maryland. Caroline Co.: 18* = 38.817N 75.748W (10, Crawford 244–253). Worcester Co.: 19* = 38.096N 75.499W (17, Crawford 186–202).
Massachusetts. Middlesex Co.: 20* = 42.504N 71.265W (15, Crawford 901–915).
North Carolina. Currituck Co.: 21* = 36.278N 75.915W (10, Crawford 118–127). Dare Co.: 22* = 35.261N 75.579W (10, Crawford 104–113). Duplin Co.: 23* = 34.926N 77.652W (10, Crawford 91–100). Hoke Co.: 24* = 35.007N 79.305W (20, Crawford 68–87).
South Carolina. Beaufort Co.: 25* = 32.377N 80.832W (10, Crawford 573–582).
Virginia. Accomack Co.: 26* = 37.912N 75.356W (20, Crawford 160–179). Northampton Co.: 27* = 37.145N 65.967W (20, Crawford 134–153). Orange Co.: 28* = 38.261N 77.980W (10, Crawford 257–266).
Nuttallanthus floridanus (8 populations)
Alabama. Baldwin Co.: 29* = 30.238N 87.882W (11, Crawford 673–683). Mobile Co.: 30* = 30.243N 88.078W (11, Crawford 657–667).
Florida. Bay Co.: 31* = 30.204N 85.847W (25, Crawford 723–747). Franklin Co.: 32* = 29.909N 84.394W (10, Crawford 467–476); 33* = 29.723N 84.890W (8, Crawford 753–760), 34* = 29.724N 84.899W (20, Crawford 772–791); 35* = 29.853N 84.664W (10, Crawford 797–806). Putnam Co.: 36* = 29.623N 81.912W (15, Crawford 837–851).
Nuttallanthus texanus (24 populations)
Alabama. Baldwin Co.: 37* = 30.414N 87.598W (14, Crawford 339–352). Mobile Co.: 38* = 30.243N 88.078W (10, Crawford 639–648).
Arkansas. Conway Co.: 39* = 35.171N 92.755W (10, Crawford 52–61). Crawford Co.: 40* = 35.528N 94.041W (10, Crawford 39–48). Logan Co.: 41 = 35.300N 93.634W (5, Crawford 270–274).
California. Monterrey Co.: 42* = 36.511N 121.942W (5, Crawford 920–924). Santa Barbara Co.: 43* = 34.044N 119.718W (5, Crawford 929–933).
Florida. Calhoun Co.: 45 = 30.464N 85.045W (4, Crawford 862–865). Franklin Co.: 46 = 29.833N 84.876W (1, Crawford 416); 47* = 29.909N 84394W (13, Crawford 431–443); 48 = 29.723N 84.890W (5, Crawford 762–766); 49 = 29.853N 84.664W (1, Crawford 811). Wakulla Co.: 50* = 30.136N 84.326W (13, Crawford 480–489, 829–831). Walton Co.: 51* = 30.309N 86.102W (7, Crawford 696–702).
Georgia. Candler Co.: 52* = 32.355N 81.989W (5, Crawford 587–591).
Louisiana. St. Landry Parish: 53* = 30.540N 92.028W (10, Crawford 296–305). St. Tammany Parish: 54* = 30.298N 89.666W (12, Crawford 309–320).
Oklahoma. Cleveland Co.: 56* = 35.214N 97.328W (10, Crawford 626–635). Garvin Co.: 57* = 34.708N 97.543W (20, Crawford 606–625); 58* = 34.745N 97.548W (25, Crawford 944–968). Johnston Co.: 59* = 34.327N 96.770W (10, Crawford 888–897).
South Carolina. Beaufort Co.: 60* = 32.377N 80.832W (10, Crawford 559–568).
Texas. Harrison Co.: 61* = 34.470N 94.595W (10, Crawford 283–292). Smith Co.: 62* = 32.469N 95.389W (15, Crawford 871–885).
FOOTNOTES
1 The authors thank G. Uno for use of controlled environment facilities and D. Hough for assistance with statistical analyses. Funding was provided by the Graduate Student Senate of the University of Oklahoma and the National Science Foundation (DEB 9303317). This paper represents a portion of a doctoral dissertation by PTC submitted to the University of Oklahoma. 
4 Author for correspondence (ptcrawford{at}ou.edu
)
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•
) refer to populations included in genetic analyses; hollow symbols (
) refer to populations sampled but not included in genetic analyses because of small sample sizes
