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(American Journal of Botany. 2004;91:1757-1766.)
© 2004 Botanical Society of America, Inc.

Genetics and Molecular Biology

Genetics and morphology in a Borrichia frutescens and B. arborescens (Asteraceae) hybrid zone1

Maria V. Cattell2 and Stephen A. Karl3

Department of Biology, SCA 110, University of South Florida, Tampa, Florida 33620 USA

Received for publication January 5, 2004. Accepted for publication July 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Interspecific plant hybridization is a common and evolutionarily important phenomenon. Here, the results of a study of hybridization in the Florida Keys between two species of sea oxeye daisy, Borrichia frutescens and B. arborescens, are reported. Nuclear and chloroplast genetic loci, log-likelihood assignment tests, and maximum likelihood estimates of genealogical class frequencies were used to identify hybrid and parent genotypes, to investigate the utility of leaf and flower morphology for hybrid identification, and to study symmetry and degree of introgression between the species. Genetic analyses confirmed the identity of the hybrid and parent plants that were used for the morphological studies. Together, leaf and flower morphology can be used to identify hybrid and parental types with moderate accuracy (4% error rate). Population genetic analyses indicate that, in spite of a significant level of hybridization, pure B. frutescens and B. arborescens are persisting in the hybrid zone. Of the nonparentals, about 18% appear to be F1 hybrids, over 50% F2 hybrids, and the remainder backcrossed individuals but only with the B. frutescens parent. It is postulated that the hybrid zone in the Florida Keys is being maintained by a combination of positive assortative mating and clonal reproduction.

Key Words: Asteraceae • chloroplast DNA • cytonuclear disequilibrium • Florida, USA • hybrid zone • single-copy nuclear DNA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Interspecific hybridization among plants is a common phenomenon in nature, and hybrid zones are model systems for the study of evolutionary processes (Barton and Hewitt, 1989 ; Rieseberg and Ellstrand, 1993 ; Arnold et al., 1999 ). The evolutionary consequences of hybridization, however, are manifold and include increased genetic diversity of one or both hybridizing parental species through introgression, novel adaptations, breakdown, or reinforcement of reproductive isolating barriers, novel ecotypes or species, and reticulate speciation from introgressive hybridization (Gallez and Gottlieb, 1982 ; Arnold et al., 1991 ; Wendel et al., 1991 ; Arnold, 1997 ). Hybridization also can threaten the persistence of rare species through gene swamping by a more common, reproductively compatible species (Rieseberg and Morefield, 1995 ; Levin et al., 1996 ; Rhymer and Simberloff, 1996 ). Ecological implications of hybridization include the influences of hybrids on the organisms with which they interact, such as competing plant species and herbivorous animals (Strauss, 1994 ; Fritz et al., 1999 ).

For plants, hybridization is thought to play a prominent role in the evolution of new taxa (Grant, 1981 ; Rieseberg, 1995 , 1997 ). Much of the evolutionary significance of hybridization, however, depends on the fate of hybrids, which in turn depends on the fitness of hybrids and their fitness relative to the parental species. Although direct estimates of fitness are difficult at best, the fate of hybrids often can, at least in part, be determined empirically through the assessment of the degree of introgression following hybridization. If the genes of one species are introgressing into another, then the fitness of hybrids must be high enough to allow the hybrids to survive to maturity and backcross with parental individuals.

The initiation of hybridization between taxonomically valid species results from a variety of forces but essentially involves the mixing of gametes of reproductively compatible species. In many instances, hybridization may be a highly transient event and of no real evolutionary consequence. Alternatively, repeated crossing between parental species or stabilization of hybrid breeding systems can result in the formation of hybrid swarms, hybrid zones, or new hybrid species (Grant, 1981 ; Rieseberg, 1997 ). If the hybrid fitness is low relative to the parental species, tension zones may be formed (Barton and Hewitt, 1985 ). Severe fitness consequences of hybrid production favor the evolution of reproductive incompatibility through reinforcement. If the fitness of the hybrids is equal to or greater than the parental species throughout the zone of hybridization, as well as in the range of the parental species, then the parental species are likely to merge. Finally, if hybrids have superior fitness, but only in specific habitats (e.g., ecologically intermediate or disturbed habitats), hybrid zones may be stably maintained (Endler, 1977 ; Moore and Buchanan, 1985 ; but see Arnold, 1997 for explanations not involving selection).

When natural selection plays a role in the maintenance of hybrid zones, the response of genes under selection is expected to be different from neutral genes. Genes from one species that are negatively selected in the alternate species are unlikely to introgress and may only be found in low frequency in the hybrid zone. Contrariwise, neutral genes are free to cross hybrid barriers and introgress into the parental species. Similarly, genes under positive selection outside of the hybrid zone may spread into the alternate parental species (Arnold, 1997 ). The direction and degree of introgression, however, is influenced by the frequency of backcrossing between the hybrids and the parental species, which may be more common than the initial production of F1 hybrids (Arnold, 1997 ).

To assess the degree of introgression, the genealogical classes (parental, F1, F2, backcrosses, etc.) must be accurately differentiated. Intermediacy (i.e., morphological or physiological) might be used in some species in the early stages of introgression to indicate hybrid status but generally is only of limited use. In a review of 46 studies of hybrid morphology, Rieseberg and Ellstrand (1993) concluded that artificially created hybrid plants were no more likely to display intermediate character states than parental ones, and 10% of morphological characters measured in F1 hybrids and 30% in later generation hybrids were shown to be novel or extreme relative to the parental species. The accurate classification of hybrid backcrosses and pure parental genotypes usually is not possible using morphological data alone. The use of molecular genetic markers has circumvented some of the limitations of morphological or physiological characters. Many of these markers are codominant and inherited in a Mendelian fashion, making unambiguous identification of hybrids possible. The use of multiple, independent molecular markers allows the discrimination of backcrossed individuals and an assessment of the degree of introgression (Nason et al., 1992 ; Nason and Ellstrand, 1993 ). Further, studying the distribution of genetic variation and genetic associations at both nuclear–nuclear and nuclear–cytoplasmic loci also provides information on patterns of mating and the direction or symmetry of introgression (Cruzan and Arnold, 1993 ; Cruzan, 1998 ).

In this study, we assess morphological and molecular markers to evaluate the extent of hybridization and introgression in Borrichia frutescens (Linnaeus) de Candolle, B. arborescens (Linnaeus, de Candolle: Asteraceae), and their hybrid, B. x cubana (Britton and Blake), in sympatry in coastal areas of the Florida Keys, Florida, USA. Borrichia is a primarily tropical genus of aster that is divided into six species, three of which occur in North America. Borrichia frutescens is a woody subshrub that generally has silvery, pubescent leaves. The plant's morphology, however, is extremely plastic and has a range of habitat-associated morphologies (P. Stiling, University of South Florida, personal communication; M. V. Cattell, personal observation). Borrichia frutescens grows in saline marshes and estuaries from Maryland to the Florida Keys (Semple and Semple, 1977 ; USDA, NRCS, 1999 ). The congener, B. arborescens, is a larger, woody shrub that can be found in two forms, either with silvery pubescent or completely glabrous leaves. Borrichia arborescens also is found along saline coasts and mangrove marshes, but ranges throughout the Greater Antilles to Bermuda and the Florida Keys and in the west central Caribbean from the Yucatan peninsula to Belize (Semple and Semple, 1977 ). In the continental United States, only the glabrous leaf form of B. arborescens is found and it is never found north of Miami, Florida, USA (Semple and Semple, 1977 ). Both species are perennial, reproduce sexually and clonally, and individuals may live in excess of five years (Antlfinger, 1981 ). In the Florida Keys, where the ranges of the two species overlap, they are known to hybridize extensively (Semple and Semple, 1977 ). The hybrids (taxonomically designated B. x cubana) generally are morphologically intermediate to the parental species; however, like B. frutescens, the hybrids exhibit a range of habitat-associated morphologies. Flowering characteristics such as timing are similar in all three species.

Here, nuclear and cytoplasmic (chloroplast) DNA variation was assessed in areas of parapatry. Symmetry of crossing and introgression were investigated using a cytoplasmic marker in conjunction with nuclear markers. Leaf, inflorescence, and stem morphological traits also were measured and examined for their utility in distinguishing hybrid and parent plants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Sample collections
Samples from 27 different locations throughout the Florida Keys (Fig. 1) were collected between 1996 and 1999. Collecting sites were primarily chosen for the presence of several individuals of each species and to thoroughly cover the geographic range of the area of sympatry. Five to 20 young leaves were collected from single individuals of each of the putative species at each location. Samples were placed in individually marked bags and stored either on dry ice or placed directly into silica gel desiccant. Frozen leaves were kept on dry ice until they were returned to the laboratory and stored at –80°C. Desiccated samples were kept cool until they were returned to the laboratory and placed at –20°C. Because these species extensively propagate clonally, each species at each site generally was sampled only once to minimize inadvertent, multiple sampling of the same individual. However, when groupings of plants (a patch) were separated by a gap of at least 3 m, individuals were sampled from multiple patches at a site. A total of 50 B. frutescens, 53 B. x cubana, and 52 B. arborescens individuals were collected. Samples from putative parental species also were collected from outside the area of overlap. Seven B. frutescens individuals were sampled at Upper Tampa Bay Park in Tampa, Florida, two individuals from Ruskin, Florida, just south of Tampa, and three from individuals on the east coast of Florida near Jacksonville, Florida. Eight B. arborescens individuals were sampled from the coast of Puerto Rico.



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  		Fig. 1. Map of the Florida Keys, Florida, USA, showing hybrid zone collection sites of Borrichia frutescens, B. arborescens, and B. x cubana. Circles with associated site names indicate the general location of plants used for both genetic and morphological studies. Unlabeled circles indicate the general location of plants included in the genetic study only

 
Molecular markers
To obtain single-copy nuclear loci that were diagnostic at the species level, a genomic DNA library was constructed and screened. Total cell DNA from a single individual from Upper Tampa Bay Park was extracted for library construction using a standard hexadecyltrimethylammonium bromide (CTAB) extraction protocol (Milligan, 1992 ) with several modifications. One to two grams of leaf tissue were ground to a fine powder in liquid nitrogen with a mortar and pestle. The macerated tissue was then added to 8 mL of 65°C CTAB extraction buffer and incubated at 65°C for 60 min. After incubation, the solution was extracted twice with 25 : 24 : 1 phenol : chloroform : isoamyl alcohol and once with 24 : 1 chloroform : isoamyl alcohol. DNA was precipitated by the addition of two-thirds volume isopropanol and incubation for 30 min at –20°C. Purified DNA was washed with 95% ethanol, dried, and resuspended in 400 µL of a 10 mmol/L tris-CL and 1 mmol/L ethylenediaminetetraacetic acid (TE) solution.

The library was prepared and screened for single-copy loci according to Karl and Avise (1993) . Briefly, total cell DNA was digested with Sau3A and fragments from 500 bp to 5000 bp were gel isolated. Size-fractioned DNA was ligated into the cloning vector pBSSK + (Stratagene; La Jolla, California, USA), transformed into bacteria, and plated on antibiotic media. Recombinant clones were screened for insert size by restriction endonuclease digestion of mini-DNA preparations (Ausubel et al., 1993 ), and genomic copy number was determined by dot blot analysis using labeled total cell DNA as a probe (Karl and Avise, 1993 ). Once single-copy clones were identified, 18 were arbitrarily chosen from the library for sequencing and primer construction. Cloned inserts were directly sequenced from mini-prep DNA using either the dideoxy chain-termination method (Sanger et al., 1977 ) with a Sequenase version 2.0 sequencing kit (USB; Cleveland, Ohio, USA) and size sorted on acrylamide gels or with an ABI Prism automated sequencing kit (Big Dye or D-rhodamine; Perkin-Elmer, Boston, Massachusetts, USA) and analyzed on an ABI 310 automated sequencer. Sequences were aligned manually using the computer program SeqEd (Applied Biosystems; Foster City, California, USA) and screened for possible polymerase chain reaction (PCR) priming sites using the computer programs OLIGO (Molecular Biology Insights; Cascade, Colorado, USA) and Primer 3 (Rozen and Skaletsky, 1997 ). Primers to 11 clones were synthesized by Cybersyn (Lenni, Pennsylvania, USA), and PCR amplification conditions were optimized using the genomic DNA used in library construction and miniprep vector DNA from the corresponding clone. Exact thermal-cycling parameters varied depending on the primers used and were determined empirically, generally by varying annealing temperature and time (Table 1).


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  		Table 1. Polymerase chain reaction annealing conditions and results of restriction enzymes survey of three individuals of Borrichia frutescens and B. arborescens from Florida USA

 
Primers to three of the 11 single-copy nuclear loci amplified reliably, producing a single product of the expected size and were screened for polymorphic restriction endonuclease sites. One microliter of genomic DNA from three individuals from each species was amplified in 50-µL reactions containing 3.0 mmol/L of each dNTP, 2.5 units Taq polymerase (Promega, Madison, Wisconsin, USA), 2.5 mmol/L MgCl2, 0.10 µmol/L bovine serum albumin (Roche Diagnostics; Indianapolis, Indiana, USA), and 0.41 µmol/L of each primer. Thermal cycling generally consisted of a 2-min denaturation at 95°C, followed by 30 cycles of 1 min at 95°C, annealing (Table 1), extension for 1 min at 72°C, and a single final extension step of 7 min at 72°C.

To check the amount and fidelity of amplification, ~5 µL of each undigested sample was electrophoresed through a 2.0% agarose gel stained with ethidium bromide. Successfully amplified DNA was used directly in restriction digestions, without further purification. Eight microliters of each amplification were digested separately in a 20-µL reaction volume with 10 units of restriction endonuclease enzyme for a minimum of 2 h, according to the manufacturer's recommended reaction conditions (F. Hoffmann-La Roche, Basel, Switzerland). Each locus was digested with up to 26 different enzymes or until a variable site was found. Digested DNA was electrophoresed in 2.5% agarose gels stained with ethidium bromide, and restriction fragment profiles were revealed using short-wave ultraviolet light. Enzymes that produced easily scored restriction fragment profiles that appeared to be species specific were used on the whole set of plant samples. As an additional nuclear locus, the universal PCR primers ITS 4 and 5 (White et al., 1990 ) were used to amplify the internal transcribed spacer region between ribosomal genes. The amplified fragment was digested as described earlier with the same set of restriction enzymes.

To identify a species-specific cytoplasmic marker, several sets of universal chloroplast primers were screened (Taberlet et al., 1991 ; Demesure et al., 1995 ). The fragment generated using the "a" and "b" primers of Taberlet et al. (1991) contained a small, apparently species-specific size difference and was used to genotype all individuals. Chloroplast fragments were digested with restriction enzymes as for the nuclear loci. When digested with HinfI, the restriction endonuclease profile consisted of several smaller fragments, which eased the scoring of the size difference. To verify the presence and size of the indel, one individual of each species was amplified, sequenced, and the sequences aligned as described before.

Individual genotypes
DNA was extracted from leaves using the CTAB procedure detailed before, with modifications as follows. The extractions were scaled down so that they could be completed in 1.5-mL microcentrifuge tubes. For these extractions, a piece of leaf, roughly 1 cm in length (smaller for dried samples) was ground in 100 µL of 65°C CTAB buffer with a small amount of sterile sand and a sterile, reusable plastic pestle. Once the tissue was well macerated, 400 µL of warm CTAB buffer was added. The sample was incubated at 65°C for 1–2 h, and the DNA was extracted organically and purified as for the library construction. The DNA was resuspended in 100 µL TE. The samples were further purified for PCR amplification by spinning them through Microcon 100 000 nominal molecular weight limit spin columns. The final sample volume was adjusted to 50 µL with TE.

Morphological measurements
Leaf samples for genetic analysis were taken from five stems per plant of each of the three putative species that were haphazardly collected from each of two individuals within five sites (Fig. 1; Card Sound, Teatable, Boot Key, Little Torch, and Rockland). Within several hours of collection; the diameter of the stem tip and the length, width, and thickness of the third leaf from the meristem were measured with dial calipers to the nearest one-hundredth of a millimeter. If flowers were present, the height of the inflorescence was measured from the base of the phyllaries to the base of the ray flowers. The width of the inflorescence was measured at the widest point, and the length of the first pair of phyllaries was measured. These morphological traits were chosen because they appeared to differ between the parent species and could be measured reliably in the field before leaves began to wilt.

Data analysis
Estimates of Hardy-Weinberg genotypic frequency equilibrium (HWE), linkage disequilibrium, FST, and log-likelihood (LL) assignment of individual genotypes to species (B. frutescens or B. arborescens) for standard, genotypic, co-dominant data with gametic phase unknown were calculated using Arlequin version 2.0 (Schneider et al., 2000 ). Program default settings were used for all the genetic analyses. Cytonuclear disequilibrium (CND; D, D1, D2, and D3) values were estimated using the CND computer package (Asmussen and Basten, 1994 ; Basten, 1996 ). Here, D measures the nuclear allelic disequilibrium, D1 measures the disequilibrium between one cytotype and one homozygous genotype, D3 measures the disequilibrium between the same cytotype and the other homozygous genotype, and D2 measures the disequilibrium between the cytotype and the heterozygous genotype (Asmussen et al., 1987 ; Arnold, 1993 ; Asmussen and Basten, 1994 ). Cytonuclear disequilibria were only calculated between the chloroplast and the nuclear loci ITS and 6G3. The 2D4 and 3F6 loci were not alternately fixed in the pure parental species making this analysis inappropriate (Arnold, 1993 ). Linear regression analysis of the LL genotype scores vs. site of collection for all individuals was used to indicate geographical localization of parental and hybrid genotypes.

The occurrence and extent of introgression and hybridization was assessed following the hierarchical hypothesis testing approach of Nason and Ellstrand (1993) . Individuals were assigned to one of seven genotypic categories based on their multilocus nuclear genotypes at loci with species-unique alleles. Individuals were classified as pure parental A or B (genealogical classes P1 and P2 for B. frutescens and B. arborescens, respectively), first filial hybrids H (F1), second filial hybrids S (F2), or backcrosses A1 or B1 (BP1 and BP2; Nason and Ellstrand, 1993 ). A series of hierarchical hypotheses was tested, in which the presence of nonparental multilocus genotype classes indicates hybridization and the presence of second filial or backcross genotype classes indicates introgression. Maximum likelihood estimates of each class were used to test for the significance between a reduced model, assuming no backcrossed individuals, and a full model, with all six classes present. These models were tested constraining the estimates to values between zero and one and unconstrained. A log-likelihood ratio tested with a chi-square statistic indicates statistical significance between the models (for computational details, see Nason and Ellstrand, 1993 ). Standard deviations of the genotype class frequencies were estimated by bootstrapping with 600 replicates.

Morphological data were tested for significant differences between species using one-way analysis of variance (ANOVA). Square root or log10 transformations were used when necessary to fit model assumptions. When assumptions could not be met, Kruskal–Wallace nonparametric tests were applied using species as the grouping variable. Morphological data were tested for reliability in discriminating plant genotypes using canonical discriminant function analyses of the means of measurements within plant patches (each patch is likely a single individual). To help determine which type of data is most informative, discriminant function analyses were applied to all of the morphological characters separately, as well as together, for flower and leaf morphological data sets. Three of the plants did not have flowering stems and were excluded from analyses. The proportion of the total variance in the discriminant scores that is not explained by the group differences (Wilks' {lambda}) was calculated and transformed to approximate a chi-square distribution test of significance of differences between group centroids. Eigenvalues (ratio of between-groups sums of squares to the error sum of squares) also were calculated to help estimate the spread of the group centroids. These statistics were done using SPSS version 7.5 (SPSS, Chicago, Illinois, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Molecular markers
Per locus variation ranged from 0.00 to 13.64% in B. frutescens and from 0.00 to 12.50% in B. arborescens (Table 2). The enzymes HaeIII, EcoRI, AluI, and RsaI produced the most reliable and easily scored species-specific polymorphisms for ITS, 6G3, 2D4, and 3F6, respectively, and were used to genotype all individuals. The chloroplast locus was size polymorphic.


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  		Table 2. Locus variability revealed by restriction endonuclease digestion

 
Screening 175 individuals at the four nuclear loci and one chloroplast locus resolved 37 unique multilocus genotypes (Table 3). Assessment of putative pure parental species outside of the Florida Keys (i.e., in parapatry) revealed that the nuclear loci ITS and 6G3 were essentially fixed for different alleles in B. frutescens and B. arborescens (Table 3). For convenience, nuclear alleles that were predominantly B. frutescens are capitalized and those that were predominantly B. arborescens are lowercase throughout the text. Loci 2D4 and 3F6 were not fixed for alternate alleles in the parent species but did exhibit significant species-specific allele frequencies (FST,2D4 = 0.448, P < 0.001; FST,3F6 = 0.334, P < 0.001). Parapatric B. frutescens individuals possessed only one 2D4 allele (D), while an alternate allele (d) was found in all parapatric and most putative B. arborescens individuals in the hybrid zone. A similar situation was true for locus 3F6 with parapatric B. arborescens individuals fixed for one allele (f), and a second allele (F) was found in all parapatric and most putative B. frutescens individuals in the hybrid zone. Both the "d" and "F" alleles were considered to be species unique. Two chloroplast haplotypes were identified: a B. frutescens haplotype found in all parapatric B. frutescens individuals and a B. arborescens haplotype found in all of the presumed pure B. arborescens individuals from the Florida Keys. Unexpectedly, the eight putatively pure B. arborescens individuals from Puerto Rico contained the B. frutescens chloroplast haplotype.


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  		Table 3. Individual genotypes and log-likelihood assignment probabilities for 175 Borrichia individuals in this study. Capital letters indicate the typically B. frutescens alleles and lowercase letters indicate the typically B. arborescens alleles. In general, lower-numbered multilocus genotypes were more B. frutescens-like and the higher-numbered ones more B. arborescens-like. Assignment probabilities were calculated excluding the chloroplast locus; therefore, individuals with identical nuclear but different chloroplast genotypes have identical log-likelihood values

 
Population genetic analysis
When all plants (i.e., sympatric and parapatric) were lumped together, there were significant deviations (P ≤ 0.001) from HWE expectations at ITS, 6G3, and 3F6. In each case, there were fewer than expected heterozygotes. Additionally, while the deviation from HWE expectation was not statistically significant for locus 2D4 (P = 0.165), the direction was the same as for the other loci (fewer than expected heterozygotes). Significant linkage disequilibrium also was found between all possible nuclear locus pairs (P ≤ 0.001). Both of these results are consistent with lumping genetically differentiated groups (e.g., pure parentals in parapatry). When morphologically classified, B. frutescens and B. arborescens species were analyzed separately regardless of collection location, no significant HWE deviations were indicated (PITS and 6G3 = 1.000 for both species, P2D4 = 0.135 for B. frutescens and 0.071 for B. arborescens, P3F6 = 0.599 for B. frutescens and 1.000 for B. arborescens). For B. x cubana, however, there were significantly more heterozygotes at ITS and 6G3 (P = 0.004) and significantly fewer heterozygotes at 3F6 (P < 0.001) than expected, but no significant deviation at 2D4 (P = 1.000). All species pairwise FST values were fairly large and statistically significant (P ≤ 0.001; B. frutescens vs. B. x cubana FST = 0.188, B. frutescens vs. B. arborescens FST = 0.788, and B. x cubana vs. B. arborescens FST = 0.446).

Significant cytoplasmic-nuclear disequilibrium was found between the B. frutescens chloroplast haplotype and the nuclear genotypes at ITS and 6G3 in the Keys individuals. The D and D1 values were significant (P ≤ 0.05) and positive (DITS = 0.1780, D6G3 = 0.1813, D1,ITS&6G3 = 0.1841) and D3 was significant and negative (D3,ITS = –0.1720 and D3,6G3 = –0.1785). D2 values (–0.0121 and –0.0056 for ITS and 6G3, respectively) were not significantly different from zero. This analysis indicates that the B. frutescens cytotype is found more often with homozygous B. frutescens nuclear genotypes (D1) and less often with homozygous B. arborescens nuclear genotypes (D3) than expected under random mating. There is no trend for the heterozygous state to be found with either chloroplast type (D2) more often than expected under random mating.

Following Nason and Ellstrand (1993) , the maximum likelihood estimates of genotypic class frequencies also indicated a significant amount of hybridization (Table 4). Both the reduced model, for which the backcross frequencies were set to zero, and the full model, which included all classes, gave similar results estimating that about 32% of the Keys population consisted of nonparental types. Relative to the likelihood of the reduced model (Ln = –16.5082), the full model was a significantly better fit to the data in both the constrained (Ln = –10.50172, P ≤ 0.01) and unconstrained (–12.8930, P ≤ 0.05) analyses. Regardless of the model, most of the nonparental individuals appeared to be second generation hybrids (F2; ~20 to ~25% of all individuals) with very few classified as first generation hybrids (F1; ~7%; Table 4). A statistically significant number of individuals was estimated to be backcrossed to B. frutescens (~10%) but not to B. arborescens (Table 4). The estimated number of hybrids backcrossed to B. arborescens was negative, but not significantly so.


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  		Table 4. Maximum likelihood frequency estimates of genealogical classes in the Florida Keys with (full model) and without (reduced) the assumption of backcrossing and with and without constraining the estimates between zero and one. Borrichia frutescens is P1 and B. arborescens is P2

 
Assignment tests
Results of the LL genotype assignment analysis are plotted in Fig. 2 and the likelihood values are in Table 3. The genotypes at the far extremes of the spread of the points are most likely pure parental genotypes. In general in the multilocus genotypes assignment, low genotype numbers are more B. frutescens-like and genotypes 1 to 3 and 7 are likely pure B. frutescens individuals. Higher numbered genotypes are more B. arborescens-like and genotypes 34 to 37 are probably of pure B. arborescens individuals. The genotypes that fall in the center of the distribution of Fig. 2 are intermediate in LL score and most likely are hybrids and backcrosses. Log-likely scores were not, however, geographically assorted, and extreme, as well as intermediate LL scores, were found at nearly all sites (Fig. 3). A linear regression of LL genotype score vs. geography for the putatively B. x cubana individuals results in an insignificant slope of 0.0051 with an r2 = 0.0037 (Fig. 3).



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  		Fig. 2. Log-likelihood of each genotype being assigned to Borrichia frutescens or B. arborescens. Assignment analysis is based on nuclear genotypes only. The numbers in the graph correspond to the numbers assigned to the genotypes in Table 3 . Individuals that are most like B. arborescens are located in the lower right corner and those most like B. frutescens are in the upper left. Individuals with log-likelihood in the center of the graph are likely hybrids and backcrosses

 


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  		Fig. 3. Plot of the B. frutescens divided by B. arborescens log-likelihood genotype assignment scores vs. geographic site of collection for samples from the Florida Keys only. Circles are individuals with nuclear genotypes identical to B. frutescens found in parapatry, squares are individuals with nuclear genotypes identical to B. arborescens found in parapatry, and triangles are all other genotypes (i.e., not pure parental)

 
Morphological characteristics
All B. frutescens that were morphologically measured had either genotype 2 or 3, the B. x cubana individuals had genotypes 9, 15, 24, 25, 28, or 29, and B. arborescens individuals had genotypes 36 or 37 (Table 3). Individuals that were genetically identified as hybrids were not always intermediate to the parents in the morphological characters. Significant differences between plant species were identified for leaf length, leaf thickness, inflorescence height, first phyllary length, flower number, and stem tip diameter (P ≤ 0.001; data not shown). Putative hybrids were on average intermediate in measurement for all measured morphological characters, except leaf length, for which hybrids were the shortest. No difference was found for leaf and inflorescence width.

Discriminant function analyses (DFA) indicate that leaf morphology provides good discrimination along function 1 for pure B. arborescens vs. pure B. frutescens or B. x cubana (Fig. 4A). Borrichia frutescens and the hybrid were better differentiated along function 2; however, six individuals were misgrouped, three genetically B. frutescens individuals were grouped with B. x cubana, and three genetic hybrids were grouped with B. frutescens (Fig. 4A). Flower morphology alone did not discriminate as well between the plant species; 11 individuals were grouped incorrectly. The B. frutescens and B. arborescens clusters were relatively well defined, but B. x cubana individuals clustered with one or the other parental species and did not form a third grouping (Fig. 4B). The spread of the group centroids (as indicated by the eigenvalues for function 1; leaf = 2.973, flower = 1.404, combined morphological = 13.967) and the proportion of the discriminant scores that are not explained by differences among groups (Wilks' {lambda} values for function 1 through 2; leaf = 0.219, flower = 0.340, combined morphological = 0.036; P < 0.05) also indicate that leaf morphological characters are better at discriminating the groups than flower morphology. Discrimination among the groups was best when all morphological data were included in the analysis (Fig. 4C). Here, only one of 27 individuals was misgrouped (a B. x cubana individual was classified as B. frutescens). The magnitudes of the eigenvalues indicate that the spread between the group centroids was greatest when using all characters. Wilks' {lambda} indicates that 96.4% of the variance in the DFA scores was explained by among-group differences.



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  		Fig. 4. Discriminant function analyses of morphological characters for (A) leaf morphology only, (B) flower morphology only, and (C) all of the morphological characters combined. Clusters of genotype groups as defined by the DFAs are enclosed in the lines with a dashed line for B. frutescens, a solid line for B. arborescens, and a dotted line for B. x cubana. Circles are B. frutescens individuals, squares are B. arborescens, and triangles are B. x cubana. Stars indicate species cluster centroids

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The application of a molecular genetic approach to hybridization and introgression in Borrichia proved quite useful. Multilocus genotypes based on one cytoplasmic and four nuclear markers were able to resolve a variety of genotypes and genotypic classes (e.g., backcrosses). Using an LL assignment to species resulted in a continuum of LL values ranging from very likely B. frutescens and unlikely B. arborescens to very unlikely B. frutescens and likely B. arborescens. The B. frutescens individuals from outside of the hybrid zone (genotypes 2, 3, and 7) corresponded to large B. frutescens LL values and small B. arborescens values (Table 3 and Fig. 2), supporting the assertion that they are pure parental types. Morphologically putative pure B. frutescens found within the hybrid zone had genotypes 1 to 3, and large B. frutescens-like LL and small B. arborescens-like LL. Borrichia arborescens individuals from outside the hybrid zone (i.e., Puerto Rico) had genotype 35, and LL scores, indicating clear B. arborescens-like genotypes. Individuals that were morphologically B. arborescens-like within the zone had genotypes 34, 36, or 37 and similarly high LL assignment to B. arborescens. Many genotypes had more moderate or nearly equal LL scores for both species assignments, indicating that they are likely hybrids (center of the graph in Fig. 2). These data clearly indicate that a considerable amount of hybridization must be taking place in the Florida Keys. It also is clear that not all individuals in the Florida Keys are hybrids and that pure parental types are persisting sympatrically with hybrids (Fig. 3).

Not all markers were alternately fixed for species-specific alleles; however, our approach as outlined by Nason and Ellstrand (1993) allowed a robust estimation of genealogical classes from the observed genotypic data. Maximum likelihood estimates of genealogical class based on observed multilocus genotypes also indicated that a significant amount of hybridization and backcrossing is taking place. In the Florida Keys hybrid zone, most individuals were either pure parental types or later generation hybrids (i.e., F2 or backcrosses; Table 4). Only about 7% of the individuals were of the F1 hybrid class. This low frequency of F1 individuals has been observed often in other plant hybrid systems and is believed to be characteristic of a system in which the formation of the initial hybrid generation (i.e., F1) is a difficult or an infrequent event (Arnold et al., 1992 , 1993 ; Nason et al., 1992 ) probably as a result of pre- and post-fertilization barriers between the parental species. Once this compatibility barrier is breached, however, further crossings are less difficult and more likely (Arnold, 1997 ). Considering the cytonuclear disequilibrium analysis, however, the insignificant D2 value indicates that there does not seem to be asymmetry in the direction of hybridization. Apparently then, the barrier to hybridization is equal regardless of which species is the maternal or the paternal parent. In addition, the maximum likelihood estimates of genotype classes indicate that backcrossing is a significant event in this hybrid zone. Unlike hybridization, backcrossing does not; however, appear to be symmetrical with respect to parental species. Of the nonparental genealogical classes estimated in the constrained full model, 55.4% of them are BP1 and none are BP2. Clearly, fertilization between hybrids and B. frutescens is more easily or frequently accomplished than with B. arborescens. It should be noted, however, that, given the small sample size (53 hybrid individuals), while backcrossing with B. arborescens clearly is less common, we cannot rule it out entirely.

The discriminant function analyses of the morphological characters suggest that hybrid plants are more morphologically distinct from B. arborescens than they are from B. frutescens (Fig. 4). In all of the analyses, the B. arborescens cluster and group centroid were set farther apart from the B. frutescens and B. x cubana clusters and centroids than the latter two were from each other. Additionally, only four individuals clustering outside their groups involved B. arborescens. It is important to note that the misclassifications occurred when considering flower morphology alone—the least differentiated of the morphological comparisons. All other misclassifications by the DFA were between B. frutescens and B. x cubana (six with leaf morphology alone, nine with flower morphology alone, one with all morphological data). Interestingly, this is consistent with the hybrid, B. x cubana, consisting of a larger portion of individuals backcrossed to B. frutescens than to B. arborescens. Finally, although all individuals with high LL assignment values were of the appropriate morphology, not all putative species assignments based on morphology were extremes in LL score. These results, when considered together, suggest that morphological characters may not provide very accurate information on the identity of Borrichia species within the region of species overlap. This is particularly true when differentiating pure B. frutescens from hybrid plants. If morphological assignments are going to be made, these data clearly indicate that several morphological characters must be measured to accurately assess identity.

There are several factors, such as the distribution of the parental plants and how long hybridization has been taking place, that likely contribute to the genetic patterns observed in the Florida Keys. While it is difficult to determine with certainty how long the Borrichia species in the Florida Keys have been hybridizing, it is possible that the plants may have been there for a long time. It has been estimated that the limestone of south Florida has been exposed for from 5000 to 8000 years, suggesting the potential for a long period of evolution in the Florida Keys (Snyder et al., 1990 ). Thus the habitat may have been available to these plants for up to 8000 years. If true, and in the absence of other evolutionary forces, hybridization and random mating likely would have led to equilibrium conditions by now, and few if any pure parental individuals would be expected in the Keys. This is clearly not the case.

If the hybrid zone is relatively young, historic parental genetic association would still exist and result in the observed high frequency of parental types. Although there is some indication that these Borrichia species have been sympatric for some time, it is possible that they have not been hybridizing for the duration of their sympatry. Semple and Semple (1977) suggest that hybridization has only recently occurred as a result of disturbance in the Florida Keys and that less disturbed habitats tended to be occupied by only one of the parent species. Additionally, even if the hybrid zone is old in terms of the number of years since hybridization began, it may be relatively young in terms of the number of generations that have passed. These Borrichia species are highly clonal and invest a relatively small amount into reproductive structures (Semple and Semple, 1977 ). Generation time for these plants is unknown and is generally difficult to establish for clonal plants; however, vegetatively reproducing genets may persist for hundreds or thousands of years (Wolf et al., 2000 ). It is therefore possible that what we are detecting is the early formation of a hybrid zone. The few putatively F1 hybrid individuals identified also may be pointing to a young age for the hybrid zone because at the onset of hybridization, only a minority of the plants would have hybrid genotypes. This is not supported, however, by prevalent occurrence of later generation hybrids and backcrosses. Given this and that suitable habitat likely has been available for quite some time, we believe that the hybrid zone is not a recent creation but that other factors are maintaining pure parentals and hybrids in sympatry.

If this were a geographically simple hybrid zone with a majority of pure parental genotypes at the extreme ends of the region of overlap and relatively few parental genotypes in the center, then selection against hybrids at the extremes and/or migration of pure parental individuals to the center could account for the observed genetic patterns. Selection against hybrid genotypes in a hybrid zone also could cause a reduction of heterozygotes and significant disequilibrium. Selection, however, would have to be acting directly on the loci under study, or closely linked loci, because neutral alleles can diffuse through hybrid zones and parental genetic associations should be disrupted by recombination (Arnold et al., 1987 ; Hewitt, 1988 ; Marchant et al., 1988 ). It is unlikely, however, that selection is acting directly on the restriction site polymorphisms at the loci used here (see Karl and Avise, 1993 , for a discussion of the neutrality of restriction site polymorphisms at single-copy nuclear loci). Additionally, they could be linked to loci under selection; however, this seems unlikely to be the case simultaneously for four different nuclear loci, as well as for a cytoplasmic one.

Migration from pure parental populations into a hybrid zone also can cause continued association of nuclear and cytoplasmic genotypes after long periods of hybridization by continually reintroducing the parental gene combinations into the zone. In this study, there was no apparent association between genotype LL score and geography throughout the hybrid zone. Individuals with genotype LL scores similar to those found in both species in parapatry (i.e., putative pure parental types) were observed at all sites along with ones that were clearly hybrids (Fig. 3). Individuals from throughout the range of LL scores were found at nearly all sites north to south, and linear regression analysis of the LL scores of putative hybrid individuals vs. collection site resulted in an insignificant slope. For migration to result in the pattern of geographic distribution and cytonuclear disequilibrium found in this study, migration rates into the Florida Keys from both pure parent plant populations would have to be similar. Equal rates of migration from pure B. frutescens and pure B. arborescens populations outside of the zone of hybridization are unlikely from geographical considerations. The Florida Keys hybrid population is contiguous with B. frutescens individuals to the north, but is clearly disjunct from B. arborescens populations in the Caribbean. The closest B. arborescens individuals are in the Bahamas and Cuba, each of which is separated from the Florida Keys by at least 80 km of ocean. It seems reasonable, therefore, to expect a greater degree of migration and gene flow from pure B. frutescens populations into the hybrid zone than from B. arborescens. The genealogical class analysis does indicate a significant amount of backcrossing to B. frutescens (9.44 ± 6.13%) but not to B. arborescens (0.00 ± 4.05%), and this may be due to the presence of the northern reserve of pure B. frutescens. Notably, although Semple and Semple (1977) successfully produce viable offspring from artificial crosses involving B. arborescens and B. x cubana, no progeny were produced from B. frutescens and B. x cubana crosses. Nonetheless, given our sample size, the large standard errors on the maximum likelihood (ML) estimates, and the overall low level of backcrossing, it is difficult to conclude true asymmetry in backcrossing or persistence of parental types in the hybrid zone due to migration.

We believe that the clonal nature of these species and predominantly positive assortative mating explain the genetic data from the Florida Keys. Clonal propagation would have two principle effects. First, if clonal propagation were the dominant form of reproduction, the opportunity for hybridization through necessarily sexual means would be reduced and the resultant merging of the parental species would be slowed. Second, and more important here, allowing a significant contribution of both sexual and clonal reproduction might simultaneously produce frequent opportunities for hybridization while increasing the longevity of the parental individuals in the hybrid zone. Clonality also allows for the persistence and growth of relatively uncommon, presumably difficult-to-create F1 hybrids, increasing the likelihood of backcrossing and production of later generation hybrids. The FST values calculated indicate that there is genetic differentiation between the three groups of plant types (pure parents and hybrid). The FST values also indicate that there is a greater degree of population differentiation between the parents than there is between both parents and the hybrid group, as expected with assortative mating. For the most part then, in spite of hybridization and backcrossing, genetic differentiation between the parental species is being maintained. Random mating between the plants in this hybrid zone, therefore, seems unlikely. The population genetic patterns found suggest that the Florida Keys population likely is exhibiting positive assortative mating of the parental species. No data, however, are currently available regarding the mechanisms of assortative mating. Considerably more information is needed on the specifics of the frequency of sexual reproduction, mechanisms of pollination, longevity of clonal units, and the geographic distribution and frequency of genotypes to fully understand the evolutionary history of this complex region of hybridization.


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  		APPENDIX. Primer sequences for nuclear loci screened in this study

 

   FOOTNOTES
 
1 The authors thank M. Asmussen, S. Bell, B. Cochrane, E. McCoy, J. Nason, A. Schnabel, P. Stiling, S. Ravdal, M. Roberts, A. Rossi, E. Severance, and J. T. Streelman for helpful suggestions on experimental design, laboratory protocols, field work, and data analysis. J. Nason kindly shared analysis programs. We also thank The Nature Conservancy for providing access to plants on Little Torch Key. This research was supported in part by a National Science Foundation Program in Systematics grant (DEB 98-06905) to SAK. Back

2 Present address: 5497 Gunbarrel Road, Longmont, Colorado 80503 USA Back

3 E-mail: karl{at}mail.cas.usf.edu Back


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