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American Journal of Botany 91(11): 1757–1766. 2004.


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

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.

Interspecific hybridization among plants is a common phe- brids must be high enough to allow the hybrids to survive to
nomenon in nature, and hybrid zones are model systems for maturity and backcross with parental individuals.
the study of evolutionary processes (Barton and Hewitt, 1989; The initiation of hybridization between taxonomically valid
Rieseberg and Ellstrand, 1993; Arnold et al., 1999). The evo- species results from a variety of forces but essentially involves
lutionary consequences of hybridization, however, are mani- the mixing of gametes of reproductively compatible species.
fold and include increased genetic diversity of one or both In many instances, hybridization may be a highly transient
hybridizing parental species through introgression, novel ad- event and of no real evolutionary consequence. Alternatively,
aptations, breakdown, or reinforcement of reproductive isolat- repeated crossing between parental species or stabilization of
ing barriers, novel ecotypes or species, and reticulate specia- hybrid breeding systems can result in the formation of hybrid
tion from introgressive hybridization (Gallez and Gottlieb, swarms, hybrid zones, or new hybrid species (Grant, 1981;
1982; Arnold et al., 1991; Wendel et al., 1991; Arnold, 1997). Rieseberg, 1997). If the hybrid fitness is low relative to the
Hybridization also can threaten the persistence of rare species parental species, tension zones may be formed (Barton and
through gene swamping by a more common, reproductively Hewitt, 1985). Severe fitness consequences of hybrid produc-
compatible species (Rieseberg and Morefield, 1995; Levin et tion favor the evolution of reproductive incompatibility
al., 1996; Rhymer and Simberloff, 1996). Ecological impli- through reinforcement. If the fitness of the hybrids is equal to
cations of hybridization include the influences of hybrids on or greater than the parental species throughout the zone of
the organisms with which they interact, such as competing hybridization, as well as in the range of the parental species,
plant species and herbivorous animals (Strauss, 1994; Fritz et then the parental species are likely to merge. Finally, if hybrids
al., 1999). have superior fitness, but only in specific habitats (e.g., eco-
For plants, hybridization is thought to play a prominent role logically intermediate or disturbed habitats), hybrid zones may
in the evolution of new taxa (Grant, 1981; Rieseberg, 1995, be stably maintained (Endler, 1977; Moore and Buchanan,
1997). Much of the evolutionary significance of hybridization, 1985; but see Arnold, 1997 for explanations not involving se-
however, depends on the fate of hybrids, which in turn de- lection).
pends on the fitness of hybrids and their fitness relative to the When natural selection plays a role in the maintenance of
parental species. Although direct estimates of fitness are dif- hybrid zones, the response of genes under selection is expected
ficult at best, the fate of hybrids often can, at least in part, be
to be different from neutral genes. Genes from one species
determined empirically through the assessment of the degree
that are negatively selected in the alternate species are unlikely
of introgression following hybridization. If the genes of one
species are introgressing into another, then the fitness of hy- to introgress and may only be found in low frequency in the
hybrid zone. Contrariwise, neutral genes are free to cross hy-
brid barriers and introgress into the parental species. Similarly,
Manuscript received 5 January 2004; revision accepted 2 July 2004.
The authors thank M. Asmussen, S. Bell, B. Cochrane, E. McCoy, J. Nason, genes under positive selection outside of the hybrid zone may
A. Schnabel, P. Stiling, S. Ravdal, M. Roberts, A. Rossi, E. Severance, and spread into the alternate parental species (Arnold, 1997). The
J. T. Streelman for helpful suggestions on experimental design, laboratory direction and degree of introgression, however, is influenced
protocols, field work, and data analysis. J. Nason kindly shared analysis pro- by the frequency of backcrossing between the hybrids and the
grams. We also thank The Nature Conservancy for providing access to plants parental species, which may be more common than the initial
on Little Torch Key. This research was supported in part by a National Science
Foundation Program in Systematics grant (DEB 98-06905) to SAK. production of F1 hybrids (Arnold, 1997).
Present address: 5497 Gunbarrel Road, Longmont, Colorado 80503 USA. To assess the degree of introgression, the genealogical clas-
E-mail: ses (parental, F1, F2, backcrosses, etc.) must be accurately dif-

Because these species exten- gener. Rie- seberg and Ellstrand (1993) concluded that artificially created hybrid plants were no more likely to display intermediate char- acter states than parental ones. USA (Semple ida. Purified DNA was washed . Molecular markers—To obtain single-copy nuclear loci that were diag- tensively (Semple and Semple. arborescens individuals were the west central Caribbean from the Yucatan peninsula to Be. and isopropanol and incubation for 30 min at 2208C. morphological or physiological) might be used in some species in the early stages of intro- gression to indicate hybrid status but generally is only of lim- ited use. Florida. is a larger. reproduce sex. then added to 8 mL of 658C CTAB extraction buffer and incubated at 658C Here. several modifications. Leaf. Collecting sites woody subshrub that generally has silvery. 1992. Borrichia arborescens also is found along However. Symmetry of crossing and 1 phenol : chloroform : isoamyl alcohol and once with 24 : 1 chloroform : introgression were investigated using a cytoplasmic marker in isoamyl alcohol. In the Florida Keys. 3 cubana. and individuals may live in excess of five pled from the coast of Puerto Rico. 1993. Borrichia frutescens grows in saline bags and stored either on dry ice or placed directly into silica gel desiccant.1758 AMERICAN JOURNAL OF BOTANY [Vol. collection sites of Borrichia frutescens. only once to minimize inadvertent. Florida. 1977). putative species at each location. In this study. individuals were sampled from multiple patches at a site. thylammonium bromide (CTAB) extraction protocol (Milligan. Florida near Jacksonville. Intermediacy (i. the solution was extracted twice with 25 : 24 : was assessed in areas of parapatry. NRCS. 1999). 1977. frutescens individuals were sampled at States. like B. One to two grams of leaf tissue were ground to a fine phologies. A total the Greater Antilles to Bermuda and the Florida Keys and in of 50 B. either with silvery pubescent or complete. they are known to hybridize ex. 1998). Florida. ly glabrous leaves. showing hybrid zone netic associations at both nuclear–nuclear and nuclear–cyto. personal communication. marshes and estuaries from Maryland to the Florida Keys Frozen leaves were kept on dry ice until they were returned to the laboratory (Semple and Semple. Flor- and it is never found north of Miami. The use of mul- tiple. arborescens.e. we assess morphological and molecular mark- ers to evaluate the extent of hybridization and introgression in Borrichia frutescens (Linnaeus) de Candolle. each species at each site generally was sampled found in two forms. B. collected. Nason and Ellstrand. making un- ambiguous identification of hybrids possible. Eight B. 3 cubana) generally are morphologically Park was extracted for library construction using a standard hexadecyltrime- intermediate to the parental species. M. The con. when groupings of plants (a patch) were separated by a gap of at saline coasts and mangrove marshes. arborescens. nuclear and cytoplasmic (chloroplast) DNA variation for 60 min. de Candolle: Asteraceae). 3 for their utility in distinguishing hybrid and parent plants. nostic at the species level. Borrichia is a primarily trop. Total cell DNA from a single individual from Upper Tampa Bay ically designated B. frutes. The macerated tissue was in all three species.. The use of molecular genetic markers has circumvented some of the limitations of morpho- logical or physiological characters. Both species are perennial. B. 1993). two individuals from Ruskin. Stiling. arborescens individuals were sam- ually and clonally. In the continental United outside the area of overlap. the hybrids exhibit a range of habitat-associated mor. 1977). USA. DNA was precipitated by the addition of two-thirds volume conjunction with nuclear markers. and stored at 2808C. USDA. USA. 1992) with cens. and 52 B. 1. Borrichia frutescens is a Florida Keys (Fig. Florida. only the glabrous leaf form of B. 1) were collected between 1996 and 1999. 3 cubana. cubana (Britton and Blake). just south of Tampa. three of Sample collections—Samples from 27 different locations throughout the which occur in North America. MATERIALS AND METHODS ical genus of aster that is divided into six species. studying the distribution of genetic variation and ge- Fig. After incubation. but ranges throughout least 3 m. Florida. 1981). 91 ferentiated. V. and three from individuals on the east coast of and Semple. Samples were placed in individually marked personal observation). Samples from putative parental species also were collected from lize (Semple and Semple. multiple sampling of the same individual. The hybrids (taxonom. In a review of 46 studies of hybrid morphology. frutescens. independent molecular markers allows the discrimination of backcrossed individuals and an assessment of the degree of introgression (Nason et al. Cattell.. and their hybrid. and B. however. Map of the Florida Keys. Unlabeled circles indicate the Arnold. Flowering characteristics such as timing are similar powder in liquid nitrogen with a mortar and pestle. woody shrub that can be sively propagate clonally. arborescens stem morphological traits also were measured and examined (Linnaeus. Five a range of habitat-associated morphologies (P. Many of these markers are codominant and inherited in a Mendelian fashion. B. in sympatry in coastal areas of the Florida Keys. Cruzan. The accurate classification of hybrid back- crosses and pure parental genotypes usually is not possible using morphological data alone. were primarily chosen for the presence of several individuals of each species The plant’s morphology. is extremely plastic and has and to thoroughly cover the geographic range of the area of sympatry. pubescent leaves. years (Antlfinger. Univer. however. general location of plants included in the genetic study only. Seven B. Further. to 20 young leaves were collected from single individuals of each of the sity of South Florida. B. 1977). arborescens is found Upper Tampa Bay Park in Tampa. inflorescence. where the ranges of the two species overlap. 53 B. 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. a genomic DNA library was constructed and screened. Desiccated samples were kept cool until they were returned to the laboratory and placed at 2208C. plasmic loci also provides information on patterns of mating Circles with associated site names indicate the general location of plants used and the direction or symmetry of introgression (Cruzan and for both genetic and morphological studies.

an ABI 310 automated sequencer. Hoffmann-La Roche. Boot Key. for 1 min at 728C. As an additional nuclear locus. and screened for possible polymerase chain reaction (PCR) priming sites using and the sequences aligned as described before.November 2004] CATTELL AND KARL—INTERSPECIFIC HYBRIDIZATION IN BORRICHIA 1759 TABLE 1. Sequences were aligned manually using the which eased the scoring of the size difference. was well macerated. Annealing Species-specific Locus temperature (8C) Annealing time (s) Fragment size (bp) No. The extractions were fication conditions were optimized using the genomic DNA used in library scaled down so that they could be completed in 1. Cleveland. a piece of leaf. 18 were To identify a species-specific cytoplasmic marker.0 sequencing kit (USB. 400 mL of warm CTAB buffer was added. or until a variable site was found. Indiana. one individual of each species was amplified. Exact For these extractions. USA). and PCR ampli. and genomic copy number amplified fragment was digested as described earlier with the same set of was determined by dot blot analysis using labeled total cell DNA as a probe restriction enzymes. 1993).5-mL microcentrifuge tubes. 1.. purified as for the library construction. b This locus was size polymorphic. Once the tissue (Table 1).. Size-fractioned DNA ily scored restriction fragment profiles that appeared to be species specific was ligated into the cloning vector pBSSK 1 (Stratagene. The fragment generated using the ‘‘a’’ and ‘‘b’’ primers of Taberlet et dideoxy chain-termination method (Sanger et al. manufacturer’s recommended reaction conditions (F. the diameter of the stem tip and the length. Ba.41 mmol/L of Morphological measurements—Leaf samples for genetic analysis were each primer. Massachusetts. pro. Wisconsin. 1977) with a Sequenase al. Digested DNA was electrophoresed in 2. Primers to 11 clones Individual genotypes—DNA was extracted from leaves using the CTAB were synthesized by Cybersyn (Lenni. reusable plastic pestle. The samples were further purified for PCR amplification by spinning them three individuals from each species was amplified in 50-mL reactions con. 1991. however. Foster City. digestion with HinfI resulted in smaller fragments more clearly indicating the size difference. If flowers were present. La Jolla. several sets of universal arbitrarily chosen from the library for sequencing and primer construction. final sample volume was adjusted to 50 mL with TE. according to the the widest point. Successfully amplified DNA was used directly in restric. enzymes screened enzyme used 3F6 52 60 312 12 RsaI 2D4 48 60 298 9 AluI ITS 52 60 771 7 HaeIII 6G3 55 45 941 3 EcoRI Taba-ba 56 45 711 3 —b 3B6 62 30 677 26 — 11C3c 50 45 740 6 — TrnM-rbcLa 58 30 2866 9 — TrnL1-L2a 58 30 472 7 — TrnH-Ka 58 45 1630 6 — a Chloroplast locus. (1991) contained a small. Eight microliters of each ampli. extension haphazardly collected from each of two individuals within five sites (Fig. Demesure et al. apparently species-specific size difference and version 2. and plated on antibiotic media. was incubated at 658C for 1–2 h. hours of collection. followed by 30 cycles of 1 min at 958C. The width of the inflorescence was measured at of restriction endonuclease enzyme for a minimum of 2 h. Card Sound. 2.5% The library was prepared and screened for single-copy loci according to agarose gels stained with ethidium bromide. procedure detailed before. Cascade. Mad. The mini-DNA preparations (Ausubel et al. Enzymes that produced eas- fragments from 500 bp to 5000 bp were gel isolated.10 mmol/L bovine serum al- bumin (Roche Diagnostics. To verify the presence and computer program SeqEd (Applied Biosystems.0% agarose gel stained with thickness of the third leaf from the meristem were measured with dial calipers ethidium bromide. transformed into bacteria. Indianapolis. Chloroplast fragments were digested acrylamide gels or with an ABI Prism automated sequencing kit (Big Dye or with restriction enzymes as for the nuclear loci. Boston. California. Polymerase chain reaction annealing conditions and results of restriction enzymes survey of three individuals of Borrichia frutescens and B. USA) and analyzed on the restriction endonuclease profile consisted of several smaller fragments. USA) and Primer 3 (Rozen and Skaletsky. and the length of the first pair of phyllaries was measured. annealing (Table 1).. When digested with HinfI. arborescens from Florida USA. Little Torch. Teatable. The sample Primers to three of the 11 single-copy nuclear loci amplified reliably. and gested sample was electrophoresed through a 2. without further purification. Cloned inserts were directly sequenced from mini-prep DNA using either the 1995). sequenced. California. and restriction fragment profiles Karl and Avise (1993).5 mL of each undi. One microliter of genomic DNA from TE.5 mmol/L MgCl2. generally by varying annealing temperature and time amount of sterile sand and a sterile. 0. roughly 1 cm in length (smaller for thermal-cycling parameters varied depending on the primers used and were dried samples) was ground in 100 mL of 658C CTAB buffer with a small determined empirically. Briefly. (Karl and Avise. USA). The taining 3. the computer programs OLIGO (Molecular Biology Insights. Perkin-Elmer. and a single final extension step of 7 min at 728C. USA) and size sorted on was used to genotype all individuals. . Pennsylvania. 1993). total cell DNA was digested with Sau3A and were revealed using short-wave ultraviolet light. USA). and resuspended in 400 mL of a 10 mmol/L tris-CL sel.. (1991) and Demesure et al. USA) size of the indel. D-rhodamine. Primer names refer to those in Taberlet et al. width. These morphological traits were chosen because they appeared to differ be- . chloroplast primers were screened (Taberlet et al. 1990) were used to clones were screened for insert size by restriction endonuclease digestion of amplify the internal transcribed spacer region between ribosomal genes.5 units Taq polymerase (Promega. dried. Recombinant the universal PCR primers ITS 4 and 5 (White et al. Ohio. c Although this locus contained an apparent interspecific restriction site polymorphism. with modifications as follows..0 mmol/L of each dNTP. Thermal cycling generally consisted of a 2-min denaturation at taken from five stems per plant of each of the three putative species that were 958C. 2. Each locus was digested with up to 26 different enzymes and 1 mmol/L ethylenediaminetetraacetic acid (TE) solution. Once single-copy clones were identified. Col- orado. with 95% ethanol. Switzerland). through Microcon 100 000 nominal molecular weight limit spin columns. height of the inflorescence was measured from the base of the phyllaries to fication were digested separately in a 20-mL reaction volume with 10 units the base of the ray flowers. The DNA was resuspended in 100 mL phic restriction endonuclease sites. to the nearest one-hundredth of a millimeter. USA). and the DNA was extracted organically and ducing a single product of the expected size and were screened for polymor. it was not used in the study because the restriction profile scoring was unreliable. and 0. the tion digestions. ison. Within several To check the amount and fidelity of amplification. (1995). were used on the whole set of plant samples. construction and miniprep vector DNA from the corresponding clone. 1997). and Rockland).

00 to 12. 91 TABLE 2. were excluded from analyses.50% in B. (1991) and Demesure et al. (Table 3). morphological data sets.59 0 0. EcoRI.16 0 0. These models were of the Florida Keys (i. sites Nucleotides Locus No. A series of hierarchical hypotheses was tested.00 (1993). Morphological data were tested for lated using Arlequin version 2.00 6G3 4 20 2.41 1 5.13 0 0. using species as the grouping variable. D1 measures the disequilibrium between one morphological characters separately. Cyto.37 1 6. for flower and leaf cytotype and one homozygous genotype. tween the parent species and could be measured reliably in the field before and Ellstrand.50 TrnM-rbcla 42 168 5. USA). Illinois. D2. respec- and Ellstrand.97 0 0. frutescens are cap- . discriminant function analyses were applied to all of the nuclear allelic disequilibrium.86 0 0. 1993.00 TrnL1-L2a 6 24 5.e. D. A log-likelihood ratio tested with a chi-square statistic indicates sta. arborescens) for stan. 1987. as well as together.38 11C3 4 16 2. frutescens or B. in parapatry) revealed that the nuclear tested constraining the estimates to values between zero and one and uncon. on their multilocus nuclear genotypes at loci with species-unique alleles. assuming no backcrossed in.0 (Schneider et al. frutescens and from 0. and a full model. Program default reliability in discriminating plant genotypes using canonical discriminant settings were used for all the genetic analyses. Square root or log10 transfor- rium (HWE).00 3B6 11 46 6. or backcrosses A1 or B1 (BP1 and BP2. Eigenvalues (ratio of nuclear loci ITS and 6G3. and 3F6.00 TrnH-Ka 10 40 2. Morphological data were tested for significant differences between species Data analysis—Estimates of Hardy-Weinberg genotypic frequency equilib.09 0 0. see Nason nuclear alleles that were predominantly B. and D3) values were estimated using the CND computer patch is likely a single individual).79 1 2. The proportion of the total variance in the sures the disequilibrium between the cytotype and the heterozygous genotype discriminant scores that is not explained by the group differences (Wilks’ l) (Asmussen et al.. 1993).97 0 0.00 2D4 5 22 7. with all six classes present. culated to help estimate the spread of the group centroids. loci ITS and 6G3 were essentially fixed for different alleles in strained. genotypic. 1994. in which tively. Assessment of putative pure parental species outside dividuals.20 1 2. arborescens.1760 AMERICAN JOURNAL OF BOTANY [Vol. arborescens (Table 3).00 Taba-ba 3 14 1. Individuals were assigned to one of seven genotypic categories based to 13.45 0 0.38 3 13.64% in B.17 11C3 2 8 1. Nason specific polymorphisms for ITS. were estimated by bootstrapping with 600 replicates.00 Borrichia arborescens 3F6 4 16 5. tion and the presence of second filial or backcross genotype classes indicates Screening 175 individuals at the four nuclear loci and one introgression. and were used to genotype all individuals. Locus variability revealed by restriction endonuclease digestion. For convenience. frutescens and B. Basten. frutescens and B.00 6G3 2 10 1.25 ITS 6 24 3. D measures the most informative.09 0 0. The chlo- the presence of nonparental multilocus genotype classes indicates hybridiza. 2D4. Standard deviations of the genotype class frequencies leaves began to wilt. AluI. first filial hybrids H (F1). Chicago.13 0 0. FST. nucleotides Sequence No. tions could not be met. and log-likelihood (LL) assignment mations were used when necessary to fit model assumptions. using one-way analysis of variance (ANOVA). and D2 mea. D1. linkage disequilibrium. Primer names refer to those in Taberlet et al. and dividuals were classified as pure parental A or B (genealogical classes P1 and RsaI produced the most reliable and easily scored species- P2 for B.11 0 0.08 1 12. tistical significance between the models (for computational details. 1993). site of collection for were done using SPSS version 7. 1996). No. 2000). Maximum likelihood estimates of each class were used to test chloroplast locus resolved 37 unique multilocus genotypes for the significance between a reduced model. Arnold. To help determine which type of data is package (Asmussen and Basten. 1993). second filial hybrids S (F2). roplast locus was size polymorphic. Here.86 0 0. was calculated and transformed to approximate a chi-square distribution test nuclear disequilibria were only calculated between the chloroplast and the of significance of differences between group centroids. In- arborescens (Table 2). co-dominant data with gametic phase unknown were calcu.45 0 0.. Cytonuclear disequilibrium function analyses of the means of measurements within plant patches (each (CND. The 2D4 and 3F6 loci were not alternately fixed between-groups sums of squares to the error sum of squares) also were cal- in the pure parental species making this analysis inappropriate (Arnold. respectively). The enzymes HaeIII. Kruskal–Wallace nonparametric tests were applied dard. RESULTS The occurrence and extent of introgression and hybridization was assessed following the hierarchical hypothesis testing approach of Nason and Ellstrand Molecular markers—Per locus variation ranged from 0.00 TrnH-Ka 10 40 2.00 TrnL1-L2a 6 24 5. Asmussen and Basten. 1994).. B. (1995). D3 measures the disequilibrium be. When assump- of individual genotypes to species (B.00 a Chloroplast locus. 6G3.00 Taba-ba 3 14 1.06 0 0. cut sites surveyed surveyed (%) polymorphic polymorphic (%) Borrichia frutescens 3F6 5 20 6.5 (SPSS.00 2D4 4 16 5. These statistics Linear regression analysis of the LL genotype scores vs.64 ITS 5 20 2. Three of the plants did not have flowering stems and tween the same cytotype and the other homozygous genotype. all individuals was used to indicate geographical localization of parental and hybrid genotypes.00 3B6 10 42 6.00 TrnM-rbcla 42 168 5.

while an nificant deviations (P # 0. frutescens individuals and a B.510 30 2 Ii Gg dd FF B. frutescens-like and the higher-numbered ones more B. Individual genotypes and log-likelihood assignment probabilities for 175 Borrichia individuals in this study. sym- P .559 22 3 Ii Gg DD ff B.510 29 6 Ii Gg Dd ff B.776 27 1 Ii Gg Dd Ff B. A similar pected heterozygotes.402 13 1 Ii GG DD ff B. 21.661 18 1 Ii GG dd ff B.587 27. 212.279 28. fru. 26.842 212. In general. Additionally.332 211.. e Eight individuals of Borrichia arborescens from Puerto Rico had this genotype. Capital letters indicate the typically B. patric and parapatric) were lumped together. 28. frutescens indi. 214. 23. 27. Unexpectedly. arborescens haplotype entals in parapatry). arb. USA.582 9 2 II Gg DD ff B.710 15 1 Ii GG Dd ff B.060 21.023 23.711 37 15 ii gg dd ff B. fru. Significant linkage viduals in the hybrid zone.023 23. fru. Population genetic analysis—When all plants (i. arborescens individuals from Puerto Rico contained the B.113 32 1 Ii gg Dd ff B. arborescens-like.792 16 1 Ii GG Dd ff B.782 2a 27 II GG DD Ff B.396 25.463 28.622 25. fru. 28. Florida. fru.776 28 5 Ii Gg Dd ff B. arborescens alleles. had this genotype. individuals with identical nuclear but different chloroplast genotypes have identical log-likelihood values. frutescens B.037 22. 26.858 29. ITS.g.559 21 1 Ii Gg DD Ff B. arb.906 26. 28.3F6 5 0. arb. arb. fru. there were sig- cens individuals possessed only one 2D4 allele (D). 210. 23.685 7c 7 II GG N/Ad FF B.130 3b 34 II GG DD ff B.476 223. 216.273 26. arb 214. 218. 28. 25. and a second allele (F) was 2D4 (P 5 0.926 22. fru. arb.165). the eight putatively pure regardless of collection location. fru.533 24.001) from HWE expectations at alternate allele (d) was found in all parapatric and most pu. c Seven individuals of Borrichia frutescens from Upper Tampa Bay Park.960 29. fru. 216. with lumping genetically differentiated groups (e. arb. 28. fru.428 26 1 Ii Gg Dd Ff B. arb.293 24 3 Ii Gg Dd FF B. 21.2D4 5 0. Two chloroplast haplotypes locus pairs (P # 0.533 24. 0.607 34 7 ii gg DD ff B. USA (just outside of Tampa). fixed for alternate alleles in the parent species but did exhibit significant species-specific allele frequencies (FST. 22.334.425 26. fru. FST.582 10 1 II Gg Dd FF B.031 31 1 Ii Gg dd ff B.906 26.711 36 28 ii gg Dd ff B. fru.646 28. and 3F6. Parapatric B. arborescens are B.396 25. 29. arb. arborescens species were analyzed separately from the Florida Keys. In each case. loci (fewer than expected heterozygotes). USA. arborescens HWE expectation was not statistically significant for locus individuals fixed for one allele (f).560 22. 0. d Genotype could not be determined. frutes.762 8 1 II Gg DD ff B. B.001). arb. Both the ‘‘d’’ and ‘‘F’’ alleles were disequilibrium also was found between all possible nuclear considered to be species unique. Locus Log-likelihood Geno- type Number ITS 6G3 2D4 3F6 cp B. 25. fru. Florida. Both of these results are consistent were identified: a B. 28.. fru. Assignment probabilities were calculated excluding the chloroplast locus. b Three individuals of Borrichia frutescens from Jacksonville.792 17 1 Ii GG dd Ff B. 26. 6G3. 29. lower-numbered multilocus genotypes were more B.395 19 1 Ii Gg DD FF B.273 26.060 21.960 29. fru. fru. pure par- patric B. arborescens individuals tescens and B. fru. lowercase throughout the text. fru. 22.368 210. 25. therefore.575 14 1 Ii GG Dd FF B. the direction was the same as for the other found in all parapatric and most putative B. fru. 212.112 24. 26.361 27. 23.911 33 1 ii Gg Dd ff B. had this genotype.587 27. while the deviation from situation was true for locus 3F6 with parapatric B. had this genotype. arborescens individuals in the hybrid zone. fru.717 11 1 II Gg Dd ff B. P .211 20 1 Ii Gg DD Ff B. 24.001. fru.799 12 1 II Gg dd ff B. 25.428 25 3 Ii Gg Dd FF B. no significant HWE devia- . there were fewer than ex- tative B.906 26.112 24.999 5 2 II GG Dd Ff B. 28. arborescens 1 2 II GG DD FF B. Loci 2D4 and 3F6 were not frutescens chloroplast haplotype. arb.494 35e 8 ii gg Dd ff B.865 4 1 II GG Dd FF B. frutescens haplotype found in all para. When morphologically classified. arb. Florida.314 a Two individuals of Borrichia frutescens from Ruskin.396 25.293 23 1 Ii Gg DD ff N/Ad 26.e. arb 26. italized and those that were predominantly B.448.347 6 1 II GG dd ff B.622 25.900 24. arb. frutescens alleles and lowercase letters indicate the typically B.001). fru- found in all of the presumed pure B. arb.November 2004] CATTELL AND KARL—INTERSPECIFIC HYBRIDIZATION IN BORRICHIA 1761 TABLE 3. 28.097 28.

arborescens.0677 0. B.0000 Constrained 0. lihood estimates of genotypic class frequencies also indicated zygotes at ITS and 6G3 (P 5 0. frutescens nuclear genotypes (D1) and less often with homozygous B.0000 0.2479 0. Assignment analysis is based on nuclear genotypes 3). 15.2479 0. 25. frutescens are in the upper characters. 3 cubana individuals results in an insignificant slope of 0.446). The geno- types that fall in the center of the distribution of Fig. P3F6 ing.10%) but not to B.0603 Constrained 0.3157 0. there were significantly more hetero. arborescens is P2. 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. 3 cubana individuals had genotypes 9. Individuals that were genetically identified as hybrids were only.1785).188. B.3157 0.000). the maximum like- B.8930. were found at nearly all sites (Fig. frutescens cytotype is found more arborescens was negative.1813. consisted of nonparental types.0051 with an r2 5 0.004) and significantly fewer a significant amount of hybridization (Table 4).0944 0. which included all classes.1780. arborescens are located not always intermediate to the parents in the morphological in the lower right corner and those most like B. Higher numbered ge- notypes are more B.0709 0. for which the backcross frequencies were set to nificant deviation at 2D4 (P 5 1. frutescens and 1. All species pairwise zero. Morphological characteristics—All B.3655 0. The genotypes at the far extremes of the spread of the points are most likely pure parental genotypes. the reduced model (Ln 5 216. frutes.01) and unconstrained (212.000 for both species.071 for B.7%. The estimated number of hybrids backcrossed to B. geography for the putatively B.ITS&6G3 5 0. D2 values (20. A linear regression of LL genotype score vs.001) than expected. Table 4). as well as intermediate LL scores.0718 0. or 29. and B.3479 0. arborescens.0121 and 20. 2 are intermediate in LL score and most likely are hybrids and back- crosses.05) and positive (DITS . D and D1 values were significant (P # 0.599 for B. and stem tip diameter (P .25% of all individuals) with very few classified as 5 0.50172. Table 3. and extreme. Both the re- heterozygotes at 3F6 (P .3655 0. significantly better fit to the data in both the constrained (Ln Significant cytoplasmic-nuclear disequilibrium was found 5 210. B. crossed to B.0842 60. tions were indicated (PITS and 6G3 5 1. arborescens FST 5 0.788. the B. frutescens individuals. most of the nonpar- clear genotypes at ITS and 6G3 in the Keys individuals. 3). The ental individuals appeared to be second generation hybrids (F2. 0. Fig. 24. but not significantly so. low genotype num- bers are more B. . brids and backcrosses. Model P1 P2 F1 F2 BP1 BP2 Reduced Unconstrained 0.0386 60.0613 60.2017 0. and B. 3 cubana vs.1720 and D3. however. Log-likelihood of each genotype being assigned to Borrichia fru.05) analyses. inflorescence height. 91 TABLE 4. 0. D1.0000 SDa 60.ITS 5 20.0056 for ITS and 6G3. 3 cubana FST 5 0. frutescens (. arborescens individuals.3081 0. B.0403 60. arborescens respectively) were not significantly different from zero. Significant differences between plant species were left. 5 0. frutescens that were morphologically measured had either genotype 2 or 3. In general in the multilocus genotypes assignment. B.3281 0. arborescens-like and genotypes 34 to 37 are probably of pure B. 3).000 for B. 2. 28.2138 0. but no sig.0000 0. geographically assorted. 2 and the likelihood values are in trend for the heterozygous state to be found with either chlo. For Following Nason and Ellstrand (1993).0037 (Fig. P # 0. and the full model.0000 Full Unconstrained 0. 3 cubana. The numbers in the graph correspond to the numbers assigned to the genotypes in Table 3. duced model. first phyllary length. P # between the B.1001 20. frutescens vs.135 for B. D6G3 5 0. often with homozygous B. frutescens chloroplast haplotype and the nu. gave sim- FST values were fairly large and statistically significant (P # ilar results estimating that about 32% of the Keys population 0.6G3 5 nificant number of individuals was estimated to be back- 20.1841) and D3 was first generation hybrids (F1. This (Table 4).001. leaf thickness. frutescens-like and genotypes 1 to 3 and 7 are likely pure B. There is no analysis are plotted in Fig. however.0295 60. arborescens nuclear ge. Individuals with log-likelihood in the center of the graph are likely hy.0709 0. Borrichia frutescens is P1 and B. identified for leaf length. arborescens). flower number. Individuals that are most like B. arborescens individuals had genotypes 36 or 37 (Table tescens or B. the full model was a arborescens FST 5 0. analysis indicates that the B. frutescens and 0. Regardless of the model.20 to . Log-likely scores were not. Assignment tests—Results of the LL genotype assignment notypes (D3) than expected under random mating.0405 a Standard deviations estimated by 600 bootstrap replicates.5082). Relative to the likelihood of cens vs. A statistically sig- significant and negative (D3.3267 0.1762 AMERICAN JOURNAL OF BOTANY [Vol. P2D4 roplast type (D2) more often than expected under random mat- 5 0.

not pure parental).001.4% of the variance in the DFA scores was explained by among- group differences. squares are individuals with nuclear ge- notypes identical to B. frutescens). frutescens individuals.. (B) flower morphology only. frutescens (Fig. three genetically B. 3 cubana. Circles are individuals with nuclear genotypes identical to B. data not shown). the DFAs are enclosed in the lines with a dashed line for B. The B. 3 cubana individuals clustered with one or the other parental species and did not form a third grouping (Fig.404. except leaf length. 4A). combined mor- phological 5 13.g. arborescens. 4. leaf 5 2. arborescens. and (C) all of the mor- phological characters combined. arborescens vs. arborescens found in parapatry. Borrichia frutescens and the hybrid were better dif- ferentiated along function 2. flower 5 0. six individuals were misgrouped. B. 4A). squares are B. Clusters of genotype groups as defined by The application of a molecular genetic approach to hybrid. The spread of the group centroids (as indicated by the eigenvalues for function 1. and three genetic hybrids were grouped with B. however. 4C).340.November 2004] CATTELL AND KARL—INTERSPECIFIC HYBRIDIZATION IN BORRICHIA 1763 Fig. Fig. and triangles are all other genotypes (i. pure B. only one of 27 individuals was misgrouped (a B.05) also indicate that leaf morphological characters are better at dis- criminating the groups than flower morphology. frutescens and B. solid line for B. flower 5 1. Plot of the B. 11 individuals were grouped incorrectly. but B.973. arborescens clusters were relatively well defined. Using an LL assignment to species resulted in a continuum of LL values ranging from . No difference was found for leaf and inflorescence width. leaf 5 0.. and triangles are B. a ization and introgression in Borrichia proved quite useful. for which hybrids were the shortest. frutescens individuals were grouped with B. 0. combined morphological 5 0. 3 cubana (Fig. Putative hybrids were on average intermediate in measurement for all measured morphological characters. 3. 3 clear markers were able to resolve a variety of genotypes and cubana. genotypic classes (e. # 0. frutescens. P . geographic site of collection for samples from the Florida Keys only. frutescens found in parapatry. and a dotted line for B.967) and the proportion of the discriminant scores that are not explained by differences among groups (Wilks’ l values for function 1 through 2. Wilks’ l indicates that 96. Flower morphology alone did not discriminate as well between the plant species. frutescens or B. backcrosses). Discriminant function analyses (DFA) indicate that leaf morphology provides good discrimination along function 1 for pure B.219. 3 cubana. The magnitudes of the eigenvalues indicate that the spread between the group centroids was great- est when using all characters. Stars indicate species cluster centroids. arborescens log-likelihood genotype assignment scores vs.036. Here. Discrimina- tion among the groups was best when all morphological data were included in the analysis (Fig.e. Circles are Multilocus genotypes based on one cytoplasmic and four nu. 3 cubana individual was classified as B. frutescens divided by B. Discriminant function analyses of morphological characters for (A) DISCUSSION leaf morphology only. 4B).

not all and LL scores. in the region of species overlap. we cannot rule it out entirely.e. this is putative pure B. tainty how long the Borrichia species in the Florida Keys have ses from the observed genotypic data. arborescens values (Table 3 and Fig. the B. F1) is a difficult or an infrequent event (Arnold case. 91 very likely B. These results. however. rium conditions by now. and Semple. it is possible that they have not been hybridizing the insignificant D2 value indicates that there does not seem for the duration of their sympatry.. 3 cubana (six small B. historic parental ge- pre. by prevalent occurrence of later generation hybrids group centroid were set farther apart from the B. 2). only a minority of the distinct from B. passed. appear atively young in terms of the number of generations that have to be symmetrical with respect to parental species. backcrossing does not. while backcrossing with B. Apparently suggest that hybridization has only recently occurred as a re- then. Table 4). arborescens to very portant to note that the misclassifications occurred when con- unlikely B. portion of individuals backcrossed to B. frutescens is more easily known and is generally difficult to establish for clonal plants. that likely contribute to the genetic patterns observed in alleles. the barrier to hybridization is equal regardless of which sult of disturbance in the Florida Keys and that less disturbed species is the maternal or the paternal parent. some time. arborescens-like LL. It also is clear that not all individuals in the Florida sured to accurately assess identity. we believe that the hybrid were from each other. or 37 and similarly high suggest that morphological characters may not provide very LL assignment to B.and post-fertilization barriers between the parental spe. In all of the analyses. 55. Of the non. Additionally. 1977).. F2 or backcrosses. high frequency of parental types.e. frutescens found within the hybrid zone had consistent with the hybrid. arborescens. Clearly.. frutescens and likely B. frutescens LL values and the DFA were between B. 4). Given this and that suitable habitat likely has and B. 2000). or frequently accomplished than with B. consisting of a larger genotypes 1 to 3. when considered together. our approach as outlined by Nason and Ells. however. dication that these Borrichia species have been sympatric for Considering the cytonuclear disequilibrium analysis. indicating that they are likely hybrids (center of the differentiating pure B. it may be rel- Unlike hybridization. these data clear- amount of hybridization must be taking place in the Florida ly indicate that several morphological characters must be mea- Keys. This is not supported. 1990). and few if any pure parental individ- istic of a system in which the formation of the initial hybrid uals would be expected in the Keys.. however. (Fig. have been available to these plants for up to 8000 years. even if the hybrid zone is old in terms of that backcrossing is a significant event in this hybrid zone. arborescens than they are from B. Puerto Rico) had genotype 35. however. It should however. All other misclassifications by 2. arborescens. Maximum likelihood been hybridizing. and in the absence of other evolutionary forces. frutescens plants would have hybrid genotypes.e. 36. 3. assignment values were of the appropriate morphology. the habitats tended to be occupied by only one of the parent spe- maximum likelihood estimates of genotype classes indicate cies. Additionally. 1993. putative species assignments based on morphology were ex- Individuals that were morphologically B. taining pure parentals and hybrids in sympatry. This is clearly not the generation (i. frutescens from hybrid plants. Morphologically alone. one with all morphological data). within the zone had genotypes 34.1764 AMERICAN JOURNAL OF BOTANY [Vol. Many genotypes had more accurate information on the identity of Borrichia species with- moderate or nearly equal LL scores for both species assign. 3). nine with flower morphology the assertion that they are pure parental types. frutescens and B. the number of years since hybridization began. frutescens and backcrosses. 2). the Florida Keys. Although there is some in- ther crossings are less difficult and more likely (Arnold. sidering flower morphology alone—the least differentiated of tescens individuals from outside of the hybrid zone (genotypes the morphological comparisons. and 7) corresponded to large B. Once this compatibility barrier is breached. however. Semple and Semple (1977) to be asymmetry in the direction of hybridization. Thus the habitat may or later generation hybrids (i. 1992. Interestingly. that. B. Nason et al. It is therefore individuals). If mor- graph in Fig. arborescens. fur.. given the small sample size (53 hybrid dreds or thousands of years (Wolf et al. arborescens clearly possible that what we are detecting is the early formation of is less common. This is particularly true when ments. frutescens-like LL and small B. suggesting the potential for a long period of evolution hybrid zone. Generation time for these plants is un- fertilization between hybrids and B. most individuals were either pure parental types in the Florida Keys (Snyder et al. Finally. frutescens and unlikely B. of south Florida has been exposed for from 5000 to 8000 ization and backcrossing is taking place. and large B. It has been estimated that the limestone genotypes also indicated that a significant amount of hybrid. . These Borrichia species are highly clonal and invest parental genealogical classes estimated in the constrained full a relatively small amount into reproductive structures (Semple model. arborescens cluster and however. While it is difficult to determine with cer- trand (1993) allowed a robust estimation of genealogical clas. parental plants and how long hybridization has been taking Not all markers were alternately fixed for species-specific place.4% of them are BP1 and none are BP2. 1997). such as the distribution of the sympatrically with hybrids (Fig. If Only about 7% of the individuals were of the F1 hybrid class. The B. In the Florida Keys years. 3 cubana clusters and centroids than the latter two been available for quite some time. arborescens-like tremes in LL score. hybrid- This low frequency of F1 individuals has been observed often ization and random mating likely would have led to equilib- in other plant hybrid systems and is believed to be character. indicating clear B. 1992) probably as a result of If the hybrid zone is relatively young. frutescens than to B. Borrichia arborescens individuals from arborescens. zone is not a recent creation but that other factors are main- tering outside their groups involved B. only four individuals clus. arborescens. although all individuals with high LL outside the hybrid zone (i. et al. fru. true. supporting with leaf morphology alone. 3 cubana. a hybrid zone. netic association would still exist and result in the observed cies. It is im. These data clearly indicate that a considerable phological assignments are going to be made.. Keys are hybrids and that pure parental types are persisting There are several factors. In addition. vegetatively reproducing genets may persist for hun- be noted. it is possible that the plants may have been estimates of genealogical class based on observed multilocus there for a long time. arborescens-like genotypes.. The few putatively F1 hybrid individuals iden- The discriminant function analyses of the morphological tified also may be pointing to a young age for the hybrid zone characters suggest that hybrid plants are more morphologically because at the onset of hybridization.

J. patterns found suggest that the Florida Keys population likely mic genotypes after long periods of hybridization by contin. D. 68: 399–404. G. is exhibiting positive assortative mating of the parental spe- ually reintroducing the parental gene combinations into the cies. For migration to result in the pattern of geographic distribution ANTLFINGER. In this study. Interspecific pollen competition and reproductive isolation in Iris. M. frutescens and B. AND G. Notably. however. there was no apparent association between mechanisms of assortative mating. however. Individuals from throughout the range of LL plex region of hybridization. then selection against hybrids at the extremes and/or resultant merging of the parental species would be slowed. HAMRICK. crosses involving B. Journal of Heredity from pure B. J. The genealogical class analysis does in. Oxford. hamas and Cuba. cytoplasmic genotypes. C. J. 1992. M. M. 1987. H. E.. also indicate that there is a greater degree of population dif- sion of the neutrality of restriction site polymorphisms at sin. New York. 1994. P. Annual Review from pure B. D. C. 1981. frutescens Pollen dispersal and interspecific gene flow in Louisiana irises. ROBINSON. BASTEN.05%). mechanisms of pollination. STRUHL.. M. from B. duction of later generation hybrids. migration of pure parental individuals to the center could ac. 1999. would have to be acting directly on the loci under the hybrid zone. B. K. AND J. Heredity (9. that se. Equal rates of migration ARNOLD. D. The genetic basis of microdifferentiation in natural and cytonuclear disequilibrium found in this study. are currently available regarding the zone. mation is needed on the specifics of the frequency of sexual Individuals with genotype LL scores similar to those found in reproduction. Evolution 35: 1056–1068. 1987. increasing the likelihood of backcrossing and pro- be disrupted by recombination (Arnold et al. J. arborescens populations in the Ca. USA 88: 1398–1402. Marchant et al. L. the National Academy of Sciences. A. Natural hybridization: how low can you go and still Keys by at least 80 km of ocean. Oxford University graphical considerations. Hewitt. R. as well as crossing. ARNOLD. while increasing the longevity of the parental individuals in however. BUCKNER. HEMPEL. ARNOLD. is being maintained. J. It seems reasonable. AND B. D. 1988). The Florida Keys hybrid population Press. putative pure parental types) units. Genetics 138: 1351–1363. Selection against hy. be important? Ecology 80: 371–381. there. Nonetheless. AND J.November 2004] CATTELL AND KARL—INTERSPECIFIC HYBRIDIZATION IN BORRICHIA 1765 If this were a geographically simple hybrid zone with a principle effects. R. 1993. genetic differentiation between the parental species for a cytoplasmic one. For the most part then. longevity of clonal both species in parapatry (i.. Clonality also allows for the persistence and study. this seems unlikely to be the ing. indicate that there is genetic differentiation between the three lection is acting directly on the restriction site polymorphisms groups of plant types (pure parents and hybrid).. L. 1993. as expected with assortative mat- loci under selection. ulations would have to be similar. F. Random mating between the plants in this Migration from pure parental populations into a hybrid zone hybrid zone. 1987.00 6 4. MOORE. Second. the large standard er. M. 1993. HEWITT. A. A. given our sample size. The closest B. Greene Publishing Associates We believe that the clonal nature of these species and pre. frutescens and pure B. but introgression and hybrid speciation in Louisiana irises. frutescens. J. . AND C. BRENT. Pollen mediated is contiguous with B. E. USA 84: 3946–3950. ARNOLD. dicate a significant amount of backcrossing to B. Additionally. M. ARNOLD. Sampling theory for cytonuclear all low level of backcrossing. J. SMITH. AND S. BUCKNER. for a discus. Genetics 115: 755–768. J. AVISE. in spite of hybridization and back- case simultaneously for four different nuclear loci. 1985. the hybrid zone due to migration. BURKE. frutescens populations into the hybrid zone than 84: 13–16. it is difficult to conclude true disequilibria. collection site resulted in an insignificant slope. J. It is unlikely. SHAW. WANG-IVERSON. they could be linked to parents and the hybrid group. 3 cubana cross. to expect a greater degree of migration and gene flow ARNOLD. L. C. allowing a significant con- count for the observed genetic patterns. M. Natural hybridization and evolution. J. AND B. Considerably more infor- genotype LL score and geography throughout the hybrid zone. BENNETT. if clonal propagation were the dominant majority of pure parental genotypes at the extreme ends of the form of reproduction. AND J. SEIDMAN. seems unlikely. arborescens and B. ARNOLD. ferentiation between the parents than there is between both gle-copy nuclear loci). The FST values calculated 1988. AND N. scores were found at nearly all sites north to south. BONITZ... G. CONTREARAS. Cytonuclear disequilibria in hybrid zones.44 6 6. 3 cubana. M.. Current protocols in molecular biology. New York. 1991.. L. L. fore. although Semple and Semple encoding DNA introgression across a narrow hybrid zone between two subspecies of grasshopper. Selection. arborescens (0. each of which is separated from the Florida WILLIAMS. M. Definition and prop- eny were produced from B. rors on the maximum likelihood (ML) estimates. Proceedings of the National Academy of Sci- (1977) successfully produce viable offspring from artificial ences. BULGER. arborescens. BENNETT. A. therefore. 1993. ASMUSSEN. and this may be due to the presence of the northern reserve ARNOLD. or closely linked loci. and the over. arborescens individuals are in the Ba.. frutescens individuals to the north. KINGSTON. presumably difficult-to-create through hybrid zones and parental genetic associations should F1 hybrids.13%) but not to B. Ribosomal RNA- of pure B. UK. and more important here. and the geographic distribution and frequency of geno- were observed at all sites along with ones that were clearly types to fully understand the evolutionary history of this com- hybrids (Fig.. and linear regression analysis of the LL scores of putative hybrid indi- LITERATURE CITED viduals vs. Analysis of hybrid zones. Clonal propagation would have two Review of Ecology and Systematics 16: 113–148. M. and Wiley-Interscience. 3). L. First. tribution of both sexual and clonal reproduction might simul- brid genotypes in a hybrid zone also could cause a reduction taneously produce frequent opportunities for hybridization of heterozygotes and significant disequilibrium. linity. Proceedings of is clearly disjunct from B. M. ROBINSON. 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