Heredity 68 (1992) 399—404 Received 26Apr11 1991 OThe Genetical Society of Great Britain

Pollen dispersal and interspecific gene flow in Louisiana irises

M. L. ARNOLD*, J. J. ROBINSON, C. M. BUCKNER & B. D. BENNETt Department of Genetics, University of Georgia, Athens, GA 30602, USA

An analysis of chloroplast DNA (cpDNA) variation was carried out for 106 individual plants from three natural populations of Louisiana irises. Two of the samples (59 individuals) represented I. brevicaulis populations. The third sample was from a population defined by allozyme markers as an area of contact between I. fidva, I. hexagona and I. brevicaulis. The cpDNA acts as a seed-specific genetic marker because it is inherited from the maternal parent. cpDNA markers were thus used to discriminate between (i) introgressive hybridization due to seed movement followed by pollen transfer and, (ii) introgression resulting from direct transfer of pollen between allopatric populations of the hybridizing taxa. Furthermore, the concurrent analysis of biparental and maternal markers for the same individuals allowed a test for any directionality in the introgression. A comparison of cpDNA results with data from previous nuclear analyses led to the conclusion that pollen flow occurred from allopatric populations of I. hexagona into an area of sympatry involving I. fulva and I. brevicaulis. In addition, the genotypes detected in the hybrid population indicate that 1. fulva and I. brevicaulis have acted as both pollen and seed parents to produce introgressant individuals. The results of the present study and those of previous nuclear and cpDNA analyses suggest that pollen dispersal is the most important avenue for gene flow between these Iris species.

Keywords:chioroplastDNA, introgression, Iris, plants.

Introduction patric populations of the hybridizing taxa. The mechanism of interspecific gene flow may have a signi- Oneaspect of gene flow in plants, suggested as a major ficant impact on the pattern and extent of dispersal stimulus for evolution, involves the transfer of genetic and, thus, the genetic structure of areas of overlap material between hybridizing taxa (i.e. introgression; (Barton & Hewitt, 1985; Asmussen et at., 1989). Anderson, 1949). The process of introgressive hybrid- Furthermore, the avenue of gene flow between hybrid- ization, like gene flow in general, may act to restrain izing taxa should either enhance or decrease the likeli- evolution by leading to the amalgamation of the hood of allopatric introgression (Potts & Reid, 1988; hybridizing taxa (Grant, 1963). Alternatively, intro- dePamphilis & Wyatt, 1989; 1990; Arnold eta!., 1990 gression may be evolutionarily creative by facilitating a,b; 1991). (i) an increase in the fitness of the introgressed form A classic example of introgressive hybridization (Nagle & Mettler, 1969; Bennett, 1989; Bennett & involves species from the 'Louisiana Iris' group Grace, 1990), (ii) an invasioit of extreme environments (Anderson, 1949). Some of the species in this complex by the introgressed form (Lewontin & Birch, 1966, but have geographical ranges that are closely associated see Gibbs, 1968 and Birch & Vogt, 1970) and/or (iii) with the Mississippi drainage (I. fulva and 1. brevicaulis), the process of speciation (Mecham, 1960; Levin, 1969; while others are associated with freshwater marsh Rieseberg et a!., 1990). In plants, the first phase of habitats extending from Texas to South Carolina (1. introgressive hybridization may be accomplished by hexagona; Randolph et a!., 1967; Bennett, 1989; N. seed dispersal into populations of the alternate taxon Henderson, personal communication). The habitats followed by pollen transfer (sympatric introgression) associated with each species include moist hardwood or, alternatively, by the transfer of pollen between allo- forests (1. brevicaulis), understory regions along the banks of bayous derived from the Mississippi river (I. *Correspondence tPresent address: Espey, Huston & Associates, Inc., 888 West Belt fulva) and open freshwater marshes (I. hexagona; Dr. South, Suite 200, Houston, TX 77042, USA. Viosca, 1935; Bennett, 1989). The area of geograph- 399 400 M. L. ARNOLD ETAL. ical overlap between these three species occurs in there are habitats typically occupied by either I. fu!va southern Louisiana and numerous cases of natural or I. hexagona within a few kilometres of the hybrid hybridization have been reported from this region population. (Viosca, 1935; Riley, 1938; Anderson, 1949; DNA isolation was carried out using leaf material Randolph eta!.1967;Bennett & Grace, 1989; Arnold and the procedure described by Arnold et a!. (1990b). et a!., 1 990a, b; 1991). Recent studies of this species This resulted in the collection of both nuclear and complex have determined the distribution of nuclear, cytoplasmic DNA. The identification of diagnostic genetic markers and have led to the inference of both cpDNA markers for I. brevicaulis, I. fulva and I. sympatric and allopatric introgression (Arnold et a!., hexagona involved the use of oligonucleotide primers 1 990a, b; 1991). An initial study of chloroplast DNA that were designed to anneal to the region containing (cpDNA) markers for I. fu!va and I. hexagona the ribulose-1, 5-bisphosphate carboxylase gene (i.e. suggested that introgression between these two species rbcl; Z 1, 5'-ATGTCACCACAAACAGAAACTAA- was a result of pollen being transferred between allo- AGCAAGT-3', McIntosh et a!., 1980; ORF1O6, 5'- patric populations of the taxa rather than seed disper- ACTACAGATCTCATACTACCCC-3', Hiratsuka et sal followed by pollen transfer (Arnold et a!., 1991). A a!., 1989). The Zi primer shares sequence identity test for the mechanism of gene flow between these two with the Zea mays rbc! gene from positions 1—30 species was possible because the cpDNA markers are (Mcintosh et a!., 1980). The ORF1O6 primer shares maternally inherited and, therefore, act as seed-specific sequence identity with the Oryza sativa conserved open markers (Arnold et a!., 1991). We have extended our reading frame region, ORF1O6 (Hiratsuka et a!., 1989). analysis of gene flow between iris species by examining PCR amplification of this cpDNA region was accom- cpDNA variation in populations of I. brevicaulis and in plished using the following protocol: Reaction a population containing nuclear markers characteristic volume 100 p1; reaction mix 10 mtvi Tris-HC1 (pH of I. brevicau!is, I. hexagona and I. fulva. These data 8.3), 50 mi KC1, 1.5 mtvi MgCl7, 0.001% gelatin, 0.1 were used to test for the presence of maternal lineages mM each of dATP, TTP, dCTP and dGTP, 50 pmol of originating from these three species. By comparing each primer, 1—2 g of genomic DNA and 2.5 units of data for biparentally transmitted nuclear markers Taq DNA polymerase (Perkin Elmer Cetus). The (Arnold et a!., 1990a, b; 1991) with the maternally amplification experiments were carried out using a transmitted cpDNA markers, we were able to test for Perkin Elmer Cetus DNA Thermal Cycler. The ther- introgression resulting from pollen flow between allo- mal cycler was programmed for one cycle of 1 mm at patric populations versus introgression due to seed dis- 94°C, 35 cycles of 1 mm at 94°C, 1 mm at either 55°C persal into populations of the alternate species, or 60°C, 4 min at 72°C and one cycle of 7 mm at 72°C. followed by pollen transfer. It was also possible to test The cpDNA amplification products were used in res- for any restriction on which species may act as the triction endonuclease digests to identify diagnostic res- maternal parent in interspecific crosses. triction site or length differences. Initial digests to identify diagnostic markers for I. Materialsand methods brevicau!is, I. fu!va and I. hexagona involved 10 enzymes (HphI, A!uI, BamHI, BstUl, EcoRI, HaeIII, Atotal of 106 individual iris plants from three popula- HhaI, HindJII, PstI, and RsaI; New England Biolabs). tions were examined for chioroplast DNA markers. The various fragments were resolved using 1.5% These populations include two populations identified agarosegels run at 75 V for approximately 5 h. EtBr as I. brevicaulis, and one population that had allozyme was present in the gels during electrophoresis to allow markers characteristic of I. brevicaulis, 1. fulva and 1. photography of the restriction fragments under ultra- hexagona (Arnold et aL, 1990a). The collection locali- violet light. ties for these populations are the following ('n' sample sizes): I. brevicaulis; Louisiana, St. Martin Results Parish, St. Martinville, n =20;Ascension Parish, Prairieville, n =39;Hybrid Popu!ation; Louisiana, St. Figure1 illustrates both restriction site and length var- Martin Parish, St Martinville, n =47.The hybrid iation between the rbc! amplification products from I. population was estimated to possess hundreds of indi- brevicaulis, 1. fulva and I. hexagona. Amplification of vidual plants and extends semicontinuously for over 1 this region of cpDNA from I. fulva and I. hexagona km. Populations of I. brevicaulis are known to occur resulted in the production of an approximately 3.2 kb within 5 km of this hybrid population. Populations of fragment (Fig. 1; Arnold et a!., 1991). In contrast, the both I. fulvaandI. hexagona have been sampled within amplification product from this region for I. brevicaulis approximately 70 km of this population. However, individuals was approximately 3.5 kb in length (Fig. 1; INTROGRESSION IN LOUISIANA IRISES 401 abcdefghij abcde fgh ijk Imnopq rs 2.2O 1.60— 1.56— 1.20 — 0.B3 — 0.96— 0.56 — 0.65— 0.37 — 0.47" 0.23 — 0.25' Fig.2 HhaI restriction digests of PCR amplification 0.16" products of the rbcl region from individual iris plants in an area of contact between I. fulva, 1. brevicaulis and!. hexagona. Lane a = phage DNA digested with BstYI; Fig. I PCR amplification products and restriction digests of Lanes b-s contain HhaI digested, amplified DNA from these products from I. fulva, I. brevicaulis and I. hexagona. individual iris plants. Individuals in Lanes b, c, e—g, j,1,m, Lane a =4X174 DNA digested with Rsal; lane b =rbc! q—s have the restriction fragment pattern characteristic of 1. region amplification product from I. fulva; lane c =rbcl brevicaulic. The remainder of the individuals possess a region amplification product form I. brevicaulis; lane d =rbcl restriction fragment pattern diagnostic for I. fulva. region amplification product from I. hexagona; lanes e—g are the results of Hhal restriction digestions of the rbcl amplifi- cation products from I. fulva, I. brevicaulis and I. hexagona, the HhaI digests (Fig. 1) we analysed the majority (97 respectively; lanes h—j are the results of RsaI restriction of the 107 individual plants) of the samples with this digestions of the rbcl amplification products from 1. fulva, I, enzyme alone. The restriction enzyme analysis of the brevicaulis and I. hexagona, respectively. rbcl amplification products from 59 individuals col- lected from two I. brevicaulis populations failed to M. L. Arnold unpublished data). The size difference resolve any restriction site or length variation. Thus, between I. fulva, I. hexagona and I. brevicaulis is appar- the restriction fragment length polymorphisms illu- ent for both the undigested and digested amplification strated in Fig. 1 are apparently species-specific for I. products (Fig. 1). Although the amplification products brevicau!is. In contrast to the two L brevicaulis popula- for I. fulva and I. hexagona are of similar size, these tions, the third population sampled included indi- products have a number of restriction site differences viduals that possessed one of two different haplotypes (Fig. 1; Arnold eta!., 1991). A survey of I. fulva and I. (Fig. 2). Figure 2 illustrates the HIial restriction frag- hexagona populations has indicated that these differ- ment profiles for 18 of the 47 individuals analysed ences are species-specific (Arnold et a!,, 1991). A from this population. The two haplotypes resolved difference in the size estimates between the I. were characteristic for either I. brevicau!is or I. fu!va brevicaulis and the I. fu!va and 1. hexagona digested (Figs 1 and 2). The remaining 29 individuals also amplification products is also apparent when the possessed either the I. fulva or I. brevicaulis haplotype. enzyme RsaI is used (Fig. 1). The estimates for the sizes The spatial distribution of the plants sampled from this of each of the three species, estimated from the RsaI population and the cpDNA haplotypes are shown in experiment, are approximately 0.70 kb shorter than the Fig. 3. Fourteen of the individuals surveyed had the I. estimates from the undigested products. This may be fulva haplotype with the remaining 33 possessing the explained by the presence of co-migrating bands and! haplotype characteristic for I. brevicaulis (Fig. 3). or the production of much shorter restriction frag- Figure 3 also contains data from Arnold et a!. (1 990a) ments that were unresolved in our experiments. Arnold concerning the nuclear, multilocus genotypes for these eta!. (1991) tested the pattern of inheritance of cpDNA 47 individuals. These individuals possess an array of in these Iris species by examining artificial F1 hybrids nuclear genotypes that include components from I. produced from crosses of I. fu!va and I. hexagona. fulva, I. brevicaulis and I. hexagona (Fig. 3): These authors detected only the cpDNA markers from the maternal parent in the hybrid offspring. Discussion Three populations were examined using restriction digestion of the rbcl amplification product. Initially we Thepattern of genetic structuring of plant populations examined individuals using both HhaI and RsaI is closely associated with the predominant mechanism digests. However, because of the resolution afforded by of gene flow present in a particular species (Loveless & wise, inastudyofnaturalhybridizationbetween involving theintroductionofseedsfromPhlox pollen orseedimmigrationintopopulationsofthe izmg taxawillbeaffectedbythelikelihoodofeither gene exchangebetweennaturalpopulationsofhybrid- 402 M.L.ARNOLDETAL. dePamphiis &Wyatt(1989) foundevidenceforan (Aesculus pavia,A.sylvatica andA.flava).Inthisstudy, introgression involving three speciesofbuckeye also beenreachedforan example oflong-distance birds (Potts&Reid,1988). Asimilarconclusionhas persal versuspollination by wide-ranginginsectsor structures thatmightfacilitatelong-distanceseeddis- species OfEucalyptusismostlikelyduetothelackof and seed-mediatedgeneflowbetweenthesetwo Reid, I988).Thedifferencesintheimpactofpollen- movement were4.6and82m,respectively(Potts& gene flowdistancesresultingfromseedandpollen Eucalyptus risdoniiandE.amygdalina,estimatesof detected overan8—yearperiod(Levin,1983).Like- persal fromtheinitialpointofintroductionwas drummondii ssp.drummondii,onlylimitedseeddis- drummondii ssp.mcallisteriintoapopulationofP. alternative species.Forexample,inanexperiment Hamrick, n 0 fl•D \1 -D :— — C, — ,-, ,-, C . - '-4, . '4, Z 1984). Likewise,thepatternandextentof ii C) cocccccccccccccci 17 1 tsccccccto©ssooss co-<>w

C) CSLST ee•:xn CXOIXXD j followed bypollentransfer (Arnoldetal.,1991).This movement, andnotasa resultofseeddispersal between thesetwospecies wasduemainlytopollen from thislatteranalysissuggested thattheintrogression an areaofoverlapbetween thesetwospecies.Findings natural populationsofI.fulva andI.hexagonain (1991) carriedoutananalysisofcpDNAvariation in tions oftheparentalspecies.Inaddition,Arnoldetal. hybridization inbothsympatricandallopatricpopula- the earlierworkbydetectingapparentintrogressive (Anderson, 1949).Theserecentstudieshaveextended the occurrenceof populations oftheLouisianairisspeciesI.fulva, I. 1991) havesupportedearlierhypothesesconcerning hexagona andI.brevicaulis(Arnoldetal.,1990a,b; seed versuspollendispersal. specific geneflowmaybedifferentiallyaffectedby size thewayinwhichpatternandextentofinter- (dePamphilis &Wyatt,1989).Theseexamplesempha- the knownpollinators,ruby-throatedhummingbird was correlatedwiththemigratorypatternsofone to adistanceof200km.Thispatternintrogression asymmetric transferofgenesbetweenthesespeciesup n Recent geneticanalysesofparentalandhybrid co-obw arranged besideoneanotherrepresent respectively. Thecirclesthatare dual) and48individualirisplants, in eachof47(nodataforoneindivi- and allozyme(nuclear)markerspresent Fig. 3ChioroplastDNA(cpDNA) The allozymedataarefromArnoldet individual atthelower-lefthandcorner. figure isactuallylocatednexttothe dual atthetopright-handcornerofthis along alineartransectandtheindivi- individual plant.Thesamplewastaken cpDNA andnucleardataforthesame al. (1990a). introgressive hybridization INTROGRESSION IN LOUISIANA IRISES 403 conclusion is consistent with data concerning the mode the observed introgression has come about in sympatry of sexual reproduction in these species. Thus, the or has resulted from the transfer of pollen from allo- major pollinators for this species complex are bees patric populations of the alternative species. In this from the subfamily Apinae (Viosca, 1935). Further- regard, we have detected seven individuals that have I. more, hummingbirds have been observed visiting fulva genotypes and nine individuals that appeared to flowers of I. flulva, 1. hexagona and natural hybrids (B. possess I. brevicaulis genotypes (Fig. 3). The detection D. Bennett, M. L. Arnold, personal observation). of apparently pure parental individuals of both I. fulva These pollinators include species that are capable of and I. brevicaulis supports the hypothesis that this long-distance pollination, although the major pollin- population is an area of sympatry between these two ator species (bumblebees) are known to demonstrate a species. The presence of parental genotypes in a popu- predominant pattern of near-neighbour foraging in lation containing apparent advanced generation back- other plant species (Schmitt, 1980; Thomson & cross individuals (Arnold et a!., 1990a; this study) Thomson, 1989). Direct observations concerning seed would also indicate a potential for the continued dispersal in this species complex are lacking. However, impact of immigrants on the genetic structure of a inferences can be made concerning the possibility of hybrid population. Levin (1983) has previously seed dispersal from the morphology of the fruit and demonstrated that immigration by annual plants can seed and the habitat in which these species normally indeed have a continued effect on the genetic structure occur. The seeds produced by all of the iris species in of the recipient population across several generations. this complex are contained in a large fleshy fruit In species such as the Louisiana irises, where plants are (Viosca, 1935; Randolph et al., 1967). The release of perennial and have both clonal and sexual modes of the seeds from the seed capsule apparently occurs due reproduction (Bennett, 1989), there should be the to the rotting of the capsule. Since the seeds have a opportunity for an extended impact from an immigra- corky seed coat that allows them to float (M. L. Arnold, tion episode on the genetic structure and evolution of personal observation), and the iris plants are normally hybrid populations. located in marshes, swamps or bayous (Viosca, 1935; There is incongruence between the cpDNA and Randolph eta!., 1967; Bennett, 1989), the opportunity nuclear data for this population (Fig. 3) with regard to for dispersal on water currents would seem to be avail- I. hexagona markers. Thus, we have detected nuclear able. markers, but not cpDNA markers that are charac- In the present analysis we have extended our earlier teristic for this species. This finding is particularly examination of the mechanism of gene flow between 1. germane with regard to testing for long-distance gene fulva and I. hexagona to include a hybrid population flow due to either pollen or seed dispersal. The lack of involving these two species and I. brevicaulis. Such 1. hexagona seed-specific markers (cpDNA) and the hybrid associations between these three species have presence of nuclear markers from this species, been reported previously (Randolph et a!., 1967). supports the hypothesis that I. hexagona pollen has Furthermore, hybridization between these three entered this population from allopatric 1. hexagona species is thought to have resulted in the production of individuals. I. hexagona populations are known to be the hybrid species I. nelsonii (Randolph, 1966; Arnold present within approximately 70 km of this hybrid et a!., 1990a; 1991). Figure 3 summarizes the cpDNA population. However, there are habitats within several and allozyme data for each of 47 individuals from such kilometres of this population that are characteristic of a hybrid population. The data from the nuclear and other I. hexagona populations. Therefore, it is possible cpDNA analyses support the hypothesis of localized that there are plants belonging to this species located introgression between I. fulva, I. brevicaulis and I. within 10km of the hybrid population. hexagona (Arnold et a!., 1 990a; present study). These Nuclear and cpDNA variation in allopatric and sym- data also indicate that the introgression is bidirectional patric populations of I. fulva, 1. hexagona and I. with respect to I brevicaulis and I. fulva. This conclu- brevicaulis are suggestive of pollen-mediated introgres- sion can be drawn from the finding of introgressant, sive hybridization (Arnold et al., 1991; this study). nuclear genotypes that have cpDNA haplotypes Although rare, long distance seed dispersal, followed characteristic of either I. brevicaulis (23 individuals) by pollen transfer, may occur, the main avenue for the or I. fulva (seven individuals; Fig. 3). The greater fre- interspecific gene flow appears to be the direct transfer quency of introgressant types possessing the cpDNA of pollen. Indeed, each of the cases of introgression and markers characteristic of I. brevicaulis suggests that hybrid speciation, detected within this species complex this species has predominated as the seed parent in this (Anderson, 1949; Randolph, 1966; Arnold et a!., population. 1990a, b; 1991; this study), indicate a predominant The nuclear and cpDNA data from this hybrid role for pollen dispersal in the regulation of interspe- population also allow inferences concerning whether cific gene flow. 404 M. L. ARNOLD ETAL.

DNA inversion during the evolution of the cereals. Mol. Acknowledgements Gen. Genet., 217, 185—194. LVIN, D. A. 1969. The challenge from a related species: a Weare grateful to F. Foti for permission to collect Iris stimulus for saltational speciation. Am. Nat., 103, samp1es on his property and to T. Hebert for help with 316—322. collecting the samples. This research was supported by LEVIN, D. A. 1983. An immigration-hybridization episode in a NSF grant BSR-9004242 (MLA, JL Hamrick and B. Phlox. Evolution, 37, 575-582. D. B.) and NSF REU supplement (MLA), a Faculty LEWONTIN, R. C. AND BIRCH, L. c. 1966. Hybridization as a Research Grant from the University of Georgia (MLA) source of variation for adaptation to new environments, and a grant from the American Iris Society Foundation Et.'olution, 20, 3 15—336. (BDB and MLA). LOVELESS, M. D. AND HAMRICK, J. L. 1984. Ecological deter- minants of genetic structure in plant populations. Ann. Rev. Ecol. Syst., 15, 65—95. McINTOSH, L., POULSEN, C. AND B000RAD, L. 1980. Chloroplast References gene sequence for the large subunit of ribulose bisphos- phatecarboxylase of maize. Nature, 288, 556—560. ANDERSON,E. 1949. Intro gressive Hybridization. John Wiley & MECFIAM, .S.1960. Introgressive hybridization between two Sons, Inc., New York. southeastern treefrogs. Evolution, 14, 445—457. ARNOLD, M. L., HAMRICK, J. L AND BENNETT, B. 0. 1990a. Allo- NAGLE, S. J. AND METFLER, L. E. 1969. Relative fitness of intro- zyme variation in Louisiana irises: a test for introgression gressed and parental populations of Drosophila and hybrid speciation. Heredity, 65, 297—306. mojavensis and D. arizonensis. Evolution, 23, 5 19—524. ARNOLD, M. L, BENNETT, B. D. AND ZIMMER, B. A. 1990b. Natural de PAMPHILIS, C. w. AND WYATT, R. 1989. Hybridization and hybridization between Iris fulva and I. hexagona: pattern introgression in Buckeyes (Aesculus: Hippocastanaceae): a of ribosomal DNA variation. Evolution, 44, 1512—1521. review of the evidence and a hypothesis to explain long- ARNOLD, M. L., BUCKNER, C. M. AND ROBINSON, j,j. 1991.Pollen distance gene flow. Syst. Bot., 14, 593—611. mediated introgression and hybrid speciation in Louisiana de PAMPHILIS, C. w. AND WYATT, R. 1990. Electrophoretic confir- irises. Proc. NatI. Acad. Sci., U. S. A., 88, 1398—1402. mation of interspecific hybridization in Aesculus (Hippo- ASMUSSEN, M. A., ARNOLD, J. AND AVISE, j.c.1989. The effects of castanaceae) and the genetic structure of a broad hybrid assortative mating and migration on cytonuclear associa- zone. Evolution, 44, 1295—13 17. tions in hybrid zones. Genetics, 122, 923—934. POTTS, B. M. AND REID, J. B. 1988. Hybridization as a dispersal BARTON, N. H. AND HEWITT, G.M. 1985. Analysis of hybrid zones. mechanism. Evolution, 42, 1245—1255. Ann. Rev. Ecol. Syst., 16, 113—148. RANDOLPH, L. F. 1966. Iris nelsonii, a new species of Louisiana BENNE1r,B. D. 1989.Habitat differentiation of Iris fulva Ker iris of hybrid origin. Baileya, 14, 143—169. Gawler, Iris hexagona Walter, and their hybrids. Ph. D. RANDOLPH, L. F., NELSON, I. S. AND PLAISTED, R. L. 1967. Negative Thesis, Louisiana State University. evidence of introgression affecting the stability of BENNETT, B. D. AND GRACE, J. B. 1990. Shade tolerance and its Louisiana Iris species. Cornell Univ. Agr. Expt. Stat. effect on the segregation of two species of Louisiana Iris Memoir, 398, 1—56. and their hybrids. Am. J. Bot., 77, 100—107. RIESEBERG, L. H., CARTER, R. AND ZONA, s. 1990. Molecular tests BIRCH, L. C. AND VOGT, \V. G. 1970. Plasticity of ta.xonomic of the hypothesized hybrid origin of two diploid characters of the Queensland fruit flies Dacus Ilyoni and Helianthus species(Asteraceae).Evolution,44., Dacus neohumeralis (Tephritidae).Evolution,24, 1498—15 11. 320—343. RILEY, H. P. 1938. A character analysis of colonies of Iris fulva, GIBBS, 0. w. 1968. The frequency of interbreeding between Iris hexagona var. giganticaerulea and natural hybrids. Am. two sibling species of Dacus (Diptera) in wild populations. J. Bot., 25, 727—738. Evolution, 22, 667—683. SCHMITT,J. 1980. Pollinator foraging behavior and gene dis- GRANT, v. 1963. The Origin ofAdaptations. Columbia Univer- persal in Senecio (Compositae). Evolution, 34, 934—943. sity Press, New York. THOMSON, J. D. AND ThOMSON, B. A. 1989. Dispersal of HIRATSUKA, J., SHIMADA, H., WHITHER, R., ISHIBASHI, T., SAKAMOTO, Eiythronium grandiftorum pollen by bumblebees: implica- M., MORI, M., KONDO, C., HONJI, Y., SUN, C., MENG, B., LI, Y., KANNO, tions for gene flow and reproductive success. Evolution, A., NISHIZAWA, Y., HIRAI, A., SHINOZAKI, K., AND SUGIURA, M. 43,657—661. 1989. The complete sequence of the rice (Oryza sativa) VIOSCA, P., Jr. 1935. The irises of southeastern Louisiana: a chloroplast genome: intermolecular recombination taxonomic and ecological interpretation. J. Am, Iris Soc., between distinct tRNA genes accounts for a major plastid 57, 3—56.