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PREMISE When divergent lineages come into secondary contact, reproductive isolation may be incomplete, thus providing an opportunity to investigate how speciation is manifested in the genome. The Louisiana Irises (Iris, series Hexagonae) comprise a group of three or more ecologically and reproductively divergent lineages that can produce hybrids where they come into contact. We estimated standing genetic variation to understand the current distribution of population structure in the Louisiana Irises. METHODS We used genotyping-by-sequencing techniques to sample the genomes of Louisiana Iris species across their ranges. We sampled 20 populations (n = 632 individuals) across 11,249 loci and used Entropy and PCA models to assess population genetic data. RESULTS We discovered evidence for interspecific gene flow in parts of the range. Our analysis revealed patterns of population structure at odds with widely accepted nominal taxonomy. We discovered undescribed hybrid populations, designated as belonging to the I. brevicaulis lineage. Iris nelsonii shared significant ancestry with only one of the purported parent species, I. fulva, evidence inconsistent with a hybrid origin. CONCLUSIONS This study provides several key findings important to the investigation of standing genetic variation in the Louisiana Iris species complex. Compared to the other nominal species, I. brevicaulis contains a large amount of genetic diversity. In addition, we discovered a previously unknown hybrid zone between I. brevicaulis and I. hexagona along the Texas coast. Finally, our results do not support the long-standing hypothesis that I. nelsonii has mixed ancestry from three parental taxa.

Taxonomic revisions using newly available molecular data can have profound consequences for identifying areas of high endemism and, therefore, high conservation priority. A good example of the connection between taxonomy, biodiversity ecology, and conservation issues is genus Phaeiris (Iris subsect. Hexagonae), an endemic taxon of the southeastern United States and in particular P. hexagona (I. hexagona) (Blue Flag), perhaps the best‐known species of this genus. Some authors recently provided evidence for the need to revise the taxonomy of the Blue Flag, which has usually been considered to consist of a single species, P. hexagona. Using molecular and bioclimatic analyses of Blue Flags from Florida and Louisiana, collected at their loci classici, we challenge the notion that P. hexagona is a single species, referring to the almost forgotten taxonomic context of Phaeiris as established by John K. Small and Edward J. Alexander nearly 90 yr ago. Our vision of the P. hexagona complex reinforces the current treatment of Southern Coastal Plain as a biodiversity hotspot. Our results also argue for the complex nature of endemism on the Florida Peninsula, and elsewhere on the Southern Coastal Plain, including a new interpretation of the well‐studied Louisiana irises. We also propose that the valleys of Florida rivers and lakes are likely to harbor additional hidden biodiversity. Accurate taxonomy proved to be a heuristic tool to characterize better the actual level of biodiversity within Southern Coastal Plain as well as to describe the different areas endemism. The newly re‐documented taxonomic diversity in broadly defined P. hexagona should also be considered in future conservation efforts.

The formation of hybrids among closely related species has been observed in numerous plant taxa. Selection by pollinators on floral traits can act as an early reproductive isolating barrier and may be especially important when there is overlap in distribution and flowering time. In this study, we use Quantitative Trait Locus (QTL) mapping based on 293 codominant SNP markers in an F2 population (n = 328) to assess the size, magnitude, and location of the genetic regions controlling floral traits known to be important for pollinator attraction in 2 species of Lousiana Irises, Iris fulva and Iris hexagona. We also evaluate correlations among F2 traits and identify transgression in the hybrid population. Overall, we observe that differences in most floral traits between I. fulva and I. hexagona are controlled by multiple QTLs and are distributed across several linkage groups. We also find evidence of transgression at several QTL, suggesting that hybridization can contribute to generating phenotypic variation, which may be adaptive in rapidly changing environments.

PREMISE OF THE STUDY Iris pseudacorus spread rapidly into North America after introduction from Europe in the 1800s and now co-occurs with native I. hexagona in freshwater Louisiana wetlands. Native irises support and interact with multiple trophic levels, whereas I. pseudacorus is classified an invasive pest because it grows aggressively, reduces biodiversity, and displaces native vegetation. Salinity levels are increasing in coastal wetlands worldwide. We examined how salt-stress affects competitive interactions between these conspecifics. METHODS We established a three-way full-factorial common-garden experiment that included species (I. pseudacorus, I. hexagona), competition (no competition, intraspecific competition, and interspecific competition), and salinity (0, 4, 8 parts per thousand NaCl), with six replicates per treatment. KEY RESULTS After 18 mo, Iris pseudacorus produced much more biomass than the native species did (F1, 92 = 71.5, P < 0.0001). Interspecific competition did not affect the introduced iris, but biomass of the native was strongly reduced (competition × species interaction: F2, 95 = 76.7, P = 0.002). Salinity significantly reduced biomass of both species (F2, 92 = 21.8, P < 0.0001), with no species × salinity interaction (F2, 84 = 1.85, P = 0.16). CONCLUSIONS Our results demonstrate that salt stress strongly reduced clonal reproduction in native and introduced irises; however, the introduced iris had a competitive advantage over the native, regardless of environmental salinity levels. Based on patterns in clonal reproduction, the introduced iris could potentially threaten native iris populations. We are currently investigating seed production and mortality during competition and stress because both clonal and sexual reproduction must be considered when predicting long-term population dynamics.

Hybrid speciation represents a relatively rapid form of diversification. Early models of homoploid hybrid speciation suggested that reproductive isolation between the hybrid species and progenitors primarily resulted from karyotypic differences between the species. However, genic incompatibilities and ecological divergence may also be responsible for isolation. Iris nelsonii is an example of a homoploid hybrid species that is likely isolated from its progenitors primarily by strong prezygotic isolation, including habitat divergence, floral isolation and post-pollination prezygotic barriers. Here, we used linkage mapping and quantitative trait locus (QTL) mapping approaches to investigate genomic collinearity and the genetic architecture of floral differences between I. nelsonii and one of its progenitor species I. hexagona. The linkage map produced from this cross is highly collinear with another linkage map produced between I. fulva and I. brevicaulis (the two other species shown to have contributed to the genomic makeup of I. nelsonii), suggesting that karyotypic differences do not contribute substantially to isolation in this homoploid hybrid species. Similar to other studies of the genetic architecture of floral characteristics, at least one QTL was found that explained >20% variance in each color trait, while minor QTLs were detected for each morphological trait. These QTLs will serve as hypotheses for regions under selection by pollinators.

 Premise of the study : Saltwater intrusion is one of the most widespread environmental threats to freshwater wetlands. Iris species worldwide are important members of these plant communities. Wetland irises reproduce clonally and sexually, which permits populations to spread and disperse in benign and stressful conditions. The ability of iris populations to tolerate and adapt to elevated salinity can play an important role in determining the long-term health of wetland ecosystems.  Methods : We used microsatellite markers to evaluate population structure and genetic diversity, and we performed a common garden experiment to examine the effect of salinity on the growth and reproduction of wild Iris hexagona collected from freshwater and brackish wetlands.  Key results : Colonies were genetically distinct, with average to high heterozygosity (0.55 – 0.66) for a clonal species. Salinity had negative linear effects on leaf mass (g), clonal growth (g), root mass (g), and fl ower numbers, and it had nonlinear effects on seed numbers and seed mass (mg). The greatest sexual reproduction occurred in the intermediate-salinity (4 parts per thousand) treatment. Flowering phenology was delayed for 5 days in the highest-salinity treatment.  Conclusions : We hypothesized that irises from brackish habitats would tolerate salinity better than freshwater irises would, but no difference in iris performance existed between the two habitats. The observed salinity tolerance and genetic diversity of I. hexagona indicate that populations will persist despite moderate increases in environmental salinity.

In this review, we discuss findings from studies carried out over the past 20+ years that document the occurrence of asymmetric introgressive hybridization in a plant clade. In particular, analyses of natural and experimental hybridization have demonstrated the consistent introgression of genes from Iris fulva into both Iris brevicaulis and Iris hexagona. Furthermore, our analyses have detected certain prezygotic and postzygotic barriers to reproduction that appear to contribute to the asymmetric introgression. Finally, our studies have determined that a portion of the genes transferred apparently affects adaptive traits.

We characterize the salinity stress response of Iris hexagona, a freshwater species, by measuring three putative osmolytes, betaine, proline, and dimethylsulphonio-propionate (DMSP) in plants after short (3 days) and long-term (4–5 months) exposure to NaCl. HPLC analyses show that untreated control leaves contained 2.1, 0.2 and 3.2 mg g−1 DW of betaine, proline, and DMSP, respectively, and establish the presence of these compounds in the Iridaceae. Within 4 days of salinity stress (200 mM NaCl) betaine, proline and DMSP increased significantly. Among vegetative tissues, the highest level of proline occurred in roots; betaine and DMSP were highest in leaves. Analyses of generative tissue (flowers and flower stalks) after long-term exposure showed the highest levels (>10 mg g−1 DW) of all examined compounds in petals but only proline and betaine increased with salinity. All three substances showed a basipetal gradient in flower stalks. Although the examined compounds responded to salinity, the osmometry of the sap indicated that they comprise less than 10% of the osmotically active solutes. The temporal and spatial changes in the distribution of the analyzed compounds indicate complex responses to salinity.

We investigated genetic variation within and among 11 populations of Iris hexagona at its southern limits in the Florida peninsula by using 19 microsatellite loci. All of the populations contain varying numbers of identical multilocus genotypes, indicative of clonal reproduction. Two population samples consist largely of one clonal lineage and two clonal lineages: the first from the Caloosahatchee drainage west of Lake Okeechobee and the other from the Big Cypress Swamp. The populations are predominantly outcrossing, with high levels of heterozygosity, and show a highly significant pattern of isolation by distance that fits a modified stepping‐stone model. This pattern breaks down at the local level, however, where metapopulation dynamics or asymmetrical gene flow may exert a stronger effect on patterns of genetic diversity. Though the majority of genetic variation is within populations, 20% occurs between populations. Genetic distance resolves five clusters: four in the Caloosahatchee Valley and one in t...

Cadmium (Cd) toxictity and possible role of salicylic acid (SA) in alleviating Cd-induced toxicity were investigated on ornamental hydrophyte Iris hexagona. Compared to the control, treatments with 100 and 500 µM Cd for 7 days significantly decreased dry weight, the contents of chlorophyll, photosynthetic parameters, and increased the content of thiobarbituric acid reactive substance. Pretreatment of the roots of I. hexagona seedlings with 1 µM SA before Cd exposure may increase dry weight, photosynthetic rate, activities of antioxidant enzymes, improve the cell ultrastructure and protect plants from Cd-induced oxidative stress damage. However, SA pretreatment had no significant effect on Cd concentrations in the leaves and roots. It is suggested that SA-induced Cd tolerances in I. hexagona are likely associated with increases in antioxidant enzyme activities and vacuolar compartmentation, rather than Cd uptake.

Gene flow among widespread populations can be reduced by geographical distance or by divergent selection resulting from local adaptation. In this study, we tested for the divergence of phenotypes and genotypes among 8 populations of Iris hexagona. Using a genotyping-by-sequencing approach, we generated a panel of 750 single nucleotide polymorphisms (SNPs) and used population genetic analyses to determine what may affect patterns of divergence across I. hexagona populations. Specifically, genetic differentiation was compared between populations at neutral and nonneutral SNPs and detected significant differences between the 2 types of markers. We then asked whether loci with the strongest degree of population genetic differentiation were also the loci with the strongest association to morphology or climate differences, allowing us to test if pollinators or climate drive population differentiation or some combination of both. We found 2 markers that were associated with morphology and 1 marker associated with 2 of the environmental variables, which were also identified in the outlier analysis. We then show that the SNPs putatively under selection were positively correlated with both geographic distance and phenotypic distance, albeit weakly to phenotypic distance. Moreover, neutral SNPs were only correlated with geographic distance and thus isolation-by-distance was observed for neutral SNPs. Our data suggest that both deterministic and neutral processes have contributed to the evolutionary trajectory of I. hexagona populations.

Iris hexagona is a perennial freshwater species indigenous to Louisiana wetlands. Coastal iris populations that are exposed to saline conditions often suffer heavy floral browsing. This paper is the first to photographically document florivory by white-tailed deer (Odocoileus virginianus Zimmerman), which consume iris flowers and immature seed capsules. Severe florivory can prevent sexual reproduction in plants and eliminate habitat for floral arthropods. In this three-year study, we monitored environmental salinity, assessed natural levels of florivory, and established deer exclosures to test the effects of florivory on arthropod abundance and diversity. In two out of three years, florivory rates were positively correlated with the level of salinity at individual I. hexagona populations. In all three years, florivory in the brackish marsh was an order of magnitude greater than florivory in intermediate and freshwater sites. In 2003, the number and diversity of arthropods was significantly greate...

We used field, common garden, and laboratory studies to investigate the influence of environmental stress on a native wetland species, Iris hexagona (Iridaceae), and an iris leafminer, Cerodontha iridiphora (Diptera: Agromyzidae). There were strong effects of salinity, herbivory, and phenology on jasmonic acid (JA) and salicylic acid (SA), two important signal phytohormones. Within 48 h, foliar JA increased an order of magnitude, and SA declined 50% in plants grown under salinity stress compared to freshwater controls. Leafminer performance declined on plants exposed to the salinity treatment. Both herbivory and salinity stress induced JA and suppressed SA, indicating that plants can respond to abiotic and biotic stress through shared hormonal pathways. Surprisingly, leafminer pupae contained JA and SA, and we discuss the implications of this novel discovery. Unrelated to salinity, there was a sharp decline in foliar JA between April and September. Despite its strong impact on hormonal signaling in plants, the abiotic environment is often ignored in ecological studies of induced plant defenses. Importantly, natural seasonal cycles and/ or environmental stress can induce stronger changes in phytohormones than herbivory, and should be incorporated into the design of ecological experiments whenever possible, for a balanced understanding of phytohormonal ecology.

Summary 1 Salinization is a growing environmental stress in wetland ecosystems world-wide. Several models have been proposed that predict clonal plant responses to stress, including that environmental stress stimulates sexual reproduction. 2 We conducted a common-garden experiment to investigate the effects of salinity on 10 natural populations of Iris hexagona, a clonal perennial endemic to freshwater and brackish wetlands of the North American Gulf Coast. 3 Salinity reduced vegetative growth but either increased or had neutral effects on sexual reproduction, consistent with the clonal stress hypothesis. Salinity of 4 µg g−1 more than doubled the number of seeds produced compared with freshwater controls, but flower number and seed mass were unaffected. 4 Salinity reduced total below-ground mass by nearly 50% compared with controls, with no significant change in rhizome numbers. 5 Plants from 10 randomly selected I. hexagona populations differed dramatically in growth and reproduction, independent of salinity. Total biomass that accumulated over the 20-month experiment ranged across all treatments from 52 to 892 g, and flower numbers varied from 2.3 to 11.3 per replicate. 6 Populations did not respond differently to salinity, except with respect to above- : below-ground ratios, thus providing no conclusive evidence for local adaptation to salinity stress. 7 Our results concur with published models of plant reproductive strategies in variable environments, in that environmental stress stimulated sexual reproduction at the expense of growth. However, these models do not predict the observed sharp decline in seed production at near lethal salinity levels.

Adults and larvae of the loberine erotylid beetle Loberus impressus (LeConte) were found associated with fungi growing on corolla and seed capsule tissue of the blue flag iris, Iris hexagona. We examined adult beetle specimens using light and scanning electron microscopy to determine if specialized structures (mycangia) may function in transporting fungi. Two pairs of deep pits on the ventral aspect of the gena between the eyes and the maxillae of both sexes contained fungal spores and hyphae, suggesting a possible role as mycangia in addition to their role as glandular outlets. Inoculation from the surface of cleaned beetle specimens produced colonies of Cladosporium and Fusarium. These genera are widespread, usually air-dispersed conidial fungi that sometimes are associated with insects.

Phytohormones play critical roles in regulating plant responses to stress. We investigated the effects of salinity on abscisic acid (ABA), indole-3-acetic acid (IAA), salicylic acid (SA), and jasmonic acid (JA) in leaves, stalks, fruits, and seeds of Iris hexagona, a native wetland species. Using gas chromatography-mass spectroscopy with selected ion monitoring, our experiments demonstrated significant and different short- and long-term changes in iris phytohormones. ABA and JA generally increased and IAA and SA declined in response to salinity. We conclude that these phytohormones may have separate and interactive effects on how plants respond and adapt to stress in natural environments.

We studied pollinator visitation rates and movement patterns in experimental arrays of irises at two sites within a Louisiana iris hybrid zone. Arrays contained single-flowered stems of red-flowered I. fulva, blue-flowered I. hexagona, and purple-flowered F1 hybrids. At one site, where I. hexagona was the only wild-growing iris, queen bumble bees were the most common pollinator, and the rank order of pollinator visit rates was I. hexagonaI. fulva. At a second site, where I. fulva predominated in the wild, hummingbirds were the most common pollinators, and this order was reversed: I. hexagona

The larva of Loberus impressus LeConte is described based on adult-associated and reared specimens collected from corollas and seed pods of Iris hexagona on the gulf coast of Louisiana. The larva of L. impressus is similar to larvae of Zavaljus brunneus (Gyllenhal) Hapalips prolixus Sharp, Pharaxonotha spp., and Bolerus angulosus (Arrow), the other described larvae within the Xenoscelinae. Larval characters typical for the Loberini that are found in L. impressus include arrangement of integumental granules into rows, frayed and aciculate setae borne on spiny tubercles, and urogomphi spiny or tuberculate and recurved. The tarsungular setae of L. impressus and B. angulosus are single, in contrast to the dual setae present in other described larvae of Languriidae. Larva and adults of L. impressus were common in dried corollas and seed pods of I. hexagona that had begun to split open or had been invaded by Crematogaster ants. Fusarium and Cladosporium fungi were potential sources of food for L. impressus and other fungivorous beetles in the iris seed pods and on corollas.

The assumption of hybrid inferiority is central to the two models most widely applied to the prediction of hybrid zone evolution. Both the tension zone and mosaic models assume that natural selection acts against hybrids regardless of the environment in which they occur. To test this assumption, we investigated components of fitness in Iris fulva, I. hexagona and their reciprocal F1 hybrids under greenhouse conditions. The four cross types were compared on the basis of seed germination, vegetative and clonal growth, and sexual reproduction. In all cases, the hybrids performed as well as, or significantly better than, both of their parents. These results suggest that F1 hybrids between I. fulva and I. hexagona are at least as fit as their parents. The results of this study are therefore inconsistent with the assumptions of both the tension zone and mosaic models of hybrid zone evolution.

Ten microsatellite loci isolated from Iris hexagona are described. All these loci are polymorphic, with three to 13 alleles across 24 individuals from a single natural population. Heterozygosity ranged from 0.125 to 0.870. Three loci depart significantly from Hardy–Weinberg equilibrium in our test population. The test population shows significant heterozygote deficiency in these and two other loci. Three loci exhibit significant linkage disequilibrium. These loci will be utilized to investigate patterns of genetic variation in the species throughout the Florida peninsula.

Postpollination mechanisms can play an important role in limiting natural hybridization in plants. Reciprocal hand pollination experiments were performed to study these mechanisms in two species of Louisiana iris: Iris brevicaulis and I. fulva. Relative pollen-tube growth rates changed significantly through time, with I. fulva tubes increasingly outperforming I. brevicaulis tubes in both conspecific and heterospecific styles. However, this pattern of change in relative performance was a poor predictor of siring success: the majority of seeds sired by both maternal species was conspecific rather than hybrid. Experimental crosses and field studies show consistent asymmetric hybridization in Louisiana irises, with I. fulva being a more successful father and a more selective mother than both I. brevicaulis and a third species, I. hexagona. The cause of this pattern is not yet clear, but the pattern itself is unusual. Typically, short-styled species tend to be less successful in reciprocal crosses than long-styled relatives, but I. fulva has shorter styles than either I. brevicaulis or I. hexagona. The effects of pollen-tube competition, differential fertilization, and selective abortion in causing this pattern of asymmetric hybridization is discussed.

To elucidate the importance of hybridization in evolution, it is necessary to understand the processes that affect hybridization frequency in nature. Here we focus on postpollination, prefertilization isolating mechanisms using two hybridizing species of Louisiana iris as a study system. We compared the effects of differential pollen-tube growth on the frequency of F1 hybrid formation in experimental crosses between Iris fulva and Iris hexagona. Analyses of seed production in fruits from pure conspecific and heterospecific pollinations revealed that more seeds were produced in the top half than the bottom half of fruits for all four crosses. Heterospecific pollen was applied to flowers of each species at zero to 24 h prior to conspecific pollen, thereby giving a head start to the foreign pollen. Using diagnostic isozyme markers, the frequency of hybrid progeny was examined at the level of the whole fruit and separately for the top and bottom halves of fruits. In both species, the proportion of hybrid seeds per fruit increased significantly with increasing head starts, suggesting that differences in pollen-tube growth rates affect the frequency of hybridization. In I. fulva fruits, the increase in hybrid seeds occurred in both halves of the fruits, but in I. hexagona an increase was only detected in the top half of fruits. These findings are consistent with a model that assumes attrition of pollen tubes due to the greater length of I. hexagona styles. While pollen-tube growth rate appears to be the most important factor affecting hybridization frequency in I. fulva, both pollen-tube growth rate and pollen-tube attrition appear to be important in I. hexagona.

Pollen-tube growth and seed siring ability were measured in crosses between the Louisiana iris species Iris fulva and Iris hexagona and their F1 and F2 hybrids. Flowers of the parental species were pollinated with self, outcross intraspecific, and interspecific pollen. Pollen-tube lengths were similar for all three pollen types in L fulva, but in L hexagona interspecific pollen tubes were longer than intraspecific pollen tubes. Pollen-tube lengths also differed for F, and F2 flowers pollinated with I. fulva, I. hexagona, and hybrid pollen. For both hybrid classes L fulva pollen tubes were the shortest while pollen tubes from I. hexagona and hybrids grew the furthest. Mixtures of genetically marked pollen were used to assess the seed siring ability of intra- and interspecific pollen in the parental species by varying the proportion of each pollen type in a replacement series design. For both species, the observed proportions of hybrid seeds were lower than the expected based on the frequency of each pollen type in the mixtures across all treatments. Flowers of L fulva produced less than 10% hybrid progeny even when 75% of the pollen applied to stigmas was derived from interspecific flowers. The frequency of hybrid seed formation was somewhat greater in I. hexagona, but was still significantly lower than expected across all mixture treatments. Seed set per fruit remained constant across the mixture treatments for both species, but in L fulva fruit set decreased with an increase in the proportion of interspecific pollen. The data indicate that both pre- and postfertilization processes contribute to discrimination against hybrid seed formation.

Saltwater intrusion into wetland ecosystems has destroyed or damaged many native plant populations. Iris hexagona is a salt-sensitive species that exhibits intraspecific variation in salinity tolerance. To investigate the effect of salinity on flowering, we exposed I. hexagona collected from natural populations to salt treatments in a common garden. Experimental salinity additions strongly delayed flowering phenology, but the effect was not apparent until the second year, when less than 4 g/L NaCl delayed flowering up to 3 d. In the field, soil salinity and flowering phenology varied substantially within I. hexagona populations. Iris flowers are receptive to pollinators for 2 d or less, therefore a 3-d delay could affect outcrossing dynamics, and ultimately, the evolutionary ecology of iris populations. Salinity also had a carryover effect; prior salinity exposure delayed flowering in irises that had been replanted in freshwater conditions for 6 mo. This is an important result because it suggests that episodic stress (such as tropical storms) can influence performance well after the stress has disappeared. Our research further underscores the importance of long-term studies because a 1-yr experiment would have failed to reveal the strong effects of salinity that emerged in the second year.

Inheritance of chloroplast DNA haplotypes was determined for progeny from interspecific crosses involving Iris fulva and Iris hexagona. Polymerase chain reaction amplification of chloroplast DNA followed by restriction fragment length analysis of the amplification products was used to identify the haplotypes of 213 experimental hybrids. This analysis allowed a test for maternal, paternal, and biparental inheritance in the hybrid offspring. Two of the hybrid progeny possessed haplotypes that were combinations of those present in the pollen and seed parents. One of the offspring possessed only the paternal haplotype. The remaining 210 plants had the haplotypes characteristic of the maternal plant. Chloroplast DNA variation in iris populations has previously been used to infer not only introgressive hybridization between I. fulva and I. hexagona, but also the greater role of direct pollen transfer relative to seed dispersal as the avenue for interspecific gene flow. We reexamined the previous conclusions concerning the mode of introgressive hybridization between L fulva and I. hexagona in light of the results from the chloroplast DNA inheritance analysis. The low level of paternal and biparental inheritance detected in this analysis suggests that previous analyses using the chloroplast DNA as a seed-specific marker were robust. Furthermore, data concerning barriers to hybridization between I. fulva and I. hexagona suggest that the probability of

Populations of the "Louisiana iris" species Iris fulva, I. hexagona, and I. nelsonii were examined genetically to test for interspecific gene flow between I. fulva and I. hexagona, for pollen- versus seed-mediated introgression between these species, and for the presumed hybrid origin of I. nelsonii. Genetic markers were identified by using both a polymerase chain reaction-like method that allows the identification of random, nuclear markers and standard polymerase chain reaction experiments involving specific chloroplast DNA (cpDNA) oligonucleotides. Restriction endonuclease digestions of the cpDNA amplification products resolved diagnostic restriction site differences for I. fulva and I. hexagona. The distribution of the species-specific nuclear markers supports a hypothesis of bidirectional introgression between I. fulva and I. hexagona. Thus, individuals analyzed from a contemporary hybrid population demonstrate multilocus genotypes that are indicative of advanced-generation hybrid individuals. Furthermore, several markers from the alternate species were present in low frequency in one allopatric population each of I. fulva and I. hexagona. Data from the nuclear analysis also support the hypothesized hybrid origin of I. nelsonii from the interaction of I. fulva and I. hexagona. Finally, cpDNA data support the hypothesis that the localized and the dispersed introgression are largely due to pollen transfer. In addition to the biological implications, this study demonstrates the power of the polymerase chain reaction methodology for the rapid identification of random and specific genetic markers for testing evolutionary genetic hypotheses.

Genetic variation present in 599 individual Iris samples was examined at 50 presumptive loci (isozymes). The samples included allopatric populations of I. fulva, I. hexagona, I. brevicaulis and the putative hybrid species, I. nelsonii. In addition, we examined two populations from areas of overlap involving I. fulva with I. hexagona, and I. fulva with I. brevicaulis and I. hexagona. Allozyme data for both of these hybrid populations suggest the presence of genes from I. fulva, I. brevicaulis and I. hexagona. Bidirectional introgressive hybridization is indicated between I. fulva and I. hexagona. Individuals from a parapatric association between these two species possess various combinations of I. fulva and I. hexagona diagnostic markers. The pattern of genetic variation in this sample indicates the presence of advanced hybrid generations and localized introgression; this finding is in accord with a previous molecular genetic analysis (Arnold et al., 1990). Furthermore, currently allopatric populations of I. fulva and I. hexagona were found to have low frequencies of marker alleles diagnostic for the alternate species. These populations also contain a low frequency of alternate, diagnostic ribosomal DNA repeat length variants (Arnold et al., 1990). The allozyme (and molecular) data suggest that these allopatric samples represent previously introgressed populations. Findings from the allozyme survey indicate that I. nelsonii contains an array of markers characteristic for I. fulva, I. hexagona and I. brevicaulis. Furthermore, some of these markers are found in a present-day hybrid population involving I. fulva, I. hexagona and I. brevicaulis. These findings suggest that I. nelsonii is of hybrid origin, deriving from hybridization between I. fulva, I. hexagona and I. brevicaulis. The genetic analysis along with previous chromosomal, distributional and demographic studies of the Louisiana Iris species suggests that a number of mechanisms may have been involved in the stabilization of this hybrid derivative.

In this study, biomass and its reduction by shade as well as clonal growth have been used as partial indicators of fitness of the taxa. It should be noted that fitness is particularly difficult to estimate in long-lived, clonal perennials such as irises

We performed transplant experiments with Louisiana irises to test the assumptions of three models of hybrid zone structure: the bounded hybrid superiority model, the mosaic model, and the tension zone model. Rhizomes of Iris fulva, I. hexagona, and F1 and F2 hybrids were planted at four sites in southeastern Louisiana in 1994. Wild irises grew at all four sites, but differed in genotypic composition among sites. The sites were characterized by (1) pure I. fulva plants; (2) I. fulva-like hybrids; (3) I. hexagona-like hybrids; and (4) pure I. hexagona plants. The sites differed significantly in light availability, soil moisture and chemical composition, and vegetation. Survival of transplants was high in all sites and did not differ significantly among plant classes. Iris hexagona produced significantly more leaf material than I. fulva at the I. hexagona and I. hexagona hybrid sites. The two species did not differ in leaf production at the I. fulva and I. fulva hybrid sites. Leaf production by both classes of hybrid was as great as, or significantly greater than, both parental classes in all sites. Iris hexagona rhizomes gained mass in the I. hexagona and I. hexagona hybrid sites, but lost mass in the I. fulva and I. fulva hybrid sites. Iris fulva rhizomes lost mass in all sites. There were no significant differences in rhizome growth among classes at the I. fulva site. At all other sites, F1 rhizomes grew significantly more than all other classes except for I. hexagona at the I. hexagona hybrid site. There were no significant differences among classes in the production of new ramets. Overall blooming frequencies were 30% for I. fulva, 10% for F1 s, 3% for F2 s, and 0.7% for I. hexagona. Blooming frequency did not differ among sites for I. fulva, but significantly more F1 s bloomed at the I. hexagona site than at the I. fulva site. These results are inconsistent with all three models of hybrid zone structure. They suggest that once rhizomes become established, hybrids can reproduce by clonal growth as successfully as parents in all habitats, and can outperform them in some habitats. Clonal reproduction may ensure the long-term survival of early generation hybrids and allow the establishment of introgressed populations, despite the fact that F1 hybrids are rarely produced in nature.

We study the strength of the genetic barrier to neutral gene flow in a general one-locus, two-allele model that includes viability selection as well as fertility selection and premating isolation. We have separately considered adult migration and pollen migration. Our theoretical results suggest that selection against hybrid formation in the form of fertility selection or assortative mating is more effective in preventing introgression of neutral alleles than is reduction in hybrid viability. We argue that in experimental studies of introgression testing of F1 hybrids is as important as testing of parental forms. To illustrate the utility of this approach, we estimate the strength of the barrier to neutral gene flow between Piriqueta caroliniana and P. viridis, between Iris hexagona and I. fulva, and between Chorthippus brunneus and C. biguttulus.

Genetic variation associated with the natural hybridization of Iris fulva and I. hexagona was investigated to test for the occurrence of introgression. These species have been viewed as a classic example of the process of introgressive hybridization (Anderson, 1949). However, more recent studies have concluded that there has not been an exchange of genetic material between I. fulva and I. hexagona (Randolph et al., 1967). Our analysis has involved the examination of both allopatric and parapatric populations of I. fulva and I. hexagona with reference to diagnostic ribosomal DNA markers. The pattern of variation in the parapatric population indicates the presence of the repeated backcrossing necessary to the process of introgressive hybridization. Indeed, in the region of parapatry, we suggest that localized introgression of ribosomal sequences has occurred into both I. fulva and I. hexagona. Significantly, we have also detected the presence of the diagnostic ribosomal markers from each species in allopatric populations of the alternate species. Our findings suggest that not only is introgressive hybridization presently occurring in parapatry between I. fulva and I. hexagona, but that past hybridization between these species has resulted in introgression into areas of allopatry.

THE SHAPE of the ovaries of Iris hexagona var. giganticaerulea and of I. fulva is the result of the relative growth of the various radii of those ovaries. Differences in relative growth rates of any two radii or of the same radius at different stages of ovary development are the result chiefly of differences in the relative growth rates of the wall of the ovary at those radii (Riley, 1942a). Riley and Morrow (1942) showed that in fulva growth of any part of the ovary wall is a function of the rate and duration of cell division and of the rate of increase in cell size following cell division. The relative effects of cell division and cell enlargement during various periods of growth are discussed in that paper and are summarized in table 1 of this paper. The present paper reports a similar study of giganticaerulea and a comparison with fulva. The rate of growth of one part of the ovary wall of fulva in relation to another is due chiefly to the rate of cell division in the one part in relation to the rate of cell division in the other part. This is shown in two ways. In several instances one part of the wall will grow more rapidly than a second part, although the cells of the second part enlarge following cell division to a greater extent than do those of the first part of the wall. The more rapid growth of the first part must, therefore, be due to more frequent cell divisions. Also, it has been found that frequently the relative growth rate of two parts of the wall changes, but that the relative enlargement of the cells of the two regions does not change. The change in relative growth of the two parts of the wall must, therefore, be due to changes in the relative amount of cell division in the two parts. MATERIALS AND METHODS.-The plants used for this study were from Bayou Barataria and have been described elsewhere (Riley, 1942a). The methods of obtaining measurements of cells and averages of cell size, of plotting measurements, and of calculating the slopes of the lines are the same as those used in the study of fulva (Riley and Morrow, 1942). As in the paper on fulva, corresponding parts of the several curves are identified by the same letter. These letters correspond to similar places on the curves of radii and of their components (Riley, 1942a). Places marked "A" on the graphs indicate a time shortly after meiosis has begun. Point "B" represents a time just at the conclusion of the divisions of the nuclei of the megagametophyte and the fusion of the two polar nuclei. Point "C" occurs considerably after fertilization when the capsule has partly matured. These points cannot be determined with great accuracy, because all the ovules in one ovary are not in exactly the same stage of

THE IRISES of southern Louisiana have been of peculiar botanical interest since they were first called to the attention of botanists by the late Dr. John K. Small a little more than a decade ago. Many of these irises differ to a greater or lesser extent in the size and shape of the flower parts and in the amount and distribution of pigments in the flower, and there is so much variation among them that Small and Alexander (1931) have listed more than eighty species from within the borders of Louisiana. This interpretation of thespecies, however, has not been accepted by all botanists, for Viosca (1935), chiefly on the basis of ecological evidence, recognizes but four species from the same territory and maintains that the other types which are listed by Small and Alexander are hybrids.. In a more recent paper, Foster (1937) agrees essentially with Viosca. He considers I. virginica L., I. fuiva Ker-Gaw., and I. brevicaulis Raf. (-I. foliosa Mackenzie and Bush) to be the only species in Louisiana, regards I. giganticaerulea Small as a variety of I. hexagona Walter, and believes with Viosea that many of the remaining types are the result of natural hybridization between I. fulva and I. hexagona var. giganticaerulea (Srhall) R. C. Foster. Throughout this present paper, Foster's treatment of the genus will be followed, since it is the only comprehensive treatment of the American species of Iris. Foster's study has been largely taxonomic but has combined chromosome counts with a study of morphological characters. Cytological and ecological approaches are of great value in supplementing the methods of pure taxonomy and especially in determining the probable hybrid origin of a given type of plant. It is possible that biometric methods may in certain cases furnish even more precise estimates of hybridity, especially such methods as have been suggested by Anderson (1928, 1936). An attempt to apply these methods to some of the Louisiana irises is reported in this paper. MATERIALS AND METHODS.-This investigation represents a comparative study of seven characters on plants of four populations, which consist of a group of clones 2 of Iris hexagona var. giganticaerulea, a group of clones of I. fulva, and two groups of clones located geographically between the other two groups. These four colonies of wild plants were found growing in a. region between Thibodaux and Vacherie, which has often yielded a large number of Iris varieties. As the inhabitants of that area have been