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Population Biology |
2Department of Evolution, Ecology, and Organismal Biology, Ohio State University, 300 Aronoff Laboratory, 318 West 12th Avenue, Columbus, Ohio 43210 USA; 3School of Botany and Zoology, University of Natal, Private Bag X01, Scottsville, Pietermaritzburg, 3209, South Africa
Received for publication December 9, 2003. Accepted for publication May 13, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: ethological isolation • flowering time • hybridization • ISSR • PCA • Scrophulariaceae • Zaluzianskya
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Rieseberg and Wendel, 1993
; Rieseberg, 1995
). When hybridization is discovered between species, two key questions are: (1) what is incomplete about their isolating mechanism and (2) what effect is this having on the genetic structure of these species and their hybrids? Isolating mechanisms can occur at many levels, ranging from distributional, ecological, or structural differences between the parental species to hybrid inviability or sterility (reviewed by Stebbins, 1950
; Grant, 1994
). As a result, a breach in these barriers can occur in multiple ways. For example, isolation of parental species may be violated through range expansion or habitat alteration, either natural or anthropogenic (e.g., Levin et al., 1996
; Francisco-Ortega et al., 2000
; Runyeon-Lager and Prentice, 2000
; Wolf et al., 2001
; Capula, 2002
). Plant–animal interactions may also be an important factor in hybridization. For example, an herbivore in one part of the parental distributions could alter the relative fitness of hybrids vs. parental species in those regions, or a new pollinator could be introduced that facilitates gene exchange between two species that were previously separated by ethological isolation. Once hybridization has occurred, there is a wide range of possible consequences, ranging from a small, inert zone of hybrid individuals to the complete merging of parental species. Hybridization is often suspected because of some degree of morphological intermediacy and examination of morphological traits can be very useful in studies of hybrids. However, morphology does not always reflect the degree to which genomes have become contaminated. Molecular markers may allow a more accurate estimation of the permeability of species barriers (e.g., Hodges and Arnold, 1994
).
This study involves the putative hybridization occurring between a day- and night-flowering species of Zaluzianskya F. W. Schmidt (Scrophulariaceae sensu stricto, i.e., within "Scroph I" of Olmstead and Reeves, 1995
; tribe Manuleeae): Z. microsiphon (O. Kuntze) K. Schum. and Z. natalensis Krauss, respectively. Previous field studies have shown apparent pollinator specificity for each species, with long-proboscid flies (Nemestrinidae) pollinating Z. microsiphon and hawkmoths (Sphingidae) pollinating Z. natalensis (Johnson et al., 2002
). Despite this seeming ethological isolation, putative hybrids have been observed in an area where the two species occur sympatrically (Fig. 1; Johnson et al., 2002
). The intermediacy of these individuals in morphology and flowering time leads one to expect that they are hybrids, despite the brevity of overlap in daily flowering times for the presumed parental species. The goals of this study were to use inter-simple sequence repeat (ISSR) markers to test the hybrid status of these individuals and to investigate potential gene flow between the species, both in the area of sympatry and in several isolated populations of each species. In addition, several morphological traits were analyzed to determine if ordination of these characters also supported the hybrid hypothesis.
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). Both Z. microsiphon and Z. natalensis are placed within section Nycterinia (D. Don) Hilliard, and this placement is supported by phylogenetic analyses of both nuclear (internal transcribed spacer [ITS]) and cpDNA sequences (rpl16, trnL-trnF). However, species relationships within section Nycterinia and the monophyly of this section remain partially unresolved (J. K. Archibald et al., unpublished manuscript). Phylogenetic analyses with expanded population sampling placed some populations of Z. microsiphon, Z. natalensis, and their putative hybrids as sister taxa, whereas other populations were placed in nearby polytomies or separate clades (J. K. Archibald, unpublished data). The DNA sequence data were not sufficiently variable to clearly resolve relationships between these populations. Additionally, cladistic analyses are not able to completely resolve reticulate relationships and may not be useful for identifying hybrids (McDade, 1992
). Data from ISSRs are generally more variable than any currently used DNA sequence regions (e.g., Mort et al., 2003
). These types of molecular markers (i.e., ISSRs and RAPDs) have been used in a number of studies examining hybridization (see below).
Section Nycterinia consists of 20 species. Pollination biology for this section has been examined in five species, all of which are pollinated by insects (hawkmoths, or, in the case of Z. microsiphon, long-proboscid flies; McGregor, 1989
; Johnson et al., 2002
). Zaluzianskya microsiphon is the sole day-flowering species in the section. Members of this section are morphologically distinct from the rest of the genus, sharing a suite of floral characters including a long, narrow corolla tube, five notched petal lobes, and contrasting red and white petal coloration (Fig. 1). However, there are several notable differences between the flowers of Z. microsiphon and most of the other species within this section, including Z. natalensis (Table 1). These characteristics appear to be derived (Johnson et al., 2002
; J. K. Archibald et al., unpublished manuscript) and several seem adaptive for pollination by long-proboscid flies. Day flowering is obviously adaptive for pollination by day-flying pollinators. Additionally, several features of Z. microsiphon (e.g., lack of scent and zygomorphic flowers) are shared with unrelated species utilizing the same pollinator, such as species of Pelargonium, Gladiolus, and Disa. This suggests that these characteristics may be part of a pollination syndrome for long-proboscid flies (Goldblatt and Manning, 2000
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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20 mg) was collected on silica gel from 11–20 individuals within each population. Voucher specimens were deposited in the Ohio State University (OS) and University of Natal (NU) herbaria. DNA was extracted from leaf samples using standard hexadecyltrimethyl-ammonium bromide (CTAB) methods (Doyle and Doyle, 1987
) and cleaned using an EluKwik DNA purification kit (Schleicher and Schuell, Keene, New Hampshire, USA).
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; Wolfe et al., 1998
). Inter-simple sequence repeat markers have been used successfully in numerous population genetic studies for natural populations and also specifically in the study of hybridization (e.g., Wolfe et al., 1998
; Datwyler, 2001
). They are usually as, or more, variable than RAPDs, while being more robust to slight changes in DNA concentration and buffers (Wolfe et al., 1998
; Esselman et al., 1999
). In addition, they retain the benefits of other PCR-based techniques such as the need for very little template material. Forty-three ISSR primers were screened for the presence of variation and species-marker bands. Four primers were found suitable after optimization and used in this study: (CA)7YG, (CTC)7RC, (CA)6RG, and (AG)8RG. For simplicity, these primers will be referred to as primer I, II, III, and IV, respectively. The PCR reactions (25 µL) consisted of 1 µL DNA, 1 µmol/L primer, buffer (0.02 mol/L Tris, 0.05 mol/L KCl, and 0.001% Tween-20), 2.9 mmol/L MgCl2, 0.2 mmol/L dNTPs, and 0.5–1.25 units Taq DNA polymerase (Invitrogen, Carlsbad, California, USA; 0.5 units for I and IV, 0.75 units for III, and 1.25 units for II). An Eppendorf MasterCycler Gradient thermocycler was used with the following settings: 110 s at 94°C; 35 cycles of 45 s at 94°C, 45 s at 51°C, and 110 s at 72°C; 10 min at 72°C. All reactions were replicated and only those bands that were visible in both replicates were scored for analysis. Each thermocycler run included a negative control reaction of 25 µL with all of the reagents except for the DNA.
The ISSR reactions were electrophoresed on 1.5% agarose gels in 1% TAE (Tris-acetic acid-EDTA) buffer. The entire reaction volume was loaded into prepared wells and each gel was run until the bromophenol blue indicator dye had traveled 10 cm. Afterwards the gels were stained in ethidium bromide and visualized using UV light. An imaging system (Alpha Innotech, San Leandro, California, USA) was used to record the gels as a TIFF file that was transferred to a PowerMac 7500 and examined using Kodak 1D image analysis software (Eastman Kodak, Rochester, New York, USA). The 1-kilobase (kb) plus ladder size standard (Invitrogen) was used to estimate band sizes. Loci were designated based on fragment size; bands were scored as diallelic (1 = band present, 0 = band absent).
The ISSR data analyses
Genetic clustering of populations and species
Nei and Li's (1979)
coefficient was used to assess pair-wise individual similarity: 2NAB/(NA + NB), where NAB is the number of shared bands, NA is the total number of bands in taxon A, and NB is the total number of bands found in taxon B. This coefficient is appropriate for analyses of ISSR data because it does not include shared absences in the calculation of similarity values. As with RAPDs, absences in ISSR banding patterns can occur for a variety of reasons and thus should not be considered homologous (Wolfe and Liston, 1998
). Distance matrices were created using !WXDNL (Vera Ford, University of California, Davis, California, USA) and RAPDPLOT 3.0 (using option "S"; William C. Black, IV, Colorado State University, Fort Collins, Colorado, USA). These programs produce identical results but the former is formatted for use with NTSYS (Rohlf, 1998
) whereas the latter is formatted for PHYLIP's NEIGHBOR (Felsenstein, 1993
). A neighbor-joining analysis was run in NTSYS to determine whether there were tied trees (Backeljau et al., 1996
), but no such trees were found and the remainder of the neighbor-joining analyses were run in NEIGHBOR. RAPDPLOT has the capability of producing a distance matrix along with a set of bootstrapped distance matrices from the original data. These latter matrices can then be input into NEIGHBOR using the multiple data sets option to produce a set of bootstrap trees. After saving these trees in NEXUS format using TREEVIEW (Page, 1996
), their majority rule consensus was calculated in PAUP* 4.0b10 (Swofford, 2003
) to determine the level of bootstrap support for neighbor-joining groups. All runs of NEIGHBOR employed the jumble option to randomize taxon addition order prior to construction of the tree; this also randomizes taxon order between bootstrap replicates (Farris et al., 1996
). Neighbor-joining analyses were run with and without hybrid individuals included.
A Mantel test (Mantel, 1967
) was performed using NTSYS to determine if genetic distance was correlated with geographic distance. Geographic distances were calculated based on GPS coordinates for each population (Appendix). Genetic distances were derived from population similarity values calculated using !WAVSIML (V. Ford). A normalized Mantel statistic was used and significance was tested with the maximum number of random permutations (9999).
Hybridization and gene flow
The distribution of each ISSR locus was examined in all populations to ascertain patterns of gene flow. Ideally, species-specific marker bands (i.e., bands that are present in all individuals of one species and none of the other) would be found and morphologically intermediate individuals would be examined for additivity of these bands to test the individuals' hybrid status (e.g., Cruzan and Arnold, 1993
; Steen et al., 2000
). However, it is frequently not possible to find such marker bands, possibly due to the often-close relationship of the parental species or to introgression. Several studies of hybridization have therefore used less stringent methods for defining marker bands, employing bands that are more common in one taxon vs. the other, to some degree (Allan et al., 1997
; Wolfe et al., 1998
; Neuffer et al., 1999
; Datwyler, 2001
; Feliner et al., 2002
). In this study, gene flow was traced in three ways. First, those bands that occurred in one species in at least a 25% higher frequency than the other were considered species-typical bands. Band frequencies in the proposed hybrids were then examined to determine if they were intermediate to those found in the two species. This is a modification of the method used by Wolfe et al. (1998)
. Second, bands that occurred in only one species plus the hybrids, or sometimes also extended into the sympatric population of the other species, were noted regardless of the band frequency difference between Z. microsiphon and Z. natalensis. Third, band frequencies for each locus were inspected at the population level to determine whether there were statistically significant differences between populations. This was tested using a modification of Fisher's exact test (Raymond and Rousset, 1995
), implemented in TFPGA (Miller, 1997
) with 10 000 permutations, 50 batches, and 5000 dememorization steps.
Morphological survey of sympatric populations
Nine morphological characters were assessed in the sympatric populations of Z. microsiphon, Z. natalensis, and their putative hybrids (Table 2). These traits were chosen due to their apparent or potential distinctness in each species. Eighteen individuals of Z. microsiphon, 19 of Z. natalensis, and 11 hybrid individuals were examined (48 total, Appendix). Of these 48, ten accessions from each species and ten accessions of the putative hybrid individuals were also included in the ISSR study (30 total). Seven of the morphological traits were measured for all 48 individuals whereas two (plant height and floral orientation) were measured for about half of the individuals of each population. It was not possible to measure these two traits in the other half due to the condition of the plant collections.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A positive and highly significant correlation was found between genetic distance (Nei and Li, 1979
) and geographic distance (r = 0.73155, P = 0.0002, Mantel test). This correlation was also found when only looking at populations of Z. microsiphon (r = 0.84532. P = 0.003) and when looking at those populations plus the hybrid population (r = 0.81041, P = 0.0008). Thus, the correlation between genetic and geographic distance was seen at the population level and was not due solely to differences between the species.
Hybridization and gene flow
No completely species-specific ISSR loci were found (i.e., present in all individuals of one species and no individuals of the other). However, 15 loci occurred with at least a 25% difference in band frequencies between Z. microsiphon and Z. natalensis (Table 3); these loci were used to examine gene flow. For eight of these "species-typical" loci the putative hybrids had band frequencies intermediate to those found in the presumed parental species, corroborating the hypothesis that they are in fact hybrids. In four of the other seven loci, the hybrids had higher band frequencies than either of the two species, whereas the hybrids lacked the other three loci despite their presence in both parental species. These seven loci do not necessarily support the hybrid hypothesis although there are several possible explanations for this pattern, regardless of whether these individuals are hybrids or not (see Discussion).
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; Joliffe, 1986
; Jackson, 1993
), only the first axis, and possibly to a small extent the second axis, accounted for a significant amount of variation. The variation shown in the first axis was clearly significant, whereas the percent variation accounted for by the second axis was nearly identical to that produced under the broken-stick model and thus of marginal or no significance according to that model. Many of the variables contributed to the loading on the first axis, especially floral orientation (coefficient = –0.950), throat indumentum (0.924), floral symmetry (0.887), node length (–0.840), and tube length (0.821). Thus, individuals were divided generally into those with vertically oriented petal lobes, glabrous throats, zygomorphic flowers, longer distances between flowers on inflorescences, and shorter corolla tubes (i.e., morphology associated with Z. microsiphon) and those with the opposite: horizontally oriented petal lobes, a ring of hairs around the throat, actinomorphic flowers, shorter distances between flowers on inflorescences, and longer corolla tubes (i.e., morphology associated with Z. natalensis). Only leaf length (0.778) and plant height (0.720) had coefficients greater than 0.7 for the second axis (Table 6). A plot of the first two axes resulted in spatial separation of the two species along the first axis but not the second. The hybrids are distributed within the area between the two species but are more concentrated towards individuals of Z. natalensis (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, putative hybrids occur in the sympatric Mt. Gilboa populations of these species. These individuals appear intermediate in flowering time and general morphology (Johnson et al., 2002
; J. K. Archibald, personal observation). The results of the molecular analyses support the possibility of gene flow between the two species. These data are somewhat inconclusive due to the genetic similarity of the proposed parental species, however their combination with the morphological analyses strengthens the case for introgression. The neighbor-joining tree suggests a geographic component to genetic diversity for the two species and their putative hybrids, as would be expected if gene flow is occurring between rather than just within populations. Adjacent populations merged into mixed groups in the tree rather than forming distinct clusters, and this occurred both within and between species (Fig. 3). The correlation between genetic distance and geographic distance was verified by the highly significant results of the Mantel test. Thus, if hybridization is occurring in a few sympatric populations, the effects could be spreading to nearby allopatric populations. Alternatively, it is possible that the genetic similarity between populations could be due to recent colonization from a common ancestor. A choice cannot be made between these hypotheses of historical similarity vs. current gene flow based on these data.
Zaluzianskya microsiphon and Z. natalensis largely cluster separately within the neighbor-joining tree. However, there is some mixing, particularly in a small group of individuals from the Mt. Gilboa populations, including hybrids and members of both species. The fact that these individuals cluster together rather than with other members of their own species corroborates the hypothesis of interspecific gene flow, although this tree has very low bootstrap support. Additional hybrids are found in several groups across the tree; however, they are always found with individuals from the sympatric populations (MG and/or NG). The placement of these hybrids on the neighbor-joining tree and the morphological PCA plot were compared but no obvious pattern was apparent. Those hybrids that clustered with a particular species on the tree did not necessarily fall closest to individuals of that species on the PCA plot. Instead they were spread across the range between the two species on the PCA plot (Fig. 5).
The ISSR loci were also examined individually, first at the species level and then at the population level. Fifteen species-typical loci were found (Table 3). As explained above, band frequencies at a particular locus must differ by at least 25% between the two species for the locus to be considered species-typical. By this definition, a species-typical locus does not necessarily need to be found exclusively in one species. In the literature, species markers have been defined in a variety of ways; this is a similar, although less stringent, definition to those used by several other studies (e.g., Wolfe et al., 1998
; Neuffer et al., 1999
; Datwyler, 2001
). Ironically, the difficulty in locating more conclusive species-typical markers could be due to the very hybridization and gene flow that we are trying to detect. In fact, if the sympatric Mt. Gilboa populations are excluded from the analyses, the number of loci that are only found within a single species is raised from 10 to 19 (including two species-typical loci). In contrast, if any two other populations are removed from the analyses (one from each species), this number is only raised to 13 at the most.
However, in addition to hybridization, overlap in loci could also be due simply to a high level of genetic similarity between the species. Excluding floral characters, these species are nearly identical, and parsimony analyses of morphology place these taxa in a clade, along with another very similar species, Z. spathaceae (Johnson et al., 2002
). Regardless, it is possible to see some trends in the data at hand given the caveat that these marker bands are not truly species-specific.
Putative hybrids had intermediate band frequencies relative to Z. microsiphon and Z. natalensis for eight of the 15 marker bands (Table 3), lending support to the hypothesis of hybrid origin. The remaining seven marker bands showed "extreme" frequencies in the hybrids, that is, the hybrid band frequencies were either higher or lower than either of the putative parental species. Only the three loci with lower hybrid band frequencies were not additive in the hybrids, in that bands for those loci were completely absent in the hybrids while being present in at least some individuals of both proposed parental species. Incomplete additivity in these types of markers (i.e., RAPDs and ISSRs) for hybrids has been found in multiple previous studies (e.g., Huchett and Botha, 1995
; Smith et al., 1996
; Huang et al., 2000
; Steen et al., 2000
; Feliner et al., 2002
). The extreme band frequency values, including the absence of those three loci in hybrid individuals, could possibly be explained by several factors, regardless of whether those individuals truly are hybrids. The fact that ISSRs are dominant markers means that a heterozygous individual will show the same banding pattern as a dominant homozygous individual. Thus, some hybrids could lack a particular band due to inheriting the absent "allele" from two heterozygous parents. Probably of more importance in this study is the fact that both proposed parental species are polymorphic for all marker bands. Thus a true hybrid individual may lack a band seen in some individuals of a parental species simply because the actual parents of that plant also lacked the band. The band frequency for one population of putative hybrid individuals was compared to the band frequency across several populations of each presumed parental species. Ideally, these band frequencies would reflect hybrid intermediacy. However, due to intraspecific variation, the band frequency for each species did not always reflect population-level band frequencies. For example, the band frequencies of the two Sentinal populations of Z. microsiphon (MR and MS) were often distinct from those found in other populations of that species. This difference was statistically significant for eight of the 52 ISSR loci (results shown for III-6, see Fig. 4). After comparing the hybrid population band frequencies to those found in each population of each species, we found that all seven marker bands with supposedly "extreme" values were actually well within the normal range of population-level frequencies found in one or both parents. This was verified statistically for all seven bands (results shown for III-6, see Fig. 4). The band frequencies for the putative hybrids were statistically indistinguishable from at least some of the populations of one or both proposed parental species. Also, one or both of the sympatric populations (MG and NG) were always among those populations with similar band frequencies to the hybrids—again congruent with the possibility of hybridization. This was true for all 15 species-typical loci.
When examining populations individually, four of the 15 marker bands were found to have a statistically significant pattern relevant to hybridization (Fig. 4 and Table 5). Although, again, variation within species complicates interpretation, several trends are visible in these four loci. The band frequencies for the putative hybrids appear to be intermediate between the two species. In all four loci, the populations of Z. microsiphon have (in general) higher band frequencies than those of Z. natalensis, with the hybrids falling in between. For three of these loci (I-7, II-9, and III-6), the hybrid population is statistically indistinguishable from several populations of Z. microsiphon and the sympatric population of Z. natalensis (NG), while being significantly different from the other populations of Z. natalensis. At the other locus (II-4) the hybrid band frequency is statistically indistinguishable from one allopatric Z. natalensis population (NA) and the sympatric Z. microsiphon population, while the other populations of both species are significantly different. One potential explanation for these patterns is that gene flow is occurring, possibly preferentially from Z. microsiphon to Z. natalensis.
This interpretation is also supported by the data shown in Table 4. Although these loci (with the exception of III-6 and III-12) do not show a >25% difference in band frequencies between the species, they do demonstrate a pattern that could indicate gene flow. In nine of these 10 loci, ISSR bands were found in Z. microsiphon and in the hybrids, but not in Z. natalensis—except, in six cases, for the sympatric population of Z. natalensis (NG). This pattern could again be explained by differential gene flow from the day-flowering Z. microsiphon into the hybrids and the night-flowering Z. natalensis. One locus (IV-1) shows the reverse pattern, supporting the hypothesis of hybridization but in this case implying gene flow from Z. natalensis into the hybrids.
Hybrids were identified in the field by their generally intermediate morphology and flowering time. However, when nine morphological traits were examined more closely using PCA (Table 2), they indicated that although the putative hybrid individuals occurred across the range between the clusters of Z. microsiphon and Z. natalensis individuals, most were placed very close to individuals of the latter species (Fig. 5). This is particularly noticeable if you ignore the second axis, whose significance was questioned by comparisons with a broken-stick model. Thus, the ISSR data suggest that the hybrids are potentially more similar to Z. microsiphon whereas the morphological data indicate that many of them are more similar to Z. natalensis. Mantel tests comparing these two data sets also show a significant (P = 0.0012) negative correlation, albeit a very weak one (r = –0.19203). One possible conclusion based on the ordination analyses alone is that many of these "hybrids" are misidentified individuals of Z. natalensis. However, the differences seen in these individuals extend beyond the generally accepted range of variation for this species (Hilliard, 1994
), and this would also conflict with the ISSR data. A stronger hypothesis is that these individuals are backcrosses with Z. natalensis. It appears that the Z. natalensis-like individuals are more genetically similar to Z. microsiphon (at least in some portions of their genomes), supporting the possibility of introgression of Z. microsiphon genetic material into the night-flowering species. Perhaps introgression of morphological characters of Z. microsiphon is more strongly selected against, as seen in Hodges and Arnold's (1994)
study of Aquilegia and Goulson and Jerrim's (1997)
study of Silene. Further investigation is necessary to examine that possibility. Although additional molecular and morphological data should aid in clarifying the degree and direction of gene flow, the unidirectional nature of this gene flow is corroborated by these data.
It appears plausible that pollen flow is also unidirectional in these taxa. As noted above, long-proboscid flies pollinate Z. microsiphon while Z. natalensis is pollinated primarily by hawkmoths. Only one exception to this pollinator specificity has been observed; a single long-proboscid fly was seen probing flowers of Z. natalensis after visiting Z. microsiphon, during the brief period at dusk when flowers of both species are open (Johnson et al., 2002
). This pattern of visiting Z. natalensis after Z. microsiphon may be typical of natural crosses between these species. Flowers of Z. microsiphon become less visible than those of Z. natalensis as the light fades. These flowers are not only beginning to close, but also their upper petal surface is duller than flowers of Z. natalensis (greenish-white vs. white; Hilliard, 1994
). The lower petal surface of both species is dark red, seeming to camouflage closed and closing flowers. While Z. microsiphon flowers are closing, flowers of Z. natalensis are beginning to open and becoming clearly visible in the low-intensity light. Thus, differential pollen flow could be occurring towards Z. natalensis when late afternoon turns to evening, with some pollinators picking up pollen from Z. microsiphon before those flowers close and carrying that pollen to the increasingly more visible and prevalent flowers of Z. natalensis. The reverse flow of pollen could be less likely because as more Z. natalensis flowers are open and available for pollen-donation, more of the Z. microsiphon flowers are closed (or at least are less conspicuous). It is unlikely that these species are also hybridizing in the morning hours. Long-proboscid flies are not active in the morning until after flowers of Z. natalensis are completely closed, and flowers of Z. microsiphon do not open early enough in the morning to be affected by moth activity.
Differential pollen flow has been shown in previous studies to result in unidirectional gene flow. For example, in Aesculus, migrating hummingbirds disperse pollen far north of the range of one parental species, leading to introgression only with a single parental species (dePamphilis and Wyatt, 1989
, 1990
). Seed dispersal for these Aesculus species appears to be localized and thus does not play a role in gene flow between the species. Unidirectional gene flow in several species of Penstemon is also hypothesized to be due to differential pollen flow, as a result of both the timing of pollinator visits to different species as well as differences in floral morphology (Wolfe and Elisens, 1995
). Concluding whether an analogous mechanism is influencing gene flow in these species of Zaluzianskya requires further study of pollinator interactions between these populations. This will help to determine the potential causes for unidirectional gene flow, such as hybrids crossing more easily with individuals of Z. natalensis, possibly through differences in fruit abortion, pollen germination rates, floral mechanics, or pollinator behavior (Barton and Hewitt, 1985
; Ellis and Johnson, 1999
; Tiffin et al., 2001
).
The intermediacy of putative hybrids for some morphological and especially phenological traits could have implications for hybrid fitness. Many flowers of these individuals remain partially open throughout the day and evening, but are never fully open. This would be expected to be unattractive to either species' pollinators, possibly leading to a "dynamic-equilibrium" hybrid zone, which is maintained by a balance between dispersal and hybrid inferiority (Barton, 1979
; Arnold, 1992
). However, the preliminary data given here suggests that the hybrids may contribute to further gene flow, with some introgression of Z. microsiphon genetic material into Z. natalensis. Field studies are necessary to determine if pollinators are visiting the putative hybrids. The data in this study are consistent with the hypothesis that hybridization is occurring with introgression. However, the genetic similarity of these two species makes it difficult to more conclusively support these hypotheses over alternatives such as historical similarity. Future work will focus on increasing population sampling and developing other markers for examining these questions.
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FOOTNOTES |
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4 Present address: Department of Ecology and Evolutionary Biology, University of Kansas, 1200 Sunnyside Avenue, Lawrence, Kansas 66045 USA. jkarchibald{at}yahoo.com
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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dePamphilis C. W. R. Wyatt 1990 Electrophoretic confirmation of interspecific hybridization in Aesculus (Hippocastanaceae) and the genetic structure of a broad hybrid zone. Evolution 44: 1295-1317[CrossRef][ISI]
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= 0.05) frequencies are labeled with the same letter. Column color indicates populations of Zaluzianskya microsiphon (black), putative hybrids (gray), or Z. natalensis (white). Population abbreviations are as in the 