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Systematics |
2Department of Botany, 3190 Maile Way, University of Hawai‘i, Honolulu, Hawai‘i 96822 USA 3Center for Conservation Research and Training, 3190 Maile Way, University of Hawai‘i, Honolulu, Hawai‘i 96822 USA
Received for publication September 19, 2000. Accepted for publication February 23, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Dubautia • Hawai‘i • hybridization • introgression • RAPD • silversword alliance
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Three genera (Argyroxiphium, Dubautia, and Wilkesia) are currently recognized. Dubautia is the largest genus, with one or more of 23 endemic species found on all six of the major islands of the Hawaiian archipelago (Carr, 1985b, 1999
). Dubautia also exhibits the most unusual variety of growth forms found in the alliance, including cushion plants, mesophytic and xerophytic shrubs and trees, and lianas. The creation of new habitats during island formation has led to rapid speciation within this genus. This radiation has been accompanied by chromosomal evolution resulting in at least eight cytological genomes with either 13 or 14 pairs of chromosomes (Carr and Kyhos, 1981, 1986
; Kyhos, Carr, and Baldwin, 1990
).
The silversword alliance is now well characterized both taxonomically (Carr, 1985b, 1999
) and phylogenetically (Baldwin, 1992
; Baldwin et al., 1991
). Studies have further shown that hybridization has played an important role in the early evolution and speciation of this alliance (Baldwin, 1997
; Baldwin et al., 1998
). Recent population-based studies have also demonstrated that these species contain some of the most striking examples of recent hybridization in the Hawaiian flora (Carr and Kyhos, 1981, 1986
; Robichaux, 1984
; Crins, Bohm, and Carr, 1988
; Carr, 1995
). Because of the mosaic distribution of these species across the landscape, species from different ecological regimes are often found growing sympatrically, and hybrids are frequently found in these situations, even between morphologically and chromosomally differentiated taxa (Carr and Kyhos, 1981
). At least 30 different interspecific and five intergeneric naturally occurring hybrid combinations are known (Carr and Kyhos, 1981, 1986
; Carr, 1995
). Cross compatibility of the species is well documented and artificial hybrids have been achieved from virtually all crosses attempted (Carr and Kyhos, 1981, 1986
; Carr, 1995
). In contrast to many other examples in which hybrid derivatives are often sterile or semisterile and may contribute little to the gene pool (Stebbins, 1959
; Grant, 1985
; Randell, 2000
), hybrids among Dubautia species are often highly fertile. Mean pollen stainability of artificial and natural Dubautia hybrids ranged from 26 to 99%, and 11 of 36 hybrid combinations exhibited an average pollen stainability of 90–99% (Carr and Kyhos, 1986
). Seed-producing hybrids such as these often promote gene flow back to the parental species (Rieseberg and Gerber, 1995
; Levin, Francisco-Ortega, and Jansen, 1996
).
One extensive Dubautia hybrid zone occurs on the island of Hawai‘i, at the summit of the saddle between Mauna Loa and Mauna Kea near Pu'u Huluhulu, elevation 1980 m. Two species occur at this site: D. scabra subsp. scabra (n = 14) and D. ciliolata subsp. ciliolata (n = 13). Dubautia scabra is a decumbent mat-forming shrub with alternate leaves that remain attached to the stem after senescence and a flat-topped (corymbiform) capitulescence of white-flowered heads. Dubautia ciliolata is an erect, rigid shrub with whorled leaves that abscise from the stem following senescence and an elongate capitulescence of yellow-orange-flowered heads. Ecologically, the two species occupy very different habitats. This site is dominated by pahoehoe lava from a 1935 Mauna Loa eruption. However, the flow is discontinuous in places, forming a mosaic of "islands" (kipuka) of older substrate consisting of pahoehoe and a'a lava from an 1855 flow that has weathered to a rocky soil (Hazlett and Hyndman, 1996
). Dubautia scabra is a colonizer restricted at this site to the 1935 lava flow, whereas D. ciliolata is found almost exclusively within the islands of older substrate; a few individuals of what appears to be D. ciliolata may be found on the 1935 flow within 4 m of the 1855 flow boundary, where the younger flow tends to decrease in thickness (Robichaux et al., 1990
).
Hybrids between D. ciliolata and D. scabra occur on the newer substrate, often but not exclusively, within 4 m of the boundary between the 1935 and 1855 lava flows (Robichaux et al., 1990
). The hybrids are morphologically intermediate to the parents, and possess 2n = 27 chromosomes that appear as 12 pairs plus a chain of three at meiosis (Carr and Kyhos, 1981, 1986
). Very high chromosomal homology and an average of 82% pollen stainability in the hybrids indicate a high degree of relatedness between the parental species. A comparison of the flavonoid chemistry of an F1 plant (from controlled crosses) with the field-collected hybrids suggested that the Pu'u Huluhulu population may consist of second generation (or later) hybrids or backcrosses to either parent (Crins, Bohm, and Carr, 1988
).
This research uses random amplified polymorphic DNA (RAPD) markers to examine the population dynamics of the D. ciliolata and D. scabra individuals and assess potential for gene flow between these species. RAPD markers have been used to study hybrid progeny among several plant groups (Arnold, Buckman, and Robinson, 1991
; Smith, Burke, and Wagner, 1996
; Daehler and Strong, 1997
; Ayres et al., 1999
; De Greef and Triest, 1999
; Kuehn, Minor, and White, 1999
; Neuffer et al., 1999
; Rieseberg and Linder, 1999
; Randell, 2000
). Specific goals of this study were to (1) determine the levels of genetic variation within populations of D. ciliolata and D. scabra, (2) identify genetic markers unique to each species, (3) assess the genetic variation found among the hybrid individuals, and (4) determine whether backcrossing or further recombinant generations of hybrids are detectable.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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with some minor adjustments (Morden, Caraway, and Motley, 1996
). The standard extraction buffer consists of 2% CTAB, 100 mmol/L Tris-HCl (pH 8.0), 1.4 mol/L NaCl, and 20 mmol/L EDTA, with 0.2% ß-mercaptoethanol added immediately prior to use. Leaves were ground in hot CTAB buffer (65°C) and incubated for 15–60 min at 65°C. The slurry was extracted once with chloroform : isoamyl alcohol (24 : 1) and centrifuged for 10 min at 3000 rpm in a Sorvall tabletop centrifuge. The aqueous phase was removed to a new tube, combined with a minimum two-thirds volume of isopropanol, mixed, incubated at –20°C for at least 30 min to precipitate the DNA, and then centrifuged again for 5 min to collect the precipitate. The pellet was washed in 76% EtOH with 10 mmol/L ammonium acetate for at least 10 min and recentrifuged for 5 min to collect the precipitate. The pellets were drained and resuspended in 4 mL TE (10 mmol/L Tris-HCl pH 8.0, 1 mmol/L EDTA). DNA was purified by cesium chloride density gradient ultracentrifugation (Sambrook, Fritsch, and Maniatis, 1989
). DNA bands were recovered, ethidium bromide was removed with water-saturated butanol, and the DNA was dialyzed against four washes in TE. Isolated DNA was accessioned into the Hawaiian Plant DNA Library (HPDL) (Morden, Caraway, and Motley, 1996
) and stored at –20°C until used.
DNA samples of three individuals from each species and the hybrids were screened with 180 Operon primers of which 43 primers were selected for analysis of all individuals. Primers were selected on the basis of their ability to amplify DNA, band intensity, number of loci amplified, and reproducibility of the products. DNA amplifications were performed in 25 µL reaction volumes consisting of 4 mmol/L random 10-mer primer (Operon Technologies, Alameda, California, USA), 0.2 mmol/L dNTP, 1x polymerase buffer, 25 mmol/L MgCl2, 1 unit Taq Polymerase (Promega, Madison, Wisconsin, USA), and 
25 ng of isolated DNA overlaid with two drops of mineral oil. The samples were exposed to the following conditions on a OmniGene temperature cycler (Hybaid Limited, Franklin, Massachusetts, USA): one cycle at 94°C for 3 min, 35°C for 30 sec, and 72°C for 2 min; 43 cycles 95°C for 45 sec, 35°C for 30 sec, and 72°C for 2 min; a final cycle at 94°C for 45 sec, 35°C for 30 sec, and 72°C for 6 min. The amplification products were assayed on 1.5% agarose gels in 0.5x TBE (tris-borate-EDTA) (Sambrook, Fritsch, and Maniatis, 1989
). Gels were stained with ethidium bromide and photographed under ultraviolet light. Loci were identified based on the size of the band relative to a restriction digested pBS plasmid (Stratagene, La Jolla, California, USA). Only markers that were unambiguous, well amplified, and reproducible in replicate tests were scored.
Genetic markers (bands) resulting from the RAPD amplifications were scored for each locus based on their presence (1) or absence (0). Bands of identical size were assumed to be homologous across the individuals sampled in this analysis, and bands of different sizes were assumed to represent separate genetic loci. However, no tests to determine this have been conducted. All markers were scored for each primer sampled. A locus was considered polymorphic if any variation for expression of the marker was detected within a species. Markers that were present in all individuals of a taxon were referred to as "constant," and it is assumed that the alleles were fixed homozygous for such markers. Although fixation may not be absolute, the frequency of a "null" allele is likely to be low within the parental species. Markers that were inconsistently amplified in repeated RAPD reactions were not included in subsequent analyses. Constant markers from each species were used to create a phenotypic index for putative hybrid individuals following Ayres et al. (1999)
.
Both simple matching and Jaccard's similarity coefficients were used to assess relatedness of individuals within and among species using NTSYS (Rohlf, 1993
). Both of these methods consider two character states, the presence (1) and absence (0) of a particular DNA fragment. Jaccard's method is calculated based on positive matches (RAPD markers present in both individuals) and mismatches (marker present in one individual and absent in the other), whereas the simple matching also takes into consideration the occurrence of negative matches (markers absent in both individuals) giving equal weight to both positive and negative matches (Sneath and Sokal, 1973
; Dunn and Everitt, 1982
).
Similarity coefficients were subjected to cluster analysis using unweighted pair-group method analysis with arithmetic averages (UPGMA), and dendrograms were constructed from these values. Principal components analysis (PCA) was used to find relationships between RAPD markers and specimens without a priori division of the samples into discrete groups (Wiley, 1981
). Multivariate statistics were computed using the MINITAB statistical package (MINITAB, 1996
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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18 per primer. It became apparent from examination of the distribution of markers among the individuals that a priori identification of one plant (Motley 1496) as D. scabra was in error, and it was treated as a hybrid in all subsequent analyses.
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The mean similarity coefficients within and among the Dubautia species and hybrids were computed using Jaccard's association coefficients and simple matching (Sneath and Sokal, 1973
) and are found in Table 3. Both Jaccard's index and simple matching (SM) show that individuals of D. scabra are genetically more similar than are individuals of D. ciliolata. As would be expected, the two species are less similar to each other than the hybrid is to either D. scabra or to D. ciliolata.
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used statistical models to determine that few markers were necessary for characterizing individuals as pure species or hybrids (as few as five), although many more (as many as 70) would be required to distinguish pure species from advanced (B4 or greater) backcrosses. The number employed in our analysis is sufficient to clearly distinguish filial and backcrossed hybrid progeny and determine if advanced hybridization events have taken place. Assuming that markers within each parent were homozygous, all 40 loci of an F1 individual should be heterozygous and their marker present. Markers in a given F2 individual should sort in approximately a 3 : 1 ratio (i.e., 30 of the markers should be present). Although there would be differences in the number of markers present among later generation hybrids depending whether they are outcrossing or inbreeding, this is not a factor at the depth of the analysis presented here. Total marker presence among the hybrids ranges from 14 to 38 markers. No individual had all 40 markers that would be expected in an F1 hybrid, including the artificial hybrid (i.e., Carr 1342-8) that had only 38 of the 40 markers present. Two plants had 32 (Morden 1347) or 33 (Morden 1346) markers, and several had 34 markers. These totals, and a relatively even distribution of markers from both species, suggest they are F2 or some other product of hybridization beyond the F1. Six plants had between 35 and 37 markers present, and it is uncertain based on the information available whether these individuals represent F1 or later generation hybrids. If an individual is a backcross progeny, the constant markers of the nonrecurrent parent should decrease by one-half with each successive backcross. Thus, it is expected that a hybrid backcrossing once (B1) to D. ciliolata would lose approximately half (11–12) of the D. scabra markers. A second backcross (B2) would result in a further loss of approximately half of the remaining D. scabra markers, and so on. Each of the seven plants that clustered near D. ciliolata in the PCA have all or most of the diagnostic D. ciliolata markers and markedly fewer of the D. scabra markers. The total number of D. scabra markers still present in a B2 is expected to be 5 or 6, as found in Morden 1352 (Table 4). Hybrids Motley 1513 and Motley 1516 have two and one D. scabra markers present, respectively, while retaining all 17 D. ciliolata markers, suggesting that these plants are products of backcrossing beyond the B2. Four individuals suspected to be of hybrid origin based on their morphology (Motley 1514, 1515, 1517, and 1520) contained none of the D. scabra constant markers but at least one of the diagnostic polymorphic markers and may also represent later generation backcross individuals. However, these four plants did not have all 17 of the D. ciliolata constant markers present.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and grasses (Stiller and Denton, 1995
). Alternatively, the breeding systems of these species could result in the different levels of genetic variation observed. It is known that both D. ciliolata and D. scabra are capable of self-fertilization (Carr, Powell, and Kyhos, 1986
), but it is not known to what extent this is realized in natural populations. The percentage of markers constant in the putative hybrid is lower than in the parents. This statistic is not surprising of a hybrid suspected of advanced generations or of recent backcrossing. A true F1 should possess all 40 markers that are constant in one parent and absent in the other (17 from D. ciliolata and 23 from D. scabra) plus the 38 markers that are constant in both parents (i.e., a total of 78 fixed markers). However, only 49 markers were constant in the hybrid individuals, suggesting that many of the hybrids are later recombinants. The percentage of polymorphic markers in the hybrids is high (73%), but not surprising given the parents' relatively high polymorphism proportionate to the 241 markers scored (55% in D. ciliolata and 44% in D. scabra) and even higher polymorphism proportionate to the species markers (70% and 59%, respectively).
One surprising statistic is the high number of markers unique to the hybrids (12). This suggests that the 66 yr that have elapsed since the most recent lava flow in the Pu'u Huluhulu area (i.e., 1935; Hazlett and Hyndman, 1996
) may be sufficient time for the hybrid population to become established and to begin to differentiate from its parental species. These markers were not common among the hybrid plants (usually in only one or two individuals tested). Thus, some of the markers thought to be unique to hybrids may also occur in low frequency in the parental populations and might be detected with additional sampling.
The number of markers present among the presumed filial (nonbackcrossed) hybrids ranged from 32 to 38, and it was unclear where to draw a distinction between F1 and F2 (or later generation) plants, if at all. It is clear that the assumption of parental markers being fixed if polymorphism was not detected was violated at least once; only 38 of the 40 markers were present in the artificial hybrid (Carr 1342-8). It also seems probable that plants with only 32 or 33 of the diagnostic markers are the product of filial hybridization beyond the F1. Since F1 plants are expected to have 38–40 diagnostic markers and F2 plants 30 markers, those plants with marker numbers intermediate to the expected values (e.g., 34–37 markers) may reasonably be interpreted as the result of crosses between F1 and F2 (or later generation) plants. However, it was not possible to clearly distinguish among these various scenarios, and we have not attempted to further classify the hybrids with markers in the range of 32–38. Nevertheless, the data strongly suggest that these presumed filial hybrids include generations beyond the F1.
Results suggest the occurrence of unilateral introgressive hybridization in the direction of D. ciliolata. Six hybrid plants clustered with D. ciliolata in both the simple matching and Jaccard's analyses as well as the PCA. A seventh putative backcross plant (Morden 1352) that clustered with the other hybrids had only one-fourth of the diagnostic D. scabra markers, suggesting it is a B2 generation plant. This plant was intermediate to the hybrid and D. ciliolata groupings in the initial PCA. Two other individuals are potential B3 generation plants with only one and two D. scabra markers, respectively. An alternative explanation for the low number of D. scabra markers present in these plants is that they are the products of an F2 or later generation hybrid, with an already depauperate number of D. scabra markers, backcrossed to D. ciliolata. Similarly, the four plants with no D. scabra markers and less than the total number of D. ciliolata markers may be the result of other backcross events such as an F1 or F2 plant crossing with an introgressed plant. Initial results suggested that Motley 1496 may have been the progeny of a backcross between a hybrid and D. scabra based on both the UPGMA and initial PCA. However, it clearly clustered with other hybrids in the second PCA (with diagnostic species markers only), and the distribution of markers further suggests it may be the progeny of later filial generation hybridization rather than backcrossing.
An alternative explanation for the distribution of markers among backcrossed individuals is that there may be a deviation from neutral random assortment. Rieseberg and Linder (1999)
found that genetic markers from Helianthus petiolaris were disproportionately lost when H. annuus x H. petiolaris hybrids were successively backcrossed to H. annuus; the B2 generation included individuals that appeared to be later generation backcrosses (B3 to B7). Rieseberg and Linder (1999)
attribute this lack of introgressive neutrality to selection against H. petiolaris as a consequence of chromosomal translocations and inversions that distinguish the two species. Similarly, D. ciliolata and D. scabra are distinguished by different chromosome numbers and at least one translocation (Carr and Kyhos, 1981, 1986
). Although there was no obvious loss of either species markers in the filial crosses, the loss of D. scabra markers among the backcross progeny may be skewed in a manner similar to that found in Helianthus. As such, the genealogical categorization for these individuals hypothesized above should be viewed with caution pending analysis of additional controlled crosses among these species (now underway).
It is noteworthy that all hybrids observed were limited to the newer substrate (this study; Robichaux et al., 1990
). Curiously, the recurrent parent of all hybrids designated backcrosses in this study was D. ciliolata, the species nearly restricted to the old substrate. Although more sampling might identify hybrids resulting from backcrossing to D. scabra, the number would likely be low because our sample of field hybrids (20) included about two-thirds of those occurring at the study site; 29 hybrids were noted in approximately the same area a few years earlier (Robichaux et al., 1990
). The nature of the hybrids and their distribution could be due to randomness within the population, but we suspect other physiological or ecological parameters may be involved (see Robichaux, 1984
).
One consequence of the unidirectional hybridization occurring at the Pu'u Huluhulu site is an apparent introgression of genes from D. scabra into D. ciliolata. Genetic infusion from D. scabra appears to be allowing D. ciliolata, or at least recombinants with a morphology similar to D. ciliolata, to occupy a niche previously unavailable to the species (i.e., the new lava flow). Thus, even the low frequency of individuals of D. ciliolata reported to occur on the fringes of the 1935 flow by Robichaux et al. (1990)
may actually represent introgressed hybrids. This interpretation is supported by the demonstration in other silversword relatives that second generation backcrosses derived from an F1 hybrid between morphologically and chromosomally very distinctive parents were nearly indistinguishable from the recurrent parent (Carr, 1995
). Moreover, the reduced fertility due to chromosome pairing anomalies in the F1 disappeared by the second backcross generation. In contrast, D. ciliolata and D. scabra are morphologically and chromosomally very similar despite the difference in the chromosome number, and F1 fertility is relatively high (Carr and Kyhos, 1981
). As such, it is plausible that future generations of D. ciliolata occurring on the new substrate will continue to expand their range and blend with the population on the old substrate to the extent that their hybrid parentage would only be detectable by genetic examination. However, it is equally plausible that volcanic activity from Mauna Loa may again cover this region in the coming years and the cycle begin anew.
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FOOTNOTES |
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4 Current address: State Botanist, Department of Land and Natural Resources, Division of Forestry and Wildlife, Honolulu, Hawai‘i 96813 USA.
5 Author for reprint requests (cmorden{at}hawaii.edu
).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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= D. scabra; 
= hybrids