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Genetics and Molecular Biology |
Department of Biology, Indiana University, Jordan Hall 142, 1001 East Third Street, Bloomington, Indiana 47405 USA
Received for publication April 1, 2003. Accepted for publication July 11, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Asteraceae • Helianthus • hybrid speciation • hybridization • parallel speciation • phylogeography • sunflowers
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and to the establishment of new lineages through homoploid or polyploid hybrid speciation (Stebbins, 1950
; Grant, 1971
). The formation of a sexually reproducing species via either of these two mechanisms is largely dependent on two events: the hybrid lineage must become reproductively isolated from the parents (in parapatry or sympatry), and it must achieve a stable, fertile genotype. When fulfilled through allopolyploidy, these requirements are met by genome duplication in hybrids. Homoploid hybrid speciation is evolutionarily more difficult, but can be achieved through ecological reproductive isolating barriers or chromosomal rearrangements in the hybrid and will result in a new species with the same ploidy as the parents (Smith and Daly, 1959
; Grant, 1966a
, b
, 1971
; Gallez and Gottlieb, 1982
; Buerkle et al., 2000
). Although both modes of speciation are known to have generated stable and reproductively isolated hybrid lineages in nature, more is presently known about the frequency and evolutionary significance of allopolyploidy (reviewed in Otto and Whitton, 2000
) than of homoploid hybrid speciation.
Botanists have long recognized the importance of polyploid hybrid speciation in plant evolution (Stebbins, 1950
), and recent models demonstrate the ease with which new allopolyploid species can arise and become established (Rodriguez, 1996
). Allopolyploidy also represents the most rapid kind of speciation known, since reproductive isolation is an instantaneous by-product of genome doubling. In addition to reproductive isolation, genome doubling often is accompanied by a diverse array of morphological, life history, and physiological changes (Levin, 1983
; Thompson and Lumaret, 1992
). These phenotypic changes sometimes result in modified niche preferences, which increase the likelihood of polyploid establishment (Rodriguez, 1996
). Allopolyploid species are often polyphyletic, with individual populations arising from independent hybridization events between different populations of the same two parental species (Soltis et al., 1995
). The mechanism by which allopolyploid species arise is conducive to the formation of polyphyletic species; each F1 hybrid contains the same initial chromosomal complement consisting of one part from each parent, and chromosomal doubling will establish this genome permanently in all polyploids formed.
In contrast to the frequent documentation of allopolyploid speciation in nature, only a handful of diploid hybrid species have been thoroughly investigated and verified using molecular markers (reviewed in Rieseberg, 1997
). The creation of a homoploid hybrid species from an F1 hybrid has traditionally been considered an unusual event (Grant, 1971
). This view derives from simple models in which the origin of a fertile hybrid segregant was achieved via the stochastic sorting of chromosomal and genic sterility factors that differentiated the parental species. Even with modest levels of differentiation between the parental species, the probability of generating a fertile hybrid segregant is low. Inbreeding increases the likelihood of achieving a fertile, stable genome, so early workers predicted that homoploid hybrid speciation would be most likely in inbreeding systems (Stebbins, 1957
). Given the stochastic nature of the sorting process, it was anticipated that each hybrid speciation event resulting from hybridization between the same two parental species would produce a different genome and that the newly formed microspecies would be perpetuated through inbreeding (Stebbins, 1957
; Grant, 1958
).
The prediction that multiple homoploid hybrid speciation events involving the same parental species can result in distinct hybrid lineages is verified in the genus Helianthus, where the same parents (H. annuus and H. petiolaris) are the progenitors of three different diploid hybrid species: H. anomalus, H. paradoxus, and H. deserticola (Rieseberg et al., 1990
, 1991
; Rieseberg, 1991
). However, more recent theory has emphasized the important role that selection for fertility and viability plays in establishing stable combinations of sterility factors (Templeton, 1981
; McCarthy et al., 1995
; Buerkle et al., 2000
). Experiments by Rieseberg and colleagues (Rieseberg et al., 1996
; Rieseberg, 2000
) have shown that experimental hybrids converged toward chromosomal combinations similar to those found in natural hybrid species when selection was made for fertility alone, suggesting a more prominent role for selection than chance in the establishment of a new hybrid lineage. This indicates that multiple origins for a single homoploid hybrid species in the wild is a possibility; evidence consistent with multiple origins of a single diploid hybrid species has now been documented in several systems (Brochmann et al., 2000
; Wang et al., 2001
; Schwarzbach and Rieseberg, 2002
). Recent models and demonstrated cases of homoploid hybrid speciation further suggest that this mode of speciation may occur frequently in outcrossing species rather than being restricted to inbreeding taxa (Gallez and Gottlieb, 1982
; Arnold et al., 1990
; Wang et al., 1990
; Wang and Szmidt, 1994
; McCarthy et al., 1995
; Sang et al., 1995
; Allan et al., 1997
; Rieseberg, 1997
; Wolfe et al., 1998a
, b
; Buerkle et al., 2000
).
This study was designed to evaluate the possibility that the diploid hybrid species, H. deserticola, has arisen multiple times in the wild using evidence from variation in chloroplast DNA (cpDNA), nuclear microsatellite loci, and interpopulation crossability. Helianthus deserticola is a xerophytic species found in sandy soils and distributed in small populations located in western Nevada, west central Utah, and along the border of Utah and Arizona, USA (Fig. 1). It is a confirmed diploid hybrid species based on comparison of isozyme, nuclear ribosomal DNA, and cpDNA with its parental species, H. annuus and H. petiolaris (Rieseberg, 1991
). Helianthus annuus is distributed throughout the central and western United States and typically inhabits heavy, clay-based soils. Helianthus petiolaris, the smaller of the two parental species, is distributed mainly through the central United States and inhabits sandier soils than H. annuus. The two parental species co-occur and often hybridize throughout their range. The species are all annual, outcrossing, and have a haploid chromosome number of 17 (Heiser, 1947
; Heiser et al., 1969
; Rogers et al., 1982
).
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Finally, interpopulation crosses were made between six of the eight H. deserticola study populations, and pollen viability of the progeny was used to verify interfertility among the geographically separated populations. Uniform patterns of interfertility between geographically disparate populations would be a sign of a single origin and a common genetic composition across the species. Discontinuities in patterns of pollen viability would suggest marked genetic differentiation, attributable to extreme population subdivision and isolation or to intrinsic differences in genetic composition due to multiple origins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Because both H. anomalus and H. deserticola have the same parental species and occur in the desert Southwest, many of the same parental species populations were informative for both studies. Thus, haplotype information has been previously reported for all six H. petiolaris populations and for six of the 12 H. annuus populations in Table 1 (Schwarzbach and Rieseberg, 2002
). Chloroplast DNA PCR amplification and restriction fragment length polymorphism (cpDNA PCR-RFLP) analysis of the remaining six populations of H. annuus and of all eight populations of H. deserticola are reported here for the first time.
As described previously, primers rpoC1–195—rpoC2–1364 (Liston, 1992
), trnC—trnD (Demesure et al., 1995
), trnF—trnV, and trnV—rbcL (Dumolin-Lapegue et al., 1997
) were employed to amplify cpDNA fragments for all individuals (Schwarzbach and Rieseberg, 2002
; Welch and Rieseberg, 2002
). All amplify large regions of cpDNA containing considerable variation (Schwarzbach and Rieseberg, 2002
; Welch and Rieseberg, 2002
). The PCR reactions were performed in volumes of 50 µL containing 1 µL of template DNA, 0.08 µg of each primer, and 1 unit Taq DNA polymerase, at a final concentration of 30 mmol/L Tricine, 50 mmol/L KCl, 2 mmol/L MgCl2, and 100 µmol/L each dNTP. Reactions were performed in an MJ Research (Watertown, Massachusetts, USA) PTC-100 Thermal Cycler programmed for an initial denaturation step of 1 min at 94°C, 37 cycles of 45 s at 94°C, 45 s at 55°C, and 3 min at 72°C, with a final extension at 72°C for 7 min.
The four PCR products were digested with restriction endonucleases known to yield distinct restriction profiles for the parental species in previous studies (HhaI, AciI, DdeI, BstUI, HaeIII, MseI, TaqI) (Table 2). Digestions were performed in volumes of 15 or 20 µL, containing 5 or 10 µL of PCR product, respectively. Each digestion contained 2.5 units restriction enzyme, 1.5 µL buffer (New England Biolabs, Beverly, Massachusetts, USA), 0.15 µL of bovine serum albumin (BSA) if required and distilled water. After mixing, samples were incubated for a minimum of 3 h at either 37° or 60°C, as recommended by the supplier. Digests were run on 1.5% agarose gels, stained with ethidium bromide, and visualized under UV-light. Restriction profiles were scored as either 1 or 0 (Table 3), depending on the presence or absence of a restriction site. Restriction digests that yielded more than one site were scored separately for each site. A most parsimonious network was manually constructed based on restriction site polymorphisms and verified with PAUP* 4.0b8 (Swofford, 2002
).
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) were screened for clean amplification and codominance in the taxa included in the study, and the following 18 loci were chosen for surveys of all populations and species: ORS3*, ORS4*, ORS5*, ORS7*, ORS8*, ORS10*, ORS12*, ORS59*, ORS297, ORS299, ORS377, ORS437, ORS442, ORS484, ORS541, ORS613, ORS618, ORS733. Primer sequences can be found at http://www.css.orst.edu/knapp-lab/sunflower. Also, note that the eight loci marked with an asterisk were previously assayed in six populations of each parental species (see Table 1) by Schwarzbach and Rieseberg (2002)
. Primers were synthesized, and the 5' end of each forward primer was labeled with one of three fluorescent dyes (6FAM, HEX, or NED) by MWG Biotech (High Point, North Carolina, USA), Invitrogen (Grand Island, New York, USA), or PE Biosystems (Foster City, California, USA).
The PCR reactions were performed in volumes of 10 µL containing 20 ng DNA, 1 unit Taq DNA polymerase, and 0.0016 µg of each primer at a final concentration of 30 mmol/L Tricine, 50 mmol/L KCl, 2 mmol/L MgCl2, and 100 µmol/L each dNTP. Fragments were amplified using a "touchdown" PCR protocol, developed to reduce nonspecific primer binding and fragment amplification (Don et al., 1991
). An initial denaturing cycle of 3 min at 95°C was followed by 10 touchdown cycles (the program starts 10°C above the appropriate annealing temperature, and the temperature drops 1°C each cycle) of 30 s at 94°C, 30 s at the annealing temperature, and 45 s at 72°C. These 10 cycles were followed by 29 cycles of 30 s at 94°C, 30 s at the appropriate annealing temperature, and 45 s at 72°C, with a final elongation period at 72°C for 20 min. Annealing temperatures are as follows: 51°C for ORS3 and ORS377, 52°C for ORS7, ORS8, ORS10, and ORS12, and 55°C for ORS4, ORS5, ORS59, ORS297, ORS299, ORS437, ORS442, ORS484, ORS541, ORS613, ORS618, and ORS733.
Microsatellite fragments were assayed via electrophoresis on an ABI 3700 capillary sequencer (Applied Biosystems, Foster City, California, USA). The PCR fragments of non-overlapping size and color were pooled and diluted 1 : 20 with ddH2O. Finally, 1 µL of the diluted PCR product was added to a 10 µL mixture of 9.82 µL ddH2O and 0.18 µL of the GenSize R500 ROX size standard (GenPak, St. James, New York, USA). Samples were denatured at 95°C and snap cooled on ice before loading onto the 3700. Chromatographs of sequencer data were generated using GENESCAN 3.5 (Applied Biosystems, Foster City, California, USA) and fragment lengths were scored using GENOTYPER 3.6 (Applied Biosystems, Foster City, California, USA).
Linkage disequilibrium between all pairs of microsatellite loci and number of migrants per generation (Nem) were calculated using GENEPOP (Raymond and Rousset, 1995
; Hendrie et al., 1998
). Estimates of Nem are based on the private alleles method (Barton and Slatkin, 1986
). F statistics were calculated using ARLEQUIN 2.0 (Schneider et al., 2000
), assuming an infinite alleles model according to the methods of Weir (1996)
. Average gene diversity (He) was also calculated using ARLEQUIN 2.0. A Mantel test was used to check for correlations between geographic distance and FST, as implemented by the R PACKAGE 4.0 (Casgrain and Legendre, 2001
). Significance was tested using 999 permutations of the data.
Phylogenetic analysis of microsatellite frequency data was performed using PHYLIP 3.6 (Felsenstein, 1993
). Neighbor-joining trees (Saitou and Nei, 1987
) were constructed based on Nei's genetic distances (Nei, 1987
). Support for nodes was evaluated based on 1000 bootstrap replicates. Hybrid lineages, by definition, contain a mixture of parental loci, and it is acknowledged that reticulate evolution presents a special challenge to phylogeny reconstruction (McDade, 1990
, 1992
; Rieseberg and Ellstrand, 1993
). It has also been demonstrated that independent hybrid lineages may become fixed for the same parental loci (Rieseberg et al., 1996
). Thus, populations from two independently derived hybrid lineages may appear more similar to each other than to any populations of the parental species. One way to alleviate this problem is to construct trees based only on loci derived from one parental species (Schwarzbach and Rieseberg, 2002
). Loci in H. deserticola were assigned to one parent or another using a maximum likelihood approach (Rieseberg et al., 1998
). The value produced by this analysis is essentially a hybrid index based on allele frequencies; a hybrid index score was generated at every locus for the species as a whole and for each population individually. Two separate neighbor-joining trees were then constructed using the loci that could be assigned to a particular parent and containing populations of only that parental species and the hybrid species. The allele frequencies of the other parental species were pooled and used as an outgroup.
The divergence times among populations of H. deserticola were calculated as a proxy for time of origin of the hybrid species. Divergence times were calculated based on microsatellite allele frequency according to the methods of Zhivotovsky (2001)
, using Equation 1. Here, TD = D1/2w – V0/w; TD is the divergence time in generations, D1 is the average over all loci of the squared difference in repeat numbers for pairs of alleles drawn from different populations (from equation 2 of Goldstein et al., 1995
), w is the effective mutation rate, and V0 is the average over all loci of the variance in repeat number in the ancestral population. V0, a property of the ancestral population, is unknown, so we followed Zhivotovsky's recommendation to set V0min equal to 0, and V0max equal to the average within-population variance of the two populations being compared. This allowed us to generate a lower and upper bound for the divergence times (TDmin and TDmax, respectively). The mutation rate, w, was set at 2 x 10–4 based on estimated rates of mutation for 20 microsatellites in soybean populations of known pedigree (Diwan and Cregan, 1997
).
Crossing studies
Genetic relatedness of six of the eight H. deserticola study populations were evaluated using interpopulational crosses and pollen viability counts; no seeds were available from populations DESB and DESC, so they were excluded from the analysis. A minimum of 10 plants from each population was propagated from seed in an Indiana University greenhouse. Reciprocal crosses were made between the six populations and among individuals within each population, resulting in 36 sets of seeds representing every inter- and intrapopulational parental combination.
Seeds were germinated at the Willamette University greenhouses, and pollen viabilities of progeny resulting from the crosses were estimated by staining with a solution of 30% sucrose and 0.1% MTT (Chandler et al., 1986
). Pollen viability was scored for two plants resulting from each parental combination, and 
500 pollen grains were scored per flowering head. Means and standard errors were calculated for pollen viabilities for each cross combination. Correlations between pollen viability and either geographic distance or FST were tested using the Mantel test, implemented by the R PACKAGE 4.0 (Casgrain and Legendre, 2001
). Significance was tested using 999 permutations of the data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, and the same numerical designations are employed in both studies. Haplotypes unique to this study were assigned new numbers. Haplotypes varied considerably within and among populations of all three species, with eight haplotypes in H. annuus, (1, 6, 10, 13, 17, 18, 19, and 20), 10 in H. petiolaris (1, 5, 6, 7, 8, 9, 10, 12, 13, and 14), and four in H. deserticola: 1, 8, 13, and 16 (Table 1; Figs. 1 and 2). Of the four haplotypes present in H. deserticola, one was characteristic of H. petiolaris (8), one was characteristic for H. annuus (1), one was found in equal frequencies in both parental species (13), and one was unique to H. deserticola (16), but was more similar to H. petiolaris types than H. annuus (Fig. 2). All three species were occasionally polymorphic at the population level, with up to five different cpDNA haplotypes occurring in a single population (Table 1).
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Of the four different haplotypes present in H. deserticola, haplotype 13 occurred in seven of the eight populations surveyed. The two H. deserticola populations farthest east along the Utah–Arizona border in the Colorado River drainage basin also contained some individuals with haplotype 8 (DES1274 and DES1296). Type 16, unique to H. deserticola, occurred in only one population near the southwest corner of Utah and made up part of a population that was predominantly type 13 (DES1270). The single H. deserticola population with haplotype 1 (the typically H. annuus haplotype) occurred in the Little Sahara National Recreation Area located in west central Utah (DES1265).
Microsatellites
Analysis of linkage disequilibrium revealed that none of the 18 loci were significantly linked to any others. All loci were included in subsequent analyses. According to estimates of FST, H. deserticola showed the most extreme partitioning of genetic variation among populations (FST of 0.249), followed by H. petiolaris and H. annuus (FST of 0.194 and 0.159, respectively). In all cases, the majority of the genetic variation exists within populations, as compared to among populations or species. There was no correlation, however, between geographic and genetic distances within H. deserticola. Nem values are estimated as 0.70 for H. deserticola, 1.0 for H. annuus, and 0.73 for H. petiolaris. Average and median variation in microsatellite fragment size, average and median number of alleles per microsatellite locus, and average gene diversity (He) were calculated to assess genetic diversity of the three species (Table 4). Helianthus deserticola was generally the least variable according to all estimates, the only exception being that the mean allele size variance was slightly higher for H. deserticola than for H. annuus, but still much lower than that for H. petiolaris.
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Crossing studies
Average pollen viability for any population as maternal or paternal parents ranged from 85 to 94%. This degree of interfertility is much greater than that achieved in crosses between H. deserticola and either parental species and also suggests that there is potential for gene flow between all populations of the hybrid species (Fig. 6). There were no obvious discontinuities in the degree of interfertility, nor were there any statistically significant associations between interfertility and FST or geographical location.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Single origins with subsequent introgression
The cpDNA haplotypes from both parents are present in H. deserticola. A single origin is only in accordance with this evidence if the possibility of cytoplasmic introgression is considered. Given the current distribution of haplotypes and the constraint of a single origin, all populations of H. deserticola would likely derive from a single diploid hybrid speciation event in which the maternal population of H. petiolaris contained haplotypes 8 and 13, probably a population in the Colorado River basin (e.g., PET1283). The predominance of haplotype 13 in the southwest corner of Utah and in Nevada could be attributed to genetic drift or to founder events, while the polymorphism in eastern populations may be due to retention of ancestral variation (provided sufficient population size) or continued gene flow with neighboring populations of H. petiolaris. Population DES1265 in northern Utah could have gained haplotype 1 through introgression with H. annuus. This explanation is plausible, as haplotypes 1, 6, 10, and 13 are found in both of the parental species (e.g., PET1271 contains one individual with the H. annuus haplotype 1, and ANN1281 is fixed for an H. petiolaris haplotype 13), and cytoplasmic introgression is not an uncommon event in taxa capable of interspecific hybridization (Rieseberg, 1995
).
A striking case of polyphyly occurs when the tree based on the eight microsatellite loci largely derived from H. annuus is considered. Here, the two populations of H. deserticola from western Nevada (DESB and DESC) group more closely with a local population of H. annuus than with conspecifics. A single origin would require hybridization and nuclear introgression between these or ancestral populations, an event that seems reasonable based on the patterns of cpDNA variation noted earlier. The inclusion of ANN5 in this group is curious, given that the population is not geographically proximal; one explanation for its occurrence may be long-distance dispersal.
The case for a single origin of H. deserticola followed by dispersal or range expansion is also supported by the fact that none of the populations in the Virgin River drainage basin of Utah contain the haplotypes that are unique to local H. petiolaris populations. This suggests that the populations of H. deserticola in this area are derived from a founding population that was not native to their current geographical location. Gene flow across the mountain range separating the two river basins seems feasible because haplotype 10 occurs in populations on both sides of the divide, so it is possible that H. deserticola could have been dispersed over this geographical barrier after originating in the Colorado River drainage basin. This pattern may also be the result of sampling error or genetic divergence of the H. petiolaris populations in this area after the hybrid speciation event. One H. deserticola population from this area has apparently developed a unique haplotype since the speciation event (DES1270), suggesting that the same may have occurred in the local H. petiolaris populations.
Overall, H. deserticola has lower levels of genetic diversity than do the parental species (Table 4), the only exception being the slightly higher average variation in microsatellite repeat length found in H. deserticola as compared to H. annuus. Certainly, the hybrid species is genetically depauperate in contrast to the parental species, a pattern that may be attributed to a genetic bottleneck at its origin. The low levels of genetic variation in H. deserticola suggest that the species has not yet developed the same degree of genetic complexity as the parental species and are most consistent with a single origin.
Multiple origins
The presence of haplotypes from both parental species in different populations of H. deserticola and the polyphyletic nature of the hybrid lineage can also be explained by multiple origins for the hybrid species. As in the scenario for a single origin, the populations of H. deserticola with haplotypes 8 and 13 are likely derived from a hybrid speciation event involving a maternal population of H. petiolaris in the Colorado River basin. The single H. deserticola population with an H. annuus haplotype (haplotype 1) in northern Utah may have originated in a distinct hybridization event where H. annuus served as the maternal parent. These two hypothetical hybrid speciation events occur in localities that are geographically isolated via distance; this supports the possibility of multiple origins rather than the possibility that H. deserticola has spread to these different locations after a single hybrid speciation event. If this population of H. deserticola were, in fact, locally derived, then we might expect it to be closely related to one of the proximal populations of H. annuus based on patterns of microsatellite variation. Instead, this population appears to be more closely related to geographically distant populations of H. deserticola than to any nearby populations of the parental species according to both the neighbor-joining trees and FST values, implying that the population of H. annuus that served as the maternal parent in this speciation event (or hybridized with H. deserticola) was not sampled in our survey. The high interfertility and moderate values of Nem among populations of H. deserticola (0.70) are also relevant to this issue, however, because gene flow among populations of the species may have eroded initial differences resulting from multiple origins.
The patterns of microsatellite variation provide stronger support for an alternate hybrid speciation event; this one would have occurred in western Nevada, where there are currently two isolated populations of H. deserticola, DESB and DESC. The potential for a unique speciation event is suggested by the close phylogenetic relationship between the populations of H. deserticola and the ANNReno population based on the alleles derived from H. annuus; this suggests that ANNReno (or an ancestral population) may have served as the pollen parent in the original hybrid speciation event (Fig. 4). These populations of H. deserticola have no nearby H. petiolaris sample populations, so it is not possible to say if the type 13 cpDNA present in those populations is locally derived or the result of the proposed hybrid speciation event in the Colorado River basin. These populations are geographically distant from other populations of H. deserticola, and H. petiolaris does occur in this area historically (Rogers et al., 1982
), so an independent hybrid speciation event is a possibility.
Finally, it is interesting to consider some of the broad patterns relevant to this question. First, no two populations seem to share an identical genetic makeup. When loci were assigned an origin in either of the two parents, 12 of the 17 loci amenable to this analysis had varying patterns at the population level (Table 5). If the species was derived from a single speciation event, one might predict a greater uniformity at the genome level. The fact that populations of the hybrid species have retained loci from different parents suggests that the species is either a product of a diverse founder population or that there were multiple origins of the species, resulting in the fixation of different loci in many cases. The five loci that do not show this pattern may show the effects of selection or certain genetic constraints on the formation of H. deserticola; i.e., they may be vital to survival in the desert environment, or they may be the only possible combination of parental loci that do not contribute to negative epistasis in the hybrid. Second, there is a considerable variation in the degree of interfertility among populations of H. deserticola, ranging from 67.7% (±3.1) to 96.8% (±1.2). While this variation is not significantly correlated with geographic or genetic variation, it exceeds the level of variation normally found among populations of the parental species (Rieseberg, 2000
) and suggests that the populations are not genetically uniform.
Diploid hybrid species and their origins
The possibility of multiple origins of a diploid hybrid species has only recently been explored in the literature (Brochmann et al., 2000
; Wang et al., 2001
; Schwarzbach and Rieseberg, 2002
; Welch and Rieseberg, 2002
). In contrast, researchers have already shown that multiple origins of allopolyploids are almost as common as single origins (Soltis and Soltis, 1993
). It is possible that the potential for multiple origins of diploid hybrid species was not investigated earlier because theory has held it to be unlikely. Whatever the reason, research into this scenario is less clear-cut than is research into the origins of allopolyploids. The main difficulty lies in the potential for continued gene flow between the hybrid species and its progenitors; the cytoplasmic and nuclear introgression that result from these events can leave patterns that are essentially indistinguishable from those caused by multiple origins. The strong reproductive isolating barrier between allopolyploids and their diploid parents makes introgression less likely (although see Husband, 2000
) and the answers less equivocal.
In this study, we have shown that H. deserticola is a genetically diverse species, containing cpDNA haplotypes from both parents and showing patterns of polyphyly at 18 microsatellite loci. The occurrence of multiple cpDNA haplotypes is not unexpected because the same phenomenon is seen in other diploid hybrid species, such as Pinus densata, Argyranthemum sundingii, and H. anomalus (Wang and Szmidt, 1994
; Brochmann et al., 2000
; Schwarzbach and Rieseberg, 2002
). Para- or polyphyly for nuclear markers in a diploid hybrid lineage has also been reported previously; populations of P. densata appear to be paraphyletic, based on evidence from allozyme loci (Wang et al., 2001
). However, this is the first report of polyphyly of a hybrid species based on nuclear microsatellite loci.
Levels of genetic diversity within the hybrid species H. deserticola are similar to those present in H. paradoxus, a species with a single origin, i.e., H. deserticola is genetically depauparate when compared to its parents, suggesting a population bottleneck at its origin. This finding confirms reports based on allozyme variability, where H. deserticola and H. paradoxus generally contained a lower percentage of polymorphic loci, mean number of alleles per locus, and mean heterozygosity than the parental species. Particularly, H. anomalus, the hybrid species most likely to have originated from multiple hybridization events, yielded higher levels of genetic diversity than the other two hybrid species based allozyme data (Rieseberg et al., 1991
), and data from microsatellite loci revealed no significant difference between the levels of diversity in the hybrid compared to the parental species (Schwarzbach and Rieseberg, 2002
). Two other diploid hybrid species, Iris nelsonii and Stephanomeria diegensis, are characterized by levels of diversity that are roughly equivalent or slightly lower than those found in either parent according to allozyme markers (Gallez and Gottlieb, 1982
; Arnold et al., 1990
). Pinus densata differs from H. deserticola in that the hybrid species has a higher level of genetic diversity than either parent based on allozyme markers (Wang et al., 1990
, 2001
).
Remarkably, despite the variety of cpDNA types and polyphyletic nature of H. deserticola, the geographically disparate populations have a high degree of interfertility, especially compared to interspecific crosses, indicating that H. deserticola is a "good" species (Fig. 6). The species likely originated between 170 000 ± 12 000 and 63 000 ± 11 000 years before present, and thus its inception likely preceded human disturbance and was the result of hybridization events in the wild (Dixon, 2001
). Unfortunately, it is impossible to say which of the diploid hybrid sunflowers came first, as the other studies yield similar dates; the estimated origin of H. anomalus is between 144 000 and 116 000 years before present, while H. paradoxus likely originated between 208 000 and 78 000 years ago (Schwarzbach and Rieseberg, 2002
; Welch and Rieseberg, 2002
). The estimated maximum and minimum age of H. deserticola differ greatly, and the dates should be viewed with reservation because it was necessary to assume that the markers evolved according to the multistep mutational model employed by Zhivotovsky (2001)
and that they evolved at the same rate as SSRs in soybean (Diwan and Cregan, 1997
). However, note that the estimated dates place the origin of all three hybrid species after the colonization of North America by bison approximately 200 000 years BP (Dary, 1974
). Bison are considered to be the primary dispersal agent for sunflower and may have brought the parental species into contact and created the habitat disturbances that appear to facilitate hybridization between them (Asch, 1993
).
Conclusions and implications
The patterns of genetic variation within H. deserticola, when compared with the parental species, have two potential explanations, neither of which appears to be superior. It is possible that there was a single origin for the species, followed by cytoplasmic and nuclear introgression with different populations of the parental species. It is also possible that H. deserticola originated from up to three unique hybrid speciation events and that the present day populations are the result of parallel selection pressure and a coordinate response based on a common genetic starting point.
The recent interest in the potential for multiple origins of diploid hybrid species comes on the heels of convincing evidence for parallel speciation due to ecological factors from several laboratories (Schluter and Nagel, 1995
; Rundle et al., 2000
; Levin, 2001
). In light of these findings, it is important to consider that a diploid hybrid speciation event depends on both ecological and genomic factors to enforce reproductive isolation between the nascent hybrid and its parental species. This represents a contrast to cases of allopolyploid hybrid speciation where ploidy differences alleviate the requirement for immediate ecological divergence (although not the eventual necessity). We propose that despite their apparent rarity, homoploid hybrid species represent potentially powerful study systems for the investigation of principles basic to more common forms of speciation. For example, homoploid hybrid speciation represents a kind of speciation with gene flow. Likewise, this mode of speciation is facilitated by rapid ecological divergence, where hybrid species typically inhabit extreme environments in comparison with the parental species. This dependence on strong ecological selection in the face of ongoing gene flow is clearly pertinent to speciation as a general process, particularly given that the ancestral genotype of diploid hybrid species can be reproduced in a laboratory setting (Rieseberg et al., 1996
; Rieseberg, 2000
). Thus, diploid hybrid species represent tractable systems in which to investigate ecologically relevant traits that are of importance early in the speciation process and that diverge in the presence of gene flow (Lexer et al., 2003
, in press
).
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FOOTNOTES |
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2 E-mail: brgross{at}indiana.edu
3 Present address: Department of Biological Sciences, Kent State University, Kent, Ohio 44242 USA
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LITERATURE CITED |
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