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HORTSCIENCE 43(3):637–643. 2008. information useful in strategies for identify- ing sources of intermountain Allium germ- plasm rich in genetic diversity. Detection of Genetic Variation The genus Allium has a worldwide distri- bution consisting of 700 species, including in Wild Populations of Three Allium 80 species in North America (Brewster and Rabinowitch, 1990; Hanelt, 2001; Kamenetsky Species Using Amplified Fragment and Rabinowitch, 2006; McNeal and Ownbey, 1973; McNeal and Jacobsen, 2001). The genus includes economically important Length Polymorphisms crop species such as onions, garlic, chives, Nathan C. Phillips1 leeks, and other minor crops. Allium species Middle Tennessee State University, School of Agribusiness and Agriscience, have also been traditionally used in the or- namental industry with occasional new spe- Campus Box 5, Murfreesboro, TN 37132 cies derived from natural populations being Steven R. Larson introduced into the trade (Kamenetsky and Rabinowitch, 2006). Virtually all cultivated USDA-ARS, Forage and Range Research Laboratory, Utah State University, Alliums are derived from old world species, Logan, UT 84322-6300 and little is known about many North Amer- ican species. Considering the economic and Daniel T. Drost cultural importance of Alliums throughout Utah State University, Plants Soils and Climate, Logan, UT 84322-4820 history, and the wealth of biodiversity in the genus, it would be prudent to use genetically Additional index words. genetic diversity, germplasm, divergence, Allium acuminatum, Allium sound strategies for the collection of cur- brandegei, Allium passeyi rently unexploited Allium germplasm. Abstract. Three wild onion species native to the intermountain west in the United Rising interest in the preservation of in- States—Allium acuminatum, A. brandegei, and A. passeyi—show horticultural potential, termountain Allium germplasm has encour- but little is known about patterns of genetic diversity among localized populations and aged research to further our understanding of geographical regions. We examined amplified fragment length polymorphisms (AFLP) the genetic diversity found within and among within and among five Allium acuminatum, four A. brandegei, and three A. passeyi species (Adair et al., 2006; Hellier, 2000). collection sites in Utah. These three congeners with contrasting abundance and dis- Growers depending on wild seed collection tribution patterns provide an opportunity to investigate the role of geographic distance, would benefit from understanding genetic altitude, and rarity in patterns of genetic divergence. The collection sites were selected variation in wild populations across the along an altitudinal gradient to reflect ecogeographic variation. Individual plants from natural landscape. Knowing the extent of each of the 12 sites were genotyped using six AFLP primer combinations detecting DNA genetic diversity within and among popula- variation within and among all three species. Genetic differences between species were tions provides information that might be high enough to render comparisons among species impractical, so each species was useful in the selection of a seed collection analyzed separately for differences between populations and variability within popula- site. Intermountain Allium species can vary tions. Similarity coefficients were significantly greater within collection sites versus considerably in their distribution, occurrence, among collection sites indicating divergence between populations. Within-population and habitat. Although among-population genetic diversity was not correlated with elevation for any of the three species. Analysis of genetic variation is often known to be a result molecular variance revealed that 66% (A. acuminatum), 83% (A. passeyi), and 64% (A. of isolation by distance, the significance of brandegei) of observed variation is found within populations. Genetic divergence among correlations between geographic distribution and genetic diversity has been a topic of populations (VST) was higher in the widely distributed species, suggesting that inter- population gene flow may be negatively correlated with range size. Allium acuminatum debate among biologists (Gitzendanner and and A. brandegei individuals cluster into groups corresponding strictly to collection sites Soltis, 2000; Hamrick and Godt, 1989; Karron, based on neighbor-joining analysis of the total number of DNA polymorphisms between 1987). Recent studies have cast doubt on individual plants. Allium passeyi populations, however, had less overall genetic variation the theory that narrowly restricted or rare between populations. Genetic isolation by distance appeared responsible for much of the species necessarily exhibit lower levels of variability among populations, although there was one notable exception showing sig- among-population genetic variation in com- nificant differences between two geographically close populations in A. acuminatum. parison with widespread species (Bjerregaard, 2004). In comparing genetic variation in rare and widespread congeners compiled from the existing literature, there was no universal Efficient propagation of native inter- genetic diversity among localized popula- trend in levels of among-population genetic mountain plant species for rangeland resto- tions and geographical regions needs to be diversity in response to range size (Gitzen- ration and other commercial uses requires better understood to help identify collections danner and Soltis, 2000). The authors did some basic knowledge about natural genetic crucial for conservation and suitable for note high congeneric correlations for genetic diversity in the landscape (Adair et al., 2006; breeding efforts (Adair et al., 2006; Rio and measures, suggesting that it is useful to assess Monsen and Shaw, 2001). The patterns of Bamberg, 2002). Ideally, a crop gene bank genetic variation in relation to range size collection would contain a representative within species of the same genus. Doing so core of a species germplasm, providing stor- allows one to investigate genera-specific re- age and access to the widest range of genetic lationships between range size and genetic Received for publication 8 Aug. 2007. Accepted traits. In conjunction with evaluations of life variation within and among populations. for publication 13 Nov. 2007. history, growth characteristics, demograph- Like range size, habitat variation may also This research was supported by grants from the ics, and habitat of three intermountain Allium play a role in the partitioning of genetic vari- Utah Botanical Center, the Utah Native Plant Society, and by the Utah Agricultural Experiment species native to Utah, we used the amplified ation. Genetic differentiation in some species Station, Utah State University, Logan, UT (journal fragment length polymorphism (AFLP) tech- has also been shown to be closely related paper no. 7909). nique in investigating patterns of genetic to altitude (Wen and Hsiao, 2001), whereas 1To whom reprint requests should be addressed; diversity within and among the study pop- in other species, no such correlation was e-mail [email protected] ulations. The description of our study offers found (Bingham and Ranker, 2000). Ecotypic
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genetic variation may be detected within a understand the genetic variation that exists Data analyses. Hypothesis testing using species by sampling along an altitudinal gra- within and among common and rare Allium the similarity index (Leonard et al., 1999), dient (Clausen et al., 1940). Understanding populations. analyses of molecular variance (Excoffier how genetic diversity is partitioned within et al., 1992), and neighbor-joining genetic and among intermountain Allium populations distance analysis (Saitou and Nei, 1987) were can give insight into the mechanisms that Materials and Methods used to investigate genetic diversity within have influenced their current habitats as well and among populations. as aid in the development of propagation Plant materials and DNA extraction. Genetic distances between individual protocol, seed transfer guidelines, and con- Fresh leaf material was collected in the wild plants were determined by the numbers of servation management plans. at multiple locations for each species (Table AFLPs (Euclidean distance) and similarity Previous studies of genetic diversity in 1) in the spring of 2005. Five A. acuminatum, coefficients computed as 2Nm/(Nx +Ny)in several North American Allium species have four A. passeyi, and three A. brandegei sites which Nm is the number of pairs of bands provided information useful for germplasm were sampled. Samples were collected along matching between individuals and Nx and Ny conservation strategies. In a study examining a line transect to minimize potential clonal are the total number of bands amplified from the genetic diversity existing in three popu- influence with distances between individuals the individuals (Dice, 1945; Lynch, 1990; lations each of Allium columbianum and A. varying according to the density of individ- Nei and Li, 1979). Neither of these Euclidean fibillum, over 90% of the diversity was found uals at the site. Genomic DNA was extracted distances nor similarity coefficients relies on within-population for each species (Hellier, from leaf tissue of 192 individuals using the shared null alleles. Analyses of variances 2000). Thus, all of the analyzed populations DNeasy 96 plant DNA extraction kit (two 96- based on pairwise comparisons of these for each species can be treated as one popu- well plates) and MM300 mixer mill (Qiagen, similarity coefficients were performed using lation while still maintaining a high level of Valencia, CA). Voucher specimens from each SAS (SAS Institute, Cary, NC) as described biodiversity in the collection. A study inves- site were prepared from flowering plants in by Leonard et al. (1999). tigating the genetic diversity in two other 2006 (Table 1) and submitted to the Inter- In further analyses, the average number of intermountain Allium species also found a mountain Herbarium, at Utah State Uni- pairwise differences among individuals in the high degree of within-species genetic vari- versity, Logan, UT. sampled populations (PXY), average number ability not only in the narrowly restricted Amplified fragment length polymorphisms of pairwise differences among individuals species (A. aaseae), but also in a widespread analysis. The AFLP technique was per- within populations (PX and PY), corrected congener (A. simillimum) (Smith and Pham, formed as described by Vos et al. (1995). average pairwise differences among popula- 1996). The low levels of among-population Six selective primer combinations (E.ACCA// tions [PXY –(PX +PY)/2], and corresponding genetic variability found in these two studies M.CAG, E.ACC//M.CAT, E.AGG//M.CAT, analysis of molecular variance based on Eu- are similar to other outcrossing species E.AGT//M.CTA, E.ACCA//M.CTC, E.ACAC// clidean distances were computed using Arle- (Hamrick and Godt, 1989). In species that M.CTCT) were chosen with the universal quin (Excoffier et al., 1992). show low genetic diversity within and high EcoRI and MseI adaptor sequences. Selective Neighbor-joining trees were generated by genetic diversity among populations, the amplification reactions were replicated for PAUP* version 4.0b8 (Swofford, 2000) using source location becomes more of a determin- each preamplified polymerase chain reaction the distance matrix generated by Arlequin ing factor in seed collection to avoid genetic template. The amplification products were (Excoffier et al., 1992) with the user-defined erosion (Rogers, 2004). This also becomes an size-fractionated using an ABI3730 instru- distance coefficient equal to the total number issue when propagating plants for reclama- ment with 50-cm capillaries, POP-7 polymer, of marker differences between individual tion or revegetation. In species with high Genescan 500 LIZ internal size standards, plants. Significance testing for the trees was levels of genetic diversity between popula- and Genescan software (PE Applied Bio- performed using bootstrap resampling of tions, seed collected from one population systems, Foster City, CA). Individuals that 10,000 replicates. Graphic displays of the might not produce plants best suited for sur- produced low-quality DNA samples were ex- neighbor-joining trees were developed using vival in the area of reclamation (Lesica and cluded from analysis. A total of 104 indivi- TREEVIEW (Page, 1996). Allendorf, 1999). duals (46 A. acuminatum,39A. passeyi, and In an effort to increase our knowledge of 19 A. brandegei) presented fragments evenly Results genetic variation in intermountain Allium distributed throughout the entire profile, species, we have selected three species native adequate for scoring. These GeneScan sample Genetic diversity within and among A. to Utah for our study—A. acuminatum, A. files were visually analyzed for the pre- acuminatum collections. With the six selec- brandegei, and A. passeyi—as described in sence and absence of polymorphic DNA frag- tive primer combinations, AFLP analysis McNeal and Jacobsen (2001). These species ments, between 50 and 500 base pairs, using generated a total of 539 polymorphic loci. have widely varying ranges of distribution, Genographer version 1.5 (Benham et al., The average number of polymorphic bands rates of occurrence, and habitat preferences. 1999). per plant was significantly higher (P < 0.05) Allium acuminatum is widely distributed and the most common Allium species in western North America. It appears to tolerate a wider Table 1. Identification of the sampled populations, their site locations and global positioning satellite range of habitats, occurring at elevations from coordinates. 1210 and 2490 m (McNeal and Jacobsen, Species/ID Location Elevation (m) Latitude, longitude 2001; Shultz et al., 2006). In comparison, A. acuminatum A. brandegei occupies a more narrow range BC-1 Blue Creek, Box Elder Co., UT 1,556 41.94333 N, 112.42260 W of distribution but is relatively abundant in BC-2 Blue Creek, Box Elder Co., UT 1,652 41.93954 N, 112.41738 W high elevation alpine and subalpine mead- HR Hardware Ranch, Cache Co., UT 1,761 41.60985 N, 111.58331 W ows throughout the northwest (McNeal and LR Liberty Road, Weber Co., UT 1,870 41.35938 N, 111.90188 W Jacobsen, 2001; Neely and Barkworth, 1984; SF Swan Flat, Cache Co., UT 2,318 41.95702 N, 111.48882 W A. passeyi Shultz et al., 2006). The third species in our GS Golden Spike, Box Elder Co., UT 1,546 41.63054 N, 112.49809 W study, A. passeyi, is a rare, localized endemic AH-1 Anderson Hill, Box Elder Co., UT 1,622 41.62616 N, 112.49790 W and appears to be limited to a very specific AH-2 Anderson Hill, Box Elder Co., UT 1,638 41.81793 N, 112.44055 W habitat occurring only in five populations BC Blue Creek, Box Elder Co., UT 1,744 41.93906 N, 112.41613 W in Box Elder County in north central Utah A. brandegei (Holmgren and Holmgren, 1974; McNeal LR Liberty Road, Weber Co., UT 1,859 41.35938 N, 111.90188 W and Jacobsen, 2001; Shultz et al., 2006). SF Swan Flat, Cache Co., UT 2,342 41.95702 N, 111.48882 W The objectives of this study are to better TG Tony Grove, Cache Co., UT 2,515 41.89923 N, 111.64093 W
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in individuals from the LR site in comparison and AH-2) showed significant differences in per plant was significantly greater (P < 0.05) with the lower elevation sites (Table 2). On their similarity index despite being separated in individuals from the SF collection com- this point, plants from the highest elevation by less than 1 km. The sites separated by the pared with the TG collection. The LR plants site (SF) did not differ significantly when furthest geographical distance (GS and BC) did not differ significantly from the other compared with the LR population, and al- also exhibited a significant difference in the groups in the number of polymorphic frag- though the average number of polymorphic between-group similarity index value (Table ments (Table 2). bands in individuals from the SF site was 5). AMOVA analysis of AFLP data showed Between-group similarity coefficients were higher than all of the lower elevation pop- among-group variation accounting for only all lower than the compared groups, and in ulations, these differences were only signif- 17% of the total variation observed with 83% most cases very low, suggesting genetic dif- icant when compared with plants from the of the observed variation occurring within ferentiation between populations, but sample lowest elevation site. So we see that the two groups. The overall FST for the four A. sizes were too small to be confident of any highest elevation sites have the highest aver- passeyi populations was 0.174 with popula- significance (Table 7). AMOVA analysis of age number of polymorphic fragments out of tion pairwise FST values ranging from 0.086 AFLP data showed among-group variation the groups tested, but only the LR site dif- to 0.255 (Table 6). accounting for 36% of the total variation fered significantly in all of its comparisons The neighbor-joining tree constructed observed with 64% of the observed variation (Table 2). from the A. passeyi data (Fig. 2) reveals a occurring within groups. Population pairwise We performed a permutation test on the closer relationship between groups than that FST values were between 0.216 and 0.417 AFLP data for population subdivision using seen in the A. acuminatum and A. brandegei and had an overall mean of 0.355 (Table 8). the similarity index as the basis for the test groups. Fewer than 30 polymorphisms sepa- In the phenetic tree, each of the three col- statistic. With the exception of comparisons rate the least similar individuals. lection groups segregate out very clearly with with the BC-1 collection site, similarity Genetic diversity within and among A. no overlap with the character differences coefficients were significantly lower between brandegei collections. With the six selective being reflective of the geographical distances collection sites versus within collection sites primer combinations, AFLP analysis gener- separating the sampling sites. The geograph- (Table 3). The relatively small sample size ated a total of 539 polymorphic loci. The ically closer sites (TG and SF) are paired from the BC-1 site limited the test power for average number of polymorphic fragments more closely together (Fig. 3). comparisons with all but the largest site (HR) (Table 3). The HR group is significantly dif- ferent from all other groups, and the BC-2 Table 2. Allium populations in order of increasing elevation with sample size, within-population similarity ˆ z group is also significantly different in three coefficient (S), and the average number of observed polymorphic bands per plant (ANPB). of four comparisons (Table 3). Analysis of Species/ID Elevation N Sˆ SE ANPB SE molecular variance (AMOVA) of AFLP data A. acuminatum showed among-group variation accounting BC-1 1,556 m 5 0.577 0.013 146.0 7.0 for 34% of the total variation observed in BC-2 1,652 m 8 0.603 0.009 137.6 5.1 the A. acuminatum samples, whereas 66% of HR 1,761 m 14 0.600 0.012 147.4 5.6 the variation was observed within groups. LR 1,870 m 7 0.583 0.015 169.7 3.8 SF 2,318 m 12 0.664 0.008 158.8 3.3 The overall FST value for the sampled A. A. passeyi acuminatum sites was 0.339, and the popu- GS 1,546 m 10 0.608 0.016 130.8 4.2 lation pairwise FST values ranged from 0.235 AH-1 1,622 m 11 0.566 0.015 128.2 5.2 to 0.472 (Table 4). AH-2 1,638 m 8 0.630 0.018 147.6 4.2 Individual plants group together strictly BC 1,744 m 10 0.628 0.036 128.9 2.8 by collection site in the neighbor-joining tree A. brandegei based on pairwise comparisons of the total LR 1,859 m 7 0.604 0.010 125.1 2.6 number of DNA polymorphisms between SF 2,342 m 5 0.545 0.015 129.6 6.1 TG 2,515 m 7 0.552 0.015 114.9 4.9 individual plants (Fig. 1). Moreover, boot- z strap confidence levels provide support hier- SE values are shown for the similarity coefficients and band number. archical groups of BC-1, LR, and SF and also differentiate BC-2 from the other four col- lection sites. The most interesting aspect of Table 3. Allium acuminatum mean similarity index values within (diagonal) and between (below diagonal) this tree is the degree of differentiation be- A. acuminatum sampling sites.z tween the two BC sites despite their proxim- Population N BC-1 BC-2 HR LR SF ity to each other (600 m), indicating a lack Blue Creek 1 5 0.577 of gene flow between those two populations. Blue Creek 2 8 0.414 0.603 Genetic diversity within and among A. Hardware Ranch 14 0.330* 0.478* 0.600 passeyi collections. Only five primer combi- Liberty Road 7 0.445 0.313* 0.410* 0.583 nations were used in the A. passeyi analysis Swan Flat 12 0.439 0.311* 0.436* 0.510* 0.664 as a result of poor resolution resulting from zSignificant pairwise differences (P < 0.05) are denoted with an asterisk. one primer combination (E.ACCA//M.CAG). With the remaining five primer combina- tions, a total of 361 polymorphic bands were observed in the A. passeyi samples. The Table 4. Allium acuminatum pairwise comparisons of the average number of polymorphisms, between individual plants, within groups (PX or PY) (diagonal); the average total number of DNA average number of polymorphic fragments polymorphisms between groups (P ) (above diagonal); the corrected number of DNA per plant was significantly greater (P < 0.05) XY polymorphisms between groups (PXY –(PX1 +PX2)/2) (below diagonal); and the proportion of in the AH-2 collection compared with the DNA polymorphism among groups (FST = average corrected number of polymorphisms/average total other A. passeyi collections, which were not number of polymorphisms) (in parentheses, below diagonal). statistically different in fragment number Population N BC-1 BC-2 HR LR SF (Table 2). Blue Creek 1 5 123.2 165.8 196.1 175.1 170.9 Between-group similarity coefficients were Blue Creek 2 8 49.6* (0.305) 109.2 148.7 210.8 203.9 all lower than the within-group similarity Hardware Ranch 14 75.8* (0.391) 35.4* (0.235) 117.4 186.5 172.1 coefficients in the compared groups, indicat- Liberty Road 7 42.8* (0.239) 85.5* (0.409) 57.1* (0.317) 141.4 160.7 ing differences in genotypes between groups. Swan Flat 12 56.0* (0.340) 96.0* (0.472) 60.1* (0.348) 36.7* (0.242) 106.5 The two Anderson Hill collection sites (AH-1 *P < 0.001significant level of divergence based on permutation test described by Excoffier et al. (1992).
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Fig. 1. Neighbor-joining tree constructed from amplified fragment length polymorphism data from the five sampled Allium acuminatum collection sites. Acronyms correspond to sites listed in Table 1. The marker in the lower left-hand corner represents a distance of 10 character differences or polymorphisms. The neighbor-joining genetic distance analysis (Saitou and Nei, 1987) was based on a user-defined Euclidean distance matrix of the total number of character differences between individual plants from Arlequin (Excoffier et al., 1992) using PAUP* version 4.0b10 (Swofford, 2000). Significance testing for the trees was performed using bootstrap resampling of 10,000 replicates. A graphic display of the neighbor-joining tree was developed using TREEVIEW (Page, 1996).
Discussion of our sampling in only one region of their production, outcrossing species would tend larger distributional range. to have a larger proportion of their total ge- The sampling scheme used in this study Most Allium species, including those in netic variation occurring within populations allows us to look for patterns of within- our study, are known to produce viable seeds in comparison with selfing species (Hamrick species genetic diversity in relation to geo- (Kamenetsky and Rabinowitch, 2006). Natu- and Godt, 1989; Hellier, 2000). Because all graphical distribution, abundance, and site ral vegetative propagation also occurs in many individuals in the three species in our study elevation. The sampling of A. passeyi sites Allium species, although the rate of vegetative exhibited unique AFLP phenotypes, we can occurred over the species’ known range of reproduction is generally low (Kamenetsky assume that asexual reproduction plays no distribution, thereby providing comprehen- and Rabinowitch, 2006). All three species in more than a minor role in their mode of sive information on the patterns of genetic our study are able to reproduce clonally by reproduction. The high levels of variability diversity found in this species. However, means of bulb division, although field studies maintained within populations compared inferences drawn from the A. acuminatum tend to suggest that clonal reproduction with the between-population variability sug- and A. brandegei data are limited as a result occurs at a lower rate than sexual reproduc- gest outcrossing as the most likely mating tion (Phillips, unpublished data). It is com- system. The results of this study affirmed the mon for the mating system of a species to importance of sexual reproduction and polli- Table 5. Allium passeyi mean similarity index play a critical role in the patterns of genetic nator service in these species. values within (diagonal) and between (below diagonal) A. acuminatum sampling sites.z diversity within and among populations, but Partitioning of genetic variability by the dominant nature of AFLP markers does AMOVA revealed that the diversity in all Population N GS AH-1 AH-2 BC not provide direct information on the mating three species was largely distributed within Golden systems of the studied species. However, the populations. The percentage of variation found Spike 10 0.608 Anderson partitioning of molecular variance can give within the populations of the widespread Hill 1 11 0.517 0.566 clues to the mode of reproduction. A high congeners (A. acuminatum and A. brandegei) Anderson dependence on asexual reproduction would was estimated to be 65%, whereas the rare Hill 2 8 0.529 0.562* 0.630 naturally lead to lower levels of within- A. passeyi within-population genetic varia- Blue Creek 10 0.527* 0.512 0.503 0.628 population genetic diversity and the exis- tion accounted for 83% of its total variation. zSignificant pairwise differences (P < 0.05) are tence of genetically identical individuals. In Much of the between-population variation denoted with an asterisk. populations relying primarily on sexual re- appeared to result from isolation by distance. In general, the differences expressed in the population pairwise similarity coefficients Table 6. Allium passeyi pairwise comparisons of the average number of polymorphisms, between individual plants, within groups (P or P ) (diagonal); the average total number of DNA appear to be correlated with the geographical X Y distance between populations (Tables 3, 5, polymorphisms between groups (PXY) (above diagonal); the corrected number of DNA polymorphisms between groups (PXY –(PX1 +PX2)/2) (below diagonal); and the proportion of and 7). The neighbor-joining trees based on DNA polymorphism among groups (FST = average corrected number of polymorphisms/average total total character difference and the AMOVA number of polymorphisms) (in parentheses, below diagonal). population pairwise FSTs also supported this Population N GS AH-1 AH-2 BC correlation. In general, geographically closer Golden Spike 10 102.0 130.8 124.6 122.4 populations within each species were genet- Anderson Hill 1 11 25.3* (0.145) 108.8 120.2 137.0 ically more similar to each other than to more Anderson Hill 2 8 18.2* (0.195) 10.4* (0.086) 110.7 125.0 distant populations. Blue Creek 10 23.5* (0.192) 34.7* (0.173) 21.7* (0.255) 95.8 However, there was one significant excep- *P < 0.001 significant level of divergence based on permutation test described by Excoffier et al. (1992). tion found in the A. acuminatum populations
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Fig. 2. Neighbor-joining tree constructed from amplified fragment length polymorphism data from the four sampled A. passeyi collection sites. Acronyms correspond to sites listed in Table 1. The marker in the lower left-hand corner represents a distance of one character difference or polymorphism. The neighbor-joining genetic distance analysis (Saitou and Nei, 1987) was based on a user-defined Euclidean distance matrix of the total number of character differences between individual plants from Arlequin (Excoffier et al., 1992) using PAUP* version 4.0b10 (Swofford, 2000). Significance testing for the trees was performed using bootstrap resampling of 10,000 replicates. A graphic display of the neighbor-joining tree was developed using TREEVIEW (Page, 1996).
we sampled. The BC-1 and BC-2 populations Temporal isolation is another possible Although the differences between BC-1 and are only separated by 600 m but exhibit explanation for the genetic differentiation BC-2 appear great on the phenetic tree (Fig. markedly dissimilar genotypes (Table 3; Fig. between these two close populations. Despite 1), it is important to note that the pairwise 1). Although our data are insufficient to give the proximity of the two A. acuminatum pop- similarity coefficient is not statistically sig- definite explanations for this anomaly, we ulations, there are some distinct habitat dif- nificant (Table 3). can make some informed speculations. ferences that may impact reproductive timing The loss of genetic diversity, or genetic A recent introduction event might account at each site. Elevation and aspect differ erosion, in a species is an important consid- for the genotypic differences in these two greatly between the two populations. BC-1 eration in collection practices for restoration, populations. Their respective locations may is located on a gradual south-facing slope conservation, or breeding purposes. One way provide clues as to how this could occur. BC- nearly 1000 m lower than BC-2, which is that genetic erosion can be minimized by 1 is located on the edge of a dryland wheat found on a steep northwest-facing slope. The managers or propagators is by using practices field, and BC-2 is found on a hillside reserved elevation, grade, and aspect at each of these based on familiarity with species-specific for hunting 100 m from a dirt road. One of sites could change the microclimates enough patterns of genetic diversity (Rogers, 2004). the populations may be the result of uninten- to alter the timing of flowering in such a way The study described in this article provides tional anthropogenic seed dispersal where that interpopulation crosspollination would insight into patterns of diversity in inter- seed was transferred from another site in not occur, rendering the populations repro- mountain Alliums as related to range of dis- mud on a vehicle, farm implement, or the ductively isolated. Lack of gene flow result- tribution, geographical distance or isolation, boot of a hunter. ing from isolation by time could lead to the and altitudinal variation. divergence seen in our study. This form of When comparing species, geographical Table 7. Allium brandegei mean similarity index adaptive divergence is supported by other range of distribution appears to play an values within (diagonal) and between (below studies of flowering plant species (Hendry important role in the genetic differentiation diagonal) A. acuminatum sampling sites.z and Day, 2005). observed in this study. A measure of genetic Population N 1 2 3 It is also possible that the observed geno- divergence among populations can be quan- Liberty Road 7 0.604 typic differences in BC-1 and BC-2 are dis- tified by computing F statistics (Wright, 1965). Swan Flat 5 0.278 0.544 proportionally inflated as a result of low FST values are often used as an estimation of Tony Grove 7 0.309 0.428 0.552 sample sizes. The sampling of only five divergence among populations. A FST value zSignificant pairwise differences (P < 0.05) are individuals from BC-1 may not be adequately of zero indicates no divergence, and a FST denoted with an asterisk. representative of the population as a whole. value of one indicates a maximum divergence between populations. Wright (1965) sug- Table 8. Allium brandegei pairwise comparisons of the average number of polymorphisms, between gested that values exceeding 0.2 demonstrate low rates of gene flow among populations. individual plants, within groups (PX or PY) (diagonal); the average total number of DNA polymorphisms between groups (PXY) (above diagonal); the corrected number of DNA Limited gene flow is especially evident polymorphisms between groups (PXY –(PX1 +PX2)/2) (below diagonal); and the proportion of in A. acuminatum and A. brandegei where DNA polymorphism among groups (FST = average corrected number of polymorphisms/average total AMOVA analysis showed high genetic dif- number of polymorphisms) (in parentheses, below diagonal). ferentiation between populations. Population Population N LR SF TG pairwise FST values among these two species Liberty Road 7 99.0 183.8 165.7 ranged from 0.216 to 0.472, whereas the nar- Swan Flat 5 75.5* (0.417) 117.8 139.7 rowly distributed A. passeyi exhibited lower Tony Grove 7 64.9* (0.391) 29.4* (0.216) 102.8 values between 0.086 and 0.255. This dis- *P < 0.001 significant level of divergence based on permutation test described by Excoffier et al. (1992). parity would be expected because sampling
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Fig. 3. Neighbor-joining tree constructed from amplified fragment length polymorphism data from the three sampled Allium brandegei collection sites. Acronyms correspond to sites listed in Table 1. The marker in the lower left-hand corner represents a distance of 10 character differences or polymorphisms. The neighbor-joining genetic distance analysis (Saitou and Nei, 1987) was based on a user-defined Euclidean distance matrix of the total number of character differences between individual plants from Arlequin (Excoffier et al., 1992) using PAUP* version 4.0b10 (Swofford, 2000). Significance testing for the trees was performed using bootstrap resampling of 10,000 replicates. A graphic display of the neighbor-joining tree was developed using TREEVIEW (Page, 1996). in the A. acuminatum and A. brandegei two more common species showed enough Brewster, J.L. and H.D. Rabinowitch (eds.). 1990. populations occurred over a much larger genetic difference that there was no overlap Onions and allied crops. Vol. 3. CRC Press, geographical area than the A. passeyi sam- of individuals from different populations in Boca Raton, FL. Clausen, J., D.D. Keck, and W.M. Hiesey. 1940. ples, but when comparing pairwise FST val- the trees. However, the narrowly distributed ues between similarly distanced populations, and geographically closer A. passeyi showed Experimental studies on the nature of species. 1. Effect of varied environments on western less differentiation between populations. This we can see that the patterns of gene flow are North American plants. Carnegie Institution of not simply a function of distance, but may is again reflected in the higher pairwise Washington Publication No. 520. Washington, also reflect size of the species geographical similarity index values of A. passeyi (Table 5). DC. range. For example, less than 1 km separates AFLP data from A. acuminatum, A. pas- Dice, L.R. 1945. Measures of the amount of both of the two BC A. acuminatum popula- seyi, and A. brandegei has provided useful ecologic association between species. Ecology tions and the two AH A. passeyi populations. information regarding patterns of genetic 26:297–302. The pairwise FST values between these two variation within and among populations. Excoffier, L., P.E. Smouse, and J.M. Quattro. 1992. A. acuminatum populations is much higher The data confirm the importance of sexual Analysis of molecular variance inferred from (0.305) than that found in rare A. passeyi reproduction and pollinator service. Isolation metric distances among DNA haplotypes: populations (0.086) sampled at a comparable by distance can explain much of the observed Application to human mitochondrial DNA distance. Pairwise F values between pop- variation with only a few exceptions. Our restriction data. Genetics 131:479–491. ST Gitzendanner, M.A. and P.S. Soltis. 2000. Patterns ulations separated by 20 to 30 km are higher data also suggest that the widespread species of genetic variation in rare and widespread in three of the four A. passeyi comparisons in our study tend to have lower levels of plant congeners. Amer. J. Bot. 87:783–792. (0.255, 0.173, 0.195, and 0.145) than the interpopulation gene flow in comparison with Hamrick, J.L. and J.W. Godt. 1989. Allozyme more common A. brandegei populations the rare species. With this information, strat- diversity in plant species, p. 43–63. In: Brown, (0.216). In populations separated by 50 egies can be used in an effort to identify di- A.H.D., M.T. Clegg, and A.L. Kahler (eds.). km, the A. acuminatum again shows lower verse parental lines for breeding purposes, Plant population genetics, breeding, and ge- rates of gene flow (0.348 and 0.317) than the develop practical restoration guidelines, and netic resources. Sinauer Associates, Sunderland, rare A. passeyi (0.192). From these data we improve conservation management plans. MA. see that A. acuminatum demonstrated lower Hanelt, P. 2001. Alliaceae. In: Hanelt, P. (ed.). Mansgeld’s encyclopedia of agricultural and rates of gene flow than A. passeyi in all pair- Literature Cited wise comparisons separated by similar dis- horticultural crops. Vol. 4, 3rd ed. Springer- Adair, R., R.C. Johnson, B. Hellier, and W. Kaiser. Verlag, Vienna. tances. Comparing the rates of gene flow Hellier, B. 2000. Genetic, morphologic, and habitat between similarly distanced populations in A. 2006. Collecting tapertip onion (Allium acumi- natum Hook.) in the Great Basin using tradi- diversity of two species of Allium native to the brandegei and A. passeyi was less meaning- tional and GIS methods. Native Plants Journal Pacific Northwest, USA and their implications ful as a result of only having one separation 7:141–148. for in situ seed collection for the National Plant distance in common. These values suggest Benham, J., J.-U. Jeung, M. Jasieniuk, V. Kanazin, Germplasm System. Washington State Univer- that interpopulation gene flow may be corre- and T. Blake. 1999. Genographer: A graphical sity, Pullman WA, Master’s Thesis. lated with range size. In general, the higher tool for automated AFLP and microsatellite Hendry, A.P. and T. Day. 2005. Population struc- ture attributable to reproductive time: Isolation pairwise FST values in this study are found analysis. Journal of Agricultural Genomics 4: among the species with wide distributions. Article 4. Jan. 2007.
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Karron, J.D. 1987. A comparison of levels of June 13–15; Provo, UT. Proc. RMRS-P-21. Shultz, L.M., D.R. Ramsey, and W. Lindquist. genetic polymorphism and self-compatibility USDA-Forest Service, RMRS, Ogden, UT. 2006. Revised atlas of Utah plants. College of in geographically restricted and widespread Neely, E. and M. Barkworth. 1984. Vegetation on Natural Resources, Utah State University, plant congeners. Evol. Ecol. 1:47–58. soils derived from dolomite and quartzite in the Logan, UT. 20 Jan. 2007.
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