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Mci199 467.478

Annals of Botany 96: 467–477, 2005doi:10.1093/aob/mci199, available online at Genetic Variation in Echinacea angustifolia Along a Climatic Gradient D . W . S T I L L *, D . - H . K I M and N . A O Y A M A Department of Horticulture/Plant and Soil Science, California State Polytechnic University, Pomona, 3801 W Temple Avenue, Pomona, CA 91768, USA  Backgrounds and Aims Echinacea angustifolia is a widespread species distributed throughout the Great Plainsregion of North America. Genetic differentiation among populations was investigated along a 1500 km north–southclimatic gradient in North America, a region with no major geographical barriers. The objective of the study wasto determine if genetic differentiation of populations could be explained by an isolation-by-distance model or byassociations with climatic parameters known to affect plant growth and survival.
 Methods Historical climatic data were used to define the nature of the climatic gradient and AFLP markers wereused to establish patterns of population genetic differentiation among ten Echinacea populations collected from North Dakota to Oklahoma. A total of 1290 fragments were scored using six EcoRI/MseI and three PstI/MseI primercombinations. Assessment of the correlation between climatic, genetic and geographic distances was assessed byMantel and partial Mantel tests.
 Key Results PstI/MseI combinations produced significantly fewer fragments, but a larger percentage was uniquecompared with EcoRI/MseI markers. Using estimates of FST, populations in Oklahoma and southern Kansas wereidentified as the most divergent from the other populations. Both the neighbour-joining tree and principal co-ordinate analysis clustered the populations in a north–south spatial orientation. About 60 % of the genetic variation wasfound within populations, 20 % among populations and the remaining 20 % was partitioned among groups thatwere defined by the topology of the neighbour-joining tree. Significant support was found for the isolation-by-distance model independent of the effects of annual mean precipitation, but not from annual mean temperatureand freeze-free days.
 Conclusions Echinacea angustifolia populations exhibit genetic divergence along a north–south climaticgradient. The data support an isolation-by-distance restriction in gene flow that is independent of annual meanprecipitation.
Key words: Echinacea angustifolia, population differentiation, isolation by distance, widespread species, climatic gradient,selection pressure, AFLP.
divergence. In the absence of a barrier that restricts geneflow, adaptation to a new environment will be limited. Geo- The level of genetic diversity and its causes are of great graphical barriers and/or restricted gene flow coupled with interest in evolutionary biology as genetic diversity greatly differential selection pressures within the geographical affects the evolutionary potential of a species (Futuyma, range of the species should provide the opportunity for 1998). Abiotic and biotic processes acting upon isolated divergence. Often reports that examine the evolutionary populations are thought to be a key factor in species diver- history and distribution of a given taxa are discussed in gence (Grant, 1981; Loveless and Hamrick, 1984; Levin, the context of palaeoclimatic and geographic factors (e.g.
2003), but populations with low levels of genetic diver- Francisco-Ortega et al., 1995; Sharbel et al., 2000; Zhang sity may not be able to adapt to a changing environment et al., 2001), whereas it is clear that successful adaptation to (Ellstrand and Elam, 1993). In a widespread species, the a changing environment must exist within the context of conditions for divergence and local speciation are likely to standing genetic variation. Evidence for active speciation exist in those populations at the geographical and ecological might be detected by varying levels of genetic divergence edges of the species distribution (Levin, 2003). Although observed in populations of the species (Schultz and Soltis, geographic variation within widespread species is well 2001). Climatic factors are a major contributing factor reported in the literature (e.g. Holman et al., 2003), there in shaping the vegetation in the prairie ecosystem in are cases of widespread species exhibiting neither genetic North America (Weaver and Fitzpatrick, 1934). A number nor phenotypic differences, and thus geographic distribu- of environmental factors may exert selection pressure thus tion is not always a good predictor of genetic divergence affecting evolutionary change, and among these are freezing Processes of geographical divergence occur by isolating Echinacea angustifolia is a widespread species of mechanisms, in part due to the restriction of gene flow bet- rocky open areas in the North American plains occurring ween populations. Among subpopulations of a widespread from Texas to Saskatchewan and from western Iowa to species, different ecological environments and independent Minnesota (McGregor, 1968; Kindscher, 1989). Echinacea evolution of populations through genetic drift may lead to spp. are perennial plants and reproduce predominantly by * For correspondence. E-mail seed but also reproduce vegetatively by crown-division ª The Author 2005. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: Still et al. — Intraspecific Variation in Echinacea angustifolia T A B L E 1. Geographic distribution and climatic parameters of Echinacea angustifolia populations and the outgroup E. sanguinea All data are for the period 1931–2000 for climatic divisions from which the collection sites were located. See text for additional details.
FFD, freeze-free days; AMP, annual mean precipitation; AMT, annual mean temperature.
* USDA accession Ames 23874.
(McGregor, 1968; Li, 1998). They have a long history of use collected from each plant, and kept separate to maintain the in North America, and E. angustifolia was the most widely identity of each plant. The seeds were harvested and stored used medicinal plant of the plains Indians, and today it is a at 6 % moisture content at À20 C until needed. The seeds popular medicinal herb (Kindscher, 1989; Percival, 2000).
were germinated in Petri dishes and transferred to field plots The distribution of E. angustifolia along a 1500 km north– at Cal Poly Pomona where approx. 80 plants from each south (N–S) climatic gradient is well suited to study patterns population were established in a common nursery. Leaves of genetic differentiation and the underlying causes of harvested from these plants served as source DNA for these microevolution. Within the geographic boundaries of the experiments and no individual plant was represented more present study there are no major physical barriers limiting than once in the analysis. Because >90 % of the transplants gene flow among populations. Climatic conditions that survived it is likely the nursery population is genetically could potentially have profound effects on plant growth representative of the original populations. Voucher speci- and reproduction vary greatly within the distribution of mens were deposited at the University of California E. angustifolia (Table 1). Within the geographic range of Herbarium, Los Angeles and their taxonomic identity con- our study populations, over a period of 70 years (1931– firmed by morphological keys (B. Prigge, pers. comm.).
2000), average annual rainfall varied from 400 to 750 mm, Taxon names reported herein are consistent with the and the average number of frost-free days ranged from USDA-ARS National Genetic Resources Information Net- 118 to 209. The objectives of the present study were to: work. Echinacea sanguinea was used as an outgroup for (a) establish a pattern of genetic diversity among popula- phylogenetic rooting purposes. Previously, E. sanguinea tions collected along an approx. 1500 km N–S cline from had been shown to be a member of the sister clade of North Dakota to Oklahoma; (b) test the theory that popu- the E. angustifolia species complex (Kim et al., 2004).
lations at the geographic edges of its distribution exhibit McGregor (1968) reported that all species of Echinacea the greatest genetic divergence; (c) determine if these gen- will readily form hybrids, suggesting a close genetic rela- etic differences are correlated with a simple isolation by tionship and a previous report based on amplified frag- distance model (Wright, 1946), or alternatively; (d) deter- ment length polymorphism (AFLP) analysis concluded mine if specific climatic attributes are correlated with the that each species is genetically distinct, but closely related observed genetic variation among the populations thus (Kim et al., 2004). Seeds of E. sanguinea were obtained from the USDA Plant Introduction Station (Ames, IA, USA)and were originally collected in Louisiana. Accession, plantand population identification numbers, and geographic co-ordinates are given in Table 1 and the collection sitesare graphically represented in Fig. 1. A matrix of geographic distances (GEO) between collection sites was calculated Seeds of E. angustifolia D.C. var. angustifolia sensu using the geographic co-ordinates of each site.
McGregor were collected from ten sites within the GreatPlains region of the United States in September 2000 (Fig. 1 and Table 1). The distance between any two populationsranged from 71 to 1496 km. Each E. angustifolia population Climatic data for each collection site were obtained from consisted of 200 to 300 plants with the exception of the historical records compiled from the National Oceanic and SD-002 population, which contained 40–50 plants. At each Atmospheric Administration (NOAA, 2002). Climatic divi- site, 20–30 plants were chosen at random, the inflorescences sions, often used in agricultural and ecological modelling, Still et al. — Intraspecific Variation in Echinacea angustifolia F I G . 1. Collection areas of Echinacea angustifolia and the outgroup E. sanguinea.
are defined as regions within a state that are as climatically level for the 30-year period (NOAA, 1988). A climatic homogeneous as possible (NOAA, 2002). The average area normal is defined as the arithmetic mean of a climatological encompassed by a climatic division in the present study was element computed over consecutive decades (NOAA, 26 537 square km (range 18 109–48 764 square km). Thus, these data may be considered representative of a macro-environment. In four instances, collection sites (populations ND-007, NE-002, KS-HO and LA) were within 15 km of aclimatic division boundary, in which case these data were Genomic DNA was isolated from recently expanded averaged across those two climatic divisions.
leaves using a modified CTAB method (Doyle and Doyle, All climatic parameters, with the exception of freeze-free 1987). Freshly harvested leaves (200 mg) were homo- days data, were recorded from 1931–2000. The statistics for genized in extraction buffer using a FastPrep1 System freeze-free days were not calculated at each weather station cell disrupter (Qbiogene, Inc., Carlsbad, CA, USA). The for the entire 1931–2000 interval; however, data from 1951 quality of the extracted DNA was estimated by measuring to 1980 were available and were therefore used for this the 260 and 280 UV absorbance and the integrity was veri- climatic parameter (NOAA, 1988). The average annual tem- fied by electrophoresis on a 1Á0 % agarose gel. Only samples perature for the 1931–2000 period was computed from the with 260 : 280 ratios of 1Á8–1Á9 showing clear high mole- monthly average temperature normals which were them- cular weight bands were used for AFLP analysis.
selves computed by averaging the monthly maximum and AFLP reactions were performed as described by Vos et al.
minimum normals (NOAA, 2002). The average annual pre- (1995) with minor modifications optimized for capillary cipitation was computed as the mean of 12 monthly nor- electrophoresis as previously described (Kim et al., 2004).
mals. Freeze-free days were calculated as the median length Adapters were synthesized by Operon Technologies of time between the last 0 C temperature in spring and (Alameda, CA, USA), MseI and PstI primers were obtained the first 0 C temperature in autumn with a 90 % probability from Gibco (Invitrogen Life Technologies, Baltimore, Still et al. — Intraspecific Variation in Echinacea angustifolia T A B L E 2. Oligonucleotide adapters and primers used for increase on the leading edge of a given peak, while the AFLP analysis of Echinacea angustifolia populations relative peak height threshold (RPHT) specifies the min-imum height (relative to the second highest peak) required before being included in the fragment list. Based on pre-vious results a RPHT : ST of 5 : 10 was used. For each individual, the AFLP product was run in three separate capillaries, and a fragment was included only if its presence was detected in at least two of the three capillaries. Where discrepancies existed, the electropherograms were visually reviewed to reconcile ambiguities. Fragment sizes greater than 400 bp were less reproducible and were therefore For each pairwise comparison between individuals i and j, the 1/0 matrix was used to calculate the Dice estimate of genetic similarity [GSD = 2a/(2a + b + c)] (equivalent to 1 – Nei and Li’s genetic distance; Nei and Li, 1979), where a is the number of bands shared by i and j, b is the number of bands present in i and absent in j, and c is the number of bands present in j and absent in i for the data pooled over all primer combinations.
A genetic distance matrix (GEN), equivalent to the 1 – Dice estimate, was used to construct phenograms using the neighbour-joining (NJ) method (Saitou and Nei, 1987).
Internal branch support was evaluated by bootstrap analysis (Felsenstein, 1985) of 1000 bootstrap replicate data sets with PAUP software (Beta version 10; Swofford, 2002). To visu- alize the dispersion of individual plants in relation to thefirst two principal axes of variation, principal co-ordinateanalysis (Gower, 1966) was performed on the genetic sim-ilarity data (Dice estimates) matrix using the DCENTER MD, USA) and phosphoramidite dye-linked EcoRI primers and EIGEN modules of NTSYSpc (Rohlf, 2002).
were synthesized by Proligo Primers and Probes (Boulder, Genetic diversity within and among populations was CO, USA). All EcoRI primers were labelled with blue calculated using Arlequin (Schneider et al., 2000). The phosphoramidite dye (D-4 dye; Beckman-Coulter, Inc., analysis of molecular variance computed by the Arlequin program generates F statistics, which are F-statistic analogs Preselective primer pairs with a single selective nucleo- (Excoffier et al., 1992). Genetic variation was partitioned tide extension of the AFLP adapter were used, whereas selective PCR primers utilized three nucleotides (Table 2).
After selective amplification, 0Á5 mL of the reaction pro- SC) and within populations FST). Group designations were based on the topology of the NJ tree. The topology- ducts were mixed with 30 mL of sample loading solution based grouping method clustered populations into four (Beckman-Coulter, Inc., Fullerton, CA, USA) and 0Á5 mL groups: Group 1, ND-004, ND-007 and SD-002; Group 2, of fragment size standard was added to each sample (60– NE-001, NE-002 and NE-003; Group 3, KS-CD and KS- 600 bp; Beckman-Coulter, Inc.). Samples were separated HO; Group 4, OK-WD and OK-CO. It was assumed that using capillary electrophoresis on an automated CEQ 8000 each population was in Hardy–Weinberg equilibrium, with DNA fragment analysis/sequencer (Beckman-Coulter, Inc.) three randomly chosen individuals representing the popu- with running conditions as follows: denaturation at 90 C lation. This assumption appears to be well founded on the for 120 s, injection for 30 s at 1000 V, separation at 4800 V basis of bagging studies conducted on plants growing in a common nursery at Cal Poly Pomona. Following the bag-ging of 97 capitula (heads) from plants of each population, <1 % (38/16 146) of the achenes produced embryos, indic- Fragment sizes were automatically calculated by CEQ ating E. angustifolia is a more or less obligate out-crossing 8000 software (ver. 4.2.0) using local Southern sizing algo- rithms. Each fragment was treated as a separate character To estimate the proportion of genetic differentiation and scored as either present (1) or absent (0) across all geno- between populations explained by geographical distance, types by CeqCluster fragment analysis software (Beckman- the genetic (GEN) and geographic (GEO) distance dis- Coulter, Inc.). Inclusion or exclusion of fragments with similarity matrices were subjected to a Mantel test of asso- automated fluorescent dye capillary electrophoresis systems ciation with 1000 replications using the MXCOMP module was performed by adjusting threshold levels of signal of NTSYSpc (Rohlf, 2002). To estimate the proportion heights and slopes. The CeqCluster software slope thres- of genetic differentiation that could be associated with hold (ST) algorithm specifies the minimum rate of signal the climatic parameters annual mean temperature (AMT), Still et al. — Intraspecific Variation in Echinacea angustifolia T A B L E 3. Characteristics of fragment variation generated by nine primer combinations in the AFLP analysis of E. angustifolia (n = 30 individuals) and E. sanguinea (n = 3 individuals) EM14 EM15 EM19 EM20 EM22 EM25 PM 17 PM22 PM25 EM PM Total EM PM No. of fragment shared by all populations No. of fragments unique to one population No. of fragments present in two or more populations 136 Fragments were scored within a range of 60–400 bp.
T A B L E 4. Matrix of average genetic distance (Nei and Li, 1979) between 10 Echinacea angustifolia populations and the outgroup, The leading diagonal represents genetic distance within a population.
freeze-free days (FFD) and annual mean precipitation more fragments generated from the EM primer sets than (AMP), dissimilarity matrices were subjected to a Mantel the PM primers (Table 3). With the exception of PM25, all test of association with 1000 permutations. Each matrix was primer combinations generated fragments that were present constructed by subtracting the differences in values between in all populations. All EM and PM primer combinations populations. Likewise, the relationship between geographic generated fragments that were unique to a single population, distance and each climatic parameter was tested with a but PM primer sets produced approx. 2Á4 times more unique Mantel test, as described above. Because the climatic para- meters and geographic distance are likely to follow a com- The average genetic distance was calculated for each mon spatial structure, the partial Mantel association test population and a pairwise genetic distance matrix construc- was employed to control for such spatial autocorrelation ted (Table 4). Within each E. angustifolia population, the (MXCOMP module of NTSYSpc; Smouse et al., 1986; average genetic distance was 0Á0272, whereas the average Legendre and Fortin, 1989; Roseman, 2004). A partial pairwise distance between populations was 0Á0494, approx.
Mantel test is computed between matrices A and B while 1Á8 times the intrapopulation distance. The intrapopulation controlling for the effect of matrix C (Smouse et al., 1986; distance (located on the leading diagonal; Table 4) ranged Legendre and Fortin, 1989). The computations are accomp- from 0Á0205 (population OK-CO) to 0Á0363 (population lished by computing an A0 matrix that contains the residuals NE-003). Excluding the outgroup, the greatest genetic of the linear regression values of matrix A over matrix C; distance between two populations was between OK-WD computing a B0 matrix containing residual linear regression and NE-001 (0Á0578), whereas the closest genetic distance values of B over matrix C; computing the Mantel statistic observed was between OK-CO and OK-WD (0Á0351). The between A0 and B0 (Smouse et al., 1986; Legendre and average genetic distance between all E. angustifolia popu- lations and the outgroup E. sanguinea, was 0Á1033 (arith-metic average of the bottom row, excluding E. sanguineavalues; Table 4), approximately twice the genetic distance observed among E. angustifolia populations.
For the ten E. angustifolia populations and the outgroup The NJ tree (Fig. 2) contained four major groups, with E. sanguinea, 1290 fragments were scored (Table 3). Both each node relatively well supported by high bootstrap EcoRI/MseI (EM) and PstI/MseI (PM) primer combinations values. The ND-004, ND-007 and SD-002 populations com- were used to generate markers. There were approx. 2Á3 times prised a sister group to the NE populations, and the KS Still et al. — Intraspecific Variation in Echinacea angustifolia I G . 2. Neighbour-joining phenogram of Echinacea angustifolia and the outgroup E. sanguinea using Nei and Li’s (1979) genetic distance based on AFLP markers obtained from six EM and three PM primer combinations. Numbers shown at the node represent bootstrap values (as a percentage of 100 replicates).
populations formed a group sister to these two clades (71 % T A B L E 5. AMOVA estimates of genetic structure among bootstrap value). The OK populations formed a group sister Genetic structure among the ten E. angustifolia popula- tions was estimated by subjecting the binary fragment data to AMOVA using Arlequin. The greatest amount of vari-ation was observed within a population (approx. 60 %), which is not unexpected in an obligate out-crossing species.
The remaining variation (40 %) was equally distributed among groups and among populations within groups (Table 5). To identify genetic differentiation betweenpairs of populations, a matrix was constructed using F Each population was grouped according to the topology of the NJ tree (Fig. 2); P is the probability of having a more extreme variance component probability values (Table 6). The southern-most popula- and F-statistic than the observed values by chance alone. Mantel permu- tions, namely OK-WD, OK-CO and KS-HO, had the highest tations tested for significance where FCT represents variation among groups; number of pairwise differences in the 10 · 10 matrix of FSC, variation among populations within groups; FST, variation within ST values, suggesting a greater degree of differentiation Group 1 = ND-004, ND-007 and SD-002; Group 2 = NE-001, NE-002 and in comparison to other populations (Table 6). With increas- NE-003; Group 3 = KS-CD and KS-HO; Group 4 = OK-LO and OK-WD.
ing geographic distance there was also a trend towardsgreater genetic distance as indicated by the increasing FSTvalues within columns (Table 6). A principal co-ordinate A simple Mantel’s association test between the climatic analysis constructed from the Dice similarity coefficient parameters annual mean temperature, freeze-free days and showed a pattern of population clustering consistent with annual mean precipitation indicated significant correlations the N–S origin of the populations (Figure 3). The first between genetic diversity and each climatic parameter two co-ordinates explained approx. 24 % of the observed (Table 7). A significant positive relationship was likewise detected between genetic and geographical distance Still et al. — Intraspecific Variation in Echinacea angustifolia T A B L E 6. Genetic differentiation between pairs of populations of Echinacea angustifolia expressed by FST (significance T A B L E 7. Simple and partial Mantel tests of association among genetic distances, geographic distance and climatic variables of Echinacea angustifolia populations I G . 3. Principal co-ordinates analysis among ten Echinacea angustifolia populations and the outgroup E. sanguinea. The first two dimensions accounted for approx. 24 % of the variability.
The pattern of association in the dependent matrix A was compared with the predictor matrix B, while controlling for the effects of matrix C, using thepartial Mantel test (Smouse et al., 1986).
(Table 7). However, a strong association was detected Genetic distance was calculated using Nei and Li’s (1979) estimate.
Climatic data are averaged values from the time period 1931–2000 with between the climatic parameters and geographic distance the exception of freeze-free days, which is from the time period 1951–1980.
along the N–S cline supporting spatial autocorrelation AMT, annual mean temperature; AMP, annual mean precipitation; FFD, among these variables. This would be expected as there freeze-free days; GEN, genetic distance; GEO, geographic distance.
is a N to S trend toward increasing precipitation, freeze- * Probability that a random Z < observed Z.
free days and, especially, annual mean temperature.
y Probability that a random Z > observed Z.
Therefore, to separate the spatial autocorrelation and theinterdependence of the climatic variables with geographicdistance, a partial Mantel test was used. Once geographic geographic distances and annual mean temperature and distance (GEO) was controlled for, the correlation between freeze-free days, the combined effects of these para- the climatic parameters annual mean temperature and meters cannot be separated on genetic structure of the freeze-free days and genetic distance (GEN) was not sig- E. angustifolia populations used in these studies.
nificant (Table 7). The influence of geography on geneticstructure was reduced from 0Á54 to 0Á35 when controllingfor the influence of annual mean precipitation. The differ- ence (0Á1960) is attributed to the annual mean precipitation component of geographic distance. The partial Mantel testindicated a significant relationship between genetic and The present data show a distinct genetic pattern among geographic distance when annual mean precipitation was E. angustifolia populations that is consistent with a N–S controlled. Thus, the present data support the notion of an spatial orientation. Most (60 %) of the observed genetic isolation-by-distance influence on gene flow, independent variation occurred among plants within populations, with of the effects of annual mean precipitation. However, the remaining 40 % equally distributed among groups and because of the strong spatial autocorrelation between populations. Similar amounts of intra-population genetic Still et al. — Intraspecific Variation in Echinacea angustifolia variation have been reported in out-crossing species, includ- sufficient to assess the degree of differentiation between ing sunflower (approx. 70 %; Quagliaro et al., 2001) and populations using SSR markers (Dyer and Sork, 2001).
the arctic-alpine plant Trollius europaeus (approx. 64 %;Despres et al., 2002). Using isozyme or RAPD data, Baskauf et al. (1994) and Kapteyn et al. (2002) estimatedthat approx. 93 % and 78 % of the variability observed The AFLP method was developed using AT-rich recog- among E. angustifolia populations occurred within popula- nition sites EcoRI and MseI (EM) restriction enzymes, and tions. The higher amount of intra-population variability it has been widely reported these markers are distributed observed in their studies is most likely attributed to the throughout the genome (Vos et al., 1995; Jones et al., 1997; different markers systems used among these studies, Hansen et al., 1999). However, clustering of EM markers although sampling may also contribute to these differences.
was detected in linkage maps of A. thaliana recombinant The genetic coefficient interval between each node branch inbred lines around centromeres (Alonso-Blanco et al., of the four groups in the NJ tree is relatively short, indic- 1998). Clustering of EM-derived AFLP markers has also ating the divergence of these groups occurred within a short been reported in potato (van Eck et al., 1995), barley time period, but early in the radiation of these populations.
(Becker et al., 1995; Powell et al., 1997), soybean (Keim Conversely, the branch lengths of each population within et al., 1997) and maize (Vuylsteke et al., 2000). Generally, a major group are relatively long, again suggesting a relat- low recombination rates are observed in regions surr- ively long period of isolation following radiation.
ounding centromeres (Schnable et al., 1998) which are reportedly AT-rich (Richards and Dawe, 1998) as are telo-meric regions (Arabidopsis Genome Initiative, 2000). Clus- tering of EM markers may be a consequence of either low The use of AFLP technology in phylogenetic and eco- recombination in the heterochromatic region around the logical studies is becoming increasingly popular because centromere (Hoopen et al., 1996) or it may simply be a of its unique ability to detect polymorphisms within the result of increased restriction sites in this region. By com- genome without requiring prior sequencing information.
bining experimentally derived AFLP data with in silico Because AFLP faithfully generates many fragments per analyses using the sequence of Arabidopsis thaliana, Peters primer combination, differences can be detected between et al. (2001) provided convincing evidence that clustering parental genotypes and their segregating progeny with is at least partly due to the increased frequency of AFLP as little as two primer combinations (E. Hayashi and restriction sites in this region, as opposed to a reduced D. W. Still, unpubl. res.). Results from AFLP data sets recombination rate around the centromeric region. By repla- have been largely concordant with other molecular markers cing EM with Pst1/MseI (PM) restriction enzymes, the pro- (Powell et al., 1996) or analyses based on gene sequencing portion of recognition sites occupied by GC nucleotides (Spooner et al., 2005). Most, but not all, AFLP fragments increases from 0Á2 to 0Á4, and thus GC-rich sites are of a specific size can be considered to represent the same targeted. The GC content of arabidopsis and rice is approx.
loci (Cervera et al., 2001; Peters et al., 2001), although this 35 % and 44 %, respectively, with each chromosome in likelihood apparently decreases with an increase in genetic arabidopsis having approximately the same GC content distance between species (Mechanda et al., 2004). Large (Arabidopsis Genome Initiative, 2000; Goff et al., 2002).
data sets can offset the assumption of orthology simply by In the present study, approx. 50 % fewer fragments gener- increasing the number of independent loci sampled across a ated by PM relative to EM primer sets were observed, genome and establishing ‘correct’ phylogenetic relation- perhaps indicating a lower GC content than that reported ships among species (Rokas et al., 2003). The AFLP method for rice or arabidopsis. Fewer PM-generated fragments have largely fulfills this requirement, and any non-orthologous been reported in AFLP analyses in maize, tomato and yam, fragments detected among populations should be overcome with reductions of 25, 15 and 64 %, respectively (Haanstra by the much larger number of orthologous fragments. The et al., 1999; Vuylsteke et al., 2000; Mignouna et al., 2002).
present data, consisting of 1290 fragments, produced an NJ No differences in fragment numbers were detected in cotton tree with relatively high, to very high levels of support at between PM and EM primer sets (Liu et al., 2001). In the most nodes (Fig. 2). The fact that each node is supported by present study, a greater percentage of PM-generated frag- relatively high bootstrap values, and each plant from a given ments was observed that were unique to a single population population clustered to its proper population at the terminal compared with EM primer sets. Relative levels of poly- node, supports the robustness of the AFLP technique in morphism appears to be species-specific as a 16 % decrease accurately identifying polymorphisms. Further, because was reported for PM primer sets in tomato (Haanstra et al., the accuracy of reconstructing phenetic relationships 1999), whereas Liu et al. (2001) reported no differences in increases with the number of independent loci assayed total fragments or polymorphisms between the EM and PM (Travis et al., 1996; Rokas et al., 2003) large numbers of individuals need not be genotyped for the purpose of estab- Given the preponderance of evidence that AT- and lishing patterns of genetic differentiation among popula- GC-rich regions are not randomly and evenly distributed tions. Relatively little improvement in bootstrap values throughout the genome, it is likely that assessments of popu- was found by adding four or more individuals when con- lation genetic differentiation based on AFLP may be affec- structing the NJ tree. Others have demonstrated a sample ted by the choice of restriction enzymes. In the present size of four individuals of an out-crossing species was study, the PM primer sets produced slightly different NJ Still et al. — Intraspecific Variation in Echinacea angustifolia topologies than the EM primer sets, and many nodes had and the relationship between genetic distance and climatic bootstrap support values <50 % (data not shown). The topo- distance examined. Although climate-induced selection logy of the NJ tree constructed from the EM data set did not pressures may occur, the partial Mantel test shows that change once the PM data were added, but bootstrap values once the effect of geographic distance is removed did increase with the combined EM/PM dataset. These res- (AMT · GEN controlling GEO) most of the correlation ults are most likely due to the fact that, relative to PM primer between each of the climatic parameters and genetic dis- sets, EM primer sets produced more fragments per primer tance disappears. Neutral (AFLP) markers, by definition, are combination and fewer polymorphisms. Because bootstrap not expected to be affected by selection pressure. Instead, in procedures estimate the likelihood of a ‘correct’ topology a random mating population they may serve as marker by removing and replacing subsets of data from the dataset alleles that can provide ways to identify regions of the and reconstructing the tree, they will have a greater effect on genome that are associated with effects of adaptedness the PM data than the EM data. An AFLP analysis based on (Allard, 1996). Experimentally, sampling additional popu- combined EM/PM fragments should theoretically sample lations along an east–west transect would allow uncoupling more areas of the genome than EM primer sets and therefore of the strong N–S spatial autocorrelation with the climatic provide a more accurate assessment of genomic divergence parameters. Molecular marker candidates for adaptation may alternatively be associated with quantitative traitloci which could only be identified by transplanting hybridpopulations derived from parents taken from the extreme Isolation-by-distance vs. climatic selection pressures geographic distributions into two or more locations.
Theoretically, the basic ingredients for genetic diver- The genetic data represent standing genetic variation that gence are present within the distribution of E. angustifolia, is presented against a climatic time-frame of 70 years and, namely a wide geographic distribution and contrasting cli- although it is not possible to project climatic conditions matic conditions. It was hypothesized that those populations further than the 70-year time frame of our dataset, there at the margins of their geographic range would show the is no indication that the climate of the Great Plains has greatest amount of genetic divergence in a pairwise com- changed substantially since the last ice age, roughly parison. The NJ and principal co-ordinate analysis data of 10 000 years ago. Genetic composition of plants has been E. angustifolia exhibited clustering of each population shown to reflect climatic parameters in a relatively short consistent with their latitude of origin, indicating genetic time frame. Allard and coworkers (Allard, 1988; Perez de la divergence along their geographic distribution (Figs 2 Vega et al., 1994) have shown that within a 60-year period and 3). A trend between geographic distance and increasing genetically distinct populations grown under similar genetic distance (FST values) was observed (column 1, environments often develop similar multi-locus allelic asso- Table 6). These data show a significant isolation by distance ciations and, conversely, genetically identical material effect and the three highest FST values were observed grown under different environments develops different between ND-007 and OK-CO (0Á4661), ND-004 and multi-locus associations. Allelic frequencies may change OK-CO (0Á4826) and NE-001 and OK-CO (0Á4541), thus within a few generations in a predominantly selfing plant, supporting the hypothesis that the greatest divergence is at whereas in an obligate out-crossing species, such as the geographic edges of the distribution of this species. The E. angustifolia, discernible multi-locus associations would simple Mantel test also supports an isolation-by-distance theoretically occur much more slowly. However, in either model for genetic structure of these populations (GEN · selfing or out-crossing species, a constant selection pressure GEO, R = 0Á5423, P = 0Á001). However, because of the must remain if the assemblages are to remain intact. Con- strong association between climatic conditions and the N–S sequently, the genetic structure of a population, and assess- sampling transect, at this point it cannot be ruled out that ments of divergence among populations might be more the genetic structure is influenced by the selection pressure reflective of an ecological time scale rather than an evolu- caused by climatic parameters. Once annual mean tem- tionary time scale. Only by resampling the same Echinacea perature or freeze-free days were controlled for, there was populations over time can the potential magnitude of no support of an isolation-by-distance model (Table 7), climatic effects on genetic diversity and allelic frequencies although controlling for freeze-free days is marginally be evaluated. Our climatic data represent averaged events close to significance (GEN · GEO controlling for FFD, and the Mantel test statistic values indicate a strong spatial R = 0Á2056, P = 0Á0769). Controlling for the effects N–S association. Although the temperature–geographic of annual mean precipitation resulted in support of an autocorrelations could not be separated, it is possible that isolation-by-distance model, independent of the effects of plant adaptation, and therefore a portion of the standing genetic variation, is a reflection of more extreme events The variation in climate along the 1500 km N–S cline such as prolonged drought or untimely freezing events, suggests selection pressures among the populations could be the likelihood of occurrence of which is not expected to different within the range of E. angustifolia populations sampled in this study, especially at the margins of the In conclusion, the present data show that populations of sampled distribution. The Mantel test of association indic- E. angustifolia exhibit genetic differentiation in concord- ated a strong support for the correlation of genetic structure ance with a N–S spatial orientation. Isolation-by-distance with each climatic parameter (Table 7). Again, the partial restrictions in gene flow, independent of the influence of Mantel test allowed the geographic influence to be removed annual mean precipitation, appear to be the main factor for Still et al. — Intraspecific Variation in Echinacea angustifolia the genetic divergence observed among populations.
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Conformation of therapeutic lens, from bacterial cellulose with ciprofloxacin incorporation for medical application C. M. Caliri1, R. Marchetto1, W. R. Lustri,Y. Messaddeq. 1Instituto de Química – UNESP, Araraquara, SP, Brazil Currently, several methods have been proposed for the controlled release of ophthalmic drugs using soft contact lenses. However, the current systems do not p

Vorträge im Naturkundehaus / 1.Halbjahr 2014 BN = Bund Naturschutz LBV = Landesbund für Vogelschutz TGN = Tiergarten der Stadt Nürnberg Donnerstag, 16. Januar 2014, 19.30 Uhr, Vortragssaal TGN Die Eingewöhnung unserer Tiere in Lagune und Manati-Haus Sarah Bucherer, Andreas Fackel, Lisa Kukuk, Christiane Thiere, Tiergarten Nürnberg In drei Einzelvorträgen geb

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