| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Genetics and Molecular Biology |
2Forest and Evolutionary Genomics Laboratory, and the Center for Structural and Functional Genomics, Biology Department, Concordia University, 7141 Sherbrooke Street West, Montreal, Quebec H4B 1R6, Canada; 3Department of Forest Science, Oregon State University, 321 Richardson Hall, Corvallis, Oregon 97331-5752 USA
Received for publication March 23, 2004. Accepted for publication January 27, 2005.
ABSTRACT
Red pine (Pinus resinosa Ait.) is an ecologically and economically important forest tree species of northeastern North America and is considered one of the most genetically depauperate conifer species in the region. We have isolated and characterized 13 nuclear microsatellite loci by screening a partial genomic library with di-, tri-, and tetranucleotide repeat oligonucleotide probes. In an analysis of over 500 individuals representing 17 red pine populations from Manitoba through Newfoundland, five polymorphic microsatellite loci with an average of nine alleles per locus were identified. The mean expected and observed heterozygosity values were 0.508 and 0.185, respectively. Significant departures from Hardy-Weinberg equilibrium with excess homozygosity indicating high levels of inbreeding were evident in all populations studied. The population differentiation was high with 28–35% of genetic variation partitioned among populations. The genetic distance analysis showed that three northeastern (two Newfoundland and one New Brunswick) populations are genetically distinct from the remaining populations. The coalescence-based analysis suggests that "northeastern" and "main" populations likely became isolated during the most recent Pleistocene glacial period, and severe population bottlenecks may have led to the evolution of a highly selfing mating system in red pine.
Key Words: genetic bottleneck • genetic diversity • microsatellites • Pinus • Pleistocene refugia • population genetics • Postglacial colonization
Red pine, Pinus resinosa Ait., is one of the most extensively planted trees in the northern United States and Canada and is widely used as a source of wood for poles, cabin logs, and fuel. It is distributed throughout northeastern North America, from southeastern Manitoba, eastward through the Great Lakes/St. Lawrence region to Newfoundland, and south to West Virginia (Rudolf, 1990
). Despite its wide geographic distribution, red pine shows high morphological uniformity and is considered to be one of the most genetically depauperate conifer species in North America (Fowler and Lester, 1970
). Its genetic uniformity may have resulted from passage through a genetic bottleneck during the last glacial period (Fowler and Morris, 1977
; Walter and Epperson, 2001
, 2005
). The fossil data, dating from 16 000 to 18 000 years BP (before present), suggest that red pine populations persisted in refugia located in the southern Appalachian Mountains (Jackson et al., 2000
). Red pine may also have survived during the glacial period in refugia off the present coastline of the eastern seaboard in nonglaciated islands and extensions of the mainland (Pielou, 1991
). The highly fragmented population structure and self- compatible mating system (Fowler, 1964
, 1965
) may also have contributed to the loss of genetic variation through inbreeding and genetic drift during the post-glacial expansion from these refugia. The genetic uniformity of red pine contrasts strikingly with the high genetic variability found in most conifer species (Hamrick and Godt, 1990
). However, low genetic diversity has also been reported in several conifer species including torrey pine (Pinus torreyana Parry ex Carr; Ledig and Conkle, 1983
), red spruce (Picea rubens Sarg; Hawley and DeHayes, 1994
), and western red cedar (Thuja plicata Donn. Ex D. Don; Glaubitz et al., 2000
).
Population bottlenecks are known to reduce genetic variability, purge deleterious mutations, and lower inbreeding depression, thereby promoting evolution of self-fertilization (Charlesworth and Charlesworth, 1979
; Lande and Schemske, 1985
). The highly self-compatible mating system in red pine (Fowler, 1965
) may have evolved as a consequence of historical population bottlenecks. The low genetic variability of red pine may compromise its ability to respond to selective pressures such as changes in environmental conditions and may increase its risk of extinction from threats such as pests and disease. It has been predicted that red pine will be extirpated from the USA within the next century because of increases in temperature during the growth season (Prasad and Iverson, 1999
; Cherry, 2001
). Further decline in natural populations of red pine has been caused by harvesting without regeneration and by suppressing the fire that is necessary for seedling recruitment (Mosseler et al., 1992
). As a mitigating measure against potential threats, genetically distinct populations of red pine need to be identified and conserved to ensure long-term survival of the species.
Estimates of effective population sizes (Schoen and Brown, 1991
; Beerli and Felsenstein, 2001
), selfing rates of populations, gene flow, and mean coalescent times (Slatkin, 1995
) are needed to gain insights into the evolutionary history of red pine and to formulate genetically sound conservation plans. These parameters could be estimated using polymorphic genetic markers. However, because of low or no variability, the traditional genetic markers are of limited utility for assessing the genetic architecture of red pine populations. Lack of genetic variability in red pine has been reported in isozyme (Fowler and Morris, 1977
; Allendorf et al., 1982
; Simon et al., 1986
; Mosseler et al., 1991
) and RAPD analyses (Mosseler et al., 1992
). DeVerno and Mosseler (1997)
reported that digestion of RAPD reaction products with restriction enzymes (RAPD-RFLP analysis) allowed detection of low levels of polymorphism in red pine that was not apparent with simple RAPD analysis. Recent studies based on chloroplast simple sequence repeat (cpSSR) analysis have revealed genetic polymorphism in red pine (Echt et al., 1998
; Walter and Epperson, 2001
, 2005
). However, these markers are of limited utility because of the limited intrapopulation polymorphism and uniparental (paternal) inheritance of chloroplast genome in pines (Mitton, 1994
).
Nuclear microsatellite markers have been useful in detecting genetic variation in animals (Firestone et al., 2000
; Feldheim et al., 2001
; Bidlack and Cook, 2002
) and plants (Dayanandan et al., 1998
; (Rajora et al., 2001
; Stacy et al., 2001
; Bérubé et al., 2003
), including pines such as Pinus strobus (Echt et al., 1996
), P. radiata (Fisher et al., 1998
), P. sylvestris (Soranzo et al., 1998
), and P. taeda (Al-Rabab'ah and Williams, 2002
). These markers have been used for examining the genetic effects of different harvesting practices of forest trees (Thomas et al., 1999
), estimating population differentiation (Echt et al., 1996
), and clonal fingerprinting (Dayanandan et al., 1998
). To date, no nuclear microsatellite markers are available for red pine. Here we report the results of the first genetic study based on nuclear microsatellite loci of red pine identified through screening genomic libraries. We assayed the genetic diversity by scoring microsatellite polymorphism among populations distributed throughout its natural range, and we show that red pine populations from Newfoundland are genetically distinct from most mainland populations.
Our objectives were to (1) characterize nuclear microsatellite markers for red pine; (2) assay range-wide genetic diversity to identify patterns of isolation by distance and to detect genetically distinct populations; (3) estimate gene flow, effective population size, and coalescent time parameters to gain insight into the evolutionary history and possible historical population bottlenecks of the species; and (4) estimate mating system parameters of populations to better understand the evolution of selfing in red pine.
MATERIALS AND METHODS
Samples collected
A total of 518 individuals were sampled from 17 different red pine populations distributed throughout its natural range (Table 1, Fig. 1).
|
|
. A partial genomic library of red pine DNA fragments between 500 to 1000 bp was constructed and screened with (TC)15, (AC)15, (AT)15, (AAT)15, (AAAT)10, (TG)15, (AG)15, (AGAT)15, and (AAAG)10 oligonucleotide probes following the protocol of Dayanandan et al. (1998)
. Either single-stranded DNA (isolated using the Wizard M13 DNA purification system; Promega, Madison, Wisconsin, USA) or double-stranded DNA (isolated using the QIAprep Spin Miniprep Kit; Qiagen, Mississauga, Ontario, Canada) of positive clones were sequenced using the ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, California, USA). Oligonucleotide primers complimentary to regions flanking the repeat regions were designed. These primers were synthesized by Operon Technologies (Alameda, California, USA). The polymerase chain reaction (PCR), under optimized conditions, was used for amplifying microsatellites in the sampled individuals. Amplification reactions were performed in a total volume of 25 µl with 0.2 mM dNTP, 2.0 mM MgCl2, 2.5 µl 10x buffer, and 2.5 pmol of each primer. The thermal cycling profile of PCR reactions consisted of an initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 1 min (denaturation), 1 min annealing (at the optimal annealing temperature as given in Table 1), and 72 C for 1 min (extension). This was followed by 72°C for 4 min (final extension). The sizes of amplified products were determined using an ABI 310 genetic analyzer and Genescan software (Applied Biosystems).
Genetic diversity and mating system analyses
For each polymorphic locus, the total number of alleles per locus and per population was determined, and the distribution of allele frequencies was calculated. The average number of alleles per locus (A) for each population over all loci and unbiased values of expected (He) and observed (Ho) frequency of heterozygotes were calculated using the Genetic Data Analysis (GDA) version 1.0 software (Lewis and Zaykin, 2001
). Deviations from Hardy-Weinberg equilibrium and linkage disequilibrium were tested using Fisher exact tests with 3200 reshufflings. The heterozygote deficiency for each locus and population was tested using Genepop 3.4 software (Raymond and Rousset, 1995
) following the method of Rousset and Raymond (1995)
. Population genetic structure was analyzed through hierarchical F coefficients (Weir and Cockerham, 1984
) using the GDA software. The statistical significance of F coefficients at the 95% confidence level was tested through bootstrap analysis with 10 000 replicates. The selfing rate (S) in each population was calculated using the formula S = 2F/1 + F (Crow and Kimura, 1970
).
The standard genetic distances, DS (Nei, 1978
), were calculated using the GDA software, and RST (Slatkin, 1995
) and delta µ2 (Goldstein et al., 1995
) were calculated using RSTCALC (Goodman, 1997
). The FST and DS assume an infinite allele model (IAM), while RST and delta µ2 assume a stepwise mutation model (SMM). The concordance between FST and RST values, as well as between DS and delta µ2 distance values were tested through Mantel tests to determine if our results depended on the underlying mutation model of the analyses. The unweighted pair group method with arithmetic average (UPGMA) dendrograms based on DS and delta µ2 were constructed and visualized with the TreeView software program (Page, 1996
). The relationships between genetic distances and geographic distance were analyzed using Isolation By Distance (IBD) software (Bohonak, 2002
). The IBD software program assesses the significance between a given distance matrix and the geographic distance through Mantel's test and evaluates the strength of the relationship through regression of all pairwise genetic distances with their corresponding geographic distances. We tested the relationships between DS and delta µ2 with geographic distance, Rousset (1997)
distance with the logarithm of geographic distance, and the logarithm of Slatkin (1993)
distance with the logarithm of geographic distance.
Estimation of effective population sizes, migration rates, and coalescence times
Genetic distance analysis revealed two major clusters of populations, the Northeastern group consisting of three populations (Terra Nova and Rowsells Brook in Newfoundland, and Geary in New Brunswick) and the other group consisting of the 14 remaining populations (Figs. 2, 3). Hereafter we refer to these two groups as Northeast and Main populations, respectively. We have estimated the effective population sizes, migration rates, and coalescence times for Northeast, Main, and total (Northeast + Main) groups separately. Coalescent-theory -based maximum likelihood estimation of effective population sizes was carried out using MIGRATE software (Beerli and Felsenstein, 2001
). The value of 
was estimated using the MIGRATE software, and assuming an average mutation rate of microsatellites as 10–3 per generation, we calculated Ne as /4 x 10–3. The RST-based migration rates (MR) and coalescent times (
) were estimated following Slatkin (1995)
. We calculated the average coalescent time TR following equation 19 of Slatkin (1995)
. The coalescent time in generations was calculated as = TRx N, where N is the size of each group. We used the these estimates of effective population sizes as N, and assuming an average generation length of red pine as 50 yr, we estimated the absolute coalescent time as t = x 50 yr.
|
|
Recovery of microsatellite loci and detection of polymorphism
A partial genomic library consisting of 
13 000 clones was screened using di-, tri-, and tetra-nucleotide repeat oligonucleotide probes. The sequencing of 35 positive clones identified through screening with TC/AG probes revealed 19 microsatellites (17 TC/AG, one AT, and one AT mixed repeat), and primers were designed for nine clones. Seven primers were designed based on 23 microsatellites (12 AC/TG, three AT, three TC/AG, three AC/TG and AT mixed repeats, one TCC/AGG repeat, and one 6-bp repeat of TCCACA) identified through the sequencing of 68 clones positive for AC/TG probes. The AT/AAT/AAAT probes identified 13 positive clones, but sequencing revealed microsatellites (AT/AAT/ AAAT repeats) in nine clones, and primers were designed based on nine clones. A total of 25 oligonucleotide primer pairs complementary to flanking regions of microsatellites were designed. Optimization of PCR conditions resulted in successful amplification with 20 primer pairs. Thirteen of these 20 successfully amplified primer pairs produced amplification products consistent with a single-locus segregation pattern, and four primer pairs showed polymorphism. One of these polymorphic primer pairs had an amplification pattern consistent with two loci, and alleles of each locus were easily distinguishable for scoring. Of the remaining 12 primer pairs, seven primer pairs produced multiple amplification products, and amplification by the five primer pairs was inconsistent.
Levels of genetic variation and population genetic structure
In a total of 518 red pine individuals surveyed, 45 alleles were identified at five polymorphic microsatellite loci, with an average of nine alleles per locus. The highest diversity was found at the (AT)22 repeat (locus PRE16) with 15 alleles. The loci PRE10, PRE13, PRE24, and PRE24B had 13, 12, 3, and 2 alleles, respectively (Table 2).
|
Significant departure from Hardy-Weinberg equilibrium was observed at most loci in most populations, and significant heterozygote deficiency was evident in all populations at all loci except for PRE24B. No linkage disequilibrium was observed, indicating all five loci segregate independently of each other. The values of FIS, FIT, and FST were –0.133–0.840, 0.221– 1.000, and 0.211–0.455, respectively. The bootstrap analyses showed that overall F values were significantly (P = 0.05) greater than 0 (Table 3). The overall FST value was 0.280, while the overall RST value was 0.350. The F values ranged from 0.384 to 1.000 across all populations and were significantly greater than 0 for all populations except for the two Newfoundland populations. The selfing rates (S) ranged from 0.555 in the Beaver Lake (Nova Scotia) population to 1 in the in the Hardy County, West Virginia (Table 1).
|
|
Weak but significant positive correlations were found between geographic distance and all genetic distance measures tested (DS: r = 0.3812, P = 0.0072; delta µ2: r = 0.3323, P = 0.0144; Rousset's distance: r = 0.1320, P = 0.0291). Similarly, a negative correlation was found between geographic distance and the Slatkin's (1993)
measure of similarity (r = –0.2500, P = 0.0214).
The effective population sizes, migration rates and coalescent times of Northeast Main, and total populations are given in Table 5. The effective population sizes of Northeast, Main, and total populations were 62, 222, and 615, respectively. The migration rate within Northeast and Main populations was 0.9, and the migration between Northeast and Main populations was 0.42. The average coalescent time in the Northeast population was 69 generations (3 400 yr), and the corresponding value for the Main population was 246 generations (12 300 yr). The coalescent time of the total population was 1463 generations (73 154 yr).
|
The low recovery rate of microsatellites with a single-locus inheritance pattern in this study is typical of organisms with a large genome size such as conifers (Fischer and Bachmann, 1998
) and is in agreement with several studies (Smith and Devey, 1994
; Kostia et al., 1995
; Pfeiffer et al., 1997
; Fisher et al., 1998
; Bérubé et al., 2003
). The reason for this could be multiple sites for primer binding as a result of genome duplications, a common phenomenon in large genomes, or an increased probability of nonspecific binding sites for the designed primers (Garner, 2002
).
The greater number of TC/AG repeats relative to AC/TG repeats in red pine is consistent with findings for several tree species, including Populus tremuloides (Dayanandan et al., 1998
), Pinus radiata (Fisher et al., 1998
), and Picea glauca (Rajora et al., 2001
). An exception is white pine (Pinus strobus), in which AC/TG repeats were found to be more abundant than TC/AG (Echt et al., 1996
). Although microsatellite primers based on longer repeats are expected to demonstrate higher levels of polymorphism (Weber, 1990
), the greatest polymorphism in red pine was detected in locus PRE16 with 15 alleles, which was designed based on a clone with (AT)22 repeat, whereas the locus PRE24, which was designed based on a clone with longest repeat, (AG)29, had the least polymorphism, with only three alleles at the locus PRE24 and two alleles at locus PRE24B. This pattern may not be unusual for forest trees, however, because other studies have indicated that loci based on longer repeats do not necessarily have higher levels of polymorphism (Echt et al., 1996
; Dayanandan et al., 1998
). The microsatellite locus based on the shortest cloned repeat demonstrating polymorphism in red pine was (AT)21, whereas all primer pairs based on clones with repeats shorter than 21 were monomorphic.
The observed heterozygosity (Ho) values of 0.067–0.317 with a mean of 0.185 reported here for red pine are lower than those reported for other pine species using microsatellite markers. For example, Ho values for P. radiata ranged from 0 to 0.85 with a mean of 0.625 (Smith and Devey, 1994
), and those for P. strobus ranged from 0.125 to 0.812 with a mean of 0.515 (Echt et al., 1996
). The Ho values for red pine are much lower than expected heterozygosity (He) values (Tables 1, 2), and all populations as well as four of the five loci showed significant heterozygote deficiency. Despite the lower levels of Ho and relatively few polymorphic loci used, the average of nine alleles per locus across the five polymorphic loci characterized for red pine was comparable to or greater than microsatellite-based genetic studies of other pines, including six alleles per locus observed in P. radiata (Smith and Devey, 1994
), 5.4 alleles per locus in P. strobus (Echt et al., 1996
), and 6.7 alleles per locus in P. sylvestris (Soranzo et al., 1998
). Similar results of relatively high genetic diversity but low heterozygosity have been reported in Elymus alaskanus, and this pattern has been attributed to a high level of inbreeding (Sun and Salomon, 2003
).
Genetic diversity across red pine populations was highly variable, with He values ranging from 0.489 in Beauceville (Quebec) to 0.014 in Hardy County (West Virginia). The highest allelic diversity was found in the Delta County (Michigan) population, and the lowest in the Hardy County (West Virginia) population. The presence of unique or private alleles can be considered a measure of genetic distinctiveness. Private alleles were found in several populations in both the western and eastern extremes of the red pine range. The lower number of alleles detected in the Terra Nova (Newfoundland) population, however, may be due to smaller sample size. From a conservation perspective, the distribution pattern of alleles, the presence of unique alleles in several of the populations examined, and the distribution of genetic variation in red pine could be used as a genetic criterion to protect as many distinct populations as possible throughout its range.
The FIS and FIT values measure the deviation of genotypic frequencies from Hardy-Weinberg equilibrium within subpopulations and in the total population, respectively. The overall FIS and FIT values were significantly higher than zero and suggest a departure from Hardy-Weinberg equilibrium with deficiency of heterozygotes. The increased levels of homozygosity and the departure from Hardy-Weinberg equilibrium could be attributable to a mating system with a high level of inbreeding through self-fertilization (Fowler, 1965
) or through mating between closely related individuals. Several factors, including the presence of null alleles due to sequence variation at the primer sites that prevent amplification during PCR, and selection against heterozygotes, can also result in heterozygote deficiency. However, the pattern detected here was consistent across all loci, and therefore it is unlikely that the high levels of observed homozygosity result from null alleles or selection against heterozygotes.
The level of evolutionary divergence of populations is influenced by the opposing effects of migration, which tends to homogenize populations, and genetic drift or mutation, which leads to population differentiation. The overall FST value of 0.280 and overall RST value of 0.350 indicate that 28–35% of the genetic variation present in red pine is partitioned among subpopulations. This indicates that red pine populations are highly differentiated, an unusual feature of many conifers mainly because of long-distance pollen flow facilitated by wind pollination. For example, allozyme studies demonstrated low differentiation (FST 
0.02) among northern populations of European Scots pine (Gullberg et al., 1985
), and the proportions of genetic diversity detected among populations of several widespread conifer species are usually less than 10% (Ledig, 1998
). The high genetic differentiation among populations in red pine could be attributable to a high rate of selfing in red pine, because predominantly selfing plant species tend to show higher population differentiation than outcrossing species (Loveless and Hamrick, 1984
; Hamrick and Godt, 1990
).
The high selfing rate observed in red pine (Table 1) is unusual among members of Pinaceae, in which most species possess a primarily outcrossing mating system. The evolution of self-fertilization is promoted by population bottlenecks (Lande and Schemske, 1985
), and selfing is known to have evolved multiple times from outcrossing lineages (Stebbins, 1957
). Furthermore, Lande and Schemske (1985)
demonstrated that selfing is promoted if inbreeding depression is less than 50%. In general, the inbreeding depression values of the majority of conifers are high (Williams and Savolainen, 1996
), and therefore a selfing mating system is not favored. However, inbreeding depression in red pine is considerably low, and the lethal equivalents per diploid zygote of red pine is the lowest among pines (Fowler, 1964
), suggesting that population bottlenecks may have purged deleterious mutations, thereby reducing inbreeding depression and promoting the evolution of self fertilization in red pine.
The mutation model of microsatellites for calculating genetic distances and population differentiation remains controversial. The infinite allele model (IAM) assumes that each mutation creates a novel allele, whereas the stepwise mutation model (SMM) assumes a closer genetic relatedness between alleles of similar size. The FST (Wright, 1965
) and standard genetic distance Ds (Nei, 1978
) are based on the IAM, whereas the RST (Slatkin, 1995
) and delta µ2 are based on the SMM. Mantel test results showed no significant differences between RST and FST estimates, nor between DS and delta µ2 values, indicating that the genetic patterns detected in our study are not dependent on the underlying mutation model and confirming the robustness of our findings.
The genetic distance analyses showed that the Northeastern population group, comprising the two Newfoundland populations and Geary in New Brunswick, is distinct from the Main (western) population. The high gene flow rates within the Northeastern and Main populations and lower gene flow rate between the Northeastern and Main populations further support the genetic isolation between these two populations. The smaller effective population size of Northeastern populations compared to Main populations suggests that Northeastern populations may have experienced a more severe bottleneck than the western populations. The coalescent time estimate of 70 000 years suggests that the isolation of Northeastern from Main populations may have occurred during the most recent Pleistocene glacial period. Although the assumptions underlying these estimates (mutation rate of microsatellite loci as 10–3 and generation time of red pine as 50 yr) are debatable, these values agree with data from the current literature. In plant microsatellites, mutation rates of 1 x 10–2 and 3.9 x 10–3 in chickpeas (Udupa and Baum, 2001
), 2.4 x 10–4 in durum wheat (Thuillet et al., 2002
), and 7.7 x 10–4 in maize (Vigouroux et al., 2002
) have been reported. Seed production in red pine is known to reach maximum levels at approximately 50 yr of age (Rudolf, 1990
).
Among the red pine populations examined in this study, the area of highest genetic diversity based on expected heterozygosity values is centered in the New Brunswick–Nova Scotia region and extends to Quebec. This is consistent with the evidence from cpSSR data (Echt et al., 1998
; Walter and Epperson, 2001
, 2005
). This pattern of genetic structure could have arisen from the postglacial colonization of this area by individuals from multiple refugia, including southern Appalachian and northeastern refugia, forming an admixed population through secondary contact (Walter and Epperson, 2001
, 2005
). The presence of highest allelic diversity and greatest number of private alleles in the Michigan population suggests the possible existence of a glacial refugium in the Michigan area. Although no direct evidence exists for red pine in this area during the last glacial period, macrofossils of other conifers (e.g., Picea spp.) have been found in the area south of Michigan (Jackson et al., 2000
). Thus, based on genetic evidence, red pine may have colonized its current range from as many as three glacial refugia.
Higher genetic variation is often found in southern populations relative to northern populations, as is evident from several taxa that survived in much reduced refugial populations in Europe during the Pleistocene glacial periods (Hewitt, 2001
). A similar trend has also been demonstrated for plants in western (Soltis et al., 1997
) and eastern (Lewis and Crawford, 1995
) North America, suggesting low diversity in newly colonized northern areas. In our study, however, the lowest genetic diversity was found in the three most southern populations, a pattern inconsistent with post-glacial colonization from a single southern glacial refugium. This further supports the existence of multiple refugia. However, reduced genetic diversity in the smaller and more isolated population in West Virginia could result from lack of gene flow and genetic drift in marginal populations, because the effect of genetic drift may be two to 30 times greater near the edge of a species range because of smaller effective population sizes (Vucetich and Waite, 2003
).
In summary, these polymorphic microsatellite markers are highly valuable to assess genetic diversity and identify genetically unique populations of red pine, one of the economically and ecologically important, but genetically depauperate tree species in North America. The geographical distribution and genetic structuring of red pine populations strongly suggests dispersal from multiple refugia with an intermediate admixture zone. The high levels of population differentiation maintained in this species may result from high levels of self-pollination and inbreeding. The information obtained in this study will be valuable not only for the theoretical advancement of our knowledge on the plant genetic diversity in relation to geological history and refugial habitats during the Pleistocene era, but also valuable for designing genetically sound conservation and management programs for red pine.
FOOTNOTES
1 The authors are grateful to L. Hermanutz (Memorial University), D. McLaughlin and C. Calegoropolous (Concordia University), N. Fleury and E. Morin (Montreal Botanical Garden), R. Jordan (The Guelph University Arboretum), J. Bowen and T. George (West Virginia Division of Forestry), D. Simpson (Canadian Forest Service), R. Wolanin (Massachusetts Audubon Society), and R. Kesseli (University of Massachusetts) for providing plant samples. We thank J. Gray (Concordia University) for preparing the map, and D. McLaughlin and E. Stacy (Concordia University) and B. Epperson (Michigan State University) for comments on the manuscript. We greatly appreciate the support and generosity of S. D'eon (Petawawa Research Forest, Canadian Forest Service) for plant materials. This research was supported by grants from Concordia University, the Natural Sciences and Engineering Research Council of Canada, Le Fonds Québécois de la Recherche sur la Nature et les Technologies, and Canada Foundation for Innovation to S.D. 
4 E-mail: daya{at}alcor.concordia.ca
LITERATURE CITED
Allendorf F. W. K. L. Knudsen G. M. Blake 1982 Frequencies of null alleles at enzyme loci in natural populations of ponderosa and red pine. Genetics 100: 497-504
Al-Rabab'ah M. A. C. G. Williams 2002 Population dynamics of Pinus taeda L. based on nuclear microsatellites. Forest Ecology and Management 163: 263-271[CrossRef][Web of Science]
Beerli P. J. Felsenstein 2001 Maximum likelihood estimation of a migration matrix and effective population sizes in n subpopulations by using a coalescent approach. Proceedings of National Academy of Sciences, USA 98: 4563-4568
Bérubé Y. C. Ritland K. Ritland 2003 Isolation, characterization, and cross-species utility of microsatellites in yellow cedar (Chamaecyparis nootkatensis). Genome 46: 353-361[Medline]
Bidlack A. L. J. Cook 2002 A nuclear perspective on endemism in northern flying squirrels (Glaucomys sabrinus) of the Alexander Archipelago, Alaska. Conservation Genetics 3: 247-259[CrossRef][Web of Science]
Bohonak A. J. 2002 IBD (Isolation By Distance): a program for analyses of isolation by distance. Journal of Heredity 93: 153-154
Charlesworth B. D. Charlesworth 1979 The evolutionary genetics of sexual systems in flowering plants. Proceedings of the Royal Society, London, B 205: 513-530[Medline]
Cherry M. 2001 Options for allocating afforestation stock in Ontario with anticipated climate change. Ontario Ministry of Natural Resources, Ontario Forest Research Institute, Forest Research Paper no. 148
Crow J. F. M. Kimura 1970 An introduction to population genetic theory. Harper and Row, New York, New York, USA
Dayanandan S. K. S. Bawa R. Kesseli 1997 Conservation of microsatellites among tropical trees (Leguminosae). American Journal of Botany 84: 1658-1663[Abstract]
Dayanandan S. O. P. Rajora K. S. Bawa 1998 Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides). Theoretical and Applied Genetics 96: 950-956[CrossRef][Web of Science]
DeVerno L. L. A. Mosseler 1997 Genetic variation in red pine (Pinus resinosa) revealed by RAPD and RAPD-RFLP analysis. Canadian Journal of Forest Research 27: 1316-1320
Echt C. S. P. May-Marquardt M. Hseih R. Zahorchak 1996 Characterization of microsatellite markers in eastern white pine. Genome 39: 1102-1108[Medline]
Echt C. S. L. L. Deverno M. Anzidei G. G. Vendramin 1998 Chloroplast microsatellites reveal population genetic diversity in red pine, Pinus resinosa Ait. Molecular Ecology 7: 307-316
Feldheim K. A. S. H. Gruber M. V. Ashley 2001 Population genetic structure of the lemon shark (Negaprion brevirostris) in the western Atlantic: DNA microsatellite variation. Molecular Ecology 10: 295-303[CrossRef][Medline]
Firestone K. B. B. A. Houlden W. B. Sherwin E. Geffen 2000 Variability and differentiation of microsatellites in the genus Dasyrus and conservation implications for the large Australian carnivorous marsupials. Conservation Genetics 1: 115-133
Fischer D. K. Bachmann 1998 Microsatellite enrichment in organisms with large genomes (Allium cepa L). Biotechniques 24: 796-798[Web of Science][Medline]
Fisher P. J. T. E. Richardson R. C. Gardner 1998 Characteristics of single- and multi-copy microsatellites from Pinus radiata. Theoretical and Applied Genetics 96: 969-979[CrossRef][Web of Science]
Fowler D. P. 1964 Effects of inbreeding in red pine, Pinus resinosa Ait. I. Factors affecting natural selfing. Silvae Genetica 13: 170-177
Fowler D. P. 1965 Effects of inbreeding in red pine, Pinus resinosa Ait. II. Pollination studies. Silvae Genetica 14: 12-23
Fowler D. P. D. T. Lester 1970 Genetics of red pine. Research paper WO-8, USDA Forest Service, Washington, D.C
Fowler D. P. R. W. Morris 1977 Genetic diversity in red pine: evidence for low genetic heterozygosity. Canadian Journal of Forest Research 7: 343-347
Garner T. W. J. 2002 Genome size and microsatellites: the effect of nuclear size on amplification potential. Genome 45: 212-215[Medline]
Glaubitz J. C. Y. A. El-Kassaby J. E. Carlson 2000 Nuclear restriction fragment length polymorphism analysis of genetic diversity in western red cedar. Canadian Journal of Forest Research 30: 379-389
Goldstein D. B. A. R. Linares L. L. Cavalli-Sforza M. W. Feldman 1995 An evaluation of genetic distances for use with microsatellite loci. Genetics 139: 463-471[Abstract]
Goodman S. J. 1997 RST Calc: a collection of computer programs for calculating estimates of genetic differentiation from microsatellite data and determining their significance. Molecular Ecology 6: 881-885[CrossRef]
Gullberg U. R. Yazani D. Rudin N. Ryman 1985 Allozyme variation in Scots pine (Pinus sylvestris L.) in Sweden. Silvae Genetica 34: 193-200
Hamrick J. L. M. J. W. Godt 1990 Allozyme diversity in plant species. In A. H. D. Brown, M. T. Clegg, A. L. Kahler, and B. S. Weir [eds.], Plant population genetics, breeding and genetic resources, 43–63. Sinauer, Sunderland, Massachusetts, USA
Hawley G. J. D. H. de Hayes 1994 Genetic diversity and population structure of red spruce (Picea rubens). Canadian Journal Botany 72: 1778-1786[CrossRef]
Hewitt G. M. 2001 Speciation, hybrid zones and phylogeography—or seeing genes in space and time. Molecular Ecology 10: 537-549[CrossRef][Medline]
Jackson S. T. R. S. Webb K. H. Anderson J. T. Overpeck T. Webb III J. W. Williams B. C. S. Hansen 2000 Vegetation and environment in Eastern North America during the last glacial maximum. Quaternary Science Reviews 19: 489-508
Kostia S. S.-L. Varvio P. Vakkari P. Pulkkinen 1995 Microsatellite sequences in Pinus sylvestris. Genome 38: 1244-1248[Medline]
Lande R. D. W. Schemske 1985 The evolution of self-fertilization and inbreeding depression in plants. I. Genetic models. Evolution 39: 24-40[CrossRef][Web of Science]
Ledig F. T. 1998 Genetic variation in Pinus. In D. M. Richardson [ed.], Ecology and biogeography of Pinus, 251–273. Cambridge University Press, Cambridge, UK
Ledig F. T. M. T. Conkle 1983 Gene diversity and genetic structure in a narrow endemic, Torrey pine (Pinus torreyana Parry ex Carr). Evolution 37: 79-85[CrossRef][Web of Science]
Lewis P. O. D. J. Crawford 1995 Pleistocene refugium endemics exhibit greater allozymic diversity than widespread congeners in the genus Polygonella (Polygonaceae). American Journal of Botany 82: 141-149
Lewis P. O. D. Zaykin 2001 Genetic Data Analysis: Computer program for the analysis of allelic data, version 1.0 (d16c). Distributed by the authors at website http://lewis.eeb.uconn.edu/lewishome/software.html
Loveless M. D. J. L. Hamrick 1984 Ecological determinants of genetic structure of plant populations. Annual Review of Ecology and Systematics 15: 65-95
Mitton J. B. 1994 Molecular approaches to population biology. Annual Review of Ecology and Systematics 25: 45-69
Mosseler A. D. J. Innes B. A. Roberts 1991 Lack of allozyme variation found in disjunct Newfoundland populations of red pine (Pinus resinosa). Canadian Journal of Forest Research 21: 525-528[CrossRef]
Mosseler A. K. N. Egger G. A. Hughes 1992 Low levels of genetic diversity in red pine confirmed by random amplified polymorphic DNA markers. Canadian Journal of Forest Research 22: 1332-1337
Nei M. 1978 Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 147: 1943-1957
Page R. D. M. 1996 TREEVIEW: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12: 357-358
Pfeiffer A. A. M. Olivier M. Morgante 1997 Identification and characterization of microsatellites in Norway spruce (Picea abies K). Genome 40: 411-419[Medline]
Pielou E. C. 1991 After the Ice Age: the return of life to glaciated North America. University of Chicago Press, Chicago, Illinois, USA
Prasad A. M. L. R. Iverson 1999 A climate change atlas for 80 forest tree species of the eastern United States. Available at website http://www.fs.fed.us/ne/delaware/atlas/index.html, Northeastern Research Station, USDA Forest Service, Delaware, Ohio, USA
Rajora O. P. M. H. Rahman S. Dayanandan Mosseler 2001 Isolation, characterization, inheritance and linkage of microsatellite DNA markers in white spruce (Picea glauca) and their usefulness in other spruce species. Molecular General Genetics 264: 871-882
Raymond M. F. Rousset 1995 GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. Journal of Heredity 86: 248-249
Rousset F. 1997 Genetic differentiation and estimation of gene flow from F-statistics under isolation by distance. Genetics 145: 1219-1228[Abstract]
Rousset F. M. Raymond 1995 Testing heterozygote excess and deficiency. Genetics 140: 1413-1419[Abstract]
Rudolf R. O. 1990 Pinus resinosa Ait., red pine. In R. M. Burns and B. H. Honkala [eds.], Silvics of North America, vol. 1, Conifers, 442–455. USDA Forest Service Agriculture Handbook 654, Washington, D.C., USA
Schoen D. J. A. H. D. Brown 1991 Intraspecific variation in population gene diversity and effective population size correlates with the mating system in plants. Proceedings of National Academy of Sciences, USA 88: 4494-4497
Simon J.-P. Y. Bergeron D. Gagnon 1986 Isozyme uniformity in populations of red pine (Pinus resinosa) in the Abitibi region, Quebec. Canadian Journal of Forest Research 16: 1133-1135[CrossRef]
Slatkin M. 1993 Isolation by distance in equilibrium and non-equilibrium populations. Evolution 47: 264-279[CrossRef][Web of Science]
Slatkin M. 1995 A measure of population subdivision based on microsatellite allele frequencies. Genetics 139: 457-462[Web of Science][Medline]
Smith D. N. M. E. Devey 1994 Occurrence and inheritance of microsatellites in Pinus radiata. Genome 37: 977-983[Medline]
Soltis D. E. M. A. Gitzendanner D. D. Strenge P. S. Soltis 1997 Chloroplast DNA intraspecific phylogeography of plants from the Pacific Northwest of North America. Plant Systematics and Evolution 206: 353-373[CrossRef][Web of Science]
Soranzo N. J. Provan W. Powell 1998 Characterization of microsatellite loci in Pinus sylvestris L. Molecular Ecology 7: 1247-1263[CrossRef][Medline]
Stacy E. A. S. Dayanandan B. P. Dancik P. D. Khasa 2001 Microsatellite DNA markers for the Sri Lankan rainforest tree species, Shorea cordifolia (Dipterocapaceae), and cross-species amplification in S. megistophylla. Molecular Ecology Notes 1: 53-54
Stebbins G. L. 1957 Self-fertilization and population variability in higher plants. American Naturalist 91: 337-354[CrossRef][Web of Science]
Sun G. L. B. Salomon 2003 Microsatellite variability and heterozygote deficiency in the arctic-alpine Alaskan wheatgrass (Elymus alaskanus) complex. Genome 46: 729-737[Medline]
Thomas B. R. S. E. Macdonald M. Hicks D. L. Adams R. B. Hodgetts 1999 Effects of reforestation methods on genetic diversity of lodgepole pine: an assessment using microsatellite and randomly amplified polymorphic DNA markers. Theoretical and Applied Genetics 98: 793-801[CrossRef][Web of Science]
Thuillet A. C. D. Bru J. L. David P. Roumet S. Santoni P. Sourdille T. Bataillon 2002 Direct estimation of mutation rate for ten microsatellite loci in durum wheat, Triticum turgidum (L.) Thell. ssp. durum desf. Molecular Biology and Evolution 19: 122-125
Udupa S. M. M. Baum 2001 High mutation rate and mutational bias at (TAA) (n) microsatellite loci in chickpea (Cicer arietinum L). Molecular Genetics and Genomics 265: 1097-1103[CrossRef][Web of Science][Medline]
Vigouroux Y. J. S. Jaqueth Y. Matsuoka O. S. Smith W. D. Beavis J. S. Smith J. Doebley 2002 Rate and pattern of mutation at microsatellite loci in maize. Molecular Biology and Evolution 19: 1251-60
Vucetich J. A. T. A. Waite 2003 Spatial patterns of demography and genetic processes across the species' range: null hypotheses for landscape conservation genetics. Conservation Genetics 4: 639-645[CrossRef][Web of Science]
Walter R. B. K. Epperson 2001 Geographic pattern of genetic variation in Pinus resinosa: area of greatest diversity is not the origin of postglacial populations. Molecular Ecology 10: 103-111[CrossRef][Medline]
Walter R. B. K. Epperson 2005 Geographic pattern of genetic diversity in Pinus resinosa: contact zone between descendants of glacial refugia. American Journal of Botany 92: 92-100
Weber J. L. 1990 Informativeness of human (dC-dA)n:(dG-dT)n polymorphisms. Genomics 7: 524-530[CrossRef][Web of Science][Medline]
Weir B. S. C. C. Cockerham 1984 Estimating F-statistics for the analysis of population structure. Evolution 38: 1358-1370[CrossRef][Web of Science]
Williams C. G. O. Salvolainen 1996 Inbreeding depression in conifers: implications for breeding strategy. Forest Science 42: 102-117[Web of Science]
Wright S. 1965 The interpretation of population structure by F-statistics with special regard to systems of mating. Evolution 19: 395-420[CrossRef][Web of Science]
This article has been cited by other articles:
|
|
 |
|
  M. A. Gonzalez-Perez, P. A. Sosa, E. Rivero, E. A. Gonzalez-Gonzalez, and A. Naranjo Molecular markers reveal no genetic differentiation between Myrica rivas-martinezii and M. faya (Myricaceae) Ann. Bot., January 1, 2009; 103(1): 79 - 86. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. J. Derieg, A. Sangaumphai, and L. P. Bruederle Genetic diversity and endemism in North American Carex section Ceratocystis (Cyperaceae) Am. J. Botany, October 1, 2008; 95(10): 1287 - 1296. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. J. Colbeck, H. L. Gibbs, P. P. Marra, K. Hobson, and M. S. Webster Phylogeography of a Widespread North American Migratory Songbird (Setophaga ruticilla) J. Hered., September 1, 2008; 99(5): 453 - 463. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Mimura and S. N. Aitken Increased selfing and decreased effective pollen donor number in peripheral relative to central populations in Picea sitchensis (Pinaceae) Am. J. Botany, June 1, 2007; 94(6): 991 - 998. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
