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2 Department of Forestry, Forest Biotechnology Group, Box 7247, North Carolina State University, Raleigh, North Carolina 27695 USA; 3 Department of Botany, Box 7612, North Carolina State University, Raleigh, North Carolina 27695 USA
Received for publication March 30, 1999. Accepted for publication September 10, 1999.
ABSTRACT
Development of conservation strategies for Fraser fir (Abies fraseri) in the southern Appalachian Mountains depends in part on recognition of the extent to which Fraser fir is genetically distinct from the closely related balsam (A. balsamea) and intermediate (A. balsamea var. phanerolepis) fir. These sibling species have exhibited intergrading, clinal variation in morphological, chemical, and genetic characteristics in prior research. Chloroplast microsatellite markers were polymerase chain reaction amplified from genomic DNA samples of 78 individuals representing the geographic ranges of Fraser, balsam, and intermediate fir. Gene diversity levels at two loci ranged among taxa from 0.65 to 0.84. Allele frequencies demonstrated significant differentiation among taxa, with RST values of 0.36 and 0.10. Haplotype diversity and DSH were highest for balsam fir and lowest for intermediate fir. A haplotype network analysis based on allele size distribution for the two loci revealed two distinct clusters of haplotypes and population-specific haplotypes. Ninety-two percent of the haplotypes in one cluster were from balsam fir and intermediate fir, and 84% of the haplotypes in the other cluster were from Fraser fir and intermediate fir. The genetic differentiation of chloroplast DNA markers provides justification for the recognition of Fraser fir as a distinct Management Unit (MU) for conservation purposes, regardless of its taxonomic classification.
Key Words: Abies • chloroplast DNA • chloroplast haplotypes • chloroplast microsatellites • chloroplast SSRs • conservation • Fraser fir • Pinaceae
Eastern North American Abies (Mill.), Fraser fir [Abies fraseri (Pursh) Poir.], balsam fir [Abies balsamea (L.) Mill.], and a variety of balsam, intermediate fir (Abies balsamea var. phanerolepis Fern.), exhibit minimal reproductive isolation (Hawley and DeHayes, 1985
) and are considered closely related sibling species in the Section Balsameae (Flora of North America Editorial Committee, 1993
). Classification of these three taxa is based largely on cone bract length to subtending scale ratio, number of leaf hypodermal cells, and geographic location (Fig. 1) (Myers and Bormann, 1963
; Robinson and Thor, 1969
; Liu, 1971
; Thor and Barnett, 1974
). In recent times, populations in the southern limits of growth are found only at higher elevations along the Appalachian Mountains, due to a lack of suitable habitat. The resulting gap of several hundred kilometres in the distribution of fir contributes to the recognition of two species, balsam and Fraser fir. Based on the low level of character divergence, some authorities consider eastern North American fir a single species with three varieties (Thor and Barnett, 1974
; Jacobs, Werth, and Guttman, 1984
). Taxonomic boundaries are obscured by continuous variation in foliage, seed, and cone morphology (Robinson and Thor, 1969
), and clinal variation in terpene concentrations (Thor and Barnett, 1974
). Prior evaluation of allozyme variation among the three taxa showed nearly identical complements of alleles at 13 polymorphic loci (Jacobs, Werth, and Guttman, 1984
).
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). Declines of 44–91% of mature, reproductive Fraser fir in spatially isolated, high-elevation ecosystems are in part attributed to the presence of an introduced insect pest, the balsam woolly adelgid (Dull et al., 1988
), although environmental factors are also implicated. In situ and ex situ gene conservation efforts will be facilitated by techniques that estimate diversity and genetic discontinuity for closely related groups.
Molecular genetic markers may better reveal genetic discontinuities and distinctiveness among or between taxa with slight morphological or reproductive differentiation. At the level of nuclear genes, interbreeding and genetic exchange obscure the evidence of past demographic patterns. Characteristics of allozyme diversity have been examined for many representatives of the Pinaceae, which are typically outcrossing, wind-pollinated species. Conifers often display minimal levels of differentiation among populations and high levels of genetic diversity at loci in the nuclear genome (Hamrick and Godt, 1990, 1996
). Within the Pinaceae opposite uniparental inheritance of mitochondrial and chloroplast genomes provides opportunities to assess discontinuity within an effectively haploid genome. The alleles of loci in an organelle genome can be collectively considered as a haplotype since they are nonrecombining. In these organelles, effective population size is approximately one-half that of nuclear genes (McCauley, 1995
) and, consequently, both the time to allele fixation within populations and response time to stochastic change are reduced. In plants, mitochondrial genomes have not typically been useful for phylogenetic analysis due to a high rate of sequence reorganization (Sederoff et al., 1981
; Wu, Krutovskii, and Strauss, 1998
). Mitochondrial haplotype diversity related to sequence rearrangement has, however, shown utility in population differentiation of pine and fir taxa (Strauss, Hong, and Hipkins, 1993
; Tsumura and Suyama, 1998
). In chloroplasts, the conservation and homology of sequence make it possible to compare genes across the plant kingdom (Clegg and Zurawski, 1992
) and examine phylogenetic relationships in taxa that have diverged for hundreds of thousands to millions of years. Conversely, until hypervariable regions were located, the chloroplast genome had shown minimal utility in examining closely related taxa or intraspecific variation (McCauley, 1995
).
The recent development of microsatellite, or SSR (Simple Sequence Repeat), markers in chloroplast DNA (cpDNA) with high levels of variation (Powell et al., 1995a, b
; Vendramin et al., 1996
; Vendramin and Ziegenhagen, 1997
) has provided greater resolution in differentiation among closely related taxa. Paternal inheritance of chloroplast genomes in most conifers (Neale, Wheeler, and Allard, 1986
; Neale and Sederoff, 1989
) makes chloroplast microsatellites particularly effective markers for studying mating systems, gene flow via both pollen and seeds, and uniparental lineage. Predominant paternal inheritance of chloroplast DNA has been demonstrated in European Abies with previously characterized (Vendramin et al., 1996
) Pinus thunbergii primers at two highly variable microsatellite loci (Vendramin and Ziegenhagen, 1997
; Ziegenhagen et al., 1998
; Vendramin et al., 1999
). Intraspecific diversity and population structure in heterologous amplification with these primers have been demonstrated in many pine species as well (Morgante and Olivieri, 1993
; Powell et al., 1995a
; Cato and Richardson, 1996
; Vendramin et al., 1996
; Morgante, Felice, and Vendramin, 1998
). Chloroplast microsatellite loci have also revealed population structure in species considered genetically depauperate (e.g., P. leucodermis, Powell et al., 1995a
; P. resinosa, Echt et al., 1998
). In this study, we assessed genetic discontinuity and variability of chloroplast microsatellite loci in eastern North American Abies using fluorescent DNA fragment analysis. The geographic distribution of chloroplast haplotypes was evaluated, as well as criteria for recognizing distinct populations in eastern North American fir.
METHODS AND MATERIALS
DNA extraction and PCR amplification
Genomic DNA was extracted from fir needle tissue following the method of Doyle and Doyle (1990)
. Three range-wide samples (26 from each taxon) of Fraser, balsam, and intermediate fir were analyzed (Table 1). Intermediate fir were classified primarily by their geographic locations described in earlier studies (Robinson and Thor, 1969
; Thor and Barnett, 1974
; Jacobs, Werth, and Guttman, 1984
). Oligonucleotide primers (Vendramin et al., 1996
) were used to PCR (polymerase chain reaction) amplify genomic DNA in 10-µL reactions (Jang, Genetic Analysis Bulletin number 30, LI-COR Inc., Lincoln, Nebraska, USA) containing: 20 ng genomic DNA, 10x PCR buffer (Boehringer Mannheim, Indianapolis, Indiana, USA) [50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3)], IRD-41 labeled forward strand primer (0.8 pmol) (LI-COR Inc., Lincoln, Nebraska), reverse strand primer (1.6 pmol), 0.1 mmol/L each dATP, dTTP, dCTP, and dGTP, MgCl2 (2 mmol/L), 1 unit Taq polymerase (Boehringer Mannheim, Indianapolis, Indiana, USA) and sterile deionized H2O. A "touchdown" program on an MJ Research (Watertown, Massachusetts, USA) PTC-100 Programmable Thermal Controller was used for amplification. After an initial denaturation at 94°C x 3 min, eight cycles with descending annealing temperatures (from 56°C x 30 s at 1.0°C per cycle) were followed by a constant annealing temperature of 55°C for 28 cycles. Extension was at 72°C x 30 s for all cycles, and a final 30-min extension time at 72°C was used to ensure that the terminal transferase reaction by the polymerase was carried out to completion. Then 5 µL of stop buffer (95% formamide, 0.1 mmol/L EDTA, and 0.1% bromophenol blue) were added to each well, and reactions were denatured at 94°C for 3 min prior to electrophoresis.
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). A primer producing a monomorphic band with a size similar to that of the polymorphic alleles was included in all reactions as an internal standard in each lane. DNA from Abies nordmanniana (J. C. Raulston Arboretum, North Carolina State University, Raleigh, North Carolina, Accession number 950295) was also included in two lanes to standardize fragment sizing within and between gels. DNA fragment sizes were analyzed with the software program RFLPScan (Scanalytics, Billerica, Massachusetts, USA). Repeatability of allele assignments was assessed in two independent PCR amplifications with the samples randomly rearranged on plates. PCR products from Pt 30204 were cloned and sequenced using a pGEM T vector (Promega, Madison, Wisconsin, USA) and dRhodamine dye labeled terminator reactions (GenBank accession numbers GBAN-AF 183871–AF 183875; the prefix GBAN- has been added to link the online version of American Journal of Botany but is not part of the actual accession number). PCR products at Pt 71936 from BESS (Base Excision Sequence Scanning) (Hawkins and Hoffman, 1997
) reactions were visualized using infrared labeled primers on a LI-COR DNA 4000L automated sequencer.
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i=1n pi2) where n equals the number of alleles and pi equals the frequency of the ith allele in the population. Departure from homogeneity of allele frequencies among taxa was tested by 
2 analysis (e.g., Neter et al., 1996
). Haplotype diversity was calculated in the same manner as gene diversity, with n and pi referring to haplotypes.
Population structure was assessed at individual loci with RST (Slatkin, 1995
) as an index of genetic structure found within and among populations. RST, based on a stepwise mutation model (SMM), was calculated with the formula RST = (
- Sw)/, where Sw is the sum over all loci of twice the weighted mean of the within population variances, and is the sum over all loci of twice the variance of the combined populations (Microsat version 1.5b; Minch et al., 1995/1996
).
An analysis of molecular variance (AMOVA) (Schneider et al., 1997
) using FST as the measure of genetic distance was carried out to assess population structure of haplotypes within and among Fraser, balsam, and intermediate fir. Empirical significance of the AMOVA values was calculated with >25 000 permutations. Departure from random association of the two alleles in haplotypes was measured by exact tests for linkage disequilibrium (Fisher's exact test with 3200 permutations) using Genetic Data Analysis software (Lewis and Zaykin, 1998
).
An estimate of genetic distance among individuals within populations was calculated with the mean pairwise haplotype distance measure D2SH, also based on a stepwise mutation approach. It is the squared sum of the pairwise absolute differences in allele sizes for both loci, averaged over all possible pairs of haplotypes and was computed with the formula:
; Vendramin et al., 1998
).
A chloroplast haplotype network, based on similar representations of parsimonious relationships in chloroplast microsatellites, or nonrecombining portions of the human Y chromosome, was constructed for the three taxa (Cooper et al., 1996
; Doyle et al., 1998
; Morgante, Felice, and Vendramin, 1998
). Haplotypes were plotted directly with allele sizes found at Pt 30204 (vertical axis) and Pt 71936 (horizontal axis) and then connected to their nearest neighbors.
RESULTS
The chloroplast genomes of Fraser, balsam, and intermediate fir contain 12 sites that can be PCR amplified using the P. thunbergii microsatellite primers (Table 2). Of the 12 primer pairs, all amplified consistently under standard conditions except PT 9383, which showed poor and inconsistent amplification. All PCR reactions produced a single, robust band per primer pair; thus, there was no evidence for heteroplasmy. Fragments that differed in size by 1 bp could be reliably distinguished and assigned to allele categories for the same samples run on different gels. The mean absolute difference in estimated fragment size of gels was <0.6 bp (Pt 71936: 
= 0.20 bp, SD = 0.31; Pt 30204: = 0.5 bp, SD = 0.36). PCR products of Pt 30204 from five individuals representing the three taxa were sequenced and the presence of a (dA)n (dG)n site similar to that found in P. thunbergii was confirmed. Repeat-length variation both at this site and in a (dT)n site within the same fragment was observed. There was 91% conserved identity between the P. thunbergii and the Abies sequences.
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2 tests for departure from homogeneity in allele frequency at Pt 30204 showed highly significant differentiation between Fraser and intermediate, Fraser and balsam, and balsam and intermediate fir (P 
0.001). At Pt 71936, there was a highly significant difference in allele frequency between Fraser and intermediate fir (P 0.001), and a significant difference (P 0.05) between balsam and Fraser, and balsam and intermediate fir. A high degree of population subdivision was shown in RST for Pt 30204 (0.36) and a much lower value for Pt 71936 (0.10) (Table 4).
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0.001) and balsam fir (P 0.055) showed departure from random association of alleles at the two loci. Highly significant haplotypic differentiation was detected in an AMOVA analysis of haplotypic differentiation (FST = 0.06, P < 0.001, IAM, infinite alleles model).
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Chloroplast microsatellites at two highly variable loci provided clear evidence for genetic divergence in eastern North American Abies. The high level of population subdivision at Pt 30204 (RST > 0.3) suggests that recent gene flow between the geographically separated fir taxa is limited. If gene flow via pollen were extensive between stands, little population subdivision would be expected in paternally inherited DNA. Strong among-population subdivision at chloroplast microsatellite loci has also been demonstrated in P. halepensis (RST > 0.2) and P. brutia (RST > 0.3) at nine chloroplast microsatellite loci (Bucci et al., 1998
). More extensive population subdivision is typically found in maternally inherited mitochondrial genes in conifers, due to limited dispersal via seeds (e.g., Latta et al., 1998
; Strauss, Hong, and Hipkins, 1993
). Chloroplast DNA is maternally inherited in angiosperm species and spatial clustering of haplotypes is often pronounced related to limited seed dispersal. Petit et al. (1993)
demonstrated that genetic markers for cpDNA in oak have highly significant geographic variation and high genetic differentiation (GST = 0.895). Petit et al. (1997)
showed large patches (hundreds of square kilometers) of oak forest in France that had a single cpDNA marker haplotype. Other analyses of Quercus robur and Q. petrea in Europe have also shown nonrandom patterns of cpDNA variation that likely arose through the "resampling" process involved in northward migration of tree populations during the Holocene warming 7000 yr ago (e.g., Dumolin-Lapegue et al., 1997
; Johnk and Siegismund, 1997
; Ferris et al., 1998
).
Studies of chloroplast microsatellites in European Abies found high levels of diversity at the loci used in this study (Vendramin and Ziegenhagen, 1997
; Ziegenhagen et al., 1998
; Vendramin et al., 1999
). An examination of sequence variation in European Abies (Vendramin and Ziegenhagen, 1997
) showed a highly conserved sequence at Pt 71936. Length variation at Pt 30204 in European Abies was found in two different mononucleotide units within the DNA fragment, and in insertions and deletions in the flanking sequence. In A. alba, Pt 30204 appeared to be a "mutational hotspot" (Vendramin et al., 1999
). DNA sequence of Pt 30204 in eastern North American Abies also had two mononucleotide repeat units and insertions/deletions in the flanking sequence. Mutational processes at this locus may be different than those found in Pt 71936. Currently, little is known about the processes or rates of mutation at chloroplast microsatellite loci. Predominant paternal inheritance of Abies cpDNA was demonstrated in a study of European Abies species (Vendramin and Ziegenhagen, 1997
).
Fraser, balsam, and intermediate fir individuals within populations showed high levels of genetic variation at the level of haplotypes reflected by the elevated values found for D2SH. Balsam fir, which had the largest range, had the largest value of D2SH, 26.17. Haplotypes from Alberta, which were distinct from the balsam haplotype cluster in the network analysis, contributed to the large value. Intermediate fir, which occurs in small, isolated stands, had the lowest value, 13.90. In A. alba, values of D2SH were also high, 28.08 (Vendramin 1999
, personal communication, Instituto Miglioramento Genetico Piante Forestali). Values of mean D2SH for chloroplast microsatellite loci in P. pinaster averaged 0.51, and in P. resinosa ranged from 0.05 to 0.44 (Echt et al., 1998
; Vendramin et al., 1998
). Within populations of P. halepensis, D2SH ranged from 0.04 to 14.41 and showed evidence of two major branches within the taxon (Morgante, Felice, and Vendramin, 1998
).
A network analysis has the potential to reveal some of the haplotypic structure that results from ancestral relationships and the nonrandom distribution of mutations among lineages. Ancestry is not taken into account by analyses based on pairwise haplotypic distance measures. The construction of the haplotype networks (Goldstein et al., 1995
; Slatkin, 1995
) assumes that microsatellites add or subtract repeat units with equal probability and, in most cases, one repeat unit at a time (i.e., the stepwise mutation model, SMM). Therefore, alleles of a similar size could have a history of fewer intervening mutations and contain information about time since divergence from a common ancestor. However, identical states of microsatellite alleles can arise independently, and the potential for homoplasy must be considered when assessing relationships at these sites (Doyle et al., 1998
). Fast-evolving microsatellite loci on the human Y-chromosome have provided nonrecombining haplotypes that have been useful for interpreting the expansion and migration of human populations during the past 200 000 yr (e.g., Wilson and Balding, 1998
). The mutation rate for chloroplast microsatellite loci could be low, ~10-5 (Provan et al., in press
), which would suggest that the discontinuity in fir chloroplast haplotypes could predate the most recent glacial episode. Further research is needed to evaluate a coalescent-with-stepwise-mutation model for the nonrecombining fir microsatellite haplotypes as an explanation for the two groups revealed by the fir haplotype network analysis. The current distribution of fir haplotypes could also be related to environmental gradients and that possibility needs to be investigated as well.
The geographic distribution of the two groups of haplotypes in eastern North American fir showed a nonrandom pattern and provides evidence for the divergence of the taxa by revealing two evolutionary lineages. The number of population-specific haplotypes (balsam 65%, Fraser 75%, intermediate 55%) could make these markers useful for discrimination among the taxa. A study of 714 A. alba individuals from 17 populations revealed 90 haplotypes with evidence for spatial distribution of specific haplotypes (Vendramin et al., 1999
). Thirteen of the 17 populations in the study of A. alba were characterized by at least one population-specific haplotype. Population-specific haplotypes were found in 88% of the populations analyzed in the P. halepensis species complex (Bucci et al., 1998
). The majority of haplotypes in this study, with small sample sizes, were detected with a frequency <0.05. Further study and larger sample sizes are needed to characterize the distribution of low-frequency haplotypes within populations and the presence of population-specific haplotypes.
Some authorities believe that the divergence in eastern North American Abies taxa began after the end of the most recent glacial phase (Oosting and Billings, 1951
; Myers and Bormann, 1963
). Eastern North American Abies were present as a continuous population 8000 yr ago along the Appalachian Mountains from Georgia to Newfoundland, but the once uninterrupted range was broken into discontinuous populations with warming during the early and mid-Holocene 7000 yr ago (Delcourt and Delcourt, 1987
). Glacial phases account for 90% of the 2 million yr period of the Quaternary. Interglacial periods were relatively brief, lasting 10 000 to 15 000 yr with temperate climates similar to that of the present (Davis, 1983
). The last interglacial period prior to the present began 125 000 yr ago and lasted about 15 000 yr. This period was followed by brief cycles of warming and cooling until the beginning of the most recent glacial period 70 000 yr ago. This last interval culminated in a glacial maximum 18 000 to 20 000 yr ago, and much of the current range of balsam fir was under ice (Delcourt and Delcourt, 1987, 1991
). During this interval, many tree species most likely persisted in small, isolated population centers in refugial areas (Davis, 1983
). Evidence suggests that tree species adapt very slowly to changing climate and are forced to migrate to survive (Bradshaw and McNeilly, 1991
). In recent times, migration and population history of Abies taxa may have contributed a larger part to current patterns of diversity than has natural selection.
Regardless of the taxonomic identity of Fraser, balsam, and intermediate fir, we can recognize evolutionary divergence in cpDNA haplotypes among populations. The almost reciprocal monophyly present in haplotypes between balsam and Fraser fir provides justification for recognizing these taxa as independent Management Units according to the definition by Moritz (1994)
: "Populations with significant divergence of allele frequencies at nuclear or mitochondrial loci, regardless of the phylogenetic distinctiveness of the alleles." The naturally fragmented population centers make it critically important to understand the distribution of genetic diversity within these three taxa for the development of effective conservation strategies for Fraser fir. The highly variable repeat sequences in chloroplast genomes could reveal population history and phylogeographic structure for plant taxa similar to that revealed for animal taxa in studies of mitochondrial DNA sequence variation (Avise, 1994
; Avise and Hamrick, 1996
).
Summary
In this paper, we have shown a strong discontinuity in chloroplast microsatellite haplotypes of eastern North American Abies taxa. The level of divergence found for two chloroplast microsatellite loci was greater than previously shown in allozymes, terpenes, and morphological characteristics (Robinson and Thor, 1969
; Zavarin and Snajberk, 1972
; Jacobs, Werth, and Guttman, 1984
). These markers exhibit a high degree of among-population differentiation and significant difference in allele frequency. Population-specific haplotypes were found in all of the three taxa and could provide information useful for conservation planning, although larger sample sizes are necessary to evaluate haplotype distribution. The high level of variability found at these loci could provide a method to characterize pollen dispersal among and between populations. The large between-taxon variation also suggests geographical structure that contrasts with earlier intergrading variation. These markers show a potential, as demonstrated in larger studies of European Abies populations, to delineate species boundaries and populations with a high value for gene conservation in closely related taxa.
FOOTNOTES
1 The authors thank Giovanni Vendramin for comments on an earlier version of this manuscript; J. Frampton, NCSU, G. Hawley, UVM, L. De Verno, CFS, New Brunswick, L. Corriveau and D. Stepnisky, Weyerhaeuser Canada, J. Brown, OSU, N. Dhir, Alberta Environmental Protection, F. C. Yeh, Univ. Alberta, T. Blount, Shenandoah National Park, and K. Garrard for their assistance in obtaining samples for this study; and the Forest Biotechnology Group and the Department of Botany at North Carolina State University, and a grant-in-aid from Sigma Xi for providing funding. This work represents a portion of a Master's thesis submitted to the Department of Botany at North Carolina State University. 
4 Author for correspondence, current address: North Carolina State University, Forest Biotechnology Group, Campus Box 7247, Raleigh, North Carolina 27695 USA. (e-mail: catherine_clark{at}ncsu.edu
).
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