Clonal Fidelity in Large Colonies of
Gaylussacia brachycera Gray (Box Huckleberry)
Assessed by DNA Fingerprintings
Margaret Pooler, Rob Nicholson, and Andrew Vandegrift
Northeastern Naturalist, Volume 15, Issue 1 (2008): 67–74
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2008 NORTHEASTERN NATURALIST 15(1):67–74
Clonal Fidelity in Large Colonies of
Gaylussacia brachycera Gray (Box Huckleberry)
Assessed by DNA Fingerprinting
Margaret Pooler1,*, Rob Nicholson2, and Andrew Vandegrift1
Abstract - Gaylussacia brachycera (box huckleberry) is a slow-growing, dwarf
evergreen member of the family Ericaceae that is native to eight states in the
eastern United States. It is a rare plant with conservation status in several states
of critically imperiled (S1). Botanists have been intrigued by this enigmatic native
plant since it was discovered in 1796 in Virginia. One of the mysteries of
this species is whether plants in a colony arose from different genotypes or are
clonal. The species reproduces primarily by means of underground runners and
appears to be self-sterile, so sexual reproduction within isolated colonies could
be limited. Using molecular markers, we tested samples taken from three of the
best-known colonies in Pennsylvania and one in Tennessee. Based on 104 polymorphic
markers, we found that one of the Pennsylvania colonies contained two
genotypes among 11 samples tested; one Pennsylvania colony contained three
genotypes among five samples tested; and the other two colonies exhibited no
variation among the 8–10 samples tested. This study represents the first time that
molecular markers have been used in a systematic assay to determine the existence
of variation among individuals within a colony of box huckleberry.
Gaylussacia brachycera (Michx.) Gray (box huckleberry) is a slow-growing,
evergreen groundcover in the family Ericaceae that is native to Delaware,
Pennsylvania, Maryland, Virginia, West Virginia, Kentucky, and Tennessee
(USDA NRCS 2006), as well as North Carolina (Wilber and Bloodworth
2004). It is usually found growing in dry or well-drained acidic soils or duff
in partial shade. Discovered in 1796 by Andre Michaux near Winchester, VA,
this plant was originally named Vaccinium brachycerum (Michaux 1803).
It was described and renamed Gaylussacia brachycera by Asa Gray (Gray
1846), based on collections made in 1845 by S.F. Baird in Pennsylvania.
Awareness of the plant grew with the subsequent discovery of a large colony
in Pennsylvania by H.A. Ward in 1919 (Ward 1920), as well as smaller colonies
discovered in other states (Smith and Smith 1971).
Despite its distribution in eight states in the middle eastern US, box
huckleberry is not a common plant. The global conservation status for
box huckleberry is listed as G3 (rare), with S1 (critically imperiled)
listing for the states of Delaware, Maryland, and Pennsylvania and S2
1USDA/ARS, US National Arboretum, Floral and Nursery Plants Research Unit,
3501 New York Avenue, NE, Washington, DC 20002. 2Smith College Botanic Garden,
15 College Lane, Northampton, MA 01063. *Corresponding author - Margaret.
68 Northeastern Naturalist Vol. 15, No. 1
(imperiled) for Virginia and West Virginia (Center for Plant Conservation
2006). It is estimated that currently this species grows at 100 sites within
its native range (Crable 1999).
Box huckleberry reproduces predominantly vegetatively by means of
underground runners, with an estimated average annual spread of six inches
per year (Coville 1919). Although seed set is possible, it is rare in natural
populations due to self-incompatability in isolated clones where outcrossing
is not possible (Coville 1919, Dirr 1998, Wherry 1934). This obligatory
clonal reproduction, combined with the size of the colonies found in Pennsylvania,
has led to rampant speculation on the age of the clones. Estimates
ranging from 5000 years to 13,000 years have been made, which would
make the former 2-kilometer-long clone in Pennsylvania the oldest documented
living thing on earth (Krussmann 1977, Moldenke 1957, Wherry
1972, Willaman 1965).
The purpose of this study was to investigate the clonality of several
colonies of box huckleberry in Pennsylvania that are reputed to be
single-genotype stands that are thousands of years old. We used AFLP
markers to fingerprint plants in order to ascertain the level of diversity
among samples within each colony and to determine the level of diversity
among separate colonies.
Materials and Methods
Leaf samples were collected from three stands in Pennsylvania that
showed contiguous growth, as well as one stand in Tennessee. Approximate
colony size and locations of samples within each colony are indicated in
Figure 1. Leaf samples were sent to the US National Arboretum for DNA
Figure 1. Sampling patterns for stands of G. brachycera (box huckleberry) from Perry
County, PA (WardA, WardB, and Baird; sampled August 2004) and Morgan County,
TN (Rugby, sampled July 2000). The WardA and WardB populations are approximately
300 m apart and 16 km east of the Baird population.
2008 M. Pooler, R. Nicholson, and A. Vandegrift 69
extraction and analysis. Leaves for DNA analysis were freeze-dried and
stored at -80 ºC in a freezer until analysis.
DNA was extracted from three freeze-dried leaves of each sample using
the methods described for other species in our laboratory (Pooler et al. 2002),
with the following modification: leaves were first ground to a powder in the
lysing matrix (Bio101, Vista, CA) by dry vortexing for 2–3 minutes, and then
500 μl of prewarmed (65 ºC) grinding buffer (Wilson et al. 1992) were added.
The mixture was processed in a FastPrep machine (Bio101), centrifuged for
1 minute at top speed (≈10,000 rpm) in a microcentrifuge, and the grinding
buffer was then pipetted off. Five hundred μl of CTAB buffer (Doyle and
Doyle 1987) and 1.5 μl of beta-mercaptoethanol were added to the pelleted
ground plant material, and our standard DNA extraction procedure (Pooler et
al. 2002) using the QIAamp tissue kit (Qiagen, Inc., Valencia, CA) was followed.
Processing the leaves in the grinding buffer prior to CTAB extraction
was necessary for DNA recovery from box huckleberry.
AFLP analysis was performed as described by Vos et al. (1995) and Invitrogen
Corporation (2003), with slight modifications, noted below, to prepare
samples for analysis on an ABI 310 Genetic Analyzer (Applied Biosystems,
Foster City, CA). DNA restriction, digestion, and ligation were carried out
sequentially using approximately 0.25 μg of genomic DNA. Restriction
digestion was performed at 37 ºC for three hours, and ligations at 20 ºC for
three hours. Preselective reactions took place in 20-μl volumes containing
PCR buffer (Invitrogen Corp., Carlsbad, CA), plus 3 mM MgCl2, 100 μM
dNTP, 0.125 μM each preselective primer, 2.0 U of Taq DNA polymerase
(Invitrogen Corp.), and 3 μl diluted restriction/ligation reaction. Completed
preselective reactions were diluted 1:50 with TE, and 5 μl were used as
template for all selective reactions. Selective amplification reactions were
carried out in 20-μl volumes containing the same reagents as for preselective
amplification, except that 0.25 μM MseI primer and 0.1 μM EcoRI primers
(Table 1) were used instead of preselective primers and only 0.5 U Taq DNA
polymerase was used. The EcoRI selective primers had fluorescently labeled
5’ ends and were purchased from the Applied Biosystems Custom Oligonucleotide
Synthesis Service (Foster City, CA). Completed selective reactions
were analyzed on an ABI310 automated DNA sequencer with POP4 polymer.
Samples were prepared by mixing 1.0 μl of selective reaction, 0.07 μl Genescan
500 ROX size standard (Applied Biosystems), and 10.93 μl deionized
formamide. All reactions were replicated from DNA extraction through amplification to ensure repeatability.
Markers were visualized as peaks using Genotyper® 2.5 software (Applied
Biosystems) and scored manually as present or absent for each accession.
70 Northeastern Naturalist Vol. 15, No. 1
Similarity coefficients between each accession were calculated using the
SIMQUAL program in NTSYS-pc, version 1.70 (Rohlf 1992), using the Dice
similarity coefficient (2a / [2a + b + c], where a = total number of bands shared
by both individuals, b = bands unique to one individual, and c = bands unique
to the other individual). These data were subjected to cluster analysis using
the UPGMA method in the SAHN program of NTSYS to generate a phenogram.
Cophenetic matrices were constructed and compared with the similarity
matrices using the MXCOMP program to test the goodness of fit of a cluster
(Rohlf 1992). Bootstrap analysis using 5000 replications was performed on
the original raw data using WinBoot (Yap and Nelson 1996) to determine
confidence limits of clusters in the UPGMA-based dendrograms (Felsenstein
1985). The bootstrap value indicates the percentage of times the group to the
right of the node occurred in the bootstrap analysis.
Results and Discussion
Analysis of 13 AFLP primer pairs revealed 104 polymorphic bands out of
a total of 1682 bands (average 8 polymorphic bands per primer pair), with a
range of three to 17 polymorphic bands per primer (Table 1). Reproducibility
between replicate samples was good, with approximately 8% of markers not
used due to inconsistencies between samples or one sample that had missing
data. Monomorphic bands (typically more than 100 per primer pair) were
not scored, since they are not informative. This level of polymorphism is
consistent with a previous study in our lab where we examined genetic distances
among 24 geographically diverse box huckleberry accessions using a
slightly different AFLP protocol (Pooler et al. 2006). While the present study
complements our previous population genetic distance study, the results of
these two studies cannot be compared directly, nor can the data be merged
because different primers and a different AFLP protocol were used.
Table 1. List of AFLP selective primer extensions, total number of peaks detected, and number
of polymorphic markers per primer pair among samples from four populations of G. brachycera
(box huckleberry) accessions.
primer extension MseI selective Total number of Number of
with dye name primer extension scorable markers polymorphic markers
AAC (NED) CAC 118 15
AAC (NED) CTC 104 6
AAC (NED) CTG 139 17
ACC (FAM) CAC 110 6
ACC (FAM) CTC 179 10
ACC (FAM) CTG 168 3
ACG (NED) CAC 127 3
ACG (NED) CAG 99 11
ACG (NED) CTC 78 7
ACG (NED) CTG 123 5
AGC (HEX) CAC 138 8
AGC (HEX) CTC 155 6
AGC (HEX) CTG 144 7
2008 M. Pooler, R. Nicholson, and A. Vandegrift 71
Pairwise comparisons of accessions using the 104 polymorphic markers
were computed using the DICE coefficient. In our previous work (Pooler
et al. 2006), as well as that of others (Koopman et al. 2001; Mace et al.
1999a, b), we found that using UPGMA clustering, the DICE coefficient
resulted in identical phenograms as the Jaccard coefficient, with similar
correlation coefficients. The DICE similarity values were then used to
construct a phenogram using UPGMA clustering. A phenetic approach
using UPGMA clustering was used in this study because our primary
objective was to determine the genetic relationships among accessions
within a single colony, rather than to determine phylogenetic histories. In
addition, it has been demonstrated that branches or clusters in a cladogram
or phenogram that are well supported by bootstrap statistics will be similar
regardless of the method used (Kardolus et al. 1998, Koopman et al.
2001). The UPGMA-based phenogram using the DICE similarity data had
a cophenetic correlation coefficient (r) of 0.9985 (Fig. 2), indicating a very
Figure 2. UPGMA-derived phenogram of genetic similarity based on the Dice similarity
coefficient among G. brachycera (box huckleberry) accessions based on 104
polymorphic characters from 13 AFLP primer pairs. Cophenetic correlation coeffi-
cient (r) = 0.9985. Bootstrap confidence values are indicated to the left of each node
and represent the percentage of times that cluster appeared in the consensus tree.
72 Northeastern Naturalist Vol. 15, No. 1
good fit of the phenogram with the original distance matrix (Rohlf 1992).
In addition, the bootstrap values for most of the clusters were high, indicating
that those clusters have statistical and therefore biological significance,
and are not simply an artifact of the clustering technique (Yap and Nelson
1996). The low bootstrap value (39.9) in the WardB cluster indicates
that these groups are not well defined, probably due to the low number of
polymorphic markers between them (two out of 104). The clustering of the
samples, as well as the relative genetic similarities between clusters, was
directly related to physical proximity of the samples (Figs. 1 and 2). It is
important to note that the genetic similarity values were based on only the
polymorphic markers; therefore, like in other studies, the values reflect the
genetic similarities of these individuals and colonies relative to each other,
and not an absolute genetic similarity.
The AFLP fingerprints indicated that the Baird colony of box huckleberry
has two genotypes represented among the 11 samples collected, with one
genotype (Baird1, Baird2, and Baird3) originating from one corner of the
collection site (Fig. 1). These genotypes differ by only one polymorphism
out of 104 scored, however, which suggests that the different genotypes may
have arisen by somatic mutation rather than sexual recombination. This
colony, located near New Bloomfield, PA, was the first discovered and is
probably the best-known population of box huckleberry in Pennsylvania.
The existence of at least two genotypes in this population is also supported
by results from our previous study, which also showed molecular variation
within an albeit limited sample of two clones (Pooler et al. 2006).
The WardA population represents the second-known stand in Pennsylvania,
discovered by Ward in 1919 and approximately 16 kilometers east
of the Baird colony. WardA is what remains of the 2-kilometer-long patch
described in 1920 (Ward 1920). Much of this stand has been lost due to a
forest fire in 1963 and highway construction. The longest sampled transect
of this population was 307 meters, and all 10 samples from this site had
identical genotypes. The WardB stand was approximately 300 m from the
WardA stand, on a separate ridge with a stream in between. The five samples
from the WardB stand exhibited three different genotypes, defined by two
AFLP markers unique to the WardB-5 sample and two markers unique to
the WardB-1 and -2 samples. All three genotypes appeared along the longest
transect of 110 meters (Fig. 1).
It is not clear why there was more variability seen in the samples from
the smaller WardB colony than the larger WardA stand. Perhaps the WardB
colony was originally established by seed representing several genotypes,
and this diversity has been maintained through relatively noncompetitive
clonal reproduction. Prevailing winds or other environmental factors affecting
insect behavior could have contributed to outcrossing and subsequent
seed production in this population.
The box huckleberry population that was sampled in Rugby, TN was
used as a test population to obtain preliminary data on clonal fidelity in
2008 M. Pooler, R. Nicholson, and A. Vandegrift 73
other populations of box huckleberry. The eight samples collected from
two transects had identical genotypes. The relative genetic similarity
between this population and the cluster containing the Pennsylvania
populations was only 0.38, which is not surprising based on their physical
and geographic distance.
This study clearly illustrates the existence of genetic diversity within
and between large stands of box huckleberry in Pennsylvania. It also
lends support to, but cannot prove unequivocally, the hypothesis that
within the former 2-kilometer-long WardA stand, all plants are clonal.
Based on the large size of the WardA colony and the presence of at least
three genotypes in the nearby WardB colony, more sampling from the
WardA colony is necessary to provide strong evidence of the clonal nature
of this colony. However, the assertions that the box huckleberry clones in
Pennsylvania are the oldest living things on earth cannot be disproven.
Based on the current size of the remaining colonies and a presumed
growth rate of 15 cm per year, it can certainly be stated that these clones
are some of the oldest plants in the eastern US.
The authors wish to thank Dr. Melvin Shemluck, Quinsigamond College,
Worcester, MA for assistance in collecting samples in Pennsylvania.
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