Plant Syst. Evol.
225:85-101 (2000)
Inter- and intraspecific genetic variation in Hippophae
(Elaeagnaceae) investigated by RAPD markers
I. V. Bartish 1'3, N. Jeppsson l, G. I. Bartish 1, R. Lu 2, and H. Nybom 1
1Balsgfird-Department of Horticultural Plant Breeding, Swedish University of Agricultural Sciences,
Kristianstad, Sweden
a Chengdu Institute of Biology, Chinese Academy of Sciences, Sichuan, China
3 Present address: Institute of Botany, Academy of Sciences, Pruhonice, Czech Republic
Received October 5, 1999
Accepted September 6, 2000
Abstract. Genetic diversity has been investigated
by the application of molecular markers in, for the
first time, all the taxa recognised in recent treatises
of the genus Hippophae. RAPD (random amplified
polymorphic DNA) analyses were conducted with
9 decamer primers, which together yielded 219
polymorphic markers. We found 16 fixed RAPD
markers, i.e. markers that either occurred in all
plants of a population or were absent from all
plants. Several of these markers were useful for
analysis of interspecific relationships, whereas oth-
ers can be considered as taxon-specific markers.
Clustering of taxa and populations in our neigh-
bour-joining based dendrogram was in good agree-
ment with some recently suggested taxonomic
treatises of Hippophae. Amount and distribution
of genetic variability varied considerably between
species. Partitioning of molecular variance within
H. rhamnoides supported earlier findings that a
considerable part of the total variance resides
among subspecies (59.6%). Within-population
variability also differed considerably. Percentage
polymorphic RAPD loci and Lynch and Milligan
within-population gene diversity estimates showed
relatively high values for some species close to the
geographic centre of origin in Central Asia, e.g.
H. tibetana and the putatively hybridogenous
H. goniocarpa. Spatial autocorrelation analyses
performed on 12 populations of H. rhamnoides
Plant Systematics
and Evolution
Springer-Verlag 2000
Printed in Austria
revealed positive autocorrelation of allele frequen-
cies when geographic distances ranged from 0 to
700 km, and no or negative autocorrelation at
higher distances. At distances between 700 and
1900 km, we observed deviations from the expected
values with strongly negative autocorrelation of
allele frequencies. A corresponding relationship
between geographic and genetic distances could
not be found when the analysis instead was based
on one population from each of 8 species.
Key words: Elaeagnaceae, Hippophae, sea buck-
thorn, Systematics, taxonomy, genetic variation,
RAPD.
The genus Hippophae L. is distributed from
N o r t h e r n Europe through Central Europe
and Central Asia to China, ranging from 27
to 69N and from 7W to 122E, with a
probable centre o f origin in Central Asia
(Rousi 1971). All species are diploid
(2n = 2 4 ) ( R o u s i 1965, 1971), windpollinated
and dioecious, with gender being genetically
determined (Persson and N y b o m 1998). They
reproduce vegetatively with root suckers and
sexually with bird-dispersed seeds. Natural
habitats include sea shores, river deltas, river
terraces, valley slopes and sometimes mead-
86 I.V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae
ows, at altitudes from 0 to 5000 m (Rousi 1971,
Lian et al. 1998).
Most authors follow the taxonomy present-
ed by Rousi (1971), with the genus Hippophae
divided into three species; H. rhamnoides L.,
H. salicifolia D. Don and H. tibetana Schlecht.
The species H. rhamnoides was further divided
into nine subspecies; ssp. rhamnoides, ssp.
fluviatilis van Soest, ssp. carpatica Rousi, ssp.
caucasica Rousi, ssp. turkestanica Rousi,
ssp. mongolica Rousi, ssp. sinensis Rousi, ssp.
yunnanensis Rousi and ssp. gyantsensis Rousi.
Later described taxa include H. neurocarpa
S.W. Liu and J.N. He (Liu and He 1978),
H. goniocarpa Lian, X.L. Chen and K. Sun,
H. goniocarpa ssp. litangensis Lian and X.L.
Chert and H. neurocarpa ssp. stellatopilosa Lian
and X.L. Chen (Lian et al. 1995). Classification
has been based almost exclusively on morpho-
logical characters. However, an isozyme anal-
ysis, conducted mainly on H. rhamnoides but
with some additional samples of H. tibetana
and H. neurocarpa, supports Rousi's division
of this genus into species and subspecies (Yao
and Tigerstedt 1993). Morphologically inter-
mediate plants are found where different
subspecies of H. rharnnoides grow sympatrical-
ly, indicating inter-subspecies hybridisation
(Rousi 1971).
Sea buckthorn was mentioned as a medi-
cinal plant already in the traditional Tibetan
pharmacopoeia, completed 618-907 A.D. In
China it is also widely used to control erosion
(Lu 1992). Sea buckthorn is an important berry
crop in Russia (5.000 ha in 1987) and neigh-
bouring countries. The domestication started in
1934 in the Altai, Siberia, using local germ-
plasm of H. rhamnoides ssp. mongolica. Re-
cently, an interest for sea buckthorn as a berry
crop has emerged also in Finland, Germany,
Sweden and Canada (Vetters 1990, Yao and
Tigerstedt 1994, Wahlberg 1995, Li and Sch-
roeder 1996). The aromatic juice and the high
contents of vitamins A, C and E make these
berries very interesting for the food processing
industry. Problems with climatic adaptation
and disease susceptibility, however, necessitates
the exploitation of a wide range of taxa in plant
breeding programs to develop commercially
suitable cultivars (Kalinina and Panteleyeva
1987, Yao and Tigerstedt 1994, Burmistrov
1995, Trajkovski and Jeppsson 1999).
Both isoenzyme (Yao and Tigerstedt 1993)
and RAPD (Bartish et al. 1999a) analyses have
been applied to investigate genetic diversity and
partitioning of genetic variation in Hippophae.
The hitherto investigated plant material has,
however, been restricted mainly to some of the
subspecies of H. rharnnoides. The present
investigation was undertaken to analyse, for
the first time, inter- and intraspecific genetic
variation in the whole genus. The presently
studied material therefore included wild popu-
lations of all described taxa. We choose to use
RAPD for this study since RAPD markers
have previously been applied successfully in
Hippophae to analyse segregation ratios in
experimental crosses (Persson and Nybom
1998) and to estimate genetic variation in wild
populations (Bartish et al. 1999a).
Material and methods
Plant material. All plant material was raised from
seed except for one population (Gb) of H. rhamno-
ides ssp. rhamnoides which was collected as leaves
from plants in native stands (Table 1, Fig. 1). Seed
samples were collected in natural populations with
three exceptions: (1) One of the populations of
H. rharnnoides ssp. fluviatilis (9821) was represented
by seedlings from a cross conducted at Balsg~rd
between two plants originating from Switzerland,
(2) material of H. salicifolia (9804) was raised from
seeds collected in a population with approx. 100
individuals grown in Maoxian, Sichuan, China to
where it had been introduced from its original
location in Tibet, (3) material of H. rhamnoides
ssp. carpatica (9895-9898) was collected from open
pollinated female clones which had been trans-
ferred to Pitesti Research Institute, Romania. Seed
collection was generally undertaken from several
motherplants in each population except for
H. rhamnoides ssp. carpatica (9895-9898) and
caucasiea (9831, 9835, 9847, 9893), where seeds
were collected from a single motherplant in each
population.
DNA extraction and PCR amplification. Apices
of one-month old seedlings were frozen in liquid
I. V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae 87

0 0 0
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I o o o o o
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o~ , 0 0 0 0 0 o kO 0 0 ~ ~ ~-~ 0

0 :

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0
N
%

"~.~ ~ ~ ~
~ - ~~ ~~ .~ :E~ ~z
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88 I. V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae

H. rhamnq
ssp. fluvi,~

H. rhamnoides
ssp. turkestanic~
tanensis

H. s a l i c i ~ n t s e n s i s -~

Fig. 1. Geographical distribution of the genus Hippophae (from Hyv6nen 1996). The different subspecies of
H. rhamnoides are marked with light grey whereas the medium dark grey and A-marked area in central Asia
represents several species, namely H. neurocarpa ssp. neurocarpa, H. n. ssp. stellatopilosa, H. goniocarpa ssp.
goniocarpa and H. g. ssp. litangensis. The area marked with dark grey represents H. tibetana

nitrogen in an Eppendorf tube (1.5 ml) and stored
at -80 C until used. In two cases, leaves from four
and seven adult plants of H. rhamnoides ssp.
gyantsensis and from ssp. fluviatilis (9313), respec-
tively, were obtained by forcing twigs in a green-
house in the winter. DNA was extracted from the
frozen seedling apices or from fresh unfolded leaves
(5-10 mg in each case) following the CTAB-based
method described earlier (Bartish et al. 1999b). The
only exceptions were that plant tissue was ground
in Eppendorf tubes using a minipestle, and that
volumes of extraction buffer consequently could be
reduced to 100-250 gl per sample. For population
Gb, previously extracted DNA samples (used in
our study on H. rhamnoides ssp. rhamnoides,
Bartish et al. 1999a) were utilised. With the excep-
tion of the use of another thermocycler (PTC-100
TM, MJ Research, Inc.), and the reduction of PCR
reaction volume to 15 gl, amplification conditions
were as described previously (Bartish et al. 1999b).
We selected nine decamer primers from Operon
Technologies kits A, B, and D on the basis of
results in our previous study (Bartish et al. 1999a).
This selection was based both on the quality
(including reproducibility) of PCR amplification
products and on how these primers partitioned
genetic variability within and among populations
and/or subspecies as calculated with AMOVA
(analysis of molecular variance). Consequently,
the nine primers could be chosen according to
their previous performances (Bartish et al. 1999a)
so that three levels of differentiation were equally
well represented, i.e. three primers indicating very
high percentages of within-population variance,
three primers indicating very high percentages of
among subspecies variance, and three primers that
were intermediate between the first two groups.
Data analysis. Amplification products were
scored visually as present or absent. Bands of
identical size, amplified with the same primer, were
considered to be homologous. Bands were not
scored if they were either very faint, very rare or
very common (frequency in the whole plant sample
less than 3% or more than 97%).
To estimate genetic relatedness among popu-
lations and taxa, null allele frequencies were
calculated for all 219 scored RAPDs in accordance
with Lynch and Milligan (1994):
I. V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae
q = xl/2[1 - Var(x)/8x2] -1 ,
where q = estimated null allele frequency, x =
proportion of the N sampled individuals that do
not exhibit the marker, Var(x) = x ( 1 - x ) / N is the
sampling variance of the frequency of null homo-
zygotes. A matrix of null allele frequencies within-
populations (the original data are available from
the authors upon request) was used in a neighbour-
joining (NJ) cluster analysis based on Nei's genetic
distances (Nei 1972) (Appendix) with software
package PHYLIP, version 3.573c (Felsenstein
1993). To test robustness of the relationships
indicated in our data, a bootstrap analysis was
performed. The resulting trees were used to gener-
ate a consensus tree (by majority rule and strict
consensus tree program in PHYLIP) summarising
the relationships described by all 100 resampled
data sets.
Principal co-ordinate analysis (DECENTER
and EIGENVECTOR programs in NTSYS-pc,
version 1.8, Rohlf 1997) was applied to a matrix
of Nei's genetic distances among populations and
families to obtain additional information about
inter- and intraspecific similarities. A three-dimen-
sional plot was produced by NTSYS-pc to repre-
sent the result graphically.
Within-population gene diversity was calculat-
ed using the formulae of Lynch and Milligan (1994)
as has been described in detail in a previous study
(Bartish et al. 2000). To avoid serious underesti-
mation of gene diversity within-populations (or
families) due to a high number of taxon-specific loci
in the whole data set (Liu and Furnier 1993, Bartish
et al. 2000), two or, in some cases, three gene
diversity estimates were derived for each popula-
tion. In the first set of gene diversity estimates, all
219 scored loci were included in the calculations
according to the standard procedure for this kind
of data. A second set of gene diversity estimates
was calculated only for species with more than one
population, and includes only loci that are poly-
morphic within that species. The third set of gene
diversity estimates was calculated according to Liu
and Furnier (1993) and includes those loci that are
polymorphic within a species (population if there
was only one) but to this was also added twice the
number of loci with fixed marker presence within
that species (or population).
To estimate the partitioning of genetic variance
at different levels, a matrix of squared Euclidean
distances among individual plants for natural
89
populations (excluding half- and full-sib families
and also pop. 9313 for which sampling procedure
was not known in detail, i.e. populations from the
subspecies carpatica, caucasica and fluviatilis) was
computed from the data matrix using all 219 scored
RAPDs (SIMINT in NTSYS). This matrix was
used for an AMOVA analysis (Excoffier et al. 1992,
Huff et al. 1993) to estimate variance components.
We analysed the partitioning of molecular variance
between species/subspecies for our total plant
sample, and between subspecies within H. gonio-
carpa, H. neurocarpa and H. rhamnoides (ssp.
mongolica, rharnnoides, turkestanica) respectively.
To analyse possible relationships between
genetic and geographic distances in our plant
sample, we generated a matrix of geographic
distances between populations (Kirvan 1997):
Dij = [(111.3 ABS(lat~ - latj)) 2
2 ] 1/2
+ (111.3 ABS(lon,- lonj)) cos(lati)cos(latj)|J
where D/i is the distance between populations i and
j in kilometres, and lati, lati, loni, and loni are
latitudes and longitudes of populations i and j.
Correlation analyses were carried out with Mantel
tests (MXCOMP in NTSYS-pc, 9999 permutations
were used to compute the significance of a given
correlation) between the geographic distances ma-
trix and the above described population-based
matrix with Nei's genetic distances among RAPD
profiles. These analyses were conducted: (1) on the
entire plant material, (2) on 12 populations and
families of H. rhamnoides, and (3) on 8 popula-
tions, each representing a different Hippophae
species: H. rhamnoides was represented by popula-
tion 9810, H. neurocarpa by population 9837, and
H. goniocarpa by population 9806. Populations
9805 and 9808 were excluded from the second data
set but included in the third since their systematic
affiliation with H. rhamnoides remains doubtful. In
addition, pairwise comparisons of genetic distances
between populations were plotted against corre-
spondent geographic distances for the H. rharnnoi-
des populations and families.
A modified version of spatial autocorrelation
analysis as described by Sokal and Oden (Sokal
and Oden 1978) was conducted to further examine
the association between genetic and geographic
distances for 12 populations and families within
H. rhamnoides. A series of matrices corresponding
to different geographic distance classes were pro-
90 I.V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae

duced to represent the model to be tested. Com- Table 2. RAPD markers with fixed allele presence/
parisons between pairs of populations within a absence (i.e. the marker is present or absent in all
certain distance class were coded 0, and all other plants, or in all but one plant within the popula-
pairwise comparisons were coded 1. To achieve tion). Marker code: OPA-08.1050 denotes the am-
approximately similar numbers of pairwise com- plification with primer OPA-08 of a band which is
parisons coded as 0 in each of these matrices, the 1050 base pairs
distance range was set at 300 km in most cases
Marker Present in Absent in
(sometimes increased to 350 km or 400 km). By
populations populations
computation of Pearson correlation coefficients
between the matrix of Nei's genetic distances OPA-08.1050 9804 All others
among populations and families, and each of the OPA-08.1650 9895, 9896, All others
matrices of distance classes, we obtained altogether 9897, 9898
18 autocorrelation coefficients (Ra), which were OPA-15.440 9838, 9803 All others
plotted in a correlogram covering a distance of OPA-15.550 9801, 9810, Gb, All others
more than 6000 kin. However, correlogram pat- 9821, 9313,
terns are influenced not only by the strength of the 9847, 9893,
correlation between genetic and geographic dis- 9831, 9835,
tances but also by the spatial distribution of 9895, 9896,
pairwise geographic distances among the analysed 9897, 9898
populations. Consequently, we wanted to compare OPA-15.680 9805 All others
our actual R~ values with 'ideal' values ( = expected OPB-02.780 9805, Gb All others
values) derived from a hypothetical perfect corre- OPB-04.430 9895, 9896, All others
lation between genetic and geographic distances in 9897, 9898
our data set. To obtain these 'ideal' values, we just OPB-04.1700 All others 9895, 9896,
repeated the autocorrelation analysis but this time 9897, 9898
substituting the genetic distances with the actual OPD- 18.280 Gb All others
geographic distances. OPD-18.300 9805 All others
OPD-18.320 9804 All others
OPD-18.800 9804, 9808 All others
Results OPD-18.1000 9838, 9803 All others
R A P D profiles and fixed loci. All markers OPD-18.1100 9808, Gb, All others
scored in our R A P D analysis, 219 in total, 9821, 9313,
9847, 9893,
were polymorphic. We were able to closely
9831, 9835,
compare R A P D profiles from the present
9895, 9896,
study with previously obtained data (Bartish 9897, 9898
et al. 1999a) since the same individual plants in OPD-18.1200 9894, 9128, 9866 All others
population G b were included in both studies, OPD-18.1500 9804, 9837 All others
as well as the same set of primers. In the
present study, 109 not previously scored
markers were encountered, none of which, in all individual plants from the same popula-
however, occurred in population Gb. The tion, or in all but one individual. The ampli-
numbers of loci with markers scored in at fication of such markers differed considerably
least one individual plant within population among primers, with 50% of them generated
G b in the previous and present studies were by primer OPD-18. Three of these markers
very similar: 95 and 97, respectively. (OPA-08.1650, OPB-04.430 and OPB-04.1700)
W e found 16 markers with fixed R A P D were specific for H. rhamnoides ssp. carpatica.
allele presence or absence within all popula- Several other taxa (H. gyantsensis, H. rharn-
tions included in the analysis (Table 2). A noides ssp. rharnnoides a n d ssp. sinensis,
marker allele was regarded as fixed if the H. salicifolia, H. tibetana) had one or two
correspondent marker was present or absent taxon-specific markers.
I. V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae 91

Gene diversity within populations. Esti-
mates of gene diversity within populations
are strongly affected by whether all loci in the
data set are included or not (Table 3). How-
ever, the rank order o f populations remains
rather similar, and Pearson correlation coeffi-
cients are high for comparisons between diver-
sity values obtained in different ways (P <
0.001 in all cases). F o r instance, r = 0.882 for
comparison between gene diversity estimates
based on all loci (Hjl in Table 3) and on a
corrected number of loci (Hj3), respectively.
The correspondent comparison between esti-
mations of polymorphism yields r = 0.894.
Comparison between gene diversity and per-
centage polymorphic loci, including all loci,
yielded r = 0.966, whereas the corresponding
analysis between estimations based on the
corrected number of loci (P%3 and Hj3 in
Table 3), yielded r = 0.978.
The within-population gene diversity esti-
mates and the percentages of polymorphic loci

Table 3. Within-population (I) and within-family (II) gene diversity according to Lynch and Milligan
(1994) (Hj)* and percentage polymorphic RAPD loci (P%). Each of these parameters was calculated in at
least two ways: (1) inlcuding all 219 loci, (2) including only loci that are polymorphic within species, (3)
including loci that are polymorphic within the species (population, if there was only one) as well as two
times the number of loci that are fixed for marker presence in that species (population). Populations 9805
and 9808 are here treated as two separate species

Hjl Hj2 Hj3 P% 1 P% 2 P% 3

I
H. goniocarpa
ssp. goniocarpa (9806) 0.151 0.217 0.206 45.2 65.1 61.9
ssp. litangensis (0001) 0.166 0.239 0.228 52.1 75.0 71.3
H. gyantsensis (9128, 9866, 9894) 0.115 - 0.195 36.1 - 61.2
H. neurocarpa
ssp. neurocarpa (9837) 0.083 0.152 0.132 21.5 39.2 34.1
ssp. stellatopilosa (9807) 0.087 0.159 0.138 26.5 48.3 42.0
H. rhamnoides
ssp. fluviatilis (9313) 0.097 0.126 0.115 24.7 32.0 29.2
ssp. mongolica (9801) 0.087 0.112 0.102 26.9 34.9 31.9
ssp. rhamnoides (Gb) 0.104 0.128 0.117 31.1 40.2 36.8
ssp. sinensis (9805) 0.064 - 0.121 20.6 - 29.1
ssp. turkestanica (9810) 0.118 0.153 0.140 36.1 46.7 42.7
ssp. yunnanensis (9808) 0.076 - 0.143 26.5 - 50.0
H. salicifolia (9804) 0.058 - 0.108 19.2 - 35.6
H. tibetana (9803, 9838) 0.146 - 0.245 51.1 - 86.5
II
H. rhamnoides
ssp. carpatica (9895) 0.088 0.108 0.099 23.8 30.8 28.1
ssp. carpatica (9896) 0.104 0.105 0.096 24.2 31.4 28.7
ssp. carpatica (9897) 0.088 0.107 0.098 23.8 30.8 28.1
ssp. carpatica (9898) 0.104 0.134 0.122 28.8 37.3 34.1
ssp. caucasica (9847) 0.074 0.070 0.064 16.9 32.0 29.2
ssp. caucasica (9893) 0.072 0.082 0.075 18.3 30.2 27.6
ssp. caucasica (9831) 0.074 0.074 0.069 17.4 32.0 29.2
ssp. caucasica (9835) 0.072 0.069 0.063 14.2 30.2 27.6
ssp.fluviatilis (9821) 0.046 0.060 0.054 12.8 16.6 15.1
* Standard errors for Hjl-values were 0.00008-0.00018, and for Hj2- and Hj3-values 0.0007-0.0008
92 I.V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae

varied considerably between populations. The
highest diversities were observed in popula-
tions of H. goniocarpa (both subspecies) and
H. tibetana with Hi3 values between 0.206 and
0.245 (Table 3). Hippophae salicifolia (popula-
tion 9804) was the least diverse among species
(Hi3 = 0.108).
Cluster analysis. Relationships resolved by
the cluster analysis (Fig. 2) were in good
correspondence with one of the most recent
taxonomic treatises of Hippophae (Lian and
Chen 1993). Six of the subspecies of H. rhamno-
ides (carpatica, caucasica,fluviatilis, mongolica,
rhamnoides and turkestanica) formed a well
supported cluster (bootstrap support 96%).
The remaining two subspecies, sinensis and
yunnanensis, are more problematic with a very
weak support for their positions (21% and
31%, respectively) in the dendrogram. In
addition, H. gonioearpa showed some affinity
to the H. rhamnoides group. The two species
H. tibetana and H. salicifolia occupy relatively
isolated positions, and do not appear to be
closely related, neither to each other nor to any
other taxon.
Several clusters within the main H. rhamno-
ides group had very high values of bootstrap
support in our dendrogram (Fig. 2) and there-
fore appear quite reliable. A m o n g these are
three clusters comprising all the populations

9897 H. rhamnoidesssp. carpatica

57.0~
9896 H. rhamnoidesssp. carpatica
100.0
~ ~ ) 9898H. rharnnoidesssp. carpatica
55.0
~) 9895 H. rhamnoidesssp. carpatica
C) Gb H. rhamnoidesssp. rhamnoides

63.0 70.0 U 9847 H. rhamnoidesssp. caucasica

100.0[ - " - " ~ 9893 H. rhamnoidesssp. caucasica
9831 H. rhamnoidesssp. caucasica
9835 H. rharnnoidesssp. caucasica
96.0
100.____~ 9821 H. rhamnoidesssp. fluviatilis
9313 H. rhamnoidesssp. fluviatilis
31.0
100.0 9801 H. rhamnoidesssp. mongolica
21.0 9810 H. rhamnoidesssp. turkestanica

51.0 O 9808 H. rhamnoidesssp. yunnanensis

I
56.0 [----] 9805 H. rhamnoidesssp. sinensis
,& 9806 H. goniocarpassp. goniocarpa
68.0
i 0001 H. goniocarpassp. litangensis
47.0~"~ 9128,9894,9866 H. gyantsensis
99.0
9837 H. neurocarpassp. neurocarpa
9807 H. neurocarpassp. stellatopilosa
9803,9838 H. tibetana
9804 H. salicifolia

Fig. 2. Neighbour-joining cluster analysis of RAPD-based genetic distance data in the genus Hippophae.Node
numbers indicate the percentage of node presence in 100 bootstrap subreplicates
I. V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae
of ssp. carpatica, of ssp. caucasica, and of ssp.
fluviatilis, respectively (100%), one cluster with
ssp. mongoIica together with ssp. turkestanica
(100%), and one cluster with ssp. fluviatilis
together with ssp. caucasica (99%). By con-
trast, the support is considerably weaker
(55%) for the group with ssp. carpatica and
ssp. rhamnoides.
Finally, the two subspecies of H. neurocar-
pa have marginally good support for their
clade (58%), whereas the two subspecies of
H. goniocarpa do not seem to belong to the
same lineage (Fig. 2).
Principal co-ordinate analysis. As in the
cluster analysis, a principal co-ordinate anal-
ysis (PCO) revealed close genetic relationships
among populations from the same subspecies
(ssp. carpatica and ssp. caucasica, but note the
somewhat more distant placement of the ssp.
fluviatilis populations 9313 and 9821) and
between ssp. fluviatilis - caucasica, and ssp.
mongolica- turkestanica (Fig. 3). In accor-
dance with the cluster analysis, H. salicifolia
(9804) is clearly differentiated from all other
taxa. However, several taxa were placed con-
siderably closer to each other in the PCO
compared to in the cluster analysis. For
example, population Gb (ssp. rhamnoides)
appears very close to the group of Asiatic
H. rhamnoides subspecies (mongolica, 9801,
0.049
3
I Table 4. Analyses of molecular variance (AMOVA)
PCO3
! -0.116 -0.I22
PCO2
O86
0.047
Fig. 3. Three-dimensional representation of a princi-
pal co-ordinate analysis of genetic relationships
between populations of Hippophae (see Table 1 for
collection data). Percentage explained variability was:
PCO1 41.5%, PCO2 20.2%, and PCO3 12.1%.
Labelling of taxa as in Fig. 2
93
and turkestanica, 9810). The sample of pooled
H. gyantsensis populations was close to the
sample of pooled H. tibetana populations, and
the two subspecies ofH. goniocarpa were placed
close to each other. In contrast, the group of
H. rhamnoides ssp. carpatica populations was
rather far removed from the other groups.
AMOVA analyses. Several analyses of
molecular variance were calculated on different
levels, and including different parts of the
dataset (Table 4). First, one A M O V A was
carried out on the total plant sample (exclud-
ing populations consisting of full-sib or half-
sib families), using species as a group variable.
This indicated that 48.6% of the total vari-
ability resides between species and 51.4%
within. Another A M O V A was carried out on
the same data set but using subspecies as
a group variable. This time 59.6% of the
variability was found between subspecies and
40.4% within.
Three species were represented by more
than one subspecies and could therefore be
analysed for intraspecific partitioning of mo-
lecular variance. We found that 37.3%, 51.5%
and 42.8% of the total variance was allocated
between subspecies within H. goniocarpa,
H. neurocarpa, and H. rhamnoides, respectively
(Table 4). For H. rhamnoides, only the sub-
species mongolica, rhamnoides and turkestanica
using 219 RAPD loci, conducted among and
within species and subspecies of the entire genus
Hippophae; and among and within subspecies of
H. goniocarpa, H. neurocarpa and H. rhamnoides.
Percentual distribution of the variance components
is given for each of two levels. All estimations are
significant (P < 0.001)
Source of variation
Among Within Among Within
species species subsp, subsp.
Hippophae 48.6 51.4 59.6 40.4
H. goniocarpa 37.3 62.7
H. neurocarpa 51.5 48.5
H. rhamnoides 42.8 57.2
94 I.V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae
were included in the analysis since sinensis and
yunnanensis may not belong here altogether,
and the other three subspecies had been
sampled as full-sib or half-sib families.
Correspondence with geographic distances.
We found a significant correlation between
geographic and genetic distances when the
whole plant material was analysed (r = 0.398,
P = 0.0000) and when the 12 populations
(families) of H. rhamnoides were analysed
(r = 0.345, P = 0.0000) (excluding family
9821, which was derived from an experimental
crossing, and populations 9805 and 9808
which may not belong to this species). By
contrast, there was no significant correlation in
the analysis of eight populations from separate
species (r = -0.175, P = 0.2943).
Plotting pairwise genetic distances within
H. rhamnoides against correspondent geo-
graphic distances revealed four distinct clusters
of pairwise comparisons (Fig. 4). Cluster A
represents comparisons between populations
0.15
19
o
c'-
N
o9 0.10
"O
.o D
19
e-
19
(:J)
/
o9 j"
19 0.05 /
g Discussion
0.00
0 2000 4000 6000 8000
Geographical distance (km)
Fig. 4. Plot of pairwise comparisons of genetic and
geographic distances for 12 populations ofH. rhamnoi-
des. Regression analyses were calculated on all
comparisons (I; y = 0.072 + 0.91 - 10-Sx; R 2 =
0.119), and on comparisons included in clusters A
and C only (II; y = 0.018 + 0.25. 10-4X; R 2 =
0.922)
less than 700 km apart, whereas the other
three clusters (B-D) contain geographically
more distant comparisons. Cluster B contains
pairwise comparisons between families of
H. rhamnoides ssp. carpatica on the one hand,
and ssp. caucasica and ssp. fluviatilis on the
other hand. Cluster D contains comparisons
between two Asiatic subspecies of H. rhamno-
ides (mongolica and turkestanica) on the one
hand and two subspecies from Southern and
Northern Europe (fluviatilis and rhamnoides)
on the other hand. All other comparisons are
represented in clusters A and C except for one
outlier caused by a comparison between pop-
ulations of subspecies mongolica and turkesta-
nica, which are genetically very similar in spite
of growing 1315 km apart.
Spatial autocorrelation analysis, performed
only on populations (families) of H. rhamnoi-
des, revealed a relatively close association of
expected and observed Ra when the geographic
distances between populations (families) ran-
ged from 0 to 700 kin, and from 1900 to
4700 km (Fig. 5). Considerable deviations
from expected values of Ra were observed
within ranges from 700 to 1900 km (these
distances mostly correspond to cluster B in
Fig. 4) and above 4700 km (cluster D). This
analysis shows that pairwise comparisons
within clusters A and C represent the closest
approximation to an isolation by distance
model for our data set.
Fixed alleles. Only four out of 156 polymor-
phic RAPD loci had fixed allele presence/
absence differences between subspecies of
H. rhamnoides in a previous R A P D study of
genetic diversity in this species (Bartish et al.
199%). By contrast, 16 loci with fixed alleles
between populations were scored in the present
study (Table 2). This number may even be
underestimated, since some apparently homol-
ogous D N A fragments (bands with the same
size and amplified by the same primer) may, in
fact, be heterologous, especially when geneti-
cally distant taxa are included in the same
I. V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae 95

0,8 study, we refrain from a detailed discussion of

systematic and phylogenetic aspects and have
0,6
essentially followed the taxonomy suggested
by Rousi (1971). However, we believe it is
worthwhile to point out some results that are
0.4, i ~
---"(~--"
Observed
Expected either in accordance with previously published
studies, or disagree seriously.
_~ 0,2" Q In general, our results are in good corre-
"Q, spondence with the taxonomy suggested by
o
O Lian and Chen (1993), who considered that: i)
0,0-
< H. goniocarpa, H. gyantsensis, H. neurocarpa
and H. tibetana form a group (section Gyants-
-0.2 i i
ensis) separately from the H. rhamnoides,
' ' b" Q~. ...o..~ H. salicifolia and H. sinensis group (section
-0.4 A , ~i B i i i
C
I i
~I D U Hippophae); ii) H. rhamnoides consists of
, - ,ooooooooo oo oo o o S & oo 88
o o o o ~- o o o&
& & six subspecies (carpatica, caucasica, fluviatilis,
COI~ 0 0 ~0 03 ~o ~ kO.O0
O oooooo ooo mongolica, rhamnoides and turkestanica); iii)
.... caca ~ ,<-ao H. sinensis consists of two subspecies, sinensis
and yunnanensis, which previously were in-
Distance class
cluded into H. rhamnoides (Rousi 1971). It is
Fig. 5. Correlogram of autocorrelation coefficients clear from our data that H. gyantsensis is
for 18 distance classes in 12 populations ofH. rhamno- separate from H. rhamnoides, and we thus
ides. Filled circles indicate observed autocorrelation follow the taxonomy of Lian and Chen here
coefficients, open circles indicate expected autocorre- instead of Rousi (who regarded it as a
lation coefficients provided that genetic and geo- subspecies of H. rhamnoides). There is still a
graphic distances are perfectly correlated. Letters dispute concerning the status of ssp. sinensis
(A-D), denoting different distance ranges, corre- and ssp. yunnanensis, which were again con-
spond to those used for labelling the clusters in Fig. 4
sidered subspecies of H. rhamnoides in the
most recent systematic overview of the genus
analysis (Rieseberg 1996). Loci with fixed (Lian et al. 1998). However, our R A P D data
alleles between species or subspecies are espe- do not support the notion that ssp. sinensis and
cially valuable for phylogenetic reconstruction ssp. yunnanensis are closely related, and it is
based on character state differences, such as not clear whether they should be kept within
parsimony or maximum likelihood analyses. H. rhamnoides. These matters will be dealt with
However, we refrained from using this kind of in more detail later, incorporating data also
analyses since (1) we had no data to unambig- from amplified chloroplast D N A RFLPs (Bar-
uously support the notion that the markers tish et al., unpubl, data).
obtained with our 16 fixed loci really were There was also some correspondence be-
homologous, and (2) plant samples were tween our results and a previously published
relatively small. parsimony analysis based on morphological
T a x o n o m y of Hippophae. Phylogeny and characters (Hyv6nen 1996), like the clustering
taxonomy in Hippophae is rather complicated of ssp. rhamnoides and ssp. carpatica as well as
as can be concluded from the existence of H. gyantsensis and H. neurocarpa. However, in
several highly dissimilar taxonomic systems, our analysis, H. gyantsensis and H. rhamnoides
the latest of which were suggested by Rousi ssp. turkestanica were not closely related,
(1971), Avdeyev (1983), Lian and Chen (1993), which instead Hyv6nen's (1996) morphologi-
Hyv6nen (1996) and Lian et al. (1998). Due to cal study suggests. We also found no evidence
the restricted plant material analysed in our of a close relationship between H. salicifolia on
96 I.V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae
the one hand and the H. rhamnoides subspecies
sinensis and yunnanensis on the other hand.
Moreover, Hyv6nen's data suggest that H. tibe-
tana and H. neurocarpa are much more closely
related to each other than most other subspe-
cies of H. rhamnoides are, which is highly
unlikely according to previous morphology-
based studies (Rousi 1971), isozyme analysis
(Yao and Tigerstedt 1993), and our results.
Additional research and application of other
molecular marker systems in the genus Hip-
pophae may be necessary to clarify these
discrepancies.
To conclude, the present analysis is con-
cordant with the previous isoenzyme study by
Yao and Tigerstedt (1993) and strongly sup-
ports the systematic treatment of Hippophae
developed by Rousi (1971) and Lian et al.
(1993, 1998). By contrast, our data do not
support the treatment of Hippophae as a genus
with only one (Avdeyev 1983) or two (Hyv6-
hen 1996) species.
Reticulate evolution in Hippophae. Based
on several shared morphological characters
and a distribution which is sympatric with that
of two putative parental species, Lian and
Chen (1993) and Lian et al. (1995) suggested
that H. goniocarpa ssp. goniocarpa and ssp.
litangensis have originated from interspecific
hybridisation between H. neurocarpa ssp. neu-
rocarpa and H. rhamnoides ssp. sinensis, and
between H. neurocarpa ssp. stellatopilosa and
H. rhamnoides ssp. yunnanensis, respectively.
Our fixed marker data support this hybridisa-
tion theory for both taxa. Thus, we found
seven RAPD markers with fixed absence/
presence in one or both subspecies of H. neu-
rocarpa and which had the reciprocal absence/
presence in all populations of H. rhamnoides,
including ssp. sinensis and ssp. yunnanensis.
These seven markers had a frequency of about
0.5 within both or at least one of the popula-
tions of H. goniocarpa (9806 and 0001, data
not shown). Besides, RAPD markers, recipro-
cally fixed in the suggested parental taxa of
both subspecies (41 and 36 markers for the
putative parents of ssp. goniocarpa and ssp.
litangensis, respectively), occurred in approxi-
mately the same number in populations of
H. goniocarpa as in one or the other parent,
and many markers were not fixed. Further
pointing to a hybridogenous origin, ssp.
goniocarpa and ssp. litangensis were among
the most diverse taxa in our study regarding
percentage of polymorphic markers and with-
in-population gene diversity. However, results
of the PCO analysis (Fig. 3) indicate that
H. goniocarpa ssp. goniocarpa and ssp. litan-
gensis are closely related to one only of their
putative parental taxa: H. rhamnoides ssp.
sinensis and ssp. yunnanensis, respectively,
since they clustered much closer to these taxa
than to any other (including the other two
putative parental taxa: H. neurocarpa ssp.
neurocarpa and ssp. stelIatopilosa). Conse-
quently, we cannot exclude the possibility that
the two H. goniocarpa subspecies have been
derived mainly from the two H. rhamnoides
subspecies with only minor introgression from
e.g. the two H. neurocarpa subspecies. How-
ever, irrespective of their origination, we do
not think treating these distinctive, alleged
hybrid taxa as subspecies of one and the same
species is justifiable.
Although the evolutionary history of
H. rhamnoides ssp. sinensis and ssp. yunnanen-
sis is not clear, at least some genetic introgres-
sion to these taxa from a phylogenetically
distant group of species, like H. gyantsensis,
H. neurocarpa and H. salicifolia, can be
suspected from analysis of markers diagnostic
for differentiation between this group and
H. rhamnoides. For example, marker OPD-
18.800 was present and fixed in H. salicifolia
and in ssp. yunnanensis, but absent in all other
populations (Table 2), and marker OPD-
18.1400 was absent in H. salicifolia, in ssp.
sinensis and ssp. yunnanensis, and in H. neuro-
carpa ssp. neurocarpa, but present and fixed in
all other (except H. goniocarpa ssp. litangensis)
populations. The clustering of these two sub-
species together with H. salieifolia in a parsi-
mony analysis (Hyv6nen 1996), and separating
them from the group of remaining H. rhamnoi-
des subspecies (Lian and Chen 1993), demon-
strates how introgression is indicated also by
I. V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae
morphological characters. Moreover, analysis
of phylogenetic relationships between chloro-
plast genomes of the same group of taxa as
used in the present study (Bartish et al.,
manuscript submitted.) strongly suggests con-
siderable differentiation of ssp. sinensis from
other species. In conclusion, the systematic
position of ssp. sinensis and ssp. yunnanensis
within H. rhamnoides is not well supported, and
treating them as separate species appears to be
a better alternative.
Gene diversity estimates. The within-pop-
ulation gene diversity estimate for the
H. rhamnoides ssp. rhamnoides population Gb
was, in a previous study (Bartish et al. 1999a),
approximately twice as high as the value
obtained in the present study, when all scored
loci were included (0.201 and 0.104, respec-
tively). By contrast, there was only a slight
difference between the corresponding estimates
obtained after exclusion of loci that lacked
markers in this particular population (0.240
and 0.234, respectively).
Considerable underestimation of gene di-
versity values can thus be avoided by not
including loci with fixed marker absences. This
appears especially valid in studies of popula-
tions from highly diverse taxa (Bartish et al.,
2000). It should be kept in mind, however, that
gene diversity values may still be underesti-
mated due to the presence of loci with high
marker frequencies in small plant samples
(Lynch and Milligan 1994), and erroneously
scoring some co-dominant loci as dominant
(Bartish, unpublished). Consequently, compar-
isons of the actual gene diversity estimates
across studies or marker systems may not be
valid, whereas the relative order of estimates
obtained within a study can be considered as
more reliable. In the present study, H. gonio-
carpa and H. tibetana were the most diverse
species, and H. salicifolia the least diverse, with
an almost two-fold difference in within-popu-
lation gene diversity estimates. This is a rather
modest range of variability compared to the
10-fold differences reported in the isozyme
study by Yao and Tigerstedt (1993). In their
study, ssp. rhamnoides and ssp. sinensis were
97
represented by 9 and 11 populations, respec-
tively, and this range of variability was actu-
ally found within one subspecies (ssp.
rhamnoides). These results suggest that we
need to analyse several more natural popula-
tions from each of our species before making
safe conclusions about levels of within-popu-
lation diversity.
Partitioning of genetic variance. Applica-
tion of AMOVA in our material revealed that
a substantial part of the molecular variability
(from 37.3% to 59.6%) is allocated among
subspecies, both within the genus as a whole
and within separate species. Variability among
species within the genus is also of this same
magnitude (48.6%). Variability among sub-
species of H. rhamnoides (ssp. mongolica,
ssp. rhamnoides and ssp. turkestanica) in the
present study (42.8%) was the result reported
in an isozyme study (Yao and Tigerstedt 1993):
41.6% of the variability within H. rhamnoides
and 55.6% of the total variability was allocat-
ed among subspecies (ssp. mongolica, ssp.
rhamnoides, ssp. sinensis and ssp. turkestanica)
and species (H. neurocarpa, H. rhamnoides and
H. tibetana), respectively.
Obviously the partitioning of molecular
variance within and among populations is
directly influenced by the overall diversity of
the plant sample. Within-population variabil-
ity accounted for 85% of the total variance
within ssp. rhamnoides (Bartish et al. 1999a),
but only for 40.4% when the analysis was
enlarged to the entire genus Hippophae (pre-
sent study).
Geographic distances. The published evi-
dence for an isolation-by-distance model re-
vealed with D N A markers has increased
considerably in recent years (e.g. Dawson et al.
1995, Raybould et al. 1996, Le Corre et al.
1997, Comes and Abbott 2000, Reusch
et al. 2000). The present study shows that the
association between genetic and geographic
distances can change considerably along the
range of geographic distances (Fig. 5). Thus a
series of relatively high positive correlations at
0-700 km show good concordance between
observed and expected Ra values. These are
98 I.V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae
followed by a series of strong negative corre-
lations at 700-1900 km which are much lower
than the expected Ra values. In the range of
1900 to 4700 km, observed and expected Ra
values are instead quite similar. Finally, the
observed Ra values are much higher than the
expected Ra values for comparisons among
populations from the geographically most
distant (>4700 km) subspecies (mongolica
and turkestanica on the one hand compared
tofluviatilis and rharnnoides on the other hand;
cluster D, Fig. 4) which may indicate a devia-
tion from the isolation-by-distance model due
to relatively recent gene flow between these
subspecies. Climatic oscillations in the late
Quaternary may have caused considerable
changes in the distributional ranges of the
H. rharnnoides subspecies, and may have cre-
ated natural conditions amenable for genetic
exchange between at present geographically
distant taxa (Bartish and Jeppsson, in press).
Pairwise comparisons between families of
ssp. carpatica and population 9313 (ssp. flu-
viatilis), and between families of ssp. carpatica
and ssp. caucasica (cluster B, Fig. 4), revealed
genetic distances that are higher than expected
from an isolation-by-distance model. It should
be noted that ssp. carpatica is the only
subspecies of H. rhamnoides, which grows
both on seashores and in mountains. It is also
possible that extant populations of ssp. car-
patica, on the one hand, and ssp. caucasica and
ssp. fluviatilis, on the other hand, dispersed
from different glacial refugia. Studies of post-
glacial dispersal routes, as well as investiga-
tions of controlled crosses between ssp.
carpatica and other subspecies of H. rhamno-
ides could possibly yield important informa-
tion on these matters.
Finally, it should be mentioned that re-
moval of comparisons included in clusters B
and D (Fig. 4) yields a very close correlation
among the remaining genetic and geographic
distances in H. rhamnoides: r = 0.940, P =
0.0000. This indicates that isolation-by-dis-
tance may be considered as the prevailing
model to explain population differentiation
within this widely distributed, wind-pollinated
and outcrossing species.
No significant correlation between genetic
and geographic distances was found when
populations from different species were includ-
ed in the same analysis. This may indicate
a relatively random spatial structure in the
distribution of species, and a lack of major
interspecific gene flow within Hippophae de-
spite the evidence for interspecific hybridisa-
tion between at least some of the species (H.
goniocarpa, H. neurocarpa and H. rhamnoides)
as indicated by both morphological (Lian et al.
1995) and molecular (present study) data.
Valuable help with collection of material was
received from Mahir Kucuk, Rolv Lundheim,
Paulina Mladin and Murtazali Rabadanov. Help-
ful remarks on an earlier version of the manuscript
were obtained by two anonymous reviewers. Fi-
nancial support from the Swedish Council for
Forestry and Agricultural Research to IVB and
HN is greatly appreciated.
I. V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae 99

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tt3

e3

,e3

t~

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t~
3
c~

D
tt~

r/)

~D

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Z
c~

m.
100 I.V. Bartish et al.: Inter- and intraspecific genetic variation in Hippophae
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Address of the authors: Igor V. Bartish, Institute
of Botany, Academy of Sciences, C2-25243 Pruho-
nice 1, Czech Republic. Niklas Jeppsson, Galina I.
Bartish, Hilde Nybom, Balsgfird-Department of
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of Agricultural Sciences, Fjfilkestadsvfigen 123-1,
SE-291 94 Kristianstad, Sweden. Rongsen Lu,
Chengdu Institute of Biology, Chinese Academy
of Sciences, P.O. Box 416, Chengdu, Sichuan 610
041, China.