Journal of Fish Biology (1996) 48, 54–67

Variation in mitochondrial DNA and post-glacial

colonization of north western Europe by brown trout
R. A. H, A. F  M. A. MC
School of Biology and Biochemistry, The Queen’s University of Belfast,
Belfast BT7 INN, U.K.

(Received 12 October 1994, Accepted 7 March 1995)

A purified mitochondrial DNA (mtDNA) probe was used to examine restriction fragment
length polymorphisms produced by six restriction enzymes (Xba I, Eco RV, Ava II, Hinf I, Hae
III, Mbo I) in 915 brown trout from western Europe. A total of 20 composite haplotypes were
found with one to seven haplotypes in individual populations. Icelandic trout samples from
north, south, east, and west coast drainages showed only a single common haplotype in
contrast to the high level of polymorphism found in Irish and Scottish populations. The
phylogeny of mtDNA haplotypes and the pattern of haplotype distribution suggests that
post-glacial colonization of brown trout in NW Europe was more complex than the dual
colonization model which has been proposed on the basis of differential LDH-5* allele
distribution. For example, Lough Melvin (Ireland) appears to have been independently
colonised by three distinct types of trout. ? 1996 The Fisheries Society of the British Isles

Key words: brown trout; Salmo trutta; mitochondrial DNA; LDH-5*; post-glacial
colonization.

INTRODUCTION
Brown trout Salmo trutta L., has caught the attention of biologists by virtue of
the extensive variability and plasticity which it shows in many aspects of its
morphology, ecology and behaviour. From the time of Linnaeus about 50
species have been described for what is commonly referred to as brown trout,
including 10 species described by Günther for Britain and Ireland. The tendency
during this century has been for biologists to regard all of these forms as
belonging to a single polytypic species.
During the past two decades, electrophoretic analysis of enzyme variation has
provided extensive evidence of genetic differentiation within and among brown
trout populations (Ferguson, 1989; Skaala, 1992; Cross et al., 1992; Hansen
et al., 1993). Examination of loci common to various studies of brown trout
revealed some 60% of the observed variability to be distributed among popula-
tions in Atlantic drainages (Ferguson, 1989). Part of this considerable genetic
diversity of NW European brown trout populations could have evolved since
these waters were colonized at the end of the last Ice Age (10–15 000 years ago).
In addition, it could be the result of independent post-glacial colonization by
already distinct types of trout which diverged from each other during, or prior
to, the last glaciation. Based on the differential occurrence of two alleles at
LDH-5* it was suggested that NW Europe was independently colonized by two
races of brown trout (Ferguson & Fleming, 1983; Hamilton et al., 1989). The
first colonist was the ancestral race, so-called because of its possession of the
54
0022–1112/96/010054+14 $12.00/0 ? 1996 The Fisheries Society of the British Isles
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ancestral LDH-5*100 allele. At a later time the modern race, typified by the
LDH-5*90 allele, colonized NW Europe and replaced the ancestral race in those
waters to which it was able to gain access. The ancestral race may have spread
postglacially from a refuge in Biscay drainages while the modern race may have
evolved in a refuge in the current Baltic or White sea area.
Studies on mtDNA, as compared with nuclear coded genes, have a distinct
advantage in studying population phylogeny. During population isolation (e.g.
following glacial advance), divergence occurs in both the nuclear and
mitochondrial genomes as a result of drift and selection. However, once the
populations come into secondary contact (e.g. on glacial retreat), nuclear
divergence is liable to be lost due to gene flow and recombination. By contrast
the asexual inheritance of mtDNA permits the organization of individuals into
matriarchal lineages.
To date relatively few studies of restriction fragment length polymorphism
(RFLP) of mtDNA variation have been carried out on brown trout populations
(Palva, 1986; Gyllensten & Wilson, 1987; McVeigh & Ferguson, 1988; Hynes
et al., 1989; Ovenden et al., 1993; Bembo et al., 1994). In general, these have
been preliminary investigations aimed primarily at developing and testing new
techniques. Nevertheless, they revealed considerable mtDNA variation within
and among brown trout populations, providing corroborative evidence of the
high genetic variability demonstrated by protein studies.
As a test of the dual colonization hypothesis, this study reports on mtDNA
diversity in brown trout populations from NW Europe, using a mtDNA probe
for the rapid detection of mtDNA RFLPs in total DNA samples restricted with
four, five and six base pair cutting enzymes.

MATERIALS AND METHODS

Frozen or ethanol preserved muscle tissue was available from the brown trout samples
listed in Table I. Genomic DNA was isolated as described by Taggart et al. (1992).
Genomic DNA extracts were digested with six restriction enzymes; Xba I, Eco RV (6
base); Ava II, Hinf I (5 base); Hae III, Mbo I (4 base), according to the manufacturer’s
directions. These enzymes were chosen because (with the exception of Eco RV) they had
been found previously to be polymorphic in preliminary studies of Irish brown trout
populations involving 21 enzymes (McVeigh, 1989; Hynes et al., 1989). Eco RV was
found to be polymorphic in Norwegian brown trout (G. Dahle pers. comm.).
Xba I, Eco RV and Ava II restricted samples (2 ìg lane "1) were size separated on 0·9%
agarose gels, with up to 90 samples in three rows being analysed per 25 cm long gel. Hinf
I, Hae III and Mbo I (3 ìg lane "1) fragments were separated on 1·6% agarose gels, with
a total of 30 samples being analysed per 25 cm long gel. All electrophoresis was carried
out at room temperature in 0·5#TBE buffer (Maniatis et al., 1982), in non-recirculating
buffer tanks. Samples on 0·9% gels were electrophoresed for 4–5 h at 4 V cm "1, while
samples on 1·6% gels were electrophoresed for 20–22 h at 2·3 V cm "1. In the latter case,
buffer exhaustion was prevented by draining and mixing the buffer two to three times
during running. Following depurination, denaturation and neutralization the DNA was
transferred to Hybond-N nylon membrane (Amersham International) by Southern
blotting according to the manufacturer’s recommendations. The DNA was fixed
subsequently to the membrane by baking for 2 h at 80) C.
Approximately 10 ng of total brown trout mtDNA, purified on a caesium chloride
gradient, was 32P labelled by random oligonucleotide priming as described by Dalgleish
(1987). Contaminating nuclear DNA present in the labelled mtDNA probe was removed
56 . .    .

through the hybridization of the probe with 50 ìg of competitor DNA (i.e. brown trout
total cellular DNA), in 1·5#SSPE (0·54  NaCl, 0·03  Na2H2PO4, 3 m EDTA Na2
salt; pH 7·7), for 45 min at 65) C. Prior to use the competitor DNA was extensively
sonicated to reduce the fragment size to <2 kb.
Filters were prehybridized for a minimum of 3 h in 1·5#SSPE, 6% polyethylene glycol
(PEG), 0·5% dried milk and 1% SDS (pH 7·7), at 65) C (Dalgleish, 1987). The total 10 ng
of oligolabelled mtDNA probe was added to hybridization solution and used to
simultaneously probe one to four filters. Following overnight hybridization at 65) C,
filters were washed once in 2#SSC (0·3  NaCl 0·03  Na3 citrate; pH 7·0) followed by
two washes in 0·2#SSC, 0·1% SDS. All washes were carried out for 20–30 min at 65) C.
Filters were finally rinsed in 1#SSC at room temperature and autoradiographed for 2–5
days at "70) C in the presence of intensifying screens.
Data on LDH-5* allele frequencies were taken from Hamilton et al. (1989) or, where
not available, samples were screened by the method described in that paper.
MtDNA restriction fragment data were analysed using the REAP (Mc Elroy et al.,
1991) and PHYLIP (Version 3.3; provided by J. Felsenstein, Department of Genetics,
SK-50, University of Washington, Seattle, WA 98195, U.S.A.) computer packages using
individual programs and methods as identified in the results section.

RESULTS
Table I lists the LDH-5*100 allele frequencies in the populations examined for
mtDNA RFLP variation. Ninety mtDNA restriction fragments were observed
among the 915 individuals sampled, representing a total of 394 nucleotides. This
corresponds to sampling some 2% of the mitochondrial genome. Patterns for
each enzyme were assigned upper case letters in order of discovery. A total of 20
composite haplotypes were found in the populations screened (Table II). Other
than for the unique haplotypes present in the Spanish and Mediterranean French
samples, there is no obvious geographical pattern in the occurrence of
haplotypes.
The haplotype and nucleotide diversity values within each of the populations
were calculated using the REAP DA program (Table III). While the majority of
populations examined exhibit several haplotypes, some, e.g. Melvin ferox and all
Icelandic brown trout populations, exhibited only a single haplotype. The
nucleotide divergence between haplotypes was calculated from restriction frag-
ment data using the REAP D program, following the method of Nei & Li (1979).
These values are summarized in the form of a dendrogram (Fig. 1) produced by
UPGMA cluster analysis. As expected, the most distinct haplotypes are XVIII
and XIX from the Mediterranean sample. Haplotype XII is also distinct and
was found in trout from the northern Scottish lakes, in several Irish Sea rivers
and in the Polish sample. The remaining haplotypes form three groups with each
group comprising a mixture of populations of both high and low LDH-5*100
frequency. Haplotypes found in gillaroo occur in all three groups and sonaghen
haplotypes in two of them.
Presence/absence fragment data were bootstrapped (Felsenstein, 1985) using
the PHYLIP SEQBOOT program, and the resulting data subsequently used to
construct unrooted Wagner parsimony trees of haplotype phylogeny using the
PHYLIP MIX program. Consensus trees were obtained for each bootstrapped
sample using the PHYLIP CONSENSE program. The resulting majority-rule
consensus tree is shown in Fig. 2 with associated bootstrap values. Most
groupings show low bootstrap support, i.e. they occur less than 50% in the
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T I. Location of samples examined and associated LDH-5*100 allele frequencies

LDH-5*100
Country Locality Population Notes
frequency

Ireland Erne Cladagh 0·77 1

Ireland Erne Owengarr 0·90 1
Ireland Movanagher Hatchery 0·08 2
Ireland Melvin Ferox 0·79 3
Ireland Melvin Sonaghen 0·02 3
Ireland Melvin Gillaroo 0·01 3
Ireland Antrim Glenariff Upper 0·27 4
Ireland Antrim Glenariff Lower 1 0·06 5
Ireland Antrim Glenariff Lower 2 0·09 5
Ireland Antrim Carnlough Upper 0·11 4
Ireland Antrim Carnlough Lower 0·00 5
Ireland Antrim Glencloy 0·07 5
Ireland Antrim Glenarm 0·02 5
Ireland Antrim Glynn 0·00 5
Ireland Galway Crumlin 0·16
Scotland Loch Ness 0·16
Scotland Sutherland 2 lakes 1·00 4
Iceland Veidivötn Snjoölduvatn 1·00
Iceland Veidivötn Onytavatn 1·00
Iceland Veidivötn Litlisjór 1·00
Iceland Veidivötn Stora Fossvatn 1·00
Iceland Veidivötn Litla Fossvatn 1·00
Iceland Veidivötn Langavatn 1·00
Iceland Veidivötn Litla Skálavatn 1·00
Iceland north Gautlandalaekur 0·87
Iceland north Svartá 0·52
Iceland north Laxa 0·20
Iceland north Máslaekur 0·65
Iceland west Thingvallavatn 1·00
Iceland east Skriduvatn 0·75
Sweden Kallsjön 0·37 6
Sweden Fälpfjälltjärnarna 0·06 6
Sweden Blanktjärn 0·28
Poland Leba 0·00
Denmark Jylland 4 locations 0·00 7
France Mediterranean Tes 1·00
Spain Asturias Esqvero 1·00

Notes:
1. Rivers not known to be stocked.
2. Hatchery established in 1968 from eggs imported from England and Denmark.
3. Ferox, sonaghen and gillaroo are sympatric reproductively isolated brown trout populations in
Lough Melvin. (See Ferguson and Taggart, 1991, for details.)
4. Trout populations isolated above impassable water falls.
5. Trout populations consisting of a substantial anadromous (sea trout) component.
6. The Kallsjön and Fälpfjälltjärnarna fish were maintained in a hatchery and planted out in Lake Lilla
Bävervattnet from which these samples were obtained.
7. Samples from Harkjar Hatchery, Sejbaek, Rabis Baek and Trevad Mollebaek.
 T II. Frequency of mitochondrial DNA haplotypes in brown trout populations

Haplotype* I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII XIX XX

Population N
Ferox 37 1·00
Sonaghen 55 0·25 0·29 0·22 0·04 0·16 0·04
Gillaroo 47 0·13 0·04 0·23 0·46 0·06 0·06 0·02
Movanagher 50 0·54 0·04 0·04 0·02 0·14 0·18 0·04
Glenariff Upper 50 1·00
Glenariff Lower 1 50 0·42 0·42 0·16
Glenariff Lower 2 51 0·08 0·06 0·06 0·74 0·06
Glynn 102 0·92 0·01 0·07
Carnlough U 26 0·69 0·31
Carnlough L 28 0·64 0·15 0·21
Glencloy 15 0·40 0·40 0·20
Glenarm 39 0·51 0·10 0·39
Sutherland 32 1·00
Loch Ness 15 0·07 0·93
Iceland 130 1·0
Spain 7 0·43 0·43 0·14
Denmark 30 0·53 0·47
Sweden A+B 20 1·00
Sweden D 5 0·60 0·40
Erne 50 1·00
Leba, Poland 18 0·55 0·06 0·06 0·22 0·11
Crumlin 56 0·59 0·11 0·30
Tes, France 2 0·50 0·50
Total 915 0·19 0·02 0·02 0·002 0·01 0·39 0·02 0·003 0·02 0·06 0·01 0·18 0·05 0·01 0·003 0·003 0·001 0·001 0·001 0·02

Haplotypes comprise Xba I, Ava II, Hinf I, Hae III, Mbo I and Eco RV morphs respectively.
I, BAAAAA; II, ABAAAA; III, BADAAA; IV, BADAAB; V, ACEAAA; VI, ACAAAA; VII, ABAABA; VIII, ABAACA; IX, ACCBAB; X, ACCBAA; XI,
BABAAA; XII, ADACCB; XIII, BAADAA; XIV, ACFAAA; XV, AEACDA; XVI, CGCCAA; XVII, AGHAAA; XVIII, DFGEEA; XIX, DGGEEA; XX,
AGICAA.
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T III. Haplotype and nucleotide diversity within brown trout

populations

Population Haplotype diversity Nucleotide diversity

Ferox 0·0000&0·00000 0·000000

Sonaghen 0·7879&0·02129 0·003633
Gillaroo 0·7345&0·04822 0·003498
Movanagher 0·6645&0·06065 0·004461
Glenariff Upper 0·0000&0·00000 0·000000
Glenariff Lower 1 0·6343&0·03007 0·009271
Glenariff Lower 2 0·4369&0·08336 0·007196
Glynn 0·2462&0·06860 0·000880
Carnlough Upper 0·4431&0·07353 0·001993
Carnlough Lower 0·5397&0·08556 0·005685
Glencloy 0·6857&0·06147 0·009818
Glenarm 0·5938&0·04301 0·005203
Sutherland 0·0000&0·00000 0·000000
Lough Ness 0·1333&0·11230 0·001067
Iceland 0·0000&0·00000 0·000000
Denmark 0·5149&0·02678 0·000753
Sweden A+B 0·0000&0·00000 0·000000
Sweden D 0·6000&0·17527 0·004801
Erne 0·0000&0·00000 0·000000
Leba, Poland 0·6601&0·10196 0·006923
Crumlin 0·5591&0·04798 0·003757
Esqvero, Spain 0·7143&0·12675 0·004918
Tes, France 1·0000&0·50000 0·002188
Average 0·4325&0·00428 0·003306&0·0000004

bootstrap sampling. One pair of haplotypes share a common ancestor in

100% of trees: XVIII and XIX. These haplotypes were found only in the
Mediterranean sample. Otherwise only three clades show >85% bootstrap
support. One of these clades comprises haplotypes IX and X which are found in
a range of low LDH-5*100 populations from Denmark, Poland, Scotland,
Sweden and Ireland including gillaroo but not the sympatric sonaghen. A
second group (III and IV) is restricted to sonaghen and the fish farm which has
in the past incorporated some sonaghen in the broodstock. The other group (I,
II, III, IV, VII, VIII, XI, XII and XIII) comprises haplotypes found in mainly
Irish populations together with Scottish, Swedish and Polish ones, and includes
populations fixed for alternate LDH-5* alleles. There is no indication of a
relationship between geographical occurrence and phylogenetic relationship of
haplotypes other than those found in Mediterranean France and Spain.
As shown in Table IV, populations of low *100 frequency exhibit many more
mtDNA haplotypes than do populations with middle or high LDH-5*100
frequency. Apart from the haplotypes which were exclusive to the Spanish and
Mediterranean populations, all other populations from the high *100 frequency
group exhibited one of three haplotypes (I, VI or XII) all of which were found in
the low *100 frequency group as well. There was significant heterogeneity
(P<0·001) in mtDNA haplotype distribution between all pairwise comparisons
of low, middle and high LDH-5* groups as determined by the Monte Carlo ÷2
60 . .    .

Nucleotide divergence

0.030 0.025 0.020 0.015 0.010 0.005 0.000

I
XIII
III
IV
XI
II
VII
VIII
V
VI
XIV
IX
X
XVI
XVII
XV
XX
XII
XVIII
XIX
F. 1. Dendrogram based on UPGMA cluster analysis of nucleotide divergence values between mtDNA
haplotypes.

T IV. Presence of mtDNA lineages in populations grouped into high, middle and low
LDH-5*100 allele frequencies

LDH-5* group† Mitochondrial DNA lineages present

High I, VI, XII, XV, XVI, XVII, XVIII, XIX

Middle I, VI, X, XII
Low I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XX

†High, frequency from 0·75 to 1·00; middle, 0·26–0·74; low, 0·00–0·25.

simulation method of Roff & Bentzen (1989), using the MONTE program from
the REAP package.
The genetic relationships of the populations examined were calculated
using the REAP DA program and are shown in Fig. 3. Again other than for
 m      61

V
XIV

VII
VI
(63) II
X (40)
(17) XII
IX
VIII
(60)
(87) (55)
(33)
(93)
(45)
(25)
XX (38)
I
(25)
(50)
XVII (28) XV
(91)
(59) (47) IV

III
XVI
(100) XIII

XVIII
XIX XI
F. 2. Unrooted Wagner Parsimony majority-rule consensus tree of mitochondrial haplotypes. Values
given in brackets represent the % number of times that grouping occurred out of 1000 bootstrap
replicates.

the Mediterranean outgroup, there is no broad geographical pattern to the

population clustering, with for example Melvin ferox clustering with Swedish
brown trout populations, and Irish sea trout rivers grouping with Iceland.

DISCUSSION
The application of a mtDNA probe for the detection of mtDNA RFLP
variation permitted the use of total genomic DNA. This was readily extracted
from minimal amounts of frozen or alcohol preserved tissue, thus dispensing
with the need for fresh tissue. One disadvantage, however, of this approach
was the inability to detect small fragments. This meant that it was not possible
to determine restriction site changes for all enzymes and consequently only
fragment data were available for use in estimating phylogenetic relatedness.
The development of PCR amplification and subsequent RFLP analysis of
specific mitochondrial regions undoubtedly surpasses the speed and ease of use
of the probe method. Initial results (authors’ unpubl. data) suggest that 15 of the
20 haplotypes reported in this study can be detected through RFLP analysis of
the NADH dehydrogenase-1 (ND-1) and ND-5/6 mitochondrial regions, using
the primers of Cronin et al. (1993). One disadvantage of the PCR approach,
however, is that it does not examine variation across the entire mitochondrial
genome, consequently variants outside the selectively amplified regions remain
62 . .    .

Percent sequence divergence

2.500 2.000 1.500 1.000 0.500 0.000

Ferox
Sweden A and B
Erne
Sonaghen
Movanagher
Sweden D
Poland
Gillaroo
Glynn
Iceland
Carnlough Upper
Carnlough Lower
Glenarm
Crumlin
Glenariff Lower 1
Glencloy
Loch Ness
Denmark
Spain
Glanariff Upper
Sutherland
Glenariff Lower 2
Mediterranean
F. 3. Dendrogram based on UPGMA cluster analysis of percent sequence divergence estimates between
brown trout populations.

undetected and thus potentially useful markers remain unidentified. Also,

variants close to the ends of the amplified segments are unlikely to be detected as
the fragments would be too small.
Extensive variation was found in the mtDNA of the samples examined. This
contrasts with the distinct lack of polymorphism as revealed by sequencing
studies. Direct sequence analysis of 640 bp of the mitochondrial control region
(Bernatchez et al., 1992) failed to find any variation among brown trout
individuals from various parts of the Atlantic basin. Additional analysis of
sequence variation in segments of the cytochrome b and ATPase subunit VI
genes (Giuffra et al., 1994) did not differentiate these Atlantic samples further.
Sequencing studies revealed a maximum 1·44% sequence divergence between the
Mediterranean and Atlantic haplotypes. In this study a 3% divergence was
found between these two groups. However, this study involved only enzymes
already known to locate RFLPs. Direct sequencing of regions of the cytochrome
b and 16S rRNA mitochondrial genes revealed differentiation between S. trutta
individuals from Italy and Ireland, with a mean nucleotide sequence divergence
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of 1·1% being observed between the two groups (Patarnello et al., 1994). Thus,
overall the RFLP approach may be more appropriate for population studies
unless sequencing can be directed at the regions of the mitochondrial genome
where variation is present.
A main objective of this study was to test the LDH-5* based dual post-glacial
colonization hypothesis. The phylogenetic tree of mtDNA haplotypes does not
show any dichotomy leading to haplotypes specific to populations of low and
high LDH-5*100 frequency. Indeed the only highly supported clade, of more
than two haplotypes, contains haplotypes present in populations fixed for
alternate LDH-5* alleles. The low bootstrap support for most other clades in the
tree (Fig. 2) makes it difficult to draw reliable inferences. Three haplotypes (I, VI
and XII) are common and widespread which suggests that they were present in
the common ancestor of the Atlantic colonists. Each of the supposed ancestral
(high LDH-5*100 frequency) NW European populations appears to have
become fixed for one of these mtDNA types due to random lineage sorting. The
geographically adjacent, but now isolated, Melvin ferox and Erne populations
are fixed for the same haplotype and it is possible that these were both derived
from a common subpopulation already fixed for this haplotype. Similarly the
Glenariff river of NE Ireland has the same haplotype as that of the isolated
Scottish Sutherland populations. Again these two areas may have been colo-
nized from a common west Scottish ancestor. These common ancestors may
have been transient populations in lakes which existed in the immediate
post-glacial period only, as a result of the reduced sea level. Alternatively,
this sharing of haplotypes may, for example in the case of ferox and Erne
populations, reflect river capture (Waters et al., 1994).
Overall there is a significant difference in the occurrence and frequency of
mtDNA haplotypes in the low (putative modern race) and high (putative
ancestral race) LDH-5*100 population groups. The low mitochondrial diversity
of the presumed ancestral populations is in keeping with their lower allozyme
diversity and with postulated colonization bottlenecks (Hamilton et al., 1989).
The isolation of ancestral populations from present-day migratory trout has
prevented the acquisition of further mtDNA haplotypes by gene flow. The
elapsed post-glacial period is too short for new mutants to be detectable with the
sample sizes and proportion of the mtDNA genome studied here. The low *100
frequency (putative modern race) populations have a much greater mtDNA
diversity, in keeping with their larger population sizes and opportunities for
spread of haplotypes by interpopulation migration.
The occurrence of a single invariant mitochondrial DNA haplotype in all of
the Icelandic populations examined is of particular interest, especially as these
come from large, widely separated north, south, east and west coast drainages.
This is in contrast to the situation found in Ireland, where even small river
populations possess several haplotypes. Given the sample size examined only
rare haplotypes would have been missed. Using the equation given by Grewe
et al. (1993) it can be calculated that the sample size gives greater than 99·9%
confidence that at least one individual should have been observed of those
haplotypes present at a frequency §0·05. The absence of variation would
suggest that all of these Icelandic populations were derived from a single
ancestral population in which a severe bottleneck had occurred, and that
64 . .    .

bottlenecks subsequent to colonization have resulted in the loss of any new

mutations. Iceland does, however, possess the LDH-5*90 allele at low frequen-
cies. It is postulated that this allele comes from a second and later colonization
(Hamilton et al., 1989). It appears then that this allele can be introduced into a
population without new mtDNA haplotypes being introduced. Thus the Melvin
ferox and the native Erne populations also have low frequencies of the *90 allele
but only a single mtDNA haplotype. This is not surprising given that, with the
quarter effective population size of mtDNA compared with nuclear DNA, a rare
mitochondrial haplotype introduced into the population at a low frequency
would be more likely to be lost by drift than a nuclear allele. In addition, the
postulated selective advantage of the *90 allele (Henry & Ferguson, 1985) would
enhance its likelihood of being retained in the population. Since all of the
Icelandic populations examined show high frequencies of the *100 allele, the
mtDNA monomorphism does not exclude the possibility of a second coloniza-
tion. Further mtDNA analysis of Icelandic populations with low *100 frequen-
cies, especially anadromous populations, will be required before definitive
conclusions can be reached.
On the basis of morphological and allozyme data, it has been suggested
(Cawdery & Ferguson, 1988) that the sympatric gillaroo and sonaghen of Lough
Melvin are the result of a sympatric split of the modern race. However, these
two forms occur in separate distinct groups of the dendrogram based on
nucleotide divergence. The clades involving the gillaroo specific haplotypes IX
and X, and the sonaghen specific haplotypes III and IV are highly supported in
the Wagner tree (Fig. 2) suggesting that divergence of the haplotypes occurred
after divergence of sonaghen and gillaroo. Overall, the extent of genetic
divergence between sonaghen and gillaroo (0·08%) is indicative of a separation
time of about 80 000 years which coincides with the onset of the last glaciation.
This calculation assumes a 1% per million years (Bembo et al., 1994) rate of
divergence for brown trout mtDNA but, as pointed out by Giuffra et al. (1994),
this may be too fast for fish mtDNA, suggesting that this may be the minimum
divergence time. Irrespective of the exact divergence time, it would suggest that
the two types had differentiated prior to colonizing Lough Melvin when it
became deglaciated some 13 000 years ago. Bernatchez & Dodson (1990) offered
a similar explanation for the co-occurrence of dwarf and normal, reproductively
isolated populations of lake whitefish Coregonus clupeaformis in Cliff Lake,
Maine. Alternatively, sonaghen and gillaroo could have diverged post-glacially
from a shared polymorphic ancestor with stochastic lineage sorting resulting in
the present day pattern. A similar theory was proposed by Taylor & Bentzen
(1993) for the origin of reproductively isolated sympatric populations of smelt
Osmerus in Lake Utopia, which have a similar degree of mtDNA divergence to
the Melvin trout types. However, it is unlikely that the Melvin sonaghen and
gillaroo originated in such a way given the large number of haplotypes present in
the two types. No other contemporary natural population shows more than five
haplotypes, suggesting that no reduction in diversity has occurred in sonaghen or
gillaroo. Thus Lough Melvin appears to have been independently colonized by
three genetically distinct brown trout types. As already pointed out, the unique
characteristics of mtDNA make it more appropriate than nuclear characters for
phylogenetic studies. Several authors (e.g. Hindar et al., 1986) have argued that
 m      65

the greater allozyme similarity of sympatric populations, compared with possibly

homologous allopatric ones, indicates sympatric ‘ speciation ’. However, a
small amount of inter-breeding could produce nuclear, but not mtDNA,
convergence.
The heterogeneous but widespread distribution of mtDNA haplotypes sup-
ports earlier accounts of genetically distinct groups of brown trout in NW
Europe. However, the data suggest that the pattern of post-glacial colonization
may have been more complex than the dual colonization model proposed on the
basis of the distribution of the LDH-5* frequencies. Thus present day brown
trout populations appear to have arisen from several genetically differentiated
ancestral groups. This differentiation may have been the result of multiple
glacial, or immediate post-glacial, refugia. A similar situation occurred when
Ward et al. (1989) compared mtDNA and allozyme variation in walleye
Stizostedion vitreum Mitchill across Canada. The distribution of mtDNA
haplotypes inferred the recolonization of Canadian lakes from three separate
glacial refugia while allozyme data suggested only two.
Verification of the above suggestions and the provision of a more detailed
understanding of the pattern and timing of post-glacial colonization will require
the analysis of further brown trout populations, in particular additional popula-
tions exhibiting high LDH-5*100 frequency. In addition, sequencing directed
towards the variable mtDNA regions (ND-1, ND-5/6) would enable more
accurate phylogenetic tree construction, as well as allowing examination of a
greater part of the mtDNA genome. The latter is particularly important given
the relatively short time available for divergence. Such studies are being
undertaken currently.

We thank L. Bernatchez, S. Gudjonsson, M. Hansen, M. Jóhannson, L. Kristjánsson,

P. Moran, M. O’Farrell, P. Prodöhl, N. Ryman, A. Shine, J. Taggart, T. Tómasson and
A. Walker for providing, or for assistance in obtaining, samples. P. Prodöhl also assisted
with the data analysis. The work was supported by grant GR3/7601 from the Natural
Environment Research Council.

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