Biological Journal of the Linnran Sociely (1987) 30: 193-228. With 12 figures
Colonial breeding in Mute swans (C’gnus olor)
essociated with an allozyme of lactate
dehydrogenase
P. J. BACON*
Institute of Terrestrial Ecology, Merlewood Research Station,
Grange-over-Sands, Cumbria LA11 63U
AND
P. ANDERSEN-HARILD
Ministry of Environmenl, Amaliegade 13,
DK-1256 Kobenhavn K , Denmark
Received 9 October 1986, accepted for publication I November 1986
The Mute swan (Cygnus olor Gmelin (Anatidae)) is a common water bird of lowland freshwaters
and coastal shallows. Its typical breeding system involves lifelong monogamous pairs which
vigorously defend large breeding temtories, sometimes killing intruding swans that are unable to
escape. However, in some unusual circumstances (superabundant food coupled with limited nesting
sites) Mute swans may nest colonially. At the only two colonies in southern England a rare allele for
lactate dehydrogenase (LDH) was found to be unusually common and colony swans carrying this
allele were shown to breed more successfully. This isolated finding could, however, have originated
either from a chance ‘founder’ effect or from human management of the main colony. We now
show that this allele is also significantly commoner at two recently formed colonies in Denmark,
implying that the association between the allele and colonial breeding may be widespread and
longstanding.
KEY WORDS:-Mute swan - Cygnus olor - colonial breeding - allozyme - lactate dehydrogenase population genetics - population dynamics.
CONTENTS
Introduction . . . . . . .
General Mute swan biology . .
The British Mute swan population.
The Danish Mute swan population
The LDH polymorphism . . . .
Biochemistry . . . . . .
Routine screening ofsamples . .
Inheritance data from Britain . .
Inheritance data from Denmark .
Conclusion . . . . . .
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*To whom correspondence should be addressed.
0024-4066/87/030193+ 36 $03.00/0
I
193
0 1987 The Linnean Society of London
I94
P. J. BACON AND P. ANDERSEN-HARILD
Population genetics
British data .
Danish data .
Discussion. . .
Acknowledgements
References. . .
Appendix 1 . .
Appendix 2 . .
Appendix 3 . .
Appendix 4 . .
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INTRODUCTION
General Mute swan biology
Mute swans usually pair at 2-3 years old and breed the following year; the
single annual brood typically hatches from five to eight eggs and comprises three
to six cygnets at fledging. Breeding activities are normally confined to territories,
which comprise some 2-3 km of river or 4-5 ha of lake (Cramp & Simmons
1977). Up to 3% of swan deaths arise when intruders are unable to escape from
aggressive breeders (Ogilvie, 1967). Colonial birds may show very little
aggression (Bloch, 1970; Perrins & Ogilvie, 1981) and nest about 100 times
closer than territorial pairs, often 10-30 m apart, or less.
The British Mute swan population
The British Mute swan population went through an intense period of semidomestication during the 14th to 18th centuries (Birkhead & Perrins, 1986).
Initially Mute swans were the property of the crown, and rights to take for food
the swans living in particular areas were granted to local nobles. This led to an
elaborate system of swan-marks to identify ownership (Ticehurst, 1957) as the
birds were generally left to move around at will: the degree of mobility of the
marked birds (a high proportion of the population during the heydays of swankeeping) was however restricted by pinioning the birds so they were unable to
fly. Despite popular folklore to the contrary, it is most unlikely that the British
Mute swan population derives solely from a few introduced birds; early
manuscripts and place-names indicate swans were common well before the
middle ages, while Ticehurst (1957) shows that by 1250 the Mute swan was far
too common to have been initially introduced by Richard I around 1192. It is
much more likely that Britain has had a fair sized Mute swan population since
rising seas after the last ice-age separated the British Isles from the continent.
The present British population is certainly effectively isolated from continental
Mute swans; only in exceptionally cold winters (Hilprecht, 1972; Cramp &
Simmons, 1977; Spencer & Hudson, 1977) do a few continental Mute swans
reach British shores and those that survive normally return to the continent
without breeding (in keeping with known winter movement patterns in
Scandinavia (Mathiasson, 1973a, b; Andersen-Harild, 1981) ). Within Britain
the Mute swan is also sedentary, over 95% remain within 50 km of their natal
site (Ogilvie, 1967). With the demise of swan-keeping in the 18th century the
British swan population probably declined slowly (it was still protected from
shooting), and may have gone through a low of a few thousand breeding adults
in the 1920s (but available data are very sparse, see Birkhead & Perrins, 1986).
GENETICS OF COLONIAL MUTE SWANS
195
20 000 -
/
10 000 -
1900
/
/
I
I
I
1920
1940
1960
I
1980
t
I6O0
I200 -
Autumn
800
-
400 -
I
1900
Spring min.'
1920
1940
1960
1980
Figure I . A, Recent changes in the British Mute swan population: estimates of total population ( 0 )
and breeding adults (m) (nof pairs) for 1955 and 1978; the population is known to have increased
prior to 1950, but the level from which it rose is unknown. B, Changes in numbers of swans on The
Fleet at the Abbotsbury colony. Autumn counts by Ilchester estate employees are shown, as a solid
line, from 1928 to 1980 and the Winter maxima and Spring minima from the censuses of Perrins
and Ogilvie as dotted and pecked lines, respectively, from 1960 to 1980 (see Perrins & Ogilvie,
1981).
Thereafter the population expanded rapidly, to some 7600 breeding adults in
1955 and a total population (including non-breeders) of some 19000 (Fig. 1).
Since 1955 the population has remained at around 20000-18000 with some
4000 breeding adults, although there have been considerable regional variations
in population density not revealed by these national totals (Campbell, 1960;
El tringham, 1963; Ogilvie, 1981, 1986).
The historic colony of Mute swans at Abbotsbury in the county of Dorset is
situated at the west end of a brackish lagoon, the Fleet. The Fleet is some 14 km
long, and varies in width from 1.0 to 0.2 km, with an area of about 480 ha. The
water is mostly less than 2 m deep. There is a single narrow outlet to the sea at
the eastern end, where the water is mainly sea-water (34% salinity), decreasing
to only 1904 salinity at the western end.
196
P. J. BACON AND P. ANDERSEN-HARILD
The colony is situated on low-lying ground (some 300-600 mm above normal
water level) and if spring-tides during the nesting period are accompanied by
on-shore winds many clutches may be flooded and lost. The fragmentary
historic record from the 14th century indicates that the Abbotsbury population,
which was initially managed for food with hatched cygnets being raised in pens
(it is now a tourist attraction), has been at a similar level to its present state for
several hundred years (Perrins & Ogilvie, 1981). There is a ‘resident’
population of some 500 birds, which is increased by 400-500 immigrants during
the summer moult and in winter. The number of nesting pairs varies widely, for
reasons that are not understood, between 20 to 100 pairs a year. Details of the
present population dynamics and method of management can be found in
Ilchester (1934), Bacon (1980) and Perrins & Ogilvie (1981). Swans that had
been bred in the colony prior to our studies were identifiable as they are still
‘marked’ by a cut in the web of the foot when released by the Abbotsbury swanherd.
The Danish Mute swan population
The Danish Mute swan breeding population reached a minimum due to
hunting in about 1924 with 3 to 4 pairs. I t is suggested that considerable
numbers bred in the 16th and 17th century, mainly in the eastern part of
Denmark, but it was a scarce breeder by the middle of the 19th century. There
were large flocks of non-breeders moulting flight feathers at least until about
1800 or later, but these became rare between 1900 and 1930 (Bloch, 1971;
Dybbro, 1976). Colonial breeding has not been reported before the 20th century.
From 1926 the Mute swan was legally protected throughout the year and a
rapid increase in breeding numbers began (see Fig. 2). The increase came partly
from the small wild population, and partly from ornamental pairs in parks
whose cygnets were allowed to escape to the wild. The latter mainly occurred in
Jylland, Lolland and South Sjelland. The input to the breeding population
from migrants is unknown, but in later years could have been appreciable. A
number of counts show the increase in the breeding population: 1925, 3 or 4;
1935, 35; 1950, 385; 1954, 758; 1966, 3000. An estimate for 1978 gave 7000
pairs and this is thought to be the maximum of the population which has since
decreased slightly. Nowadays migrants to Denmark are common, both for the
summer moult (about 40000 birds) and winter feeding (about 70000), and
include swans from Germany, Poland, western U.S.S.R. and Scandinavia
(Andersen-Harild & Preuss, 1978). Colonial breeding was recorded in one to
two localities in 1943 and 1957, but in that period only 1-2% of pairs bred in
this way. In 1966 about 650 pairs ( =22y0) bred colonially, and this has since
increased to about 1500 pairs (38%) in 1978; it is estimated that the percentage
is even higher today. Nearly all such colonies are situated on small islands in salt
or brackish water areas. In 1966 the average of cygnets per pair at the age of
fledging was 1.9 for colonial breeding swans and 3.5 for solitary pairs on fresh
water lakes. In the 1970s similar calculations for a study area in North Sjelland
gave 1.1 for colonial breeders and 2.5 for solitary pairs. Mortality studies of
ringed birds show that the production of cygnets from colonial breeders is too
low to maintain, let alone increase, the colonial populations. Considerable flow
of surplus cygnets reared by solitary pairs to colonial breeding sites takes place,
and it was estimated in about 1980 that about half the birds breeding in the
GENETICS OF COLONIAL MUTE SWANS
197
I
Lo-./
'
0
0
I
0
1900
1920
1940
I
1960
1980
Figure 2. A, Changes in the numbers of breeding pairs of Mute swans in Denmark from 1925
to 1977, showing the dramatic increase from 1950 to 1980. B, Changes in numbers of breeding
pairs for three sub-regions. The number of breeding pairs on territories in the Kobenhavn area
(m-.)
has remained nearly constant (except for temporary decreases after severe winters)
while the numbers breeding at the Roskilde ( 0- - - 0 )and Ringkobing (A-A) colonies have
increased dramatically. In 1979 the numbers breeding at Ringkobing fell, to near zero by 1981,
following pollution of the fjord which destroyed nearly all the macrophyte vegetation on which the
swans feed.
colonies in North Sjaelland were reared by solitary pairs. I n the 1950s and 1960s)
when the numbers of colonially breeding birds were increasing rapidly, many
more than half the pairs nesting in colonies must have themselves been reared
by solitary, territorial pairs.
THE LDH POLYMORPHISM
Biochemistry
Horizontal starch-gel electrophoresis was used as a cheap, simple and
convenient method, which gives clear, repeatable results.
t t t f t
GENETICS O F COLONIAL MUTE SWANS
I99
Figure 4. Interpretation of the LDH patterns illustrated in Fig. 3. The patterns seen in Fig. 3 are
illustrated on the left, with their genotypes given below. O n the right standard numbering of the
bands and their composition by four monomers from the A and B LDH loci for the three genotypes,
with a polymorphism at the B locus producing the nine-banded Aa heterozygote and, potentially,
the five bands indicated for the aa homozygote. Note that bands near the insert may not show as
their mobility is so low that they often remain at the insert and are hence lost (by diffusion and
insert removal); hence the Aa heterozygote often shows only eight bands not nine, while band 3 of
the AA homozygote is sometimes unclear (it is wider, so more usually shows). The bbbb and Abbb
bands of the aa homozygote have not been clearly resolved from either of the two examples of this
genotype (see text and Fig. 3).
It was found by experimentation (Bacon, 1980) that isozymes of LDH from
avian red cells were separable in tris-citrate buffers at pH 7.0 using dilute
buffers such as those recommended by Markert & Masui (1969) in preference to
the stronger concentrations suggested by Shaw & Prasad (1970). The higher
pHs of stain buffer, around pH 8, recommended by Markert & Masui (1969)
also gave much better results than the neutral buffer recommended by Shaw
and Prasad ( 1970).
Details of the final system used are given in Appendix 1. The details of
temperature and voltage gradient were important, especially in separation of
the bands of the fainter, eight-banded phenotype. The rate of band separation
decreased with time, as the more concentrated electrode buffer moved through
the gel: because of this, and the placement of the inserts at the centre of the gel,
better separation was achieved with the same voltage gradient and time on a 18cm gel than on 12-cm gel. The banding patterns observed are illustrated in
Figure 3. Photograph of a gel stained for LDH and showing the banding patterns of the three
genotypes. The five bands for LDH-AA are shown numbered according to standard nomenclature
on the left, and the position of the insert line is marked on the right. Replicates of the same sample
are shown by underlinings beneath their adjacent patterns (13 samples, most in duplicate) and
illustrate the pattern repeatability: the genotype of each sample is given beneath these underlinings.
Note: ( i ) the shadow bands in some AA samples, particularly from bands 1 and 2, especially in the
first and last samples; (ii) the narrower bands of the Aa patterns and their clear separation from and
uniform sparing between equivalent bands of the AA pattern, in contrast to the close and joined
shadow bands in some AA samples; (iii) in the aa sample anodal bands 1 and 2 are faint or absentthe sample illustrated was slightly clotted and difficult to resolve and the only other aa sample was
from a cygnet where the anodal bands are normally weaker.
200
P.J. BACON AND P. ANDERSEN-HARILD
Figs3 & 4, with Fig. 4 describing the terminology for the bands. The fivebanded phenotype called A A is consistent with the known tetrameric structure
of LDH, as derived from four monomers from two loci (A and B) (see Markert
& Masui, 1969). Samples from very young cygnets (7-20 days from hatching)
showed a pattern with LDH 5 staining most intensively, with activity decreasing
through to LDH 1. Conversely, older cygnets (60 days or more) and adults show
the most intense staining in LDH 1, decreasing through to LDH 5. This agrees
with the known ontogeny of LDH: A chain tetramers work best in anaerobic
conditions, and A monomers are preferentially produced in late embryos,
whereas B tetramers work best in aerobic conditions and are preferentially
produced in adults (and tissues with high 0, potentials) (see Lindy &
Rajalsami 1966; Markert & Ursprang 1962; Markert & Masui, 1969).
Markert & Masui (1969) showed that some possible associations between
LDH subunits fail to produce detectable activity. If one assumes that there is a
polymorphism at either the A or B locus (both are known, but the former is
more common) and that tetramers having A and a or B and b subunits are
inactive, a heterozygote would produce a nine-banded zymogram as in Fig. 4. If
active tetramers could be formed composed of both subunit products from the
same locus, then additional bands should occur but are not observed in Mute
swans. In practice, as isozyme bands move both forwards and backwards at this
pH, one of these nine has near zero mobility, usually remains on the insert and
is rarely seen (this sometimes happens for LDH 3 on five-banded phenotypes
also). Thus the putative heterozygous pattern usually shows only eight bands
not nine. It should be noted here that either a faster mutant of Mute swan LDH
A locus or a slower mutant of the B locus could produce the eight (nine)-banded
phenotype observed. Only two examples of the third phenotype are known from
some 2500 samples: both of these show a strong band corresponding to LDH 5,
the AAAA tetramer, which indicates any genetic polymorphism should be at the
B locus, as a homozygote for a mutated A locus gene, A' would not produce any
A monomers but only A' monomers (see Fig. 4). One of the two aa phenotypes
(both found in Denmark in 1983) was the cygnet of only the second A a x Aa
mating and brood ever found and sampled and this itself indicates that genetic
inheritance is a reasonable hypothesis for the different LDH phenotypes.
Although the banding patterns of the two aa phenotypes indicate that any
genetic polymorphism is at the LDH B locus, not the LDH A locus, the
terminology AA, Aa and aa for the phenotypes (genotypes) is retained here, both
for consistency with previous publications and also in reference to the
Abbotsbury colony from which the variant was first described.
Routine screening of samples
Experience has shown the electrophoresis system to give reliably repeatable
results; accordingly single aliquots of samples are initially run and their LDH
phenotypes recorded. Samples that are clearly A A are not repeated; samples
which seem to be AA but are slightly indistinct, and all samples that are thought
to be, or even are clearly, Aa are always repeated in duplicate. However, when
repeats of samples are run, duplicates of samples initially recorded as clearly AA
are run as controls alongside every repeated sample. Random selection of these
controls and the standard repeating of Aa phenotypes allows us to be confident
GENETICS OF COLONIAL MUTE SWANS
20 1
in the reliability of our scoring; several hundred such controls have been re-run
over the years and have always matched their initial scores. Indistinct samples
usually result from: (i) slight technical irregularities-which
will normally be
corrected on repeating; (ii) low activity giving faint bands-corrected by using
more sample per slot when repeating; (iii) streaking of bands-often corrected
by diluting sample when repeating. Exceptionally weak activity in a few
samples (often ones which had clotted slightly) may be correctable by freezedrying and re-constituting. Of some 2500 samples only three have given
inconsistent scorings, due to streaking that was probably caused by either the
bird’s physiological state or difficulties in taking the initial blood sample.
Anyone intending to repeat the method is warned that highly active AA
patterns may show ‘shadow bands’ in front of some, or all, of the five main
bands-these are fainter than the main bands, so that the pattern is clearly
distinct from the narrower and uniformly fainter bands of the Aa phenotype.
However, we emphasize this point as we know that other workers looking at
different species (and lacking family data) have mistaken such artefacts for
genetic polymorphisms; the Aa genotype is generally rare in Mute swans (see
below) and ‘shadow band’ artefacts are more likely to be encountered than the
true polymorphism. ‘Shadow bands’ usually disappear if aliquots of such
samples are diluted about 1 : 1 to 1 : 3 and re-run.
Inheritance data from Britain
As the Aa phenotype is rare outside Abbotsbury/Weymouth, very few
complete family data were available from other areas to indicate its inheritance.
However, over the years more than 100 breeding pairs and all their cygnets
have been sampled from territorial sites (see below): we will conservatively only
consider a single brood for each such family. I n a single one of these families the
male was Aa, the female AA, one and five cygnets, respectively, Aa and AA. I n
all other families both parents and all cygnets were AA (there are usually three
to five cygnets in broods from which some cygnets fledge). T h e expected
frequency of 0, 1, 2 . . . Aa cygnets in families cannot be simply calculated, due
to the variations in family size. However, if the Aa phenotype occurred a t
Table 1. Inheritance data for LDH phenotypes, based on complete
family data for 102 cygnets from 19 families at the Abbotsbury
colony in 1978
0bserved
Genetic hypothesis
Cygnet
genotypes
Expected
ratio
x2
( P W
Parental
Mating
AA
Aa
AA:Aa:aa
AA x AA
45
0
1 :o:o
NA
A A x Aa
22
16
l:l:O
0.95
(0.5>P>0.3)
Random hypothesis
Freq. Aa
X2
P>
15
20
25
7.8
11.2
15.0
0.005
0.001
0.001
15
20
25
21.9
11.6
5.9
0.001
0.005
0.02
(Yo)
P. J. BACON AND P. ANDERSEN-HARILD
202
random, the probability of an Aa cygnet given one, or more, Aa adults would
be independent, so for the single family having an Aa parent we may estimate
the chances of one or more Aa cygnets in the brood of 6: from binomial theory
this is (1-( 1-0.006)6)=0.04, where 0.006 is the Aa frequency estimated for
territorial swans at moult catches (the probability becomes 0.03 if we take
Aa = 0.005 = 1/200 breeding adults, as observed).
Inheritance data from the Abbotsbury colony are more conclusive. In 1970
102 cygnets from 19 complete families were sampled, and their LDH
phenotypes are given in Table 1 according to the mating type of their parents.
The data strongly reject the hypothesis of random distribution of Aa phenotypes
within families over the whole range of likely Aa phenotype frequencies in the
breeding population (15-2574, but are not significantly different from the
expected frequencies for genetic inheritance of two co-dominant alleles at an
autosomal locus. Further data from the Abbotsbury colony for previous (Bacon,
1980) and subsequent (unpublished) years support the genetic inheritance of the
AA and Aa patterns, but are not here included as many pairs have been sampled
in successive years and those data are not fully independent.
Inheritance data f r o m Denmark
Further data on inheritance of the Aa phenotype are available from the
Danish samples, but again the rarity of the phenotype in the Kobenhavn area
and the difficulty of catching both adults of families at Roskilde fjord make the
resulting data sparse. The complete family data are as follows:
No. of cygnets
Mating
tY Pe
AA x AA
AA x Aa
Aa x Aa
No. of families
sampled
AA
Aa
M
35
3
I
143
0
3
1
0
0
I
6
I
Apart from the presence of an aa homozygote in the brood from the single
Aax Aa mating we may calculate that the probability of one or fewer AA
cygnets in a brood of three, given random occurrence in broods and an
estimated AA frequency of 0.962 as observed, is P < 0.0043. The information on
families for which only one parental genotype is known added little to our
knowledge of Aa heritability, but were entirely consistent with genetic
inheritance. However, the small sample sizes prevent their refuting chance
sampling effects on their own.
Conclusion
In conclusion, we note that independent data sets from Denmark and from
territorial sites in England show that the Aa phenotype does not occur at
random within families (P~0.0043
and P<0.04,respectively). Data from 19
complete families of 102 cygnets from the Abbotsbury colony in England reject
the hypothesis of random Aa distribution in families (P<0.005)but are
consistent with Mendelian inheritance (0.5 >P > 0.3), as are similar data from
Denmark, We accordingly conclude the AA, Aa and aa phenotypes are inherited
GENETICS OF COLONIAL MUTE SWANS
203
as products of co-dominant alleles at the (autosomal) LDH-B locus, and
hereafter refer to these as genotypes.
POPULATION GENETICS
British data
The genotypic frequencies found at nine sites in Britain are given in Table 2
and illustrated in Fig. 5. The data usually refer to samples of swans caught by
rounding up moulting flocks; the great majority of non-flying birds are usually
caught. Such flocks comprise juveniles, non-breeders and failed breeders from
the surrounding area, and the data presented here are restricted to a sample in a
single year for each area. Family data (related individuals whose genotypes do
not constitute an independent random sample) are completely excluded in every
case, while detailed studies of both LDH and an exterase polymorphism in Mute
swans (Bacon, 1980) show that samples of moulting birds are representative of
genotype frequencies in the breeding population also. I t can be seen that the Aa
heterozygote comprises less than 1yo of birds from territorial nesting areas, but
around 15% frequency at two adjacent sites in Dorset, namely Abbotsbury and
Weymouth, where the swans nest in a dense and a loose colony, respectively.
This difference in gene frequencies between colonial and territorial areas is very
highly significant, P = 2 . 8 x 10- * 4 . The association between Aa frequency and
colonial nesting becomes even more striking when sub-sections of the
populations from these areas are considered. At Weymouth there is a significant
difference in genotype frequencies between (i) resident breeders and their adult
offspring that were hatched at Radipole lake and (ii) swans frequenting
Radipole lake, some breeding there, but hatched elsewhere (Fisher exact,
P=0.002) as shown in the inset of Fig. 5. For the population on The Fleet at
Table 2. LDH genotype frequencies for seven territorially breeding populations
and two colonially breeding populations in Britain; numbers in the second
column refer to Pie chart No. in Fig. 4.
LDH genotype (numbers of swans)
Site
Breeding
system
Colonial
Territorial
____
Chart in
Fig. 4
-~
. . .~
Name
s*
I
2
Abbotsbury
Weymouth
14
8
3
4
5
6
7
8
9
Christchurch
Salisbury
Oxford
Midlands
Hebrides
Strathbeg
Montrose
1
0
2
I
0
I
0
c*
s*
92
22:
40
5:
I35
24
217
109
40
182
102
Notional
Yo Aa
AA
Aa
xz for rowst
C*
s*
c*
S*
c*
132:
17
13
14.3%
34
42
76
809:
I
2
0
1
3
I
0
I
0
1
0.6%
1
15
I
3
3
x:=91t
~
Each site separately, c=site data combined for comparison.
?The expected frrquencies of Aa at most sites are so low ( < 5 ) that the xz test is not strictly valid. An
alternative test of the genotype/colony differences, based on the totsls: gives Fisher Exact probability
*s=
P=2.8~
P.J. BACON AND P. ANDERSEN-HARILD
204
I
I
\ 3 /
---t
Abbotsbury
Wevrnouth
0
u
'Territorial' sites
mmigronts
m
Residents
Figure 5. Frequency of LDH genotypes at nine sites in Britain, emphasizing the difference in Aa
frequency between the two colonial breeding sites and the seven territorial ones. At both colonial
sites the LDH-Aa frequency is higher in resident and breeding sections of the populations than in
immigrants. See Tables 2 and 3 for further details of sites and differences between them. The
frequency data are shown superimposed, with permission, on a map of present Mute swan breeding
distribution from Sharrock (1976).
Abbotsbury the frequency of the Aa heterozygote increases significantly
(P<0.025) from 8% in the peripheral flock swans which moult on The Fleet
but rarely come to the colony, to 13% in birds that frequent the colony and to
19% in birds which breed in the colony, as detailed in Fig. 5 and Table 3. (No
data from cygnets within families, which are not independent samples, are
included in any estimate of genotype frequencies for 'breeders'.) It should be
noted that the 8% frequency for the peripheral flock includes some swans
hatched at the colony but rarely seen there and is thus not typical of
GENETICS OF COLONIAL MUTE SWANS
205
(overestimates) the heterozygote frequency in immigrants to The Fleet. Given
these striking differences in genotype frequencies, it is necessary to consider how
they may have arisen. There are two main hypotheses: firstly random drift (see,
for example, Gale, 1980) and/or a ‘founder’ effect (Berry, 1975, 1977) and
secondly selection for some genetic attribute linked to the a allele at the LDH-B
locus (the ‘hitch-hiking effect’, Maynard Smith & Haigh, 1974).
Possible ‘chance’ causes
In small isolated populations stochastic changes in gene frequencies can be of
considerable importance and may occasionally lead to elevated frequencies, or
even fixation, of initially rare genes (random drift, for example see Gale, 1980).
These effects may be enhanced by selection on initial combinations militating
against the subsequent incorporation of rare immigrant genes (Berry, 1975,
1977). Neither of these mechanisms is likely to have operated at the Abbotsbury
colony, as this population is not genetically isolated. Some 25% of adults nesting
at the Abbotsbury colony are immigrants (Bacon, 1980; Perrins & Ogilvie,
1981) and these breed as successfully as the residents (Bacon, 1980; Perrins &
Ogilvie, 1981); thus 25% of cygnets fledged in the colony have a n immigrant
parent not itself hatched at Abbotsbury but drawn from a territorial population
with a wide catchment area (Perrins & Ogilvie, 1981). A deterministic
calculation hence shows that, being diluted by 25% per generation, the Aa
frequency would be expected to fall from a level of 20% to the 1% level in
surrounding territorial populations in about 10 generations or some 80 years,
This period of about 80 years is of importance: Perrins & Ogilvie (1981) give
the fluctuations in winter numbers (their table 2) since 1928 and breeding
numbers since 1969 (their table3a); the effect of the severe winter in 1963 is
evident in their former Table (winter numbers drop from about 1000 to 550,
but recover to 760 by 1969) and similar severe effects are reputed to have
resulted from very severe winters in 1865 and 1880 (Ilchester 1934) which
severely depleted the swans’ food supply and resulted in widespread starvation
and dispersal. Hence, even if stochastic chance had locally elevated the Aa
frequency at Abbotsbury in the late 1800s to around 20%, we would by now
(late 1900s) expect it to have declined to a low level, about lyo, given the
observed proportion of immigrant AA breeders. We further note that in a longlived species such as the Mute swan with overlapping generations and a social
system favouring survival of mature adults in harsh conditions and when food is
Table 3. LDH genotype frequencies within
different subsections of the flock found on The Fleet
at the Abbotsbury colony
Pie chart
No. in
Fig. 4
la
lb
Ic
LDH Genotypes
Subsections of
Abbotsbury flock
Peripheral flock
Resident flock
Breeders
=6.6, 0.05 >I‘
>0.025.
An
AA
%An
17
14
16
188
92
69
8
13
19
206
P. J. BACON AND P. ANDERSEN-HARILD
sparse (Andersen-Harild, 1981) such total counts probably exaggerate the
‘genetic bottle-neck’ indicated by the annual changes in numbers.
A rigorous estimate of the probability that the elevated Aa frequency at
Abbotsbury could have arisen by chance would require accurate biological data
(survival; life-time productivity variance), census data (population trends over
some 100 years or more) and genetic data (initial gene frequencies) which we
cannot get. However, we present below a stochastic simulation which we believe
gives a fair estimate of that probability.
A stochastic simulation of ‘drift’ efects at Abbotsbury
The available evidence suggests that the Abbotsbury Mute swan population
has been in approximate equilibrium in a similar environment for several
hundred years. For the purpose of assessing drift of a neutral allele it seems
reasonable to consider the stochastic sampling effects for the effective population
size, 2Ne, where 2Ne is the effective number of alleles (haploid model). The
available data do not indicate assortative mating or other effects which would
invalidate this approach, indeed the neutral hypothesis requires there be none.
Following methods given in Falconer (1960) and Hill (1972) we use the
following procedure. We estimate the geometric means of population sizes by
combining data from tables 1 and 2 in Perrins & Ogilvie (1981) to estimate
changes per generation over six generations or 50 years. We use combined
estimates of mortality and ages of first breeding from various British swan
studies, and a variance of life-time family size intermediate between the
observed annual variance of family size, and the common assumption that this
variance in a stable population is the result of a Poisson process and has a mean
and variance of 2.0. Both the mortality and variance of family size values used
probably result in under-estimates of Ne, and will hence accentuate the
stochastic effects. This leads to an estimated value of Ne= 72 individuals. If we
exaggerate the effect of the 1962/63 hard winter by substituting the lowest
census figure for that period instead of the mean (within that generation), the
estimated Ne only decreases to 65. We note that these values are comparable to
the annual number of breeding birds at Abbotsbury during 1969-1980,
usually 30-50 pairs (Perrins & Ogilvie, 1981, table 3). This acts as a useful
cross-check that the census and mortality data are not seriously biased, as Bacon
(1980) found similar correspondence between calculated Ne for the British
population (which is isolated) and the censused total of breeding adults. The
simulation model has four input parameters: (i) the effective population size, Ne,
which is taken as constant once given; (ii) the a gene frequency in the founding
(Abbotsbury) population; (iii) the a gene frequency in the surrounding
population from which the immigrants are drawn; (iv) the proportion of
immigrant breeder per generation. The model performs S simulations each of G
generations duration: each simulation starts with the initial conditions as
specified and calculates a new a gene frequency using a random-number
generator and the present a gene frequency in the breeding population; in the
next and subsequent generations the new a gene frequency, modified by the
proportion of immigrant breeders with fixed a frequency, is used to calculate the
next a gene frequency, as indicated in the flow-diagram of Fig. 6. An output
graph from the model is shown in Fig. 7, where 100 simulations (representing
GENETICS O F COLONIAL MUTE SWANS
207
V
INPUT
Number of simulations
Generations Der simulation
Effective po&otion size
u frequency founding popn.
u frequency immigrant
010 Immigrant bteeders&Etype
JI
FOR each of S simulations
I
Resident
0
S
G
N.
F
I
p ~ (as
l proportion)
I
freq. = founding freq.; R = F ]
I
\ P R I N T summary statistics table
/
U,
Figure 6. Flow-diagram of the simulation model to assess the probability that drift of a neutral
allele in the Abbotsbury population could have caused a difference in genotype frequencies similar
to that found between LDH genotypes at Abbotsbury and surrounding areas. As the Abbotsbury
population has remained stable for some centuries the model uses random numbers to assess the
likely changes due to sampling error of gene frequencies in subsequent generations given their
present frequencies, in resident and immigrant breeders, in the present generation. For each allele
(either resident or immigrant) random numbers determine whether its replacement in the next
generation is A or a depending on current frequencies R (variable) and I (fixed for each run of
model).
100 independent random ‘drifts’ of gene frequency) are superimposed on each
other in respect of time during 80 generations since initial conditions, showing
the resulting heterozygote frequencies in those generations. It can clearly be seen
that the great majority of generations are well below the observed heterozygote
frequencies in the Abbotsbury flock, and tabular output from the model (not
given here) shows that, for these likely initial conditions, the probability of a
sample taken in one generation having a n a gene frequency as high as the lower
-
- ....
20
........
..
..
-
-
.
40
.
.
-. .
....
Generations
.
.
.-.-.
..
..
60
.....
.
..
..
SO
---..--._. _._
14
- 19
H
0.0014
0.0124
0.0796
0.1892
S
I
0
0.0000
"
H
represents
Lower
95% C.L.
brrrdrrs
I
Proportion of
gomatione
aboveH
12
19
27
'Yo H
in
popn.
Interpretation
Figure 7. An example of the effects of drift at Abbotsbury as predicted by the simulation model. Each of 100 (superimposed) lines represents a simulation
lasting 80 generations (about 600 years since the early records of colony numbers). The vertical axis represents numbers of An heterozygotes, and
threshold values of these are shown as horizontal lines. These numbers relate to a total effective populations size of Ne = 72 swans. To the right of the
figure the percentage heterozygote frequency (H/Nc)is given, along with the proportion of 8000 generations for which the predicted level of H in the
population was above these values; the levels of H corresponding to the observed LDH-An frequency at Abbotsbury and its lower 95% confidence limit
are also given.
.........
--
a = 0.003 {Immigrants)
100 Simulations, each of 80 generations.
16% Immigrant brooders
Foundrrr
b
m
2
N
CO
0
GENETICS OF COLONIAL MUTE SWANS
209
95% confidence limit for the observed a gene in Abbotsbury breeders is
P<O.OOl.
However, the actual initial conditions are not precisely known, so a sensitivity
analysis was performed to investigate model sensitivity to variations of its input
parameters. The results are presented as 3-dimensional graphs in Figs 8 and 9.
In each graph, the vertical axis (response variable) is the probability that the
heterozygote Aa frequency exceeded the threshold level of nine individuals. The
horizontal axes are, respectively, the driving variables .Ne and proportion of
immigrant breeders, and are shown over a wide range of possible values: the
proportion of immigrant breeders is deliberately extended to zero as, during the
height of swan-keeping in Britain, the proportion of unpinioned birds would
have been low for long periods. As the simulation is stochastic, many repeated
runs for each set of conditions have been used; every point on the response
surface represents 100 simulations of 80 generations each. The scenarios of Figs 8
and 9 differ only in the assumed proportion of Aa swans in the founding and
immigrant populations: these are a=0.003, as observed in Britain, for Fig. 8
but a much higher value, a=0.030 for Fig. 9. T o keep the stochastic effects
comparable between different simulation runs, an integer threshold of nine Aa
swans has been used in each case: accordingly there is a small change in
heterozygote frequency from 9/60= 15% to 9/84= 11yo as Ne varies from 60 to
84. Note, however, that this ‘threshold’ of nine is continually as close or closer to
the lower 95% confidence limit of the observed Aa proportion in Abbotsbury
breeders (19% f 4 % ) than to the observed level (0.19), so the illustrated
probabilities are conservative.
The first scenario outcome, with a=0.003 gives probabilities P<0.02 for all
situations, even with zero immigration: with realistic immigration in the range
15-300/, these probabilities fall to between 0.005 >P>O.OOl. In these
circumstances the observed Aa frequency at Abbotsbury is clearly unlikely to be
due to chance (P<0.005). The most unlikely assumption of this scenario is
actually that a in the immigrant ‘British’ population would have remained close
to 0.003 for the 80 generations of each simulation. With a British Mute swan
population having Ne N 2400 (Bacon, 1980) we would only expect 14 Aa
heterozygotes on average. We may use the simulation model to show that about
half theoretical populations of this size would lose the a allele by chance in 80
generations. In scenario 2, illustrated in Fig. 9 we have envisaged a 10-fold
increase of a to 0.030 giving a probability of 95% that a will persist well above
0.003 for 80 generations. This scenario unrealistically assumes a in immigrants is
continually 0.030, which is most certainly untrue at present, and, further, that
N, was continually ~ 2 4 0 0 whereas
,
swan densities during the periods of swankeeping may have been very much higher (even 10 times higher, see Birkhead &
Perrins, 1986). Figure 9 shows that the probabilities of exceeding the threshold
of nine Aa swans are now also about a factor of 10 higher. For immigration rates
in the range 15-30y0 the probabilities are below 0.10. Remember, however,
that H = 9 is about the lower 95% limit of the Aa frequency at Abbotsbury: on
the edges the block-diagram of Fig. 9 we also illustrate the trend for H = 14, the
observed levels of Aa in breeders ( = 19%) which are well below P=0.05 for the
whole range of likely immigration rates.
Accordingly we conclude that random drift in the Abbotsbury population is
unlikely (P<0.05) to have permitted Aa frequencies to rise to the observed 19%
210
P. J. BACON AND P. ANDERSEN-HARILD
Figure 8. Sensitivity analysis of the simulation model for drift at Abbotsbury, using observed
frequencies of LDH-a allele=0.003 for founders and immigrants. Each point on the response
surface is the proportion of generations above H-9 Aa heterozygotes in a population of about
Nc= 72 swans for 100 separate simulations lasting 80 generations each, and with actual values of Nc
and percentage of immigrant breeders as given on the horizontal axes. The predicted probabilities
for Aa exceeding H=9 swans are continually below 0.01 for immigration rates above 8y0,and are
in the region of 0.001 for immigration rates of about 25%, as observed.
even if the proportion of Aa immigrants has, historically, continually been 10 times
higher than at present and, further, that if the present proportion of Aa swans in
immigrants is typical historically (there is about 50% chance of this being true)
the probability of 19% Aa due to random drift at Abbotsbury is about
0.005<P<0.001.
These simulations confirm the results of some simple calculations which help
understand the outcomes predicted by the model. With a gene frequencies of
0.003 or 0.030 in a population of Ne=72 we would expect, respectively, 0.4 or
4.2 Aa heterozygotes. Let us consider A a = 4 of 7 2 individuals, typical for the
latter case and to which the former (a=0.003) might occasionally rise by
chance. The expected changes in a single generation are given by a Poisson
process with mean 4/144 genes: this distribution is such that in a single
generation the number of a genes is slightly more likely (P=0.63) to decline or
stay constant than to increase (P=0.37). The standard deviation of this change
is + 2 ; hence every other generation (on average) we can expect two extra Aa
swans by chance (and also two less, giving a mean change of zero). However,
with 25% immigrant breeders we can expect 1/4= 1 Aa breeders to be replaced
by an AA breeder every generation, so the stochastic increase is continuously
countered by a deterministic decrease of very similar magnitude.
Possible selective efects
The previous section has established that the observed LDH genotype
differences between Abbotsbury/Weymouth and other British swan populations
GENETICS O F COLONIAL MUTE SWANS
211
Figure 9. As for Fig. 8, a sensitivity analysis, but with the (only) change that the founding and
immigrant populations are given an LDH-a allele frequency of 0.030, 10 times that presently
observed, to give 95% certainty that the LDH-a allele would not become extinct due to drift in the
British population (N,= 2400) during the 600 years represented by each simulation run. The
response surface again represents proportion of generations above H = 9 Aa heterozygotes, the lower
95% confidence limit of LDH-Aa frequency at Abbotsbury, which is between 0.10 and 0.03 for the
likely range of Ne and immigration rates. However, the probability of observing more than H = 14
LDH-Aa heterozygotes, representing the observe LDH-Aa frequency, is shown as a solid line on the
edge of the block graph, and this is continually below 0.03 for all likely values of Nc and percentage
immigrants (including those not illustrated).
are not at all likely to be due to chance. We now examine the possibility that
differential selection between the genotypes could be the cause. As we have
established that Aa swans need to survive better, or breed more successfully, we
use one-tailed significance tests in the following discussion. As Bacon (1980)
showed that there were no significant mortality differences between the
genotypes, nor any assortative mating, we will here confine our discussion to
numbers of cygnets fledged per Abbotsbury flock swan per year. The data of
Table3 have already indicated that Aa swans are commoner in the breeding
population than the general flock. In 1977 an independent catch of adults at the
colony in August, all being swans who had stayed at the colony during the
breeding season and could have bred, showed five breeders of 14 Aa swans and
12 breeders of 92 A A swans, (P=0.047, Fisher’s exact test). The proportions of
Aa breeders in the three years 1976, 1977 and 1978 were continually above the
resident flock average of 13%, being, respectively, 17, 27 and 19% (Bacon,
1980). In 1978 a large comprehensive sample of most breeding adults and all
cygnets fledged was obtained, and productivity data are presented in Tables 4
and 5 . In Table 4 the data are divided both by LDH genotype and by natal
origin (Abbotsbury colony/elsewhere) of the adults. It can be seen that the
fledging success of Abbotsbury bred A A swans is low (43% fail to fledge any
cygnets) compared with other categories (14% or less fail). This difference is
largely responsible for the low number of cygnets per nesting adult for
P. J. BACON AND P. ANDERSEN-HARILD
212
Table 4. Production of cygnets per breeding adult
(=brood size divided by 2 and ascribed to both
adults) according to the LDH genotypes and natal
origins of those parents. Abbotsbury natal origin
denotes those with an ‘Ilchester’ web-nick
Within each cell: stage C/a=cygnets per nestin’g
parent ( = brood/2) including broods of zero; stage
YoF= percentage failing to fledge any cygnets; stage
AvB = average brood size of broods greater than
zero
Natal origin
LDH
genotype
AA
Stage
Abbotsbury
Elsewhere
C/a
53.5137 = 1.45
16/37= 43%
53.5121 = 2.55
15.517 =2.22
1 / 7 = 14%
15.5/6=2.58
16.5/7 =2.38
1/7 = 14%
16.516 =2.75
8.5/3 = 2.84
0/3= O(;G
8.5/3 = 2.84
Yo F
AvB
C/a
Aa
“/OF
AvB
Test for differences between categories of adults on Cygnets per parent
Test I : Ho= no significant differences at all
pl =0.16
Kruskal-Wallis H = 5 . 2 4
Test for differences between genotypes
Test 2: Ho= Aa adults fledge more irrespective of natal origin
p2=pl ~ 0 . 5 ~ 0 . 5 = 0 . 0 4
Abbotsbury A A swans (see Table 4). A straightforward test for differences in
average cygnets fledged for the categories of Table4 yields a probability of
P=O.16, above formal significance, but as both the comparisons of interest to us
(AA versus Aa irrespective of natal origin) show Aa to be more successful as our
null hypothesis requires the appropriate probability for our specific null
hypothesis is 0.16 x 0.5 x 0.5 = 0.04, a value just below formal significance.
Table 5. Probability that a pair of swans nesting at
Abbotsbury will fledge some cygnets (brood at fledging
greater than zero) according to LDH mating type. The
data are presented irrespective of natal origin of parents
as residents and immigrants pair randomly
LDH mating type
Fledging success
Number of pairs fledging
Number of pairs nesting
Percentage of pairs fledging
A A x AA
11
21
52y0
Test I : Ho=no differences within table. Fisher Exact pl=0.104
Test 2: H o = A a is more successful than A A . p2=pl x0.5=0.052
A A x Aa
9
I1
82yL
GENETICS OF COLONIAL MUTE SWANS
213
Lest anyone should think this calculation a little contrived, we present it in
more straightforward fashion for a similar data set in Table 5 . The main
difference indicated in Table 4 was the probability of fledging some cygnets: as
colony bred and immigrant birds mate randomly together (Bacon, 1980) we can
simplify the comparison to A A x A A pairs versus A A x Aa pairs (due to the rarity
of Aa swans, Aa x Aa matings hardly ever occur and did not in 1978). The
calculated probabilities of fledging are A A x A A = 52% and A A x Aa =82%,
with a probability of P=O.O52. A calculation for the mean size of successful
broods done in like fashion gives A A x A A = 5 . 0 0 and A A x Aa=5.56, a
difference which is not significant (P=O. 16), but indicates no countering
advantage favouring A A x A A pairs that succeed in fledging some young. The
mean weights of A A and Aa cygnets, which affect their winter survival prospects,
are not significantly different xi=O.4, P>0.80, Bacon, 1980). As yet we are
unable to assess life-time production for the genotypes as the study has not
continued long enough, but the indications are that Aa swans breed more often
than A A swans. In ideal circumstances, one would control for the effects of
different families of origin when comparing A A and Aa breeders, but this is not
practicable at Abbotsbury. The parents of many swans are presently unknown,
and those whose parents are known often breed in different years; numbers
breeding per year vary considerably and density-dependent fledging success
prevents a simple comparison. As the reasons for the annual fluctuations can not
presently be accounted for (Perrins & Ogilvie, 1981) more complex comparisons
are not fruitful with present data either. For the present we simply note the
following three points: (i) half the cygnets of an A A x Aa mating are AA, and
such birds will occur in the present comparison anyway; (ii) the small size of the
Abbotsbury population means relatedness between ‘resident’ birds will probably
be high irrespective of their immediate ancestry; (iii) the probable higher
proportion of Aa breeders in years of low nesting number but high fledging
success may further increase the advantage associated with the Aa birds.
Table 6. Estimated relative fitness from increased cygnet
productivity of LDH A A compared to LDH Aa for three sequential
aspects of breeding success
LDH Genotype
Factor
( i ) Probability
of nesting
(ii) Probability
of fledging
some cygnets
(iii) Mean brood
(for broods
greater than zero)*
Total advantage
AA
Aa
Ratio AaIAA
Individual
significance
0.14
0.19
1.35
0.047
0.52
0.82
1.58
0.052
5.00
5.00
5.56
1.13
2.40
0.160 n.s.
If the non-significant factor
(iii) is excluded:
Total advantage
*Hence (iii) is independent of (ii).
2.13
214
P. J. BACON AND P. ANDERSEN-HARILD
Summary of British Population genetics
The LDH A a genotype is significantly commoner at the colonies of
Abbotsbury and Weymouth compared to populations which nest territorially
(14% versus 1yo;P = 2.8 x 10- 14). This large difference in genotype frequencies
is unlikely to have arisen by random drift in the Abbotsbury population
( P < 0.01). However, Aa swans show increased breeding success compared with
A A swans, with a relative advantage of a factor of between 2.1 and 2.4
(P<0.05) see Table6. This estimated advantage is of the right magnitude to
counter dilution of L D H - a genes by 25% immigrants, mostly AA, from
surrounding territorial populations.
Danish data
The British data had established an association between the LDH Aa
heterozygote and colonial breeding, and that the Aa heterozygotes bred more
successfully in the Abbotsbury colony. As the a allele was not present at other
similar coastal sites, it seemed that any possible cause was more likely to be
related to colonial breeding than to any obscure habitat effects, although human
management could have been a n influence. As Abbotsbury and Weymouth
(closely adjacent) were the onb colonial or loose colonial sites in Britain there
was no possibility of seeing if the association with colonial breeding was more
widespread.
In Denmark, colonial breeding is relatively common; some 38% of all pairs
presently nesting in dense colonies, entirely free from human interference. Two
such colonies at Ringkobing and Roskilde fjords have been censused and ringed
since the 1960s. In 1983 we collected blood samples from five sites in Denmark
(see map, Fig. 10). Samples were collected in two ways. At four sites, flightless
birds from moulting flocks of several hundred swans were caught from
speedboats (the round-up technique used in Britain is generally impractical on
the large open Danish fjords); these birds comprise independent samples from
an adult population and the data can be used directly to estimate gene
frequencies. At two other sites families were caught. At Abbotsbury (and many
English sites), it is usually possible to catch both adults with the family, and use
the adult’s genotypes as independent data to estimate gene frequencies. In
Denmark, however, especially on the fjords, it is often difficult to catch both
adults: accordingly for the Danish family data we have estimated population
gene frequencies and their errors by utilizing data from cygnets but only for those
families where both parents were not also caught, and have then used the
method of Finney (1948) to derive unbiased estimates of gene frequencies and
their standard errors when such inter-related cygnet data are included; the
calculations for these adjustments are given in Appendices 2-4..
The data collected in 1983 are shown in Fig. 10 and Table 7. Of the samples
from moulting flocks the Ringkobing flock comprises almost entirely birds from
the colony at that fjord, the moulting flocks at Roskilde are approximately an
equal mix of birds from the Roskilde colonies and birds, mainly juvenile nonbreeders, from surrounding territorial sites which congregate on Roskilde for the
moult; while the flocks at Stryno and Saltholm are predominantly drawn from
territorial populations. Due to small sample sizes (resulting in less than five
expected Aa individuals at Stryno and Saltholm which invalidates x 2 test) the
GENETICS O F COLONIAL MUTE SWANS
215
Table 7. LDH genotype frequencies recorded from various sites in Denmark in
1983 according to site and method of capture and indicating composition of
sample according to the swans having been bred colonially or territorially
Site
Numbers of LDH genotypes
sampled
~~
No. in
Fig. 9
Name
Moult flocks
1
Ringkobing
Flock
composition
Data
C:T* type*
Colonial
I :O
AA
M
Aa
Roskilde
Stryno
Mixed
Territorial
1:1
0:I
M
M
64
32
4
Saltholm
Territorial
0: I
M
29
Colonial
Territorial
1: O
F
F
122
147
0: I
0.10 k0.03
15
9
6Im
ua
17
19m
I1
3
2a
3
a
20
8
31
95"
Families
2b
Roskilde
5
Kobenhavn
%Aa
Gene frequencies
ferror:
0.07 f0.02
0.04 f 0.02
4m
6
1
3
I1
4
8
3
0.02 f 0.02
Family estimates:
0.042 k0.017:
0.019f0.013$
*Flock composition. All moult flocks are mixtures of colonial and territorial swans: the approximate ratios
for the origins of flock birds are given as C=colonial : T = territorial.
?Independence of data: M = independent individuals from moulting flock, F = related individuals in
families.
mFisher Exact test gives: P , = 0.033 for genotype comparison: PM =0.037 for gene frequency comparison.
:Gene frequencies for families, and standard errors, calculated according to untabulated data on genotypes
1.02, one-tailed PF=0.15.
of individuals within families, following Finney, 1948.
Overall comparison, Territorial versus colonial swans, gives P, x PF=0.037 *O. 15 = 0.0055.
<=
1983 data were grouped as shown in Table 7, conservatively assuming the
Roskilde flock to be entirely colonial. A Fisher exact test gave a probability of
P = 0.033 that the observed genotype differences were due to random sampling
errors. It should be noted from Table 7 that there is a striking correspondence
between Aa frequency at a site and the proportion of 'colonial' birds in the
moulting flock.
Family data collected in 1983 from families at Roskilde fjord and the adjacent
territorial families in the Kobenhavn area also showed slightly more Aa swans in
Table 8. LDH genotype frequencies in Denmark: summary of 1983 and 1985
results. CR is the coloniality rank for moulting populations
Moulting
flocks
1983 Data:
Genotypes
.-
Site
name
Ringkobing
Roskilde
Stryno
Saltholm
CR
4
3
2
I
AA
An
31
8
64
I1
3
2
3
29
I
Ringkobing, Roskildr, Saltholm only
All four sitcs
Combined data
1985 Data:
Genotypes
u'oAa
20
15
6
3
AA
Aa
20
4
120
12
108
6
~~
Genotypes
Gene frequency
a+s.E.
?&An
AA
An
%Aa
16
9
51
184
32
137
12
23
3
7
19
I1
6
5
5
I ; = 10.1 P40.01
= 10.3 P<0.02
0.095k0.026
0.055_+0.011
0.043f0.024
0.024f0.009
P.J. BACON AND P. ANDERSEN-HARILD
216
t
‘Territorial’ sites
Figure 10. Frequency of LDH genotypes at five sites in Denmark in 1983, emphasising the
difference in Aa frequency between the two colonial breeding sites and the three territorial ones. See
Table 7 for details of flock composition by tenitorial/colonial origin (sites 1, 2a, 3, 4), and Table 8
and Figure 11 for summary information. The frequency data are shown superimposed, with
permission, on a map of present Mute swan breeding distribution from Dybbro (1976).
colony families than territorial ones (8% us. 3y0, see Table 7 and Fig. 10)
although this difference was not significant (<= 1.02, one-tailed P= 0.15).
We took these results as strong evidence to reject the null hypothesis “that the
LDH-Aa genotype was associated with colonial breeding only at
Abbotsbury/Weymouth” (see Bacon, 1980) and collected further samples in
1985 to permit more accurate analysis. The 1983 and 1985 moulting-flock data
are presented separately and combined in Table 8; bad weather prevented a
repeat catch at Stryno in 1985, but all other sites have adequate combined data
for a fully valid x2 test of between-site differences, giving xi = 10.3, P<0.02.
Again, there is a striking correspondence between coloniality rank of the
moulting flock and Aa frequency. As coloniality proportion and Aa frequency
for Stryno and Saltholm are very similar we now assume they are not different:
there are factorial 4 (=4!) ways of arranging four ranks in order and only four
orders in which the two lowest ranks could both be at the bottom, so the
probability that the observed order of Aa frequency in Denmark would match
the colonial rankings of the moulting flocks is 4/4! = 0.17. Hence the probability
that differences of the observed magnitude in Aa frequency between sites would
also be so correctly associated with the coloniality rank is <0.17 x 0.02 <0.003.
217
GENETICS O F COLONIAL MUTE SWANS
LDH-Aa
frequency
-
0.0
0.I
I
I
I
L
0.2
,
I
0.3
,
,
I
I
.
,-c-
4families
-s-
Ringkobing moull
] Roskilde
Stryno moult
Saltholm moult
4Kobenhovn families
Figure 11. LDH-An frequency and its standard error for six estimates at five sites in Denmark. The
sites are arranged from top to bottom in order of ‘colonialty rank’ depending on their estimated
composition by natal origin. Note their corresponding order of LDH-An frequency.
We further note that the moulting flocks are not only the correct order of Aa
frequency for their coloniality ‘rank’, but that the Aa frequency at Roskilde,
where the flock is known to be roughly equally composed of territorial and
colonially bred birds, has an Aa frequency of 11yo which is very close to that
predicted from an equal mix of ‘predominantly colonial’ (Ringkobing)
and ‘predominantly
territorial’
(Stryno and
Saltholm), namely
(19+5.5)/2= 12.25%.
Further samples from families from the Roskilde colony and territorial
families from the Kobenhavn area were also collected in 1985, and showed a
smaller difference in Aa frequency than in 1983 (8% us. 5%). The family data
Table 9. Summary of LDH family data for Kobenhavn and Roskilde families
for 1983, 1985 and combined. See Appendices 2-4 for explanation of Effective
number of (independent) alleles, as calculated by the method of Finney (1948)
Estimated
frequency
of
a allele
Effective
allele
numbers*
Kobenhavn
1983
I985
Combined
Roskilde
1983
1985
Combined
Assuming
random
mating
Aa genotype
Number of
families
WY
All
2ws
frequency
S.E.
yo Frequency
31
28
N.A.
2
3
5
107
99
I87
0.0186
0.0282
0.0255
0.0 I30
0.0166
0.0115
3.6
5.5
4.9
2.5
3.2
2.3
35
33
N.A.
5
5
130
117
242
0.0416
0.0405
0.0420
0.0175
0.0182
0.0 I29
8.0
7.7
8.0
3.3
3.5
2.5
U
10
-
*See Appendices 2 - 4 for calculation of wy and 2 w ~allele scores.
S.E.
Yo
218
P. J. BACON AND P. ANDERSEN-HARILD
Figure 12. Distribution of Mute swan nests at the Langholm island colony in Roskilde fjord,
showing the dates of nest establishment, and the locations of the few pain that were able to
maintain defended territories around their nests throughout incubation; those birds with nesting
territories were more successful.
for Roskilde and Kobenhavn in each year are shown separately and combined
in Table9; in combining the family data for the two years we have
appropriately excluded repeated data for the few individual adults (or their
subsequent cygnets) which were sampled in both years (see Appendices 2-4).
The combined family data for 1983 and 1985 give estimated Aa frequencies of
0.0805 f0.0247 for Roskilde and 0.049k 0.0228 for Kobenhavn, and these
frequencies are not significantly different (<=0.92, one-tailed P = 0.10, see
Appendix 4). The estimated Aa frequencies and their standard errors are shown
in Fig. 11 with the different sites and samples arranged by coloniality rank.
There are no indications that brood sizes at fledging differ between pairs with
and without an Aa member, but sample sizes for this comparison are small as
both parents are rarely caught at Roskilde. However, we should recall that
there was no significant difference in brood size at fledging for pairs that fledged
some cygnets at Abbotsbury either. At Abbotsbury the difference in breeding
success between genotypes was manifest between nesting and hatching, and as
yet we have no data on the frequency of the Aa swans when nesting starts at the
Roskilde colony. It is likely, however, that the dynamic picture may be different
in any case: the Abbotsbury colony has been established for hundreds of years;
pairs nesting there are notably unaggressive (Perrins & Ogilvie, 1981; Fair,
1985) and no eggs are lost due to aggression at the nest-site (Fair, 1985, and
pers. comm.). The colony at Roskilde however is very recent, some 25 years or
GENETICS OF COLONIAL MUTE SWANS
219
three generations old, and still expanding. Moreover, aggression at the nest site
is common at Roskilde, nearly all early nesting birds try and establish
territories, but few are able to maintain them due to intense pressure from other
swans moving in to nest nearby, see Fig. 12. Colonial swans at Roskilde lose
42% of their eggs during incubation, and most of these are broken during fights
at the nest (this is more than double the egg loss of 18% for territorial pairs in
Denmark: Andersen-Harild, 1978). There are clearly major differences in
ecology, social behaviour and lack of human management between the Danish
colonies and the Abbotsbury colony, so selective pressures and any resulting
effects on gene frequencies may well not be identical.
DISCUSSION
The LDH-a allele has been found to be significantly commoner at colonial
rather than territorial breeding places in both England and Denmark. Colonial
breeding is rare in England, 501, of pairs, but common in Denmark, some 38%
of pairs: in territorial circumstances the LDH-Aa heterozygote is also
significantly rarer in England than Denmark, 1% us. 5y0, 2: =23.76, P<<O.OOl).
A simulation model of drift at the Abbotsbury colony shows that the elevated
Aa frequency there is very unlikely to have arisen by random drift of a neutral
allele, but analysis of the data on breeding success shows a significantly
increased productivity for Aa swans at the colony. We have not presented a
similar calculation for the nearby ‘loose colony’ at Weymouth, where the Aa
frequency is also high, as many of the Weymouth swans probably derive from
“a number of pairs of Abbotsbury swans presented to Weymouth corporation
(by Lord Ilchester) in 1870” (Ilchester, 1934) and are hence not an
independent example.
In Denmark the swan colonies have arisen in the last 25 years, or three
generations: they were originally founded by the offspring of territorial breeders,
and owing to the low average productivity of colonial birds, the increase in
numbers of colonial breeders must itself be largely due to further immigration of
territorially bred swans. Data on the origins of known colonial breeding
individuals suggest 50% of breeders were themselves territorially bred, a n
immigration rate twice that shown to prevent divergence due to drift at
Abbotsbury. Thus, although circumstances in the Danish colonies are changing
too rapidly for an adequate simulation model of drift to be parameterized we
may conclude drift is most unlikely to have caused the elevated Aa frequencies
in the Danish colonies either. Although our present data do not indicate that Aa
swans produce larger broods in Danish colonies we lack information for the Aa
frequency among Danish nesting swans, and it is during incubation that the
advantage to Aa swans is most apparent at Abbotsbury.
The higher Aa frequencies at colonies occur despite differing ecologies
between the populations and irrespective of human management, as at
Abbotsbury, or not, as in Denmark. The main consistent factor between the sites
is that Mute swans nesting at them tolerate other swans nesting much closer to
them than is normal. It is true that the degree of tolerance varies from almost
total in the 600-year or older Abbotsbury colony to only partial in the 25-yearold Danish colonies, but it is unlikely that complete tolerance could be achieved
220
P.J. BACON AND P. ANDERSEN-HARILD
in three generations, irrespective of whether any increased tolerance came from
environmental experience or genetic selection, especially with about 50% of
colonial breeders having themselves been raised on territories. We thus incline to
the view that the null hypothesis suggested by Bacon (1980) “that there are no
significant differences in aggressive responses between LDH AA and Aa swans”
is the next most worthwhile to try and refute. With the additional information
from Denmark that the Aa allele is indeed generally associated with colonial
breeding it may now be justifiable to try to undertake the difficult task of
assessing aggressive levels in field situations which are largely uncontrollable.
It seems highly unlikely that two separate mutations at the LDH-B locus
would occur which produce indistinguishable Aa patterns, and a common origin
seems much more likely. The present British population is effectively isolated
from continental populations by the English Channel, which separated the
British Isles from Europe some 8000 years or 1000 swan generations ago. While
some Mute swans have been imported into England from Europe most of these
would have been imminently destined to be eaten at banquets; historical records
of such imports are rare, and the heavy tax on imported swans (Ticehurst, 1957)
suggests importation was discouraged to promote the prestige value of swan
meat. If the LDH-a allele has indeed been linked to some trait associated with
colonial breeding for 1000 generations (assuming the mutated a allele has not
recently been introduced to Britain by natural or human means) the degree of
linkage must be extremely close. We cannot however suggest a likely selective
advantage that might be conferred by a variant of the LDH enzyme itself: the
most likely speculation along these lines would seem to be that an LDH-Aa
heterozygote might cope better with variable oxygen and temperature changes
during its incubation as an egg, but such a mechanism, should it exist, would
also be of benefit to territorial breeders (even if their incubation were
interrupted less often) and it seems most unlikely that such a general advantage
would only be frequently manifest in colonial circumstances.
One apparent anomaly remains: if the Aa frequency is higher in Danish
colonies, but the Aa colonial breeders are not markedly more successful, how did
the increased frequency come about? A straightforward explanation, consistent
with our views on modified aggression, would be that the Aa swans hatched at
territorial sites are more likely to stay within the moulting flock at Roskilde and
subsequently attempt to breed at the colony, while their AA siblings may be
more likely to return and breed in territorial sites. Such selective movements
could elevate Aa frequency at a colony without increased breeding success.
ACKNOWLEDGEMENTS
The genetic study started as a SERC studentship at the Edward Grey
Institute at Oxford and continued as an Institute of Terrestrial Ecology project.
The English colonial sites were studied in collaboration with Drs C. M. Perrins
and M. A. Ogilvie; Lady Theresa Agnew, Weymouth Town Corporation and
the RSPB kindly permitted visits to field sites and their wardens John Fair,
Peter Leighton and Douglas Ireland provided much help. Other British samples
were obtained in collaboration with A. E. Coleman, D. Stone and Drs C. M.
Perrins and C. J. Spray; we thank numerous local land owners and bird-ringers
GENETICS O F COLONIAL MUTE SWANS
22 1
for co-operation. The Danish study was supported by the Bird-Ringing Centre,
Zoological Museum, Kobenhavn: E. Hansen, E. Fritze and R. Farmer kindly
helped collect the samples and Betty King and Dr Me1 Tonkin assisted with the
electrophoresis. The Danish visit was supported by the G . W. Trust and the
Ernest Kleinwort Charitable Trust, and Starstedt UK Ltd donated their special
collecting syringes. We thank Lizzie Bacon, D. K. Lindley, Drs Brenda Howard,
I . Newton and C. M. Perrins for discussions and comments, and Chris Benefield
and Allan Nelson for help with the illustrations.
REFERENCES
ANDERSEN-HARILD, P., 1978. Knopsuanen. Hoke, Denmark: Skarv Nature Publications.
ANDERSEN-HARILD, P., 1981. Migration of Cygnus olor ringed in Denmark in winter and during moult. In
G. T. V. Matthews & M. Smart (Eds), Proceedings of the Second International Swan Symposium, Sapporo, Japan:
120-131. Slimbridge, U.K. 11: International Waterfowl Research Bureau.
ANDERSEN-HARILD, P. & PREUSS, N. O., 1978. Optaelling of Ynglende Knopsvaner i 1970.
Feltornitologen, 20: 34-35.
BACON, P. J., 1980. Population genetics of the Mute swan (Cygnus olor). D.Phil. Thesis, University of Oxford.
D32193/80 (BLLD F).
BACON, P. J. & COLEMAN, A. E., 1986. An analysis on weight changes in the Mute swan (Cygnus olor).
Bird Study, 33: 145- 158.
BERRY, R. J., 1975. O n the nature of genetical distance and island races of Apodemus syluaticus. journal of
< o o ~ o ~176:
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293-296.
BERRY, R. J,, 1977. Inheritance B Natural History: 50-60. London: Collins.
BIRKHEAD, M. & PERRINS, C., 1986. The Mute Swan. Beckenham, U.K.: Croom Helm.
BLOCH, D., 1970. Knopsvaner som kolonifugl i Danmark. Dansk Ornithologisk Forenings Tidssknyt, 64:
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BLOCH, D., 1971. Ynglebastanden of Knopsvane (Cygnw olor) i Danmark i 1966. Dansk Vildfundersogelser, 16:
1-47. (Vildbilogisk Station, 1971).
CAMPBELL, B., 1960. The Mute swan census in England and Wales 1955-56. Bird Study, 7: 208-23.
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Staffordshire. Wildfowl, 31, 22-28.
CRAMP, S. & SIMMONS, K. E. L., 1977. Handbook of the Birds of Europe, the Middle East and North Africa; the
Birds of the Western Palearctic: I. Oxford: Oxford University Press.
DYBBRO, T., 1976. De danske ynglejiugls udbredelse. Kobenhavn: Dansk ornithologisk Forening.
EL'IRINGHAM, S. K., 1963. The British population of the Mute swan in 1961. Bird Sludy, 10: 10-20.
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FAIR, J., 1985. The Mute swan. Limington, U.K.: Gavin Press.
FINNEY, D. J., 1948. The estimation of gene frequencies from family records. I. Factors without dominance.
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GALE, J . S., 1980. Population Genetics. Glasgow: Blackie.
HILL, W. G., 1972. Effective size of populations with overlapping generations. Theoretical Population Biology 3:
278-289.
HILPRECHT, A,, 1972. Hockerschwan, Singschwan, Zwergschwan. Wittenberg: Neue Brehm-Bucherei.
ILCHESTER, The Right Hon. the Earl of, 1934. The Abbotsbury swannery. Proceedings of the Dorset Natural
Histoy B Archeological Sociely, 55: 154-164.
LINDEY, S. & RAJALSAMZ, M., 1966. Lactate dehydrogenase of chick embryo; responses to variations in
ambient oxygen tension. Science, 53: 1401-1403.
MARKERT, C. L. & URSPRANG, H., 1962. The ontogeny of isozyme patterns of lactate dehydrogenase in
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MARKERT, C. L. & MASUI, Y., 1969. Lactate dehydrogenase isozymes of the penguin Pygosalis adeliae.
Journal of Experimental <oology, 172: 121-146.
MA'I'HIASSON, S., 1973a. Moulting grounds of the Mute swan (Cygnus olor) in Sweden, their origin in
relation to the population dynamics of Mute swans in the Baltic area. Viltrey, 8: 339-452.
MATHIASSON, S., 1973b. A moulting population of non-breeding Mute swans with special reference to
flight feather moult, feeding ecology and habitat selection. Wildfowl, 24: 45-53.
MAYNARD SMITH, J. & HAIGH, J., 1974. The hitch-hiking effect of a favourable gene. Genetical Research
23: 23-35.
OGILVIE, M. A,, 1967. Population changes and mortality of the Mute swan in Britain. Wildfowl, 18: 64-73.
OGILVIE, M. A,, 1981. The Mute swan in Britain, 1978. Bird Study, 28: 87-106.
222
P. J. BACON AND P. ANDERSEN-HARILD
OGILVIE, M. A,, 1986. The Mute swan Cygnw olor in Britain in 1983. Bird Study, 33: 121-137.
PERRINS, C. M. & OGILVIE, M. A., 1981. A study of the Abbotsbury Mute swans. Wildfowl, 32: 3547.
SHARROCK, J. T. R., 1976. The Atlas OfBreeding Birds in Britain and Ireland. Waterhouses, U.K.: Poyser.
SHAW, C. R. & PRASAD, R., 1970. Starch gel electrophoresis of enzymes: a compilation of recipes.
Biochemical Genetics, 4: 297-320.
SPENCER, R. & HUDSON, R., 1977. Report on Bird Ringing for 1975. Special supplement to Bird Study, 24.
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APPENDIX 1.
TECHNICAL DETAILS ON SAMPLE COLLECTION,
SAMPLE PREPARATION, ELECTROPHORESIS AND
STAINING PROCEDURES
Further details of all procedures can be found in Bacon (1980).
Sample collection
Samples were collected into 5-ml Li-heparin Sarstedt Monovettes (05.276/001; these are syringes which also
functioned as centrifuge tubes) using 4-cm 21 g needles. Samples were taken from the foot (tarsal vein, and
puncture closed with cotton wool held in place with a turn and a half of Sellotape) or the wing near the
shoulder (where the vein runs along the underside of the humerus at the base of the lowest, proximal underwing coverts).
The samples were kept cool on melting ice, centrifuged and separated into plasma and red-cells and frozen
within 6-12 h (occasionally 24 h). Plasma was stored neat in 2-ml NA2L Sterilin tubes: red-cells were lysed
with an equal volume of O.Olyo Triton-X-100 (Sigma T-6878) in 0.9% NaCI. In contrast to LDH from
chickens (Callus domesticrrs) which is denatured on freezing, Mute swan LDH is still active after storage at
-20°C for many months (1-3 years even).
Sample preparation
Batches of samples were prepared in U-well microtitre trays (Sterelin). For ease of processing, aliquots of
samples (four to six drops from a Pasteur pipette) were added to two drops (from a Pasteur pipette) of the
following preservative/stabilizing fluid:
Sucrose
K H,PO,
K,HPO,
2-Phenoxy ethanol
Distilled water
85.6 g
2.27 g
14.51 g
15.0 ml (Sigma P-1126)
Adjusting volume to 1 litre
The use of Clelland’s reagent (0.1 g of Di-thioThreitol in 12.0 ml distilled water) either alone or in
conjunction with the above solution gave no useful improvement for Mute swan LDH.
Electrophoresis
Gel moulds
Plates for horizontal starch-gel electrophoresis were made as follows. Moulds were constructed on a base
plate of 3 mm thick glass (12x31.5cm, occasionally 1 8 ~ 3 1 . 5cm) with glass strip side formers 6 m m
deep x 10 mm wide. A 3 mm thick glass top-plate of the same dimensions was also used (see below).
Gel buffer
A stock solution of Trislcitrate buffer was made up as follows:
Tris
Citric acid
Distilled water
1.52 g (Sigma T- 1503)
0.68 g (BDH or Fisons; Analar grade)
Adjusting volume to I .O litre
The pH was carefully adjusted to 7.0 with conc. HCI or conc. NaOH.
GENETICS OF COLONIAL MUTE SWANS
223
Electrode buffer
This buffer was very weak, and should only be used for one gel; each gel requires 2 I of buffer as follows:
Tris
Citric acid
Distilled water
9.08 g
4.04 g
Adjusting volume to 2.0 litre
The pH was carefully adjusted to 7.0 with conc. HCI or conc. NaOH.
Gel preparation (12 x 31.5 cm gels)
The glass plates were carefully cleaned and wiped with acetone and the side-formers fixed to the bottom
plate with Silicone vacuum grease.
Exactly 30.0 of hydrolysed starch (Sigma S-4501) were shaken vigorously in 250 ml of gel buffer (12% w/v
solution) in a I-litre conical side-arm vacuum flask and then stirred vigorously and continuously on a heated
magnetic stirrer. Care was taken that (i) no undissolved starch remained to form lumps or burn and that (ii)
as the viscosity increased as the solution approached boiling point the magnetic stirrer had to be carefully
adjusted or else the rotor-arm became stuck and the unstirred solution degraded the starch.
When the solution reached boiling point it was degassed under vacuum (using a Buchner pump evacuating
a 500 ml conical flask reservoir); when boiling vigorously under vacuum (no small bubbles left) the vacuum
was quickly broken and the hot solution carefully and immediately poured into the gel mould, ensuring an
even covering with no entrapped air ( a few small bubbles sometimes appeared in the gel-if these were few
and small a Pasteur pipette was used to remove them). The top plate was then placed with one long edge
resting on a side-former and very carefulb lowered across the gel, allowing surface-tension to create a smooth
contact line and prevent air being trapped as bubbles between the plate and the gel. (Such bubbles seriously
disrupt the even conductivity required.)
The gel was then wrapped in polythene film (‘Cling film’) to prevent desiccation and keep the top plate in
position, allowed to cool and placed in a refrigerator at 4°C to set and cure; the gels had to be allowed to cure
for at least 2 h, preferably 4 h.
Buffer trays
Electrophoresis took place in a cold-room (refrigerator) and about I litre of Electrode buffer (to within
8 mm of tops of the trays) was allowed to cool in each of a pair of buffer trays (constructed from 3 mm glass,
36 x 6 x 6 cm external measurements) having platinum electrodes.
Wicks of eight thicknesses of ‘J-cloths’ (Jeyes Ltd, ‘J-soft Cloths’) were soaked in the electrode buffer at this
time (four 34 x 36 cm strips folded into 34 x 18 cm).
Preparing lhe gel
Just prior to loading the samples the gel was taken from the fridge, the edges trimmed to remove thin starch
between the top plate and side-formers, and the surface blotted dry.
Insert positions
Slots for the paper inserts (5 x 5 mm Whatman No. 3 MM chromatography paper) were cut with a metal
‘comb’ having 27 5-mm wide ‘teeth’. This line of slots was positioned centrally across the gel 4.5 cm from the
cathodal side-former (not plate edge) (6.5 cm for 18-cm gels).
Loading the samples
Twenty-seven 5 x 5 mm squares of filter paper were placed in the sample solutions in the micro-titre trays (it
was found to be useful to put the first and last in duplicate, with one of each duplicate having two insert
papers per slot, i.e. 25 distinct samples per gel).
When the papers had adsorbed the solution their surfaces were blotted dry, and they were carefully inserted
in the slots. Care was taken to ensure that the slot edges were not torn, to avoid streaking.
Electrophoresis
The gel was ‘run’ for 12 h, and this was conveniently done overnight, using a time-switch to turn off the
power-pack(s) as necessary. The gel was placed on the edges of the buffer trays, and the wicks lifted up so they
overlapped the starch by I .5 cm along the whole length of the anodal and cathodal edges. T h e weight of the
gel required support by a wooden block below its centre.
Power suppb
A high voltage high power DC unit was used (Heathkit Heath Zenith Model SP-2717A); this required a
warm up time of 30 min before use. T h e units were set to provide Constant Voltage and were not placed inside
the cold room.
Voltage
The voltage gradient was measured across the surface of the gel, and the power-pack current adjusted until
there was a gradient of 10.0 V/cm, measured near the centre of the gel (50 V across a 5 cm ‘Bridge Gap’ was
convenient). When the voltage was set, a glass rod was placed close to the insert line and the gel surface
covered with a thick sheet of polythene (the glass rod prevented a liquid bridge forming between adjacent
sample slots).
224
P. J. BACON AND P. ANDERSEN-HARILD
Typical current and voltage readings for the power-pack out-puts should initially be 85-100 mA and
200-300 V, falling to 65-85 mA and 200-220 V after adjustment. If such values could not be obtained, then
either the levels in the electrode buffer trays had to be altered, the cooling had to be improved or the buffer
concentrations or pH were probably incorrect.
Cooling the gel
The gel should be cooled with a layer of melting ice chips, 5 cm thick, held over its entire surface within a
polythene sheet on a wooden frame.
Voltage adjustments
After 30 and 60 min the voltage gradient was checked, and adjusted back (usually down) to 10 V/cm. After
60 min the glass rod near the insert line was also removed.
The power-pack and ice-cooled gel were then left for 11 h.
Staining
Preparing Ihc gel f o r staining
The gel was removed from the power-pack, its surface blotted and one corner marked by a cut to identify
the orientation; if several gels were being prepared together, each was marked with nicks to avoid confusion.
Using 3 mm glass runners, held in place with silicone grease and a ‘cheese wire’ on a metal frame, the gel
was cut into two 3 mm thick slices. Care andpactice w e n needed. A top-plate was used to keep the gel flat during
cutting. The top slice was carefully removed and kept moistened with buffer in case it was needed (if the
bottom slice was accidentally spoiled).
The lower slice was soaked in 100 ml of double strength stain buffer for 20-30 minutes (dilute stock stain
buffer 1 : 1).
L D H stain chemicals-stock solutions
Stock L D H stain buffer: 0.2 M Tris/HCI-24.228 g Tris, distilled water to 1.0 litre, pH adjusted to 8.2 with
conc. HCI.
Substrate: 1 M Na-lactatt8.25 ml lactic acid syrup (Sigma L-1250), distilled water to 91.75 ml, pH adjusted
to 7.0 with conc. NaOH.
Agar overlay: 2 g of Agar (Sigma A-7002) was boiled in 100 ml distilled water (2% w/v), cooled and kept in a
water-bath at 56°C.
Stock stain chemicals: these were stored in the dark at 4OC: 8-NAD (Sigma N-7004) 5 mg/ml (50 mg in 10 mi),
M T T (Sigma M-2128) 2 mg/ml (20 mg in 10 ml), PMS (Sigma P-9625) 2 mg/ml (20 mg in 10 ml).
Stain procedure
Incubation buffer was poured off the gel and the surface blotted dry. Glass strips were arranged on surface
of gel to leave a rectangle of surface having some 3 cm cathodal of the insert line and 5 anodal to it.
The stain had to be made up quickly, adding chemicals in order: 10 ml stock stain buffer, 2 ml stock Lactate
substrate, 2 ml stock NAD, 2 ml stock M T T and 2 ml stock PMS, then the mixture was stirred and the
following quickly added, 20 ml 2% agar solution; stir and quickly pour resulting mixture onto the gel surface
(within the rectangle) ensuring an even thickness of cover.
The agar over-lay solution, was allowed to set and the gel was covered with ‘Cling-film’ and placed in an
incubator (over a water-bath) at 40°C in the dark. The gel was left for several ( 2 4 ) hours, until blue bands
were fully developed, and was removed from incubator before back-ground started turning blue. The banding
patterns were recorded. The stained gel could be placed in a polythene bag and stored in a fridge for several
months.
The electrode buffer was never re-used on subsequent gels.
APPENDIX 2.
FINNEY’S METHOD CALCULATION
FOR ESTIMATING GENE FREQUENCY
AND ITS ERROR FROM FAMILY DATA FROM D E N M A R K , 1983
Brief Description: For adults the scores w a n d y are simple counts of the numbers of (independent) genes, w
being the total number and y the total of the rare allele. For cygnets, weighted scores w and y are used
depending on (i) an initial estimate of the allele frequency, (ii) the family sizes, F,, from which the samples came,
(iii) the genotype of the single parents, where known, and (iv) the genotypic distribution within aN families of
that size (from single parents of that genotype where appropriate). Maximum likelihood scores for w and
formulaey are given in detail by Finney (1948).
GENETICS OF COLONIAL M U T E SWANS
225
Kobenhavnfamiliej
Data from adult breeders:
Numbers of genotypes
Genes
A
0
aa
0
0
2
0
88
AA
32
11
Aa
Both adults
Single adults
'I'otal adults
43
2
Scores
a
swy
S2ws
_ _ _ _ _ _ _ _ _ _ _ _
Data from Cygnets Ibr whom only one parent was also sampled:
Numbers of
No. of
Cygnets
Weights
Sibship
Single
size
ad u It's
families
per gene
.N,
A A Aa aa
W
genotype
I;,
2
2 0 0
0.490
1
AA
5
10 0 0
0.325
2
AA
3
AA
2
6 0 0
0.246
4
AA
2
8 0 0
0.197
2
90
2
Initial estimate 2/[email protected]
Score
Y
2s
genes
F, x N ~ 2x
4
20
12
16
0.0
0.0
0.0
0.0
-~
0.500
1.96
6.50
2.95
3.15
Sum (2ws) = 14.56
Sum (wy) =O.O
Data from cygnets for whom neither parent was caught:
none
1
3 0 0
3
2ws
0.0
3.00
6
~
~
Sum(2ws)= 3.00
Sum (wy)=O.O
Estimated tl gene frequency for Kobenhavn
freq. (I-freq.)
Sumwy
2+0+0
- 0.0186
Var. freq.=
=
Est. freq. =Sum 2ws 90+ 14.56+3
107.56
Sum 2ws
Kobenhavn Frequency* S . E . =O.O186f 0.0 130
0.0182
- 0.000 17
107.56
=__-
Roskilde families
Data from adult breeders:
Numbers of genotypes
Genes
Scores
~~
AA
24
Both adults
Single adults
aa
0
0
A
17
Aa
0
2
41
2
0
84
a
S2ws
swy
____________
'I'otal adults
Data from cygnets for whom only one parent was also sampled:
Numbers of
Sibship
Single
No. of
Cygnets
Weights
size
adult's
families
per gene
I;,
genotype
A A Aa aa
W
I
AA
3 0 0
0.490
2
AA
5
10 0
0
0.320
Aa
I
2 0 0
0.320
3
AA
6
14 4 0
0.246
4
AA
I
4 0 0
0.197
I
3 2 0
0.164
5
Aa
2
86
2
Initial estimate 2/86 =0.0232
Score
Y
Y
0.0
0.0
-0.092
8.0
0.0
-0.046
Sum(wy) = 1.932
Data from cygnets for whom neither parent was caught:
1
none
3
3 0 0
3
none
3
6 3 0
6
none
I
6 0 0
7
none
1
7 0 0
1 .ooo
0.0
3.0
0.0
0.0
0.500
0.286
0.250
Sum (wj) = 1.5
2s
2ws
genes
F, Nfx 2
6
10
4
36
a
5
2.94
6.40
1.28
8.86
1.58
I .64
Sum(2ws) =22.69
6
la
12
14
6.00
9.00
3.40
3.50
Sum (2ws) =21.93
Estimated a eene freauencv for Roskilde
freq. (I-freq.) -~
0.398
Var. freq.=
' '
' 'Sum wy '
2 1.932 1.5 - 5.43
Sum 2ws
130.62
Est. freq. =
- 0.0416
Sum 2ws 86+22.69+21.93
130.63
= 0.00030
Roskilde Frequencyfs.~.=0.0416+0.0175.
+
~
+
~~
~
P. J. BACON AND P. ANDERSEN-HARILD
226
Comparison between Kobenhavn and Roskilde
a Fre
Kobenhavn
Roskilde
~s.E.
0.0188’ f0.13
0.0416 f0.17
Weighted proportion P’
+n2)
= ( 0 . 1 8 6 ~107.6+0.0146 x 130.6)/(107.6+130.6)
= 7.431238.2
=0.312
=pl -pS/SQRT(P’( l P ) ( I / n l l/n2))
=0.023/SQRT(0.0052)
=1.0176
= (pl x n l +p2 x n2)/(nl
<
+
giving one-tailed P=O.15
APPENDIX 3
FINNEY’S METHOD CALCULATION FOR 1985 DATA
ON LDH GENE FREQUENCIES IN DENMARK
Kobenhavnfamilies
Data from adult breeders:
Both of pair
Single from pair
Failed breeders
AA
25
6
5
Aa
I
2
0
~~
Total
36
0
A
55
0
0
10
10
aa
,
WY
I
2
0
0
75
3
78
3
Initial estimate 3/78=0.0385
Genotype frequencies of cygnets within families
Data from cygnets for whom only one parent was also sampled:
Nos. of cygnets
Allele scores
by genotypes
Adult
No of
Sibship
Aa
aa
size
genotype families AA
W
Y
WY
0.4810
0.0000
0.0000
AA
1
1
0
0
I
0.4810 -0.0770 -0.0370
Aa
1
1
0
0
4
AA
4
14
2
0
0.1960 -0.9240 -0.1810
O.OOO0
O.OOO0
0.1630
5
AA
1
5
0
0
0.1630 +0.0770
0.0130
Aa
1
2
3
0
Sum(wy)= -0.2050
Data from cygnets for whom only one parent was also sampled:
I
None
2
2
0
0
1.OO00
0.0000
0.0000
2
None
2
2
0
0
0.6667
O.OOO0
0.0000
None
I
3
0
0
0.5000
O.OOO0
O.OOO0
3
Sum(wy) =O.OOO0
Estimated gene frequency Kobenhavn
Sum(wy)
2.795
-- 3.0-0.205+0.00 = = 0.0282
Sum(2ws) 78.0+ 11.456+9.6667 99.123
2ws
a
~~~~
~
3
Scores
Genes
Numbers of genotypes
Total gene
scores
s
2s
I
1
16
2
5
5
2
32
10
10
2sw
0.9620
0.9620
6.2720
1.6300
1.6300
Sum(2ws)= 11.456
2
2
3
4
4
6
4.0000
2.6667
3.0000
Sum(2ws)= 9.6667
Estimated standard error Kobenhavn
freq. (1-freq.) = 0.0274 ~
Var. =
- 0.000276
Sum(2ws)
99.123
Hence S.E. =SQRT (Var.)= SQRT (0.000276)
=0.0 166
Kobenhavn 1985 Frequency f8.e.f =0.0282f0.0166
227
GENETICS O F COLONIAL M U T E SWANS
Roskildefamilies
Data from adult breeders:
Numbers of genotypes
AA
Aa
0
18
Both of pair
Single from pair
~
19
~
1
aa
0
0
_
A
36
39
2ws
a
0
I
WY
75
0
1
_
Scores
_
~
_
I
76
1
Initial estimate 1/76=0.0132
However, examining data from families shows at least 7/136 ‘adult genes’ were LDH-a as more accurate
initial estimate, so this was used giving 7/136=0.0500.
37
Total
~
Genes
Genotype frequencies of cygnets within families
Data from rygnets for whom only one parent was also sampled:
Nos of cygnets
by genotypes
Sibship
size
2
3
4
5
Adult
genotype
NO of
families
AA
Aa
AA
AA
AA
8
2
3
6
1
Total gene
scores
Allele scores
~
AA
Aa
aa
13
1
6
24
4
3
3
3
0
1
0
0
0
0
0
W
Y
0.3207
0.3207
0.2421
0.1964
0.1627
6.00
0.20
6.00
0.00
2.00
s
2s
2sw
16
4
9
24
5
32
8
10.2624
2.5656
4.3578
9.3408
1.6270
WY
1.9242
0.0641
1.4526
O.Ooo0
0.3254
Sum(wy)= +3.7663
18
48
10
Sum(2ws)= 28.1536
Data from cygnets for whom only one parent was also sampled:
I
2
3
4
I
2
None
None
None
None
1
4
3
4
1
1
0
0
0
0
0
1.0000
0
0.6667
0.5000
0.40000
0
0
0.00
0.00
0.00
0.00
1
4
3
4
0.00
0.00
0.00
0.00
2
~
~
Sum(wy)=O.OO
Sum(2ws)= 13.5336
~~
Estimated gene frequency Roskilde
Sum(wy) - I .O 3.7663 0.00 =-- 4.7663 - 0.0405
Sum(2ws) 76+28.1536+ 13.5336 117.6872
+
+
2.0000
5.3336
3.0000
3.2000
8
6
8
~
~~
Estimated Standard error Roskilde
freq. (I-freq.) - 0.0389
Var. =
--=
0.0003302
Sum (2ws)
117.6872
hence S.E. = S Q R T (Var.) =SQRT(0.0003302)
=0.0182
Roskilde 1985 Frequencyfs.~.=0.0405 k0.0182.
APPENDIX 4.
COMBINATION O F 1983 AND 1985 DANISH FAMILY DATA
FOR ESTIMATIONS O F OVERALL ALLELE FREQUENCIES AND
DIFFERENCES BETWEEN SITES COMPARISON
Some adults sampled in 1983 were again sampled in 1985, and their effects on the overall totals need
adjusting so they do not contribute twice. In general such adjustments were most easily made to the 1985 data
h u t in a few rases were most simply done by adjusting 1983 scores.
_
_
P. J. BACON AND P. ANDERSEN-HARILD
228
1983Adjuslmenls
Kobenhaun: One adult whose cygnets included in 1983 but was herself sampled in 1985; remove cygnets'
contribution to 1983 total: Subtracted 2ws= 1.482; wy=O.O.
Roskilde: None needed.
1985 Adjustments
Kobenhavn: Eight adults also sampled in 1983: Subtracted Pws= 16; wy=O.O. Brood of four whose single
parent's mate had been sampled in 1983: Subtracted 2ws= 1.568; wy=O.O. Totals: 2ws= 17.568; wy=O.O.
Roskilde: Two adults also sampled in 1983: Subtracted 2ws=4; wy=O.O. Brood offour whose single parent's
mate had been sampled in 1983: Subtracted 2ws= 1.5568; wy=O.O. Totals: 2urs=5.5568; wy=O.O.
Combined calculations
Kobenhavn
1983
total
2.0
107.56
I983
correction
-0.0
- 1.481
1985
total
2.795
+99.123
+
1985
correction
0.0
-17.568
+
4.795
- 187.634
=0.0255
Variance =0.0255( 1-0.0255)/187.634
=0.0249/ 187.634
=0.000133
S.E. =SQRT (variance)=SQRT(0.000133)
=0.0115
Kobenhavn: LDH-a allele frequency =0.0255 f0.0115
Roskilde
1983
total
5.43
~130.62
1983
correction
+o.o
+o.o
+
+
1985
total
4.7663
1 17.6872
1985
correction
0.0
-5.5568
+
10.1963
= 242.7504
=0.0420
Variance = 0.0420( l-O.0420)/242.7504
=0.0402/242.7504
=0.000166)
s.E.=SQRT (variance) =SQRT (0.000166)
= 0.0129
Roskilde: LDH-a allele frequency =0.0420f0.0129
Comparison of Kobenhaun u e m s Roskilde
P'= Combined proportion
P'= [(PI x n l +p2 x n2)]/(nl+ n 2 )
=[(0.0255 x 187.63)+(0.0420~
242.75)]/(187.63+242.75)
=[4.79+ 10.19]/(430.38)
= 14.97/430.38
=0.0348
< =Normal deviate
<=[pl-p2]/SQRT[P'(l-P') x (I/nl +l/n2)]
= [0.0420-0.0255]/SQRT[0.0348( 14.0348) x (1/187.63+ 1/242.75)]
=0.0165/SQRT[0.0336 x 0.00951
= 0.0 165/SQRT[0.0003191
=O.Ol65/0.0179
=0.9218
Giving one-tailed probability P=O.18
One-tailed test as deviation is in predicted direction colony LDH-n > territory LDH-a.