Preliminary Analysis of Common Loon (Gavia immer) Genetic Structure
in Northeast North America Based on Five Microsatellite Loci
Amy M. McMillan, State University of New York College at Buffalo; Mark J. Bagley, United States Environmental Protection
Agency, Cincinnati, Ohio; and David C. Evers, BioDiversity Research Institute, Gorham, Maine
Courtesy of BioDiversity Research Institute
This study seeks to determine fine-scale genetic structure of Common Loon breeding
populations in order to link wintering birds with their breeding regions. Common Loons are large
piscivorous birds that breed in lakes of northern North America and Iceland. Loons are highly
philopatric and territorial in breeding areas and are susceptible to mercury poisoning, lake
acidification and other threats across much of this region. Wintering loon populations originate
from a mix of breeding regions. In North America, wintering populations are found primarily in
nearshore coastal environments and these birds are susceptible to oil spills. Loons also are
threatened by the current botulism poisoning outbreak, which has killed thousands of loons in the
Great Lakes. Despite significant demographic data, little is known about the population genetic
structure of Common Loons. Preliminary analysis using five polymorphic microsatellite loci
demonstrated strong differentiation between loons in eastern and western North America (R X C
exact test X2 = 48.14, df = 10, P = 0.000). Differentiation among five putative eastern loon breeding
populations was also identified. Differences were found in four of ten pairwise comparisons. The
information developed on loon population structure will be crucial for understanding year-round
impacts on these birds.
Results
Materials and Methods
Abstract
 Common Loon samples collected from western (California, Nevada, Washington States;
n=70) and Eastern (Maine, Massachusetts, New Hampshire, New York, Vermont, Virginia
States; New Brunswick, Nova Scotia, Quebec Provinces; n=323) North America between
1997-2001
 DNA extracted from blood samples using Qiagen DNeasy tissue kit
 Five polymorphic microsatellite loci (Table 1) used to determine population structure (McMillan
et al. 2004)
 15 µl PCR reactions: 3 pmol each labeled forward and unlabeled reverse primer, 250 μM
each dNTP, 1.5 mM MgCl2, 0.6 U Taq DNA polymerase, and 15 ng template DNA in buffer
containing 20 mM Tris-HCl and 50 mM KCl (pH 8.0)
 Touch-down thermal regime: 1 min at 95○C; 12 cycles of 30 sec at 95○C, 30 sec at 64○C,
dropping 0.8○C for each subsequent cycle, 45 sec at 72○C; 23 cycles of 30 sec at 95○C, 30
sec at 54○C, 45 sec at 72○C; 5 min at 72○C; 10○C hold
 Two to seven alleles were present at each locus. Allele frequencies for each locus varied from
nearly monomorphic (GimE11EPA) to highly polymorphic (Table 2).
 All populations were in Hardy-Weinberg equilibrium at all loci except for locus GimE11EPA
(POPGENE, Chi-square test). Fewer birds with allele 2 were seen than expected in the East coast
birds and in the Vermont/Northern New Hampshire population.
 Breeding populations from the eastern and western portions of North America were genetically
distinct (TFPGA, R X C exact test X2 = 48.14, df = 10, P = 0.000; Raymond and Rousset 1995).
 Eastern birds showed a complicated pattern of relationships (Table 3). For this preliminary
study, we anticipated potential breeding populations based on geographic features that did not
support breeding loons (i.e., mountain ranges, extensive areas without breeding-quality lakes, etc.).
Population differentiation was tested based on these putative populations (Figure 2). Four out of 10
pairwise exact tests for population differentiation were found statistically different (Table 2) although
these differences did not follow any obvious spatial pattern.
 Alleles visualized with MJ Research Basestation and sized with Cartographer software
BioDiversity Research
Institute (www.briloon.org)
 Data analysis with TFPGA (Miller 1997) and POPGENE (Yeh et al. 1997)
Locus
GimA12EPA
Repeat
sequence
No.
alleles
Allele size
(range)
HEX-TTCATTGAGTGTAATAATGGCGA
TTGGAGGGATGGATGGACGG
(CCAT)8
7
GimC5EPA
FAM-AGTGATGCAGAAGAGGGTGG
GGTTTGGGTCACAGCTGAAT
(GT)4GG
(GT)7
4
108
(102-112)
GimE11EPA
TET-GGGAGTATTAACAGCCAGCC
CCCCATCTCCTGTTTTCTCA
(GA)8(TA)3 2
(CA)7
146
(146-148)
GimD9EPA
FAM-TTAGGTGGAACAGCTCTGGG
CTTCTTGCCCTGATCTCCAG
(GT)9
169
(173-181)
TET-CACAGCACAAGAACAAGCGT
TTATGGGGCTTTTGTATGGC
(GT)7
GimA9EPA
Figure 1. Range and migratory routes of the
Common Loon in North America.
Primer sequence (5’ to 3’)
Upper: Forward, Lower: Reverse
138
(129-153)
4
3
147
(143-147)
Table 1. Characteristics of five polymorphic microsatellite loci in Gavia
immer. Primer sequences are listed with the fluorescent dye label
indicated. Allele size is the predicted size (bp) of the cloned allele with
the observed allelic size distribution for each locus in parentheses.
1
2
3
4
5
1
****
0.2294
0.0334
0.0175
0.3983
Common Loons have been studied as a primary indicator of the impacts of methyl mercury
(MeHg) and other stressors on ecological health for over a decade (Burgess et al. 1998, Evers et
al. 1998, Meyer et al. 1998) with sampling primarily concentrated in northeastern North America.
This has resulted in a voluminous database on loon density and distribution, behavior and natural
history (based primarily on marked loons), and characterization of the exposure and hazards of
mercury and other environmental stressors. Because loons are a high trophic predator and
environmental risks are well-documented, the U.S. Environmental Protection Agency (USEPA) is
using them to model wildlife risk. In order to place the environmental risk assessment of loons into
a spatial framework, the genetic structure of Common Loons in their breeding territories must be
understood.
The summer breeding range of the Common Loon encompasses northern North America and
wintering areas include coastal waters (Figure 1). Adult loons return to the same breeding area and
usually the same lake (mean dispersal distance = 2km); juveniles disperse only slightly further
(mean = 13km; Evers 2000). This suggests a metapopulation structure where breeding loons are
divided into relatively discrete habitat “areas” separated by large areas of unsuitable habitat (Figure
2). Previous work with RAPD markers suggests that loons from the midwest and northeast are
genetically distinct (Dhar et al. 1997) but no other studies have been published using molecular
methods to define loon populations.
The primary objective of this study is to define breeding populations of Common Loons using
microsatellite markers. Genetic subdivision between loons from different breeding areas may
manifest into differences in stressor-response profiles for these birds. Geographic information on
reproductive success, fitness measurements (e.g., feather weight asymmetry), demographic
processes, toxicological exposure, and non-chemical stressors will be placed in a spatially-explicit
framework for stressor-response models if loon subpopulations can be defined. Loon populations
can be artificially defined by lake regions, topological features such as mountain ranges, or by
convenient distances. However, whether birds within our defined areas interact, more specifically,
interbreed or exchange genes, is of utmost importance to understand stressor effects.
Locus
All
Allele West East
1
2
3
4
5
70
48
56
17
119
45
n
GimA9EPA
1
2
3
325
Courtesy of BioDiversity Research Institute
1
2
3
4
5
0
0.594
0.355
0.007
0.044
0.003
0.642
0.325
0
0.031
0
0
0
0
0.660 0.616 0.618 0.702
0.266 0.384 0.353 0.290
0.064
0 0.029 0.008
0.011
0
0
0
0.011
0.611
0.356
0.022
0
GimC5EPA
1
2
3
4
0.007
0.174
0.710
0.109
0.005
0.157
0.739
0.099
0.011
0
0 0.004
0.152 0.111 0.219 0.140
0.750 0.796 0.688 0.763
0.087 0.093 0.094 0.093
0.011
0.182
0.693
0.114
GimA12EPA
1
2
3
4
5
6
7
0.219
0.391
0.156
0.156
0.031
0.016
0.031
0.052
0.487
0.162
0.214
0.046
0.018
0.021
0.054
0.380
0.141
0.326
0.054
0
0.044
0.049
0.524
0.183
0.195
0.012
0.037
0
GimE11EPA
1
2
0.978 0.995 1.000 1.000 0.906 1.000 1.000
0.022 0.005
0
0 0.094
0
0
0.046
0.482
0.130
0.232
0.065
0
0.046
0.100
0.467
0.167
0.133
0.033
0.100
0
0.036
0.554
0.153
0.176
0.050
0.018
0.014
Table 2. Sample size (n) and allele frequencies for each locus by
region. Eastern region numbers correspond to Figure 2 where
1=New York, 2=Massachusetts-southern New Hampshire (NH),
3=Vermont and northwestern NH, 4=northeastern NH and
northern Maine (ME), 5=southeastern ME, Nova Scotia, and New
Brunswick.
4
5
3
5
****
0.0057
0.2418
0.1719
****
0.0060
0.3707
1
****
0.5058
2
****
Figure 2. Putative metapopulation of Common Loons in
northeastern North America.
Discussion
0.435 0.488 0.532 0.509 0.618 0.445 0.522
0.399 0.485 0.457 0.491 0.353 0.508 0.456
0.167 0.028 0.011
0 0.029 0.046 0.022
GimD9EPA
3
Table 3. Matrix of combined probabilities for each pairwise comparison
between areas in the eastern region (exact test for population
differentiation, Raymond and Rousset 1995). Heading numbers
correspond to Figure 2 where 1=New York, 2=Massachusetts-southern
New Hampshire (NH), 3=Vermont and northwestern NH,
4=northeastern NH and northern Maine (ME), 5=southeastern ME,
Nova Scotia, and New Brunswick.
Eastern Region
Introduction
2
4
These are preliminary findings in a long-term study of Common Loon genetic structure. This
study will help to determine the spatial scale in which to model loon environmental impacts,
particularly MeHg. Our results suggest a relatively long-term split between the eastern and western
geographic regions of North America and show that the Pacific and Atlantic migratory routes
represent genetically distinct populations (Figure 1). Within the eastern portion of North America it
appears there are genetic difference among regions but that that these relationships need further
exploration. For this preliminary study we assumed populations based on geographic structures or
areas that would seem to prevent dispersal between breeding areas. Further investigations will
consider other possible population boundaries and a mixed stock analysis (e.g., Pritchard et al.
2000), which will help determine the number of populations represented in this region.
This study suggests that locus GimE11EPA may not be useful in distinguishing populations
since it is relatively monomorphic and does not meet Hardy-Weinberg expectations in some cases.
Overall variability in this study is not very high. Further development of microsatellite markers or
additional, more variable markers (i.e., AFLP) may be used to help define the Common Loon
metapopulation structure.
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