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Conservation genetics and genetic mating system of the yellow-shouldered blackbird (Agelaius
xanthomus), an endangered island endemic
Submitted to Conservation Genetics
Irene Liu
Duke University
Department of Biology
irene.a.liu@gmail.com
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Results without Qm10
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Calculation and comparison of genetic diversity: Summary statistics for genetic
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diversity across nine loci in the yellow-shouldered blackbird are shown in Table S2a. The
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bootstrap showed that yellow-shouldered blackbirds had lower allelic diversity, Shannon
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diversity indices, and expected heterozygosity than any of the red-winged blackbird populations
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(P ≤ 0.0001 for all measures, Table S2b, Fig. S1). This comparison included the Bahamas
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population, which itself had significantly lower diversity than that of the continental populations
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(Fig. S1; Liu et al. in revision). The yellow-shouldered blackbirds also had lower average allelic
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diversity across loci (5.43 alleles/locus) than the Bahamas red-winged blackbirds (12.71
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alleles/locus), despite the yellow-shouldered blackbird’s greater raw allelic diversity at Pca3
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(data not shown). By contrast, the yellow-shouldered blackbirds did not have significantly
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different levels of inbreeding when compared to the Bahamas red-winged blackbirds (t = 0.87, df
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= 6, P = 0.42) or with all red-winged blackbird populations (F = 1.03, df = 8, P = 0.43).
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Estimate of effective population size: Estimates for the LD method in NeEstimator for
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the yellow-shouldered blackbird are shown in Table 2. Estimated Ne for the Bahama red-winged
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blackbirds was significantly larger than the values for the yellow-shouldered blackbird (95%
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confidence intervals did not overlap). Separately, ONeSAMP calculated a mean Ne of 70.2 (95%
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CI = 61.5, 85.5), while COLONY calculated an Ne of 89 (95% CI = 64, 122) for the yellow-
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shouldered blackbird. The average estimate of Ne for the yellow-shouldered blackbird was 87.2
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±16.2 SD. This quantity ranges from 18-26% of the 2012 pre-breeding census size of 400 birds
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in southwestern Puerto Rico.
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Evidence of bottleneck: Under two of three models, BOTTLENECK detected
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significant heterozygosity excess (higher observed than expected heterozygosity, P < 0.01 for
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both Wilcoxon and sign tests), indicating a recent bottleneck. Whereas the IAM and TPM
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predicted heterozygosity excess at all nine loci, the SMM predicted heterozygosity deficiency at
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two loci. Thus, while predicting significance using the Wilcoxon test (P = 0.04), it did not
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predict significance using the sign test (P = 0.27). BOTTLENECK reported a normal L-shaped
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distribution for all models, indicating that there was no mode-shift in the allele frequency
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distribution (i.e., no deviation from allele frequencies predicted by drift-mutation equilibrium).
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Similar but weaker evidence of a bottleneck was revealed for the Bahamas red-winged
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blackbird population. Using the sign test, the number of loci showing heterozygosity excess
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versus heterozygote deficiency was significant under the IAM. Using the Wilcoxon test, the
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proportion was significant under both the IAM and TPM (all P < 0.02). Predictions under the
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SMM were not significant in either test.
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Figure S1 Genetic diversity, omitting Qm10, as measured by allelic diversity, expected
heterozygosity, and Shannon diversity estimated from bootstrap simulations for six continental
red-winged blackbird (“Cont. RWBL”, black) populations, one island red-winged blackbird
(“Bah. RWBL”, black) population, and the yellow-shouldered blackbird (“YSBL”, gray)
population. Bootstrap resample size was 20. The yellow-shouldered blackbirds have significantly
less genetic diversity than both red-winged blackbird populations
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Table S1. Primers used in the study. L = allele length, n = number of alleles.
Locus
Aph54
Forward/reverse primer sequences
Dye
Repeat motif
GCTGCTGTCTCTATGTCAC
6-FAM
ATT
GACACCTTTCACCAGACC
LTMR6
GCCATGCCACAGGAGTGAGTC
HEX
GT
AGTCATCTCCATCMGGGCAT
Qm10
GGAATTCCAGTATGTGAATGAGTC
NED
AAT
ATTGCAAAAAACAGAAGCATTTTAAC
Dpµ16
ACAGCAAGGTCAGAATTAAA
6-FAM
AC,GC
AACTGTTGTGTCTGAGCCT
Pca3
GGTGTTTGTGAGCCGGGG
NED
GT
TGTTACAACCAAAGCGGTCATTTG
Ap38
GGAGGGAGACCTCTTAATAC
HEX
CATC
CGACAGAGCTGGTGTCAAAA
Ap79
CCACTTCTGCTGAACATAGGG
NED
AAC
GTGCTGCAATTGTGGTCTTG
Ap107
GAAACATCCAAACCTGGCTTG
6-FAM
AGAT
AATGGACGTGCAGCCCTTC
Ap144
TCCATAACACAGTTGTCAGAG
HEX
AGAT
CTTACACAGGCACACAAACC
L
n
165-253 35
GenBank
AY928531.1
Reference
Westneat and Mays (2005)
183-213 14
FM201465.1
McDonald and Potts (1994)
211-250 16
AF013235.1
Hughes et al. (1998)
150-167 11 AM262982.1
Dawson et al. (1997)
155-179 11
AJ279805.1
Dawson et al. (2000)
268-284
5
JF907496.1
Barker et al. (2011)
218-274 15
JF907499.1
Barker et al. (2011)
207-280 27
JF907500.1
Barker et al. (2011)
187-252 31
JF907502.1
Barker et al. (2011)
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Table S2. Summary of population genetics, omitting Qm10. Sample size (N) and means for raw
(Na) and effective (Nea) number of alleles, Shannon diversity index (I), observed (Ho) and
expected (He) heterozygosity, and inbreeding coefficient (FIS) for (a) eight loci in yellowshouldered blackbirds and (b) seven loci in yellow-shouldered blackbirds (YSBL) and redwinged blackbirds (Liu et al. in revision). Numbers in parentheses are standard error.
(a)
N
63
YSBL
59
60
61
Na
5.38
(1.21)
Nea
3.53
(0.70)
I
1.29
(0.18)
Ho
0.66
(0.06)
He
0.66
(0.05)
FIS
-0.005
(0.017)
(b)
N
YSBL
63
Bahamas
66
KY
32
MI
51
NY
31
Ontario
13
PA
60
WA
31
WI
22
Na
5.43
(1.39)
12.71
(3.75)
18.29
(2.85)
20.86
(3.12)
16.86
(2.40)
12.14
(1.24)
21.57
(3.62)
15.29
(2.46)
16.43
(2.15)
Ne
3.58
(0.81)
7.00
(1.93)
10.85
(1.79)
11.56
(1.91)
10.43
(1.73)
8.13
(1.11)
11.22
(2.11)
9.69
(1.78)
11.06
(1.79)
I
1.28
(0.21)
1.87
(0.33)
2.51
(0.19)
2.57
(0.20)
2.46
(0.18)
2.23
(0.14)
2.55
(0.21)
2.36
(0.19)
2.49
(0.17)
Ho
0.66
(0.06)
0.74
(0.07)
0.86
(0.02)
0.84
(0.03)
0.83
(0.03)
0.88
(0.04)
0.87
(0.03)
0.83
(0.04)
0.88
(0.04)
He
0.65
(0.06)
0.76
(0.07)
0.88
(0.03)
0.89
(0.03)
0.88
(0.03)
0.86
(0.02)
0.89
(0.02)
0.87
(0.03)
0.89
(0.02)
FIS
-0.004
(0.020)
0.018
(0.018)
0.024
(0.021)
0.054
(0.023)
0.055
(0.022)
-0.026
(0.040)
0.012
(0.021)
0.039
(0.021)
0.015
(0.037)
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Table S3. Estimates of Ne, omitting Qm10, in the yellow-shouldered blackbird (YSBL) vs. redwinged blackbird populations using the LD method in NeEstimator. 95% confidence intervals
are calculated using the jackknife on loci method. Ne in YSBL was estimated using eight loci,
while Ne in RWBL was estimated using nine loci.
Lowest allele frequency:
YSBL
Bahamas
KY
MI
NY
Ontario
PA
WA
WI
0.05
Ne
80.9
343.1
926.1
Infinite
449.2
Infinite
242.1
141.4
100.5
CI
Ne
42.5, 261.2
108.6
125.2, infinite
445.2
88.7, infinite Infinite
236.6, infinite Infinite
77.2, infinite
444.6
83.6, infinite Infinite
122.1, 1942.3
719.3
57.1, infinite
236.8
39.6, infinite
193.9
0.01
CI
55.4, 461.7
192.0, infinite
719.3, infinite
775.9, infinite
168.0, infinite
Infinite, infinite
307.1, infinite
115.6, 8570.3
85.9, infinite
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85
86
87
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Table S4. Microsatellite ascertainment bias is not an issue, shown by effective allelic diversity
(Nea) for each of the eight loci that were amplified in both red-winged blackbird (RWBL) and
yellow-shouldered blackbird (YSBL). The Ontario population was excluded when calculating
the mean allelic diversity of the continental populations (in bold), because lower measurements
in Ontario almost certainly stem from missed alleles and would have artificially increased the
distribution about the mean.
Differences in allelic diversity between the two species are indeed larger in the RWBL-derived
loci than the loci derived from other species. However, YSBL have the lowest allelic diversity
across six loci, indicating the signal of lower diversity is not an artefact of species-specific
polymorphism. The two exceptions are LTMR6 and Qm10, two non-RWBL-derived loci, where
the Bahamas RWBL population has the lowest allelic diversity. Furthermore, at five other loci,
both the YSBL and the Bahamas RWBL population have estimated levels of allelic diversity
falling outside the mean ± SD of the continental populations. These results suggest the markers
are detecting true variation within and across species, and that this variation can be reliably
identified and compared with a closely related species.
Populations with similar sample sizes are highlighted. These populations show the same trend
that the YSBL have lower allelic diversity at all loci but Qm10.
N
KY
32
MI
51
NY
31
Ontario
13
PA
60
WA
31
WI
22
Mean_excl_Ont
SD_excl_Ont
Bahamas
YSBL
66
63
RWBL-derived loci
Non-RWBL-derived loci
Aph54 Ap107 Ap144 Ap79 LTMR6 Qm10 Dpu16 Pca3
16.38 13.47 16.25 8.94
9.99
8.19
6.94 3.94
16.21 16.11 16.83 10.49
10.42
6.76
6.98 3.86
15.38 10.22 16.86 10.74
9.56
7.84
6.28 4.00
12.52 10.24
9.14 8.45
7.19
4.69
5.73 3.63
16.71 16.33 17.73 8.94
8.77
8.96
5.31 4.73
15.75 13.93 13.73 6.77
6.24
6.99
7.88 3.53
18.26 12.41 15.37 9.31
10.30
8.27
7.39 4.38
15.89 13.24 15.13 9.09
8.93
7.39
6.64 4.01
1.62
2.30
2.72 1.23
1.51
1.30
0.85 0.39
14.21
4.59
11.34
7.89
11.27
3.55
4.87
2.00
2.39
2.92
2.88
8.47
2.92
2.20
2.00
1.90
89
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