Online Supporting Information
Appendix S1. Molecular methods
DNA extraction, PCR and Sequencing
DNA from all blood samples was extracted using the Isolate Genomic DNA Mini Kit
(BioLine) following the ‘DNA isolation from animal tissue’ instructions provided with the
kit, with a minor modification: 40μl instead of 25μl proteinase K was used. Typically, 25μl
PCRs were prepared, comprising the following reagents: 1μl DNA extract, 2μl of each of the
forward and reverse primers (10μM each), 12.5μl My Taq HS Red Mix (Bioline) and 7.5μl
UV sterilised DNA grade distilled water (dH2O). PCR amplification of target regions was
performed on a G-STORM GS1 thermal cycler with: initial denaturation (1 min at 95°C); n
cycles (marker-specific; Table 1) of denaturation (15 sec at 95°C), annealing (15 sec at
marker-specific temperature; Table 1) and extension (10 sec at 95°C); final 10 min extension
at 72°C. PCR results were verified by agarose gel electrophoresis with SybrSAFE staining
and visualised using a Bio-Rad Gel DocTM EZ Imager (software: Bio-Rad Image Lab 3.0).
PCR products were purified and sequenced by Macrogen (Europe and South Korea).
Sequence reads were manually checked and edited using the programmes FINCHTV 1.4
(Geospiza), BIOEDIT 7.2.0 (Hall 1999) and CODONCODE ALIGNER 4.2.4 (CodonCode
Corporation, Dedham, MA). Consensus sequences were aligned using the programme
CLUSTALX 2.1.12 (Larkin et al. 2007).
Molecular sexing
Individuals that could not be sexed from their plumage or after examination of gonads were
sexed by PCR amplification using P8 (5'-CTCCCAAGGATGAGRAAYTG-3') and P2 (5'TCTGCATCGCTAAATCCTTT-3') primers as per Griffiths et al. (1998). PCR cycling
conditions were 95oC for 1 minute followed by 40 cycles of 94°C for 45 seconds, 48°C for 45
seconds and 72°C for 45 seconds and a final elongation step of 72°C for 5 minutes.
Amplification volumes of 10ul contained 0.5ul DNA, 5ul My Taq HS Red mix, 0.2ul of each
primer and 4.1ul of dH20. All amplicons were examined via electrophoresis on a 3% agarose
gel to score sex by PCR product size (males produce a 350 bp product; females produce a 350
bp and 387 bp product).
Appendix S2. Details of the ABC analyses
ABC analyses
Goodness of fit was checked visually by comparing the distance between observed and
simulated datasets using Principal Component Analysis on summary statistics (using the
gfitpca function from the ABC package; Fig. S3). To assess whether ABC allowed alternative
models to be distinguished, we performed a cross-validation step. This step allows estimating
model misclassification by considering a given simulated dataset as the pseudo-observed
dataset and estimating posterior probabilities for each model from it. Then, the ability for
ABC to assign the pseudo-observed dataset to its model of origin can be assessed. One
hundred pseudo-observed datasets were picked from each scenario and assigned to the model
with the highest posterior probability to build the confusion matrix. The estimation of
posterior probabilities for each scenario was performed through a regression step (neuralnet
algorithm) on the 50,000 simulated data sets closest to the observed one. To assess whether
the best models were able to accurately estimate parameters, we performed another crossvalidation step on parameter estimations and estimated prediction error rates.
For posterior distribution of parameters we took the 10,000 (1%) simulated datasets
closest to the observed dataset from the best scenario after applying a log transformation to
the parameter values.
Table S1. Biometric measurements ± s.e. for male and female fodies: body mass, tarsus length, head-bill length, bill width and bill height (both
measured at the centre of the nostrils). As these measurements differ considerably between different groups of fodies, they were used by the field
team as a cue, in combination with visual and behavioural differences, to identify birds as F. aldabrana, F. madagascariensis or putative hybrids
(i.e., F1 or recent hybrids). For all measurements, F. aldabrana are largest, F. madagascariensis are smallest and putative hybrids are
intermediate.
Group
Body type
Male
(non-breeding
plumage)
26.6 ± 0.7
(n=11)
22.6 ± 0.2
(n=11)
34.4 ± 0.3
(n=11)
17.9 ± 0.3
(n=11)
10.1 ± 1.2
(n=8)
12.0 ± 0.4
(n=11)
24.8 ± 1.5
(n=6)
Putative F. aldabrana x F. madagascariensis hybrids
21.8 ± 0.3
(n=6)
32.9 ± 0.8
(n=6)
16.8 ± 0.6
(n=6)
8.6 ± 0.5
(n=4)
11.3 ± 0.6
(n=6)
Both types*
19.9 ± 0.8
(n=10)
20.4 ± 0.4
(n=9)
31.4 ± 0.4
(n=10)
15.9 ± 0.3
(n=9)
7.5 ± 0.2
(n=9)
9.7 ± 0.3
(n=9)
Monotonous,
‘cheeping’
18.0 ± 1.5
(n=3)
20.2 ± 0.8
(n=3)
30.8 ± 1.0
(n=3)
15.4 ± 0.7
(n=3)
7.9 ± 0.5
(n=3)
9.9 ± 0.2
(n=3)
Monotonous,
‘cheeping’
16.8 ± 0.3
(n=13)
18.9 ± 0.2
(n=6)
29.0 ± 0.2
(n=13)
13.9 ± 0.2
(n=5)
6.9 ± 0.1
(n=6)
8.9 ± 0.3
(n=6)
Monotonous,
‘cheeping’
16.3 ± 0.8
(n=9)
18.6 ± 0.5
(n=6)
28.8 ± 0.5
(n=9)
14.2 ± 0.4
(n=5)
6.9 ± 0.3
(n=6)
8.9 ± 0.3
(n=6)
Plumage
Song
Voluptuous
Bill height
Yellowish brown
Yellowish brown
Female
Male
(breeding
plumage)
Male
(non-breeding
plumage)
Tarsus
length
F. aldabrana
Orange breast and yellow belly, separated by a distinct
straight band.
Brown streaked feathers on the back and wings (without
red)
Melodious
Male
(breeding
plumage)
Morphological measurements
Head-bill
Bill
Bill width
length
length
(culmen)
Body
mass
Monotonous,
‘cheeping’
Mixed features of F. aldabrana and F. madagascariensis, e.g., voluptuous
with dark red (F. madagascariensis-like) plumage
Female
F. madagascariensis
Dark red, no yellow patch on the belly (but can have a pale
brown patch near the legs when moulting into breeding
plumage).
Red/brown streaked feathers on the back and wings.
Male
(breeding
plumage)
Male
(non-breeding
plumage)
Female
Lean
Pale brown
Pale brown
* The different groups have been observed to imitate / take over the song of the other group.
Figure S1. Histograms of model misclassification. (a) nuclear dataset, and (b) mitochondrial
+ nuclear dataset. For each scenario, 100 pseudo-observed datasets are sampled from
simulations and model choice is applied. Bars represent the number of times each scenario
displayed the highest posterior probability, given that the pseudo-observed datasets were
sampled from scenario 1 (first bar) or from scenario 2 (second bar).
(a)
(b)
Figure S2. Bayesian analyses of Foudia concatenated mitochondrial (ATPase 6 & 8 and
ND3) data, constructed as per Fig. 3, but without fodies from the Takamaka region. All
‘pure’ (non-Takamaka) Aldabra fodies (n=27) form a monophyletic group with 100%
bootstrap and Bayesian branch support. All outgroups and Foudia reference samples from the
Warren et al. (2012) dataset that were used for the analysis are marked with an asterisk.
Figure S3. Goodness of fit plots using Principal Component Analysis on summary
statistics for (a) the nuclear and (b) the complete (nuclear + mitochondrial) simulations. The
cross indicates the observed dataset. Scenario 1: Invading birds come from Assumption and
hybridise with F. aldabrana between 5 and 37 generation (years) ago; Scenario 2: Invading
birds come from a Madagascar population and hybridise after the split between F.
aldabrana and F. madagascariensis (see Fig. 2).
(a)
(b)
References
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