Appendix S3
We sampled 292 male spring peepers across two additional contact zones in the breeding season
of 2005: (A) A complex contact zone among Western, Interior and Eastern lineages spanning
Missouri, Iowa and Wisconsin (6 locales; N=119). (B) A contact zone between Western and
Interior lineages spanning Missouri, Illinois and Indiana (9 locales; N=173). The sampling
locations of these two new transects (A and B) are described in Table S1-1. All males were hand
captured and either toe-clipped and released at site of capture or euthanized with MS222 and
liver tissue harvested for later DNA work. All samples were preserved in 95% ethanol until
genetic analysis. Specimens were deposited in the Cornell University Museum of Vertebrates
(sample vouchers CUMV 14262-14568). Total genomic DNA was extracted using the QIAGEN
DNEasy tissue kit (QIAGEN, Valencia, CA) following the manufacturer’s protocol. We
amplified a portion of the mitochondrial cytochrome b gene using the primers MVZ 15L and
MVZ 18H (Moritz et al., 1992) following the methods outlined in Austin et al. (2002). We also
screen samples for variation at four highly polymorphic DNA microsatellites (Pcru32, Pcru11,
Pcru10, Pcru05; see Table 2 for primer details). We used the Bayesian clustering program
Structure v2.3.4 (Pritchard et al., 2000) implementing the admixture ancestry model, correlated
allele frequencies, with information on the provenance of each individual. We explored values of
K=1 through 5, conducting ten iterations per K value, each consisting of 2,000,000 generations
following a burn-in of 500,000. The most likely K values (K = 3 and 2, for contact zones A and
B, respectively) were determined following methods described in Evanno et al. (2005).
References
Austin JD, Lougheed SC, Neidrauer L, Chek AA, Boag PT (2002) Cryptic lineages of a small
frog: the post-glacial history of the spring peeper, Pseudacris crucifer (Anura: Hylidae). Mol
Phylogenet Evol 25: 316-329.
Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the
software STRUCTURE: a simulation study. Mol Ecol 14: 2611-2620.
Moritz C, Schneider CJ, Wake DB (1992) Evolutionary relationships within the Ensatina
eschscholtzii complex confirm the ring species interpretation. Syst Biol 41: 273-291.
Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus
genotype data. Genetics 155: 945-959.
Figure S3-1. Bar plots of admixture coefficients resulting from separate STRUCTURE analysis
for each transect. Individuals are represented as vertical bars the height of which is proportional
to the estimated membership in diagnosed clusters. Sampling locales are delineated by black
vertical lines. Pie charts above show proportion of mitochondrial cytochrome b haplotypes for
each sampled locale with “contact zone” (CZ) indicated based on presence of more than one
mtDNA lineage within a sampled site. (A) A complex contact zone among Eastern (green),
Interior (red) and Western (blue) lineages spanning Missouri, Iowa and Wisconsin. (B) A contact
zone between Western (red) and Interior (green) lineages spanning Missouri, Illinois and
Indiana. Sample sizes are indicated in Table S1-below.
Table S3-1: Genetic sampling information of populations from 2005 across 2 additional contact
zones within the USA (comprising (A) Western – Interior, and (B) Western – Interior – Eastern).
GPS coordinates are in decimal-degrees (Latitude, Longitude).
STRUCTURE
Sample Pop Type GPS
Coordinates
Pop #
Size
Western
Kirksville, MO
22
40.1763466
1 (A)
-92.649964
Contact
Lacey-Keosauqua SP, IA
21
40.6983669
2 (A)
-91.99037
Contact
Yellowstone Lake SP, WI
19
42.7687897
3 (A)
-89.985792
Eastern
Lodi Marsh, WI
19
43.2940013
4 (A)
-89.536729
Eastern
Wiese Slough, IA
14
41.5469127
5 (A)
-91.238059
Eastern
Big Mill Creek WMA, IA
24
42.2684651
6 (A)
-90.51474
Western
PRCA 2, MO
17
37.0404911
1 (B)
-91.204893
Webb Creek, MO
19
Western
37.1374063
2 (B)
-90.871602
MT Site 1, MO
22
Western1
37.4672281
3 (B)
-90.244327
Shannon’s Neighbour, MO
20
Western
37.6218723
4 (B)
-89.930712
Contact
Johnson’s Creek, IL
24
37.8390316
5 (B)
-89.51489
Contact
Ledford, IL
17
37.6922107
6 (B)
-88.597896
Interior
Route 1, IL
16
37.5565843
7 (B)
-88.178727
Interior
Sugar Ridge, IN
22
38.2554908
8 (B)
-87.160129
Interior
Chandler, IN
16
38.0878032
9 (B)
-87.421723
1
A single individual possessed an Interior mtDNA haplotype. This may be real or due to labeling
error but here we here diagnose this population as Western because the locale is geographically
distant from other obvious contact zone sites.
Site Name