Supporting information
Characterising a Hybrid Zone between a Cryptic Species Pair of Freshwater
Snail
Simit Patel, Tilman Schell, Constanze Eifert, Barbara Feldmeyer, Markus Pfenninger
Supplementary Methods
COI PCR conditions
PCR reactions were performed in a final volume of 10µl, with 1× reaction buffer (Molgene GmbH,
Butzbach, Germany), 2.5mM MgCl2, 0.2mM each dNTP, 0.2µM of each primer, 0.5U Taq Polymerase
(MOLPol DNA Polymerase, Molgen) and 1µl of genomic DNA that had been diluted 1:50 in water.
The forward primer was LCO1490 5’-GGTCAACAAATCATAAAGATATTG-3’ (Folmer et al. 1994) and the
reverse was 5’-TGTTGATATTAAAATAGGATC-3’(Cordellier & Pfenninger 2008). Reactions were
incubated in an Arktik Thermal Cycler (Thermo Scientific) under the following temperature profile:
94°C for 15 mins; 32-42 cycles of 94 °C for 30s, 40°C for 50s, 72°C for 60s; then 72 °C for 10mins. PCR
success was checked by agarose gel electrophoresis. Prior to performing the sequencing reaction,
PCR products were diluted 1:40 in water. Sequencing reactions were performed using the BigDye®
Terminator v3.1 chemistry (Applied Biosystems) and run on an ABI PRISM 3730 automated
sequencer (Applied Biosystems). In cases where the forward or reverse sequence was of poor
quality, only the opposite sequence was used for base calling, if it was of high enough quality.
Microsatellite PCR cycling conditions
Multiplex PCRs were performed using the Qiagen Type-it™ Microsatellite PCR kit. In slight deviation
from the manufacturer’s protocol, the reactions were performed in a final volume of 6.25µl, with
3.125µl of master mix; 0.625µl of primer mix; 0.625µl of Q solution and 1.25µl template DNA that
has been diluted 1:50 in water. Two primer mixes were used: one for the primers from Salinger &
Pfenninger (2009) (RBA1, RBA2, RBA3, RBA5, RBA6) and one for the newly designed primers (Table
1). Reactions were incubated in an Arktik Thermal Cycler (Thermo Scientific) under the following
temperature profile: 95°C for 15mins; 30 cycles of 94°C for 30s, 54°C for 90s, 72°C for 60s; then 60°C
for 30mins. Prior to fragment analysis, PCR products were diluted 1:100 in water. Fragment analysis
was performed on an ABI PRISM 3730 and visualised using GeneMapper® v4.0 (Applied Biosystems).
Table 1. Forward (F) and reverse (R) primer sequences and expected fragment sizes for newly
developed microsatellite markers. 1from reference sequence. 2from preliminary testing
Expected Fragment Size 1
Expected Fragment Size2
F: GGAAACCAGAAAAACCGACA
R: GCACAGGACGATGAGACGAT
244
215 - 233
RBP14
F: TTGCCTATTTCCATCCCTTG
R: GCGTGTGTGCGGTATGTATCT
183
172 - 196
RBP16
F: GGTAAACGATGGCTGGAGTG
R: TATGCTGACCAATAGTGACAAAG
379
354 - 404
RBP29
F: CTGAAGGGTTTCCAGACCAA
R: ACCCCAGATATTCCACTTGAC
325
301 - 355
Locus
Sequence (5'-3')
RBP01
1
Coding gene PCR conditions
PCR conditions were the same for all three coding genes. Reactions were performed using the same
conditions as described for COI barcoding. The primers used for each gene are shown in table 2.
The thermal cycling conditions were 95°C for 5mins; 35 cycles of 95°C for 30s, 58°C for 30s, 72°C for
90s; then 72°C for 10mins. Sanger sequencing was performed as described above for COI barcoding.
Table 2. Forward (F) and reverse (R) primer sequences for the three coding genes
Gene
RBact1a
RBHSPA2
RBPsmd2
Sequence (5'-3')
F: TGCCCCTGACTCAGTGTATTC
R: TGTACAAAGCGGGTCTCTCA
F: TGCTAACAAAAATTCATATTAAAAAGC
R: GAGATGATTTCCTCGGGTGA
F: GGCTCTTGGGGTCATGAGTA
R: GAATATGCAGCATCATCCACA
2
Supplementary Results
Below is a summary of the preparatory analyses for the nuclear marker data (microsatellites and
coding gene sequences). See Table S4 for a summary of the nuclear markers sample sizes per
population.
Genetic diversity of microsatellite markers
The fragment sizes were in the expected range for all nine microsatellite markers (Table S5), but the
number of alleles was slightly higher than expected for all nine loci, except RBA1 (five alleles
compared to seven in Salinger and Pfenninger 2009). RBA2 was particularly variable (41 alleles, p <
0.01, Grubbs’ test). Fourteen individuals were genotyped at microsatellite loci, for which there was
no mitochondrial data. Removal of these 14 individuals made no difference to downstream analyses,
so they were included anyway.
FIS was significantly deviated from Hardy-Weinberg equilibrium in 14 out of 29 populations tested, all
of which were positive (homozygote excess, Table S6). Deviations from Hardy-Weinberg equilibrium
were detected at at least one microsatellite locus in 22 out of 29 populations tested (Table S6).
Amongst these 22 populations, one to four loci showed significant deviation from Hardy-Weinberg
equilibrium, per population. RBP01 was the only locus to show no significant deviation in any
population, all other loci showed deviation in at least two populations. Most significantly deviated
loci had positive FIS values (homozygote excess), the only exceptions being RBP29 in population CPG
and RBP14 in population FCT, which were negative (heterozygote excess). HO was systematically
lower than HE at all microsatellite loci, over all populations (mean HO = 0.440, mean HE = 0.757, Table
S5). Potential null alleles were detected in some populations, although these are limited to loci also
showing deviations from Hardy-Weinberg equilibrium (Table S6).
Deviations from Hardy-Weinberg equilibrium and low HO have been reported before and can be
attributed to homozygote excess caused by a mixed mating system and partial selfing (Salinger &
Pfenninger 2009; Haun et al. 2012), as indicated in our data by widespread positive FIS. The null allele
detection method used here relies on patterns of homozygote excess too (van Oosterhout et al.
2004), so the potential null alleles detected here could be false signals caused by genuine, biological
homozygote excess. Overall Fst was 0.372, with pairwise Fst ranging from 0.056 to 0.758, all of
which were significant (Table S7 and Fig. S5).
Nuclear coding gene sequences
The number of single nucleotide polymorphism (SNP) sites was high in HSPA2 and Psmd2 coding
gene fragments (47 and 46, respectively) compared to act1a (14 SNPs) (Table S5). Indels were found
in Psmd2 at 16 sites in total. On closer inspection, the indels were restricted to the first half of the
alignment (between positions 50 and 175) and a search for untranslated motifs in ungapped Psmd2
sequences revealed upstream open reading frames and other 5’ untranslated elements in the first
half of the sequence. When Psmd2 sequences were searched using tblastx, the hits were for the
second half (>180bp) of the coding gene region. This means that the first half of the Psmd2
sequences is likely to be an untranslated region, with indel mutations that are unlikely to cause
frameshifts on the second half of the sequence, which is protein coding. SNP data from the whole
sequence was used for downstream analysis.
Strong recombination was detected in HSPA2 (Rm = 11, p = 0.000; ZZ = 0.1326, p = 0.001, Table S8).
Positive, yet non-significant recombination was found in Psmd2 (Rm = 4, p = 0.334; ZZ = 0.0185, p =
0.268). No recombination was detected in act1a (Rm = 0, p = 0.822; ZZ = -0.0188, p = 0.648).
Reducing HSPA2 and Psmd2 data sets to non-recombining (NR) blocks (herein referred to as HSPA2NR and Psmd2-NR data sets) slightly reduced the samples sizes, fragment lengths and number of
SNPs (Table S5b). The number of unique haplotypes was reduced from 56 to 31 and 54 to 30 for
3
HSPA2 and Psmd2, respectively. No loops were present in the haplotype networks for act1a and
HSPA2-NR (Fig. S4). Grouping haplotypes separated by one mutation step resulted in 13 NH groups
for HSPA2-NR and Psmd2-NR data sets. See Fig. S4 for NH groupings and Table S9 for haplotype
frequencies. NH grouping was not necessary for act1a, as this would have resulted in too few NH
groups to be informative.
4
Supplementary Figures
(a)
(b)
N
50km
(c)
Core Population Set
Extended Population Set
COI Barcode
MOTU2
MOTU3
Nuclear Cluster
C1
C2
Fig. S1 (a) Relative frequencies of MOTU2 (orange) and MOTU3 (purple) mitochondrial clades per
population. (b) Relative membership coefficients for C1 (blue) and C2 (red) nuclear clusters, as
inferred by STRUCTURE assuming K=2 (the most likely number of clusters) averaged across
individuals per population. Note the similar geographic restriction of MOTU 3 (purple) and K2 (red)
in (a) and (b). (c) STRUCTURE diagram showing relative membership coefficients for C1 and C2 per
individual in the core and extended population sets. Each vertical bar represents a different
individual. The pie charts below indicate the relative frequencies of mitochondrial clades per
population – note that C1 populations (blue) tend to be associated with R. balthica (orange),
although this is not absolute. (d) A more detailed look at the nuclear cluster membership coefficients
and mitochondrial assignments for each individual in the seven populations with mixed
mitochondrial clades. Maps and pie charts for (a) and (b) were generated using the software genGIS
(Parks et al. 2013).
5
(a)
(b)
Nuclear Cluster
C1
C2
Fig. S2 STRUCTURE results based on microsatellite data only (excluding three coding genes). (a)
Relative membership coefficients for C1 (blue) and C2 (red) nuclear clusters, as inferred by
STRUCTURE assuming K=2 (the most likely number of clusters) averaged across individuals per
population. (b) STRUCTURE diagram showing relative membership coefficients for C1 and C2 per
individual in the core and extended population sets. Each vertical bar represents a different
individual. See Fig. S1 for sample site labels.
6
Fig. S3 Compressed neighbor-joining tree based on COI sequences. The values at the nodes show
bootstrap support values from 500 replicates. The tree was constructed using the T92+Γ, Tamura 3
substitution model, which was chosen using jModelTest (Posada 2008) and Phyml (Guindon &
Gascuel 2003). See Table S2 for outgroups used.
7
5
Fig. S4 Statistical parsimony haplotype networks generated in TCS for (a) act1a (b) HSPA2-NR (nonrecombining data) and (c) Psmd2-NR. The connection limit was set to 8 for HSPA2-NR and Psmd2-NR
to include divergent haplotypes into the network and the default 95% limit was sufficient for act1a.
Different haplotypes are represented by circles and numbered. The size of the circle is proportional
to the frequency of sequences (see Table S9 for frequencies). Haplotype 1 has the highest outgroup
probability in all three networks. The lines connecting haplotypes represent mutations and the small
open circles represent missing haplotypes. Grey shading in (b) and (c) represent nested haplotype
(NH) groupings and are numbered (NH numbers are in square dotted outlines). The broken lines in
(c) represent ambiguous connections (loops) that were broken.
8
30
25
20
15
0
5
10
Frequency
0
0.1
0.2
0.3
0.4
0.5
0.6
Pairwise Fst
Fig. S5. Frequency distribution of pairwise Fst values shown in Table S7.
9
0.7
0.8
Supplementary Tables
Table S1. Sampling site locations and number of individuals identified as different Radix MOTUs and other snails by COI barcoding.
Code
Sampling site name
ALS
BLL
BSC
CMT
CPG
Aillas
Bielle / Castet
Biescas
Caumont
Campagne /
Galargues
Darnius
Dunes /
Caudecoste
Espiet / Lestrille
Foncouverte
Feuilla
Viviers
Gironella
Hernani / Ereñozu
Isle-Saint-Georges
Jurançon
L'Esquirol / Sant
Martí de Sescorts
Le Mouzy
Montbazin
Orísoain
Puyoô / Bellocq
DAS
DUN
ESP
FCT
FEL
FVI
GNL
HEN
ISG
JRC
LES
LMY
MBZ
ORN
PYO
Country
Latitude
Longitude
No.
R.balthica/MOTU3
Pyrenees (core population set)
0.07222
12/0.42444
11/1
0.32139
-/12
1.08389
5/3
No.
MOTU4/MOTU5
Galba
sp.
Pseudosuccinea
columella
-/-/-/-/-
-
-
France
France
France
France
44.47417
43.06306
42.62778
43.03222
France
43.78056
4.02694
20/-
-/-
-
-
Spain
42.35028
2.86806
15/-
-/-
3
-
France
44.10639
0.75694
5/3
-/-
-
-
France
France
France
France
Spain
Spain
France
France
44.81833
43.16694
42.93056
44.48184
42.03833
43.24444
44.72444
43.27889
0.25472
2.69167
2.90833
4.69706
1.88000
-1.94722
0.47222
0.38889
18/-/20
2/18
7/-/12
2/6
13/5/9
-/-/-/-/-/-/-/-/-
6
-
-
Spain
42.01528
2.33417
9/-
-/-
-
-
France
France
Spain
France
44.15833
43.51333
42.60583
43.52056
1.25139
3.69556
-1.60500
0.91250
3/10
20/10/6/8
-/-/-/-/-
-
-
10
2.15944
-2.03306
2.67111
3.92139
-1.56056
No.
R.balthica/MOTU3
-/2
10/2
-/14
20/6/3
No.
MOTU4/MOTU5
-/-/-/-/-/-
Galba
sp.
-
Pseudosuccinea
columella
-
0.83833
-/10
-/-
-
-
Total (core)
199/133
0/0
9
0
Northern Europe (extended population set)
47.77180
5.98470
1/46.49700
7.05000
48/47.35680
5.14590
17/54.36500
10.31600
6/48.64725
7.69000
4/50.00700
9.15601
8/-
-/-/-/-/-/-/-
-
-
France
47.15900
-1.48200
3/-
-/-
-
-
Great Britain
54.32000
-1.51200
Total (ext)
8/95/0
-/0/0
0
0
Total
(core+ext)
294/133
0/0
9
0
6/16/-/14
-/-
-
-
Code
Sampling site name
Country
Latitude
Longitude
RSL
SSB
TLR
TYR
URG
VDB
Raissac-sur-Lampy
Zubieta
Talarain
Teyran
Urguri
Vallfogona de
Balaguer /
Balaguer
France
Spain
France
France
France
43.27583
43.27417
43.04528
43.68083
43.35639
Spain
41.79583
ABO
CSV
DIJ
DSP
LAB
MBK
POR
SWA
AGL
BGC
BZY
FCA
Aboncourt
Lessoc
Dijon
Passade
Lampertheim
Glattbach
Vertou / La
Barbinière
Morton-on-Swale
L'Agly / Claira
Bergerac / Tresses
Buzy
Les Cabanes
France
Switzerland
France
Germany
France
Germany
France
France
France
France
Pyrenees (not used in further analysis)
42.75139
2.95278
-/44.84667
0.48861
-/43.12972
0.46139
-/44.09673
4.72692
2/11
1.31056
No.
R.balthica/MOTU3
-/-
No.
MOTU4/MOTU5
9/-
Galba
sp.
-
Pseudosuccinea
columella
-
43.07333
0.39333
-/-
-/11
-
-
France
France
Spain
44.13583
43.02694
42.00444
0.66611
1.01861
-1.50111
-/-/-/-
5/-/12
3/-
9
-
-
Spain
41.86000
0.83306
-/-
5/-
-
-
France
43.06583
2.60750
-/1
-/-
-
-
Spain
28.29217
-16.86137
-/-
-/-
-
12
France
42.64056
2.90417
-/-
21/-
-
-
Spain
41.87389
0.77583
-/-
4/-
1
-
Code
Sampling site name
Country
Latitude
Longitude
GRD
LBN
Grenade
La Barthe-de-Neste
/ Bas Mour
Layrac
Prat-Bonrepaux
Ribaforada
Sant Llorenc de
Montagai / Xalets
de la Solana
St-Pierre-desChamps
Teneriffa / Masca
Villeneuve-de-laRaho
Zuera / La Estación
France
43.75083
France
LYC
PBX
RIB
SLM
SPC
TEN
VNV
ZUE
12
Table S2. Taxon and GenBank Accession numbers for 14 mollusc COI sequences used as outgroups
for the neighbour joining tree.
Taxon
Galba truncatula
GenBank-ID
FR797873
FR797874
FR797875
Lymnaea stagnalis
FR797865
FR797866
FR797867
FR797868
Myxas glutinosa
EU818798
Planorbarius corneus
FR797857
FR797858
Pseudosuccinea
columella
JN614404
JN614405
JN614406
AY227366
13
Table S3. Evvano table from Structure Harvester, summarising results used to infer the most likely
number of nuclear genetic clusters (K). LnP(K) = posterior likelihood probability; Ln’(K) = mean rate
of change of the likelihood distribution across replicates; |Ln''(K)| = mean absolute value of the 2nd
order rate of change of the likelihood distribution; ΔK =|Ln''(K)|/sd(L(K)). K=2 (highlighted) has the
highest ΔK.
Standard
Mean
Deviation
K
Replicates
LnP(K)
LnP(K)
Ln'(K)
|Ln''(K)|
ΔK
1
10
-16967
0.1826
-
-
-
2
10
-15386.34
96.1767
1580.66
679.97
7.070011
3
10
-14485.65
199.424
900.69
216.45
1.085376
4
10
-13801.41
383.1357
684.24
19.1
0.049852
5
10
-13098.07
309.0838
703.34
-
-
14
Table S4. Sample sizes for nine microsatellite loci and three coding genes, per population. Sample sizes for HSPA2 and Psmd2 are the number of individuals
in the non-recombinant (NR) data set. The total sample sizes before NR filtering are shown in brackets.
Core Population Set
ALS
BLL
BSC
CMT
CPG
DAS
DUN
ESP
FCT
FEL
FVI
GNL
HEN
ISG
JRC
LES
LMY
MBZ
ORN
PYO
RSL
SSB
TLR
TYR
URG
RBA1
RBA2
RBA3
RBA5
RBA6
11
12
12
8
16
16
8
15
20
13
8
12
8
13
11
9
13
13
10
13
2
10
11
20
11
7
12
12
8
14
16
8
14
20
13
8
12
7
12
12
9
12
14
10
13
2
10
11
20
9
11
12
12
8
16
16
6
15
19
12
8
12
8
12
10
9
13
14
10
13
2
10
11
20
11
11
12
8
8
13
16
6
13
20
13
8
12
8
13
12
9
12
14
10
13
2
10
11
20
11
11
12
12
8
16
15
8
15
20
10
8
12
8
13
12
9
13
14
10
13
2
10
11
20
11
RBP01 RBP14 RBP16 RBP29 RBact1a
11
12
12
8
16
16
8
15
20
13
7
12
8
13
12
9
13
14
10
9
2
10
11
9
11
15
11
12
12
8
16
16
8
15
19
13
8
12
8
13
12
9
13
14
10
13
2
7
11
20
11
11
12
11
8
16
16
8
15
20
13
8
12
8
13
12
9
13
14
9
13
2
9
9
20
11
11
12
12
6
16
16
8
15
20
13
7
12
8
12
12
2
13
14
9
13
2
10
9
20
11
12
12
12
7
16
6
16
1
10
10
12
8
10
13
8
8
13
10
12
2
10
8
15
7
RBHSPA2
RBPsmd2
12
11
11
5
15
14
7
15
18
8
9
11
8
13
13
9
11
8
10
7
2
11
11
13
7
4
12
6
3
11
8
6
6
12
7
5
6
6
8
11
2
7
4
5
11
3
12
17
7
VDB
Total (core)
Extended Population Set
ABO
CSV
DIJ
DSP
LAB
MBK
POR
SWA
Total (ext)
Total (core+ext)
RBA1
10
305
RBA2
10
295
RBA3
10
300
RBA5
10
295
RBA6
10
303
1
51
13
4
3
7
3
8
90
395
50
13
4
3
7
2
4
83
378
1
52
13
4
3
7
3
8
91
391
1
50
11
3
3
7
3
8
86
381
1
52
13
4
3
7
3
8
91
394
RBP01 RBP14 RBP16 RBP29 RBact1a
10
10
10
10
291
303
302
293
238
1
48
12
3
3
4
3
8
82
373
16
1
52
13
4
3
6
3
8
90
393
1
52
13
4
3
7
3
8
91
393
1
52
13
3
3
7
2
8
89
382
1
21
9
3
1
2
37
275
RBHSPA2
10
269 (280)
RBPsmd2
6
185 (189)
27
7
4
7
3
5
53 (55)
322 (335)
19
6
2
2
7
3
4
43 (43)
228 (232)
Table S5. Genetic diversity for (a) microsatellite loci and (b) coding gene sequences. N=sample size; bp = base pair; HE=expected heterozygosity over all
populations; HO=observed heterozygosity over all populations; H=number of unique haplotypes; NH=number of nested haplotypes. NR=values for nonrecombinant data sets (for HSPA2 and Psmd2).
(a) Microsatellite Locus
N
No. Populations
Fragment size range (bp)
HE
HO
No. Alleles
RBA1
395
34
146-156
0.355
0.225
5
RBA2
378
34
293-381
0.957
0.532
41
RBA3
391
34
228-244
0.655
0.325
9
RBA5
381
34
141-207
0.875
0.402
15
RBA6
394
34
357-395
0.832
0.510
15
RBP01
373
34
197-272
0.548
0.448
12
RBP14
393
34
160-212
0.819
0.489
12
RBP16
393
34
352-424
0.887
0.539
18
RBP29
382
34
262-370
0.883
0.487
22
Over all loci
386.7
-
-
0.757
0.440
16.6
(b) Coding Genes
N (NR)
No. Populations
Fragment length in bp (NR)
No. SNPs (NR)
H (NR)
NH
RBact1a
275
30
359
14
13
-
RBHSPA2
335 (332)
32
389 (352)
47 (41)
56 (31)
13
RBPsmd2
232 (228)
32
346 (331)
46 (34)
54 (30)
13
17
Table S6. FIS values per microsatellite locus per population and per population over all loci.
Population (Pop) codes as in Table S1. *indicates significantly positive or negative deviation in FIS
from Hardy-Weinberg expectations (P≤0.05) as computed in FSTAT. NA = monomorphic loci; - =
population excluded; § = potential null alleles detected by Micro-Checker.
Pop
RBA1
RBA2
RBA3
RBA5
RBA6
RBP01
RBP14
RBP16
RBP29
All
ALS
0.024
1*§
0.137
0.612*§
0.225
-0.176
0.29
-0.071
0.268
0.237*
BLL
NA
0.263
0.766*
-0.055
0.193
NA
0.035
-0.082
0.542*§
0.241*
BSC
0.203
-0.274
1*§
0.774
0.006
0.061
0.353
0.828*§
0.035
0.286*
CMT
0
-0.225
0.067
-0.195
CPG
-0.034
0.792*§
DAS
0.455
DUN
0.211
-0.2
-0.4
-0.292
0.394
-0.079
NA
NA
0.783*§
-0.132
-0.304
-0.08
-0.414*
0.082
0.13
-0.034
NA
0.751*§
NA
-0.034
NA
0.257
0.283*
-0.424
-0.191
0.75*
0.167
-0.135
0
-0.167
-0.273
0.731*§
0.04
ESP
-0.167
0.3*
0.865*§
0.64*§
0.132
-0.143
0.162
-0.201
0.018
0.19*
FCT
NA
0.17
0.182
0.309
-0.073
0.022
-1*
NA
-0.107
-0.076
FEL
NA
0.723*§
0.072
0.446
0.845*§
-0.333
-0.143
-0.412
0
0.24*
FVI
NA
-0.077
0.622*
0.103
0.25
-0.358
0.114
-0.094
0.368*
0.12
NA
0.006
GNL
0
NA
-0.266
0.126
0.267
1*
0.189
0.118
HEN
1*§
-0.224
1*§
0.475*
-0.296
-0.091
-0.14
-0.191
0.164
0.151*
ISG
-0.091
0.342*§
0.447*§
0.556*§
-0.175
-0.021
-0.143
0.238
0.009
0.193*
JRC
NA
0.161
-0.043
0.316*§
0.241
0
0.137
0.091
-0.07
0.118*
LES
NA
NA
NA
-0.053
-0.143
0.543
0.059
1
0.368
LMY
0.25
NA
0.579*§
0.497*
0.614*§
-0.116
0
1*
0.138
-0.067
0.314*
MBZ
-0.043
0
-0.13
-0.011
0.107
-0.064
-0.258
0.576*
0
0.031
ORN
NA
NA
NA
0.617
NA
-0.231
0.142
NA
PYO
NA
NA
-0.2
0.385*§
0.277
-0.108
0.168
-0.057
0.146
0.169
0.488*§
0.188*
RSL
-
-
-
-
-
-
-
-
-
-
SSB
-0.339
-0.118
0.419
0.143
-0.083
0.217
0.586*§
0.529*§
0.074
0.132*
TLR
NA
-0.429
NA
TYR
-0.204
0.048
0.2
-0.364
URG
-0.053
0.111
0.067
0.444*§
VDB
NA
0.154
0.217
0
NA
NA
NA
0.103
-0.376
-0.186
0.186
-0.412
-0.118
-0.234
0.266
-0.055
0.342*
-0.268
0.31
-0.296
0.344*§
0.143*
-0.2
-0.143
0.64
0.386
0.15
0.289
0.145
ABO
-
-
-
-
-
-
-
-
-
-
CSV
0.21
0.133*
-0.085
-0.12
0.072
0.008
-0.008
-0.023
0.114
0.032
DIJ
0.662*
-0.169
0
0.2
-0.017
0.043
-0.009
-0.286
0.138
0.035
DSP
-
-
-
-
-
-
-
-
-
-
LAB
-
-
-
-
-
-
-
-
-
-
MKB
POR
SWA
NA
NA
0.5
-0.532
0.333
1
0.294
0.02
0.192
-
-
NA
-
NA
-
-
-
-
-
-
0.217
-0.037
0.659*§
-0.091
0.051
0.116
0.091
0.143
0.168*
18
Table S7. Pairwise FST values
Core Population Set
SSB
HEN ORN URG ALS
ESP
ISG
LMY DUN JRC
BLL
PYO CMT BSC
Extended
VDB GNL LES
DAS
TLR
FCT
FEL
MBZ TYR
CPG FVI
SWA DIJ
CSV
HEN 0.232
ORN 0.376 0.347
URG 0.229 0.355 0.498
ALS 0.260 0.312 0.543 0.308
ESP 0.220 0.319 0.441 0.216 0.232
ISG 0.218 0.325 0.460 0.249 0.230 0.056
LMY 0.394 0.377 0.610 0.389 0.296 0.291 0.342
DUN 0.283 0.316 0.598 0.296 0.271 0.239 0.264 0.217
JRC 0.158 0.281 0.426 0.157 0.247 0.163 0.189 0.284 0.176
BLL 0.283 0.413 0.482 0.285 0.410 0.285 0.305 0.468 0.423 0.233
PYO 0.147 0.259 0.365 0.162 0.259 0.140 0.146 0.277 0.201 0.101 0.187
CMT 0.230 0.294 0.433 0.284 0.298 0.191 0.216 0.396 0.321 0.232 0.367 0.204
BSC 0.243 0.250 0.517 0.283 0.316 0.236 0.230 0.355 0.285 0.275 0.308 0.185 0.243
VDB 0.316 0.351 0.536 0.326 0.351 0.302 0.309 0.315 0.273 0.259 0.393 0.184 0.327 0.298
GNL 0.402 0.465 0.601 0.405 0.405 0.310 0.371 0.441 0.386 0.335 0.500 0.335 0.321 0.423 0.281
LES 0.457 0.504 0.710 0.432 0.423 0.341 0.404 0.487 0.382 0.343 0.533 0.359 0.468 0.468 0.315 0.196
DAS 0.526 0.579 0.720 0.514 0.523 0.471 0.516 0.575 0.501 0.449 0.586 0.447 0.561 0.581 0.448 0.450 0.249
TLR 0.475 0.542 0.758 0.516 0.411 0.442 0.423 0.387 0.462 0.400 0.553 0.278 0.541 0.471 0.388 0.577 0.693 0.700
FCT 0.450 0.508 0.671 0.456 0.424 0.385 0.396 0.431 0.384 0.367 0.519 0.328 0.442 0.353 0.364 0.492 0.529 0.644 0.484
FEL 0.463 0.486 0.679 0.387 0.358 0.334 0.362 0.238 0.305 0.330 0.484 0.261 0.439 0.404 0.216 0.413 0.431 0.507 0.390 0.408
MBZ 0.388 0.480 0.606 0.361 0.436 0.358 0.399 0.479 0.398 0.318 0.438 0.273 0.421 0.441 0.392 0.493 0.508 0.545 0.569 0.568 0.470
TYR 0.305 0.404 0.549 0.296 0.317 0.216 0.236 0.443 0.345 0.296 0.377 0.255 0.332 0.292 0.375 0.457 0.424 0.504 0.517 0.468 0.449 0.337
CPG 0.409 0.403 0.602 0.426 0.472 0.418 0.463 0.544 0.506 0.439 0.442 0.353 0.445 0.368 0.483 0.574 0.615 0.648 0.654 0.627 0.608 0.439 0.380
FVI
0.257 0.340 0.537 0.215 0.269 0.199 0.193 0.344 0.275 0.205 0.335 0.081 0.248 0.273 0.237 0.373 0.428 0.475 0.377 0.415 0.294 0.303 0.276 0.438
SWA 0.176 0.307 0.463 0.291 0.315 0.228 0.219 0.392 0.302 0.181 0.307 0.118 0.249 0.272 0.291 0.407 0.472 0.555 0.425 0.426 0.444 0.378 0.322 0.451 0.191
DIJ
0.243 0.348 0.512 0.361 0.359 0.276 0.289 0.382 0.317 0.262 0.285 0.194 0.359 0.305 0.363 0.448 0.490 0.571 0.431 0.505 0.463 0.382 0.372 0.344 0.273 0.231
CSV 0.182 0.255 0.354 0.314 0.328 0.271 0.272 0.356 0.282 0.219 0.283 0.207 0.242 0.264 0.277 0.363 0.393 0.471 0.393 0.407 0.406 0.350 0.356 0.356 0.247 0.221 0.256
MBK 0.346 0.429 0.559 0.443 0.444 0.376 0.400 0.428 0.447 0.326 0.426 0.231 0.417 0.419 0.440 0.534 0.628 0.672 0.552 0.588 0.564 0.527 0.476 0.578 0.377 0.363 0.377 0.295
19
Table S8. Recombination tests for coding gene sequences performed in DNAsp . Average number of
site difference (theta) and recombination per gene (R) were used for coalescent simulations. Rm =
minimum number of recombination events; P[Rm] = probability of obtaining Rm ≥ observed, given
theta and R. ZZ = test statistic for intragenomic recombination (average LD across pairwise
comparisons between adjacent sites – average LD across all pairwise comparisons). P[ZZ]= probability
of obtaining ZZ ≥ observed, given theta and no recombination.
Gene
theta
R
Rm
P[Rm]
ZZ
P[ZZ]
RBact1a
1.855
6.3
0
0.822
-0.0188
0.648
RBHSPA2
6.76
2.9
11
0
0.1326
0.001
RBPsmd2
3.801
20.1
4
0.334
0.0185
0.268
Table S9. Haplotype frequencies and nested haplotype (NH) assignments. NH group assignments
were for HSPA2-NR (non-recombinant data) and Psmd2-NR only. Frequencies are the number of
phased sequences.
act1a
HSPA2 (NR)
Haplotype Frequency Haplotype Frequency
1
273
1
204
2
2
2
2
3
52
3
4
4
2
4
17
5
7
5
6
6
18
6
5
7
42
7
10
8
57
8
3
9
4
9
2
10
4
10
8
11
56
11
16
12
13
12
1
13
20
13
29
14
11
15
1
16
50
17
5
18
1
19
4
20
3
21
1
22
19
23
2
24
7
25
1
26
1
27
44
28
19
29
84
30
1
31
59
Total
550
Total
620
NH
1
1
1
2
2
3
3
4
4
5
6
6
7
7
8
9
9
9
9
10
10
11
11
11
11
11
12
12
13
13
13
20
Haplotype
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Psmd2 (NR)
Frequency
17
4
14
81
5
1
1
10
1
1
10
4
17
5
6
11
6
7
10
36
2
32
60
3
44
6
15
8
6
17
Total
440
NH
1
1
1
2
2
2
2
3
3
3
4
4
5
5
5
5
6
6
7
7
8
9
10
10
10
11
11
12
12
13
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21