SUPPLEMENTAL MATERIAL:
1.
DETAILED FIELD STUDIES:
We address natural intrapopulational variation over time in this species through
a consideration of two populations: Navafría (NAV) (Sistema Central, Spain), a
genetically and geographically isolated pure Spanish population of Cpe, and Sallent de
Gállego (SAL) (Pyrenees, Spain), a population inside the HZ (Fig. S1). We have
selected these two populations because of their different environmental conditions, and
because both are well established populations in which the incidence of Wolbachia has
been extensively studied.
1.1.
Field Collections
Three random samples of the endemism Chorthippus parallelus erythropus were
collected from the Iberian population of Navafría (NAV; 42°45'53.31"N;0°20'25.34"W,
1710 m; n = 283) in July, August and September 2005, to analyze the variation in
infection frequency in early-, mid- and late-summer populations. These organisms have
an annual life cycle, with one generation disappearing by the end of the summer and the
next appearing by the end of June of the following year. Similar three-stage sampling
was carried out in the hybrid Pyrenean population of Sallent de Gállego (SAL;
42°45'53.31"N;0°20'25.34"W, 1343 m; n = 319) (Fig. 1 and table I). Gonads were
dissected and fixed in 100% ethanol.
1.2.
DNA Extraction
DNA was extracted from whole fixed ovaries and testicles. Each sample was
individually dried and immediately frozen in liquid nitrogen before crushing in a mill
(Retsch, Germany). Each sample was homogenized in 600 µl of TNES buffer (50 mM
Tris-HCl pH 8.0; 400 mM NaCl; 20 mM EDTA pH 8.0; 0.5% SDS) containing
proteinase K (0.03% p/v) and incubated at 37ºC overnight. Phenol/chloroform
extraction was carried out, followed by ethanol precipitation. Samples were dissolved in
Tris-EDTA (tris-HCl 1 mM pH 8.0; EDTA 1 mM pH 8.0) and incubated with RNase A
(5 μg/ml) for 2 hours. The final DNA concentration was 50 ng/µl (Martínez et al., 2009;
Zabal-Aguirre et al., 2010)
1.3.
Wolbachia Detection and Sequencing
Wolbachia was detected by PCR amplification of a Wolbachia 16S rRNA gene,
using Wolbachia-specific primers (Zabal-Aguirre et al., 2010), followed by a second,
nested PCR amplification using strain-specific primers (Martínez-Rodríguez et al.,
2013). We performed this highly sensitive technique to limit false-negative data because
of the low bacterial densities present during some host ontogenetic stages (Arthofer et
al., 2009).
PCR reactions were performed in 50 µl containing 2 mM of MgCl2, 0.2 mM of
dNTP, 30 pmoles of each primer, 1.25 U of Taq Biotools DNA polymerase, and 4 µl of
DNA solution at 50 ng/µl. Thermal cycling conditions were: 30 s at 95°C, followed by
35 cycles of 30 s at 95°C, 1 min at 54°C (for the generalist primers) or 68°C (for the
strain-specific primers) and 1 min at 72°C (Zabal-Aguirre et al., 2010; MartínezRodríguez et al., 2013). After these cycles there was a final elongation stage of 10 min
at 72°C. 10 µl of each amplification product were electrophoretically separated on 0.7%
agarose gel. Gels were stained with ethidium bromide.
To confirm the strain characterization, the Multilocus Sequence Typing (MLST)
and wsp (Wolbachia surface protein) gene characterization systems were used. GatB,
coxA, hcpA, ftsZ, fbpA and wsp genes were amplified as previously described (Baldo et
al., 2006), with modifications: PCR reactions were performed in 50 µl containing 2 mM
of MgCl2, 0.2 mM of dNTP, 30 pmoles of each primer, 1.25 U of Taq BIOTAQ™ DNA
polymerase (Bioline) and 4 µl of DNA solution (50 ng/µl).
Amplified genes were purified by Illustra GFX PCR DNA and Gel Band
Purification Kit (GE Healthcare) or the ExoSAP-IT method (GE Healthcare).
Sequencing was performed by Stabvida (www.stabvida.com). The 16S rRNA, MLST,
and wsp sequences generated in this study have been deposited in the GenBank database
under accession numbers JN698878- JN698882 and JN701984- JN701990.
Bayesian likelihood was inferred using a Markov Chain-Monte Carlo variant run
under the MrBayes 3.2.1 program (Ronquist & Huelsenbeck, 2003). Phylogenies based
on single and concatenated MLST genes and wsp were reconstructed. jModeltest
(Posada, 2008) was used to distinguish the appropriate model of evolution, the best
likelihood score being evaluated by AIC criteria (Akaike, 1974). The model selected
were GTR+I+G (General time reversible model, including Gamma and Proportion
Invariant corrections) for concatenated MLST, ftsZ, gatB, and wsp; GTR+G for coxA
and hcpA, and HKY+I+G (Hasegawa, Kishino and Yano model, including Gamma and
Proportion Invariant corrections) for fbpA. Bayesian analysis was carried out with 106
generations and a sample frequency of 100. The first 5,000 trees were discarded as
burn-in.
1.4.
Statistical Analysis and Temperature Data
Data were analyzed with SPSS Statistics 19.0. Wilson’s correction was used to
estimate the infection proportions and their corresponding SEs (Wilson, 1927).
Proportions of crosstabulated variable categories were compared using the χ2 test (table
II). Temperatures at NAV were provided by the INM (Spanish National Meteorological
Institute), and those from SAL were kindly provided by José A. Villacampa from
ARAMON S.L. (Formigal, Spain). Mean temperatures were compared with a one-way
ANOVA.
The mean temperatures for July and August were different for NAV and SAL,
whilst those for September were similar (fig. 8). NAV temperatures frequently exceeded
30ºC during July and August, occasionally rising above 35ºC. Temperatures in SAL
were significantly lower than in NAV, except September (ANOVA: Global data: F =
55.23; d.f. = 1, 152; p ≈ 0.000. July: F = 26.608; d.f. = 1, 60; p ≈ 0.000. August: F =
62.840; d.f. = 1, 60; p ≈ 0.000. September: F = 0.496; d.f. = 1, 28; p = 0.487), varying
between 20°C and 25°C. (Fig. S4). Infection proportions did not change over the
summer (see above).
SUPPLEMENTAL FIGURES :
Figure S1: Populations of Chorthippus parallelus: Navafría (NAV) and Sallent de Gállego (SAL).
Figure S2: Rooted Bayesian tree of the Wolbachia 16s rRNA sequences analyzed in this study. Notice
that Wolbachia strains (grey) belong to the F and B supergroups.
Figure S3: Unrooted Bayesian trees of the Wolbachia MLST genes and Wsp gene analyzed in this study.
Notice that Wolbachia strains (grey) belong to the F and B supergroups.
Figure S4: Temperatures in the Chorthippus parallelus population locations: Navafría (NAV, black) and
Sallent de Gállego (SAL, grey).
Progression of single infection in C. parallelus. Notice that infected and
uninfected proportions are complementary. a) Change in proportions of uninfected
males after 4000 generations (stable equilibrium) as a function of the initial uninfected
male frequency (Pm0) and maternal transmission (where µ = 0 represents perfect
transmission). The initial female frequency was P f0 = 0.5. b) Change in proportions of
uninfected females as a function of initial uninfected female (Pf0) frequency and
maternal transmission. Initial male frequency was Pm0 = 0.5. c) and d) Dependence of
threshold value on H (where H = 0 represents total incompatibility) and µ (where µ = 0
represents perfect transmission). Initial male and female proportions were Pm0 = 0.5 and
Pf0 = 0.5. Note the discontinuity in the predicted infection proportions for parameter
values. This discontinuity appears irrespective of the initial infection proportions.
Figure S5:
SUPPLEMENTAL TABLES
Population
Latitude
Longitude
Altitude
(m)
n
Inf F
Inf B
Inf FB
Uninfected
NAV
40° 59´02´´N
3° 49´00´´ W
1710
283
0.64 ± 0.06 0.10 ± 0.04
0.22 ± 0.05
0.04 ± 0.02
SAL
42° 45´57´´ N
0° 20´ 33´´ W
1343
321
0.02 ± 0.02 0.23 ± 0.05
0.73 ± 0.05
0.02 ± 0.02
Table S-1: Wolbachia infection proportions ± 2 SE in Navafría (NAV) and Sallent de Gállego (SAL) populations of Chorthippus parallelus, independent of sex and month of
sampling. Let n be the number of individuals. Wilson’s correction was used to estimate the infection proportions and the corresponding SEs (Wilson, 1927).
Sex
Infection
Kind
All
individuals
Males
Females
July
August
Strain
Proportion
B
F
BF
0.34 ± 0.10
0.99 ± 0.04 0.02 ± 0.04
92
0.63 ± 0.10
Uninfected
0
B
Infected
F
BF
0.01 ± 0.04 0.01 ± 0.04
0.45 ± 0.15
0.98 ± 0.07 0.00 ± 0.06
Infected
Uninfected
0
B
Infected
F
BF
Uninfected
0
n
0.53 ± 0.15
0.02 ± 0.07 0.02 ± 0.07
0.22 ± 0.13
1.00 ± 0.06 0.04 ± 0.09
0.73 ± 0.14
0.00 ± 0.06 0.00 ± 0.06
47
45
September
Proportion
0.99 ± 0.03
n
0.01 ± 0.03
0.01 ± 0.03
0.23 ± 0.12
1.00 ± 0.05
0.02 ± 0.06
0.75 ± 0.12
0.00 ± 0.05
0.00 ± 0.05
0.23 ± 0.11
0.99 ± 0.05
0.00 ± 0.04
0.75 ± 0.11
0.01 ± 0.05
Proportion
n
0.23 ± 0.08
0.15 ± 0.07
0.99
±
0.03
0.01 ± 0.03
0.02 ± 0.04
122
107
0.75 ± 0.08
0.82 ± 0.08
0.01 ± 0.05
0.01 ± 0.03 0.02 ± 0.04
0.20 ± 0.12
0.98 ± 0.07 0.04 ± 0.08
57
49
0.75 ± 0.13
65
0.02 ± 0.07 0.02 ± 0.07
0.10 ± 0.08
1.00 ± 0.05 0.00 ± 0.05
0.90 ± 0.09
58
0.00 ± 0.05 0.00 ± 0.05
Table S-2: Wolbachia infection proportions ± 2 SE in the Sallent de Gállego (SAL) population of Chorthippus parallelus. Let n be the number of individuals. Wilson’s
correction was used to estimate the infection proportions and the corresponding SEs (Wilson, 1927).
Sex
Infection
Kind
July
Strain
B
All individuals
Infected
Uninfected
Males
Females
Infected
F
BF
0
B
F
BF
Uninfected
0
B
Infected
F
BF
Uninfected
0
August
Proportion
n
0.06 ± 0.06
1.00 ± 0.06* 0.57 ± 0.10
0.40 ± 0.10*
0.00 ± 0.06* 0.00 ± 0.03*
0.02 ± 0.07
1.00 ± 0.05* 0.58 ± 0.14*
0.4 ± 0.14*
0.00 ± 0.05* 0.00 ± 0.05*
0.10 ± 0.10
1.00 ± 0.05 0.56 ± 0.14
0.33 ± 0.14
0.00 ± 0.05 0.00 ± 0.05
September
Proportion
n
0.11 ± 0.07
98
50
48
0.98 ± 0.04* 0.68 ± 0.10
0.19 ± 0.08*
0.02 ± 0.04* 0.02 ± 0.04*
0.08 ± 0.09
0.98 ± 0.07* 0.79 ± 0.12
0.11 ± 0.10
0.02 ± 0.07* 0.02 ± 0.07
0.14 ± 0.10
0.98 ± 0.07 0.58 ± 0.14
0.26 ± 0.12
0.02 ± 0.07 0.02 ± 0.04
Proportion
n
0.13 ± 0.08
0.91 ± 0.07* 0.68 ± 0.10
88
0.10 ± 0.07*
0.09 ± 0.07* 0.09 ± 0.07*
0.07 ± 0.10
0.85 ± 0.13* 0.73 ± 0.14*
47
41
0.05 ± 0.09*
97
50
0.15 ± 0.13* 0.15 ± 0.12*
0.17 ± 0.12
0.96 ± 0.08 0.63 ± 0.14
0.15 ± 0.11
47
0.04 ± 0.08 0.04 ± 0.08
Table S-3: Wolbachia infection proportions ± 2 SE in the Navafría (NAV) population of Chorthippus parallelus erythropus. Asterisks denote significant differences between
infection levels through the summer. Let n be the number of individuals. Wilson’s correction was used to estimate the infection proportions and the corresponding SEs
(Wilson, 1927).
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