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AUTHOR'S PROOF

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1 Article Title Genetic Relationships and Spatial Genetic Structure Among

Populations of Rhodnius prolixus (Hemiptera: Reduv iidae) in
Colombia and Venezuela Based on Mitochondrial Cytochrome-b
Sequences
2 Article Sub- Title
3 Article Copyright - Sociedade Entomológica do Brasil 2016
Year (This w ill be the copyright line in the final PDF)
4 Journal Name Neotropical Entomology
5 Family Name Ruiz-García
6 Particle
7 Given Name M
8 Suffix
9 Corresponding Organization Pontificia Univ Javeriana
Author
10 Division Lab de Genética de Poblaciones Molecular y
Biología Evolutiva, Unidad de Genética, Depto de
Biología, Facultad de Ciencias
11 Address Cra 7ª No 43-82, Bogotá, DC
12 e-mail mruiz@javeriana.edu.co
13 Family Name Luna-Marín
14 Particle
15 Given Name KP
16 Suffix
17 Organization Univ Industrial de Santander (UIS)
18 Division Centro de Investigaciones en Enfermedades
Tropicales (CINTROP)
Author
19 Address Bucaramanga
20 Organization Pontificia Univ Javeriana
21 Division Lab de Genética de Poblaciones Molecular y
Biología Evolutiva, Unidad de Genética, Depto de
Biología, Facultad de Ciencias
22 Address Cra 7ª No 43-82, Bogotá, DC
23 e-mail
24 Family Name Angulo-Silv a
Author
25 Particle
AUTHOR'S PROOF

26 Given Name VM
27 Suffix
28 Organization Univ Industrial de Santander (UIS)
29 Division Centro de Investigaciones en Enfermedades
Tropicales (CINTROP)
30 Address Bucaramanga
31 e-mail
32 Family Name Hernández-Torres
33 Particle
34 Given Name J
35 Suffix
36 Author Organization Univ Industrial de Santander (UIS)
37 Division Lab de Biología Molecular (CINBIN), Escuela de
Biología
38 Address Bucaramanga
39 e-mail
40 Received 8 December 2015
41 Schedule Revised
42 Accepted 15 November 2016
43 Abstract One hundred twenty Rhodnius prolixus (Stal) (Hemiptera:
Reduviidae) specimens from 6 Colombian Departments and 1
Venezuelan State had 594-bp of the mitochondrial cytochrome-b
gene sequenced to improve the understanding of evolutionary
processes that shape the main vector of Chagas disease. The levels
of genetic diversity for this species were low-medium with reference
to other bugs. The genetic heterogeneity among the populations
was very limited which means there has been extensive gene flow
and/or very recent split processes. The overall sample as well as
some individual populations showed evidence of recent population
expansions (with the exception of Arauca, which yielded evidence
of a bottleneck for a mismatch distribution). This expansion (11,000
or 2000–25,000 year ago depending of two procedures employed)
coincides with the ending of the last intense glacial conditions
during the Pleistocene and the beginning of the Holocene that had
a warmer and wetter climate. Some of our autocorrelation analyses
(AIDA and Genetic Landscape Interpolation Analysis) indicated
local patches of high genetic similarity but no globally significant
spatial structure. We did show an original haplotype distributed
throughout the entirety of the geographical area studied.
44 Keywords Vector of Chagas disease - genetic diversity - population expansion
separated by ' - ' - spatial autocorrelation - DNA
45 Foot note Edited by Patrícia J Thyssen – UNICAMP
information
 AUTHOR'S PROOF JrnlID 13744_ArtID 470_Proof# 1 - 25/11/2016

Neotrop Entomol
DOI 10.1007/s13744-016-0470-2
1
3 PEST MANAGEMENT
2

4 Genetic Relationships and Spatial Genetic Structure

5 Among Populations of Rhodnius prolixus (Hemiptera: Reduviidae)
6 in Colombia and Venezuela Based on Mitochondrial Cytochrome-b
7 Sequences
8
9 KP LUNA-MARÍN1,2, VM ANGULO-SILVA1, J HERNÁNDEZ-TORRES3, M RUIZ-GARCÍA2

F
Q1 10
1
Centro de Investigaciones en Enfermedades Tropicales (CINTROP), Univ Industrial de Santander (UIS), Bucaramanga, Colombia

O
11 2
Lab de Genética de Poblaciones Molecular y Biología Evolutiva, Unidad de Genética, Depto de Biología, Facultad de Ciencias, Pontificia Univ
12 Javeriana, Bogotá, DC, Colombia

O
13 3
Lab de Biología Molecular (CINBIN), Escuela de Biología, Univ Industrial de Santander (UIS), Bucaramanga, Colombia
14

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15
16
17
19 Keywords D Abstract
20
35 Vector of Chagas disease, genetic diversity, One hundred twenty Rhodnius prolixus (Stal) (Hemiptera: Reduviidae)
21 population expansion, spatial
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36 specimens from 6 Colombian Departments and 1 Venezuelan State had
22 autocorrelation, DNA
37 594-bp of the mitochondrial cytochrome-b gene sequenced to improve
23
38 Correspondence the understanding of evolutionary processes that shape the main vector of
EC

24
39 M Ruiz-García, Lab de Genética de Chagas disease. The levels of genetic diversity for this species were low-
25 Poblaciones Molecular y Biología Evolutiva,
40
26 Unidad de Genética, Depto de Biología, medium with reference to other bugs. The genetic heterogeneity among
27
41 Facultad de Ciencias, Pontificia Univ the populations was very limited which means there has been extensive
R

28
42 Javeriana, Cra 7ª No 43-82, Bogotá, DC, gene flow and/or very recent split processes. The overall sample as well as
29 Colombia; mruizgar@yahoo.es
R

43 some individual populations showed evidence of recent population expan-

30
44 Edited by Patrícia J Thyssen – UNICAMP sions (with the exception of Arauca, which yielded evidence of a bottle-
O

45
31 Received 8 December 2015 and accepted 15 neck for a mismatch distribution). This expansion (11,000 or 2000–
32
46
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November 2016 25,000 year ago depending of two procedures employed) coincides with
33
47 the ending of the last intense glacial conditions during the Pleistocene and
N

34
48 * Sociedade Entomológica do Brasil 2016 the beginning of the Holocene that had a warmer and wetter climate.
U

49 Some of our autocorrelation analyses (AIDA and Genetic Landscape

50 Interpolation Analysis) indicated local patches of high genetic similarity
51 but no globally significant spatial structure. We did show an original hap-
52 lotype distributed throughout the entirety of the geographical area
53 studied.
54 55
56 Introduction infection percentages in Arauca (21%) and Casanare (10%). 66
Another 3.5 million people are at risk of being infected (Guhl 67
57 Some triatomine bugs (Hemiptera: Reduviidae: Triatominae) & Lazdins-Held 2007). 68
58 are vectors of Trypanosoma cruzi which causes Chagas dis- All species of the Triatominae subfamily can be considered 69
59 ease. This disease affects around 15 million people in Latin as probable vectors of T. cruzi, but only some species are 70
60 America. Another 28 million people are at high risk of being epidemiologically significant to the human beings. 71
61 infected, making Chagas one of the most important parasitic The species of great epidemiological importance are those 72
62 disease in Latin America (Moncayo & Silveira 2009, Tarleton which have adapted to living in close contact with humans 73
63 et al 2007). It is remarkable that only about 5% the human and colonizing rural dwellings (Schofield & Galvao 2009). Out 74
64 population within endemic areas of this vector in Colombian of the 26 Triatominae species present in Colombia, 15 have 75
65 are infected. This is around 1 million people, with the highest been known to have natural infections of T. cruzi 76
 AUTHOR'S PROOF
JrnlID 13744_ArtID 470_Proof# 1 - 25/11/2016

Luna-Marín et al

77 [Panstrongylus geniculatus (Latreille); Panstrongylus compared three domestic populations of R. prolixus. They 130
78 lignarius (Walker); Panstrongylus rufotuberculatus were from the Tolima and Cundinamarca Departments, the 131
79 (Champion); Triatoma dimidiata (Latreille); Triatoma dispar Sierra Nevada of Santa Marta in the Northern Caribbean 132
80 (Lent); Triatoma maculata (Erichson); Triatoma venosa (Stal); Colombia, and one sylvatic population from the Casanare 133
81 Rhodnius prolixus (Stal); Rhodnius brethesi (Matta); Rhodnius Department. The results showed low genetic differentiation 134
82 colombiensis (Moreno, Jurberg & Galvao); Rhodnius (FST = 0.06–0.15) and high genetic similarity by gene flow 135
83 pallescens (Barber); Rhodnius pictipes (Stal); Eratyrus among the intra-domiciliary populations of the three popu- 136
84 cuspidatus (Stal); Eratyrus mucronatus (Stal); and lations analyzed. The authors found some clear genetic dif- 137
85 Cavernicola pilosa (Barber)] (Guhl & Lazdins-Held 2007). ferentiation between the intra-domiciliary and sylvatic pop- 138
86 In Colombia and Venezuela, R. prolixus is the primary vec- ulations of the Casanare Department. The second study by 139
87 tor in the transmission of this disease (Fitzpatrick et al 2008), Fitzpatrick et al (2008) in Venezuela confirmed the presence 140
88 while T. dimidiata is the second most important vector in of R. prolixus in palms. The sylvatic bugs colonized houses, 141
89 Colombia (Grisales et al 2010). This last species also plays with house and palm specimens sharing seven mitochondrial 142
90 an important role in Central America together with other cytochrome-b gene (mt Cyt-b) haplotypes. They also detect- 143

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91 secondary vectors such as in Panama (R. pallescens) and in ed mitochondrial introgression between Rhodnius robustus 144
92 Northern Peru [R. ecuadoriensis (Lent & Leon)]. In southern (sylvatic form) and R. prolixus, which agrees well with hybrid- 145

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93 South America, the main vector is Triatoma infestans (Klug). ization events. The results with ten polymorphic microsatel- 146

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94 However, it has been eliminated over much of the area due lite loci also revealed a lack of genetic structure between 147
95 to control efforts (Fitzpatrick et al 2008, Schofield et al sylvatic and domestic ecotopes and an unrestricted gene 148

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96 2006). The same has occurred in Central America with flow. Rhodnius prolixus showed low to medium levels of ge- 149
97 R. prolixus (Hashimoto & Schofield 2012). Due to the lack of netic diversity in Venezuela. 150
98 a vaccine against this disease, the more effective control
D In the current study, we employed the mt Cyt-b gene to 151
99 measure is the spraying of infested houses with insecticides analyze different aspects of the population genetics of 152
100 153
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(Schofield et al 2006). Nevertheless, the control of R. prolixus R. prolixus in Colombia and part of Venezuela. The mt Cyt-b
101 in Colombia and Venezuela has not been very effective gene has often been used to investigate evolutionary signif- 154
102 (Fitzpatrick et al 2008). icant units, population structure, and to resolve taxonomic 155
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103 Rhodnius prolixus is a very efficient vector of T. cruzi due conflicts in many animal groups (Avise 2000). This includes 156
104 to its intra-domiciliary and peri-domiciliary habits, its high differentiation of R. prolixus and R. robustus (Larousse) 157
105 dispersion rates, high susceptibility of infection by T. cruzi, (Monteiro et al 2003, Pavan & Monteiro 2007) and determi- 158Q3
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106 short time of defecation, and short life cycle (Molina et al nation of genetic structure in different triatomines, such as 159
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107 2000). In Colombia, this species has been found in intra- Triatoma brasiliensis (Neiva) (Almeida et al 2008) and 160
108 161
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domiciliary, peri-domiciliary, and sylvatic environments in T. dimidiata in Costa Rica (Blandón et al 2010).
109 three different biogeographic regions (Caribbean area, The main aims of the current work are as follows: (1) To 162
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110 Andean area, and Eastern Llanos) (Angulo et al 2012a). In determine the levels of genetic diversity in seven populations 163
111 164
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the Caribbean area (Sierra Nevada de Santa Marta), of R. prolixus (six in Colombia and one in Venezuela); (2) to
112 R. prolixus is easily detectable within indigenous huts. Some determine the degree of genetic differentiation among these 165
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113 of these indigenous communities were not enclosed within populations and about the genetic differentiation among 166
114 the control vector programs and this probably favored high intra-domiciliary, peri-domiciliary, and sylvatic populations; 167
115 levels of infestations (Montilla et al 2011, Parra–Henao et al (3) to determine possible demographic changes during the 168
116 2009). In the Andean area, this species is strictly intra- natural history of this species; and (4) to analyze the possible 169
117 domiciliary and the control vector programs seem to be spatial genetic structure of this insect in the geographical 170
118 more effective (Angulo et al 2012a). In the Eastern Llanos, area studied. 171
119 this vector is found in three cited environments (Angulo et al
Q2 120 2012b, Guhl et al 2005 ). In this geographical area,
121 D’Alessandro et al (1981, 1984) reported R. prolixus living in Material and Methods 172
122 palms (Arecaceae). High infection rates have also been doc-
123 umented in the wine palm (Attalea butyracea; Angulo et al One hundred twenty individuals from 7 populations of 173
124 2012b) and in the African oil palm (Elaeis guineensis) (Guhl R. prolixus had a 594-bp fragment of the mt Cyt-b gene 174
125 et al 2005). sequenced. Six populations were analyzed in Colombia and 175
126 Only two works have attempted to provide data on the one in Venezuela (Table 1 and Fig 1). These seven populations 176
127 genetic structure and molecular population relationships of included three biogeographic areas: Northern Colombian 177
128 R. prolixus in our study area. López et al (2007), in Colombia, coast-Santa Marta (Magdalena and Guajira Departments), 178
129 using ITS-2 of ribosomal DNA by PCR/RFLP and RAPDs, Andes (Santander and Tolima), and Colombian and 179
AUTHOR'S PROOF JrnlID 13744_ArtID 470_Proof# 1 - 25/11/2016

Population Genetics of Rhodnius prolixus in Colombia and Venezuela

t1:1 Table 1 Sample localities, sample

t1:2 sizes, habitat of the samples Country Department or Locality Sample size Habitat Latitude Longitude
employed, and coordinates of State
the samples studied.
t1:3 Colombia Guajira Dibulla 12 Intra-domiciliary 11°09′ 73°18′
t1:4 Magdalena Santa Marta 9 Intra-domiciliary 11°08′ 73°20′
t1:5 Santander Albania 4 Intra-domiciliary 5°45′ 73°55′
Macaravita 4 Intra-domiciliary 6°28′ 72°36′ t1:6
Capitanejo 3 Intra-domiciliary 6°31′ 72°39′ t1:7
Molagavita 1 Intra-domiciliary 6°35′ 72°49′ t1:8
t1:9 Tolima Coyaima 15 Intra-domiciliary 3°47′ 75°12′
t1:10 Arauca Arauca 10 Intra-domiciliary 6°58′ 71°12′
Arauca 7 Sylvatic 6°58′ 71°07′ t1:11
t1:12 Casanare Maní 10 Intra-domiciliary 4°50′ 72°21′
Nunchía 1 Intra-domiciliary 5°36′ 72°00′ t1:13

F
Paz 4 Intra-domiciliary 5°50′ 71°35′ t1:14

O
Maní 5 Peri-domiciliary 4°24′ 72°21′ t1:15
Nunchía 2 Peri-domiciliary 5°36′ 72°00′ t1:16

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Maní 20 Sylvatic 4°24′ 72°05′ t1:17
t1:18

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Nunchía 2 Sylvatic 5°36′ 72°01′
t1:19 Venezuela Barinas Bolívar 10 Intra-domiciliary 8°47′ 70°30′
Ezequiel Z 1 Intra-domiciliary 7°52′ 71°14′ t1:20
D
The sequences obtained were deposited in GenBank (provisional accession numbers: KF556068 to KF556188).
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180 Venezuelan Eastern Llanos (Casanare, Arauca, Barinas). the traditional manual collection method including capture 184
181 Individuals were collected in intra-domiciliary, peri-domicili- by homeowners and the use of dislodging spray were used in 185
EC

182 ary, and sylvatic ecotopes (Table 1). Samples were collected domiciliary and peri-domiciliary collections (Gurtler et al 186
183 with live-baited traps (Angulo & Esteban 2011). Moreover, 1995). 187
R
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Fig 1 Map with the Departments

in Colombia (Tolima, Magdalena,
Guajira, Santander, Casanare, and
Arauca) and one state in Venezuela
(Barinas) where individuals of
Rhodnius prolixus were sampled.
AUTHOR'S PROOF
JrnlID 13744_ArtID 470_Proof# 1 - 25/11/2016

Luna-Marín et al

188 Morphological characters were used to identify corresponded probabilities (Weir & Hill 2002). These genetic 234
189 R. prolixus-like specimens using Lent & Wygodzinsky’s typo- diversity and differentiation statistics were undertaken with 235
190 logical key (1979) and kept in 70% ethanol until being proc- the Arlequin 3.5.1.2 Software (Excoffier & Lischer 2010). 236
191 essed for DNA extraction, but their taxonomy were con- We also determined the differences among the individ- 237
192 firmed through molecular analysis. The individuals were not uals analyzed, and the distributions of these differences, by 238
193 deposited in any entomological collection because they were means of the Kimura 2P genetic distance (Kimura 1980) using 239
194 used for additional analyses not within the scope of this the DARwin v. 6.0 Software (Perrier & Jacquemoud-Collet 240
195 pa per (bo dy an d an tenn al mo rph ometrics, DNA 2006). 241
196 microsatellites, natural infection of T. cruzi, and food
197 preferences). Phylogenetic analyses 242

The Modeltest 3.7 Software (Posada & Crandall 1998) and 243
198 Molecular Analyses the Mega 6.06 Software (Tamura et al 2013) were applied 244
to determine the best evolutionary substitution model for 245

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199 DNA was obtained from three legs of each individual that the sequences analyzed. Akaike information criterion (AIC; 246
200 were triturated with a plastic pestle in nitrogen liquid within Akaike 1974) and the Bayesian information criterion (BIC; 247

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201 a 1.5-μl tube. For DNA extraction, we used the Wizard Schwarz 1978) were used to determine the best evolutionary 248

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202 Genomic Purification Kit (Promega Madison, WI). The DNA substitution model. 249
203 was stored at −20°C until being processed. A phylogenetic tree was obtained using a maximum like- 250

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204 A fragment of 594 base pairs (bp) from the mt Cyt-b was lihood (ML) procedure using the PAUP*4.0b8 Program 251
205 amplified by means of the primers employed by Monteiro (Swofford 2002). We employed as outgroups, one sequence 252
206 et al (2003). PCR reactions were carried out in a Bio-Rad
D of R. robustus (Monteiro et al 2003), one sequence of 253
207 thermocycler. PCR reactions were performed in a 50 μl total Rhodnius barretti (Abad-Franch, Palomeque & Monteiro) 254
208 volume, including 0.2 μM of each primer, 1.5 mM MgCl2, 255
TE
(Abad-Franch et al 2013), one sequence of R. pictipes (Justi
209 200 μM dNTP mix, 2.5 U of Taq polymerase, and 50– et al unpublished), and two sequences of T. dimidiata and 256
210 100 ng of template DNA. We used the following tempera- Triatoma phyllosoma (Burmeister) (sequenced by us). 257
EC

211 tures and cycles: 94°C for 5 min, 30 cycles of 30 s at 94°C, Additionally, we constructed a median joining network (MJ) 258
212 30 s at 47°C and 1 min at 72°C, and a final extension time of (Bandelt et al 1999) using the Network 4.2.0.1 Software 259
213 10 min at 72°C. We checked all the amplifications, including (Fluxus Technology Ltd) to estimate the relationships among 260
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214 positive and negative controls, in 1% agarose gels. We puri- the haplotypes found in R. prolixus. The networks are more 261
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215 fied the amplified samples with filters Montage™ PCR, appropriate for intraspecific phylogenies than tree algo- 262
216 263
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Millipore, and directly sequenced the double-stranded DNA rithms because they explicitly allow for the co-existence of
217 (sequencing reaction with Big Dye Terminator kit) in a 3100 ancestral and descendant haplotypes, whereas trees treat all 264
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218 (ABI) automated DNA sequencer in both directions. The con- sequences as terminal taxa (Posada & Crandall 2001). 265
219
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sensus sequences were edited and assembled using the

220 Seqman Program (DNASTAR Inc.) and were aligned using Demographic evolution 266
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221 Clustal W implemented in the MEGA 6.06 Software

222 (Tamura et al 2013). The sequences obtained are being de- We used three procedure sets to determine possible histor- 267
223 posited in GenBank (provisional accession numbers: ical population changes for different R. prolixus populations 268
224 KF556068 to KF556188). analyzed. First, the mismatch distribution (pairwise sequence 269
differences) was obtained following the method of Rogers & 270
Harpending (1992) and Rogers et al (1996). We used the 271
225 Data Analyses raggedness rg statistic (Harpending et al 1993, Harpending 272
1994) to determine the similarity between the observed and 273
226 Genetic and heterogeneity population analyses the theoretical curves. Second, we used the Fu FS statistic (Fu 274
1997) and the R2 statistic of Ramos-Onsins & Rozas (2002) to 275
227 We used the following statistics to determine the genetic determine possible population size changes in the R. prolixus 276
228 diversity within R. prolixus populations: number of polymor- populations we analyzed (Simonsen et al 1995, Ramos-Onsins 277
229 phic sites (S), haplotypic diversity (Hd), nucleotide diversity & Rozas 2002, Ruiz-García et al 2012). The two procedures 278
230 (π), average number of nucleotide differences (k), and θ by were carried out with the DNAsp 5.10 Software (Librado & 279
231 sequence (Hudson et al 1992). Rozas 2009), and the significances were estimated with 280
232 To determine possible differences among the R. prolixus 10,000 coalescence permutations (Press et al 1992). Third, 281
233 population pairs, we used the F ST statistic with their we used a Bayesian skyline plot analysis (BSP) to determine 282
 AUTHOR'S PROOF JrnlID 13744_ArtID 470_Proof# 1 - 25/11/2016

Population Genetics of Rhodnius prolixus in Colombia and Venezuela

283 possible demographic changes across the natural history of that fall within a specified DC. It takes on a value of 0 when 332
284 the overall R. prolixus population analyzed as well as for each all individuals within a DC are genetically identical and takes 333
285 one of the geographical areas studied. It was obtained by on a value of 1 when all individuals within a DC are complete- 334
286 means of the BEAST v. 1.6.2 and Tracer v1.5 Software ly dissimilar. The probability for each DC was obtained via 335
Q4 287 (Drummond & Rambaut 2007). The Coalescent-Bayesian sky- 1000 randomizations. We defined distance classes of 4 and 336
288 line option in the tree priors was selected with five steps and carried out the analysis with AIS Software (Miller 2005). 337
289 a piecewise-constant skyline model with a relaxed molecular We used AIDA as a third spatial autocorrelation procedure 338
290 clock with two independent runs (40,000,000 generations (Bertorelle & Barbujani 1995). To connect the individuals se- 339
291 with the first 4,000,000 discarded as burn in) were com- quenced within each distance class, the Gabriel-Sokal net- 340
292 bined with LogCombiner v1.6.2 software (Drummond & work (Gabriel & Sokal 1969, Matula & Sokal 1980, Ruiz- 341
293 Rambaut 2007). We plotted the likelihood versus generation García 1993, 1994, 1997) and the Delaunay’s triangulation 342
294 and estimated the effective sample size (ESS > 200) of all with elimination of the crossing edges (Ripley 1981, Upton 343
295 parameters across the two independent analyses, showing & Fingleton 1985, Isaaks & Srivastava 1989) were used. The 344
296 that the analysis was done correctly. The marginal densities Bonferroni (Oden 1984) and the Kooijman’s tests were esti- 345

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297 of temporal splits were analyzed in Tracer v1.5 and we select- mated to determine the statistical significance of the auto- 346
298 ed the Bayesian Skyline reconstruction option for the trees correlation coefficients. One AIDA run was carried out with 347

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299 log file. A stepwise (constant) Bayesian skyline variant was five DC (1 DC: 0–272 km; 2 DC: 272–345 km; 3 DC: 345– 348

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300 selected with the maximum time as the upper 95% higher 537 km; 4 DC: 537–778 km; 5 DC: 778–858 km). 349
301 posterior density (HPD). Finally, a genetic landscape interpolation analysis (GLIA) 350

PR
was performed by means of the “inverse distance weighted” 351
302 Spatial pattern analyses procedure (Miller 2005) to view in a tridimensional perspec- 352
D tive the possible spatial genetic structure of the data 353
303 A Mantel’s test (Mantel 1967) was used to detect possible analyzed. 354
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304 overall relationships between a genetic matrix among indi-
305 viduals (Kimura 2P genetic distance) and the geographic dis-
306 tance matrix among the individuals analyzed. In this study, Results 355
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307 Mantel’s statistic was normalized according to Smouse et al

308 (1986). This procedure transforms the statistic into a corre- Genetic diversity and differentiation 356
309
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lation coefficient. The geographic distances were measured

310 with the Spuhler’s (1972) procedure, where The T92 substitution model (Tamura 1992) was the best nu- 357
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   cleotide substitution model in our data (4541.239) for the 358

 
D ¼ arcos cos XðiÞ : cos Xð jÞ þ sin XðiÞ : sin Xð jÞ cos YðiÞ – Yð jÞ  ; 359
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BIC, while the HKY + G model (Hasegawa et al 1985 and G =

gamma distributed rate variation among sites) was the best 360
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311
312 where X(n) and Y(n) are the latitude and longitude of the nth nucleotide substitution model in our data (2337.286) for the 361
362
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313 individual sampled, respectively. The significance of the cor- AIC. This last model was employed for the ML tree and for
314 relations obtained was tested using a Monte Carlo simulation the BSP procedure. For the overall sample studied, the var- 363
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315 with 1000 permutations. We performed three different runs iable sites were 16, with 10 singleton variable sites (2 variants) 364
316 of the normal data, log geographic distance, and log both and 6 parsimony informative sites (2 variants). The maximum 365
317 geographic and genetic distances with NTSYS v. 2.1 likelihood estimate of Transition/Transversion bias was 1.79 366
318 Software (Rohlf 2000). (−1598.725). Fifteen mutations were synonymous and 1 was 367
319 For the first spatial autocorrelation analysis, we created not synonymous. 368
320 distograms based on Gregorius’s (1978) genetic distance for In the overall sample R. prolixus, we detected 18 haplo- 369
321 all the individuals studied (Degen & Scholz 1998, Vendramin types with medium to low levels of genetic diversity 370
322 et al 1999). Fifteen distance classes (DC) were used for this (H d = 0.559 ± 0.054, k = 1.056 ± 0.706, and π = 0.00178 371
323 analysis. We used 1000 Monte Carlo simulations to deter- ± 0.00024) following other empirical and theoretical studies 372
324 mine the significance of distograms, and autocorrelation co- (for instance, Nei 1987). By population, the highest and low- 373
325 efficients (Manly 1997) with an estimated confidence interval est genetic diversity values were those of Barinas (Hd = 0.800 374
326 of 95% (Streiff et al 1998). We used the SGS version 1.0d ± 0.114, k = 1.891 ± 1.166, and π = 0.00318 ± 0.00094) and 375
327 Software to determine the significance of these autocorrela- Tolima and Guajira (no genetic diversity was detected in ei- 376
328 tion coefficients (Degen et al 2001). ther population, respectively) (Table 2). 377
329 The second spatial autocorrelation analysis used the Ay The genetic differentiation among the seven populations 378
330 statistic (Miller 2005) for each DC. Ay can be interpreted as studied in Colombia and Venezuela was small (Table 3). Only 379
331 the average genetic distance between pairs of individuals the Arauca population was significantly differentiated from 380
AUTHOR'S PROOF
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Luna-Marín et al

t2:1 Table 2 Genetic diversity statistics for the different Rhodnius prolixus populations studied at the mt Cyt-b gene were the number of haplotypes
(NH), the haplotypic diversity (Hd), the nucleotide diversity (π), the average number of nucleotide differences (K), and the θ statistic (= Neμ; Ne =
effective female population size; μ = mutation rate per generation) by sequence.

t2:2 Populations of R. prolixus NH Hd π K θ

t2:3 Overall population 18 0.559 ± 0.054 0.0018 ± 0.0002 1.056 ± 0.706 2.985 ± 1.003
t2:4 Tolima 1 0 0 0 0
t2:5 Santander 2 0.303 ± 0.147 0.0005 ± 0.0002 0.303 ± 0.237 0.331 ± 0.331
t2:6 Magdalena 3 0.556 ± 0.165 0.0010 ± 0.0004 0.611 ± 0.530 0.736 ± 0.563
t2:7 Guajira 1 0 0 0 0
t2:8 Arauca 2 0.485 ± 0.079 0.0025 ± 0.0004 1.456 ± 0.928 0.887 ± 0.571
t2:9 Casanare 10 0.751 ± 0.054 0.0023 ± 0.0003 1.351 ± 0.853 2.069 ± 0.881
t2:10 Barinas 6 0.800 ± 0.114 0.0032 ± 0.0009 1.891 ± 1.166 2.731 ± 1.389

F
381 the Guajira, Magdalena, Santander, and Tolima populations differentiated from R. robustus, R. barretti, and R. pictipes. 402

O
382 by using the FST statistic. Also, the values of the Kimura 2P The MJN procedure (Fig 3) showed the existence of one 403
383 404

O
genetic distance were small among the populations, with the original haplotype distributed throughout the entire geo-
384 highest value only at 0.003 (Table 4). The distribution of graphical area. Other haplotypes were more specific to some 405

PR
385 these genetic distances, included two outgroups areas but differentiated from the widely distributed haplo- 406
386 (T. phyllosoma and T. dimidiata) with the DARwin Software, type by only one to three mutations. This suggests very re- 407
387 showed that the major part of the comparisons among all of cent haplotype diversification. The star-like form of this MJN 408
388 D
the individuals analyzed of R. prolixus was around 0.001– is related to recent and sudden population expansion. 409
389 0.002. In contrast, the comparisons among the individuals
TE
390 of R. prolixus and the two outgroups reached values of 0.011.
Demographic evolution 410
391 Also, the genetic distances were small among the intra-do-
392
EC

miciliary, peri-domiciliary, and sylvatic populations of

The overall sample showed mismatch pairwise distribution, 411
393 R. prolixus (0.001–0.003).
Fu’s Fu, and R2 tests supporting a population expansion 412
(Table 5). From the mismatch pairwise distribution analysis, 413
R

394 Phylogenetic relationships we estimated that this population expansion began around 414
R

11,700 years ago (YA) with an initial female effective popula- 415
395 The ML tree (Fig 2) showed that many haplotypes were in- 416
O

tions size of around 34,000 individuals and a final size

396 dependently related with geographical areas. The H1 was around 54.5 million individuals. Barinas and Santander 417
C

397 extended throughout all the populations of Colombia and showed two analyses related with population expansion 418
398 Venezuela studied. The two most differentiated haplotypes (mismatch pairwise distribution and R2 test, respectively). 419
N

399 were the H5 from Barinas (Venezuela) and the H12 from Casanare showed two analyses indicating population expan- 420
U

400 Casanare (Colombia). Clearly, all our sequences analyzed
401 belonged to R. prolixus and they were perfectly
t3:1 Table 3 Population FST pairs among the different populations of
Rhodnius prolixus studied in Colombia and Venezuela at the mt Cyt-b
gene obtained with the Arlequin 3.5.1.2 Software.
sion (mismatch pairwise distribution and Fu’s Fu test). In 421
Table 4 Kimura (1980) 2P genetic distances among the different t4:1
Rhodnius prolixus populations studied in Colombia and Venezuela at
mt Cyt-b. Below, genetic distance values in percentages (%); above,
standard errors in percentages (%).

t3:2 Populations 1 2 3 4 5 6 7 Populations 1 2 3 4 5 6 7 t4:2

t3:3 1 – 1 – 0 0 0 0.1 0.1 0.1 t4:3

t3:4 2 0.0833 – 2 0 – 0 0 0.1 0.1 0.1 t4:4
t3:5 3 0.0606 0.0432 – 3 0.1 0.1 – 0.1 0.1 0.1 0.1 t4:5
t3:6 4 0.0000 0.0833 0.0606 – 4 0 0 0.1 – 0.1 0.1 0.1 t4:6
t3:7 5 0.3127* 0.2576* 0.2644* 0.3125* – 5 0.2 0.2 0.2 0.2 – 0.1 0.1 t4:7
t3:8 6 0.1227 0.1074 0.1050 0.1227 0.0804 – 6 0.1 0.1 0.2 0.2 0.3 – 0.1 t4:8
t3:9 7 0.0926 0.0951 0.0920 0.0980 0.0128 0.0170 – 7 0.2 0.2 0.2 0.2 0.3 0.3 – t4:9

1 = Guajira; 2 = Magdalena; 3 = Santander; 4 = Tolima; 5 = Arauca; 6 = 1 = Tolima; 2 = Guajira; 3 = Magdalena; 4 = Santander; 5 = Arauca; 6 =
Casanare; 7 = Barinas. * P < 0.01. Casanare; 7 = Barinas.
AUTHOR'S PROOF JrnlID 13744_ArtID 470_Proof# 1 - 25/11/2016

Population Genetics of Rhodnius prolixus in Colombia and Venezuela

H4-1-BAR

H18-1-CAS

H1-79-TOL-GUA-MAG-SAN-ARA-CAS-BAR

H9-2-SAN

H6-1-BAR

H11-5-CAS
11
66 H14-1-CAS
H2-1-BAR

66 H17-1-CAS
H8-2-MAG
20
H13-7-CAS

H3-1-BAR

H7-1-MAG
37
50 H15-1-CAS

F
H16-1-CAS
96

O
H5-1-BAR
Fig 2 Maximum likelihood tree
99 H12-6-CAS

O
obtained for the 18 haplotypes of
Rhodnius prolixus found at the H10-7-ARA-CAS
100

PR
mt Cyt-b. Bootstrap percentages Rhodnius-robustus
are at the nodes. Number after Rhodnius-barretti
haplotype (H) is the number of
Rhodnius-pictipes
individuals found at that
haplotype; TOL Tolima, MAG D Triatomadimidiata
Magdalena, GUA Guajira, SAN 100 Triatomaphyllosoma
TE
Santander, ARA Arauca, CAS
Casanare, BAR Barinas. 0.05
EC

422 contrast, Arauca yielded the three analyses agreeing well Santander. But the Arauca zone exhibited evidence of a bot- 427
423 with a bottleneck event (mismatch pairwise distribution, tleneck event affecting this R. prolixus population. 428
R

424 Fu’s Fu and R2 tests). Thus, there is clear evidence of a pop- The BSP analysis for the overall sample showed an out- 429
425 430
R

ulation expansion when the entire geographical area is con- standing population expansion in the last 2000 years
426 sidered together and in the areas of Casanare, Barinas, and (Fig 4A). Casanare yielded an important population 431
O
C
N
U

Fig 3 Median joining network

(MJN) with haplotypes found at
the mt Cyt-b gene for the 120
Rhodnius prolixus individuals
analyzed. Yellow circles
individuals sampled in Tolima,
blue circles individuals sampled in
Magdalena, green circles
individuals sampled in Guajira,
black circles individuals sampled
in Barinas, gray circles individuals
sampled in Arauca, pink circles
individuals sampled in Casanare,
brown circles individuals sampled
in Santander, red circles indicate
missing intermediate haplotypes.
AUTHOR'S PROOF
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Luna-Marín et al

t5:1 Table 5 Tests for possible

t5:2 historical demographic Fu’s Fs Raggedness rg R2
expansions or contractions in the
t5:3 different populations of Rhodnius Overall sample P[Fs ≤ −14.3] = 0.00000** P[rg ≤ 0.0536] = 0.04497* P[R2 ≤ 0.033] = 0.0422*
t5:4 prolixus studied in Colombia and Santander P[Fs ≤ 0.297] = 0.577 P[rg ≤ 0.247] = 0.547 P[R2 ≤ 0.151] = 0.018*
t5:5 Venezuela at the mt Cyt-b gene Magdalena P[Fs ≤ −0.532] = 0.205 P[rg ≤ 0.204] = 0.413 P[R2 ≤ 0.185] = 0.170
(*P < 0.05 and **P < 0.01,
t5:6 significant population Arauca P[Fs ≤ 3.869] = 0.042* P[rg ≤ 0.736] = 0.021* P[R2 ≤ 0.243] = 0.045*
t5:7 expansions). Casanare P[Fs ≤ −4.07] = 0.013* P[rg ≤ 0.041] = 0.046* P[R2 ≤ 0.072] = 0.135
t5:8 Barinas P[Fs ≤ −1.707] = 0.079 P[rg ≤ 0.024] = 0.004** P[R2 ≤ 0.127] = 0.056

All the significant values were correlated with population expansions. Only in the case of Arauca, did the
significant values agree quite well with a bottleneck event.

432 expansion in the last 25,000 YA (Fig 4B). Barinas, Magdalena, corresponded to the individuals sampled in Arauca, Casanare, 471
433 and Santander showed a slight but continuous population and Barinas, which agrees with a small but important differen- 472

F
434 expansion in the last 25,000–30,000 YA (Fig 4C–E), although tiation of the Colombian Eastern Llanos and Barinas in regard to 473
435 no so clear as Casanare. Arauca did not show any demo- the other populations along the Northern Coast and within the 474

O
436 graphic trend with this procedure. Andean Colombian areas. Nevertheless, there is no clear spatial 475

O
pattern within the data analyzed. 476
437 Spatial structure Therefore, if we only use the existence of different haplo- 477

PR
types, a significant spatial pattern is recovered for R. prolixus 478
438 Three Mantel’s tests were carried out to determine the over- at the mt Cyt-b gene in the areas of Colombia and Venezuela 479
439 all relationship between the genetic and geographic dis-
D we studied. However, if we add the nucleotide differences 480
440 tances. None of the three analyses showed significant corre- among the haplotypes (more complete information), some 481
441 482
TE
lation between genetic and geographical distances (normal local clusters can be observed but there is an absence of any
442 data: r = −0.0057, P = 0.5124; log geographic distance: spatial pattern at the global level. This is correlated with the 483
443 r = 0.0334, P = 0.1668; log both geographic and genetic dis- limited amount of genetic differences among the individuals 484
EC

444 tances: r = 0.0264, P = 0.2097). of the different areas. 485

445 We performed three spatial autocorrelation analyses. The
446 first analysis only considered different haplotypes without
R

447 considering the nucleotide differences among the different Discussion 486
R

448 haplotypes. For the Gregorious (1978)’s distance (Fig 5), the 1
449 487
O

and 4 DCs showed significantly positive autocorrelations Genetic diversity, heterogeneity among populations,
450 (P < 0.001 and 0.001, respectively), whereas the 8, 9, 10, 11, and the question of different ecotope populations 488
C

451 13, and 14 DCs showed significantly negative autocorrelations

452 489
N

(P < 0.008, 0.000, 0.012, 0.002, 0.036, and 0.010, respec- This is the first study with mitochondrial sequences trying to
453 tively). This analysis did not show the last DC as significant shed new light on the population history of the main vector 490
U

454 because the two extreme populations (Tolima and Guajira) for the transmission of the Chagas’s disease in Colombia. 491
455 were fixed for the same haplotype. The first relevant results to discuss are the low to 492
456 The second and third analyses took into account the dif- medium genetic diversity levels and the low levels of 493
457 ferences in the nucleotide composition of each haplotype. genetic differentiation among the populations. These 494
458 The spatial autocorrelation with the Ay statistic with four levels of genetic diversity are determined as low to me- 495
459 DCs and equal DC sizes did not show any significant DC dium following the patterns showed by other authors 496
460 (V = 0.00024; P = 0.289) (Fig 6). The AIDA procedure we used (Fitzpatrick et al 2008, Nei 1987, Patton & Feder 1981, 497
461 is in Table 6. Five DCs were defined and only the 5 DC was Patton & Smith 1992, Patton & Yang 1977, Patton et al 498
462 significantly positive (P < 0.01), showing that the most distant 1979, 1994, Ruiz-García & Alvarez 2000, Wright 1978). 499
463 individuals were more closely related genetically. This sup- The fact that the levels of genetic diversity are low to 500
464 ports that the individuals of the two most distant populations medium could be related to a recent origin of these pop- 501
465 (Tolima and Guajira) were fixed for the same haplotype. ulations, with a rapid population growth from a small 502
466 The GLIA (Fig 7) ratified the existence of some local clusters original population which expanded throughout the en- 503
467 of similarity among their individuals which conformed peaks tirety of the geographical area with punctuated muta- 504
468 because they presented the highest genetic distances with re- tions in certain geographical areas (Avise 2000). Our re- 505
469 gard to other individuals within other areas of the geographical duced levels of genetic diversity agree quite with the 506
470 range we analyzed. Some of the peaks we detected results of López et al (2007) for Colombia and with the 507
AUTHOR'S PROOF JrnlID 13744_ArtID 470_Proof# 1 - 25/11/2016

Population Genetics of Rhodnius prolixus in Colombia and Venezuela

A Overall sample (population expansion) C Barinas (population expansion)

F
B Casanare (population expansion) D Magdalena (poopulation expansion)

O
O
PR
D
TE
EC

E Santander (population expansion)

R
R
O
C
N
U

Fig 4 Bayesian skyline plot (BSP) Analyses to determine possible demographic changes in the samples of Rhodnius prolixus analyzed: A Overall
sample. B Casanare. C Barinas. D Magdalena. E Santander. These were the populations which showed population expansions.

508 results of Fitzpatrick et al (2008) for Venezuela. The first 0 (Lara State). Indeed, our highest genetic diversity 517
509 study did not detect polymorphisms in the length of the values were for the sample from the Barinas State in 518
510 fragments of the ITS-2 region with the digestion of the Venezuela (they detected 11 haplotypes with seven hap- 519
511 amplified fragment with the Nhe I, Rsa I, Dde I, and Hpy lotypes unique to that State; we detected six haplotypes, 520
512 188 I enzymes. The second study, using the same molec- five exclusive to that State). This could be interpreted as 521
513 ular marker that we used, also reported genetic diversity the Colombian populations having even lower genetic 522
514 values very similar to our own. With the exception of the diversity than the Venezuelan populations. In our case, 523
515 Portuguesa State (π = 0.03), the other five Venezuelan two populations, Guajira and Tolima, only showed one 524
516 states ranged from 0.01 (Trujillo and Cojedes states) to haplotype (null genetic diversity). Fitzpatrick et al 525
 AUTHOR'S PROOF
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Luna-Marín et al

et al 2003), which suggests that this is the original haplotype 539

Distogram using mean Genetic Distance (Gregorius 1978)
in this species and that the dispersion of this species is a 540
1.0
relatively recent event both in South and Central America. 541
observed The level of genetic heterogeneity we detected was rela- 542
0.9
tively small (ranging from 0 to 12% of relative genetic differ- 543
entiation, FST, with the exception of the Arauca population). 544
0.8 95% CI The same was reported by López et al (2007) and Fitzpatrick 545
D et al (2008). The first work, using RAPDs markers, estimated 546
0.7 population differentiation FST pairs ranging from 0.062 to 547
95% CI 0.102 (average FST = 0.107). These values are very similar to 548
0.6 what we estimated for a different kind of molecular marker. 549
The second study estimated a value of FST = 0.15 at the mt 550
0.5
Reference/ Mean Cyt-b and an FST = 0.07 at the microsatellite loci among six 551
8 16 24 32 40 48 56 64 72 80 88 96 104112120 Venezuelan states. They were able to analyze the genetic 552

F
Spatial distance differentiation in greater detail within these Venezuelan 553
states than we were since they sampled more localities and 554

O
Fig 5 Distogram, using the Gregorious distance (1978) with 15 distance
classes, to analyze the possible spatial genetic structure in Rhodnius more individuals. They observed heterogeneity and differen- 555

O
prolixus at the mt Cyt-b gene.
tiation occurring within some of these states. For instance, 556
they demonstrated a more pronounced heterogeneity struc- 557

PR
526 (2008) also detected the sample from the Lara State ture within the Portuguesa State (11% of the population is 558
527 fixed for the same unique haplotype, which was present differentiated, FST values) compared to the Barinas State (3% 559
528 in 68% of the specimens they studied and in 65.83% of
D differentiated). They claimed that Portuguesa State popula- 560
529 the samples we analyzed. The finding of this haplotype tions were collected in mountainous terrain, which could 561
530 562
TE
(H1) as the most frequent, even for distinct regions (also enhance greater population isolation. In contrast, within
531 in distinct ecotopes) is expected because it is the ancient the Barinas State, the populations were situated on flat lands 563
Q5 532 one (Dobzhansky 1971), which agrees with its central po- (Venezuelan Llanos) that could allow for easier mixing of 564
EC

533 sition in the MNJ analysis. populations. However, this strongly contrasted with the fact 565
534 In the current study, the diverse haplotypes originated that the Arauca population (Colombian Eastern Llanos) was 566
535 from this original and highly dispersed haplotype were the Colombia’s most differentiated population (FST = 0.25– 567
R

536 differentiated from one to three mutations. Fitzpatrick et al 0.31), with the exception of its relationship with Casanare 568
R

537 (2008) showed this differentiation to be from one to four (FST = 0.08; small genetic differentiation). Thus, in our study, 569
538 570
O

mutations. This haplotype (H1) is also in Honduras (Monteiro there were a closer genetic relationships among populations
C

Spatial Autocorrelation Analysis Results

N

0.002
0.002
U

0.002
0.002
0.002
0.002
0.002
0.002
0.001
0.001
0.001
Ay

0.001
0.001
0.001
0.001
0.001
0.001
0.000
0.000
0.000
Fig 6 Spatial autocorrelation 0.000
analysis with the Ay statistic with 0
four distance classes for 0
Rhodnius prolixus at the mt Cyt-b 0 50 100 150 200 250 300 350
gene. Geographical Distance
AUTHOR'S PROOF JrnlID 13744_ArtID 470_Proof# 1 - 25/11/2016

Population Genetics of Rhodnius prolixus in Colombia and Venezuela

t6:1 Table 6 Spatial autocorrelation

t6:2 using the AIDA procedure with Distance classes 0–272 km 272–345 km 345–537 km 537–778 km 778–858 km
the II index for five distance
t6:3 classes. II 0.0181 −0.0179 −0.0333 −0.0361 0.0712*

*P < 0.01.

571 of different mountainous areas compared to any mountain- from different departments were intermixed. These authors 591
572 ous area with the Arauca population. determined a high genetic similarity among populations of 592
573 Fitzpatrick et al (2008) stated that the strong difference Tolima, Cundinamarca, and Sierra Nevada de Santa Marta 593
574 between the domestic population from Trujillo State and all with the sample of Casanare only slightly differentiated. 594
575 the other populations indicated that the Andes mountain They concluded that high levels of gene flow by passive mi- 595
576 range may act as a barrier to gene flow. However, the pop- gration (associated to human movements) occurred be- 596
577 ulations of Tolima, Santander, Magdalena, and Guajira were tween the Central and Eastern Andean cordilleras in 597
578 separated by extensive Andean areas and were highly relat- Colombia throughout the Magdalena Valley. The small 598
579 599

F
ed. Thus, it is not clear that the Andean mountain range acts genetic differentiation of Casanare could be caused by the
580 as an effective barrier to the dispersion of R. prolixus, at least Eastern Cordillera, which separates this last population from 600

O
581 in Colombia. However, what is clear is that the two previous all others. A similar result was supported by Esteban et al 601

O
582 works and the current one showed this insect to have a weak (2005) with a morphological trait (antennas), who found 602
583 genetic structure in Northwestern South America. some differences between the Casanare and other Andean 603

PR
584 Only one individual sampled in Barinas, seven individuals bug populations. Our mitochondrial results are practically 604
585 sampled in Casanare, and six individuals from Arauca (all of identical to all of these herein commented. 605
586 them from Colombian and Venezuelan Llanos) showed some In our limited sample, the genetic differentiation among 606
587
D
limited divergence with reference to the other specimens intra-domiciliary, peri-domiciliary, and sylvatic populations 607
588 608
TE
analyzed. The MJN analysis showed individuals from differ- from Casanare is practically null. For this question, our data
589 ent populations sharing haplotypes. This situation was also also agrees with that reported by Fitzpatrick et al (2008), 609
590 reported by López et al (2007), who showed that individuals whom showed that pairwise FST values support a lack of 610
EC
R
R
O
C
N
U

50
45
40
35
30
50 48 46 25 X (Southern edge)
44 42 40 38 20
36 34 32 30 15
28 26 24 22 10
20 18 16 14 5
Y (Western edge) 12 10 8 6 4 2

Fig 7 Genetic landscape interpolation analysis (GLIA) for the mt Cyt-b gene sequences studied in Rhodnius prolixus in Colombia and in the Barinas
state in Venezuela.
AUTHOR'S PROOF
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Luna-Marín et al

611 population structure between four adjacent house and palm geographic distances. No significant correlation was detected 662
612 populations (FST = 0–0.05) in different Venezuelan States. among states. Thus, although in Venezuela a higher spatial 663
613 These authors detected sylvatic R. prolixus in palms within structure was detected relative to Colombia, the spatial 664
614 all states, except for Trujillo and Lara, unequivocally showing structure was very limited in both countries for R. prolixus. 665
615 that R. prolixus is present in sylvatic habitats in Venezuela. We estimated that population expansion for the overall 666
616 This correlated with Sánchez-Martin et al (2006), who deter- sample of R. prolixus began around 11,700 YA for the mis- 667
617 mined that infested palms within 100 m of a house were a match pairwise distribution, although the BSP analyses de- 668
618 risk factor for intra-domiciliary and peri-domiciliary infesta- tected this population expansion around 2000–25,000 YA. 669
619 tion. Relatedly, the morphometric study by Feliciangeli et al This coincides with the ending of the last intense glacial pe- 670
620 (2007) could not differentiate sylvatic specimens from pre- riod during the Pleistocene and the beginning of the 671
621 and post-spray intra-domiciliary and peri-domiciliary speci- Holocene when there was a warmer and wetter climate. 672
622 mens. In contrast to our findings and those from Fitzpatrick From 30,000 YA to 11,000 YA, there were many periods of 673
623 et al (2008), López et al (2007) claimed that the sylvatic extreme dry and cold conditions. For example, from 30,000 674
624 Casanare specimens were differentiated from the domestic YA to 23,000 YA, there was a very cold period in Europe 675

F
625 specimens of this Colombian Department. named Dryas I. During this period, the Fuquene Lagoon near 676
626 Due to the fact that the intra-domiciliary and peri- the current Bogota (Colombia) dried (29,000 YA; Van der 677

O
627 domiciliary populations are extremely similar from a genetics Hammen 1992). MacNeish (1979) determined changes in 678

O
628 point of view as well as many sylvatic populations are rela- the soil acidity and the types of pollen at the Pikimachay 679
629 tively similar to the first ones, effective chemical control Cove found within Peru which are related to the extreme 680

PR
630 could be carried out in Colombia as it was demonstrated in cold 23,000 YA. Later, around 19,000–16,000 YA, the last 681
631 Venezuela (Feliciangeli et al 2003) and in Central America glacial maximum (LGM or Dryer II) occurred, which had the 682
632 (Zeledón 2004). However, the sylvatic populations of
D most extensive distribution of snow and ice in the Central 683
633 R. prolixus could be a continuous source of re-invasion of Andes within the last 200,000 YA. Metivier (1998) estimated 684
634 685
TE
insecticide-treated intra-domiciliary and peri-domiciliary hab- that about 18,000 YA, the glacial extension in the North and
635 itats (Guhl et al 2005). Central Andes was around 371,306 km2, whereas it is cur- 686
rently about 3220 km2 (only 1% of the LGM). Also, there were 687
EC

636 Spatial genetic structure and demographic changes intense cold periods from 14,000 YA to 11,000 YA (with al- 688
ternating hot periods). Rodbell & Seltzer (2000) found mo- 689
637 We found significant autocorrelations in some DCs as well as raines (the front of a glacial) up to 3170 masl (12,000 YA) at 690
R

638 in the overall distograms when only the existence of different the Cordillera Blanca (San Martin Department, Peru). 691
R

639 haplotypes was considered (Gregorius’ distance distogram). Comparatively, today, the glacial front is 4600 masl. The 692
640 693
O

This analysis yielded a typical cline. Nevertheless, the other importance of Pleistocene events on the genetic structure
641 analyses, including the differences for nucleotide composi- of many Neotropical species have been reported in many 694
C

642 tion, offered a different perspective because no significant works (Ruiz-García et al 2006, 2012). 695
643 696
N

overall correlograms were detected. This probably means Once these colder climatic characteristics were over, the
644 that the spatial autocorrelation, only taking into account dif- populations of R. prolixus rapidly expanded. This expansion 697
U

645 ferent haplotypes, can more quickly detect false spatial pat- was particularly clear in the Casanare population. However, 698
646 terns. AIDA detected some local patches of high similarity, since only a very short time has elapsed, only a very limited 699
647 which is the exact pattern we observed in the tridimensional number of mutations has appeared since then. 700
648 GLIA and in the phylogenetic tree. Thus, our data revealed The bottleneck event detected in Arauca, only with the 701
649 that AIDA and GLIA are possibly the best procedures to de- mismatch pairwise distribution and Fu FS and R2 statistics, 702
650 tect spatial structure. The existence of local patches of ge- could be the effect of recent fumigations (Instituto 703
651 netic similarity is related with the fact that some haplotypes Nacional de Salud from Colombia, personal communication). 704
652 are only found in Barinas, Arauca, and Casanare. However, Also, the unique haplotype in the Guajira and Tolima 705
653 there is no globally significant autocorrelation because one Departments could have been helped by the Indigene move- 706
654 high-frequency haplotype occurred throughout the entire ments during the last thousands of years or by expeditions of 707
655 geographical area. Spaniards and other European conquerors (Schofield & 708
656 In Venezuela, Fitzpatrick et al (2008) used IBD tests to Dujardin 1999) transporting small propagules of bugs. There 709
657 detect possible spatial patterns. At the population and local- are even also many more recent transportation events in the 710
658 ity levels, they found that geographic distances explained last decades. Zeledón (2004), using historical registers, 711
659 around 6% of the genetic distances found. Also within showed that Central American populations of R. prolixus 712
660 Portuguesa (7%) and Barinas (2%) States, a minimal signifi- (fixed for the unique haplotype we observed in Guajira and 713
661 cant correlation was estimated between genetic and Tolima Departments) derived from an accidental leak of bugs 714
AUTHOR'S PROOF JrnlID 13744_ArtID 470_Proof# 1 - 25/11/2016

Population Genetics of Rhodnius prolixus in Colombia and Venezuela

715 from a laboratory in El Salvador in 1915. This colony was D’Alessandro A, Barreto P, Thomas M (1981) Nuevos registros de 773
triatominos domiciliarios y extradomiciliarios en Colombia. Colomb 774
716 constituted by bugs originating from La Guayra
Méd 12:75–85 775
717 (Venezuela). Thus, many of the population genetics parame- D’Alessandro A, Barreto P, Saravia N, Barreto M (1984) Epidemiology of 776
718 ters of R. prolixus could be of recent origin. Trypanosoma cruzi in the oriental plains of Colombia. Am J Trop Med 777
719 More molecular markers as well as additional populations Hyg 33:1084–1095 778
720 and specimens need to be studied to confirm our results. We Degen B, Scholz F (1998) Spatial genetic differentiation among popula- 779
tions of European beech (Fagus sylvatica L.) in Western Germany as 780
721 only employed sequences of a unique mitochondrial gene identified by geostatistical analysis. For Genet 5:191–199 781
722 and therefore we could only determine some evolutionary Degen B, Petit R, Kremer A (2001) SGS—spatial genetic software: a 782
723 events which occurred in the female lineages. Thus, it is nec- computer program for analysis of spatial genetic and phenotypic 783
724 essary to analyze nuclear autosomic gene and Y chromosome structures of individuals and populations. J Hered 92:447–448 784
Esteban L, Angulo VM, Feliciangeli MD, Catalá S (2005) Analysis of an- 785
725 gene sequences to obtain a more comprehensive evolution- tennal sensilla patterns of Rhodnius prolixus from Colombia and 786
726 ary perspective of R. prolixus in Colombia. Venezuela. Mem Inst Oswaldo Cruz 100:909–914 787
Excoffier L, Lischer HEL (2010) Arlequin suite ver 3.5: a new series of 788
727 Acknowledgements Thanks go to Dr. Gustavo Vallejo, Dr. M. D. programs to perform population genetics analyses under Linux and 789
728 Feliciangeli and the human communities from Magdalena, Santander, Windows. Mol Ecol Resour 10:564–567 790

F
729 Arauca, Casanare, and Barinas for their assistance in collecting the in- Feliciangeli MD, Campbell-Lendrum D, Martinez C, Gonzales D, Coleman 791

O
730 sects. Special thanks to Dr. E. M. Dotson from the jump CDC, Atlanta, P, Davies C (2003) Chagas disease control in Venezuela: lessons for 792
731 Georgia, USA for helping to sequence. This study was supported by the Andean region and beyond. Trends Parasitol 19:44–49 793

O
732 COLCIENCIAS. Thanks also to Pontificia Universidad Javeriana at Feliciangeli MD, Sanchez-Martin M, Marrero R, Davies C, Dujardin JP 794
733 Bogotá for logistical support to M.R.-G. to carry out the population (2007) Morphometric evidence for a possible role of Rhodnius 795

PR
734 genetics analyses. Thanks to Dr. J. M. Shostell (from Math, Science prolixus from palm trees in house re-infestation in the State of 796
735 and Technology Department, University of Minnesota Crookston, USA) Barinas (Venezuela). Acta Trop 101:169–177 797
736 for English corrections. Fitzpatrick S, Feliciangeli MD, Sanchez-Martin MJ, Monteiro FA, Miles 798
737 D MA (2008) Molecular genetics reveal that sylvatic Rhodnius prolixus 799
do colonize rural houses. PLoS Negl Trop Dis 2:e210 800
Fu YX (1997) Statistical tests of neutrality of mutations against popula- 801
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AUTHOR'S PROOF
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