Morphometric and molecular evidence of intraspecific biogeographical differentiation of Rhodnius pallescens (HEMIPTERA: REDUVIIDAE: RHODNIINI) from Colombia and Panama

Infection, Genetics and Evolution 12 (2012) 1975–1983

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Morphometric and molecular evidence of intraspecific biogeographical differentiation of Rhodnius pallescens (HEMIPTERA: REDUVIIDAE: RHODNIINI) from Colombia and Panama Andrés Gómez-Palacio a,⇑, Nicolás Jaramillo-O. a, Harling Caro-Riaño a, Sebastián Diaz a, Fernando A. Monteiro b, Ruben Pérez c, Francisco Panzera c, Omar Triana a a

Grupo de Biología y Control de Enfermedades Infecciosas – BCEI, Sede de Investigación Universitaria – SIU, Instituto de Biología, Universidad de Antioquia, Medellín, Colombia Laboratório de Genética Molecular e Microorganismos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil c Sección de Genética Evolutiva, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay b

a r t i c l e

i n f o

Article history: Received 7 December 2011 Received in revised form 27 March 2012 Accepted 5 April 2012 Available online 24 May 2012 Keywords: Triatominae Rhodnius pallescens Chagas disease Cytochrome b Morphometrics Genome size

a b s t r a c t Rhodnius pallescens is considered the main vector of Chagas disease in Panama and a relevant secondary vector in northern Colombia. Previous data reported that this species presents cytogenetically heterogeneous populations, which are probably biogeographically segregated. To provide new information on the diversity of R. pallescens, we compared several populations from Colombia and Panama based on the morphometric analyses of wings, mitochondrial cytochrome b (cyt b) gene sequencing, and genomic DNA measurements. Although no differences in DNA amount were detected, significant differences in cyt b sequences as well as wing size and shape were identified among populations. The results obtained in this work indicate R. pallescens comprises two evolutionary lineages with genetic and morphological differences that could be explained by their geographic isolation in distinct ecological zones. These results provide new insight into R. pallescens population diversity and the underlying biological processes that shape its evolution. Ó 2012 Published by Elsevier B.V.

1. Introduction The subfamily Triatominae (Hemiptera–Reduviidae) includes 141 species of hematophagous insects taxonomically grouped into five tribes and 15 genera (Jurberg et al., 2009; Schofield and Galvão, 2009). Most species are potential vectors of the protozoan parasite Trypanosoma cruzi, the causative agent of American trypanosomiasis, or Chagas disease. In Central America, Rhodnius pallescens is the only endemic Rhodnius species, because R. prolixus is thought to have been accidentally introduced by humans (Zeledón, 2004). R. pallescens is widely distributed across Central America and Colombia (Galvão et al., 2003), where it occurs under a wide range of climatic conditions and ecological zones (Gottdenker et al., 2011; Jaramillo et al., 2000). Although this species inhabits sylvatic palm trees, adult insects frequently invade human dwellings, attracted by artificial light, a behavior that increases its importance in disease transmission (Calzada et al., 2006; Cantillo-Barraza et al., 2010; Gottdenker et al., 2011; Zeledón et al., 2006). ⇑ Corresponding author. Address: Grupo de Biología y Control de Enfermedades Infecciosas – BCEI, Sede de Investigación Universitaria – SIU, Instituto de Biología, Universidad de Antioquia, Calle 69 # 52-59, Medellín, Colombia. Tel.: +57 4 2196681; fax: +57 4 2196520. E-mail address: [email protected] (A. Gómez-Palacio). 1567-1348/$ - see front matter Ó 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.meegid.2012.04.003

Rhodnius pallescens is the main vector in Panama (Calzada et al., 2010, 2006) and is one secondary vector in Costa Rica, Nicaragua (Zeledón et al., 2006), and the Colombian Caribbean region (Cantillo-Barraza et al., 2010; Guhl et al., 2007), where R. prolixus and T. dimidiata are the main vectors (Guhl, 2007). Therefore, this species was included as an important target to be controlled in the Central American Initiative for the interruption of vectorial transmission (Ponce, 1999a,b). An apparent epidemiological heterogeneity between Panamian and Colombian R. pallescens populations seems to suggest a possible biological differentiation between them. Thus, whereas in Panama R. pallescens have often been found inside dwellings infected with T. cruzi and therefore have been the principal Chagas vector there (Calzada et al., 2010, 2006), in Colombia it is assumed to be a sporadic vector related to accidental T. cruzi transmission to humans and is therefore considered a secondary vector (CantilloBarraza et al., 2010; Guhl et al., 2007). The variation in epidemiological relevance in R. pallescens could result from several endogenous factors such as vectorial capacities of biological or evolutionary divergent entities (i.e., different populations’ structure could involve differentiation of certain niche attributes such as bloodmeal sources or the ability to colonize artificial habitats, etc.) as well as exogenous factors such as human activities (i.e., landscape use, particularly palm trees) (Gottdenker et al., 2011)

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and interspecies competition with intradomiciliary species such as R. prolixus or T. dimidiata. Interestingly, although many recent studies in Rhodnius systematics have shown the macroevolution diversity of the species, few inter- or intra-population studies have been conducted so far on Rhodnius. Thus the phylogeography and population structure of R. pallescens are only beginning to be elucidated. The finding of two different chromosomal cytotypes (called A and B cytotypes) in different frequencies in populations from Colombia and Panama could indicate the existence of potentially important genetic differentiation, possibly related to biogeographical factors such as the altitude and ecological zone of their original populations (Gómez-Palacio et al., 2008). Studies on triatomine genetic and phenotypic variation are essential for the understanding of the natural mechanisms responsible for their adaptation to living in close contact with humans, and therefore accurate information on population structure and dispersal potential of triatomine species is necessary for optimum vector control. In this paper, we analyzed 19 R. pallescens populations to provide new information on the biological variability in this species. We compared these populations with the cytotype groups defined by Gómez-Palacio et al. (2008), using mtDNA sequences and morphometric approaches, to obtain information on population structure and diversity among populations with different epidemiological behaviors in Colombia and Panama. 2. Materials and methods 2.1. Insects R. pallescens of sixteen populations from Colombia and three populations from Panama were used for morphometrics (n = 93), cyt b DNA sequencing (n = 52), and genomic DNA content measurements (n = 51) (Table 1). Individuals were field-collected from Attalea butyracea and Elaeis oleifera palm trees combining live-bait traps (Noireau et al., 1999) and manual capture during the years 1997–2006. All capture sites were georeferenced using DIVA-GIS software v.5.2 (Hijmans et al., 2005). The geographical distribution of the R. pallescens populations was subdivided into the three regions proposed by Gómez-Palacio et al. (2008) according to the frequencies of the A and B cytotypes: the Colombia-North (98%

cytotype A), Colombia-South (100% cytotype B), and ColombiaWest and Panama-West regions (30% and 71% cytotype B, respectively). These geographic regions included different ecological zones according to the Terrestrial Ecoregions System proposed by the World Wildlife Foundation (WWF) (Olson et al., 2001) that were used as the biogeographical location of the populations analyzed (Table 1). Details on the biogeographical origin and number of individuals analyzed per population for each technique are shown in Table 1 and Fig. 1. Most of insects used in this study were previously analyzed using cytogenetic techniques and classified into the two chromosomal groups (or cytotypes) according to the number of heterochromatic autosomes: Group A, 4–12 autosomes with C-blocks, and Group B, 14–16 autosomes with C-blocks (Gómez-Palacio et al., 2008). 2.2. Geometric morphometric analysis Right wings were dissected and mounted using standard techniques as reported elsewhere (Ayala et al., 2011), and photographed with a Nikon 990 digital camera fitted to a Nikon SMS 800 stereomicroscope. The wings were placed in the center of the visual field to reduce the risk of optical distortion. Ten landmarks were selected and the geometric coordinates of each landmark were digitalized using tpsDIG version 2.16 (Rohlf, 2010). To compare wing size among groups, the ‘‘centroid-size’’ was used as isometric estimator of overall size derived from coordinate data (Bookstein, 1996). It is defined as the square root of the sum of squared distances between the center of the configuration of landmarks and each individual landmark (Bookstein, 1991). Differences in size were tested by ANOVA and a post-hoc pairwise comparison was performed, based on Tukey’s HSD (honestly significant difference) test, when ANOVA showed significant inequality of the means. The Generalized Procrustes Analysis (GPA) superimposition algorithm implemented in the tpsRelw v.1.11 (Rohlf, 1997) was used to obtain the wing shape variables. Shape variation was analyzed using the principal components of shape variables or relative warps (Rohlf, 1997). Differences in shape were tested by MANOVA. A post-hoc analysis by a pairwise Hotelling test (with Bonferroni corrections) was performed when the MANOVA showed a significant overall difference between groups. A principal component analysis was performed to produce a scatter plot of specimens

Table 1 Biogeographical origin according to Olson et al. (2001) and summary of R. pallescens populations from Colombia and Panama analyzed in this study. Country

Region (ecoregion)

State/locality-abbreviation (number in map)

Colombia

North (Magdalena Uraba moist forest)

Magdalena/San Sebastián de Buenavista-SSb (1) Magdalena/San Zenón- Sze (2) Bolívar/Mompós-Mom (3) Bolívar/San Fernando-Sfe (4) Córdoba/San Bernardo del Viento-SBv (5) Sucre/Galeras-Gl (6) Cesar/Aguachica-Agu (7) Sucre/San Onofre-SO (8) Antioquia/Vegachi-Veg (9) Caldas/Norcasia-Nor (10) Santander/San Vicente del Chucurí-Svi (11) Santander/Bucaramanga-Bug (12) Norte de Santander/El Carmen-Elc (13) Antioquia/Necoclí –Nec (14) Antioquia/Turbo-Tur (15) Chocó/Acandí-Aca (16) Estado de Panamá/Chepo –Che (17) Estado de Panamá/La Chorrera-Cho (18) Veraguas/Santa Fe-SF (19)

North (Guajira Barranquilla xeric scrub) South (Magdalena Valley montane forest)

South (Catatumbo moist forest) West (South Caribbean mangroves)

Panama

West (Isthmian-Atlantic moist forests) West (Talamancan montane forest)

Total

Number of individuals studied by Morphometry

cyt b

Flow cytometry

20 0 3 0 4 3 0 2 5 15 0 0 0 10 0 0 0 0 31 93

3 2 4 1 1 0 4 0 4 4 3 2 2 3 2 5 4 3 5 52

16 0 0 0 0 0 0 0 6 6 0 0 0 5 0 0 0 0 18 51

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Fig. 1. Map of the biogeographical location of Rhodnius pallescens populations from Colombia and Panama analyzed in this study. Circles (d): Colombia-North region; squares (j): Colombia-West region; pentagons ( ): Panama-West; triangles (N): Colombia-South region. Numbers on localities and their respective ecological zone is described in Table 1.

along the first two component axes, producing maximal and second to maximal separation between all groups. Additionally, a discriminant analysis (DA) reclassification test based on the first two discriminant functions (DF) to each observation was designed and the Mahalanobis distances among the shape’s centroids for each pair of population groups (Colombia-North, Colombia-South, Colombia-West, and Panama-West; see Table 1) were compared. All the computational statistics were performed using the free software PAST v.1.94b (Hammer et al., 2001), and the statistical significance of pairwise comparisons was tested by a null model using 1000 permutations with PADwin v.81a (Dujardin, 2006). 2.3. DNA sequence analyses Genomic DNA was extracted from legs of individual insects as described elsewhere (Lyman et al., 1999). For each specimen, a 682-bp fragment of the cyt b gene was PCR-amplified using primers 7432F (50 -GGACGWGGWATTTATTATGGATC-30 ) and 7433R (50 -GCWCCAATTCARGTTARTAA-30 ) (Monteiro et al., 2003). PCR reactions were conducted in a final volume of 35 ll using 2 ll of 50-ng/ll templates DNA, 3.5 ll of 10X PCR buffer (0.1 M Tris– HCl, 0.5 M KCl, and 0.015 M MgCl2, pH 8.3), 4.5 ll of 2-mM dNTP, 1.5 ll of each 10-lM primer, 3.5 ll of 50 mM MgCl2 and 1 U/ll of Taq DNA polymerase (FermentasÒ). After an initial denaturation of 95 °C for 5 min, PCR reactions were 35 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 60 s, followed by a final extension of 72 °C for 10 min. The PCR products were sent to Macrogen Inc., Korea for DNA purification and sequencing service. For all samples, sequencing was conducted in both forward and reverse directions. Forward and reverse sequences from specimens were used to generate a consensus sequence with a previously pairwise alignment by CLUSTAL W algorithm (Thompson et al., 1997) implemented in Bioedit v. 7.0.5 (Hall, 1999). To compare among sequences groups, we evaluated the number of segregating sites (S), nucleotide diversity (p), number of haplotypes (h) and

haplotype diversity (Hd) using DnaSP v.5.10 (Librado and Rozas, 2009). The genetic differentiation among populations groups both at nucleotide sequence and haplotype level was estimated by Hudson´s statistics Kst and Hst (Hudson et al., 1992), defining the statistical significance (p < 0.001) with a permutation test of 1.000 replicates using in DnaSP v.5.10 (Librado and Rozas, 2009). Also, genetic distances among population groups were estimated according to the best-fit substitution model based on the Akaike information criterion (AIC) (Akaike, 1974) implemented in MEGA v.5.04 (Tamura et al., 2011). Spatial analysis of molecular variance (SAMOVA) (Dupanloup et al., 2002) was performed to estimate the structure among population groups according to their geographical distances by Fct statistical calculations using SAMOVA v.1.0 (Dupanloup et al., 2002). Fct values were estimated for simulated population groups from k = 2 to k = 4 in 1000 iterations of the data set, which corresponds to the minimum number of populations groups between Colombia and Panama (k = 2) until number of populations groups suggested in this work (k = 4). Neighbor-joining (NJ) and maximum likelihood (ML) phylogenetic analyses were implemented in MEGA v.5.04 (Tamura et al., 2011) with a statistical support for tree topology obtained by bootstrapping (Efron et al., 1996) using 1000 iterations. NJ and ML trees were performed based on the best-fitted nucleotide substitution model estimated as mentioned above. To build the ML topology, the nearest-neighbor-interchange (NNI) heuristic search method was used and the estimated NJ tree was used as the initial tree. Topologies were adjusted using Figure Tree v.1.3.1 software (http://tree.bio.ed.ac.uk/). Finally, a median joining (MJ) haplotype network was used to examine interhaplotype relationships using Network 4.6.0.0 software (http://www.fluxus-engineering.com). The MJ method is based on the genetic distance among haplotypes linked by the number of mutation steps. Under a parsimony criterion, hypothetical median vectors (which represent missing intermediates) are

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added to the network in sequential steps from more internal haplotypes to external or tip haplotypes. The MJ method handles large data sets and multistate characters in non-recombining biomolecules (i.e., mtDNA) and is an exceptionally fast method that can analyze thousands of haplotypes in a reasonable amount of time. To build a haplotype network, default parameters were used (equal character weight = 10; epsilon value = 10; transversions/transitions weight = 1:1 and connection cost as a criterion) and to eliminate unnecessary median vectors and links, a maximum parsimony post-processing was conducted using Network 4.6.0.0 software (http://www.fluxus-engineering.com). 2.4. Genome size in R. pallescens cytotypes The total nuclear DNA content was measured from gonadal cells of male specimens of each R. pallescens population group and cytotype: North (cytotype A, n = 16), South (cytotype B, n = 12), Colombia-West (cytotype A, n = 4 and cytotype B, n = 1) and Panama-West (cytotype A, n = 4 and cytotype B, n = 14) following conditions and procedures described by Panzera et al. (2007). The cell DNA content was measured on an EPICS XL-MCL flow cytometer (Coulter Electronics, Hialeah, FL, USA) with an air-cooled argon-ion laser tuned to 488 nm and 15 mW. Propidium fluorescence (FL3), proportional to DNA content, was collected through a 650-nm DL dichroic filter fitted with a 625-nm BP band-pass filter. The DNA content in single cells was determined from FL3 linear histograms. For each sample, information for a minimum of 10,000 nuclear events was acquired using the System II software program (Beckman Coulter Inc., Brea, CA, USA). To evaluate the DNA content in picograms of DNA, a sample of normal human lymphocytes was fixed in ethanol/acetic acid and used as the standard reference

(2C = 7.0 pg of DNA according to the Animal Genome Size Database (http://www.genomesize.com/)). The absolute DNA amount was calculated with the ratio of the mean channel of the insect haploid peak to the mean channel of the human lymphocyte diploid G0/G1 peak. 3. Results 3.1. Wing size and shape variation Significant differences in size were detected among all populations (F = 106.6, p < 0.001). After a Tukey HSD post-hoc test, the smallest insects from Colombia-North were differentiated from the largest ones from the Colombia-South and Panama-West regions, and from the intermediate population from Colombia-West. No significant differences in size were detected between the largest insects from Panama-West and Colombia-South (Fig. 2 and Table 2). The first two axes of relative warps explained 23.3% and 18.4% of the variation, respectively, corresponding to 41.7% of the total variation of the specimens’ wing shape, dismissing the use of other axes. The MANOVA test allowed detecting significant differences in shape among all populations (F = 14.36, p < 0.001). After Hotelling post-hoc analysis, Bonferroni corrected, significant differences in shape were detected between populations from Panama West and all the populations from Colombia. Also, shape differences were detected between Colombia-North and Colombia-South populations (Fig. 3 and Table 3). Reclassification of specimens based on all discriminant factors resulted in the correct classification of 92.47% of the individuals to their attributed region of origin. Reclassification was perfect for the individuals from Panama-West (100%), followed by Colombia-South (95%), Colombia-North (87%), and finally those from the Colombia-West region (80%). These results were consistent with Mahalanobis distance pairwise comparisons, which were significantly different among all four regions studied (Table 4). 3.2. DNA Sequence analysis

Fig. 2. Wing size differences in specimens from four R. pallescens populations groups. The arithmetic median can be observed as a line that divides the boxes into two. The ends of the boxes correspond to the 25% and 75% quintiles; the vertical lines show the maximum and minimum value of the distribution. The localities are presented in Fig. 1.

The mt cyt b 682-bp fragment base composition was A-T rich (68.9%) and no insertions or deletions were present in any sequence, as expected for protein-coding insect mitochondrial DNA (DeSalle et al., 1987). The 66 variable nucleotide positions detected represent 9.68% of the total number of sites. With the exception of the specimens from the Vegachí locality (Colombia-South region) and those from the Aguachica locality (Colombia-North region), conserved nucleotide substitutions were observed among individuals from the attributed regions⁄⁄ (Table S1). Gene diversity showed the lowest nucleotide diversity value in Colombia-West and the highest values in Colombia-South and Panama-West

Table 2 ANOVA and Tukey HSD pairwise comparisons based on wing size for R. pallescens populations from the Colombia-North, Colombia-South, Colombia-West, and Panama-West regions. Bold values indicate significant differences (p < 0.001).

ANOVA Among regions Within regions Total

Sum of sqrs

df

Mean square

F

p-Value

54.9464 15.2918 70.2382 Colombia-North

3 89 92 Colombia-South

18.3155 0.171818

106.6

2.313E29

Colombia-West

Panama-West

0.0001445 –

0.00015 0.0001445 –

0.0001445 0.503 0.0001445 –

Tukey’sHSD (honestly significant difference) test Colombia-North – Colombia-South Colombia-West Panama-West

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Table 4 Mahalanobis distances derived from wing shape analysis among R. pallescens populations.

Colombia-North Colombia-South Colombia-West *

Colombia-South

Colombia-West

Panama-West

3.42*

3.23* 3.37*

3.89* 6.11* 4.94*

p < 0.05.

Table 5 Summary of genetic diversity indices and pairwise comparison based on haplotype sequences (Hst above diagonal) and nucleotide sequences (Kst below the diagonal) of the cyt b (682-bp) gene segment in R. pallescens populations from the ColombiaNorth, Colombia-South, Colombia-West, and Panama-West regions. Notations: N: number of localities; n: number of cyt b sequences; S: site variables; p (±SD): nucleotide diversity (standard deviation); h: number of haplotypes and Hd (±SD): Haplotype diversity (standard deviation); ns: not significant and ⁄⁄⁄p < 0.001.

Fig. 3. Morphometric analysis of R. pallescens male wings. Differences in shape among the R. pallescens populations are shown. The polygons represent the shape of the population projected on the first (x-axis) and second relative warp (y-axis), which were derived from a relative warp analysis. RW1 explains 23.3% of the variance while RW2 explains 18.4%. For easy visualization of populations, lines connect the most external individuals of each population (for details see Table 1).

(Table 5). Moreover, haplotype diversity was similarly high in Colombia-North and Colombia-South but not in Colombia-West and Panama-West populations (Table 5). Hudson’s statistics of nucleotide diversity (Kst = 0.654; p > 0.001) as well as haplotype diversity (Hst = 0.201; p < 0.001) among all population groups were significant, indicating a high gene differentiation. However, pairwise comparisons showed low and no significant Hst and Kst values between Colombia-North and Colombia-South population groups, and Kst no significant difference between the ColombiaWest and Panama-West population groups (Table 5). After the Akaike criterion, T92 plus Gamma (T92 + G) distribution was the best-fit nucleotide substitution model and therefore it was selected to estimate average genetic distances among population groups. Thus T92 + G-based genetic distances indicated that the greatest genetic distances were estimated between ColombiaNorth and both Panama-West and Colombia-West (d = 0.063 similar in both), and Colombia-South and both Panama-West and Colombia-West populations (d = 0.061 and 0.059, respectively). The lowest genetic distances were estimated between the Colombia-North and Colombia-South (d = 0.013) populations, and then between the Colombia-West and Panama-West (d = 0.016) populations. SAMOVA analysis showed a maximized Fct value associated to population structure in k = 4 (Fct = 0.874, p < 0.001). These groups were moderately consistent with the four proposed regions of

Region

N

n

S

p (± SD)

h

Hd (± SD)

Gene diversity Colombia-North Colombia-South Colombia-West Panama-West Total

6 5 3 3 17

15 15 10 12 52

18 24 8 24 66

0.009 0.013 0.005 0.018 0.033

9 8 2 4 22

0.848 0.876 0.467 0.636 0.924

ColombiaNorth

(0.002) (0.001) (0.002) (0.003) (0.001)

ColombiaSouth

ColombiaWest

(0.088) (0.060) (0.132) (0.128) (0.020)

PanamaWest

Haplotype-based (Hst)/Nucleotide-based (Kst) differentiation statistics Colombia-North 0.018 ns 0.189⁄⁄⁄ 0.147⁄⁄⁄ Colombia-South 0.072 ns 0.179⁄⁄⁄ 0.137⁄⁄⁄ Colombia-West 0.758⁄⁄⁄ 0.689⁄⁄⁄ 0.292⁄⁄⁄ Panama-West 0.622⁄⁄⁄ 0.558⁄⁄⁄ 0.137 ns

Colombia-North, Colombia-South, Colombia-West and PanamaWest, excepting those from Chepo and La Chorrera (Panama-West) and Vegachi (in the Colombia-South region), which were grouped with those of Colombia-West and Colombia-North regions, respectively (see Table 1). The NJ and ML phylogenetic trees showed identical topologies and bootstrap values (Fig. 4), supporting nucleotide sequences clearly separated into two main groups corresponding to the Colombia-North and the Colombia-South clade, and the Colombia-West and Panama-West clade. Secondary clades were also observed, corresponding to the Colombia-North region (excluding those from the Aguachica locality) and including haplotypes from the Vegachí and El Carmen localities (both in the Colombia-South region), and two clades that included the Panama-West and Colombia-West populations. A similar result was detected in the haplotype network, indicating a clear differentiation due to more than 17 mutation steps among haplotypes from Colombia-North and Colombia-South with Colombia-West and Panama-West regions (Fig. 5). As in phylogenetic trees, in the haplotype network haplotypes from the Vegachi locality were clustered into the

Table 3 MANOVA and Hotelling pairwise comparisons based on shape variation for R. pallescens populations from the Colombia-North, Colombia-South, Colombia-West, and PanamaWest regions. Hotelling p-values are given above the diagonal, while Bonferroni corrected values are given below the diagonal. Bold values indicate significant differences (p < 0.001). Wilk’s Lamda

df1

df2

F

p

MANOVA 0.01483

48

220.9

14.36

8.676 E46

Colombia-South

Colombia-West

Panama-West

4.20E07 – 0.0705457 2.63E14

0.0186272 0.0117576 – 7.98E06

8.90E10 4.38E15 1.33E06 –

Colombia-North Hotelling’s pairwise comparisons, Bonferroni corrected Colombia-North – Colombia-South 2.52E06 Colombia-West 0.111763 Panama-West 5.34E09

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Fig. 4. Deduced ML phylogenetic tree obtained for individuals of R. pallescens (n = 22) from Colombia and Panama based on analysis of a 682-bp segment of the mt cyt b gene. The bold numbers represent the bootstrap values supporting each clade in the NJ tree and ML. R. pictipes and R. prolixus (Accession numbers FJ887792 and EF011723.1, respectively) species were included as an outgroup. Taxon names are described in Table 1.

Colombia-North region; haplotypes from Aguachica were grouped in the Colombia-South region and those from Chepo and La Chorrera were connected to the Colombia-West haplotypes. 3.3. Genome size in R. pallescens cytotypes A DNA flow cytometric profile was obtained for each sample (data not shown). All samples studied presented a haploid DNA content (C value) of 0.73 ± 0.046 pg. No significant differences in DNA content were observed between the two R. pallescens cytotypes, or among regions (p > 0.05). 4. Discussion The genetic and morphological evidence presented here indicates that R. pallescens is divided into two evolutionary lineages

with different geographic distributions: Colombian (including populations of Colombia-North and Colombia-South) and Central American lineages (including Colombia-West and Panama-West). In turn, intralineage variability related to the genetic and morphological differences between them were also observed, which indicates that biological diversity in R. pallescens could be a consequence of both evolutionary lineage diversification and environmental attributes of their biogeographical region of origin. Therefore, different evolutionary rates in the chromosomal, molecular, and wing geometry traits of R. pallescens must be analyzed taking into account its geographical dispersion, local environmental conditions, as well as its population structure. The Colombian lineage is distributed from the central inter-Andean valleys to the northern Caribbean plains, occupying diverse ecological zones ranging from dry and warm zones, near sea level (for instance in the Guajira Barranquilla xeric scrub ecological zone), and across several moist forests dispersed from the wide

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1981

Fig. 5. Median joining haplotype network of R. pallescens population groups from Colombia and Panama. Node size is proportional to haplotype frequencies, and nine hypothetical haplotypes or median vectors are indicated as mv1-9. Numbers between connected haplotypes are the mutated positions. Bold numbers summarize mutated steps among each region’s haplogroups. Cross-fit nodes represent Colombia-North haplotypes; solid black Colombia-South; hatched Colombia-West, and solid gray PanamaWest haplotypes. ⁄Indicates haplotypes from the Chepo and La Chorrera localities; ⁄⁄indicates haplotypes from the Vegachí locality and ⁄⁄⁄haplotypes from the Aguachica locality (see Table 1 for details).

Caribbean plain to the humid zones in premontane and montane Andean valley ecosystems in central Colombia (southern limit for the species known so far). Thus, the Colombian lineage was found at altitudes ranging from 3 m above sea level (masl) such as in the San Bernardo del Viento locality to 1009 masl such as in the Bucaramanga locality, which indicates in turn that great environmental differences can be involved in genetic and morphological variation among populations. On the other hand, the Central American lineage is distributed from around of the Gulf of Urabá in Colombia (called here the Colombia-West region) associated with hyper-humid zones such as the ecological zones of the South Caribbean Mangroves and Isthmian-Atlantic moist forests to the Talamancan mountain forest in Costa Rica and Panama. Similar to the Colombian lineage, the Central American lineage could occupy broad ecosystems, including human dwellings, as a consequence of rural landscape use (Gottdenker et al., 2011). Heterogeneous environmental factors as well as the wide geographical distribution of this lineage could involve the intralineage differentiation detected here. Cytogenetic analysis in Colombian lineage R. pallescens populations suggests a cytotype differentiation between individuals from northern and southern populations, and indicates that the amount of the constitutive heterochromatin may be directly proportional to altitude and related to individuals’ ecological zone of origin (Gómez-Palacio et al., 2008). This hypothesis has been suggested for other triatomine species such as T. infestans, with the amount of heterochromatin seemingly higher in individuals from Andean highland populations than those from non-Andean lowlands (Panzera et al., 2004a). However, unlike T. infestans, in R. pallescens the variation of autosomal heterochromatin between cytotypes A and B did not show a statistically significant difference in DNA content (which is reported for first time in R. pallescens), indicating perhaps the differences in the autosomal heterochromatin between the two cytotypes are too small to be detected by flow cytometry DNA quantification. On the other hand, the presence of both cytotypes in two populations of the Central America lineage in different frequencies was suggested as evidence of a heterogeneous mixture of these

northern and southern Colombian forms, and two alternative hypotheses were proposed to explain this structure: a possible hybrid zone or a dispersed origin of the species (Gómez-Palacio et al., 2008). The results indicate that a third explanation can be postulated and that the variation in the amount of autosome heterochromatin in R. pallescens must be taken more as a reflection of local environmental factors such as elevation and ecological zones than as an trait related with its phylogeographical pattern (Abad-Franch and Monteiro, 2005). A lack of correspondence between the amount of autosomal heterochromatin and molecular-based phylogenetic inferences in Triatominae has been detected in other species. For instance, in T. dimidiata, chromosomal studies identified three possible cryptic species (Panzera et al., 2006), but only two of them were also supported by nuclear (ITS-2) (Bargues et al., 2008) and mitochondrial markers (cyt b) (Dorn et al., 2009). Similarly, in T. infestans, the sylvatic melanic form known as the ‘‘dark morph’’ of the Bolivian Chaco (Noireau et al., 1997) was included within the non-Andean genetic group (low DNA content) by cytogenetic and nuclear DNA sequence analyses (Bargues et al., 2006; Noireau et al., 2000; Panzera et al., 2004a), while cyt b sequence studies showed that these individuals are more phylogenetically related to the Bolivian highlands, which belong to the Andean genetic group (high DNA content) (Monteiro et al., 1999; Quisberth et al., 2011; Waleckx et al., 2011). Thus autosomal heterochromatin variations in R. pallescens seem to be associated with environmental factors, such as altitude or ecological zones (Gómez-Palacio et al., 2008; Panzera et al., 2004b), while cyt b reflects evolutionary inter- and intralineage divergence. Cyt b molecular analyses in part supported differentiation between northern and southern Colombian populations. Phylogenetic topologies, haplotype network results, as well as SAMOVA showed moderate differentiation between them; however, similar gene diversity values, the lowest genetic distance, as well as a nonsignificant population structure based on nucleotide and haplotypes (Hst and Kst) suggests that no clear genetic or geographical barriers separate them and possible intermediate populations can be found in these geographical areas. For instance, the haplotypes

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from the Vegachi and El Carmen localities (located above 700 masl) and the Aguachica locality (161 masl) were not grouped with their respective assigned Colombia-South and Colombia-North region. Similarly, within the Central America lineage two apparent population groups were detected on molecular analyses: one integrated by Colombia-West populations plus the Chepo and La Chorrera Panamian localities and the other only by the Santa Fe locality. However additional Central American populations (i.e., from Costa Rica and northern Panama) must be included in the Central American lineage population differentiation analyses. Moderated intralineage differentiation was also detected at the morphometric level. As has been suggested for other Triatominae species, wing geometry is a quantitative trait, and its polymorphism is expected to respond to both environmental and genetic variation (Dujardin et al., 2007, 2009, 1999b). Thus we suggest that morphological diversity detected in R. pallescens is a consequence of both evolutionary lineage diversification and environmental attributes of their biogeographical region of origin, and therefore both aspects should be taken into account to further its systematic study. In addition, the wing size variation of R. pallescens described here is consistent with Bergmann’s rule: a positive correlation between the intraspecific body size and latitude, altitude, and climate, originally formulated for endothermic organisms, but also reported in ectotherms such as insects (Arnett and Gotelli, 1999; Marcondes et al., 1998; Partridge et al., 1994). Among conspecific populations of triatomines, as in other insects, this rule has been mainly related to temperature and elevation (Ayala et al., 2011; Dujardin et al., 2009). Thus, R. pallescens wing size analysis indicated that the smaller individuals were those from the ColombiaNorth region, individuals from the Colombia-South region (similar to those from Panama-West region) had the largest wing size, and Colombia-West individuals showed intermediate size. In Triatomiae, it has been shown that body size is larger for insects collected in sylvatic conditions compared to those cultivated in controlled conditions in the lab (Riaño et al., 2009), and there is considerable evidence that wing size and shape are targets of natural selection (Ayala et al., 2011; Soto et al., 2006). Also, it is well known that in Triatominae there is a strong sexual size dimorphism, with females larger than males (Lent and Wygodzinsky, 1979). Moreover, wing size has been used to describe this characteristic in many Triatominae species (Dujardin et al., 1999a; Gurgel-Gonçalves et al., 2011; Jaramillo-Ocampo et al., 2002). Finally, the hypothesis of the influence of evolutionary and biotic factors in the differentiation of R. pallescens should be tested on the basis of a phylogeographic and population structure analysis, with additional both nuclear and mitochondrial genetic markers often used in insects. Additionally, for a clear demarcation of its biogeographical distribution and knowledge of its evolutionary trends, geographical dispersion and adaptive radiation, additional analyses within and between R. pallescens lineages are necessary, including other populations, mainly from Central America and southern Colombian distribution.

5. Conclusion This work is a first approach to intraspecific variation of R. pallescens. The results suggest this species shows two evolutionary lineages within which a complex of moderated divergent population groups exists, evidenced by molecular and morphometric analyses related with their biogeographical origin. The Colombian lineage seems to be subdivided into a north and south population group cline where altitudinal and ecological zones could act in the morphological, chromosomal, and molecular differentiation detected. The Central American lineage seems to show similar

intralineage division but integrated by populations from the Colombia and Panama border to a northern distribution of the Central America distribution. However, we consider additional Central America populations must be analyzed by an elicited reliable intralineage subdivision. Therefore, we conclude that the differences in chromosomal, mitochondrial, and morphometric rates of change shown among R. pallescens of Colombia and Panama unveils a complex process of intraspecific differentiation, in which lineage evolution as well as biogeographical factors play an essential role. However, we consider that R. pallescens systematics and its relationship with ecoedpimiological attributes of lineages as well as within them still requires further clarification to access the biological causes that underlie the apparent heterogeneity in its epidemiological relevance as a Chagas vector. Acknowledgements Special thanks are extended to Drs. Azael Saldaña, José Calzada, and Lorenzo Caceres from Instituto Conmemorativo Gorgas de Estudios de la Salud (ICGES), Panama City, Panama, for their generous provision of Panamian R. pallescens individuals used in this study. This work was funded by the ECLAT network, CSIC and PEDECIBA from Uruguay, European Community (Project Contract No. CDIA-ICA4-CT-2003-10049 and ATU-SSA-CT-2004-515942) and by CODI (project CPT-0909), Universidad de Antioquia, Medellin, Colombia. Finally we want thank to reviewers by their kindly and pertinent comments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2012. 04.003. These data include Google maps of the most important areas described in this article. References Abad-Franch, F., Monteiro, F., 2005. Molecular research and the control of chagas disease vectors. Annals of the Brazilian Academy of Sciences 77, 437–454. Akaike, H., 1974. A new look at the statistical model identification. IEEE Transactions on Automatic Control 19, 716–723. Arnett, A.E., Gotelli, N.J., 1999. Bergmann’s rule in the ant lion Myrmeleon immaculatus DeGeer (Neuroptera: Myrmeleontidae): geographic variation in body size and heterozygosity. Journal of Biogeography 26, 275–283. Ayala, D., Caro-Riaño, H., Dujardin, J.P., Rahola, N., Simard, F., Fontenille, D., 2011. Chromosomal and environmental determinants of morphometric variation in natural populations of the malaria vector Anopheles funestus in Cameroon. Infect. Genet. Evol. 11, 940–947. Bargues, M.D., Klisiowicz, D.R., Gonzalez-Candelas, F., Ramsey, J.M., Monroy, C., Ponce, C., Salazar-Schettino, P.M., Panzera, F., Abad-Franch, F., Sousa, O.E., Schofield, C.J., Dujardin, J.P., Guhl, F., Mas-Coma, S., 2008. Phylogeography and genetic variation of Triatoma dimidiata, the main Chagas disease vector in Central America, and its position within the genus Triatoma. PLoS Negl. Trop. Dis. 2, e233. Bargues, M.D., Klisiowicz, D.R., Panzera, F., Noireau, F., Marcilla, A., Perez, R., Rojas, M.G., O’Connor, J.E., Gonzalez-Candelas, F., Galvão, C., Jurberg, J., Carcavallo, R.U., Dujardin, J.P., Mas-Coma, S., 2006. Origin and phylogeography of the Chagas disease main vector Triatoma infestans based on nuclear rDNA sequences and genome size. Infect. Genet. Evol. 6, 46–62. Bookstein, F.L., 1991. Morphometric Tools for Landmark Data: Geometry and Biology. Cambridge University Press, New York. Bookstein, F.L., 1996. Biometrics, biomathematics and the morphometric synthesis. Bull. Math. Biol. 58, 313–365. Calzada, J.E., Pineda, V., Garisto, J.D., Samudio, F., Santamaria, A.M., Saldaña, A., 2010. Human trypanosomiasis in the eastern region of the Panama Province. new endemic areas for Chagas disease. Am. J. Trop. Med. Hyg. 82, 580–582. Calzada, J.E., Pineda, V., Montalvo, E., Alvarez, D., Santamaría, A.M., Samudio, F., Bayard, V., Cáceres, L., Saldaña, A., 2006. Human trypanosome infection and the presence of intradomicile Rhodnius pallescens in the western border of the Panama Canal, Panama. Am. J. Trop. Med. Hyg. 74, 762–765. Cantillo-Barraza, O., Gómez-Palacio, A., Salazar, D., Mejía-Jaramillo, A.M., Calle, J., Triana, O., 2010. Distribution and ecoepidemiology of the triatomine fauna (Hemiptera: Reduviidae) in Margarita Island, Bolívar, Colombia. Biomedica 30, 382–389.

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