Morphological and phylogenetic analyses of Lutzomyia migonei from three Brazilian states

Accepted Manuscript Title: Morphological and phylogenetic analysis of Lutzomyia migonei from three Brazilian states Authors: Pietra Lemos Costa, Reginaldo Pec¸anha Brazil, Andressa Alencastre Fuzari, Maria Stefania Latrofa, Giada Annoscia, Viviana Domenica Tarallo, Gioia Capelli, Domenico Otranto, Sinval Pinto Brand˜ao-Filho, Filipe Dantas-Torres PII: DOI: Reference:

S0001-706X(17)31429-8 https://doi.org/10.1016/j.actatropica.2018.07.027 ACTROP 4732

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

30-11-2017 8-7-2018 24-7-2018

Please cite this article as: Costa PL, Brazil RP, Fuzari AA, Latrofa MS, Annoscia G, Tarallo VD, Capelli G, Otranto D, Brand˜ao-Filho SP, Dantas-Torres F, Morphological and phylogenetic analysis of Lutzomyia migonei from three Brazilian states, Acta Tropica (2018), https://doi.org/10.1016/j.actatropica.2018.07.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Morphological and phylogenetic analysis of Lutzomyia migonei from three Brazilian States

Pietra Lemos Costaa, Reginaldo Peçanha Brazilb, Andressa Alencastre Fuzarib, Maria

Domenico Otrantoc, Sinval Pinto Brandão-Filhoa, Filipe Dantas-Torresa,c,*

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Stefania Latrofac, Giada Annosciac, Viviana Domenica Taralloc, Gioia Capellid,

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* Corresponding author at: Department of Immunology, Aggeu Magalhães Institute,

Oswaldo Cruz Foundation (Fiocruz), Av. Prof. Moraes Rego s/n, 50740465 Recife,

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Pernambuco, Brazil.

Departamento de Imunologia, Instituto Aggeu Magalhães, Fiocruz, Recife,

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E-mail address: [email protected] (F. Dantas-Torres).

Pernambuco, Brazil

Laboratório de Doenças Parasitárias, Instituto Oswaldo Cruz, Fiocruz, Rio de

Janeiro, Brazil

Dipartimento di Medicina Veterinaria, Università degli Studi di Bari “Aldo Moro”,

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b

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Valenzano, Bari, Italy d

Laboratorio di Parassitologia, Istituto Zooprofilattico Sperimentale delle Venezie,

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Legnaro, Padova, Italy

Highlights

Lutzomyia migonei is a vector of Leishmania braziliensis in South America;

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The existence of isolated populations of L. migonei has been hypothesized.

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Morphological analysis indicated the existence of two distinct populations;

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Molecular analysis also indicated the existence of two monophyletic clades;

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We suggest the existence of two different populations of L. migonei in Brazil.

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Abstract

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Lutzomyia migonei is incriminated as a vector of Leishmania braziliensis, the main

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causative agent of cutaneous leishmaniasis in Brazil. Recently, this phlebotomine sand

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fly species has been suggested as a vector for Leishmania infantum, which causes

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zoonotic visceral leishmaniasis. Considering the widespread distribution of Lu. migonei in South America, the existence of isolated populations has been hypothesized. Three Lu.

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migonei populations, two from north-eastern Brazil (Machados, Pernambuco State, and Baturité, Ceará State) and other from the south-eastern region (Niterói, Rio de Janeiro

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State) were analysed both morphologically and genetically. Though no significant

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morphological differences were found amongst the sand fly specimens analysed, discriminant analysis based on specific morphometric characters (i.e., length of wing, antennal segment 3 and coxite for males, and length of wing and antennal segment 3 for

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females), showed that specimens from Machados were closer to Baturité than to Niterói. The molecular analysis of cytochrome c oxidase subunit I gene sequences also supported this observation by the distinct separation of two monophyletic clades, grouping specimens from Machados and Baturité separately from those of Niterói. Our results suggest the existence of different populations within the distribution range of Lu. migonei.

Whether these populations are reproductively isolated and/or present differences in terms of vector competence/capacity for L. braziliensis and L. infantum needs to be further investigated.

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Keywords: Migonemyia, cytochrome c oxidase subunit I gene, genetic variability.

1. Introduction

Phlebotomine sand flies are vectors of several disease agents, including protozoan

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parasites of the genus Leishmania, which may cause a group of diseases generally referred

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to as leishmaniasis. Out of ~1,000 sand fly species known to date, less than 100 have been

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humans (Maroli et al., 2013).

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regarded as proven or putative vectors of Leishmania spp. causing disease in animals and

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Studies have demonstrated that some sand fly species display remarkable specificity for the Leishmania spp. that they transmit and are referred to ‘specific vectors’. This is

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the case of Phlebotomus papatasi and Phlebotomus sergenti, the vectors of Leishmania major and Leishmania tropica, respectively (Kamhawi, 2006). On the other hand, most

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sand fly vectors tested to date support the development of multiple Leishmania spp. and are referred to as ‘permissive vectors’ (Volf and Myskova, 2007). As an example,

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Phlebotomus arabicus is a vector of L. tropica (Jacobson et al., 2003), but is highly susceptible to both L. major and Leishmania infantum (Myskova et al., 2007). Incidentally, L. infantum is a species that may develop in different sand fly species. In the Neotropical region, this parasite is primarily transmitted by Lutzomyia longipalpis, although other sand fly species have been regarded as putative vectors in different

countries (Brazil et al., 2015). For instance, a recent study has demonstrated the development of metacyclic promastigotes (infective forms) of L. infantum in Lutzomyia migonei (Guimarães et al., 2016), confirming the role of this sand fly as a permissive vector. Lutzomyia migonei is also considered to be a vector of Leishmania braziliensis, based on both field (Queiroz et al., 1994) and laboratory evidence (Nieves and Pimenta,

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2000). This sand fly is widespread in South America, occurring in Argentina, Bolivia, Brazil, Colombia, Paraguay, Peru, Trinidad and Tobago, and Venezuela (Shimabukuro et

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al., 2017).

The occurrence of differentiation and speciation due to geographical isolation and local adaptation has been hypothesized for different sand fly species, including Lu.

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migonei (Vigoder et al., 2010a). However, to our knowledge, no morphological,

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morphometric or molecular analyses have been provided to assess the existence of

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different Lu. migonei populations in South America. In this context, we assessed

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geographical locations in Brazil.

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morphologically and genetically three populations of Lu. migonei from different

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2. Materials and methods 2.1. Sand fly collection

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Lutzomyia migonei were collected in three different municipalities in Brazil (Fig. 1):

Machados, Pernambuco State (north-eastern Brazil) (07º41’09”S, 35º30’54”W); Baturité,

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Ceará State (north-eastern Brazil) (04º19’44”S, 38º53’06”W), and Niterói, Rio de Janeiro State (south-eastern Brazil) (22º52’58”S, 43º06’14”W). Machados is a highland, situated 416 metres above the sea level. It possesses a tropical climate (As type) and most of its original vegetation coverage has been replaced by crop plantations, particularly banana

trees. The average annual temperature is 23.5C and average annual precipitation is 1,197 mm. Baturité is situated 175 metres above the sea level and the climate is tropical (Aw type). The hills are mainly covered by Atlantic forest remnants, surrounded by caatinga. In Baturité, Lu. migonei is more abundant in high areas of the hill, although it is also

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collected in the caatinga (Queiroz et al., 1994). The average annual temperature is 25.8C and average annual precipitation is 1,065 mm.

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Niterói is situated at the sea level and has also a tropical climate (Aw type). The natural landscape is mainly composed by Atlantic forest remnants, also with some areas

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of resting forests and mangroves. The average annual temperature is 23.4C and average

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annual precipitation is 1,204 mm.

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In the collection sites, most of the primary vegetation coverage has been replaced by

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crop plantations, human houses and animal shelters. From October 2015 to February 2016, phlebotomine sand flies were collected using standard CDC light traps, installed

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monthly, for four consecutive nights, between dusk and dawn, from 5:00 pm to 6:00 am. Traps were placed at ca. 1 m above the ground in different habitats, including chicken

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coops and plantations. Captured sand flies were processed and identified morphologically

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using the taxonomic key proposed by Young and Duncan (1994).

2.2. Morphometric study

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From each population, 15 males and 15 females identified as Lu. migonei were used

for morphological analysis. The individuals were measured using imaging software integrated with an automated microscope and digital camera (Leica Application Suite; LAS-Leica, Australia). The following structures measured (see Tables 1 and 2): wing length (WL) and width (WW), as well as the lengths of the labrum (L), antennal segment

3 (A3), genital pump plunger (PL), coxite (C) and style (S). Measurements are expressed as mean  standard deviation and provided in micrometres.

2.3. Genetic study Genomic DNA was extracted as described elsewhere (Latrofa et al., 2017), using a

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representative number of Lu. migonei specimens (n = 46), according to gender and

sampling area. Partial region of cytochrome c oxidase subunit I gene (cox1, 710 bp) were

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amplified using primers LCO1490 and HCO2198 (Folmer et al., 1994). Each PCR reaction consisted of 4 μl genomic DNA (~100 ng) and 46 μl of PCR mix containing 2.5

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mM MgCl2, 10 mM Tris-HCl (pH 8.3) and 50 mM KCl, 250 μM of each dNTP, 100 pmol

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of each primer and 1.25 U of AmpliTaq Gold (Applied Biosystems). The cox1 gene was

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amplified using the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C

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for 1 min, 56°C for 1 min and 72°C for 1 min, and a final extension at 72 °C for 7 min. PCR products were examined on 2% agarose gels stained with GelRed (VWR

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International PBI, Milano, Italy) and visualized on a GelLogic 100 gel documentation system (Kodak, New York, USA). The amplicons were purified and sequenced, in both

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directions using the same primers as for PCR, employing the Taq Dye Deoxy Terminator

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Cycle Sequencing Kit (v.2, Applied Biosystems), in an automated sequencer (ABIPRISM 377). All representative haplotypes of Lu. migonei obtained were deposited in

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GenBank (accession numbers: MF784908-MF784937).

2.4. Data analyses The differences among the mean of the morphometric measurements in both males and female sand flies were tested by analysis of variance (ANOVA), upon the assumption of normality distribution of the data (assessed by Kolmogorov-Smirnov test). In order to

better understand which morphometric variable would be more effective in predicting category membership (intended here as “population”), morphometric data were assessed by discriminant function analysis, using the relative function available in the software SPSS, version 13.0 for Windows. Sequences of cox1 gene were aligned using ClustalW program (Larkin et al., 2007)

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and compared with those available in GenBank database by BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Genetic distance (expressed in %) among all

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haplotypes identified, was calculated using the Kimura 2 Parameter substitution model with gamma distributed (G) rates (Kimura, 1980), implemented in the MEGA6 software

(Tamura et al., 2013). The polymorphisms across haplotypes were showed using

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WebLogo Version 2.8 (Gavin et al., 2004). The phylogenetic relationships were inferred

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by maximum likelihood (ML) based on general time reversible model (GTR), gamma

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distributed with invariant sites (G+I), selected by best-fit model (Nei and Kumar, 2000),

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and tested with 4,000 bootstrap replications, using MEGA6 software (Tamura et al.,

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2013). Homologous sequence from Phlebotomus perniciosus and Lutzomyia spp. were

3. Results

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used as outgroups (accession numbers of outgroup sequences are reported in Fig. 2).

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Comparisons of general morphology and morphometric data did not reveal any

remarkable difference among males and females belonging to different populations

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assessed herein (Tables 1 and 2). However, males and females from Machados and Baturité showed similar length of wings and antennal segment 3 as compared with specimens from Niterói. Average measurements recorded for males and females from each population, along with the statistical data are reported in Tables 3 and 4. For males, the lower and upper

limits of the 95% confidence intervals for wing length, antennal segment 3 and coxite did not overlap with those of the other populations. Therefore, these measurements were useful to discriminate males from Niterói from those from Machados and Baturité (Table 3). For females, wing length appeared to be useful to discriminate Machados from the others as well as length of antennal segment 3 to discriminate Niterói from the others

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(Table 4).

Discriminant analysis showed higher coefficients for the same abovementioned

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measurements, with the addition of wing width for females (Table 5). Indeed, using the

measurements and coefficients derived by discriminant analysis, 80% of the males were classified as belonging to the correct original population. Similarly, 86.7% of the females

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Niterói and females from Machados (Table 6).

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were correctly classified, with 100% predicted group membership achieved for male from

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The BLAST analyses revealed high nucleotide identities (up to 98%) with a sequence

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of Lu. migonei available in GenBank (accession number: GU909508). Overall, 30 cox1

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haplotypes (H1-H30), aligned over 658 sites, were identified, being H1 and H2 the most commonly retrieved (25.6%) (Table 7, Fig. 3). Except for three non-synonymous

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nucleotide substitutions (Tyr-Asn in H27; Ile-Val in H10; and Arg-Gln, in H21), all the others were silent mutations. The genetic distance calculated amongst all haplotypes

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identified was up to 3.15%, revealing the existence of two distinct lineages, designated herein as A (north-eastern) and B (south-eastern). The mean nucleotide distance within

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lineages A and B was 0.75% and 0.6%, respectively (Table 7). All specimens from Baturité and Machados belonged to lineage A, whereas those from Niterói were assigned to lineage B (Table 7). The ML tree grouped all representative haplotypes of each lineage in two monophyletic clades, supported by high bootstrap value (i.e., 99%), with the exclusion of

other Lutzomyia spp. (Fig. 2). Representative cox1 haplotypes here generated herein were deposited in GenBank, under accession numbers MF784908–MF784937.

4. Discussion In the present study, we compared morphologically and genetically three populations

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of Lu. migonei in Brazil. As expected, there was no gross morphological difference between the studied specimens. However, discriminant analysis conducted based on key

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morphometric features (i.e., length of wing, antennal segment 3 and coxite for males, and

length of wing and antennal segment 3 for females) revealed that specimens from Machados and Baturité displayed similar measurements as compared to those of Niterói.

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With few exceptions, males and females from north-eastern Brazil (Machados and

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Baturité) presented larger measurements as compared to those of south-eastern Brazil

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environmental conditions.

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(Niterói). The reasons for these differences are unknown but may be related to

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While located over 500 km apart, Machados and Baturité present similar climate and vegetation, even if most of the original vegetation coverage in Machados has been

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replaced by crop plantations. In the same way, Machados and Baturité are located 416 and 175 metres above the sea level, whereas Niterói is located at the sea level. Based on

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our results, it may be speculated that the lineages of Lu. migonei identified herein have currently a patchy distribution in Brazil, probably driven by abiotic factors, such as

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altitude. Of note, the distribution of these lineages is probably not related to climate, as all three study sites present a tropical climate, with similar average annual temperature and precipitation. Genetic analysis based on cox1 sequences was concordant in showing the existence of two well-defined lineages within the populations examined. Indeed, as a corollary of

our morphometric analysis, Lu. migonei specimens of Baturité and Machados formed a distinct genetic group, which was clearly separated from the group formed by eight haplotypes found exclusively in Niterói. This genetic structure may suggest that an abiotic barrier between these populations may have contributed to their segregation, as demonstrated for other sand fly species in Brazil (Esseghir et al., 1997; Arrivillaga et al.,

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2002; Coutinho-Abreu et al., 2008; Souza et al., 2017). Whether these two lineages of Lu. migonei are reproductively isolated remains to be known. Further studies are needed to

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investigate the distribution of these lineages in different Brazilian states. The existence of areas of sympatry should be assessed as well.

The existence of distinct genetic lineages within the same morphospecies has been

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demonstrated for Lu. longipalpis (Lima Costa et al., 2015) using the period gene (525 pb)

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and, more recently, for Lu. umbratilis. Souza Freitas et al. (2015, 2016) observed the same

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results using cytochrome oxidase I (597 bp) and the period clock gene (489 bp) genes,

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indicating the presence of two distinct clades (North and Northeast populations) under

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the name Lu. umbratilis. Whether these lineages represent cryptic species, with different ecological behaviour (e.g., blood feeding pattern; Sales et al., 2015) or even with different

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vector competence/capacity for distinct Leishmania spp. remains to be investigated. The hypotheses that Lu. longipalpis would represent a complex of cryptic species

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was raised after the observation of variations in the number of whitish abdominal spots, with either a single pair on the fourth tergite (one spot - 1S) or two pairs, on the third and

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fourth tergites (two spots - 2S) (Mangabeira, 1969). Years later, Bauzer et al. (2002) provided the first molecular evidence in Brazil for the existence of two sympatric, genetically different populations, corroborating early results of Ward et al. (1983, 1988). These finding were later supported by analysis of male sex pheromones and the

copulatory courtship songs, as well as by microsatellite markers and speciation genes (Vigoder et al., 2010b). In conclusion, our coupled morphometric and genetic analyses indicate the existence of at least two distinct lineages of Lu. migonei in Brazil. Further studies are needed to better understand the role (if any) of these genetically distinct populations in the

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transmission of Leishmania spp. (e.g., L. braziliensis and L. infantum) to animals and

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humans.

Funding

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível

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Superior (CAPES) (grant number 1520/2011).

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Declarations of interest: none

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Acknowledgments

Thanks to Fernando José da Silva, Débora Elienai de Oliveira Miranda and Vanessa

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Cristina Fitipaldi Veloso Guimarães for their support with the field work.

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Figure legend

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Fig. 1. Location of study areas in Brazil.

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Fig. 2. Maximum likelihood tree based on cox1 sequences of Lutzomyia migonei here generated with those of other Lutzomyia spp. available from GenBank. Bootstrap values are based on 4,000 replicates and only bootstraps >50% are indicated. Accession numbers

A

of all Lutzomyia spp. and P. perniciosus cox1 sequences used as outgroups were reported in the Supplementary file S1.

IP T SC R U N A M ED PT

Fig. 3. Alignment of representative cox1 haplotypes of Lutzomyia migonei here generated.

CC E

A schematic representation of polymorphisms identified across haplotypes prepared

A

using Weblogo is shown in Supplementary file 2.

Table 1 Morphometric data of Lutzomyia migonei males Populations Structures Baturité

Niterói

1440.05 (±23.67) 1469.13 (±34.58) 1387.48 (±37.64)

Wing width

436.21 (±17,68 )

444.43 (±14.30)

431.51 (±14.59)

Length of the labrum

161.71 (±6.92)

166.58 (±7.54)

158.59 (±7.53)

Antennal segment 3

150.74 (±7.87)

148.75 (±4.05)

137.38 (±8.88)

Genital pump plunger

113.20 (±4.87)

114.29 (±6.26)

109.98 (±6.97)

Coxite

248.33 (±8.72)

248.66 (±6.55)

225.70 (±7.30)

Style

117.65 (±5.15)

118.60 (±5.13)

114.34 (±3.45)

A

N

SC R

IP T

Wing length

U

Machados

M

Table 2

ED

Morphometric data of Lutzomyia migonei females

Structures

Wing length

Baturité

Niterói

1794.43 (±23.20) 1729.93 (±39.16) 1711.37 (±27.53) 555.40 (±35.35)

532.80 (±24.69)

538.64 (±27.58)

Length of the labrum

241.98 (±9.24)

241.14 (±7.56)

241.39 (±5.68)

Antennal segment 3

182.50 (±7.54)

175.39 (±8.49)

165.58 (±8.68)

A

CC E

Wing width

PT

Machados

Populations

I N U SC R

Table 3

Morphometric data recorded for males belonging to each population. Variances in population means (assessed by ANOVA) are also reported Population

Mean

Standard

A

Variable

M

deviation

Standard

95% CI for mean

error

lower limit

upper limit

Minimum

Maximum

Baturité

1469.13

34.58

8.93

1449.98

1488.27

1387.53

1508.90

length

Machados

1440.05

23.68

6.11

1426.94

1453.16

1381.58

1474.36

Niterói

1387.48

37.65

9.72

1366.64

1408.33

1308.11

1429.62

PT

Wing

ED

Wing

Baturité

444.43

14.29

3.69

436.51

452.35

419.06

470.70

Machados

436.20

17.67

4.56

426.42

445.99

410.53

468.61

Niterói

431.51

14.58

3.77

423.43

439.59

410.19

455.75

Lengths of

Baturité

166.59

7.54

1.95

162.40

170.75

157.53

181.00

the labrum

Machados

161.71

6.92

1.79

157.88

165.55

150.16

178.30

Niterói

158.59

7.53

1.94

154.41

162.76

144.08

172.09

Baturité

148.75

4.05

1.045

146.50

150.99

141.21

153.78

A

CC E

width

Antennal segment 3

Machados

150.74

7.87

2.03

146.38

155.09

139.22

165.97

ANOVA F

P value

24.287

0.000

2.638

0.083

4.516

0.017

14.880

0.000

I Baturité

114.29

6.26

Machados

113.20

4.87

plunger

Niterói

109.98

6.96

Coxite

Baturité

248.66

Machados Niterói

142.29

120.76

153.87

1.61

110.82

117.75

107.80

125.37

1.26

110.50

115.90

104.66

121.49

1.79

106.13

113.84

90.60

123.88

6.54

1.69

245.04

252.29

239.57

259.49

248.33

8.72

2.25

243.50

253.15

229.04

263.11

225.69

7.29

1.88

221.66

229.74

208.67

238.73

Baturité

118.60

5.13

1.32

115.76

121.44

112.63

128.28

Machados

117.65

5.15

1.33

114.79

120.49

107.23

123.76

114.34

3.459

0.89

112.42

116.24

108.65

119.46

CC E

Style

132.46

Niterói

A

ED

PT

pump

N U SC R

8.88

Genital

2.29

A

137.37

M

Niterói

2.021

0.145

45.298

0.000

3.483

0.040

I N U SC R

Table 4

Morphometric data recorded for females belonging to each population. Variances in population means (assessed by ANOVA) are also reported Population

Mean

Standard

A

Variable

M

deviation

Standard

95% CI for mean

error

lower limit

upper limit

Minimum

Maximum

Baturité

1729.93

39.16

10.11

1708.25

1751.62

1668.05

1788.41

length

Machados

1794.43

23.19

5.99

1781.59

1807.28

1752.90

1828.41

Niterói

1711.37

27.52

7.11

1696.13

1726.61

1667.17

1777.52

PT

Wing

ED

Wing

Baturité

532.79

24.68

6.37

519.12

546.47

491.36

584.80

Machados

555.40

35.35

9.12

535.83

574.98

500.88

622.38

Niterói

538.64

25.58

6.60

524.47

552.80

491.43

593.56

Lengths of

Baturité

241.14

7.56

1.95

236.95

245.32

224.57

254.08

the labrum

Machados

241.97

9.24

2.39

236.86

247.09

226.63

252.74

Niterói

241.39

5.68

1.47

238.25

244.54

230.17

251.42

Baturité

175.39

8.49

2.191

170.69

180.09

163.28

189.09

A

CC E

width

Antennal segment 3

Machados

182.49

7.54

1.95

178.32

186.67

165.77

202.21

ANOVA F

P value

30.231

0.000

2.466

0.097

0.047

0.954

15.882

0.000

I 8.68

N U SC R

165.58

A

CC E

PT

ED

M

A

Niterói

2.24

160.78

170.39

152.49

183.33

Table 5 Standardized canonical discriminant function coefficients for males and females of Lutzomyia populations Males

Females

Function 2

Function 1

Function 2

Wing length (WL)

0.429

0.818

0.956

0.420

Wing width (WW)

-0.135

0.116

-0.362

Lengths of the labrum (L)

-0.169

0.256

Antennal segment 3 (A3)

0.308

-0.751

Genital pump plunger (PL)

-0.129

0.143

-

-

Coxite (C)

0.813

-0.342

-

-

Style (S)

-0.207

0.311

-

-

SC R

0.949

-0.573

-0.305

0.765

-0.868

U

A

M ED PT CC E A

IP T

Function 1

N

Variables

Table 6 Classification results of population membership derived by the functions described in Table 5

Females

Predicted group membership Machados

Niterói

n (%)

n (%)

n (%)

Baturité

10 (66.7)

5 (33.3)

Machados

3 (20.0)

11 (73.3)

1 (6.7)

Niterói

0

0

15 (100)

Baturité

11 (73.3)

2 (13.3)

2 (13.3)

0

15 (100)

N

Machados

2 (13.3)

A

CC E

PT

ED

M

A

Niterói

IP T

Baturité

0

SC R

Males

Original population

U

Sex

0

0 13 (86.7)

Table 7 Geographical origin, number of Lutzomyia migonei specimens examined, representative haplotypes identified and intra-genogroup nucleotide differences (% mean, min-max) retrieved amongst cox1 haplotypes. Geographical

No. of

Haplotypes (no. of specimens)

origin

specimens

Mean (min-max) nucleotide

IP T

Lineages

differences (%)

Baturité/

34 (15F;

I (6); II (5); III (2); IV (2); V

Machado

19M)

(2); VI (1) VII (1); VIII (1); IX

SC R

A

(1); X (1); XI (1); XII (1); XIII

U

0.75 (2.35-0.15)

N

(1); XIV (1); XV (1) XVI (1);

A

XVII (1); XVIII (1); XIX (1);

Niteroi

9 (5F;

XXIII (1); XXIV (1); XXV

4M)

(1); XXVI (1); XXVII (1);

PT

ED

B

M

XX (1) XXI (1); XXII (1)

A

CC E

F, female. M, male.

0.6 (1.4-0.3) XXVIII (1); XXIX (1); XXX (2)