Phylogeny and biogeography of Triatominae (Hemiptera: Reduviidae): molecular evidence of a New World origin of the Asiatic clade

MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 23 (2002) 447–457 www.academicpress.com

Phylogeny and biogeography of Triatominae (Hemiptera: Reduviidae): molecular evidence of a New World origin of the Asiatic clade V
aclav Hypsa,a,b,* David F. Tietz,a,b Jan Zrzav
y,a,c Ryan O.M. Rego,a,b Cleber Galvao,d and Jos
e Jurbergd Faculty of Biological Sciences, University of South Bohemia, Cesk
e Bud ejovice, Czech Republic b Institute of Parasitology, Academy of Sciences, Cesk
e Bud ejovice, Czech Republic c Institute of Entomology, Academy of Sciences, Cesk
e Bud ejovice, Czech Republic Laborat
orio Nacional e Internacional de Refer^ encia em Taxonomia de Triatom
ıneos, Departamento de Entomologia, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, Brazil a

d

Received 24 July 2001; received in revised form 12 December 2001

Abstract The most representative sample of molecular data, especially 16S and 12S rDNAs, is used to study the phylogeny and evolution of 57 species of three tribes, Rhodniini, Linshcosteini, and Triatomini, of the subfamily Triatominae. For the first time both New World and Old World species are brought together in a single phylogenetic analysis. Maximum-parsimony and distance estimation place both the Asiatic representatives, Linshcosteus and Triatoma rubrofasciata, as sister groups. The Linshcosteus–T. rubrofasciata clade nests firmly within Triatomini, in most analyses branching as a basalmost lineage, thus supporting a monophyletic origin of Triatominae. A paraphyly of ‘‘Triatoma’’ with respect to Linshcosteus, Dipetalogaster, Eratyrus, and Panstrongylus and the paraphyly of ‘‘Rhodnius’’ with respect to Psammolestes is observed in most of the analyses. Reinterpretation of triatomine biogeography points to the origin of Triatominae in northern areas of South America, in Central America, or in the southern region of North America. A few taxonomic changes are proposed: (1) reinclusion of Linshcosteus in Triatomini, (2) inclusion of Psammolestes in Rhodnius, (3) elevation of the ‘‘T. flavida complex’’ to the full genus Nesotriatoma (including N. flavida, N. bruneri, and N. obscura), (4) inclusion of the ‘‘T. spinolai complex’’ in Mepraia (including M. spinolai, M. gajardoi, M. eratyrusiformis, and M. breyeri), and (5) inclusion of ‘‘T.’’ dimidiata in Meccus (M. dimidiatus). Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Triatominae; Phylogeny; Biogeography; 16S rRNA

1. Introduction Although generally recognized as a monophyletic subfamily of the Reduviidae, adapted to a blood-feeding strategy (Usinger, 1944; Lent and Wygodzinsky, 1979; Clayton, 1990; Schuh and Slater, 1995), the subfamily Triatominae poses several serious phylogenetic questions with respect to its origin and geographical distribution. The current classification of Triatominae rests mainly on

*

Corresponding author. Fax: +420-38-5300388. E-mail address: [email protected] (V. Hyps˘a).

an extensive review by Lent and Wygodzinsky (1979), followed by several considerable modifications by Lent et al. (1994) (elevation of Mepraia to full genus), Jurberg and Galvao (1997) (elevation of Hermanlentia), Carcavallo et al. (1998) (description of Torrealbaia), and Carcavallo et al. (2000) (elevation of Meccus, separation of Linshcosteus as a tribe). Six tribes with 18 genera (Triatomini: Triatoma, Meccus, Dipetalogaster, Mepraia, Eratyrus, Panstrongylus, Hermanlentia, Paratriatoma; Linshcosteini: Linshcosteus; Rhodniini: Rhodnius, Psammolestes; Cavernicolini: Torrealbaia, Cavernicola; Bolboderini: Bolbodera, Belminus, Parabelminus, Microtriatoma; Alberproseniini: Alberprosenia) composed

1055-7903/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 5 5 - 7 9 0 3 ( 0 2 ) 0 0 0 2 3 - 4

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of 133 species, the great majority of them found in the New World, are now recognized. While several species representing 2 genera also inhabit continental India (5 spp. of Linshcosteus) and south and southeast Asia to New Guinea and northern Australia (8 spp. of the Triatoma rubrofasciata complex), no autochthonous endemic species are found in Africa. Trying to solve this biogeographical puzzle, Schofield (1988, 2000) proposed a concept of polyphyletic origin of the triatomines. According to his view, the Asiatic fauna is composed of at least two independent lineages. The first Asiatic lineage consists of several species of Triatoma, which had evolved from the originally New World species T. rubrofasciata after its introduction into the Old World. The other lineage is represented by the genus Linshcosteus, a supposedly autochthonous Asiatic lineage of bloodfeeding reduviids. Although frequently discussed, the question of Triatominae origin and evolution had never been seriously addressed using a rigorous cladistic approach until a recent analysis combining morphological, ecological, geographical, behavioral, and molecular characters across all Triatominae (Tietz, 2000). The results of this study nested both Linshcosteus and the T. rubrofasciata complex within the monophyletic Triatomini and placed an origin of Triatominae in the area between southwest North America and central Mexico. Recently, several phylogenetic analyses using molecular data have been published (Garcia and Powell, 1998; Stothard et al., 1998; Lyman et al., 1999; Garc
ıa, 1999; Monteiro et al., 2000; Marcilla et al., 2001). Although based on limited taxon sampling, and hence unable to solve the key questions of triatomine phylogeny, these analyses indicated that the morphological studies may overestimate the phylogenetic significance of conspicuous morphological autapomorphies. In such cases, phylogenetic affiliations of highly derived and morphologically aberrant species or lineages may not be properly recognized, resulting in taxonomic isolation of such taxa and their classification at a high taxonomical rank. This phenomenon is well documented by the obvious paraphyly of the two largest genera, i.e., Rhodnius and Triatoma, with respect to Psammolestes and other genera of the Triatomini, respectively (Lyman et al., 1999; Monteiro et al., 2000; Marcilla et al., 2001). In this paper, we use the most representative sample of molecular data published so far to study the phylogeny and evolution of 57 species of Triatominae representing the tribes Rhodniini, Linshcosteini, and Triatomini. For the first time, both New World and Old World species are brought together in a single phylogenetic analysis, thus allowing us to address the question of Triatominae monophyly. Based on the phylogenetic analyses, the origin, evolution, and biogeography of triatomines are reinterpreted and several revisions of the generic level classification are proposed.

2. Materials and methods 2.1. DNA extraction, PCR amplification, and sequencing Total DNA was extracted and purified from homogenized thoracic muscles using the Dneasy Tissue kit (Qiagen). Primer pairs mt32–mt34 and mt35–mt36 (insect mtDNA set; Biotechnology Laboratory, University of British Columbia) were used to amplify approx. 500and 400-bp-long fragments of 16S rDNA and 12S rDNA, respectively. PCR products were ligated either into the pGem-T Easy vector (Promega) or the pCRTOPO vector (Invitrogen) and sequenced using T7 and SP6 as forward and reverse primers, respectively, in an ABI PRISM sequencer (Perkin–Elmer Model 310). The species analyzed and sequences’ accession numbers are listed in Table 1. 2.2. Alignments and phylogenetic analysis Since the complete 16S rDNA matrix included 62 species and covered a large taxonomic span, the matrices were likely to be affected by misalignment due to a number of homoplasies, particularly within the variable regions. To address this problem, four matrices differing in taxa sampling were designed and analysed. (1) The ‘‘16S’’ matrix included partial 16S rDNA sequences of 57 Triatominae species representing the tribes Rhodniini, Linshcosteini, and Triatomini; Fulgora laternaria, Halobates matsumarai, Pseudovelia tibialis, and 2 nontriatomine reduviid species, Arilus cristatus and Reduvius personatus, were used as outgroups. (2) The ‘‘Rhodnius’’ matrix included partial 16S rDNA of 12 Rhodnius and 2 Psammolestes species; 4 species representing the Triatomini and 2 nontriatomine reduviid species were used as outgroups. (3) The ‘‘SA’’ matrix included 16S rDNA of all South American species of Triatoma, Panstrongylus, and Mepraia; 3 Asiatic and North American species (T. rubrofasciata, T. dimidiata, and T. rubida) were used as outgroups. (4) The ‘‘combined’’ matrix included all species for which at least two of five genes (Table 1) were available. Since the ITS-2 genes of Rhodniini and Triatomini show a very low level of homology and since only three ITS-2 sequences are available for Rhodniini, the Rhodniini ITS-2s were not included in this matrix, to avoid an increase in number of homoplastic characters. The matrices were aligned either by the MALIGN program (Wheeler and Gladstein, 1994; Rhodniini) or by ClustalX (16S and SA). In ClustalX, each matrix was aligned under nine different combinations of parameters (Ts/Tv ratios 1:1, 1:2, and 1:3; gap opening/gap extension penalties 5/3, 8/5, and 12/8). In MALIGN, a heuristic algorithm ‘‘build, score 4’’ was performed with 10 random rearrangements of sequence input order followed by branch-swapping procedures ‘‘treeswap’’ and

V. Hypsa et al. / Molecular Phylogenetics and Evolution 23 (2002) 447–457

‘‘alignswap.’’ Substitution cost to (internal) gap cost ratios were 6:3, 4:3, 3:3, 3:4, and 3:6; leading and trailing gap costs were set to 20; the Ts/Tv ratios were set to 1:1, 1:2, and 1:3. In the combined matrix, only one arbitrarily selected alignment was used for each noncoding gene, to avoid an exponential increase with number of matrices. Multiple alignments were analyzed using PAUP* 4.0b4 (Swofford, 1998). Maximum-parsimony (MP) analyses were performed by the TBR algorithm with 10 randomizations of sequence order. Each matrix was analyzed under the 1:1, 1:2, and 1:3 Ts/Tv ratios. Four different models (uncorrected distance (p); Hasegawa et al., 1985 (HKY85); Tamura and Nei, 1993 (TamNei); and log-determinant (LogDet)) were used in distance estimation. In all analyses, gaps were treated as missing data.

3. Results 3.1. 16S, Rhodnius, and SA matrices The length of the 16S rDNA sequences varies from 505 to 510 bp; after aligning with 24 additional 16S rDNAs retrieved from GenBank (Table 1), the resulting alignment is 533–557 positions long, depending on gap penalties and Ts/Tv ratio setting. Accordingly, the number of variable and parsimony-informative positions varies from 302 to 315 and from 213 to 225, respectively. The topologies obtained by MP analysis varied in dependence on parameters and produced only poorly resolved consensus trees (Fig. 1). All analyses recognized Rhodniini and Triatomini–Linshcosteini as monophyletic sister clades. Within Rhodniini, both Psammolestes species clustered together with the Rhodnius prolixus complex. The R. pallescens complex was a basalmost lineage of Rhodniini in the majority of the trees. MP analysis of the Rhodnius matrix provided results fully compatible with those inferred from the 16S matrix (Rhodniini in Fig. 4). The three complexes (R. pictipes, R. pallescens, and R. prolixus complexes) were recognized as monophyletic clades by all analyses. Of three possible topologies, (R. pallescens (R. pictipes + R. prolixus)) was supported by a majority of the trees. Psammolestes, R. neivai, and R. domesticus clustered within the R. prolixus complex. In Triatomini, a large polytomy formed a basal portion of the MP tree without any clear rooting tendency. More consistent results were obtained when the 16S matrix was analyzed under four distance estimation models (p, HKY85, TamNei, and LogDet; Fig. 2). Both semistrict and majority-rule consensus trees of the distance analyses placed the Linshcosteus–T. rubrofasciata clade as the sister group of all New World species. Within the New World Triatomini, the South American species of Triatoma, with the exceptions of T. vitticeps

449

and T. tibiamaculata, formed a robust monophyletic clade (SA Triatoma clade) present in all analyses. An internal topology of this clade was compatible with that obtained by MP and contained several well-supported species complexes. MP analysis of the SA matrix produced a tree compatible with the results of 16S analysis, but provided better resolution. While the position of the Panstrongylus species remained uncertain, T. eratyrusiformis + Mepraia spinolai constituted a sister lineage of the SA Triatoma clade. Within the SA Triatoma clade, three robust clades designated the T. infestans, T. sordida, and T. circummaculata complexes, were recognized by all analyses (Fig. 4). 3.2. Combined matrix MP analysis of conservative, unequivocally aligned sites produced 44 trees. The strict consensus was well resolved, retaining three large monophyletic clades corresponding to Rhodniini, North American Triatomini, and South American Triatomini (including T. vitticeps and Panstrongylus megistus; Fig. 3). If the equivocally aligned sites were also included in the analysis, the only change of the topology was a switch of P. megistus to the base of the North American branch, supported by a low Bremer index (Fig. 3). The preferred tree discussed below (Fig. 4) was constructed as follows. Inner topologies of Rhodniini and SA Triatoma clade are based on Rhodnius and SA matrices, respectively. The basal position of the Linshcosteus–T. rubrofasciata clade corresponds to one of three possible rootings found by MP and is, moreover, supported by the majority-rule consensus of distance analyses of the 16S matrix. A basal split into two monophyletic branches represented by North American and South American species is adopted from the combined matrix and is compatible with the majority-rule consensus trees of both MP and distance analyses of 16S matrix. The position of the Panstrongylus clade within the South American branch is preferred due to a better fit with the biogeography and the distribution of symbiotic bacteria (see below). The monophyly of Panstrongylus is based on results obtained after combination of the 16S matrix with morphological data from Tietz (2000).

4. Discussion 4.1. Origin of the Triatominae: monophyly versus polyphyly A consensus of all analyses performed in this study is consistent with the scenario held by most authors who consider the Triatominae a monophyletic clade of reduviids adapted to a blood-feeding strategy (Usinger,

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Table 1 List of species and accession numbers of genes analyzed in this study Tribe

Group

Subgroup

Complex-LW

Complex-C

Triatomini

N N N N N T. circummaculata

Dipetalogaster maximus Eratyrus mucronatus Linshcosteus sp. Triatoma protracta Triatoma nitida Triatoma lecticularia Triatoma sanguisuga Triatoma rubrofasciata Triatoma rubida Meccus pallidipennis Meccus mazzottii Meccus picturatus Triatoma dimidiata Mepraia spinolai Triatoma eratyrusiformis Triatoma flavida Triatoma bruneri Triatoma infestans Triatoma platensis Triatoma delpontei Triatoma melanosoma Triatoma rubrovaria Triatoma maculata Triatoma pseudomaculata Triatoma sordida Triatoma garciabezi Triatoma patagonica Triatoma guasayana Triatoma klugi Triatoma jurbergi Triatoma matogrossensis Triatoma guazu Triatoma williami Triatoma tibiamaculata Triatoma costalimai Triatoma arthurneivai Triatoma brasiliensis Triatoma vitticeps Triatoma circummaculata

AY035442 AY035450 AF394595 AY035444 AF045702
AY035443 AF045696
AY035468 AY035445 AF045697
AY035446 AY035447 AY035448 AY035467 AY035466 AY035451 AF394594 AF021198
AF021201
AF028745
AY035462 AF021203
AY035465 AY035461 AF021209
AY035455 AY035464 AF021192
AY035463 AY035456 AY035454 AY035457 AY035458 AY035453 AY035459 AY035460 AF021183
AF021218
AF021188


P. lignarius N N N

Panstrongylus Panstrongylus Panstrongylus Panstrongylus

AY035452 AJ243336
AF394593 AY035449

T. protracta T.lecticularia

T. rubrofasciata

T. rubrofasciata T. infestans

T. phyllosoma

N N N N Meccus

T. flavida

T. dimidiata Mepraia T. breyeri T. flavida

T. infestans

T. infestans

T. spinolai

T. maculata T. sordida

T. oliveirai

N

T. circummaculata

16S rDNA AF158057
AB026607
AB026613
AF045712
AY035436

herreri megistus geniculatus lutzi

Cyt. b

COI

ITS2

12S rDNA

AF045729
AF394517 AF045728




AJ286887




AF394524

AF045727
AF045723
AF045725
AF394523 AF394522

AF045724


AJ286882
AJ286885


AF045726


AJ286880


AF301594


AJ289876


AF021197
AF021200


AF045721


AF021199
AF021202


AF021204


AF045730


AF021210


AF021207


AJ293589


AF021193


AF021208


AF021196


AF394521

AF045722


AF021184
AF021219


AJ293591


AF021187
AF021217
AF021190


AF021179


AJ286886


AF0211178


V. Hypsa et al. / Molecular Phylogenetics and Evolution 23 (2002) 447–457

T. protracta

Species Fulgora laternaria Halobates matsumarai Pseudovelia tibialis Arilus cristatus Reduvius personatus

AF045714

AF045719


AF394518


AF394520

AF045716
AF045717


AF045718


AF045713


N N

Rhodnius stali Rhodnius prolixus R. prolixus

Rhodnius nasutus Rhodnius neglectus Rhodnius robustus Rhodnius domesticus Rhodnius brethesi Rhodnius neivai Psammolestes coreodes Psammolestes tertius

Rhodnius colombiensis Rhodnius pictipes R. pictipes

Note: The informal taxonomic groupings (‘‘groups,’’ ‘‘subgroups,’’ and ‘‘complexes’’) were accepted from Lent and Wygodzinsky (1979) (‘‘complex-W’’) and Carcavallo et al. (2000) (‘‘complex-C’’). N, taxon not assigned to any of the complexes. * Sequences retrieved from GenBank.

Rhodniini

R. pallescens

Rhodnius pallescens Rhodnius ecuadorensis

AF045706
AF028746
AF045711
AY035438 AF045709
AF028748
AY035437 AF045707
AF028747
AF028749
AF045704
AF045705
AY035440 AF045710
AY035441 AF045708
AY035439

AF045720
AF045715


AF394519

V. Hypsa et al. / Molecular Phylogenetics and Evolution 23 (2002) 447–457

451

Fig. 1. Majority-rule consensus obtained from strict consensus trees of all MP analyses of the 16S dataset. The numbers at nodes are bootstrap supports obtained by 1000 replications of MP analysis of one, arbitrarily selected 16S alignment under Tv:Ts ¼ 1:1 assumption (TL ¼ 1310, CI ¼ 0.37, RI ¼ 0.61, RC ¼ 0.22).

1944; Lent and Wygodzinsky, 1979; Clayton, 1990; Schuh and Slater, 1995). A dissenting view considering Triatominae a polyphyletic assemblage of several reduviid lineages has been proposed by Schofield (1988, 2000), based predominantly on ecological and biogeographical arguments. An independent origin of bloodfeeding strategy, at least in Rhodniini and Triatomini, was recently reported to receive support from several molecular and physiological studies. The presence of different antihemostatic components in Rhodniini and

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Fig. 3. Strict consensus of MP analyses of the combined dataset. The line width is proportional to Bremer (decay) indices shown at individual nodes (numbers above lines are Bremer indices for complete alignment; numbers below lines are Bremer indices for conservativeregions-only alignment). Two alternative positions of P. megistus are shown by dashed lines.

Fig. 2. Majority-rule and strict consensus (boldface lines) of distance estimation analyses of the 16S dataset.

Triatomini (Ribeiro et al., 1998) and the sequence diversity observed between the two tribes are considered the most significant arguments in favor of the Triatominae polyphyly and an independent origin of hematophagy (for a review see Carcavallo et al., 2000). In his review on biosystematics of Triatominae, Schofield (1988) argues that not only Rhodniini, but even some other genera classified within Triatomini (such as Dipetalogater, Panstrongylus, and Eratyrus), may represent unrelated groups that originated from different reduviids. This scenario consistently produces an apparent geographical disjunction of triatomines with the occurrence of Linshcosteus and the T. rubrofasciata complex in Asia and the absence of any sylvatic triatomines in Africa (Schofield, 1988, 2000; Gorla et al., 1997). This view was not confirmed nor supported by any of several molecular analyses published in the last few years. On the contrary, the paraphyly of Rhodnius with respect to

Psammolestes was unequivocally demonstrated in two papers (Lyman et al., 1999; Monteiro et al., 2000). This finding provides a good example of how rapid morphological and ecological changes result in a loss of the ‘‘diagnostic’’ characters that are expected to be shared by members of a given monophyletic group. We suppose that similar processes may have occurred within the tribe Triatomini, thus giving rise to several morphologically and bionomically distinct forms of triatomines. Indeed, the paraphyletic character of the genus Triatoma with respect to at least some other Triatomini genera, particularly to Panstrongylus and Dipetalogaster, has become obvious (Marcilla et al., 2001). Although there is no unequivocal proof for Triatominae monophyly at present, the following morphological characters pointed out by Lent and Wygodzinsky (1979) should be seriously discussed as possible autapomorphies of the Triatominae: (1) hematophagous feeding habit, (2) elongate and nearly straight labium with a flexible membranous connection between segments 3 and 4, allowing upwardly pointed distal rostral segment when the rostrum is in feeding position, and (3) loss of dorsal abdominal scent glands in nymphs. All these characters are primary homologies sensu de Pinna (1985) ( ¼ putative homology statements prior to tree construction) and should be tested by confrontation with other, e.g., molecular characters,

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453

Fig. 4. A preferred phylogeny of the Triatominae (see Discussion). Newly proposed taxonomic combinations are boldfaced (for discussion on the generic names used in the tree see Systematic implications). SA, SA Triatoma clade; 1, T. infestans complex; 2, T. circummaculata complex; 3, T. sordida complex.

because the test of congruence of shared derived characters (i.e., establishment of the secondary homology) is the ultimate arbiter of the hypotheses on homology. To the best of our knowledge, no single putative apomorphy shared by some triatomines and some nontriatomines has been published so far. In addition to the putative morphological synapomorphies discussed above, the monophyly of Triatominae is also supported by the observations derived from the present molecular study. The genera Panstrongylus, Dipetalogaster, and Eratyrus and the Asiatic clade cluster within the genus Triatoma and their sequences do not display significantly higher distances than those

observed among Triatoma species. Consequently, at least the hypothesis of separate origins of individual genera within the Triatomini is falsified. The transpacific distribution of the Triatominae (see Schuh and Slater, 1995, p. 40) is not exceptional in the Hemiptera—Colobathristidae, Thaumastocoridae, and the reduviid subfamilies of Physoderinae, Peiratinae, and Vesciinae share this type of distribution. In some hemipterans (smaller clades within Miridae and Rhyparochromidae), the sister groups of the transpacific clades inhabit Africa (Schuh and Slater, 1995). This seems to support the vicariant origin of the transpacific patterns (that are abundant in the flowering plants, e.g., Chloranthaceae).

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4.2. Phylogeny of Rhodniini Recently, Schofield and Dujardin (1999) published an evolutionary scenario for the origin and evolution of the Rhodniini based on two assumptions: (1) the tribe represents a monophyletic group of reduviids which has developed a blood-feeding lifestyle independently of the tribe Tratomini, and (2) an ancestor of Rhodniini, represented by an extant widespread species, R. pictipes, originated in arboreal habitats of the Amazon–Orinoco rainforests and then dispersed northwest and south to give rise to the R. pallescens and R. prolixus complexes. In contrast to this view, the R. pictipes complex was never found as the most primitive branch in any of our analyses. Depending on parameters used, two competing topologies were obtained, placing the R. pictipes complex as sister group of either the R. pallescens or the R. prolixus complex. Both topologies have been recently published as results of molecular analyses (Lyman et al., 1999; Monteiro et al., 2000). Although the available molecular data do not allow for a convincing discrimination between these two topologies, the tree with R. pallescens complex as a basal branch was preferred by a majority of the analyses performed in this study. These findings indicate that an occurrence of widespread species, such as R. pictipes, R. prolixus, and R. robustus, in Amazonia may reflect a secondary invasion of this area by highly successful species rather than the origin of the whole tribe. This scenario is well compatible with the general concept of triatomine invasion into the central parts of South America from the northern parts of the subcontinent or even from the southern parts of North America (Tietz, 2000; for further discussion see below), and with the supposedly recent origin of the Amazon fauna compared to other parts of South America (Nores, 1999, and references therein; see also Zrzav
y and Nedved, 1999). 4.3. Higher-level phylogeny of Triatomini and Linshcosteini: the rooting problem, biogeography, and ecology The Asiatic species Linshcosteus sp. and T. rubrofasciata form a monophyletic clade, branching within the Triatominae. In MP analyses, the position of this clade within Triatominae is unstable and varies with the parameters (Fig. 1). The Linshcosteus–T. rubrofasiata clade is recognized as one of three possible roots of the Triatomini, and this position is supported by a majority of distance-based analyses (Fig. 2). In addition to the Linshcosteus–T. rubrofasiata clade, two alternative roots are indicated by MP analyses, one between Meccus and the rest of Triatomini and the other between the T. protracta group and the rest of Triatomini. The rooting by T. rubrofasciata–Linshcosteus (shown in Figs. 2 and 4) is thus the only arrangement supported by both distance analyses and some MP. It has also been favored

over the T. protracta-rooted topologies by ITS-2 analysis where T. barberi (representing the T. protracta complex) is the sister group of Dipetalogaster maximus (Marcilla et al., 2001). It is important to stress that regardless of the variable position of the T. rubrofasciata– Linshcosteus clade, the monophyly of this group and its clear affinity to all other Triatomini strongly favor the monophyly of both Triatomini (including Linshcosteus): the extant Asiatic species seem likely to represent a lineage which dispersed to Asia at an early stage of the Triatomini radiation. The three possible rootings differ in evolutionary interpretation of the relationships between the North and the South American faunas of the Triatominae. Whereas the Linshcosteus–T. rubrofasciata-rooted trees tend to show the basal split situated between the North and the South American groups, with an uncertain position of the ‘‘Panstrongylus clade’’ (see below), the other two topologies make the North American species paraphyletic with respect to the South American clade. 4.4. T. phyllosoma complex–Meccus Recently, a genus Meccus was revalidated for large Mexican species with several morphological synapomorphies (Carcavallo et al., 2000). Lent and Wygodzinsky (1979), although recognizing a close relationship and unique features of these species, treated them as members of the genus Triatoma (as T. phyllosoma complex, including T. phyllosoma, T. picturata, T. mazzotiii, T. longipennis, and T. pallidipennis). They further stated that ‘‘T. dimidiata . . . is superficially somewhat similar to phyllosoma group and agrees in the features of the abdomen of the fifth instar nymph.’’ In contrast to this view, Carcavallo et al. (2000) argued that T. dimidiata differs morphologically from other species in Mexico and the United States and established a separate T. dimidiata complex with putative origin in northern South America. The results that we obtained by analyzing the 16S and combined matrices strongly support the former view and place T. dimidiata near Meccus, usually as its sister group. Clustering of T. dimidiata together with the species of Meccus was previously demonstrated in sequence analyses of the mitochondrial large subunit rRNA and cytochrome b (Lyman et al., 1999) and of ITS-2 (Marcilla et al., 2001), although on a more restricted taxon sample. Another species clustering within, or as a sister group of, Meccus in a majority of analyses was Triatoma sanguisuga. However, while the basal position of this species with respect to the T. dimidiata–Meccus clade is recognized by most MP analyses, the distance estimates place T. sanguisuga deep into Meccus. As the result of MP analysis better fits morphological arguments and since only short fragments of the 16S rRNA gene are available for T. sanguisuga and M. pallidipennis, we assume

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that the incongruence between the MP and the distance method may be due to incorrect distance calculation. 4.5. Panstrongylus clade In addition to four Panstrongylus species, this clade unexpectedly includes three species of Triatoma, viz., T. flavida, T. bruneri, and T. tibiamaculata. Although never formally suggested, a close relationships of T. flavida and T. bruneri on the one hand and Panstrongylus (namely, P. lignarius complex) on the other hand is supported by the overall color pattern and by the pronotum with an unusually wide posterior lobe (Lent and Wygodzinsky, 1979). In this paper, P. herreri is the only species representing the P. lignarius complex. It should be noted that in both distance estimation and MP, P. herreri is the closest relative of the T. flavida complex, either as a single-species branch or together with P. megistus (Figs. 2 and 4). A reliable cladogram of Panstrongylus is needed to decide whether these morphological similarities reflect phylogenetic relationship. Within the Panstrongylus clade, the relationships of individual species are poorly resolved, without any clear tendency of Panstrongylus spp. to cluster as a monophyletic group. This observation is difficult to explain, but since the genus Panstrongylus is a morphologically well-defined taxon, we speculate that the failure of Panstrongylus species to form a monophyletic clade could reflect the short time between the origin of Panstrongylus and its subsequent radiation, rather than the nonmonophyly of Panstrongylus. The position of the Panstrongylus clade within the Triatomini is sensitive to parameters used during the alignment process—it clusters as the basalmost offshoot of either the South American or the North American group. A comparison of all-sites and conservative-sites-only analyses shows a low support for either topology. Indirect evidence supporting close relationships between Panstrongylus and the South American clade comes from 98% 16S rDNA similarity of Arsenophonus triatominarum, the Triatoma infestans’s intracellular symbiotic bacterium (Hypsa and Dale, 1997), with the Panstrongylus megistus’s symbiont, whereas no symbiotic bacteria have been detected in Rhodniini and Dipetalogaster maximus (unpublished results). The symbiont distribution thus supports monophyly of Panstrongylus + the South American clade, unless secondary losses of the symbionts in other Triatomini are hypothesized. Based on analysis of ITS-2 sequences, Marcilla et al. (2001) reported a basal dichotomy between North American species on the one hand and Panstrongylus + South American species on the other, with R. prolixus used as an outgroup. However, the authors acknowledge a ‘‘substantial variation in the alignment of all triatominae ITS-2 sequences’’ and emphasize a great dissimilarity between the sequences of Rhodniini and Triatomini. Further, a comparison of

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ITS-2 genes of all available triatomines shows clearly that while there are diagnostic features characterizing both North American species and species representing the SA Triatoma clade, the P. megistus sequence differs considerably from both groups. Thus, using Rhodnius with its highly divergent ITS-2 sequence as an outgroup may be misleading and may cause an incorrect rooting of the triatomine tree. In our opinion, although an exact position of the Panstrongylus clade remains to be verified, an occurrence of several Panstrongylus species in Central America and/or northern parts of South America (P. chinai, P. geniculatus, P. humeralis, P. lignarius, and P. rufotuberculatus) and the Greater Antillean distribution of the T. flavida group suggest that an ‘‘intermediate’’ position of the Panstrongylus clade may well fit the basal dichotomy between North and South American species. 4.6. Mepraia and the T. spinolai complex Lent and Wygodzinsky (1979) characterized the T. spinolai complex, viz. T. eratyrusiformis, T. breyeri, and T. spinolai as a taxonomically and geographically isolated group of species that inhabit semiarid areas in southern South America. Based on several morphological characters, Lent et al. (1994) removed the last species from the genus Triatoma, revalidating the genus Mepraia Mazza, Gajardo & Jorg, 1940 (later an additional species, M. gajardoi, was described by Frias et al. (1998)). In our analysis, Mepraia spinolai and T. eratyrusiformis form a stable and well-supported branch placed within the South American clade, often as a most basal lineage of the SA Triatoma clade (MP analyses; Fig. 1) or as a sister group of the Panstrongylus clade (majority of distance analyses; Fig. 2). 4.7. The SA Triatoma clade The SA Triatoma clade represents one of the most robust clades, present in all analyses performed. It encompasses 20 species, recognized by Lent and Wygodzinsky (1979) on morphological grounds as members of the T. infestans, and T. circummaculata complexes (Table 1). However, 2 species previously considered members of the T. infestans complex, T. tibiamaculata, and T. vitticeps, display an unstable position, always branching outside the SA Triatoma clade. T. tibiamaculata shows a tendency to cluster in the vicinity of the Panstrongylus clade (see above) while T. vitticeps’s unstable position is reflected by a separate position of this species within a large polytomy encompassing all North American species in addition to the monophyletic SA Triatoma clade (Fig. 1). Forcing T. vitticeps to form a monophyletic group together with the SA Triatoma clade produces a tree only one step longer and places this species at the base of the clade. The same position is obtained without

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any constraint by analyzing a matrix combining 16S rDNA data with the other genes (Fig. 3). Moreover, calculation of Bremer indices for the combined matrix reveals T. vitticeps + SA Triatoma clade as one of the most robust clades in the tree. In this respect, it is pertinent to note that while all species clustering within the SA Triatoma clade share the same karyotype (20 + XY), two different patterns, 20 + XXXY and 20 + XXY, have been identified for T. vitticeps and T. tibiamaculata, respectively (Schreiber and Pellegrino, 1950; Schreiber et al., 1972; Panzera et al., 1996). Within the SA Triatoma clade, two species, T. brasiliensis and T. maculata, display quite unstable positions while the rest of species form three well-supported groups, We adopt the wellestablished terms T. infestans, T. circummaculata, and T. sordida complexes to name these groups (Fig. 4), although they differ considerably in their species contents from those suggested recently on morphological grounds (Carcavallo et al., 2000). 4.8. Systematic implications Within the Rhodniini, it is evident that Psammolestes spp. represent aberrant species of Rhodnius. Splitting the traditional (i.e., paraphyletic) genus Rhodnius into several monophyletic genera seems inappropriate because all Rhodnius s. str. species are conspicuously similar to one another morphologically and because there are no generic-rank names proposed by earlier authors. Consequently, we propose here to include all the Psammolestes species into Rhodnius, as R. arthuri (Pinto, 1926) comb. n., R. coreodes (Bergroth, 1911) comb. n., and R. tertius (Lent and Jurberg, 1965) comb. n. The interrelationships among genera and species of the Triatomini are shown as quite unstable in this study. However, the tribal separation of Linshcosteus proposed by Carcavallo et al. (2000) is not supported by the phylogenetic relationships because T. rubrofasciata, the type species of Triatoma Laporte, 1832, is an immediate sister group of Linshcosteus. The paraphyletic nature of ‘‘Triatoma’’ is evident. In all analyses, the ‘‘Triatoma’’ flavida–‘‘T.’’ bruneri clade groups as a close relative to Panstrongylus. Generic name Nesotriatoma (Usinger, 1944), has been proposed for ‘‘T.’’ flavida, a taxonomic decision followed here. The genus Nesotriatoma is a well-characterized Greater Antillean clade including three species, N. flavida (Neiva, 1911) (type species), N. bruneri (Usinger, 1944), and N. obscura Maldonado and Farr, 1962. The case of ‘‘T.’’ tibiamaculata is more problematic: this species tends to group near to the Panstrongylus– Nesotriatoma clade and might eventually be elevated to the generic rank (Eutriatoma Pinto, 1926). However, some of the species that were sometimes (in a rather chaotic manner) referred to as Eutriatoma spp. are included in the present study, and all except for Neso-

triatoma and ‘‘T.’’ tibiamaculata group within the SA Triatoma clade. At present, we do not propose to classify Eutriatoma as a full genus because of its uncertain species content. Monophyly of Meccus and its distinct phylogenetic position within the Triatomini is supported by this study. The results indicate that ‘‘T.’’ dimidiata should be included in this genus also, as Meccus dimidiatus (Latreille, 1811) comb. n. The phylogenetic position of ‘‘T.’’ sanguisuga and its possible inclusion in Meccus has to be reinvestigated when more molecular data are available. ‘‘T.’’ eratyrusiformis groups with no exceptions as a sister species of Mepraia spinolai, forming the arid southern South American T. spinolai complex (Lent and Wygodzinsky, 1979) monophyletic. Because the position of this species couple is quite uncertain (and basal in combined trees), we have decided to classify this clade as a full genus including four species, M. spinolai (Porter, 1934), M. gajardoi Frias, Henry and Gonzalez, 1998, M. eratyrusiformis (Del Ponte, 1929) comb. n., and M. breyeri (Del Ponte, 1929) comb. n. The other ‘‘Triatoma’’ spp. should be left unclassified at present. It is probable that at least three separate groups hidden under a common generic name are represented in this study—(i) the probably paraphyletic ‘‘T’’. sanguisuga–protracta–lecticularia–nitida–rubida group, possibly related to (or even congeneric with) Dipetalogaster; (ii) T. rubrofasciata (closely related to Linshcosteus); and (iii) SA Triatoma clade, a monophyletic clade including the rest of ‘‘Triatoma’’ species.

Acknowledgments Randall T. Schuh (New York) and an anonymous reviewer helped to improve the manuscript. This work was supported by Grants No. A6022801 (Grant Agency of the Academy of Sciences of the Czech Republic) and No. 96066 and MSM 123100003 (Ministry of Education, Czech Republic) and by Conselho Nacional de Desenvolvimento Cient
ıfico e Tecnol
ogico, CNPq and Fundacßa o Nacional de Sa
ude, Brazil.

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