Molecular Evidence Confirms the Taxonomic Separation of Lutzomyia tihuiliensis from Lutzomyia pia (Diptera: Psychodidae) and the Usefulness of Pleural Pigmentation Patterns in Species Identification

Abstract The phlebotomine sand flies Lutzomyia pia (Fairchild & Hertig 1961) and Lutzomyia tihuiliensis Le Pont, Torrez-Espejo & Dujardin 1997 (Diptera: Psychodidae) belong to the pia series of the Lu. verrucarum species group, which includes several species that bite humans in Andean foci of leishmaniasis. The females of these two species exhibit isometry and isomorphism in anatomical structures of the head and terminalia commonly used in taxonomic identification of sand flies. They can only be differentiated based on subtle differences in the pigmentation of the pleura. In Lu. tihuiliensis, this is restricted to the basal portions of the katepimeron and katepisternum, whereas in Lu. pia both structures are totally pigmented. Taking into account the subtle morphological differences between these species, the objective of the current study was to evaluate the specific taxonomic status of Lu. tihuiliensis with respect to Lu. pia. A 475-bp portion of the mitochondrial genome was sequenced, composed of the 3′ end of the cytochrome b gene, intergenic spacer 1, the transfer RNA gene for serine, intergenic spacer 2, and the 3′ end of the gene NAD dehydrogenase 1. Genetic analysis confirms that Lu. tihuiliensis and Lu. pia constitute two distinct species and this is supported by four strong lines of evidence, i.e., the paired genetic distances, size differences and amino acid composition of the cytochrome b protein, presence and absence of intergenic spacer one and divergence observed in the sequence of the transfer RNA gene for serine. It also confirms the validity of the pleural pigmentation pattern as a species diagnostic character and the importance of performing a detailed examination of this character during morphological determination of phlebotomine sand flies in the series pia.

To date, four species of the series pia have been recorded from Colombia: Lu. pia, Lu. limafalcaoae, Lu. emberai, and Lu. tihuiliensis. The last of these species was recorded from specimens collected in the Valle de Aburrá (Bejarano et al. 2006), department of Antio-quia, where it had originally been identiÞed as Lu. pia (Agudelo et al. 2002), due to its close morphological similarity. It is important to emphasize that the females of Lu. pia and Lu. tihuiliensis exhibit isometry and isomorphism in anatomical structures of the head and terminalia, used frequently as diagnostic characters for phlebotomine species. Their identiÞcation is only possible by the subtle differences in the pigmentation of the pleura, which in Lu. tihuiliensis is restricted to the basal portion of the katepimeron and katepisternum, whereas in Lu. pia both structures are totally pigmented (Fig. 1).
To determine whether the morphological differences between Lu. pia and Lu. tihuiliensis have a genetic basis, genetic variation between these taxa was estimated by molecular characterization of a portion of the mitochondrial genome. The mitochondrial genes most used to date in studies of the genus Lutzomyia are cytochrome b, cytochrome oxidase I, NAD dehydrogenase 4, and the small ribosomal subunit (Ready et al. 1997;Suguri et al. 1997;Uribe et al. 1998;Ishikawa et al. 1999;Bejarano 2001;Rojas 2001;Arrivillaga et al. 2002;Testa et al. 2002;Beati et al. 2004). These molecular markers have proved to be useful for analysis of both interspeciÞc and intraspe-ciÞc genetic variability, including separation of morphologically similar species. The genomic region selected for the current study extends from the 3Ј end of the cytochrome b gene to the 3Ј end of NAD dehydrogenase subunit one gene. Nucleotide sequence analysis conÞrmed the speciÞc status of Lu. tihuiliensis and the validity of pleural pigmentation as a diagnostic character for species of the series pia.

Materials and Methods
Collection of Phlebotomines. Specimens of Lu. pia were collected in the municipality of Marsella (04Њ 55Ј N, 05Њ 58Ј W), in the Colombian department of Risaralda, using two CDC light traps operated from 1800 to 0600 hours and a Shannon trap operated from 1800 to 2200 hours. Specimens of Lu. tihuiliensis were collected in a secondary forest in Vereda El Vallano, municipality of Envigado (06Њ 08Ј N, 75Њ 34Ј W) in the neighboring department of Antioquia. The latter species could only be captured by human-landing catches made from 1800 to 2200 h. Specimens were transported dry to the PECET Laboratory of the Universidad de Antioquia and stored at Ϫ4ЊC.
Taxonomic Determination. Sand ßies were examined under the stereomicroscope and dissected by separating the head and the Þnal segments of the abdomen, which include the female genitalia. These structures were cleared in lactophenol (1:1 lactic acid: phenol) for 24 h, and then they were mounted on slides in HoyerÕs medium. Species determination was performed using the keys and descriptions of Young and Duncan (1994)), Wolff and Galati (2002), Galati (2003b), and Bejarano et al. (2004), based principally on the length of the labro-epipharynx and second palpomere, ratio of the lengths of the common and individual sperm ducts, and the pleural pigmentation pattern.
Extraction, Amplification and Sequencing of DNA. Total DNA was extracted from the thorax and proximal segments of the abdomen by using the protocol described by Collins et al. (1987) with the following modiÞcations: maceration in 60 l of lysis buffer (0.08 NaCl, 0.16 M saccharose, 0.06 M EDTA, 0.5% SDS, and 0.1 M Tris-HCl, pH 7.5), precipitation with 200 l of absolute ethanol, and resuspension in 30 l of sterile water. AmpliÞcation was performed by polymerase chain reaction (PCR), by using the forward primer 5Ј-CA(T/C)ATTCAACC(A/T)GAATGATA-3Ј and the reverse primer 5Ј-GGTA(C/T)(A/T)TTGCCTC-GA(T/A)TTCG(T/A)TATGA-3Ј (Ready et al. 1997). These primers amplify a region composed of the 3Ј end of the cytochrome b gene (Cyt b), intergenic spacer 1 (IG1), transfer RNA gene for serine (UCN) (tRNA Ser ), intergenic spacer 2 (IG2), and the 3Ј end of the NAD dehydrogenase 1 gene (NAD1). PCR was carried out in 50 l of reaction mix containing PCR 1ϫ buffer (Promega, Madison, WI), 1.5 mM MgCl 2 , 0.2 mM of a mixture of deoxyribonucleotides (Promega), 0.3 M of each primer, 1.5 U of TaqDNA polymerase (Promega), and 6 l of DNA solution.
A three-stage thermic ampliÞcation proÞle was used consisting of 1) denaturation at 94ЊC for 3 min; 2) 35 cycles of denaturation for 1 min at 90ЊC, annealing for 1 min at 50ЊC and extension at 72ЊC for 1 min; and 3) a Þnal extension at 72ЊC for 10 min. The PCR products were separated by electrophoresis in 1.5% agarose gel. They were stained with 0.5 mg/ml ethidium bromide and visualized in a UV light transilluminator. They were then puriÞed using a Wizard PCR Preps DNA PuriÞcation System kit (Promega). Nucleotide sequences of the puriÞcation products were determined on an ABI Prism 3730xl electrophoresis capillary automatic sequencer (Applied Biosystems, Foster City, CA) using ßuorescent chain terminators (Big Dye Terminator Cycle Sequencing Ready Reaction kit, Applied Biosystems).
Sequence Analysis. Nucleotide sequences were deposited into GenBank with accession numbers EF033642ÐEF033648. Using the nucleotide-nucleotide search option of the BLAST program available on-line (Altschul et al. 1990) the position of the new sequences was determined with respect to the mitochondrial genome of Anopheles gambiae Giles (Beard et al. 1993). Multiple alignments of the sequences were carried out using the Clustal W program (Thompson et al. 1994) integrated with DAMBE 4.2.13 (Xia and Xie 2001). Nucleotide sequences were translated to amino acids on the MEGA 3.1 program (Kumar et al. 2004), which also was used to determine the polymorphic sites and paired genetic distances (p) between the two species. The distances between Lu. pia and Lu. tihuiliensis were then compared with a reference standard constituted by intra-and interspe-ciÞc genetic distances for Lu. verrucarum group species (Bejarano 2001, Rojas 2001. A Lu. pia homologous sequence (GenBank accession no. AF403486) also was included in the analysis, from a specimen collected in Salamina (5Њ 24Ј N, 75Њ 28Ј W) in the Colombian department of Caldas (Rojas 2001). The secondary structure of the tRNA Ser was inferred using the tRNAscan-SE 1.21 program (Lowe and Eddy 1997), and secondary structure diagrams were manually edited.

Results
Fourteen females belonging to the genus Lutzomyia were identiÞed during the study (11 Lu. pia and three Lu. tihuiliensis), from which seven 475-bp nucleotide sequences were obtained of the genes Cyt b, IG1, tRNA Ser , IG2, and NAD1. The Þrst and last nucleotides of these sequences are homologous to no. 11232 and 12575, respectively, of the Cyt b and NAD1 gene of the An. gambiae mitochondrial genome (Genbank accession no. NC 002084). A single nucleotide haplotype was found to be shared by Þve Lu. pia specimens from Marsella, Risaralda, which was identical to isolate Lpicsal1 (accession no. AF403486) from Salamina, Caldas, deposited in GenBank by Rojas (2001). Thus, a single haplotype for the two Lu. tihuiliensis specimens sequenced during the study was obtained, the Þrst known for this species. The nucleotide alignments of all the sequences analyzed are shown in Fig. 2.
Each haplotype is 475 bp, except for a single Lu. tihuiliensis 421-bp sequence. The haplotypes isolated are conformed by a partial sequence of the 3Ј end of the Cyt b gene, which in the alignment (Fig. 2) ex-Fig. 2. Multiple alignment of the nucleotide sequences of the 3Ј end of the cytochrome b gene, intergenic spacer 1, the transfer RNA gene for serine (UCN), intergenic spacer 2, and the 3Ј end of the gene NAD dehydrogenase derived from adult females of Lu. tihuiliensis (isolates Lticenv1 and Lticenv2) and Lu. pia (isolates Lpicsal1, Lpicmar1, Lpicmar2, Lpicmar3, Lpicmar4, and Lpicmar5). Dots denote homology, dashes denote missing data, and letters denote the respective nucleotide. tends from nucleotides 1 to 318 in Lu. pia and from 1 to 321 in Lu. tihuiliensis. This demonstrates an important difference between Lu. pia and Lu. tihuiliensis with respect to the size of the Cyt b protein, conferring on the latter species the ability to code for an additional amino acid on the carboxyl-terminal end (Fig.  3). This is due to the substitution of the Þrst nucleotide of the stop codon, situated at position 316, which corresponds to thymine in Lu. pia and cytosine in Lu. tihuiliensis. In the latter species, this substitution changes the TAA stop codon (UAA for messenger RNA) to the CAA codon that codes for glutamine.
A noncoding DNA sequence was located on IG1 that is only present in Lu. pia, apparently conformed by two nucleotides (cytosine and adenine) at positions 319 and 320 of the multiple alignment (Fig. 2). It is important to emphasize that Lu. tihuiliensis lacks IG1, another important difference between the two species.
The tRNA Ser gene lies between positions 321 and 387 of the alignment (Fig. 2), consisting of 67 nucleotides in both species. In Lu. pia the nucleotides of this gene are for exclusive coding, whereas in Lu. tihuiliensis the gene shares its primer nucleotide with the third position of the Cyt b stop codon. This suggests that there is an overlap between the Cyt b and tRNA Ser genes in Lu. tihuiliensis. Furthermore, Þve divergent sites were found in the nucleotide constitution of the gene tRNA Ser gene, at positions 334, 343, 368, 370, and 371 of the alignment. When the secondary structures ( Fig. 4) inferred for this molecule were compared, it was observed that the anticodon arm contains a nonÐ Watson-Crick U-U base pair in Lu. tihuiliensis, and a nonÐWatson-Crick C-U base pair in Lu. pia. All this demonstrates the existence of type-speciÞc tRNA Ser molecules in Lu. tihuiliensis and Lu. pia.
Adjacent to this gene is IG2, a noncoding DNA sequence that extends from positions 388 Ð 403 of the alignment (Fig. 2). IG2 has a constant length of 16 nucleotides in the two species, although it exhibits some differences in the nucleotide constitution. Finally, a partial sequence of the 3Ј end of the NAD1 gene is presented between positions 404 and 475 of the mitochondrial fragment (Fig. 2), which was not included in the analysis because of its size and the lack of information for one of the Lu. tihuiliensis specimens.
Multiple alignment revealed the presence of 38 nucleotide substitutions between Lu. pia and Lu. tihuiliensis at the 3Ј end of the Cyt b gene. When the amino acid sequence coded by this gene was inferred, the most important changes were found to occur at the Þrst position of the codon, provoking nine nonsynonymous substitutions on the amino acid sequence (Fig. 3). The matrix of paired genetic distances (p) was calculated from a 318-bp fragment of Cyt b, homologous with the mitochondrial region sequenced in other species of the Lu. verrucarum group (Bejarano 2001, Rojas 2001. The results provide a value of 0.1164 between Lu. tihuiliensis and Lu. pia.

Discussion
The phlebotomine sand ßies Lu. pia and Lu. tihuiliensis belong to the pia series of the Lu. verrucarum species group, which includes several species that bite humans in Andean foci of leishmaniasis. Although both species are anthropophilic, small differences in behavior have been observed in Colombia. Warburg et al. (1991) found that L. pia in Valle del Cauca did not attempt to bite immediately after settling, whereas Zuleta (2002) observed that in Valle de Aburrá L. tihuiliensis began to bite as soon as it landed on human skin (Bejarano et al. 2004). These subtle bionomic differences could favor the vectorial role of Lu. tihuiliensis over Lu. pia, and subsequently make it necessary to distinguish them.
The results of the current study conÞrm Lu. pia and Lu. tihuiliensis as being two distinct species, demonstrating the validity of the pleural pigmentation pattern as a species diagnostic character. The speciÞc status of Lu. tihuiliensis is supported by four strong lines of evidence, including 1) paired genetic distances, 2) differences in size and amino acid compo- The paired genetic distance between Lu. pia and Lu. tihuiliensis (0.1164) lies within the range of interspe-ciÞc distances for the Lu. verrucarum group species, e.g., that between Lutzomyia evansi (Nunez-Tovar, 1924) and Lutzomyia ovallesi (Ortiz 1952) (0.1195Ð 0.1384). It greatly exceeds the genetic distances between other morphologically similar species, such as Lutzomyia spinicrassa Morales, Osorno, Osorno &Munoz, 1969, andLutzomyia longiflocosa Osorno, Morales, Osorno &Munoz, 1970 (0.0629 Ð 0.0660) (Rojas 2001), and it is much higher than the intraspeciÞc distances seen for this group. For example, the values between Lu. evansi populations from Colombia and Venezuela vary from 0.0252 to 0.0377 (Bejarano 2001).
The second line of evidence to support the speciÞc status of Lu. tihuiliensis derives from the amino acid sequence inferred for the Cyt b gene. The protein product of this gene in Lu. tihuiliensis has an additional amino acid at the 3Ј end. This type of difference does not occur within a single species but it has been found between distinct species, e.g., Lutzomyia columbiana (Ristorcelli & Van Ty, 1941) and Lu. ovallesi (Rojas 2001). The latter presents a glutamine at the carboxylterminal end of the Cyt b gene, resulting in a larger protein product. Such differences in the size of this protein may reßect an important evolutionary divergence between species (Bejarano 2001). In addition, nine divergent amino acid sites were observed between Lu. pia and Lu. tihuiliensis, explaining the differences between protein products coded by the Cyt b gene of these species. These results contrast with the 38 substitutions found at the nucleotide level, denoting a high rate of neutral mutation at the third position of the different codons, as expected for genomic coding regions (Futuyma 1998).
The presence and absence of IG1 in Lu. pia and Lu. tihuiliensis, respectively, is another important difference between the two species. The IG1 is a noncoding DNA sequence, which ranges in size from 0 to 10 bp within the genus Lutzomyia (Bejarano 2001, Rojas 2001. Some studies on comparative genomics have shown that length variations of intergenic spacers are one of the causes of differences in size of the mitochondrial genome of insects. Moreover, it is well known that a drastic mitochondrial genome size reduction has occurred through the evolutionary history.
The fourth line of evidence corresponds to the Þve divergent sites presented by the nucleotide sequence of the tRNA Ser gene and differences in the conÞguration of the secondary structure inferred from this molecule, including the nonÐWatson-Crick base pairs of the anticodon arm. Although this gene tends to be highly conserved in Lutzomyia, apparently exhibiting only interspeciÞc differences (Rojas 2001, Vivero et al. 2007, Þve polymorphic sites were found in the DHU and TC loops, and the anticodon stem. Given that the mutational dynamics and evolutionary rate of the tRNA Ser gene are low in phlebotomines, these differences may reßect a marked evolutionary divergence between the species.
The genetic evidence derived from the current study supports the separation of Lu. tihuiliensis and Lu. pia, conÞrming the validity of the pleural pigmentation pattern as a diagnostic character that should be examined carefully during taxonomic determination of these species. Finally, it conÞrms the usefulness of mitochondrial genes as molecular tools in differentiating morphologically similar species of the genus Lutzomyia.