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Molecular identification of vectors of Leishmania in Colombia: Mitochondrial introgression in the Lutzomyia townsendi series
Abstract and Figures
The identity of the sandfly vectors of Leishmania braziliensis in Valle del Cauca Department, Colombia, was originally given as Lutzomyia townsendi, but then changed to L. youngi, another member of the L. townsendi series (Verrucarum group) with isomorphic females. To identify members of this series in Valle del Cauca, we analyzed the nuclear gene elongation factor-alpha (EF-alpha) and the mitochondrial gene cytochrome b (Cyt b). DNA sequences from the L. verrucarum series (L. columbiana, L. evansi and L. ovallesi) were used as outgroups. Flies from two locations on the western cordillera of the Andes were identified as L. townsendi s.s., according to male morphology and distinctive gene lineages. In the third location, on the central cordillera of the Andes, most specimens were identified as belonging to a geographical population of L. youngi, according to male morphology, an EF-alpha lineage shared with L. youngi from the Venezuelan-type locality, and a distinctive Cyt b sub-lineage. All other specimens were identified as L. youngi with the introgressed Cyt b sequences of L. townsendi. Such interspecific introgression implies that vectorial traits and ecological associations may no longer be viewed as fixed properties of different morphospecies.
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... El análisis de ADN proporciona una manera más precisa para identificación de las especies y el uso de los datos moleculares en combinación con los métodos morfológicos, ha permitido tener una aproximación que permite resolver preguntas taxonómicas en diferentes niveles jerárquicos (Hanel & Sturmbauer, 2000;Herran de la R et al., 2001). Secuencias de genes ribosomales (18SrDNA y el dominio D2 de 28SrDNA) se utilizaron para reevaluar las relaciones al interior de la familia Phlebotominae (Aransay et al., 1999) y dentro del género Lutzomyia (Beati et al., 2004;Kato et al., 2007;Kato et al., 2008).Secuencias de genes con una tasa de evolución más rápida, mitocondriales (por ejemplo, CytB y ND4) y nucleares (por ejemplo, ITS2 factor de elongación α), se han utilizado para resolver las relaciones intraespecíficas y subgenérico (Ishikawa et al., 1999;Depaquit et al., 2000;Uribe et al., 2001;Testa et al., 2002;Hodgkinson et al., 2003;Torgenson et al., 2003;Vivero et al., 2008;Kuwahara et al., 2009;Vivero et al., 2009). ...
... One of these sandflies was Pintomyia youngi, which is a confirmed vector of L. braziliensis, and a potential vector of L. amazonensis. Due to difficulty in taxonomic identification procedures, Pintomyia youngi could have been misidentified as Lutzomyia townsendi [31,101,102], which means that covariate and/or vector status data might be assigned to the wrong species. This can lead to misrepresentation in the model (that already relies on sparse data), which might explain the low probability score assigned to Pintomyia youngi. ...
The incidence of vector-borne diseases is rising as deforestation, climate change, and globalization bring humans in contact with arthropods that can transmit pathogens. In particular, incidence of American Cutaneous Leishmaniasis (ACL), a disease caused by parasites transmitted by sandflies, is increasing as previously intact habitats are cleared for agriculture and urban areas, potentially bringing people into contact with vectors and reservoir hosts. Previous evidence has identified dozens of sandfly species that have been infected with and/or transmit Leishmania parasites. However, there is an incomplete understanding of which sandfly species transmit the parasite, complicating efforts to limit disease spread. Here, we apply machine learning models (boosted regression trees) to leverage biological and geographical traits of known sandfly vectors to predict potential vectors. Additionally, we generate trait profiles of confirmed vectors and identify important factors in transmission. Our model performed well with an average out of sample accuracy of 86%. The models predict that synanthropic sandflies living in areas with greater canopy height, less human modification, and within an optimal range of rainfall are more likely to be Leishmania vectors. We also observed that generalist sandflies that are able to inhabit many different ecoregions are more likely to transmit the parasites. Our results suggest that Psychodopygus amazonensis and Nyssomia antunesi are unidentified potential vectors, and should be the focus of sampling and research efforts. Overall, we found that our machine learning approach provides valuable information for Leishmania surveillance and management in an otherwise complex and data sparse system.
... (Esseghir et al., 2000), the population genetics of Ph. ariasi (Mahamdallie et al., 2011), and the molecular identification of Ph. (Larroussius) spp. and Pintomyia (Pifanomyia) spp. of the Townsendi series (Testa et al., 2002;Absavaran et al., 2009). ...
The taxonomy and systematics of sand flies (Diptera, Psychodidae, Phlebotominae) are one of the pillars of research aimed to identifying vector populations and the agents transmitted by these insects. Traditionally, the use of morphological traits has been the main line of evidence for the definition of species, but the use of DNA sequences is useful as an integrative approach for their delimitation. Here, we discuss the current status of the molecular taxonomy of sand flies, including their most sequenced molecular markers and the main results. Only about 37% of all sand fly species have been processed for any molecular marker and are publicly available in the NCBI GenBank or BOLD Systems databases. The genera Phlebotomus, Nyssomyia, Psathyromyia and Psychodopygus are well-sampled, accounting for more than 56% of their sequenced species. However, less than 34% of the species of Sergentomyia, Lutzomyia, Trichopygomyia and Trichophoromyia have been sampled, representing a major gap in the knowledge of these groups. The most sequenced molecular markers are those within mtDNA, especially the DNA barcoding fragment of the cytochrome c oxidase subunit I (coi) gene, which has shown promising results in detecting cryptic diversity within species. Few sequences of conserved genes have been generated, which hampers higher-level phylogenetic inferences. We argue that sand fly species should be sequenced for at least the coi DNA barcoding marker, but multiple markers with different mutation rates should be assessed, whenever possible, to generate multilocus analysis.
... Sin embargo, utilizando el método de sales, desde muestras de flebotomíneos con talla pequeña como, Lutzomyia youngi (Feliciangeli y Murillo 1987), L. evansi (Nuñez-Tovar, 1934), L. nunez tovari (Ortiz, 1954), L. spinicrassa Morales, Osorno de Osorno y Hoyos, 1970, L. trinidadensis (Newstead, 1922), L. cayenensis (Floch and Abonnenc, 1941), se logra la extracción de ADN (Testa et al. 2002;Vivero et al. 2007), pero la efectividad en las extracciones extensivas es del 10% (muestras preservadas en etanol) y 20% (muestras frescas), con rendimientos menores a 0,1, lo cual limita los estudios poblacionales. En la presente nota se señala una modificación del protocolo de Arrivillaga et al. (2003) en varios pasos con la finalidad de proporcionar un método ajustado a muestras de flebotomíneos de pequeña talla corporal para estudios poblacionales. ...
... Las secuencias de nucleótidos son una herramienta molecular que se ha usado en los flebotomíneos para identificar especies indistinguibles en uno de los sexos (Testa et al. 2002), estudiar las relaciones filogenéticas (Torgerson et al. 2003;Beati et al. 2004;Vivero et al. 2007) y analizar la estructu-ra genética de las poblaciones (Ready et al. 1997;Ishikawa et al. 1999), en especial cuando se sospecha la existencia de complejos de especies (Uribe et al. 2001;Arrivillaga et al. 2002;Hodgkinson et al. 2003). Aunque por su costo, las secuencias están aún distantes de implementarse en el diagnóstico rutinario de especie, son una alternativa que permite, además de sortear las limitaciones inherentes a los caracteres morfológicos, establecer las bases para el futuro desarrollo de claves moleculares. ...
Los caracteres morfológicos se emplean para la determinación taxonómica tradicional de flebotomíneos del género Lutzomyia, sin embargo, su utilidad es limitada en algunas especies. En este trabajo se caracterizó la región del genoma mitocondrial que codifica para el extremo carboxilo terminal de la proteína citocromo b en siete especies de Lutzomyia: L. trinidadensis, L. panamensis, L. cayennensis cayennensis, L. dubitans, L. gomezi, L. rangeliana y L. evansi. Se detectaron 134 sitios polimórficos (40,9%) en el gen y 29 sitios (26,6%) en la proteína. El alto polimorfismo en citocromo b comprendió el reemplazo de aminoácidos, empleo de distintos codones de parada y diferencias en el tamaño de la proteína. Se discute la utilidad de la región estudiada en la identificación de especies de Lutzomyia.
... These data documented the power of ITS2 loci in the differentiation of these two morphotypes, too. CytB-NADH1 and especially EF-1α showed more diversity even at intraspecific level (Testa et al. 2002, Sonnenberg et al. 2007), but the ribosomal marker is much more conserved and it is worthy that ITS2 could separate these two morphotypes from each other. The proximity of GenBank sequences of P. neglectus to our morphotypes showed more support for the existence of this species in Iran. ...
The idea of the existence of Phlebotomus (Larroussius) neglectus (Diptera: Psychodidae) Tonnoir, 1921 in Iran and the skepticism about the existence of Phlebotomus major s.str. Annandale, 1910 had been grown recently in the country. This study reports a combined analysis of mitochondrial and ribosomal DNA target regions of P. major s.l.Annandale, 1910, specimens collected from different parts of Iran. Two different morphotypes were found among the collected samples based on the shape of the aedeagus, ventrally located hairs of the coxite, and parameral sheets. One morphotype seemed similar to P. neglectus Tonnoir 1921 or P. major krimensis Perfiliv1966 (called here MI.N.K.); the other one was similar to P. neglectus and to some extent to P. notus Artemiev & Neronov 1984 (here called MII.N.NO). Cytochrome B, elongation factor 1-alpha, and internal transcribed spacer II loci were amplified, sequenced, and characterized. High sequence homology (98-100%) was observed between P. neglectus and these morphotypes, and phylogenetic analysis was also concordant. Phlebotomus neglectus sequences available in GenBank are located as the sister group of sequences here, particularly near to morphotype MII.N.NO. Moreover, ITS2 locus provides the maximum resolution for differentiation of two morphotypes. Based on achieving results, although a strong support for the presence of P. neglectus was provided, but it is too early to say that P. major s.str. does/does not exist in Iran. This question could be resolved by studying more samples and, most importantly, by comparing the topotypes of P. neglectus and P. major s. str. if possible in the future.
... Sand flies were not directly targeted however, those flies in malaria endemic areas in the dry zone, which covers two-thirds of the country may have been regularly exposed to insecticides [9]. The presence of kdr mutation which is located at the position of 1014 with the amino acid changes from leucine to phenylalanine in a notable proportion of flies in Sri Lanka [10] will spread from one population to another by means of gene flow in sand flies similar to the case of mosquitoes [11][12][13][14]. ...
Phlebotomus argentipes is the vector of Leishmania donovani which causes the disease leishmaniasis, a neglected tropical disease and a growing health problem in Sri Lanka. A proper understanding of the population genetic structure of sand fly vectors is considered important prior to planning and implementation of a successful vector control program. Thus, the present study was conducted to determine the population genetic structure of sand fly vectors in Sri Lanka. Two mitochondrial genes namely Cytochrome c oxidase subunit 1 (Cox 1) and Cytochrome b (Cytb), and the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization. Analyses included maximum likelihood method, network analysis and DNA polymorphisms. The outcome revealed unique sequences of all genomic regions studied except the cox 1 gene had a relationship with sand flies isolated previously from Sri Lanka, India and Israel and cytb gene of 4 sand flies that aligned with those isolated earlier from Sri Lanka and 3 from Madagascar. Furthermore, cox 1 gene and ITS 2 region analyses based on FST values indicated a possible gene flow between the study sites whereas cytb gene analysis favoured the existence of genetically distinct populations of P. argentipes in each of the study sites. Poor population differentiation of P. argentipes, a possible consequence of a gene flow, is indeed of concern due to the risk imposed by promoting the spread of functionally important phenotypes such as insecticide resistance across the country, making future vector control efforts challenging.
Sand flies called Phlebotomus papatasi are the primary vector of cutaneous leishmaniasis, which is an endemic disease in Al‐Madinah Al‐Munawarah, Saudi Arabia. The molecular characterization of sand fly species is a helpful tool because genetic variability and population structure within and between populations might influence vectoral capability, necessitating the assessment of management strategies to control the spread of cutaneous leishmaniasis. Therefore, in this study, the structure of the P. papatasi population in Al‐Madinah region was investigated. The samples of P. papatasi were collected from Al‐Madinah region, identified, and subjected to mitochondrial cytochrome b (Cytb) gene sequencing ( n = 45). Analysis of the sequences revealed 13 substitutions. There were 12 different haplotypes identified. Nucleotide diversity and haplotype diversity were low estimated to be 0.0017 and 0.574, respectively. The neutrality indices determined using Tajima's D and Fu and Li's F * were all negative and statistically significant. This indicated that the population has an excess of singletons and low‐frequency variations. Network analysis showed one main haplotype and shared haplotypes at different nodes. According to this study, the P. papatasi population in the Al‐Madinah region is homogeneous. The knowledge obtained in this study contributes to a better understanding of the genetic geographic distribution of P. papatasi populations in relation to the location of Leishmania major foci, which could aid in the development of control strategies.
New World sandflies are vectors of leishmaniasis, bartonellosis, and some arboviruses. A classification based on 88 morphological characters was proposed 27 years ago when the New World phlebotomines were organized into two tribes Hertigiini and Phlebotomini. The latter was structured into four subtribes (Brumptomyiina, Sergentomyiina, Lutzomyiina, and Psychodopygina) and 20 genera. The subtribe Psychodopygina, including most of the American vectors of tegumentary Leishmania comprises seven genera from which no molecular work has been produced to support this classification. Here, we carried out a molecular phylogeny based on combined sequences (1,334 bp) of two genes: partial 28S rDNA and mtDNA cytochrome b from 47 taxa belonging to the Psychodopygina. The Bayesian phylogenetic reconstruction agreed with the classification based on morphological characters, supporting the monophyly of the genera Psychodopygus and Psathyromyia, whereas Nyssomyia and Trichophoromyia seemed to be paraphyletic. The paraphylies of the two latter groups were exclusively caused by the doubtful position of the species Ny. richardwardi. Our molecular analysis provides additional support to adopt the morphologic classification of Psychodopygina.
The study investigated the relationship between the polymorphism of the IL-9 gene at
position -6676 for the variation rs1799962 for tonsillitis among patients in Baquba, which
included 31 male samples with an average age of (15.65 ± 1.79) years compared to 5 healthy
males with an average age (14.66 ± 3.43) years enrolled in this study the polymorphism of
IL9-6676 was data waved by polymerase chain reaction-specific sequence primer (PCR-SSP)
assay. As the results of the electrophoresis of the IL- 9 gene at position -6676 showed a
comparison genotypes & alleles between tonsillitis patients & controls frequencies of AA
genotype & A allele, showed that a significant increase in patients with tonsillitis (45.16%
and 72.58% P =1.000 respectively) Compared to the control (70% and 40% P= 0.431
respectively) and associated RR rates were(8.6% and 38.0% respectively.) and And the
related EF values were (1.24 and 1.75). In contrast AG genotype & G allele (35.48 vs. 17.74,
P =0.357 respectively). Decreased frequency was observed in patients compared to the
control (30% and 60% P =0.431) , and the related PF values were (0.37 and 0.57
respectively). The results of the study indicate that IL9-6676 SNP may have a role in the
mechanism of etiopathogenic. It was observed that it has a negative and positive role in the
samples with tonsillitis in the samples of Iraqi patients
The repellency and insecticidal efficacy of Nopikex®, a soap formulation containing 20% diethyl toluamide and 0.5% permethrin, was evaluated against a laboratory colony of phlebotomine sand flies (Lutzomya longipalpis). The repellency of Nopikex soap was also compared with that of a placebo soap against another species (Lu. Youngi) in a forest near Tulua, Valle del Cauca, Colombia. In laboratory trials of the soap, no reduction in repellency was seen 4 hr after application, but within 8 hr, repellency decreased significantly to 67.0% of the initial value (P
Genomic DNA fragments from males of Psychodopygus wellcomei were isolated and shown to be useful as sensitive diagnostic probes for positively separating individuals of this species from those of Ps. complexus. These two members of the Ps. squamiventris series are found sympatrically in foci of cutaneous leishmaniasis in the hill forests of southern Pará State. Of the two species, only Ps. wellcomei is thought to be an important vector of Leishmania braziliensis sensu stricto, but this is based on circumstantial evidence because of the difficulties of identifying female sandflies within the series. The diagnostic probes were isolated from a library of Ps. wellcomei built by ligating short fragments of Sau 3A-restricted, genomic DNA into the plasmid vector pUC 18. Differential screening of 1316.library clones with total genomic DNA of Ps. wellcomei and Ps. complexus identified 5 recombinants, with cross-hybridizing inserts of repetitive DNA, that showed strong specificity for Ps. wellcomei. As little as 0.4% of the DNA extracted from an individual sandfly (= ca. 0.5 nanograms) was specifically detected. The diagnostic probes were used to identify as Ps. wellcomei a wild-caught female sandfly found infected with L. braziliensis s.s., providing only the second positive association between these two species.
The phylogeny and mode of speciation of Mediterranean Phlebotomus of the subgenusLarroussius were inferred by comparative sequence analyses of a fragment of mitochondrial DNA (Cytochrome b) and of a nuclear gene (Elongation factor alpha). The molecular phylogenies were congruent basally, where their clades matched the species complexes defined by a few genitalic characters of each sex. Reticulate evolution was suggested for the most derived species complex (Phlebotomus perniciosus): the molecular phylogenies were incongruent, and mitochondrial-marker distribution was consistent with introgressive hybridizations not between sister species but between species whose ranges now overlap or abut. By considering the molecular phylogenies, the mitochondrial molecular clock and the ecological niches of the species, as well as the historical biogeography and palaeoecology of the Mediterranean subregion, we propose that the derived lineages arose from a sequential series of speciation events associated with habitat shifts promoted by progressive aridification. This «taxon pulse»-like speciation occurred in the Pliocene, later than previously proposed in a vicariance hypothesis that invoked only tectonic events, but too early for Pleistocene Ice-age refugia to have played any role other than the isolation of geographical races. Speciation occurred before the proposed divergence of members of the Leishmania donovani complex and this helped to rule out any vector-parasite co-speciation or co-cladogenesis.
— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
Nearly 400 species and subspecies of phlebotomine sand flies in the medically important genus Lutzomyia are identified by taxanomic keys to the adults and by associated illustrations. References to existing and new information on their geographic distribution (with maps), biology, and disease relationships are included. The following 9 new species in the genus are described: caminoi Young and Duncan, christenseni Young and Duncan, clitella Young & Perez, falcata Young, Morales, and Ferro, gibba Young & Arias, nemorosa Young & Perez reinerti Young and Duncan, sinuosa Young & Duncan, and torvid Young, Morales & Ferro. The previously unknown females of moucheti, omagua and pelloni are described. New junior synonyms include spinosa Floch & Abonnenc (=damascenoi Mangabeira) and tikalensis Leon (=permira Fairchild & Hertig). The status of nuneztovari anglesi Le Pont & Desjeux as a subspecie of n. nuneztovari is rejected. The subgenus Xiphomyia of Artemiev 1991 is a junior homonym of Xiphomyia of Alexander 1916. The subspecies name, cruzi of Gonzalez & Garcia- Avila 1981, is a junior homonym of cruzi of Mangabeira 1938. Lutzomyia paulwilliamsi Martins, Falcao & Silva is a junior synonym of Lutzomyia monticola (Costa Lima). Sand flies, Diptera, Psychodidae, LutzoMyia.
The phylogcny and mode of speciation of Mediterranean Phlebotomus of the subgenus Larroussius were inferred by comparative sequence analyses of a fragment of mitochondrial DNA (Cytochrome b) and of a nuclear gene (Elongation factor alpha). The molecular phytogenies were congruent basally, where their clades matched the species complexes defined by a few genitalic characters of each sex. Reticulate evolution was suggested for the most derived species complex [Phlebotomus perniciosus): the molecular phytogenies were incongruent, and mitochondrial-marker distribution was consistent with introgressive hybridizations not between sister species but between species whose ranges now overlap or abut. By considering the molecular phytogenies, the mitochondrial molecular clock and the ecological niches of the species, as well as the historical biogeography and palaeoecology of the Mediterranean subregion, we propose that the derived lineages arose from a sequential series of speciation events associated with habitat shifts promoted by progressive aridification. This ‘taxon pulse’-like speciation occurred in the Pliocene, later than previously proposed in a vicariance hypothesis that invoked only tectonic events, but too early for Pleistocene Ice-age refugia to have played any role other than the isolation of geographical races. Speciation occurred before the proposed divergence of members of the Leishmania donovani complex and this helped to rule out any vector-parasite co-speciation or co-cladogenesis.
The series townsendi and verrucarum of the verrucarum species group of the phlebotomine genus Lutzomyia França are reviewed. Lutzomyia nadiae, new species, from the mountains of Yacambú, Lara State, is described. Distribution maps, illustrated keys, notes on medical importance, taxonomy, and ecology are presented for species in these series known from Venezuela.
Potential vectors of Leishmania braziliensis Vianna were assessed at four study sites in the mountainous Valle del Cauca, western Colombia, from March to June 1989.
In an active focus of transmission at 1450 m altitude, a coffee plantation at Versalles, there were high densities of anthropophilic phlebotomines: Lutzomyia columbiana (Ritorcelli & Van Ty) and Lu.townsendi (Ortiz), both in the verrucarum species group, and of Lu.pia (Fairchild & Hertig). At a comparable altitude in a forest reserve at Yotoco where leishmaniasis is unknown, Lu.pia was the prevalent species and Lu.townsendi was absent.
In two localities at 1150 m altitude, there were plentiful Lu. lichyi (Floch & Abonnenc) plus both species in the verrucarum group, but Lu.pia was absent. One of these localities, a coffee plantation at Villa Hermosa where a leishmaniasis outbreak occurred in 1986, was compared with a leishmaniasis-free, partly wooded nature reserve at Mateguadua.
No natural infections of Leishmania were found in a total of 1896 wild-caught female phlebotomines belonging to at least seven species. It remains unclear why Leishmaniasis transmission is associated with coffee plantations in this part of Colombia.
Laboratory-bred Lu. lichyi females were invariably autogenous, and blood-seeking females of this species were always parous. Parity rates in wild-caught females of other species were 55%Lu.pia, 24%Lu.columbiana and 14%Lu.townsendi.
Female Lutzomyia infected artificially with Le.braziliensis promas-tigotes developed peripylarian infections. Higher proportions of Lu.townsendi (96%) and Lu.columbiana (78%) became infected but these species developed lower rates of stomodaeal infections (P<0.1) than Lu.lichyi (37%) or Lu.pia (44%). Only 33% of a Colombian strain of Lu.longipalpis (Lutz & Neiva) became infected.
Isozyme data were used to identify populations of certain Lutzomyia taxa in the verrucarum species group, mostly in the series townsendi. Lutzomyia youngi Feliciangelis and Murillo and L. spinicrassa Morales, Osorno, Osorno, and Hoyos each have diagnostic allomorphs for phosphogluconate dehydrogenase (6PGDH) and fumarate hydratase (FUM). The 6PGDH and FUM data and those from 6-phospho-fructokinase and phosphoglucomutase distinguish Lutzomyia sp., a new species from Columbia to be described and named later. Data from these enzymes and glucose phosphate isomerase will separate L. townsendi (Ortiz) from the others and from L. longiflocosa Osorno, Morales, Osorno, and Hoyos, L. quasitownsendi Morales, Osorno, Osorno, and Hoyos, and L. sauroida Osorno, Morales, and Osorno (three species that are inseparable using enzyme data). Three other species, L. serrana (Damasceno and Arouck) (series serrana) and L. columbiana (Ristorcelli and Van Ty) and L. andina Osorno, Osorno, and Morales (both in series verrucarum), are morphologically distinct using conventional characters and have fixed diagnostic differences at several enzyme loci. Statistical analyses of the enzyme data using genetic identities (I), differences (D), and the amount of genetic variation among these taxa indicated that such statistics can be as useful in the study of sand fly phylogeny and population genetics as they have been for other organisms. I and D values indicated that L. longiflocosa, L. quasitownsendi, and L. sauroida are very similar (I = 0.991 and D = 0.010) and possibly are populations of the same conspecific species. The levels of divergence, based on combined enzyme data for up to 21 gene loci among the taxa, are discussed, and a dendrogram based on genetic distance is presented. The genetic data confirmed established phylogenetic relationships among the sand fly taxa based on structural similarities.