Forest disturbance and vector transmitted diseases in the lowland tropical rainforest of central Panama.

OBJECTIVE: To explore possible changes in the community attributes of haematophagous insects as a function of forest disturbance. We compare the patterns of diversity and abundance, plus the behavioural responses of three epidemiologically distinct vector assemblages across sites depicting various levels of forest cover. METHODS: Over a 3-year period, we sampled mosquitoes, sandflies and biting-midges in forested habitats of central Panama. We placed CDC light traps in the forest canopy and in the understorey to gather blood-seeking females. RESULTS: We collected 168 405 adult haematophagous dipterans in total, including 26 genera and 86 species. Pristine forest settings were always more taxonomically diverse than the disturbed forest sites, confirming that disturbance has a negative impact on species richness. Species of Phlebotominae and Culicoides were mainly classified as climax (i.e. forest specialist) or disturbance-generalist, which tend to decrease in abundance along with rising levels of disturbance. In contrast, a significant portion of mosquito species, including primary and secondary disease vectors, was classified as colonists (i.e. disturbed-areas specialists), which tend to increase in numbers towards more disturbed forest habitats. At pristine forest, the most prevalent species of Phlebotominae and Culicoides partitioned the vertical niche by being active at the forest canopy or in the understorey; yet this pattern was less clear in disturbed habitats. Most mosquito species were not vertically stratified in their habitat preference. CONCLUSION: We posit that entomological risk and related pathogen exposure to humans is higher in pristine forest scenarios for Culicoides and Phlebotominae transmitted diseases, whereas forest disturbance poses a higher entomological risk for mosquito-borne infections. This suggests that the Dilution Effect Hypothesis (DEH) does not apply in tropical rainforests where highly abundant, yet unrecognised insect vectors and neglected zoonotic diseases occur. Comprehensive, community level entomological surveillance is, therefore, the key for predicting potential disease spill over in scenarios of pristine forest intermixed with anthropogenic habitats. We suggest that changes in forest quality should also be considered when assessing arthropod-borne disease transmission risk.

OBJECTIF: Explorer les changements possibles dans les attributs communautaires des insectes hématophages en fonction de la perturbation des forêts. Nous comparons les modèles de diversité et d'abondance, ainsi que les réponses comportementales de trois assemblages de vecteurs épidémiologiquement distincts sur des sites illustrant divers niveaux de couverture forestière. MÉTHODES: Au cours d'une période de trois ans, nous avons échantillonné des moustiques, des phlébotomes et des moucherons piqueurs dans les habitats forestiers du centre de Panama. Nous avons placé des pièges à lumière CDC dans la canopée de la forêt et dans le sous-étage pour recueillir les femelles en quête de sang. RÉSULTATS: Nous avons collecté un total de 168.405 diptères hématophages adultes, dont 26 genres et 86 espèces. Les environnements de forêt intacts étaient toujours plus diversifiés du point de vue taxonomique que les sites forestiers perturbés, confirmant que les perturbations avaient un impact négatif sur la richesse en espèces. Les espèces de phlébotome et Culicoïdes étaient principalement classés comme climax (spécialiste de la forêt) ou généralistes de perturbation, qui ont tendance à diminuer en abondance parallèlement aux niveaux croissants de perturbation. En revanche, une partie importante des espèces de moustiques, y compris les vecteurs primaires et secondaires de maladies, a été classée dans la catégorie des colons (c'est-à-dire spécialistes des zones perturbées), qui ont tendance à se multiplier vers des habitats forestiers plus perturbés. Dans la forêt vierge, les espèces de phlébotomes et Culicoïdes les plus répandues cloisonnaient la niche verticale en étant actives dans la canopée de la forêt ou dans le sous-étage; pourtant, cette tendance était moins nette dans les habitats perturbés. La plupart des espèces de moustiques n'étaient pas stratifiées verticalement dans leur préférence d'habitat. CONCLUSION: Nous estimons que le risque entomologique et l'exposition associée des agents pathogènes à l'homme est plus élevé dans les scénarios de forêt vierge pour les maladies transmises par les phlébotomes et Culicoïdes, alors que la perturbation des forêts pose un risque entomologique plus élevé pour les infections transmises par les moustiques. Cela suggère que l'hypothèse de l'effet de dilution ne s'applique pas dans les forêts tropicales humides où se reproduisent très abondamment les insectes vecteurs, mais non reconnus, et où des maladies zoonotiques négligées surviennent. Une surveillance entomologique approfondie au niveau de la communauté est donc la clé pour prédire le potentiel de propagation des maladies dans des scénarios de forêt vierge mélangée à des habitats anthropiques. Nous suggérons que les changements dans la qualité des forêts soient également pris en compte lors de l'évaluation du risque de propagation de maladies transmises par les arthropodes.

Disturbance and mosquito diversity in the lowland tropical rainforest of central Panama.

The Intermediate Disturbance Hypothesis (IDH) is well-known in ecology providing an explanation for the role of disturbance in the coexistence of climax and colonist species. Here, we used the IDH as a framework to describe the role of forest disturbance in shaping the mosquito community structure, and to identify the ecological processes that increase the emergence of vector-borne disease. Mosquitoes were collected in central Panama at immature stages along linear transects in colonising, mixed and climax forest habitats, representing different levels of disturbance. Species were identified taxonomically and classified into functional categories (i.e., colonist, climax, disturbance-generalist, and rare). Using the Huisman-Olff-Fresco multi-model selection approach, IDH testing was done. We did not detect a unimodal relationship between species diversity and forest disturbance expected under the IDH; instead diversity peaked in old-growth forests. Habitat complexity and constraints are two mechanisms proposed to explain this alternative postulate. Moreover, colonist mosquito species were more likely to be involved in or capable of pathogen transmission than climax species. Vector species occurrence decreased notably in undisturbed forest settings. Old-growth forest conservation in tropical rainforests is therefore a highly-recommended solution for preventing new outbreaks of arboviral and parasitic diseases in anthropic environments.

Enzootic Arbovirus Surveillance in Forest Habitat and Phylogenetic Characterization of Novel Isolates of Gamboa Virus in Panama.

Landscape changes occurring in Panama, a country whose geographic location and climate have historically supported arbovirus transmission, prompted the hypothesis that arbovirus prevalence increases with degradation of tropical forest habitats. Investigations at four variably degraded sites revealed a diverse array of potential mosquito vectors, several of which are known vectors of arbovirus pathogens. Overall, 675 pools consisting of 25,787 mosquitoes and representing 29 species from nine genera (collected at ground and canopy height across all habitats) were screened for cytopathic viruses on Vero cells. We detected four isolates of Gamboa virus (family:Bunyaviridae; genus:Orthobunyavirus) from pools of Aedeomyia squamipennis captured at canopy level in November 2012. Phylogenetic characterization of complete genome sequences shows the new isolates to be closely related to each other with strong evidence of reassortment among the M segment of Panamanian Gamboa isolates and several other viruses of this group. At the site yielding viruses, Soberanía National Park in central Panama, 18 mosquito species were identified, and the predominant taxa included A. squamipennis,Coquillettidia nigricans, and Mansonia titillans.

Novel genetic diversity within Anopheles punctimacula s.l.: phylogenetic discrepancy between the Barcode cytochrome c oxidase I (COI) gene and the rDNA second internal transcribed spacer (ITS2).

Anopheles punctimacula s.l. is a regional malaria vector in parts of Central America, but its role in transmission is controversial due to its unresolved taxonomic status. Two cryptic species, An. malefactor and An. calderoni, have been previously confused with this taxon, and evidence for further genetic differentiation has been proposed. In the present study we collected and morphologically identified adult female mosquitoes of An. punctimacula s.l. from 10 localities across Panama and one in Costa Rica. DNA sequences from three molecular regions, the three prime end of the mitochondrial cytochrome c oxidase I gene (3' COI), the Barcode region in the five prime end of the COI (5' COI), and the rDNA second internal transcribed spacer (ITS2) were used to test the hypothesis of new molecular lineages within An. punctimacula s.l. Phylogenetic analyses using the 3' COI depicted six highly supported molecular lineages (A-F), none of which was An. malefactor. In contrast, phylogenetic inference with the 5' COI demonstrated paraphyly. Tree topologies based on the combined COI regions and ITS2 sequence data supported the same six lineages as the 3' COI alone. As a whole this evidence suggests that An. punctimacula s.l. comprises two geographically isolated lineages, but it is not clear whether these are true species. The phylogenetic structure of the An. punctimacula cluster as well as that of other unknown lineages (C type I vs C type II; D vs E) appears to be driven by geographic partition, because members of these assemblages did not overlap spatially. We report An. malefactor for the first time in Costa Rica, but our data do not support the presence of An. calderoni in Panama.

Amblyomma tapirellum (Acari: Ixodidae) collected from tropical forest canopy.

Free-ranging ticks are widely known to be restricted to the ground level of vegetation. Here, we document the capture of the tick species Amblyomma tapirellum in light traps placed in the forest canopy of Barro Colorado Island, central Panama. A total of forty eight adults and three nymphs were removed from carbon dioxide-octenol baited CDC light traps suspended 20 meters above the ground during surveys for forest canopy mosquitoes. To our knowledge, this represents the first report of questing ticks from the canopy of tropical forests. Our finding suggests a novel ecological relationship between A. tapirellum and arboreal mammals, perhaps monkeys that come to the ground to drink or to feed on fallen fruits.

The Great American Biotic Interchange in frogs: multiple and early colonization of Central America by the South American genus Pristimantis (Anura: Craugastoridae).

The completion of the land bridge between North and South America approximately 3.5-3.1 million years ago (Ma) initiated a tremendous biogeographic event called the Great American Biotic Interchange (GABI), described principally from the mammalian fossil record. The history of biotic interchange between continents for taxonomic groups with poor fossil records, however, is not well understood. Molecular and fossil data suggest that a number of plant and animal lineages crossed the Isthmus of Panama well before 3.5 Ma, leading biologists to speculate about trans-oceanic dispersal mechanisms. Here we present a molecular phylogenetic analysis of the frog genus Pristimantis based on 189 individuals of 137 species, including 71 individuals of 31 species from Panama and Colombia. DNA sequence data were obtained from three mitochondrial (COI, 12S, 16S) and two nuclear (RAG-1 and Tyr) genes, for a total of 4074 base pairs. The resulting phylogenetic hypothesis showed statistically significant conflict with most recognized taxonomic groups within Pristimantis, supporting only the rubicundus Species Series, and the Pristimantis myersi and Pristimantis pardalis Species Groups as monophyletic. Inference of ancestral areas based on a likelihood model of geographic range evolution via dispersal, local extinction, and cladogenesis (DEC) suggested that the colonization of Central America by South American Pristimantis involved at least 11 independent events. Relaxed-clock analyses of divergence times suggested that at least eight of these invasions into Central America took place prior to 4 Ma, mainly in the Miocene. These findings contribute to a growing list of molecular-based biogeographic studies presenting apparent temporal conflicts with the traditional GABI model.

Evolutionary history of Cuban crocodiles Crocodylus rhombifer and Crocodylus acutus inferred from multilocus markers.

Among crocodilians, Crocodylus rhombifer is one of the world's most endangered species with the smallest natural distribution. In Cuba, this endemic species coexists with the American crocodile (Crocodylus acutus). Hybridization between these two species is well known in captivity and might occur in the wild, but has never been demonstrated genetically. Here, we combined molecular data with environmental, geographic, and fossil data to infer the evolutionary history of Crocodylus in the Cuban Archipelago, and to evaluate genealogical support for species boundaries. We analyzed seven microsatellite loci plus DNA sequence data from nuclear (RAG-1) and mitochondrial (cytochrome b and cytochrome oxidase I) genes from 89 wild-caught individuals in Cuba, Grand Cayman Island, Jamaica, and Central America, and two samples from zoo collections. Microsatellites showed evidence of introgression, suggesting potential hybridization among Cuban groups. In Cuba, C. acutus contained one mitochondrial DNA (mtDNA) haplotype, whereas C. rhombifer contained two haplotypes. MtDNA data showed that C. acutus is paraphyletic with respect to C. rhombifer, revealing 1% sequence divergence between species within Cuba vs. 8% divergence between Cuban forms and mainland C. acutus. We suggest that hybridization has been a historical as well as a current phenomenon between C. acutus and C. rhombifer. These findings suggest that long-term conservation of crocodiles in Cuba will require identification of genetically pure and hybrid individuals, and a decrease in anthropogenic activities. We also recommend more extensive morphological and genetic analyses of Cuban population to establish clear boundaries of the hybrid zone between C. acutus and C. rhombifer.

Late Pleistocene environmental changes lead to unstable demography and population divergence of Anopheles albimanus in the northern Neotropics.

We investigated the historical demography of Anopheles albimanus using mosquitoes from five countries and three different DNA regions, the mitochondrial cytochrome oxidase subunit I gene (COI), the single copy nuclear white gene and the ribosomal internal transcribed spacer two (ITS2). All the molecular markers supported the taxonomic status of a single species of An. albimanus. Furthermore, agreement between the COI and the white genes suggested a scenario of Pleistocene geographic fragmentation (i.e., population contraction) and subsequent range expansion across southern Central America.

Ubiquity of the pathogenic chytrid fungus, Batrachochytrium dendrobatidis, in Anuran communities in Panamá.

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis, has been implicated as the main driver of many enigmatic amphibian declines in neotropical sites at high elevation. Batrachochytrium dendrobatidis is thought to be a waterborne pathogen limited by temperature, and the extent to which it persists and causes disease in amphibians at lower elevations in the neotropics is not known. It also is unclear by what mechanism(s) B. dendrobatidis has emerged as a pathogenic organism. To test whether B. dendrobatidis is limited by elevation in Panamá, we sought to determine the prevalence and intensity of B. dendrobatidis in relation to anuran abundance and diversity using quantitative PCR (qPCR) analyses. Sites were situated at varying elevations, from 45 to 1215 m, and were at varying stages of epizootic amphibian decline, including pre-epizootic, mid-epizootic, 2 years post-epizootic, and 10 years post-epizootic. Batrachochytrium dendrobatidis was found in all sites regardless of elevation or stage of epizootic decline. Levels of prevalence and infection intensity were comparable across all sites except at the mid-epizootic site, where both prevalence and intensity were significantly higher than at other sites. Symptoms of chytridiomycosis and corresponding declines in amphibian populations were variably seen at all elevations along a post-epizootic gradient. Because it is inherently difficult to prove a negative proposition, it can neither be proven that B. dendrobatidis is truly not present where it is not detected nor proven that it is only recently arrived where it is detected. Thus, there will always be doubts about whether B. dendrobatidis is enzootic or invasive. In any case, our results, coupled with current knowledge, suggest most clearly that the disease, chytridiomycosis, may be novel and invasive, and that the pathogen, B. dendrobatidis either is, or is becoming, globally ubiquitous.

Molecular systematics of the neotropical scorpion genus Tityus (Buthidae): the historical biogeography and venom antigenic diversity of toxic Venezuelan species.

We provide a mitochondrial DNA-based phylogenetic hypothesis for 21 Tityus species collected in Venezuela, Trinidad, Brazil and Panama, including 12 taxa known to be toxic to humans. Our phylogenetic reconstruction is based on 850 nucleotides of the combined cytochrome oxidase subunit I and 16S rRNA genes for most species, and centered on Venezuelan scorpions owing to the detailed taxonomic and biogeographic information available for Tityus in this region. The principal phylogenetic result was the strong support for mtDNA clades representing geographical groupings associated with the Perijá mountain range, the Mérida Andes, or the central and eastern coastal ranges in Venezuela, suggesting that vicariance has been a potent force in the diversification of local scorpions. Venezuelan Tityus species have been organized by González-Sponga into three artificial morphological groups, "androcottoides", "discrepans", and "nematochirus", based on the array of ventral carinae in metasomal segments II-IV. We also incorporated a fourth morphological group ("Tityus clathratus"), recently documented in Venezuela. Our results do not support the clustering of the species in the "androcottoides" and "discrepans" morphological groups, which include the majority of taxa of medical importance, but provided support for the "nematochirus" species group. T. clathratus was found to cluster with the Brazilian T. serrulatus and T. bahiensis. Divergence times of most clades are consistent with major events in the geological history of northern Venezuela and suggest that many Venezuelan Tityus species formed in the late Miocene and the Pliocene. In turn, we used the Tityus mtDNA phylogeny to determine the potential utility of phylogenetic systematics to predict Tityus venom antigenic reactivity by testing the recognition of T. nororientalis, T. discrepans, T. zulianus, T. perijanensis, and T. clathratus venoms by anti-T. discrepans horse antibodies. Cross-reactivity was significantly higher for the closely related eastern (T. nororientalis) and central coastal (T. discrepans) species in comparison to the distantly related Andean (T. zulianus) and Perijá (T. perijanensis) species. Reactivity of T. clathratus low mol. mass toxic components towards anti-T. serrulatus and anti-T. discrepans antivenoms was low, suggesting that venom components produced by the subgenus Archaeotityus (which encompass "clathratus" species) diverge antigenically from other Tityus scorpions.

Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama.

The assembly of DNA barcode libraries is particularly relevant within species-rich natural communities for which accurate species identifications will enable detailed ecological forensic studies. In addition, well-resolved molecular phylogenies derived from these DNA barcode sequences have the potential to improve investigations of the mechanisms underlying community assembly and functional trait evolution. To date, no studies have effectively applied DNA barcodes sensu strictu in this manner. In this report, we demonstrate that a three-locus DNA barcode when applied to 296 species of woody trees, shrubs, and palms found within the 50-ha Forest Dynamics Plot on Barro Colorado Island (BCI), Panama, resulted in >98% correct identifications. These DNA barcode sequences are also used to reconstruct a robust community phylogeny employing a supermatrix method for 281 of the 296 plant species in the plot. The three-locus barcode data were sufficient to reliably reconstruct evolutionary relationships among the plant taxa in the plot that are congruent with the broadly accepted phylogeny of flowering plants (APG II). Earlier work on the phylogenetic structure of the BCI forest dynamics plot employing less resolved phylogenies reveals significant differences in evolutionary and ecological inferences compared with our data and suggests that unresolved community phylogenies may have increased type I and type II errors. These results illustrate how highly resolved phylogenies based on DNA barcode sequence data will enhance research focused on the interface between community ecology and evolution.

Anopheles darlingi (Diptera: Culicidae) in Panama.

We report Anopheles darlingi in Darien Province in eastern Panama. Polymerase chain reaction-restriction fragment length polymorphism profiles of the single copy nuclear white gene and sequence comparisons confirmed the presence of 66 specimens of the northern lineage of An. darlingi. The parsimony network depicted 5 CO1 haplotypes in 40 specimens of An. darlingi, which connected through 7-8 mutational steps with sequences from Central and South America. Furthermore, the presence of haplotypes in Biroquera, Darien Province identical to those previously published from northern Colombia suggests that Panamanian samples originated in Colombia. Results of neutrality tests (R(2) and Fu's F(S)) were not significant and the mismatch distribution was multimodal and did not fit the model of sudden population growth. These findings may indicate a long and stable presence of An. darlingi in eastern Panama.

Mitochondrial DNA phylogeography of Caiman crocodilus in Mesoamerica and South America.

The Neotropical crocodylian species, Caiman crocodilus, is widely distributed through Mesoamerica, northern South America, and the Amazon basin. Four subspecies are recognized within C. crocodilus, suggesting some geographic variation in morphology. In this study, we utilized mitochondrial DNA (mtDNA) sequence data from 45 individuals of C. crocodilus throughout its range to infer its evolutionary history and population structure, as well as to evaluate genealogical support for subspecies and their geographic distributions. Our molecular phylogenetic results identified five mtDNA haplotype clades with a mean sequence divergence of 3.4%, indicating considerable evolutionary independence among phylogeographic lineages. Our results were also broadly consistent with current subspecific taxonomy, with some important additional findings. First, we found substantial genetic structuring within C. c. fuscus from southern Mesoamerica. Second, though we confirmed the existence of a widespread Amazonian clade, we also discovered a cryptic and divergent mtDNA lineage that was indistinguishable from C. c. crocodilus based on external morphology. Third, we confirm the status of C. c. chiapasius as a distinct evolutionary lineage, and provide evidence that C. c. fuscus may be moving northward and hybridizing with C. c. chiapasius in northern Mesoamerica. Finally, our results parallel previous phylogeographic studies of other organisms that have demonstrated significant genetic structure over shorter geographic distances in Mesoamerica compared with Amazonia. We support conservation efforts for all five independent lineages within C. crocodilus, and highlight the subspecies C. c. chiapasius as a unit of particular conservation concern.

Evolutionary and biogeographical patterns within Iberian populations of the genus Squalius inferred from molecular data.

Origins and biogeography of the chub, genus Squalius (formerly within the genus Leuciscus), in the Iberian Peninsula were inferred from comparison between patterns of geographic distribution and phylogenetic relationships among populations belonging to 14 European Squalius species. The phylogeny recovered was based on the complete sequence of the mitochondrial cytochrome b gene. Squalius species were grouped into three major clades. The basal clade included species distributed across the ancient Paratethys Sea. The second clade included species from Central and East Europe and the northern areas of the Mediterranean basin towards Minor Asia. The third clade included species from the Mediterranean Peninsulas (Iberian, Italy, and Balkans). The Iberian Squalius species do not constitutes a monophyletic group. Our data indicate that the Iberian Peninsula was colonized at least twice by two different monophyletic lineages, a meridional group and a Central Europe group. The amount of species diversity found in the Iberian Peninsula and the phylogenetic relationships among these species, together with their geographic distribution, suggest that the Central Europe lineage colonized the Iberian Peninsula at a latter time. Our data indicate that the northeastern Iberian lineage is phylogenetically close to Greek populations of Squalius cephalus, while the second lineage formed a monophyletic group including Squalius pyrenaicus, Squalius carolitertii, Squalius aradensis, and Squalius torgalensis. The speciation process that generated these species and the geographic structure of their populations, principally in S. pyrenaicus, can be attributed to paleogeographical events like the ancient endorrheism and the development of hydrographic basins.

Historical biogeography of the livebearing fish genus Poeciliopsis (Poeciliidae: Cyprinodontiformes).

To assess the historical biogeography of freshwater topminnows in the genus Poeciliopsis, we examined sequence variation in two mitochondrial genes, cytochrome b (1140 bp) and NADH subunit 2 (1047 bp). This widespread fish genus is distributed from Arizona to western Colombia, and nearly half of its 21 named species have distributions that border on the geologically active Trans-Mexican Volcanic Belt (TMVB), a region that defines the uplifted plateau (Mesa Central) of Mexico. We used the parametric bootstrap method to test the hypothesis that a single vicariant event associated with the TMVB was responsible for divergence of taxa found to the north and south of this boundary. Because the single-event hypothesis was rejected as highly unlikely, we hypothesize that at least two geological events were responsible for divergence of these species. The first (8-16 million years ago) separated ancestral populations that were distributed across the present TMVB region. A second event (2.8-6.4 million years ago) was associated with northward dispersal and subsequent vicariance of two independent southern lineages across the TMVB. The geological history of this tectonically and volcanically active region is discussed and systematic implications for the genus are outlined.

Phylogenetic relationships among domesticated and wild species of Cucurbita (Cucurbitaceae) inferred from a mitochondrial gene: Implications for crop plant evolution and areas of origin.

We have investigated the phylogenetic relationships among six wild and six domesticated taxa of Cucurbita using as a marker an intron region from the mitochondrial nad1 gene. Our study represents one of the first successful uses of a mtDNA gene in resolving inter- and intraspecific taxonomic relationships in Angiosperms and yields several important insights into the origins of domesticated Cucurbita. First, our data suggest at least six independent domestication events from distinct wild ancestors. Second, Cucurbita argyrosperma likely was domesticated from a wild Mexican gourd, Cucurbita sororia, probably in the same region of southwest Mexico that gave rise to maize. Third, the wild ancestor of Cucurbita moschata is still unknown, but mtDNA data combined with other sources of information suggest that it will probably be found in lowland northern South America. Fourth, Cucurbita andreana is supported as the wild progenitor of Cucurbita maxima, but humid lowland regions of Bolivia in addition to warmer temperate zones in South America from where C. andreana was originally described should possibly be considered as an area of origin for C. maxima. Fifth, our data support other molecular results that indicate two separate domestications in the Cucurbita pepo complex. The potential zone of domestication for one of the domesticated subspecies, C. pepo subsp. ovifera, includes eastern North America and should be extended to northeastern Mexico. The wild ancestor of the other domesticated subspecies, C. pepo subsp. pepo, is undiscovered but is closely related to C. pepo subsp. fraterna and possibly will be found in southern Mexico.