Eastern Colombian Páramo Liodessus Guignot, 1939 diving beetles are genetically structured, but show signs of hybridization, with description of new species and subspecies (Coleoptera, Dytiscidae).

We studied Liodessus diving beetles from six eastern Colombian Páramo areas, as well as from the Altiplano. We discovered a highly characteristic new species, based on male genital morphology, Liodessussantarositasp. nov., in the Páramo de Guantiva-Rusia. Specimens from the Altiplano around Bogotá, and the Páramos of Almorzadero, Chingaza, Matarredonda, Rabanal y Rio Bogotá and Sumapaz form one clade of genetically similar populations based on mitochondrial Cox1 sequence data. The individuals of this clade are sub-structured according to their geographic distribution. The populations differ from each other mainly in terms of body size and coloration and, at most, subtly in their genital morphology. In two cases, we find putative hybrid populations between Altiplano and Páramo areas. We suggest that the different Páramo populations are in an early phase of speciation, and perhaps already genetically isolated in some cases. They are here assigned subspecies status to highlight these ongoing processes pending more comprehensive geographic sampling and use of genomic data. We refer to this clade as the Liodessusbogotensis complex, containing Liodessusb.bogotensis Guignot, 1953; Liodessusb.almorzaderossp. nov.; Liodessusb.chingazassp. nov.; Liodessusb.lacunaviridis Balke et al., 2021, stat. nov.; Liodessusb.matarredondassp. nov., and Liodessusb.sumapazssp. nov.

Disentangling Leucocytozoon parasite diversity in the neotropics: Descriptions of two new species and shortcomings of molecular diagnostics for leucocytozoids.

Avian communities from South America harbor an extraordinary diversity of Leucocytozoon species (Haemosporida, Leucocytozoidae). Here, of 890 birds sampled, 10 (1.2%) were infected with Leucocytozoon parasites. Among them, two new species were discovered and described. Leucocytozoon grallariae sp. nov. and Leucocytozoon neotropicalis sp. nov. were found in non-migratory highland passeriforms belonging to the Grallaridae and Cotingidae, respectively. They both possess gametocytes in fusiform host cells. However, due to combining microscopic examination and molecular detection, it was revealed that these parasites were present in co-infections with other Leucocytozoon species, which gametocytes develop in roundish host cells, therefore exhibiting two highly distant parasite lineages isolated from the same samples. Remarkably, the lineages obtained by cloning the mtDNA genomes were not captured by the classic nested PCR, which amplifies a short fragment of cytochrome b gene. Phylogenetic analyses revealed that the lineages obtained by the classic nested PCR clustered with parasites possessing gametocytes in roundish host cells, while the lineages obtained by the mtDNA genome PCR protocol were closely related to Leucocytozoon parasites possessing gametocytes in fusiform host cells. These findings suggest problems with the sensitivity of the molecular protocols commonly used to detect Leucocytozoon species. A detailed analysis of the primers used in the classic nested PCR revealed a match with DNA sequences from those parasites that possess gametocytes in roundish host cells (i.e., Leucocytozoon fringillinarum), while they differ with the orthologous regions in the mtDNA genomes isolated from the samples containing the two new species. Since these are mixed infections, none of the lineages detected in this study can be assigned accurately to the new Leucocytozoon morphospecies that develops in fusiform host cells. However, phylogenetic analyses allowed us to hypothesize their most probable associations. This study highlights the need for developing detection methods to assess the diversity of Leucocytozoon parasites accurately.

Primers targeting mitochondrial genes of avian haemosporidians: PCR detection and differential DNA amplification of parasites belonging to different genera.

Haemosporida is a diverse group of vector-borne parasitic protozoa, ubiquitous in terrestrial vertebrates worldwide. The renewed interest in their diversity has been driven by the extensive use of molecular methods targeting mitochondrial genes. Unfortunately, most studies target a 478 bp fragment of the cytochrome b (cytb) gene, which often cannot be used to separate lineages from different genera found in mixed infections that are common in wildlife. In this investigation, an alignment constructed with 114 mitochondrial genome sequences belonging to four genera (Leucocytozoon, Haemoproteus, Plasmodium and Hepatocystis) was used to design two different sets of primers targeting the cytb gene as well as the other two mitochondrial DNA genes: cytochrome c oxidase subunit 1 and cytochrome c oxidase subunit 3. The design of each pair of primers required consideration of different criteria, including a set for detection and another for differential amplification of DNA from parasites belonging to different avian haemosporidians. All pairs of primers were tested in three laboratories to assess their sensitivity and specificity under diverse practices and across isolates from different genera including single and natural mixed infections as well as experimental mixed infections. Overall, these primers exhibited high sensitivity regardless of the differences in laboratory practices, parasite species, and parasitemias. Furthermore, those primers designed to separate parasite genera showed high specificity, as confirmed by sequencing. In the case of cytb, a nested multiplex (single tube PCR) test was designed and successfully tested to differentially detect lineages of Plasmodium and Haemoproteus parasites by yielding amplicons with different sizes detectable in a standard agarose gel. To our knowledge, the designed assay is the first test for detection and differentiation of species belonging to these two genera in a single PCR. The experiments across laboratories provided recommendations that can be of use to those researchers seeking to standardise these or other primers to the specific needs of their field investigations.

Leucocytozoon Diversity and Possible Vectors in the Neotropical highlands of Colombia.

Studies of the lowland avifauna in the Neotropical Region have shown a paucity of Leucocytozoon species. However, surveys conducted in the Colombian highlands revealed a great diversity of these parasites infecting resident birds. To further investigate the relationship between Leucocytozoon diversity, the potential vectors, and altitudinal distribution, birds from 41 families were sampled at low and high elevations in Colombia. Blood samples were screened by microscopy, and a fragment of cytochrome b was amplified from Leucocytozoon-positive samples. The complete mitochondrial genome was also obtained for each morphospecies of Leucocytozoon. Leucocytozoon species were detected in resident birds, with various degrees of host specificity, at elevations from 2,400 to 3,950 meters above sea level, where five new host-parasite associations were discovered. Phylogenetic analysis based on the cytochrome b fragment suggested that two nominal taxa, L. fringillinarum and L. majoris, are species complexes. Blood sources of Simuliidae revealed generalist-feeding habits that included avian and mammalian hosts. Molecular analysis of parasites in black flies indicated a close relationship with the parasites found in birds. Our investigation provides further evidence that the distribution and transmission of Leucocytozoon species in the Neotropics are influenced by elevation, with the highest prevalence between 2,400 and 3,200 m asl.

Haemoproteus erythrogravidus n. sp. (Haemosporida, Haemoproteidae): Description and molecular characterization of a widespread blood parasite of birds in South America.

The great diversity of birds and ecosystems in the Andean mountains has been understudied in terms of their parasite species. We describe a new Haemoproteus parasite, H. (Parahaemoproteus) erythrogravidus infecting Zonotrichia capensis (Rufous-Collared Sparrow) in South America. The description of this blood parasite species is supported by morphological and molecular data based on a fragment of cytochrome b gene (cyt b) and complete mitochondrial genome sequences. The new species is closely related to H. (Parahaemoproteus) coatneyi, and it can be readily distinguished from the latter parasite due to morphology of its blood stages, particularly 1) the formation of a marked protrusion on envelope of infected erythrocytes by the majority of developing gametocytes, a feature which is unique for this Haemoproteus species and 2) the extremely attenuated width of the growing dumbbell-shaped macro- and microgametocytes. Additionally, Haemoproteus erythrogravidus is shown to be a monophyletic taxon that diverges from Haemoproteus coatneyi at the molecular level. We provide the complete mitochondrial DNA genome for both H. coatneyi and H. erythrogravidus. Molecular and morphological evidences indicate that H. erythrogravidus is present in Ecuador and Colombia, and genetic lineages with 100% of identity for the cyt b gene were reported in Chile, Perú, and Venezuela. Our study also indicates that H. erythrogravidus and H. coatneyi are sympatric sister taxa sharing Z. capensis as a host species across its distribution, which could be the result of sympatric speciation or complex biogeographic processes. Further studies on the distribution and evolutionary history of Z. capensis and its parasites H. erythrogravidus and H. coatneyi insight for our better understanding of the factors and dynamics driving parasite speciation.

Avian haemosporidians from Neotropical highlands: Evidence from morphological and molecular data.

Avian haemosporidian parasites have been scarcely studied in the Neotropical highlands despite the high avian diversity reported and the uniqueness of these ecosystems. The aims of this study were to examine Haemoproteus and Plasmodium diversity based on morphological and molecular data, as well as to explore the concordance between these two approaches, when identifying species. We sampled 1487 birds belonging to 166 species, in localities of the Colombian Andean region at elevations ranging from 2100 to 4000 m above sea level. Here, we report twelve morphological parasite species, of which five are undescribed. Thirty parasite cytochrome b lineages are reported, 17 of which for the first time. We provide morphological information and illustrations, as well as, cytochrome b lineages for six morphospecies: Haemoproteus columbae, Haemoproteus witti, Haemoproteus coatneyi, Haemoproteus vireonis, Plasmodium lutzi, and Plasmodium unalis. This is the first report to provide a linkage between morphology and a molecular lineage for H. witti. Cytochrome b gene proved to be useful for species determination as DNA barcoding. Differences in parasite composition between lowlands and highlands in Colombia suggest a replacement of avian Plasmodium fauna. Parasite lineages restricted to either Colombian resident or Nearctic migratory birds were found; but a single lineage common in both has not been recorded in Nearctic non-migratory birds. We generated valuable information by using both morphological and molecular data representing competent host-parasite relationships which are based on observation of gametocytes in circulation; and increased the taxon sampling of avian haemosporidian.

Integrated taxonomy of a new species of black fly in the subgenus Trichodagmia (Diptera: Simuliidae) from the Páramo Region of Colombia.

A new species of simuliid from the Andean Mountains of Colombia is described on the basis of females, males, pupae, larvae, polytene chromosomes, and COI and ITS2 DNA sequences. Simulium (Trichodagmia) chimguazaense new species is structurally, chromosomally, and molecularly distinct from its nearest relatives, S. muiscorum Bueno, Moncada & Muñoz de Hoyos and S. sumapazense Coscarón & Py-Daniel. 

Leucocytozoon pterotenuis sp. nov. (Haemosporida, Leucocytozoidae): description of the morphologically unique species from the Grallariidae birds, with remarks on the distribution of Leucocytozoon parasites in the Neotropics.

We describe morphologically unique Leucocytozoon pterotenuis sp. nov. (Haemosporida, Leucocytozoidae), the first reported leucocytozoid species developing in fusiform host cell found in a Neotropical passeriform bird. The type host of this parasite is the Chestnut-crowned Antpitta (Grallaria ruficapilla, Grallariidae), an elusive native passerine bird whose natural history remains, to a large degree, unexplored. This bird was captured in Palacio forest in the damping zone of Chingaza National Natural Park, Cundinamarca, Colombia, at 2900 m above sea level (asl). Gametocytes of the new species develop both in roundish and fusiform host cells. This parasite is readily morphologically distinguishable from the described Leucocytozoon species because its host cells possess the narrow (needle-like) spindle-shaped processes, which length markedly exceeds their width. Additionally, the host cell nucleus markedly extends into the processes. Phylogenetic relationships were constructed based on a fragment of the mitochondrial cytochrome b gene and the complete mitochondrial genome. Phylogenetic analysis placed the lineage of L. pterotenuis in different positions depending on the length of the sequence analyzed that is likely due to poor sampling of Leucocytozoon species, especially from rare or non-passerine hosts, as well as a paucity of complete mitochondrial sequences of these parasites. Available data indicate that Leucocytozoon parasites are distributed mainly in mountain regions of the Neotropics where unique morphological forms have been recently discovered. To a better knowledge of the diversity of Leucocytozoon spp. and their host-vector-parasite interactions in Neotropical countries, additional deep and intensive samplings are needed, particularly in orders different to Passeriformes.

Description of Leucocytozoon quynzae sp. nov. (Haemosporida, Leucocytozoidae) from hummingbirds, with remarks on distribution and possible vectors of leucocytozoids in South America.

We describe Leucocytozoon quynzae sp. nov. (Haemosporida, Leucocytozoidae), which is the first Leucocytozoon parasite identified to species level in hummingbirds. It was found in the Amethyst-throated Sunangel (Heliangelus amethysticollis, Trochilidae, Apodiformes) captured in the Palacio Forest, which belongs to the damping zone of Chingaza National Natural Park, Cundinamarca, Colombia, at 2,900 m above sea level where the transmission occurs; the new species were found both in the high Andean forest and Paramo ecosystem. This parasite is described based on the morphology of its blood stages, a fragment of the mitochondrial cytochrome b gene, and the complete mitochondrial genome. Illustrations of blood stages of the new species are given, and the phylogenetic analysis places this lineage in a well-supported clade with other lineages of unidentified to species level leucocytozoids reported in the Trochilidae birds elsewhere. The new species possess gametocytes in roundish host cells; it can be readily distinguished from other similar leucocytozoids, primarily due to (1) a comma-like shape of the host cell nucleus, which extended one half or less of the circumference of the gametocyte and (2) a large number of prominent volutin granules in the cytoplasm. Identical mitochondrial cytochrome b sequence of Leucocytozoon quynzae was found in different hummingbird species at the type locality and also was reported in one passerine bird at the highlands of Peru. Leucocytozoon quynzae is the first leucocytozoid parasite described from South American birds; its transmission occurs both at low temperatures and high elevations. We discuss some patterns of distribution of avian leucocytozoids in South America and the role of Gigantodax spp. (Diptera, Simuliidae) as potential vectors of Leucocytozoon parasites in the Andean Region.

Leucocytozoon fringillinarum and Leucocytozoon dubreuili in Turdus fuscater from a Colombian Páramo ecosystem.

Leucocytozoon spp. infections have been rarely studied in Neotropical countries. The apparently low prevalence of these parasites compared to the Nearctic regions suggests the absence of competent vectors; however, a 21.3% overall prevalence has recently been reported in non-migratory birds from the páramo region of Chingaza National Natural Park (NNP), where Turdus fuscater (Great Thrush) is the species most frequently infected by these parasites. The present study provides the descriptions of the Leucocytozoon spp. detected in Great Thrushes trapped in Chingaza NNP. The parasites were confirmed by microscopic examination and PCR of blood, histopathology was also done. Leucocytozoon dubreuili and L. fringillinarum gametocytes were observed in blood smears. The corresponding cytochrome b (cyt b) lineages obtained of L. fringillinarum were closely related to lineages previously found in individuals infecting turdiid species sampled elsewhere. This is one of the few reports analyzing Leucocytozoon spp. infections in resident birds from a Neotropical country.

Description of Culicoides lisicarruni (Diptera: Ceratopogonidae), a new species from Cundinamarca, Colombia

A new species of Culicoides of the subgenus Diphaomyia Vargas from high altitudes of the Andes in Colombia is described and photographied. The species is compared with its similar congener Culicoides marinkellei Wirth & Lee. Data on the collecting site and notes on the species daily activity are also provided.

Description of Culicoides lisicarruni (Diptera: Ceratopogonidae), a new species from Cundinamarca, Colombia.

A new species of Culicoides of the subgenus Diphaomyia Vargas from high altitudes of the Andes in Colombia is described and photographied. The species is compared with its similar congener Culicoides marinkellei Wirth & Lee. Data on the collecting site and notes on the species daily activity are also provided.