Polymorphism in trypomastigotes of Trypanosoma (Megatrypanum) minasense in the blood of experimentally infected squirrel monkey and marmosets.

Experimental infections by Trypanosoma (Megatrypanum) minasense were performed in primates - Saimiri sciureus and Callithrix penicillata - with the objective of searching for morphological variations of the blood trypomastigotes with respect to hosts and time of infection. We carried out morphological and morphometric analysis of blood trypomastigotes. Illustrations are given. Both the squirrel monkey and marmoset became infected after the injection of blood trypomastigotes of T. minasense, although the parasitaemia were briefer in the squirrel monkey. The parasites detected in the later host were narrower and shorter than those found in the inoculated marmoset. In the marmoset, the blood stream parasites derived from culture metacyclic trypomastigotes were considerably smaller than those derived from the inoculation of infected blood. Stronger evidence of polymorphism was found when, at the same time of infection, the blood trypomastigotes found in squirrel monkey had smaller length, body width and the distance from posterior end of the body to the kinetoplast almost four times smaller than the parasite found in the marmoset. Therefore, conflicting results on morphology and morphometry of T. minasense obtained by previous investigators could be due to polymorphism.     

Molecular and morphometric identification of Trypanosoma (Megatrypanum) minasense in blood samples of marmosets (Callithrix: Callithrichidae) from the city of Rio de Janeiro,Brazil.

Callithrix jacchus and C. penicillata marmosets are invasive to the state of Rio de Janeiro, Brazil, threatening the native and vulnerable C. aurita. Both invasive species can be hosts of Trypanosoma cruzi, T. minasense, T. rangeli and T. devei. We aim to investigate the occurrence of trypanosomatids in Callithrix sp. from Jardim Botânico do Rio de Janeiro, located in a central and populous area of the city. Fifteen marmosets were captured. Blood samples were collected for light microscopy and molecular genetics analysis. Parasites morphometric values were evaluated for species identification. DNA was extracted from blood samples by phenol-chloroform method, for partial amplification of the 18S rRNA gene. PCR products were sequenced and aligned using BLAST®. A maximum likelihood phylogenetic tree was constructed to analyze the proximity between the observed sequences. By light microscopy, trypomastigotes were detected in five of the fifteen marmosets. Morphometric measurements and size polymorphism corresponded to those previously described for T. minasense. The DNA sequences of approximately 600 base pairs of the 18S rRNA gene were obtained for three samples with 99% identity with T. minasense sequence, forming a cluster in the phylogenetic tree and corroborating morphometric analysis. Trypanosoma minasense is a highly specific parasite to non-human primates considered as non-pathogenic. There is no evidence of infection in humans and these parasite findings from invasive marmosets do not support additional risks for the native species.     

Meiotic characterization and sex determination system of neotropical primates: Bolivian squirrel monkey Saimiri boliviensis (primates: Cebidae)

The chromosomal sex determination system differs among platyrrhine monkeys more than any other group of primates. Although a number of studies have investigated mitotic chromosomes across platyrrhine species, the meiotic chromosomes of many genera have not yet been described. The goal of this study was to characterize the sex determination system of Saimiri boliviensis. We described for the first time the meiotic cycle, confirming the sexual system in germ cells from testicular biopsies of four adult male S. boliviensis. All specimens were weighed and testicular volume was measured. We observed 22 bivalents corresponding to 2N = 44, and a "human-like" XY bivalent was found in diakinesis/metaphase I. In addition, mitotic studies from blood samples of both sexes were performed and G- and C-banding patterns agreed with previously reported karylogy of S. boliviensis boliviensis. Further meiotic studies should be performed in New World primates based on the great value of those studies for systematic evolutionary biology and conservation programs.     

Evolutionary disequilibrium and activity period in primates: a bayesian phylogenetic approach

Activity period plays a central role in studies of primate origins and adaptations, yet fundamental questions remain concerning the evolutionary history of primate activity period. Lemurs are of particular interest because they display marked variation in activity period, with some species exhibiting completely nocturnal or diurnal lifestyles, and others distributing activity throughout the 24-h cycle (i.e., cathemerality). Some lines of evidence suggest that cathemerality in lemurs is a recent and transient evolutionary state (i.e., the evolutionary disequilibrium hypothesis), while other studies indicate that cathemerality is a stable evolutionary strategy with a more ancient history. Debate also surrounds activity period in early primate evolution, with some recent studies casting doubt on the traditional hypothesis that basal primates were nocturnal. Here, we used Bayesian phylogenetic methods to reconstruct activity period at key points in primate evolution. Counter to the evolutionary disequilibrium hypothesis, the most recent common ancestor of Eulemur was reconstructed as cathemeral at ～9-13 million years ago, indicating that cathemerality in lemurs is a stable evolutionary strategy. We found strong evidence favoring a nocturnal ancestor for all primates, strepsirrhines and lemurs, which adds to previous findings based on parsimony by providing quantitative support for these reconstructions. Reconstructions for the haplorrhine ancestor were more equivocal, but diurnality was favored for simian primates. We discuss the implications of our models for the evolutionary disequilibrium hypothesis, and we identify avenues for future research that would provide new insights into the evolution of cathemerality in lemurs.     

Cysteine proteases of Trypanosoma (Megatrypanum) theileri: Cathepsin L-like gene sequences as targets for phylogenetic analysis,genotyping diagnosis

Although Trypanosoma theileri and allied trypanosomes are the most widespread trypanosomes in bovids little is known about proteolytic enzymes in these species. We have characterized genes encoding for cathepsin L-like (CATL) cysteine proteases from isolates of cattle, water buffalo and deer that largely diverged from homologues of other trypanosome species. Analysis of 78 CATL catalytic domain sequences from 22 T. theileri trypanosomes disclosed 6 genotypes tightly clustered together into the T. theileri clade. The CATL genes in these trypanosomes are organized in tandem arrays of ～ 1.7 kb located in 2 chromosomal bands of 600–720 kb. A diagnostic PCR assay targeting CATL sequences detected T. theileri of all genotypes from cattle, buffaloes and cervids and also from tabanid vectors. Expression of T. theileri cysteine proteases was demonstrated by proteolytic activity in gelatin gels and hydrolysis of Z-Phe-Arg-AMC substrate. Results from this work agree with previous data using ribosomal and spliced leader genes demonstrating that CATL gene sequences are useful for diagnosis, population genotyping and evolutionary studies of T. theileri trypanosomes.     

Pollen morphology and phylogenetic relationships in neotropical Phyllanthus (Euphorbiaceae)

In order to provide new insights into phylogenetic relationships among the neotropical taxa of Phyllanthus , 28 illustrations are provided of the pollen grains of 22 selected species studied from 11 sections of the subgenera represented in the neotropics. Special attention has been given to subgenus Conami because of its variability in pollen morphology: of eight species illustrated, the apertures are diploporate colpi in three species and pores in five species; exine ornamentation is vermiculate in two species and pilate in the other six species. The six species in the neotropical sections Pityrocladus and Microglochidion (subgenus Emblica ) are characterized by prolate grains with an increased number of colpi (4–8). Of particular interest are species in which the pollen exine is clypeate (with exine shields); clypeate pollen grains are illustrated in two species of subgenus Xylophylla and in one species of section Cyclanthera that has unique exine shields with single central pila. The pollen of the one Brazilian phylloclade-bearing species illustrated (in section Choretropsis ) has 3-colporate grains with reticulate exine, typical for subgenus Phyllanthus , and very different from the clypeate grains of the West Indian phylloclade-bearing species in section Xylophylla . This pollen evidence clearly demonstrates homoplasy in the origin of phylloclades in Phyllanthus . Pollen morphological data suggest that the neotropical taxa of Phyllanthus have arisen following colonization from Africa (subgenus Kirganelia ) and Asia (subgenus Emblica ). © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 138 , 325–338.     

Evidence for a role of the host-specific flea (Paraceras melis) in the transmission of Trypanosoma (Megatrypanum) pestanai to the European badger

We investigated the epidemiology of Trypanosoma pestanai infection in European badgers (Meles meles) from Wytham Woods (Oxfordshire, UK) to determine prevalence rates and to identify the arthropod vector responsible for transmission. A total of 245 badger blood samples was collected during September and November 2009 and examined by PCR using primers derived from the 18S rRNA of T. pestanai. The parasite was detected in blood from 31% of individuals tested. T. pestanai was isolated from primary cultures of Wytham badger peripheral blood mononuclear cells and propagated continually in vitro. This population was compared with cultures of two geographically distinct isolates of the parasite by amplified fragment length polymorphism (AFLP) and PCR analysis of 18S rDNA and ITS1 sequences. High levels of genotypic polymorphism were observed between the isolates. PCR analysis of badger fleas (Paraceras melis) collected from infected individuals at Wytham indicated the presence of T. pestanai and this was confirmed by examination of dissected specimens. Wet smears and Giemsa-stained preparations from dissected fleas revealed large numbers of trypanosome-like forms in the hindgut, some of which were undergoing binary fission. We conclude that P. melis is the primary vector of T. pestanai in European badgers.     

Anatomy and phylogenetic analysis of the neotropical callichthyid catfishes (Ostariophysi, Siluriformes)

Based mainly on morphological characters, the phylogenetic relationships among genera and some species groups of the neotropical family Callichthyidae were examined. A study of the osteology of a generalized callichthyid, Callichthys callichthys (Linnaeus), with detailed comparisons among representatives of the remaining genera in the family, is presented and used as a basis for the phylogenetic analysis. A single most parsimonious tree supported the monophyly of the family Callichthyidae based on 28 derived features and the division of the family in the subfamilies Corydoradinae and Callichthyinae. In the subfamily Corydoradinae, the genus Aspidoras is the sister-group of the clade formed by Corydoras plus Brochis. Five derived features support the monophyly of this clade and four support the monophyly of Brochis. No characters, however, were found to support the genus Corydoras. In the subfamily Callichthyinae, Dianema and Hopbstemum are sister-taxa. Megalechis represents the sister-group of Dianema plus Hoplosternum and Lepthoplosternum represents the sister-group to Megalechis plus Dianema plus Hopbstemum. Finally, Callichthys is considered the least derived member of the subfamily, and is hypothesized as the sister-group of the remaining species. A key to all callichthyid genera is provided.     

Speciation timing and neotropical biodiversity: the Tertiary-Quaternary debate in the light of molecular phylogenetic evidence

The evolutionary origin of extant species in the Neotropics, one of the most biodiverse regions of the world, has been widely debated. One hypothesis is that neotropical species emerged primarily during the Quaternary (the last ~2 million years), favoured by alternating glacial/interglacial climates. An opposite view proposes an older Tertiary origin linked primarily to palaeogeographical changes. Here, a thorough review of the available literature on DNA molecular dating shows that the Tertiary–Quaternary debate no longer makes sense. Indeed, the > 1400 neotropical species whose origin has been dated have appeared in a continual fashion since the late Eocene/early Oligocene (~39 million years before present) to the Quaternary. Palaeogeographical mechanisms of speciation are relatively well accepted, but diversification processes linked to climate are still controversial. These results are important to unravel both the origin of present-day biodiversity patterns at both local and global scales and the genetic and environmental mechanisms involved, which are two crucial aspects for suitable biodiversity conservation strategies.     

Release vocalizations in neotropical toads (Bufo): ecological constraints and phylogenetic implications

The release vocalizations of four nominal neotropical toad taxa ( Bufo achalensis , Bufo limensis , Bufo spinulosus , Bufo arenarum ) which compose three sympatric species pairs, were quantitatively analysed and homologous call types statistically compared. The first three taxa are closely related members of the mainly Andean Bufo spinulosus species group in which advertisement calls are absent. The specific vocal repertoire consisted of a uniform, unpulsed release call and one (in B. arenarum, two) pulsed release trill(s) which were given either singly or in series of up to three single calls. The uniform call was similar in structure and dominant (= basic) frequency in all taxa and probably represents an unspecific acoustic signal which is used to avoid heterospecific amplexus between sympatric toads. The release trills significantly differed in most call features among the taxa and, using discriminant analyses, even single calls were correctly assigned to sympatric pairs of species. In contrast, calls of allopatric pairs of species were confounded at rates of up to 36% indicating that selection towards species-specific signals increases when different species live in sympatry. The release trills of two allopatric populations (Perú, Argentina) assigned to B. spinulosus were similar in structure, but nevertheless features of pulse train permitted an unequivocal distinction, suggesting a long-lasting independent evolution. The taxonomic significance of this finding remains to be evaluated in further investigations. The complex vocal repertoire also offered the opportunity to assess phylogenetic relationships among the taxa. The extra-Andean B. achalensis seems to be closer related to the Andean B. spinulosus than this species is to the widely sympatric B. limensis – a species with several primitive character states indicating an early separation from the ancestral stock.     

Some problems in the study of the origin of neotropical biodiversity using palaeoecological and molecular phylogenetic evidence

This study contends that progress in the understanding of the origin and maintenance of extant neotropical biodiversity by means of empirical evidence is hampered by the persistence of anachronistic conceptual approaches, notably the adherence to closed paradigms or ruling theories. The topic is discussed from three main perspectives: (1) the tendency to shift from one paradigm to another, (2) the use and abuse of broad generalizations from a single or a few case studies and (3) the use of inadequate phylogenetic dating (usually crown dating alone) for a sound appraisal of diversification timing. The origin of neotropical biodiversity is a complex subject that requires an open-minded attitude to be fully captured. The solution proposed is fairly easy, somewhat trivial, and rooted in the classical multiple working hypotheses (MWH) approach. The MWH seeks to explore any explanation possible for observed phenomena and develop every testable hypothesis in relation to their possible causes. The MWH approach promotes thoroughness, suggests lines of inquiry that might otherwise be overlooked and develops the habit of parallel and complex thought that, unfortunately, is not fully developed in the empirical study of neotropical biodiversity.     

Multilocus phylogeographical analysis of Trypanosoma (Megatrypanum) genotypes from sympatric cattle and water buffalo populations supports evolutionary host constraint and close phylogenetic relationships with genotypes found in other ruminants

Species of the subgenus Trypanosoma (Megatrypanum) have been reported in cattle and other domestic and wild ruminants worldwide. A previous study in Brazil found at least four genotypes infecting cattle (Bos taurus), but only one in water buffalo (Bubalus bubalis). However, the small number of isolates examined from buffalo, all inhabiting nearby areas, has precluded evaluation of their diversity, host associations and geographical structure. To address these questions, we evaluated the genetic diversity and phylogeographical patterns of 25 isolates from water buffalo and 28 from cattle from four separate locations in Brazil and Venezuela. Multigene phylogenetic analyses of ssrRNA, internal transcribed spacer of rDNA (ITSrDNA), 5SrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH), mitochondrial cytochrome b (Cyt b), spliced leader (SL) and cathepsin L-like (CATL) sequences positioned all isolates from sympatric and allopatric buffalo populations into the highly homogeneous genotype TthIA, while the cattle isolates were assigned to three different genotypes, all distinct from TthIA. Polymorphisms in all of these sequences separated the trypanosomes infecting water buffalo, cattle, sheep, antelope and deer, and suggested that they correspond to separate species. Congruent phylogenies inferred with all genes indicated a predominant clonal structure of the genotypes. The multilocus analysis revealed one monophyletic assemblage formed exclusively by trypanosomes of ruminants, which corresponds to the subgenus T. (Megatrypanum). The high degree of host specificity, evidenced by genotypes exclusive to each ruminant species and lack of genotype shared by different host species, suggested that the evolutionary history of trypanosomes of this subgenus was strongly constrained by their ruminant hosts. However, incongruence between ruminant and trypanosome phylogenies did not support host-parasite co-evolution, indicating that host switches have occurred across ruminants followed by divergences, giving rise to new trypanosome genotypes adapted exclusively to one host species.     

Refined molecular characterization of the breakpoints in small inv dup(15) chromosomes

Inv dup(15) is the most common supernumerary marker chromosome in humans. To investigate the mechanism responsible for this frequent chromosome rearrangement, we characterized the breakpoints in 18 individuals with small inv dup(15) chromosomes [i.e., negative for the Prader-Willi (PWS)/Angelman syndrome (AS) critical region]. Since two proximal breakpoint regions (“hotspots”) for PWS/AS deletions have been previously identified with the most proximal 15q markers D15S541/S542 and S543, we hypothesized that formation of the small inv dup(15) chromosomes may involve one or both of these breakpoint hotspots. By analysis with S542, both breakpoint regions were found to be involved in approximately equal frequencies. In ten cases, the inv dup(15) was negative for S542 (Class I), indicating the breakpoint is between the centromere and the most proximal marker on chromosome 15. For the other eight cases, S542 was positive by fluorescence in situ hybridization (5/5) and/or microsatellite analysis (7/7), but S543 was negative (Class II). These two breakpoint regions appear to be the same as the two proximal breakpoints reported in the common PWS/AS deletions. To initiate cloning and sequencing of the Class II breakpoint, the gap in the yeast artificial chromosome (YAC) contig between S541/S542 and S543 was filled by screening the CEPH YAC and mega-YAC libraries. YACs 705C2 and 368H3 were found to bridge this gap, and therefore contain the more distal breakpoint region. The finding of consistent breakpoints in small inv dup(15), like that found in PWS/AS deletions, provides strong evidence for hotspots for chromosome breakage in this region. In addition, our results show that two extra copies (tetrasomy) of the region from 15cen to the euchromatic region containing S542 are present in individuals with Class II breakpoints. Since most individuals carrying a small inv dup(15) are phenotypically normal, the euchromatin region included in the small inv dup(15) chromosomes does not appear to contain genes with clinically significant dosage effects. Received: 23 May 1996 / Revised: 7 August 1996     

A molecular phylogeny of warbling-finches (Poospiza): paraphyly in a neotropical Emberizid genus

We investigated the phylogenetic relationships of 12 species within a single genus of neotropical passerine (Poospiza) using 849 bp (283 codons) of the cytochrome b mitochondrial gene. We further explored evolutionary affinities of these taxa using sequence from an additional 47 thraupine (tanagers) and 7 emberizine (sparrows and buntings) genera, members of the predominantly New World family Emberizidae. Poospiza have traditionally been considered part of the emberizine radiation. However, our analyses suggest that members of this genus are more closely related to some thraupine lineages than they are to the other neotropical emberizine genera included in our study (Atlapetes, Embernagra, Melopyrrha, Phrygilus, Saltatricula, Tiaris). Although member taxa are closely related, the genus Poospiza appears to be paraphyletic with representatives of 6 thraupine genera (Cnemoscopus, Cypsnagra, Hemispingus, Nephelornis, Pyrrhocoma, Thylpopsis) interspersed among four well-supported Poospiza clades. The majority of species within this Poospiza-thraupine clade have geographic ranges that are exclusive to, or partially overlap with, the Andes Mountains. It is probable that these mountains have played an important role in driving cladogenesis within this group. Sequence divergence (transversions only; mean 4.7+/-1.3%) within the clade suggests that much of this diversification occurred within the late Miocene and Pliocene, a period coincident with major orogenic activity in central-western South America.     

Developmental Stages of Trypanosoma (Megatrypanum) freitasi Rego, Magalhaes & Siqueira, 1957 in the Opossum Didelphis marsupialis (Marsupialia, Didelphidae)

Trypanosoma (Megatrypanum) freitasi, a parasite of marsupials of the genus Didelphis, has been found to undergo in the lumen of the scent (anal) glands of its vertebrate host, a cycle such as usually occurs in the intestinal tract of the insect vectors of trypanosomatids and similar to what has been reported for Trypanosoma (Schizotrypanum) cruzi. The invertebrate host of Trypanosoma freitasi is still unknown. Developmental stages of the trypanosome in its mammalian host, especially the dividing epimastigotes, multinucleate plasmodial forms and rosettes found in the lumen of the scent glands of a naturally infected Didelphis marsupialis are described and illustrated.     

Trypanosoma (Megatrypanum) melophagium in the sheep ked Melophagus ovinus from organic farms in Croatia: phylogenetic inferences support restriction to sheep and sheep keds and close relationship with trypanosomes from other ruminant species

Trypanosoma (Megatrypanum) melophagium is a parasite of sheep transmitted by sheep keds, the sheep-restricted ectoparasite Melophagus ovinus (Diptera: Hippoboscidae). Sheep keds were 100% prevalent in sheep from five organic farms in Croatia, Southeastern Europe, whereas trypanosomes morphologically compatible with T. melophagium were 86% prevalent in the guts of the sheep keds. Multilocus phylogenetic analyses using sequences of small subunit rRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase, spliced leader, and internal transcribed spacer 1 of the rDNA distinguished T. melophagium from all allied trypanosomes from other ruminant species and placed the trypanosome in the subgenus Megatrypanum. Trypanosomes from sheep keds from Croatia and Scotland, the only available isolates for comparison, shared identical sequences. All biologic and phylogenetic inferences support the restriction of T. melophagium to sheep and, especially, to the sheep keds. The comparison of trypanosomes from sheep, cattle, and deer from the same country, which was never achieved before this work, strongly supported the host-restricted specificity of trypanosomes of the subgenus Megatrypanum. Our findings indicate that with the expansion of organic farms, both sheep keds and T. melophagium may re-emerge as parasitic infections of sheep.