The roles of vicariance and dispersal in the differentiation of two species of the Rhinella marina species complex.

The high levels of Neotropical biodiversity are commonly associated with the intense Neogene-Quaternary geological events and climate dynamics. Here, we investigate the evolutionary history of two species of Neotropical closely related amphibians (R. horribilis and R. marina). We combine published data with new mitochondrial DNA sequences and multiple nuclear markers, including 12 microsatellites. The phylogenetic analyses showed support for grouping the samples in two main clades; R. horribilis (Central America and Mexico) and R. marina (South America east of the Andes). However, the phylogenetic inferences also show an evident mito-nuclear discordance. We use Approximate Bayesian Computation (ABC) to test the role of different events in the diversification between the two groups recovered. We found that both species were affected primarily by a recent Pleistocene divergence, which was similar to the divergence estimate revealed by the Isolation-with-Migration model, under persistent bidirectional gene flow through time. We provide the first evidence that R. horribilis is differentiated from the South American R. marina at the nuclear level supporting the taxonomic status of R. horribilis, which has been controversial for more than a century.

Phylogeography of endemic Xantus' hummingbird (Hylocharis xantusii) shows a different history of vicariance in the Baja California Peninsula.

Studies of phylogeographic patterns provide insight into the processes driving lineage divergence in a particular region. To identify the processes that caused phylogeographic breaks, it is necessary to use historical information and a set of appropriate molecular data to explain current patterns. To understand the influence of geological or ecological processes on the phylogeography of the only species of hummingbird endemic to the Baja California Peninsula, Hylocharis xantusii, mitochondrial DNA sequences of three concatenated genes (Cyt-b, COI and ND2; 2297bp in total) in 100 individuals were analyzed. The spatial analyses of genetic variation showed phylogeographic structure consisting of a north, central and south regions. According to estimated divergence times, two vicariant events are supported, permanent separation of the peninsula and formation of the Gulf of California at 5mya and temporary isolation of the southern region at the Isthmus of La Paz at 3mya. The temporal frame of genetic differentiation of intraspecific haplotypes indicates that 90% of haplotypes diverged within the last 500,000years, with a population expansion 80,000years ago. Only four haplotypes diverged ∼2.2 my and occurred in the south (Hxan_36, 38 and 45), and north (Hxan_45 and 56) regions; only haplotype 45 is shared between south and north populations. These regions also have the most recent haplotypes from 12,500 to 16,200years ago, and together with high levels of genetic diversity, we suggest two refuge areas, the Northern and Southern regions. Our results indicate that the phylogeographic pattern first results from vicariance processes, then is followed by historical and recent climate fluctuations that influenced conditions on the peninsula, and it is also related to oases distribution. This study presents the first investigation of phylogeography of the peninsular' endemic Xantus' hummingbird.

Sensitivity of Phakopsora pachyrhizi towards quinone-outside-inhibitors and demethylation-inhibitors, and corresponding resistance mechanisms.

BACKGROUND: Since the invasion of Phakopsora pachyrhizi (Syd. & P. Syd.) in Brazil, there have been detrimental yield losses of soybeans [Glycine max (L.) Merr.]. Disease management is mainly based on fungicide treatment. The sensitivity of single P. pachyhrizi isolates towards different demethylation-inhibitors (DMIs) and quinone-outside-inhibitors (QoI) was surveyed and the corresponding resistance mechanisms were analysed. RESULTS: The QoI-response remained stable, while a loss of sensitivity towards DMIs occurred. Molecular analyses of cytochrome b showed an intron after codon 143 which is reported to prevent the development of a G143A mutation. Analysis of cyp51 revealed that point mutations and overexpression are involved in the sensitivity reduction towards DMIs. Of the detected mutations, Y131F and Y131H, respectively, and K142R are likely homologous to mutations found in other pathogens. As suggested by modelling studies, these three mutations as well as additional mutations F120L, I145F and I475T correlate to increased effective doses of 50%, ED50 -values, towards all tested DMIs. Furthermore, a constitutive up-regulation of the cyp51-gene up to ten-fold was noticed in some of the DMI-adapted isolates, while all sensitive isolates responded as the wild type. CONCLUSION: The G143A mutation is thought to result in significant as well as stable resistance factors towards QoIs, while other mutations play only a minor role. Since G143A development is prevented in Phakopsora pachyhrizi, a stable control of soybean rust with QoIs in future is rather likely. In contrast, a shifting in sensitivity towards DMIs has been observed, which is due to multiple independent mechanisms.

Discordant genetic diversity and geographic patterns between Crassicutis cichlasomae (Digenea: Apocreadiidae) and its cichlid host, "Cichlasoma" urophthalmus (Osteichthyes: Cichlidae), in Middle-America.

Genetic analyses of hosts and their parasites are key to understand the evolutionary patterns and processes that have shaped host-parasite associations. We evaluated the genetic structure of the digenean Crassicutis cichlasomae and its most common host, the Mayan cichlid "Cichlasoma" urophthalmus, encompassing most of their geographical range in Middle-America (river basins in southeastern Mexico, Belize, and Guatemala together with the Yucatan Peninsula). Genetic diversity and structure analyses were done based on 167 cytochrome c oxidase subunit 1 sequences (330 bp) for C. cichlasomae from 21 populations and 161 cytochrome b sequences (599 bp) for "C." urophthalmus from 26 populations. Analyses performed included phylogenetic tree estimation under Bayesian inference and maximum likelihood analysis, genetic diversity, distance and structure estimates, haplotype networks, and demographic evaluations. Crassicutis cichlasomae showed high genetic diversity values and genetic structuring, corresponding with 4 groups clearly differentiated and highly divergent. Conversely, "C." urophthalmus showed low levels of genetic diversity and genetic differentiation, defined as 2 groups with low divergence and with no correspondence with geographical distribution. Our results show that species of cichlids parasitized by C. cichlasomae other than "C." urophthalmus, along with multiple colonization events and subsequent isolation in different basins, are likely factors that shaped the genetic structure of the parasite. Meanwhile, historical long-distance dispersal and drought periods during the Holocene, with significant population size reductions and fragmentations, are factors that could have shaped the genetic structure of the Mayan cichlid.

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.

Molecular and catalytic properties of the aldehyde dehydrogenase of Gluconacetobacter diazotrophicus, a quinoheme protein containing pyrroloquinoline quinone, cytochrome b, and cytochrome c.

Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone.

Pleistocene climatic cycling and diversification of the Andean treefrog, Hypsiboas andinus.

Our understanding of the causes of diversification of Neotropical organisms lags behind that of Northern Hemisphere biota, especially for montane and temperate regions of southern South America. We investigated the mitochondrial DNA genealogical patterns in 262 individuals of the frog Hypsiboas andinus from 26 sites across the eastern ranges of the Andes Mountains in Argentina and Bolivia. Our phylogenetic analyses indicate at least three distinct lineages: one representing H. andinus from Northwestern Argentina and southern Bolivia, at least one H. andinus lineage from northern Bolivia, and one clade containing both H. andinus (from the southern portion of the species range) and its putative sister taxon Hypsiboas riojanus. Hypsiboas andinus samples from northern Bolivia are well differentiated and may represent distinct species. The northern Argentine H. andinus lineage and southern H. andinus/H. riojanus lineage likely diverged between 2 and 6 million years ago; their current sympatry may be the result of secondary contact due to range expansion after isolation during Andean uplift or may reflect cryptic species. Within the geographically extensive northern H. andinus clade, we found significant geographical structuring consistent with historical fragmentation and subsequent range expansion. The timing of this fragmentation and range expansion coincide with the Pleistocene, a time of extensive climatic cycling and vegetational shifts. Average divergence among clades is lower than those found for other Neotropical taxa, highlighting the potential importance of recent climatic history in diversification in the southern Andes.

Molecular paleoparasitological diagnosis of Ascaris sp. from coprolites: new scenery of ascariasis in pre-Colombian South America times.

Paleoparasitological studies using microscopy showed that Ascarisand Trichuris trichiura are the human intestinal parasites most found in archaeological sites. However, in pre-Columbian South American archaeological sites, Ascaris is rare. In this work we standardized a molecular methodology for Ascaris diagnosis directly from ancient DNA retrieved from coprolites. Using cythochrome b gene (142 bp) target, ancient DNA sequences were retrieved from South American samples, negative by microscopy. Moreover, the methodology applied was sensitive enough to detect ancient DNA extracted from 30 Ascaris eggs from an European coprolite. These results revealed a new scenery for the paleodistribution of Ascaris in South America.

Co-amplification of mitochondrial pseudogenes in Calomys musculinus (Rodentia, Cricetidae): a source of error in phylogeographic studies.

In a previous phylogeographic study of the rodent Calomys musculinus, 24 haplotypes of the mitochondrial DNA D-loop region were detected using the restriction fragment length polymorphism technique (PCR-RFLP). Seven percent of the individuals showed patterns in which the sum of the sizes of the restriction fragments exceeded the size of the original PCR product. In the present paper we analyze possible causes of these atypical haplotypes. PCR products were cloned, and two or three different clones from a single individual were detected by their RFLP patterns. Nine clones with different restriction patterns were selected for sequence analyses. A maximum parsimony phylogenetic analysis revealed two well-supported paraphyletic groups. One group comprised sequences showing low nucleotide divergence compared with the most common haplotypes detected in the phylogeographic study. The other group was basal to the three species of Calomys other than C. musculinus included in the study; the mutations in the short portion of the cytochrome b gene amplified corresponded to 12 amino acid substitutions. The results suggest that two independent insertions of mtDNA sequences into the nucleus occurred; these sequences would co-amplify in the PCR procedure. Identification of pseudogenes is crucial to obtain reliable reconstruction of the intraspecific genealogy in phylogeographic studies.

Molecular paleoparasitological diagnosis of Ascaris sp. from coprolites: new scenery of ascariasis in pre-Colombian South America times

Paleoparasitological studies using microscopy showed that Ascarisand Trichuris trichiura are the human intestinal parasites most found in archaeological sites. However, in pre-Columbian South American archaeological sites, Ascaris is rare. In this work we standardized a molecular methodology for Ascaris diagnosis directly from ancient DNA retrieved from coprolites. Using cythochrome b gene (142 bp) target, ancient DNA sequences were retrieved from South American samples, negative by microscopy. Moreover, the methodology applied was sensitive enough to detect ancient DNA extracted from 30 Ascaris eggs from an European coprolite. These results revealed a new scenery for the paleodistribution of Ascaris in South America.

Population genetic studies of Alouatta belzebul from the Amazonian and Atlantic Forests.

Cytochrome b DNA sequence data (ca. 1,140 bp) of 66 Alouatta belzebul from the Amazonian and the Atlantic Forests of Brazil were used for phylogenetic reconstructions and population studies. Our sample consisted of 60 specimens from the Amazonian Forest (captured in 1984 and 1998 in Pará-PA state) and six specimens from the Atlantic Forest (Paraíba-PB state). We found 32 haplotypes, 23 in PA-1984 (with 12 present in more than one individual), 11 in PA-1998 (with two present in more than one individual), and a single haplotype in the PB sample. Animals from PA-1984 and PA-1998 shared three haplotypes while animals from Pará and Paraíba did not share any haplotype. We found 57 variable sites, consisting of 53 transitions and four transversions, with most replacements occurring at third codon position (77.19%) and less frequently at first and second positions (10.53 and 12.28%, respectively). Genetic distance between all haplotypes varied between 0 and 1.2%. Nucleotide diversity estimates between PA-1984 haplotypes and PA-1998 haplotypes were the same (π=0.01), and haplotype diversity estimates were very similar (h=0.96 and 0.93 for PA-1984 and PA-1998, respectively). Maximum parsimony, median-joining, split decomposition, and TCS showed that PA and PB haplotypes had not drastically diverged and that subsequent radiation within these regions was not apparent. No temporal structure was found between PA-1984 and PA-1998. The sum of square deviation estimate for PA-1984 equaled 0.01 (P=0.23), in agreement with a hypothetical model of sudden expansion contrary to PA-1998 whose sum of square deviation estimate (0.40; P=0.04) was not compatible with this model, although the small sample size of PA-1998 as well as the smaller area of capture could have also accounted for this result. Fu's F(s) and R(2) statistical neutrality tests corroborated these propositions. Lack of drastic differentiation was attributable to the once existing connection between the Atlantic and the Amazonian forests at a non-distant past.

Establishment of a mass screening method of sand fly vectors for Leishmania infection by molecular biological methods.

Surveillance of the prevalence of Leishmania and its vector, sand fly species, in endemic and surrounding areas is important for prediction of the risk and expansion of leishmaniasis. In this study, a method for the mass screening of sand flies for Leishmania infection was established. This method was applied to 319 field-captured specimens, and 5 positive sand flies were detected. Sand fly species were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of the18S rRNA gene, and all the positive flies were Lu. hartmanni. Furthermore, cytochrome b (Cyt b) gene sequence analyses identified all the parasites as Endotrypanum species including a probable novel species. Because the method requires minimum effort and can process a large number of samples at once, it will be a powerful tool for studying the epidemiology of leishmaniasis.

Comparative landscape genetics and the adaptive radiation of Darwin's finches: the role of peripheral isolation.

We use genetic divergence at 16 microsatellite loci to investigate how geographical features of the Galápagos landscape structure island populations of Darwin's finches. We compare the three most genetically divergent groups of Darwin's finches comprising morphologically and ecologically similar allopatric populations: the cactus finches (Geospiza scandens and Geospiza conirostris), the sharp-beaked ground finches (Geospiza difficilis) and the warbler finches (Certhidea olivacea and Certhidea fusca). Evidence of reduced genetic diversity due to drift was limited to warbler finches on small, peripheral islands. Evidence of low levels of recent interisland migration was widespread throughout all three groups. The hypothesis of distance-limited dispersal received the strongest support in cactus and sharp-beaked ground finches as evidenced by patterns of isolation by distance, while warbler finches showed a weaker relationship. Support for the hypothesis that gene flow constrains morphological divergence was only found in one of eight comparisons within these groups. Among warbler finches, genetic divergence was relatively high while phenotypic divergence was low, implicating stabilizing selection rather than constraint due to gene flow. We conclude that the adaptive radiation of Darwin's finches has occurred in the presence of ongoing but low levels of gene flow caused by distance-dependent interisland dispersal. Gene flow does not constrain phenotypic divergence, but may augment genetic variation and facilitate evolution due to natural selection. Both microsatellites and mtDNA agree in that subsets of peripheral populations of two older groups are genetically more similar to other species that underwent dramatic morphological change. The apparent decoupling of morphological and molecular evolution may be accounted for by a modification of Lack's two-stage model of speciation: relative ecological stasis in allopatry followed by secondary contact, ecological interactions and asymmetric phenotypic divergence.

Phylogeography and demography of Guianan harlequin toads (Atelopus): diversification within a refuge.

We investigated the genetic structure of populations of Guianan harlequin toads (genus Atelopus) and their evolutionary affinities to extra-Guianan congeners. Phylogenetic analysis of mitochondrial cytochrome b (cyt b) and NADH dehydrogenase subunit 2 (ND2) gene sequences produced well-supported clades largely corresponding to the four recognized taxa in the Guianas (Atelopus spumarius hoogmoedi, Atelopus spumarius barbotini, Atelopus franciscus, and Atelopus flavescens). Our findings suggest that the Guianan A. spumarius represent distinct evolutionary lineages that merit distinction from Amazonian conspecifics, and that the status of A. flavescens and A. franciscus is somewhat less clear. Approximately 69% of the observed genetic variation is accounted for by differences between these four recognized taxa. Coalescent-based estimates of gene flow between taxa suggest that these lineages are largely isolated from one another. Negligible rates of migration between populations and significant divergence within such close proximity suggests that although the region inhabited by these taxa is almost entirely undisturbed, significant habitat heterogeneity exists as to have produced a remarkable diversification of Atelopus within the eastern Guiana Shield. These results contradict the commonly held view of the Guiana Shield as a 'refuge' whose stability during late Tertiary and Quaternary climatic fluctuations served as a biotic reservoir. Instead, we provide evidence that climatic fluctuations during this time had a diversifying effect within the Guianan region.

Cryptic Neogene vicariance and Quaternary dispersal of the red-spotted toad (Bufo punctatus): insights on the evolution of North American warm desert biotas.

We define the geographical distributions of mitochondrial DNA (mtDNA) lineages embedded within a broadly distributed, arid-dwelling toad, Bufo punctatus. These patterns were evaluated as they relate to hypothesized vicariant events leading to the formation of desert biotas within western North America. We assessed mtDNA sequence variation among 191 samples from 82 sites located throughout much of the species' range. Parsimony-based haplotype networks of major identified lineages were used in nested clade analysis (NCA) to further elucidate and evaluate shallow phylogeographic patterns potentially associated with Quaternary (Pleistocene-Holocene) vicariance and dispersal. Phylogenetic analyses provided strong support for three monophyletic lineages (clades) within B. punctatus. The geographical distributions of the clades showed little overlap and corresponded to the general boundaries of the Peninsular Desert, and two continental desert regions, Eastern (Chihuahuan Desert-Colorado Plateau) and Western (Mojave-Sonoran deserts), geographically separated along the Rocky Mountains and Sierra Madre Occidental. The observed divergence levels and congruence with postulated events in earth history implicate a late Neogene (latest Miocene-early Pliocene) time frame for separation of the major mtDNA lineages. Evaluation of nucleotide and haplotype diversity and interpretations from NCA reveal that populations on the Colorado Plateau resulted from a recent, likely post-Pleistocene, range expansion from the Chihuahuan Desert. Dispersal across historical barriers separating major continental clades appear to be recent, resulting in secondary contacts in at least two areas. Given the observed contact between major clades, we speculated as to why the observed deep phylogeographic structure has not been eroded during the multiple previous interglacials of the Pleistocene.