Specific Clades Research Articles

Two-stage microbial community experimental design

Microbial community samples can be efficiently surveyed in high throughput by sequencing markers such as the 16S ribosomal RNA gene. Often, a collection of samples is then selected for subsequent metagenomic, metabolomic or other follow-up. Two-stage study design has long been used in ecology but has not yet been studied in-depth for high-throughput microbial community investigations. To avoid ad hoc sample selection, we developed and validated several purposive sample selection methods for two-stage studies (that is, biological criteria) targeting differing types of microbial communities. These methods select follow-up samples from large community surveys, with criteria including samples typical of the initially surveyed population, targeting specific microbial clades or rare species, maximizing diversity, representing extreme or deviant communities, or identifying communities distinct or discriminating among environment or host phenotypes. The accuracies of each sampling technique and their influences on the characteristics of the resulting selected microbial community were evaluated using both simulated and experimental data. Specifically, all criteria were able to identify samples whose properties were accurately retained in 318 paired 16S amplicon and whole-community metagenomic (follow-up) samples from the Human Microbiome Project. Some selection criteria resulted in follow-up samples that were strongly non-representative of the original survey population; diversity maximization particularly undersampled community configurations. Only selection of intentionally representative samples minimized differences in the selected sample set from the original microbial survey. An implementation is provided as the microPITA (Microbiomes: Picking Interesting Taxa for Analysis) software for two-stage study design of microbial communities.

Secondary structure models of 18S and 28S rRNAs of the true bugs based on complete rDNA sequences of Eurydema maracandica Oshanin, 1871 (Heteroptera,Pentatomidae).

The sequences of 18S and 28S rDNAs have been used as molecular markers to resolve phylogenetic relationships of Heteroptera for two decades. The complete sequences of 18S rDNAs have been used in many studies, while in most studies only partial sequences of 28S rDNAs have been used due to technical difficulties of amplifying the complete lengths. In this study, we amplified the complete 18S and 28S rDNA sequences of Eurydema maracandica Oshanin, 1871, and reconstructed the secondary structure models of the corresponding rRNAs. In addition, and more importantly, all of the length variable regions of 18S rRNA were compared among 37 families of Heteroptera based on 140 sequences, and the D3 region of 28S rRNA was compared among 51 families based on 84 sequences. It was found that 8 length variable regions could potentially serve as molecular synapomorphies for some monophyletic groups. Therefore discoveries of more molecular synapomorphies for specific clades can be anticipated from amplification of complete 18S and 28S rDNAs of more representatives of Heteroptera.

Molecular evolution and expression of archosaurian β‐keratins: Diversification and expansion of archosaurian β‐keratins and the origin of feather β‐keratins

The archosauria consist of two living groups, crocodilians, and birds. Here we compare the structure, expression, and phylogeny of the beta (β)-keratins in two crocodilian genomes and two avian genomes to gain a better understanding of the evolutionary origin of the feather β-keratins. Unlike squamates such as the green anole with 40 β-keratins in its genome, the chicken and zebra finch genomes have over 100 β-keratin genes in their genomes, while the American alligator has 20 β-keratin genes, and the saltwater crocodile has 21 β-keratin genes. The crocodilian β-keratins are similar to those of birds and these structural proteins have a central filament domain and N- and C-termini, which contribute to the matrix material between the twisted β-sheets, which form the 2-3 nm filament. Overall the expression of alligator β-keratin genes in the integument increases during development. Phylogenetic analysis demonstrates that a crocodilian β-keratin clade forms a monophyletic group with the avian scale and feather β-keratins, suggesting that avian scale and feather β-keratins along with a subset of crocodilian β-keratins evolved from a common ancestral gene/s. Overall, our analyses support the view that the epidermal appendages of basal archosaurs used a diverse array of β-keratins, which evolved into crocodilian and avian specific clades. In birds, the scale and feather subfamilies appear to have evolved independently in the avian lineage from a subset of archosaurian claw β-keratins. The expansion of the avian specific feather β-keratin genes accompanied the diversification of birds and the evolution of feathers.

Assessing phylogenetic relationships among galliformes: a multigene phylogeny with expanded taxon sampling in Phasianidae.

Galliform birds (relatives of the chicken and turkey) have attracted substantial attention due to their importance to society and value as model systems. This makes understanding the evolutionary history of Galliformes, especially the species-rich family Phasianidae, particularly interesting and important for comparative studies in this group. Previous studies have differed in their conclusions regarding galliform phylogeny. Some of these studies have suggested that specific clades within this order underwent rapid radiations, potentially leading to the observed difficulty in resolving their phylogenetic relationships. Here we presented analyses of six nuclear intron sequences and two mitochondrial regions, an amount of sequence data larger than many previous studies, and expanded taxon sampling by collecting data from 88 galliform species and four anseriform outgroups. Our results corroborated recent studies describing relationships among the major families, and provided further evidence that the traditional division of the largest family, the Phasianidae into two major groups (“pheasants” and “partridges”) is not valid. Within the Phasianidae, relationships among many genera have varied among studies and there has been little consensus for the placement of many taxa. Using this large dataset, with substantial sampling within the Phasianidae, we obtained strong bootstrap support to confirm some previously hypothesized relationships and we were able to exclude others. In addition, we added the first nuclear sequence data for the partridge and quail genera Ammoperdix, Caloperdix, Excalfactoria, and Margaroperdix, placing these taxa in the galliform tree of life with confidence. Despite the novel insights obtained by combining increased sampling of taxa and loci, our results suggest that additional data collection will be necessary to solve the remaining uncertainties.

The impact of paralogy on phylogenomic studies - a case study on annelid relationships.

Phylogenomic studies based on hundreds of genes derived from expressed sequence tags libraries are increasingly used to reveal the phylogeny of taxa. A prerequisite for these studies is the assignment of genes into clusters of orthologous sequences. Sophisticated methods of orthology prediction are used in such analyses, but it is rarely assessed whether paralogous sequences have been erroneously grouped together as orthologous sequences after the prediction, and whether this had an impact on the phylogenetic reconstruction using a super-matrix approach. Herein, I tested the impact of paralogous sequences on the reconstruction of annelid relationships based on phylogenomic datasets. Using single-partition analyses, screening for bootstrap support, blast searches and pruning of sequences in the supermatrix, wrongly assigned paralogous sequences were found in eight partitions and the placement of five taxa (the annelids Owenia, Scoloplos, Sthenelais and Eurythoe and the nemertean Cerebratulus) including the robust bootstrap support could be attributed to the presence of paralogous sequences in two partitions. Excluding these sequences resulted in a different, weaker supported placement for these taxa. Moreover, the analyses revealed that paralogous sequences impacted the reconstruction when only a single taxon represented a previously supported higher taxon such as a polychaete family. One possibility of a priori detection of wrongly assigned paralogous sequences could combine 1) a screening of single-partition analyses based on criteria such as nodal support or internal branch length with 2) blast searches of suspicious cases as presented herein. Also possible are a posteriori approaches in which support for specific clades is investigated by comparing alternative hypotheses based on differences in per-site likelihoods. Increasing the sizes of EST libraries will also decrease the likelihood of wrongly assigned paralogous sequences, and in the case of orthology prediction methods like HaMStR it is likewise decreased by using more than one reference taxon.

Hydra of Hawaii: phylogenetic relationships with continental species

The presence of freshwater hydra in the Hawaiian Islands, over 3,700 km from the nearest continental margin, provokes the question of how these animals could reach the islands. We examined three islands for hydra and found two species (the brown hydra, Hydra vulgaris Pallas 1766, and green hydra, H. viridissima Pallas 1766) present at multiple locations on Oahu and Kauai, and at a single site on Hawaii. Phylogenetic analysis based on the internal transcribed spacer (ITS1 and ITS2) regions of ribosomal DNA indicates that all collected strains of brown hydra were closely related to each other—consistent with a single introduced origin. The analysis also shows that all sampled Hawaiian brown hydra fall within a specific clade of H. vulgaris. This clade is sister to a North American clade and nested within a deeper North/Central America clade. The clade with all the Hawaiian brown hydra includes also individuals from Southern California, suggesting a Californian origin for the brown Hawaiian hydra. Hawaiian hydra were probably transported to the islands by man.

Theropod Tooth from the Upper Cretaceous Himenoura Group in the Koshikijima Islands, Southwestern Japan

Abstract. A theropod tooth found in the Upper Cretaceous Himenoura Group in the Koshikijima Islands, Kagoshima Prefecture, is described. Recent paleontological work in the region suggests a middle Campanian age for the specimen. Lacking a feature diagnostic of any specific theropod clade, this specimen is considered merely as Theropoda incertae sedis. The present finding confirms the potential of the Himenoura Group as a new source of information on the diversity of Theropoda during the Campanian.

Host specificity and coevolution of Flavobacteriaceae endosymbionts within the siphonous green seaweed Bryopsis

The siphonous green seaweed Bryopsis harbors complex intracellular bacterial communities. Previous studies demonstrated that certain species form close, obligate associations with Flavobacteriaceae. A predominant imprint of host evolutionary history on the presence of these bacteria suggests a highly specialized association. In this study we elaborate on previous results by expanding the taxon sampling and testing for host–symbiont coevolution Therefore, we optimized a PCR protocol to directly and specifically amplify Flavobacteriaceae endosymbiont 16S rRNA gene sequences, which allowed us to screen a large number of algal samples without the need for cultivation or surface sterilization. We analyzed 146 Bryopsis samples, and 92 additional samples belonging to the Bryopsidales and other orders within the class Ulvophyceae. Results indicate that the Flavobacteriaceae endosymbionts are restricted to Bryopsis, and only occur within specific, warm-temperate and tropical clades of the genus. Statistical analyses (AMOVA) demonstrate a significant non-random host–symbiont association. Comparison of bacterial 16S rRNA and Bryopsis rbcL phylogenies, however, reveal complex host–symbiont evolutionary associations, whereby closely related hosts predominantly harbor genetically similar endosymbionts. Bacterial genotypes are rarely confined to a single Bryopsis species and most Bryopsis species harbored several Flavobacteriaceae, obscuring a clear pattern of coevolution.

Editorial Commentary: Does Infection With Specific Clostridium difficile Strains or Clades Influence Clinical Outcome?

A number of DNA banding–based typing systems for classifying Clostridium difficile isolates have been devised including polymerase chain reaction (PCR) ribotyping, restriction endonuclease analysis (REA), and pulsed-field gel electrophoresis (PFGE) [1–4]. PCR ribotyping was developed in Wales and is commonly used in Europe, whereas REA and PFGE have been used more commonly in North America. In addition, toxinotyping, which is based on amplification of regions within the pathogenicity locus (PaLoc) for toxins A and B, has been used to describe “toxin variant” strains whose PaLoc genes differ from the standard or toxinotype “0” PaLoc to which most PCR ribotypes, REA groups, and PFGE types belong [5]. The aforementioned DNA-banding typing systems are invaluable for discriminating isolates from each other, but do not provide meaningful evolutionary analyses. Multilocus sequence typing (MLST), in which a selected number of housekeeping gene loci (7 in the case of C. difficile) are amplified and sequenced to yield sequence types (STs), can be used to group STs by evolutionary relationships into clades [6]. Members of a clade are considered to be derived from the same common ancestor and can be organized into evolutionary trees using phylogenetic or cladistic analyses. MLST classification of C. difficile clades agrees with previous classification based on DNA microarray comparative genomics and suggests that genotypes clustered by MLST may be an accurate proxy for whole-genome sequencing analysis [7]. STs correlate well with PCR ribotypes; however, as with toxinotyping, the vast majority of STs are clustered in a single clade, clade 1, with only 1 or 2 STs (ribotypes) in each of the remaining 4 clades (Table 1).

Phylogenomics of Annelida revisited: a cladistic approach using genome-wide expressed sequence tag data mining and examining the effects of missing data.

We present phylogenomic analyses of the most comprehensive molecular character set compiled for Annelida and its constituent taxa, including over 347000 aligned nucleotide sites for 39 taxa. The nucleotide data set was recovered using a pre-existing amino acid data set of almost 48000 aligned sites as a backbone for tBLASTn searches against NCBI. In addition, orthology determinations of the loci in the original amino acid data set were scrutinized using an All vs All Reciprocal Best Hit approach, employing BLASTp, and examining for statistical interdependency among the loci. This approach revealed considerable sequence redundancy among the loci in the original data set and a new data set was compiled, with the redundancy removed. The newly compiled nucleotide data set, the original amino acid data set, and the new reduced amino acid data set were subjected to parsimony analyses and two forms of bootstrap resampling. The last-named data set also was analysed using a maximum-likelihood approach. There were two main objectives to these analyses: (i) to examine the general topology, including support, resulting from the analyses of the new data sets and (ii) to assess the consistency of the branching patterns across optimality criteria by comparison with previous probabilistic approaches. The phylogenetic hypotheses resulting from analyses of the three data sets are largely unsupported, reflecting the continued difficulty of finding numerous, reliable, and suitable loci for a group as ancient as Annelida. Resulting parsimonious hypotheses disagree, in some respects, with the previous probabilistic approaches; Sedentaria and, in most cases, Errantia are not supported as monophyletic groups but Pleistoannelida is recovered as a (unsupported) monophyletic group in one of the three parsimony analyses as well as the likelihood analysis. In addition, we performed missing data titration studies to estimate the impact of missing data on overall support and support for specific clades.

Characterization and Comparison of Bacterial Communities Selected in Conventional Activated Sludge and Membrane Bioreactor Pilot Plants: A Focus on Nitrospira and Planctomycetes Bacterial Phyla

A pilot-scale membrane bioreactor (MBR) and a conventional activated sludge system (CAS) were in parallel operated to investigate the impact of the separation technology on the structure and functionality of the selected microbial community. Microbial communities as well as nitrogen removal efficiency of the biomass were characterized. Kinetics and microbial community structure turned out to be duly correlated. The impact of the separation technology on selective conditions and, in particular, the higher variability of solid separation efficiency in CAS with respect to MBR pilot plant possibly represented the main factor influencing the selection of bacterial communities. Concerning nitrifiers, bacteria of the genus Nitrospira were predominant in the MBR. This was in accordance with kinetics of nitrite-oxidizing bacteria that suggested the presence of k-strategists, while r-strategists were selected in the CAS plant, possibly because of the presence of transient higher concentrations of nitrite (in the range of 0.05-0.18 and of 0.05-4.4mg[Formula: see text]-N L(-1) in the MBR and CAS effluents, respectively). An unexpectedly high presence of bacteria belonging to two specific phylogenetic clades of Planctomycetes was found in both reactors.

Human Immunodeficiency Viruses Appear Compartmentalized to the Female Genital Tract in Cross-Sectional Analyses but Genital Lineages Do Not Persist Over Time

Whether unique human immunodeficiency type 1 (HIV) genotypes occur in the genital tract is important for vaccine development and management of drug resistant viruses. Multiple cross-sectional studies suggest HIV is compartmentalized within the female genital tract. We hypothesize that bursts of HIV replication and/or proliferation of infected cells captured in cross-sectional analyses drive compartmentalization but over time genital-specific viral lineages do not form; rather viruses mix between genital tract and blood. Eight women with ongoing HIV replication were studied during a period of 1.5 to 4.5 years. Multiple viral sequences were derived by single-genome amplification of the HIV C2-V5 region of env from genital secretions and blood plasma. Maximum likelihood phylogenies were evaluated for compartmentalization using 4 statistical tests. In cross-sectional analyses compartmentalization of genital from blood viruses was detected in three of eight women by all tests; this was associated with tissue specific clades containing multiple monotypic sequences. In longitudinal analysis, the tissues-specific clades did not persist to form viral lineages. Rather, across women, HIV lineages were comprised of both genital tract and blood sequences. The observation of genital-specific HIV clades only in cross-sectional analysis and an absence of genital-specific lineages in longitudinal analyses suggest a dynamic interchange of HIV variants between the female genital tract and blood.

Genome-wide analysis of the auxin response factor gene family in cucumber

Auxin response factors (ARFs) participate in the transcriptional regulation of a series of biological processes related to growth and development. The ARF genes comprise a large multigene family in plants. Recently, a draft of the full cucumber (Cucumis sativus) genome assembly has been released; however, none of the ARF genes have been characterized. We made a comprehensive analysis of ARF genes in this species. Fifteen ARF genes were identified and could be divided into three classes. Intron presence and position were conserved to some extent within one phylogenetic group. Based on genome distribution analysis, tandem duplication appears to have contributed to ARF gene expansion. Specific motifs were selectively distributed among specific clades. Putative cis-elements involved in auxin response, light signaling responses, phytohormones, and defense responses were identified in promoter regions of the ARF genes. Almost all of the ARF genes exhibited a constitutive expression pattern, implying that there are functional redundancies among the ARF proteins.

Discovery of Platyhelminth-Specific α/β-Integrin Families and Evidence for Their Role in Reproduction in Schistosoma mansoni

In all metazoa, the response of cells to molecular stimuli from their environment represents a fundamental principle of regulatory processes controlling cell growth and differentiation. Among the membrane-linked receptors mediating extracellular communication processes are integrin receptors. Besides managing adhesion to the extracellular matrix or to other cells, they arrange information flow into the cells by activating intracellular signaling pathways often acting synergistically through cooperation with growth factor receptors. Although a wealth of information exists on integrins in different model organisms, there is a big gap of knowledge for platyhelminths. Here we report on the in silico detection and reconstruction of α and β integrins from free-living and parasitic platyhelminths, which according to structural and phylogenetic analyses form specific clades separate from each other and from further metazoan integrins. As representative orthologs of parasitic platyhelminths we have cloned one beta-integrin (Smβ-Int1) and four alpha-integrins (Smα-Int1 - Smα-Int4) from Schistosoma mansoni; they were characterized by molecular and biochemical analyses. Evidence is provided that Smβ-Int1 interacts and co-localizes in the reproductive organs with known schistosome cellular tyrosine kinases (CTKs), of which the Syk kinase SmTK4 appeared to be the strongest interaction partner as shown by yeast two-hybrid analyses and coimmunoprecipitation experiments. By a novel RNAi approach with adult schistosomes in vitro we demonstrate for the first time multinucleated oocytes in treated females, indicating a decisive role Smβ-Int1 during oogenesis as phenotypically analyzed by confocal laser scanning microscopy (CLSM). Our findings provide a first comprehensive overview about platyhelminth integrins, of which the parasite group exhibits unique features allowing a clear distinction from the free-living groups. Furthermore, we shed first lights on the functions of integrins in a trematode model parasite, revealing the complexity of molecular processes involved in its reproductive biology, which may be representative for other platyhelminths.

Molecular elements of low‐oxygen signaling in plants

Oxygen and its limitation are emerging as a crucial factor in plant fitness, growth and development. Recent studies revealed the mechanisms by which oxygen is perceived by plant cells. This sensory system partly relies on an oxygen-mediated branch of the N-end rule pathway for protein degradation acting on a specific clade of ethylene responsive transcription factors (ERF-VII). A complementary regulative step is provided by aerobic sequestration of an ERF-VII protein at the plasma membrane and its timely release when hypoxia occurs. Complete absence of oxygen triggers the transient accumulation of reactive hydrogen peroxide and induces an additional set of reactive oxygen species-related genes involved in both signaling and attenuation of oxidative stress. Moreover, temporary hypoxic environments that are built up as consequence of dense cell packing have been demonstrated to trigger cell-fate determination in maize anthers. Similarly, limited oxygen delivery in bulky fruit or tuber tissues growing in aerobic conditions were shown to stimulate anaerobic-like responses. These advances in low-oxygen signaling and its effect on cell development highlight the importance of taking hypoxia into account in agronomical practices as well as in breeding programs.

Reconstructing the diversity of early terrestrial herbivorous tetrapods

Terrestrial herbivorous tetrapods first appear in the fossil record during the Late Carboniferous (306.5Ma). The diversification of herbivores is a key aspect of the transition to the modern trophic structure of terrestrial vertebrate ecosystems, because it allowed tetrapods to exploit terrestrial (i.e. non-aquatic) primary productivity. However, the palaeodiversity dynamics of the earliest terrestrial vertebrate herbivores have received relatively little attention, apart from a few studies that focus on specific clades. A new dataset containing 287 species occurrences of herbivorous tetrapods including the major Palaeozoic and Early Triassic clades Anomodontia, Archosauromorpha, Bolosauridae, Captorhinidae, Caseidae, Cynodontia, Dinocephalia, Diadectomorpha, Edaphosauridae, Pareiasauria, Poposauroidea, Procolophonoidea, Rhynchosauria, Silesauridae and Therocephalia is used to analyse palaeodiversity from the Late Carboniferous to the Middle Triassic (~306.5–~236Ma), taking into account the effects of potential sampling biases by using the number of tetrapod-bearing formations as a proxy. The results support a gradual increase in taxic diversity from the Late Carboniferous to the Wordian, followed by a dip in diversity during the Guadalupian (Middle Permian), and an increase to a peak in the Late Permian at the Wuchiapingian/Changhsingian boundary. Herbivorous tetrapods were strongly affected by the end-Permian mass extinction with both the herbivorous Pareiasauria and Captorhinidae becoming extinct and the observed number of anomodont species decreasing by up to 80%. The drop in observed diversity at the end Permian is dampened slightly because of the radiation of new herbivorous forms during the Early Triassic. A strong biological signal is apparent even after correcting for sampling.

Reconstruction of the Evolutionary Dynamics of the A(H1N1)pdm09 Influenza Virus in Italy during the Pandemic and Post-Pandemic Phases

The aim of this study was to reconstruct the evolutionary dynamics of the A(H1N1)pdm09 influenza virus in Italy during two epidemic seasons (2009/2010 and 2010/2011) in the light of the forces driving the evolution of the virus. Nearly six thousands respiratory specimens were collected from patients with influenza-like illness within the framework of the Italian Influenza Surveillance Network, and the A(H1N1)pdm09 hemagglutinin (HA) gene was amplified and directly sequenced from 227 of these. Phylodynamic and phylogeographical analyses were made using a Bayesian Markov Chain Monte Carlo method, and codon-specific positive selection acting on the HA coding sequence was evaluated. The global and local phylogenetic analyses showed that all of the Italian sequences sampled in the post-pandemic (2010/2011) season grouped into at least four highly significant Italian clades, whereas those of the pandemic season (2009/2010) were interspersed with isolates from other countries at the tree root. The time of the most recent common ancestor of the strains circulating in the pandemic season in Italy was estimated to be between the spring and summer of 2009, whereas the Italian clades of the post-pandemic season originated in the spring of 2010 and showed radiation in the summer/autumn of the same year; this was confirmed by a Bayesian skyline plot showing the biphasic growth of the effective number of infections. The local phylogeography analysis showed that the first season of infection originated in Northern Italian localities with high density populations, whereas the second involved less densely populated localities, in line with a gravity-like model of geographical dispersion. Two HA sites, codons 97 and 222, were under positive selection. In conclusion, the A(H1N1)pdm09 virus was introduced into Italy in the spring of 2009 by means of multiple importations. This was followed by repeated founder effects in the post-pandemic period that originated specific Italian clades.

Doubling of marine dinitrogen-fixation rates based on direct measurements

Biological dinitrogen fixation provides the largest input of nitrogen to the oceans, therefore exerting important control on the ocean's nitrogen inventory and primary productivity. Nitrogen-isotope data from ocean sediments suggest that the marine-nitrogen inventory has been balanced for the past 3,000 years (ref. 4). Producing a balanced marine-nitrogen budget based on direct measurements has proved difficult, however, with nitrogen loss exceeding the gain from dinitrogen fixation by approximately 200 Tg N yr−1 (refs 5, 6). Here we present data from the Atlantic Ocean and show that the most widely used method of measuring oceanic N2-fixation rates underestimates the contribution of N2-fixing microorganisms (diazotrophs) relative to a newly developed method. Using molecular techniques to quantify the abundance of specific clades of diazotrophs in parallel with rates of 15N2 incorporation into particulate organic matter, we suggest that the difference between N2-fixation rates measured with the established method and those measured with the new method can be related to the composition of the diazotrophic community. Our data show that in areas dominated by Trichodesmium, the established method underestimates N2-fixation rates by an average of 62%. We also find that the newly developed method yields N2-fixation rates more than six times higher than those from the established method when unicellular, symbiotic cyanobacteria and γ-proteobacteria dominate the diazotrophic community. On the basis of average areal rates measured over the Atlantic Ocean, we calculated basin-wide N2-fixation rates of 14 ± 1 Tg N yr−1 and 24 ±1 Tg N yr−1 for the established and new methods, respectively. If our findings can be extrapolated to other ocean basins, this suggests that the global marine N2-fixation rate derived from direct measurements may increase from 103 ± 8 Tg N yr−1 to 177 ± 8 Tg N yr−1, and that the contribution of N2 fixers other than Trichodesmium is much more significant than was previously thought.

Prevalence of avian haemosporidian parasites and their host fidelity in the central Philippine islands

We examined the prevalence and host fidelity of avian haemosporidian parasites belonging to the genera Haemoproteus, Leucocytozoon and Plasmodium in the central Philippine islands by sampling 23 bird families (42 species). Using species-specific PCR assays of the mitochondrial cytochrome b gene (471base pairs, bp), we detected infections in 91 of the 215 screened individuals (42%). We also discriminated between single and multiple infections. Thirty-one infected individuals harbored a single Haemoproteus lineage (14%), 18 a single Leucocytozoon lineage (8%) and 12 a single Plasmodium lineage (6%). Of the 215 screened birds, 30 (14%) presented different types of multiple infections. Intrageneric mixed infections were generally more common (18 Haemoproteus/Haemoproteus, 3 Leucocytozoon/Leucocytozoon, and 1 Plasmodium/Plasmodium) than intergeneric mixed infections (7 Haemoproteus/Leucocytozoon and 1 Haemoproteus/Leucocytozoon/Plasmodium). We recovered 81 unique haemosporidian mitochondrial haplotypes. These clustered in three strongly supported monophyletic clades that correspond to the three haemosporidian genera. Related lineages of Haemoproteus and Leucocytozoon were more likely to derive from the same host family than predicted by chance; however, this was not the case for Plasmodium. These results indicate that switches between host families are more likely to occur in Plasmodium. We conclude that Haemoproteus has undergone a recent diversification across well-supported host-family specific clades, while Leucocytozoon shows a longer association with its host(s). This study supports previous evidence of a higher prevalence and stronger host-family specificity of Haemoproteus and Leucocytozoon compared to Plasmodium.

Cenozoic imprints on the phylogenetic structure of palm species assemblages worldwide

Despite long-standing interest in the origin and maintenance of species diversity, little is known about historical drivers of species assemblage structure at large spatiotemporal scales. Here, we use global species distribution data, a dated genus-level phylogeny, and paleo-reconstructions of biomes and climate to examine Cenozoic imprints on the phylogenetic structure of regional species assemblages of palms (Arecaceae), a species-rich plant family characteristic of tropical ecosystems. We find a strong imprint on phylogenetic clustering due to geographic isolation and in situ diversification, especially in the Neotropics and on islands with spectacular palm radiations (e.g., Madagascar, Hawaii, and Cuba). Phylogenetic overdispersion on mainlands and islands corresponds to biotic interchange areas. Differences in the degree of phylogenetic clustering among biogeographic realms are related to differential losses of tropical rainforests during the Cenozoic, but not to the cumulative area of tropical rainforest over geological time. A largely random phylogenetic assemblage structure in Africa coincides with severe losses of rainforest area, especially after the Miocene. More recent events also appear to be influential: phylogenetic clustering increases with increasing intensity of Quaternary glacial-interglacial climatic oscillations in South America and, to a lesser extent, Africa, indicating that specific clades perform better in climatically unstable regions. Our results suggest that continental isolation (in combination with limited long-distance dispersal) and changing climate and habitat loss throughout the Cenozoic have had strong impacts on the phylogenetic structure of regional species assemblages in the tropics.