Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii

Abstract Background Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. Results We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. Conclusion We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.

Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes

Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries-a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5' untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5' UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females.

Molecular phylogeny, morphology and their implications for the taxonomy of Eriocaulaceae

The pantropical family Eriocaulaceae includes ten genera and c. 1,400 species, with diversity concentrated in the New World. The last complete revision of the family was published more than 100 years ago, and until recently the generic and infrageneric relationships were poorly resolved. However, a multi-disciplinary approach over the last 30 years, using morphological and anatomical characters, has been supplemented with additional data from palynology, chemistry, embryology, population genetics, cytology and, more recently, molecular phylogenetic studies. This led to a reassessment of phylogenetic relationships within the family. In this paper we present new data for the ITS and trnL-F regions, analysed separately and in combination, using maximum parsimony and Bayesian inference. The data confirm previous results, and show that many characters traditionally used for differentiating and circumscribing the genera within the family are homoplasious. A new generic key with characters from various sources and reflecting the current taxonomic changes is presented.

Molecular phylogeny and morphometric analyses reveal deep divergence between Amazonia and Atlantic Forest species of Dendrophryniscus

Dendrophryniscus is an early diverging clade of bufonids represented by few small-bodied species distributed in Amazonia and the Atlantic Forest. We used mitochondrial (414 bp of 12S, 575 bp of 16S genes) and nuclear DNA (785 bp of RAG-1) to investigate phylogenetic relationships and the timing of diversification within the genus. These molecular data were gathered from 23 specimens from 19 populations, including eight out of the 10 nominal species of the genus as well as Rhinella boulengeri. Analyses also included sequences of representatives of 18 other bufonid genera that were publically available. We also examined morphological characters to analyze differences within Dendrophryniscus. We found deep genetic divergence between an Amazonian and an Atlantic Forest clade, dating back to Eocene. Morphological data corroborate this distinction. We thus propose to assign the Amazonian species to a new genus, Amazonella. The species currently named R. boulengeri, which has been previously assigned to the genus Rhamphophryne, is shown to be closely related to Dendrophryniscus species. Our findings illustrate cryptic trends in bufonid morphological evolution, and point to a deep history of persistence and diversification within the Amazonian and Atlantic rainforests. We discuss our results in light of available paleoecological data and the biogeographic patterns observed in other similarly distributed groups. (C) 2011 Elsevier Inc. All rights reserved.

Ontogenetic and evolutionary change of external morphology of the neotropical shrimp potimirim (Holthuis, 1954) explained by a molecular phylogeny of the genus

A phylogenetic analysis of a fragment of the mitochondrial gene 16S was used to test the monophyletic status of Potimirim. Existing doubts on the taxonomic status of brasiliana (once P glabra) and P potimirim (once P mexicana) were clarified. Potimirim mexicana and P potimirim are distinct species according to molecular data and appendix masculina morphology. A new species (Potimirim sp. 1) from Puerto Rico was revealed with molecular data, and it is evolutionarily related to P potimirim and P mexicana according to our analysis. We found out three distinct species under the name P glabra. Then, we recommend the application of the name P glabra for the populations of the Pacific slope of Central America and revalidation of P brasiliana for the Brazilian ones. The need for a new name to those "P glabra" of the Caribbean is highlighted, and it was provisionally referred as Potimirim sp. 2. The ontogenetic (juveniles to adults) development of the appendix masculina of P brasiliana was observed and compared to the other species of Potimirim (adults). In the light of our phylogenetic hypothesis, we postulate a pattern of character addition for the evolution of the appendix masculina of Potimirim. This hypothesis is plausible for two key reasons. First. Potimirim is a monophyletic group according to our hypothesis. Second, the shape of appendix masculina found in adults of P. americana is similar and comparable to those found in the earliest juvenile stages of P brasiliana, a derived species according to our phylogeny (P americana, ((P mexicana, Potimirim sp. 1. P potimirim), (P glabra, (brasiliana, Potimirim sp. 2)))). As so, the basal P americana retain the ancestral morphological state of the appendix masculina when compared to the other species of Potimirim. In our interpretation the ontogeny of the appendix masculina recapitulated the proposed phylogeny, giving further support to it.

From Amazonia to the Atlantic forest: Molecular phylogeny of Phyzelaphryninae frogs reveals unexpected diversity and a striking biogeographic pattern emphasizing conservation challenges

Documenting the Neotropical amphibian diversity has become a major challenge facing the threat of global climate change and the pace of environmental alteration. Recent molecular phylogenetic studies have revealed that the actual number of species in South American tropical forests is largely underestimated, but also that many lineages are millions of years old. The genera Phyzelaphryne (1 sp.) and Adelophryne (6 spp.), which compose the subfamily Phyzelaphryninae, include poorly documented, secretive, and minute frogs with an unusual distribution pattern that encompasses the biotic disjunction between Amazonia and the Atlantic forest. We generated >5.8 kb sequence data from six markers for all seven nominal species of the subfamily as well as for newly discovered populations in order to (1) test the monophyly of Phyzelaphryninae, Adelophryne and Phyzelaphryne, (2) estimate species diversity within the subfamily, and (3) investigate their historical biogeography and diversification. Phylogenetic reconstruction confirmed the monophyly of each group and revealed deep subdivisions within Adelophryne and Phyzelaphryne, with three major clades in Adelophryne located in northern Amazonia, northern Atlantic forest and southern Atlantic forest. Our results suggest that the actual number of species in Phyzelaphryninae is, at least, twice the currently recognized species diversity, with almost every geographically isolated population representing an anciently divergent candidate species. Such results highlight the challenges for conservation, especially in the northern Atlantic forest where it is still degraded at a fast pace. Molecular dating revealed that Phyzelaphryninae originated in Amazonia and dispersed during early Miocene to the Atlantic forest. The two Atlantic forest clades of Adelophryne started to diversify some 7 Ma minimum, while the northern Amazonian Adelophryne diversified much earlier, some 13 Ma minimum. This striking biogeographic pattern coincides with major events that have shaped the face of the South American continent, as we know it today. (C) 2012 Elsevier Inc. All rights reserved.

Orobothriurus (Scorpiones: Bothriuridae) phylogeny, Andean biogeography, and the relative importance of genitalic and somatic characters

The genus Orobothriurus Maury, 1976 (Bothriuridae Simon, 1880) displays an Andean pattern of distribution, most of its species occurring at high altitudes (over 2000-2500 m to a maximum altitude record of 4910 m) from central Peru to Argentina. The recent discovery of several new species and the uncertain phylogenetic position of Orobothriurus lourencoi Ojanguren Affilastro, 2003, required a reanalysis of Orobothriurus phylogeny. Thirty bothriurid taxa, including all species of Orobothriurus and Pachakutej Ochoa, 2004, were scored for 65 morphological characters and analysed with parsimony under equal and implied weighting. The resulting topology justifies the establishment of a new genus, Rumikiru Ojanguren Affilastro et al., in press, for O. lourencoi and a closely related, new species, Rumikiru atacama Ojanguren Affilastro et al., in press. It also offers new insights about the phylogeny and biogeography of Orobothriurus and related genera. Characters from the male genitalia (i.e. hemispermatophore), comprising approximately 26% of the morphological matrix, were found to be less homoplastic than those from somatic morphology, contradicting suggestions that genitalia are uninformative or potentially misleading in phylogenetic studies.

Patterns and processes of diversification in a widespread and ecologically diverse avian group, the buteonine hawks (Aves, Accipitridae)

Buteonine hawks represent one of the most diverse groups in the Accipitridae, with 58 species distributed in a variety of habitats on almost all continents. Variations in migratory behavior, remarkable dispersal capability, and unusual diversity in Central and South America make buteonine hawks an excellent model for studies in avian evolution. To evaluate the history of their global radiation, we used an integrative approach that coupled estimation of the phylogeny using a large sequence database (based on 6411 bp of mitochondrial markers and one nuclear intron from 54 species), divergence time estimates, and ancestral state reconstructions. Our findings suggest that Neotropical buteonines resulted from a long evolutionary process that began in the Miocene and extended to the Pleistocene. Colonization of the Nearctic, and eventually the Old World, occurred from South America, promoted by the evolution of seasonal movements and development of land bridges. Migratory behavior evolved several times and may have contributed not only to colonization of the Holarctic, but also derivation of insular species. In the Neotropics, diversification of the buteonines included four disjunction events across the Andes. Adaptation of monophyletic taxa to wet environments occurred more than once, and some relationships indicate an evolutionary connection among mangroves, coastal and varzea environments. On the other hand, groups occupying the same biome, forest, or open vegetation habitats are not monophyletic. Refuges or sea-level changes or a combination of both was responsible for recent speciation in Amazonian taxa. In view of the lack of concordance between phylogeny and classification, we propose numerous taxonomic changes. (C) 2009 Elsevier Inc. All rights reserved.

Phylogeny of Lundia (Bignoniaceae) based on ndhF and PepC sequences

The circumscription of genera belonging to tribe Bignonieae (Bignoniaceae) has traditionally been complex, with only a few genera having stable circumscriptions in the various classification systems proposed for the tribe. The genus Lundia, for instance, is well characterized by a series of morphological synapomorphies and its circumscription has remained quite stable throughout its history. Despite the stable circumscription of Lundia, the circumscription of species within the genus has remained problematic. This study aims to reconstruct the phylogeny of Lundia in order to refine species circumscriptions, gain a better understanding of relationships between taxa, and identify potential morphological synapomorphies for species and major clades. We sampled 26 accessions representing 13 species of Lundia, and 5 outgroups, and reconstructed the phylogeny of the genus using a chloroplast (ndhF) and a nuclear marker (PepC). Data derived from sequences of the individual loci were analyzed using parsimony and Bayesian inference, and the combined molecular dataset was analyzed with Bayesian methods. The monophyly of Lundia nitidula, a species with a particularly complex circumscription, was tested using Shimodaira-Hasegawa (SH) test and the approximately unbiased test for phylogenetic tree selection (AU test). In addition, 40 morphological characters were mapped onto the tree that resulted from the analysis of the combined molecular dataset in order to identify morphological synapomorphies of individual species and major clades. Lundia and most species currently recognized within the genus were strongly supported as monophyletic in all analyses. One species, Lundia nitidula, was not resolved as monophyletic, but the monophyly of this species was not rejected by the AU and SH tests. Lundia sect. Eriolundia is resolved as paraphyletic in all analyses, while Lundia sect. Eulundia is monophyletic and supported by the same morphological characters traditionally used to circumscribe this section. The phylogeny of Lundia contributed important information for a better circumscription of species and served as basis the taxonomic revision of the genus.

Phylogeny and cryptic diversity in geckos (Phyllopezus; Phyllodactylidae; Gekkota) from South America's open biomes

The gecko genus Phyllopezus occurs across South America's open biomes: Cerrado, Seasonally Dry Tropical Forests (SDTF, including Caatinga), and Chaco. We generated a multi-gene dataset and estimated phylogenetic relationships among described Phyllopezus taxa and related species. We included exemplars from both described Phyllopezus pollicaris subspecies, P. p. pollicaris and P. p. przewalskii. Phylogenies from the concatenated data as well as species trees constructed from individual gene trees were largely congruent. All phylogeny reconstruction methods showed Bogertia lutzae as the sister species of Phyllopezus maranjonensis, rendering Phyllopezus paraphyletic. We synonymized the monotypic genus Bogertia with Phyllopezus to maintain a taxonomy that is isomorphic with phylogenetic history. We recovered multiple, deeply divergent, cryptic lineages within P. pollicaris. These cryptic lineages possessed mtDNA distances equivalent to distances among other gekkotan sister taxa. Described P. pollicaris subspecies are not reciprocally monophyletic and current subspecific taxonomy does not accurately reflect evolutionary relationships among cryptic lineages. We highlight the conservation significance of these results in light of the ongoing habitat loss in South America's open biomes. (C) 2011 Elsevier Inc. All rights reserved.

Cryptic species in Iphisa elegans Gray, 1851 (Squamata: Gymnophthalmidae) revealed by hemipenial morphology and molecular data

Iphisa elegans Gray, 1851 is a ground-dwelling lizard widespread over Amazonia that displays a broadly conserved external morphology over its range. This wide geographical distribution and conservation of body form contrasts with the expected poor dispersal ability of the species, the tumultuous past of Amazonia, and the previously documented prevalence of cryptic species in widespread terrestrial organisms in this region. Here we investigate this homogeneity by examining hemipenial morphology and conducting phylogenetic analyses of mitochondrial (CYTB) and nuclear (C-MOS) DNA sequence data from 49 individuals sampled across Amazonia. We detected remarkable variation in hemipenial morphology within this species, with multiple cases of sympatric occurrence of distinct hemipenial morphotypes. Phylogenetic analyses revealed highly divergent lineages corroborating the patterns suggested by the hemipenial morphotypes, including co-occurrence of different lineages. The degrees of genetic and morphological distinctness, as well as instances of sympatry among mtDNA lineages/morphotypes without nuDNA allele sharing, suggest that I. elegans is a complex of cryptic species. An extensive and integrative taxonomic revision of the I. elegans complex throughout its wide geographical range is needed. (c) 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 361376.

Comparative neurocranial morphology of angelsharks from the south-western Atlantic Ocean (Chondrichthyes, Elasmobranchii, Squatinidae): implications for taxonomy and phylogeny

Neurocrania of three species of angelsharks from the south-western Atlantic Ocean, occurring off south-eastern and southern Brazil, are described. A detailed morphological description is provided of the neurocranium of Squatina guggenheim and compared with S. argentina and S. occulta. Despite being generally conservative, the neurocranium of Squatina presents significant differences among these species which aid in their identification, which is otherwise problematical. The main distinctions were found in rostral projections, anterior fontanellae, supraorbital crests, upper and lower postorbital processes, otic capsules, suborbital crests, and pterotic processes. Squatina guggenheim and S. occulta share more neurocranial characters when compared to S. argentina. No basal angle was found, but we confirm the presence of a very much reduced and barely noticeable basioccipital fovea in Squatina; systematic implications within elasmobranchs of these and other features are discussed.

Morphological and molecular evidence demonstrates the amphi-Atlantic distribution of Laurencia catarinensis (Ceramiales, Rhodophyta)

We performed morphological and molecular studies of Laurencia catarinensis from the Canary Islands. This species has an entangled habit, cushion-like tuft formation, cortical cell walls, slightly to markedly projecting near the apex, and lacking lenticular thickenings in medullary cells. We inferred its phylogenetic position by analyzing the chloroplast-encoded rbcL gene sequences from 41 samples. The results demonstrate that specimens of L. catarinensis from the Canary Islands, where it is referred to as L. intricata, and those from Brazil (including specimens from the type locality in Santa Catarina) form a monophyletic clade with low genetic divergence (0-0.9%). In contrast, specimens of L. intricata from the type locality in Cuba, Mexico, and the USA were clearly distinct from L. catarinensis collected in Brazil and the Canary Islands, as shown by high genetic divergence values (4.9-5.7%). The type material of L. catarinensis from Brazil allowed us to identify all samples from the Canarian Archipelago as L. catarinensis. These findings expand the known geographical distribution of L. catarinensis to the eastern Atlantic Ocean and demonstrate an amphi-Atlantic distribution of the species.

Molecular phylogeny of the New World Dipsadidae (Serpentes: Colubroidea): a reappraisal

We present a phylogenetic analysis of the New World dipsadids based on an expanded data matrix that includes 246 terminal taxa including 196 dipsadids. The species are sampled for eight genes (12S, 16S, cytb, nd2, nd4, bdnf, c-mos, rag2). The data are explored using two distinct optimality proceduresmaximum parsimony and maximum likelihoodand two alignment strategiesdynamic homology and static homology. Two previously unsampled dipsadid genera, Sordellina and Rhachidelus, are now included in the analysis. The definitions of the genera, Erythrolamprus, Clelia, Hypsirhynchus, Philodryas and Phimophis, and the tribes Alsophiini, Echinantherini and Conophiini, are revised. In order to maintain monophyly, the genus Umbrivaga is synonymized with Erythrolamprus, and two new genera are erected to accommodate Phimophis iglesiasi and Clelia rustica, as well as their closely related species. The West Indian genera Schwartzophis, Darlingtonia, Antillophis and Ocyophis are resurrected. (c) The Willi Hennig Society 2012.

Is Drosera meristocaulis a pygmy sundew? Evidence of a long-distance dispersal between Western Australia and northern South America

South America and Oceania possess numerous floristic similarities, often confirmed by morphological and molecular data. The carnivorous Drosera meristocaulis (Droseraceae), endemic to the Neblina highlands of northern South America, was known to share morphological characters with the pygmy sundews of Drosera sect. Bryastrum, which are endemic to Australia and New Zealand. The inclusion of D. meristocaulis in a molecular phylogenetic analysis may clarify its systematic position and offer an opportunity to investigate character evolution in Droseraceae and phylogeographic patterns between South America and Oceania. was included in a molecular phylogenetic analysis of Droseraceae, using nuclear internal transcribed spacer (ITS) and plastid rbcL and rps16 sequence data. Pollen of D. meristocaulis was studied using light microscopy and scanning electron microscopy techniques, and the karyotype was inferred from root tip meristem. The phylogenetic inferences (maximum parsimony, maximum likelihood and Bayesian approaches) substantiate with high statistical support the inclusion of sect. Meristocaulis and its single species, D. meristocaulis, within the Australian Drosera clade, sister to a group comprising species of sect. Bryastrum. A chromosome number of 2n approx. 3236 supports the phylogenetic position within the Australian clade. The undivided styles, conspicuous large setuous stipules, a cryptocotylar (hypogaeous) germination pattern and pollen tetrads with aperture of intermediate type 78 are key morphological traits shared between D. meristocaulis and pygmy sundews of sect. Bryastrum from Australia and New Zealand. The multidisciplinary approach adopted in this study (using morphological, palynological, cytotaxonomic and molecular phylogenetic data) enabled us to elucidate the relationships of the thus far unplaced taxon D. meristocaulis. Long-distance dispersal between southwestern Oceania and northern South America is the most likely scenario to explain the phylogeographic pattern revealed.