Ronald M. Clouse

Diversification and Expression of the PIN, AUX/LAX, and ABCB Families of Putative Auxin Transporters in Populus.

Intercellular transport of the plant hormone auxin is mediated by three families of membrane-bound protein carriers, with the PIN and ABCB families coding primarily for efflux proteins and the AUX/LAX family coding for influx proteins. In the last decade our understanding of gene and protein function for these transporters in Arabidopsis has expanded rapidly but very little is known about their role in woody plant development. Here we present a comprehensive account of all three families in the model woody species Populus, including chromosome distribution, protein structure, quantitative gene expression, and evolutionary relationships. The PIN and AUX/LAX gene families in Populus comprise 16 and 8 members respectively and show evidence for the retention of paralogs following a relatively recent whole genome duplication. There is also differential expression across tissues within many gene pairs. The ABCB family is previously undescribed in Populus and includes 20 members, showing a much deeper evolutionary history, including both tandem and whole genome duplication as well as probable gene loss. A striking number of these transporters are expressed in developing Populus stems and we suggest that evolutionary and structural relationships with known auxin transporters in Arabidopsis can point toward candidate genes for further study in Populus. This is especially important for the ABCBs, which is a large family and includes members in Arabidopsis that are able to transport other substrates in addition to auxin. Protein modeling, sequence alignment and expression data all point to ABCB1.1 as a likely auxin transport protein in Populus. Given that basipetal auxin flow through the cambial zone shapes the development of woody stems, it is important that we identify the full complement of genes involved in this process. This work should lay the foundation for studies targeting specific proteins for functional characterization and in situ localization.

Molecular Phylogeny of Coral-Reef Sea Cucumbers (Holothuriidae: Aspidochirotida) Based on 16S Mitochondrial Ribosomal DNA Sequence

Members of the Holothuriidae, found globally at low to middle latitudes, are often a dominant component of Indo–West Pacific coral reefs. We present the first phylogeny of the group, using 8 species from the 5 currently recognized genera and based on approximately 540 nucleotides from a polymerase chain reaction–amplified and conserved 3′ section of 16S mitochondrial ribosomal DNA. Parsimony and likelihood analyses returned identical topologies, permitting several robust inferences to be drawn. Several points corroborated the Linnean classification. Actinopyga and Bohadschia each appear monophyletic and Pearsonothuria is sister to Bohadschia. Other aspects of our phylogeny, however, were not in accord with the taxonomy of Holothuriidae or previous speculations about the group’s evolutionary history. Most notably, the genus Holothuria appears paraphyletic. Actinopyga and Bohadschia, sometimes held to be closely related to one another because of certain morphologic similarities, are only distantly related. The morphologically distinct Labidodemas, even thought to warrant separation at the family level, is nested well within Holothuria. A maximum parsimony reconstruction of ancestral ossicle form on the phylogeny indicated that, in addition to a probable bout of elaboration in ossicle form (the modification of rods or rosettes to holothuriid-type buttons), at least 2 rounds of ossicle simplification also transpired in which buttons reverted to rods or rosettes. Cuvierian tubules, defensive organs unique to numerous members of Holothuriidae, were probably present before the initial radiation of the family, but the reconstruction is ambiguous as to their ancestral function.

Across Lydekker's Line – first report of mite harvestmen (Opiliones : Cyphophthalmi : Stylocellidae) from New Guinea

Opiliones (harvestmen) in the suborder Cyphophthalmi are not known to disperse across oceans and each family in the suborder is restricted to a clear biogeographic region. While undertaking a revisionary study of the South-east Asian family Stylocellidae, two collections of stylocellids from New Guinea were noted. This was a surprising find, since the island appears never to have had a land connection with Eurasia, where the rest of the family members are found. Here, 21 New Guinean specimens collected from the westernmost end of the island (Manokwari Province, Indonesia) are described and their relationships to other cyphophthalmids are analysed using molecular sequence data. The specimens represent three species, Stylocellus lydekkeri, sp. nov., S. novaguinea, sp. nov. and undescribed females of a probable third species, which are described and illustrated using scanning electron microscope and stereomicroscope photographs. Stylocellus novaguinea, sp. nov. is described from a single male and it was collected with a juvenile and the three females of the apparent third species. Molecular phylogenetic analyses indicate that the new species are indeed in the family Stylocellidae and they therefore reached western New Guinea by dispersing through Lydekker’s line – the easternmost limit of poor dispersers from Eurasia. The New Guinean species may indicate at least two episodes of oceanic dispersal by Cyphophthalmi, a phenomenon here described for the first time. Alternatively, the presence in New Guinea of poor dispersers from Eurasia may suggest novel hypotheses about the history of the island.

Return of chloroquine-sensitive Plasmodium falciparum parasites and emergence of chloroquine-resistant Plasmodium vivax in Ethiopia

BackgroundIncreased resistance by Plasmodium falciparum parasites led to the withdrawal of the antimalarial drugs chloroquine and sulphadoxine-pyrimethamine in Ethiopia. Since 2004 artemether-lumefantrine has served to treat uncomplicated P. falciparum malaria. However, increasing reports on delayed parasite clearance to artemisinin opens up a new challenge in anti-malarial therapy. With the complete withdrawal of CQ for the treatment of Plasmodium falciparum malaria, this study assessed the evolution of CQ resistance by investigating the prevalence of mutant alleles in the pfmdr1 and pfcrt genes in P. falciparum and pvmdr1 gene in Plasmodium vivax in Southern and Eastern Ethiopia.MethodsOf the 1,416 febrile patients attending primary health facilities in Southern Ethiopia, 329 febrile patients positive for P. falciparum or P. vivax were recruited. Similarly of the 1,304 febrile patients from Eastern Ethiopia, 81 febrile patients positive for P. falciparum or P. vivax were included in the study. Of the 410 finger prick blood samples collected from malaria patients, we used direct sequencing to investigate the prevalence of mutations in pfcrt and pfmdr1. This included determining the gene copy number in pfmdr1 in 195 P. falciparum clinical isolates, and mutations in the pvmdr1 locus in 215 P. vivax clinical isolates.ResultsThe pfcrt K76 CQ-sensitive allele was observed in 84.1% of the investigated P.falciparum clinical isolates. The pfcrt double mutations (K76T and C72S) were observed less than 3%. The pfcrt SVMNT haplotype was also found to be present in clinical isolates from Ethiopia. The pfcrt CVMNK-sensitive haplotypes were frequently observed (95.9%). The pfmdr1 mutation N86Y was observed only in 14.9% compared to 85.1% of the clinical isolates that carried sensitive alleles. Also, the sensitive pfmdr1 Y184 allele was more common, in 94.9% of clinical isolates. None of the investigated P. falciparum clinical isolates carried S1034C, N1042D and D1246Y pfmdr1 polymorphisms. All investigated P. falciparum clinical isolates from Southern and Eastern Ethiopia carried only a single copy of the mutant pfmdr1 gene.ConclusionThe study reports for the first time the return of chloroquine sensitive P. falciparum in Ethiopia. These findings support the rationale for the use of CQ-based combination drugs as a possible future alternative.

A phylogenetic analysis for the South-east Asian mite harvestman family Stylocellidae (Opiliones : Cyphophthalmi) – a combined analysis using morphometric and molecular data

In an effort to place type specimens lacking molecular data into a phylogenetic framework ahead of a taxonomic revision, we used morphometric data, both alone and in combination with a molecular dataset, to generate phylogenetic hypotheses under the parsimony criterion for 107 members of the South-east Asian mite harvestman family Stylocellidae (Arachnida: Opiliones: Cyphophthalmi). For the morphometric analyses, we used undiscretised characters, analysed for independence and collapsed by principal components analysis (PCA) when dependent. Two challenges not previously encountered in the use of this method were (a) handling terminals with missing data, necessitated by the inclusion of old and damaged type specimens, and (b) controlling for extreme variation in size. Custom scripts for independence analysis were modified to accommodate missing data whereby placeholder numbers were used during PCA for missing measurements. Size was controlled in four ways: choosing characters that avoided misleading size information and were easily scaled; using only locally scaled measurements; adjusting ratios by y-intercepts; and collapsing dependent characters into one. These steps removed enough size information that miniaturised and large species, suspected from molecular and discrete morphological studies to be closely related, were closely placed using morphometric data alone. Both morphometric and combined analyses generated relationships that positioned type specimens in agreement with taxonomic expectations and our knowledge of the family from prior studies. The hypotheses generated here provide new direction in linking molecular analyses with established taxonomy in this large group of South-east Asian arachnids.

Breaking out of biogeographical modules: range expansion and taxon cycles in the hyperdiverse ant genus Pheidole

Abstract Aim We sought to reconstruct the biogeographical structure and dynamics of a hyperdiverse ant genus, Pheidole, and to test several predictions of the taxon cycle hypothesis. Using large datasets on Pheidole geographical distributions and phylogeny, we (1) inferred patterns of biogeographical modularity (clusters of areas with similar faunal composition), (2) tested whether species in open habitats are more likely to be expanding their range beyond module boundaries, and (3) tested whether there is a bias of lineage flow from high‐ to low‐diversity areas. Location The Old World. Methods We compiled and jointly analysed a comprehensive database of Pheidole geographical distributions, the ecological affinities of different species, and a multilocus phylogeny of the Old World radiation. We used network modularity methods to infer biogeographical structure in the genus and comparative methods to evaluate the hypotheses. Results The network analysis identified eight biogeographical modules, and a suite of species with anomalous ranges that are statistically more likely to occur in open habitat, supporting the hypothesis that open habitats promote range expansion. Phylogenetic analysis shows evidence for a cascade pattern of colonization from Asia to New Guinea to the Pacific, but no ‘upstream’ colonization in the reverse direction. Main conclusions The distributions of Pheidole lineages in the Old World are highly modular, with modules generally corresponding to biogeographical regions inferred in other groups of organisms. However, some lineages have expanded their ranges across module boundaries, and these species are more likely to be adapted to open habitats rather than interior forest. In addition, there is a cascade pattern of dispersal from higher to lower diversity areas during these range expansions. Our findings are consistent with the taxon cycle hypothesis, although they do not rule out alternative interpretations.

Molecular phylogeny of Indo-Pacific carpenter ants (Hymenoptera: Formicidae, Camponotus) reveals waves of dispersal and colonization from diverse source areas

Ants that resemble Camponotus maculatus (Fabricius, 1782) present an opportunity to test the hypothesis that the origin of the Pacific island fauna was primarily New Guinea, the Philippines, and the Indo‐Malay archipelago (collectively known as Malesia). We sequenced two mitochondrial and four nuclear markers from 146 specimens from Pacific islands, Australia, and Malesia. We also added 211 specimens representing a larger worldwide sample and performed a series of phylogenetic analyses and ancestral area reconstructions. Results indicate that the Pacific members of this group comprise several robust clades that have distinctly different biogeographical histories, and they suggest an important role for Australia as a source of Pacific colonizations. Malesian areas were recovered mostly in derived positions, and one lineage appears to be Neotropical. Phylogenetic hypotheses indicate that the orange, pan‐Pacific form commonly identified as C. chloroticus Emery 1897 actually consists of two distantly related lineages. Also, the lineage on Hawaiʻi, which has been called C. variegatus (Smith, 1858), appears to be closely related to C. tortuganus Emery, 1895 in Florida and other lineages in the New World. In Micronesia and Polynesia the C. chloroticus‐like species support predictions of the taxon‐cycle hypothesis and could be candidates for human‐mediated dispersal.

Descriptions of two new, cryptic species of Metasiro (Arachnida: Opiliones: Cyphophthalmi: Neogoveidae) from South Carolina, USA, including a discussion of mitochondrial mutation rates

Specimens of Metasiro from its three known disjunct population centers in the southeastern US were examined and had a 769 bp fragement of the mitochondrial gene cytochrome c oxidase subunit I (COI) sequenced. These populations are located in the western panhandle of Florida and nearby areas of Georgia, in the Savannah River delta of South Carolina, and on Sassafras Mt. in South Carolina. This range extends over as much as 500 km, which is very large for a species of cyphophthalmid harvestmen and presents a degree of physical separation among populations such that we would expect them to actually be distinguishable species. We examined the morphology, including the spermatopositors of males, and sequences from 221 specimens. We found no discernible differences in the morphologies of specimens from the different populations, but corrected pairwise distances of COI were about 15% among the three population centers. We also analyzed COI data using a General Mixed Yule Coalescent (GMYC) model implemented in the R package SPLITS; with a single threshold, the most likely model had four species within Metasiro. Given this level of molecular divergence, the monophyly of the population haplotypes, and the number of exclusive COI nucleotide and amino acid differences distinguishing the populations, we here raise the Savannah River and Sassafras Mt. populations to species status: M. savannahensis sp. nov., and M. sassafrasensis sp. nov., respectively. This restricts M. americanus (Davis, 1933) to just the Lower Chattahoochee Watershed, which in this study includes populations along the Apalachicola River and around Florida Caverns State Park. GMYC models reconstructed the two main haplotype clades within M. americanus as different species, but they are not exclusive to different areas. We estimate COI percent divergence rates in certain cyphophthalmid groups and discuss problems with historical measures of this rate. We hypothesize that Metasiro began diversifying over 20 million years ago.

A novel phylogeny and morphological reconstruction of the PIN genes and first phylogeny of the ACC-oxidases (ACOs).

The PIN and ACO gene families present interesting questions about the evolution of plant physiology, including testing hypotheses about the ecological drivers of their diversification and whether unrelated genes have been recruited for similar functions. The PIN-formed proteins contribute to the polar transport of auxin, a hormone which regulates plant growth and development. PIN loci are categorized into groups according to their protein length and structure, as well as subcellular localization. An interesting question with PIN genes is the nature of the ancestral form and location. ACOs are members of a superfamily of oxygenases and oxidases that catalyze the last step of ethylene synthesis, which regulates many aspects of the plant life cycle. We used publicly available PIN and ACO sequences to conduct phylogenetic analyses. Third codon positions of these genes in monocots have a high GC content, which could be historical but is more likely due to a mutational bias. Thus, we developed methods to extract phylogenetic information from nucleotide sequences while avoiding this convergent feature. One method consisted in using only A-T transformations, and another used only the first and second codon positions for serine, which can only take A or T and G or C, respectively. We also conducted tree-searches for both gene families using unaligned amino acid sequences and dynamic homology. PIN genes appear to have diversified earlier than ACOs, with monocot and dicot copies more mixed in the phylogeny. However, gymnosperm PINs appear to be derived and not closely related to those from primitive plants. We find strong support for a long PIN gene ancestor with short forms subsequently evolving one or more times. ACO genes appear to have diversified mostly since the dicot-monocot split, as most genes cluster into a small number of monocot and dicot clades when the tree is rooted by genes from mosses. Gymnosperm ACOs were recovered as closely related and derived.

Elongation factor-1α, a putative single-copy nuclear gene, has divergent sets of paralogs in an arachnid

Identification of paralogy in candidate nuclear loci is an important prerequisite in phylogenetics and statistical phylogeography, but one that is often overlooked. One marker commonly assumed to be a single-copy gene and claimed to harbor great utility for inferring recent divergences is elongation factor-1α (EF-1α). To test this hypothesis, we systematically cloned EF-1α in three disjunct populations of the harvestman Metasiro americanus. Here we show that EF-1α has a large number of paralogs in this species. The paralogs do not evolve in a concerted manner, and the paralogs diverged prior to the population divergence. Moreover, the paralogs of M. americanus are not comparable to the highly divergent EF-1α paralogs found in bees and spiders, which are easily recognized and separated through the use of specific primers. We demonstrate statistically that our detection of paralogs cannot be attributed to amplification error. The presence of EF-1α paralogs in M. americanus prevents its use in statistical phylogeography, and the presence of out-paralogs argues against its use in phylogenetic inference among recently diverged clades. These data contradict the common assumption that EF-1α is for most or all taxa a single-copy gene, or that it has a small number of paralogs that are homogenized through gene conversion, unequal crossing over, or other processes.