A Phylogenetic Study of the Anopheles punctulatus Group of Malaria Vectors Comparing rDNA Sequence Alignments Derived from the Mitochondrial and Nuclear Small Ribosomal Subunits

A phylogenetic study of the members of the Anopheles punctulatus group was performed using structural and similarity-based DNA sequence alignments of the small ribosomal subunit (SSU) from both the nuclear and the mitochondrial genomes. The mitochondrial SSU gene (12S, 650 bp) proved to be highly restricted by its secondary structure and displayed little informative sequence variation. Consequently, it was considered unsuitable for a phylogenetic study of these closely related mosquito species. A structural alignment of the nuclear ribosomal DNA SSU (18S, 2000 bp) proved to be more informative than similarity-based alignments. Analyses showed the A. punctulatus group to be monophyletic with two major clades; a Farauti clade containing members displaying an all-black-scaled proboscis (A. farauti 1–3 and 5–7) and the Punctulatus clade containing members displaying extensive white scaling on the apical half of the proboscis (A. farauti 4, A. punctulatus, and An. sp. near punctulatus). Anopheles koliensis was positioned basal to the Farauti clade.     

Distribution and evolution of the Anopheles punctulatus group (Diptera: Culicidae) in Australia and Papua New Guinea

The members of the Anopheles punctulatus group are major vectors of malaria and Bancroftian filariasis in the southwest Pacific region. The group is comprised of 12 cryptic species that require DNA-based tools for species identification. From 1984 to 1998 surveys were carried out in northern Australia, Papua New Guinea and on islands in the southwest Pacific to determine the distribution of the A. punctulatus group. The results of these surveys have now been completed and have generated distribution data from more than 1500 localities through this region. Within this region several climatic and geographical barriers were identified that restricted species distribution and gene flow between geographic populations. This information was further assessed in light of a molecular phylogeny derived from the ssrDNA (18S). Subsequently, hypotheses have been generated on the evolution and distribution of the group so that future field and laboratory studies may be approached more systematically. This study suggested that the ability for widespread dispersal was found to have appeared independently in species that show niche-specific habitat preference (Anopheles farauti s.s. and A. punctulatus) and conversely in species that showed diversity in their larval habitat (Anopheles farauti 2). Adaptation to the monsoonal climate of northern Australia and southwest Papua New Guinea was found to have appeared independently in A. farauti s.s., A. farauti 2 and Anopheles farauti 3. Shared or synapomorphic characters were identified as saltwater tolerance (A. farauti s.s. and Anopheles farauti 7) and elevational affinities above 1500 m (Anopheles farauti 5, Anopheles farauti 6 and A. farauti 2).     

The Anopheles punctulatus group of mosquitoes in the Solomon Islands and Vanuatu surveyed by allozyme electrophoresis

Abstract. Four species within the Anopheles punctulatus group of mosquitoes (Diptera: Culicidae) were identified by allozyme analysis of samples collected from thirty-three localities in Guadalcanal, Makira, Malaita, Temotu and Western Provinces in the Solomon Islands and six localities on Efate, Espiritu Santo, Maewo and Malekula Islands in Vanuatu. Three of these species are members of the An. farauti complex. A key is given to identify five species of the An. punctulatus group known to occur in the Solomon Islands using their isoenzyme characteristics.
An. farauti No. 1 was widespread in coastal areas of the Solomon Islands and was the only species detected in Vanuatu, including Efate Island (where Faureville is the type locality of An. farauti Laveran sensu stricto). An. farauti No. 2 and An. punctulatus were common in the Solomon Islands in more inland areas. An. farauti No. 7, reported here for the first time, was found as larvae in freshwater at six localities on north Guadalcanal. Three other members of the An. punctulatus group which have been reported previously from the Solomon Islands: An. koliensis, An. renellensis and an electrophoretic variant of An. farauti sensu lato, were not found in our samples.
Previously recognized vectors of malaria and bancroftian filariasis in the Solomon Islands are An. farauti No. 1 (i.e. An. farauti s.s. ), An. koliensis and An. punctulatus s. s. Adult females of An. farauti No. 2 and An. farauti No. 7 were not attracted to human bait in areas where their larvae occurred, indicating that these two species are not anthropophilic and therefore unlikely to transmit human pathogens.     

Independent terrestrial origins of the Halosphaeriales (marine Ascomycota)

A phylogenetic study of marine ascomycetes was initiated to test and refine evolutionary hypotheses of marine-terrestrial transitions among ascomycetes. Taxon sampling focused on the Halosphaeriales, the largest order of marine ascomycetes. Approximately 1050 base pairs (bp) of the gene that codes for the nuclear small subunit (SSU) and 600 bp of the gene that codes for the nuclear large subunit (LSU) ribosomal RNAs (rDNA) were sequenced for 15 halosphaerialean taxa and integrated into a data set of homologous sequences from terrestrial ascomycetes. An initial set of phylogenetic analyses of the SSU rDNA from 38 taxa representing 15 major orders of the phylum Ascomycota confirmed a close phylogenetic relationship of the halosphaerialean species with several other orders of perithecial ascomycetes. A second set of analyses, which involved more intensive taxon sampling of perithecial ascomycetes, was performed using the SSU and LSU rDNA data in combined analyses. These second analyses included 15 halosphaerialean taxa, 26 terrestrial perithecial fungi from eight orders, and five outgroup taxa from the Pezizales. In these analyses the Halosphaeriales were polyphyletic and comprised two distinct lineages. One clade of Halosphaeriales comprised 12 taxa from 11 genera and was most closely related to terrestrial fungi of the Microascales. The second clade of halosphaerialean fungi comprised taxa from the genera Lulworthia and Lindra and was an isolated lineage among the perithecial fungi. Both the main clade of Halosphaeriales and the Lulworthia/Lindra clade are supported by the data as being independently derived from terrestrial ancestors.     

The Anopheles punctulatus complex: DNA probes for identifying the Australian species using isotopic, chromogenic, and chemiluminescence detection systems

Isotopic and enzyme-labeled species-specific DNA probes were made for the three known members of the Anopheles punctulatus complex of mosquitoes in Australia (Anopheles farauti Nos. 1, 2, and 3). Species-specific probes were selected by screening total genomic libraries made from the DNA of individual species with 32P-labeled DNA of homologous and heterologous mosquito species. The 32P-labeled probes for A. farauti Nos. 1 and 2 can detect less than 0.2 ng of DNA while the 32P-labeled probe for A. farauti No. 3 has a sensitivity of 1.25 ng of DNA. Probes were then enzyme labeled for chromogenic and chemiluminescence detection and compared to isotopic detection using 32P-labeled probes. Sequences of the probe repeat regions are presented. Species identifications can be made from dot blots or squashes of freshly killed mosquitoes or mosquitoes stored frozen, dried, and held at room temperature or fixed in isopropanol or ethanol with isotopic, chromogenic, or chemiluminescence detection systems. The use of nonisotopic detection systems will enable laboratories with minimal facilities to identify important regional vectors.     

Shared salinity tolerance invalidates a test for the malaria vector Anopheles farauti s.s. on Guadalcanal, Solomon Islands

Among the Punctulatus Group of Anopheles mosquitoes (Diptera: Culicidae), first-instar larvae of the medically unimportant freshwater Anopheles farauti species No. 7 survives a seawater tolerance test (SST) that was previously thought to be diagnostic for the saltwater-tolerant malaria vector species, An. farauti Laveran s.s. Salt tolerance in these two closely related isomorphic species appears to be a shared derived character within the Farauti Complex. Failure to differentiate An. farauti s.s. from An. farauti No. 7 will overestimate potential malaria vector numbers and waste limited larval control resources. Use of the SST should therefore be discontinued on Guadalcanal and other techniques such as allozyme electrophoresis used instead.     

Shared salinity tolerance invalidates a test for the malaria vector Anopheles farauti s.s. on Guadalcanal, Solomon Islands [corrected

Among the Punctulatus Group of Anopheles mosquitoes (Diptera: Culicidae), first-instar larvae of the medically unimportant freshwater Anopheles farauti species No. 7 survives a seawater tolerance test (STT) that was previously thought to be diagnostic for the saltwater-tolerant malaria vector species, An. farauti Laveran s.s. Salt tolerance in these two closely related isomorphic species appears to be a shared derived character within the Farauti Complex. Failure to differentiate An. farauti s.s. from An. farauti No.7 will overestimate potential malaria vector numbers and waste limited larval control resources. Use of the STT should therefore be discontinued on Guadalcanal and other techniques such as allozyme electrophoresis used instead [corrected].     

Molecular phylogeny of the Myzorhynchella Section of Anopheles (Nyssorhynchus) (Diptera: Culicidae): genetic support for recently described and resurrected species

Phylogenetic relationships among species of the Myzorhynchella Section of Anopheles (Nyssorhynchus) were investigated using the nuclear ribosomal DNA second internal transcribed spacer (ITS2), the nuclear whitegene and mitochondrial cytochrome oxidase subunit I (COI) regions. The recently described Anopheles pristinus and resurrected Anopheles guarani were also included in the study. Bayesian phylogenetic analyses found Anopheles parvus to be the most distantly related species within the Section, a finding that is consistent with morphology. An. pristinus and An. guarani were clearly resolved from Anopheles antunesi and Anopheles lutzii, respectively. An. lutzii collected in the same mountain range as the type locality were found within a strongly supported clade, whereas individuals from the southern state of Rio Grande do Sul, tentatively identified as An. lutzii based on adult female external morphology, were distinct from An. lutzii, An. antunesi and from each other, and may therefore represent two new sympatric species. A more detailed examination of An. lutzii sensu latoalong its known geographic range is recommended to resolve these anomalous relationships.     

Molecular phylogenetics and the evolution of host plant associations in the nematode genus Fergusobia (Tylenchida: Fergusobiinae)

Fergusobia nematodes (Tylenchida: Fergusobiinae) and Fergusonina flies (Diptera: Fergusoninidae) are putative mutualists that develop together in galls formed in meristematic tissues of many species of the plant family Myrtaceae in Australasia. Fergusobia nematodes were sampled from a variety of myrtaceous hosts and gall types from Australia and one location in New Zealand between 1999 and 2006. Evolutionary relationships of these isolates were inferred from phylogenetic analyses of the DNA sequences of the nuclear ribosomal DNA near-full length small subunit (up to 1689bp for 21 isolates), partial large subunit D2/D3 domain (up to 889bp for 87 isolates), partial mitochondrial cytochrome oxidase subunit I (618 bp for 82 isolates), and combined D2/D3 and mtCOI (up to 1497bp for 66 isolates). The SSU data supported a monophyletic Fergusobia genus within a paraphyletic Howardula. A clade of Drosophila-associated Howardula, including Howardula aoronymphium, was the closest sequenced sister group. Phylogenetic analysis of sequences from D2/D3 and mtCOI, separately and combined, revealed many monophyletic clades within Fergusobia. The relationships inferred by D2/D3 and mtCOI were congruent with some exceptions. Well-supported clades were generally consistent with host plant species and gall type. However, phylogenetic analysis suggested host switching or putative hybridization events in many groups, except the lineage of shoot bud gallers on the broad-leaved Melaleuca species complex.     

基于核内核糖体小亚基序列的蝗总科系统发育关系分析

用核糖体SSURdna全序列对蝗总科（Acridoidea）进行了分子系统学研究。依据测定的8种蝗虫的SSU Rdna全序列 (平均 1.844 bp),并从GenBank中选取了6种内群种类和2种外群种类的SSU Rdna同源序列,进行序列分析。利用Clustal、MEGA 和 PHYLIP 软件构建分子系统树（距离邻接法Neighbor-Joining,NJ;最小进化法 Minimum Evolution）。结果显示： (1) 蝗总科是一个单系类群;(2) 锥头蝗科（Chrotogonidae）和瘤锥蝗科（Pyrgomorphidea）亲缘关系较近,为蝗总科最原始的类群;(3) 网翅蝗科（Arcypteridae）和槌角蝗科（Gomphoceridae）有较近的亲缘关系; (4) 斑翅蝗科 （Oedipodidae）为最进化的类群; (5) SSU Rdna序列保守性强,转换transition）取代的速率大于或接近颠换（transversion）取代的速率;(6) 在系统树中,总科首先分离,大多数同科不同属的类群以高置信度聚合在一起,说明SSU Rdna序列适合用于蝗总科的系统发育关系分析。     

Genetic diversity in two sibling species of the <Emphasis Type="Italic">Anopheles punctulatus</Emphasis> group of mosquitoes on Guadalcanal in the Solomon Islands

Background  

The mosquito Anopheles irenicus, a member of the Anopheles punctulatus group, is geographically restricted to Guadalcanal in the Solomon Islands. It shows remarkable morphological similarities to one of its sibling species, An. farauti sensu stricto (An. farauti s.s.), but is dissimilar in host and habitat preferences. To infer the genetic variations between these two species, we have analyzed mitochondrial cytochrome oxidase subunit II (COII) and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences from Guadalcanal and from one of its nearest neighbours, Malaita, in the Solomon Islands.     

Amplification of mitochondrial small subunit ribosomal DNA of polypores and its potential for phylogenetic analysis

There has been a systematic need to seek adequate phylogenetic markers that can be applied in phylogenetic analyses of fungal taxa at various levels. The mitochondrial small subunit ribosomal DNA (mt SSU rDNA) is generally considered to be one of the molecules that are appropriate for phylogenetic analyses at a family level. In order to obtain universal primers for polypores of Hymenomycetes, mt SSU rRNA genes were cloned from Bjerkandera adusta, Ganoderma lucidum, Phlebiopsis gigantea, and Phellinus laevigatus and their sequences were determined. Based on the conserved sequences of cloned genes from polypores and Agrocybe aegerita, PCR primers were designed for amplification and sequencing of mt SSU rDNAs. New primers allowed effective amplification and sequencing of almost full-sized genes from representative species of polypores and related species. Phylogenetic relationships were resolved quite efficiently by mt SSU rDNA sequences, and they proved to be more useful in phylogenetic reconstruction of Ganoderma than nuclear internal transcribed spacer (ITS) rDNA sequences.     

Molecular systematic analysis of the order Proteocephalidea (Eucestoda) based on mitochondrial and nuclear rDNA sequences

Two ribosomal DNA sequences were used to infer phylogenetic relationships among the Eucestoda order Proteocephalidea. A 437 bp portion of the 16S mitochondrial and a 1149 bp 5' portion of the nuclear large sub-unit rRNA molecule were sequenced for 53 proteocephalidean cestodes (representing nine subfamilies and 22 genera) and for one outgroup species. Parsimony and distance-based analyses of the two databases, alone and combined, failed to support the monophyly of the two traditionally accepted families, of numerous subfamilies (with the exception of the Rudolphielliinae and Othinoscolescinae which were validated in our analysis) and of various genera, including the genus Nomimoscolex (Woodland), Ophiotaenia (La Rue) as well as the type genus Proteocephalus (Weinland). Palaearctic Proteocephalus species nevertheless constituted a well-defined clade. The two genes globally yielded compatible results; however, the nuclear ribosomal gene provided a better resolution of relations among Proteocephalidea.     

Toward understanding Anophelinae (Diptera,Culicidae) phylogeny: insights from nuclear single-copy genes and the weight of evidence

A phylogeny of the mosquito subfamily Anophelinae was inferred from fragments of two protein-coding nuclear genes, G6pd (462 bp) and white (801 bp), and from a combined data set (2,136 bp) that included a portion of the mitochondrial gene ND5 and the D2 region of the ribosomal 28S gene. Sixteen species from all three anopheline genera and six Anopheles subgenera were sampled, along with six species of other mosquitoes used as an outgroup. Each of four genes analyzed individually recovered the same well-supported clades; topological incongruence was limited to unsupported or poorly supported nodes. As assessed by the incongruence length difference test, most of the conflicting signal was contributed by third codon positions. Strong structural constraints, as observed in white and G6pd, apparently had little impact on phylogenetic inference. Compared with the other genes, white provided a superior source of phylogenetic information. However, white appears to have experienced accelerated rates of evolution in few lineages, the affinities of which are therefore suspect. In combined analyses, most of the inferred relationship were well-supported and in agreement with previous studies: monophyly of Anophelinae, basal position of Chagasia, monophyly of Anopheles subgenera, and subgenera Nyssorhynchus + Kerteszia as sister taxa. The results suggested also monophyletic origin of subgenera Cellia + Anopheles, and the white gene analysis supported genus Bironella as a sister taxon to Anopheles. The present data and other available evidence suggest a South American origin of Anophelinae, probably in the Mesozoic; a rapid diversification of Bironella and basal subgeneric lineages of Anopheles, potentially associated with the breakup of Gondwanaland; and a relatively recent and rapid dispersion of subgenus Anopheles.     

Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton

Analyses of small subunit ribosomal RNA genes (SSU rDNAs) have significantly influenced our understanding of the composition of aquatic microbial assemblages. Unfortunately, SSU rDNA sequences often do not have sufficient resolving power to differentiate closely related species. To address this general problem for uncultivated bacterioplankton taxa, we analysed and compared sequences of polymerase chain reaction (PCR)-generated and bacterial artificial chromosome (BAC)-derived clones that contained most of the SSU rDNAs, the internal transcribed spacer (ITS) and the large subunit ribosomal RNA gene (LSU rDNA). The phylogenetic representation in the rRNA operon PCR library was similar to that reported previously in coastal bacterioplankton SSU rDNA libraries. We observed good concordance between the phylogenetic relationships among coastal bacterioplankton inferred from SSU or LSU rDNA sequences. ITS sequences confirmed the close intragroup relationships among members of the SAR11, SAR116 and SAR86 clades that were predicted by SSU and LSU rDNA sequence analyses. We also found strong support for homologous recombination between the ITS regions of operons from the SAR11 clade.     

Phylogeny of the lichen genus Placopsis and its allies based on Bayesian analyses of nuclear and mitochondrial sequences

The phylogenetic relationships of the lichen genus Placopsis and related genera in the Agyriales were analyzed using molecular data. We obtained a total of 66 new sequences from the nuclear ITS, LSU and the mitochondrial SSU rDNA. Phylogenetic analyses were conducted in a Bayesian and a maximum-parsimony framework. Our analyses show that Placopsis is paraphyletic with members of Orceolina nesting within the genus. A morphological character supporting the Placopsis-Orceolina clade is the non-amyloid ascus. The section Aspiciliopsis as defined by sunken fruiting bodies is not supported, but the type species of Aspiciliopsis is more closely related to Orceolina. This clade shares apothecia with reduced amphithecia as apomorphic character. We suggest resurrecting the generic name Aspiciliopsis. Trapelia is the sister genus to Placopsis and Aspiciliopsis/Orceolina.     

Assessing the phylogenetic utility of four mitochondrial genes and a nuclear intron in the asian pit viper genus,Trimeresurus: separate,simultaneous, and conditional data combination analyses

A number of methods have been proposed for addressing how to optimize the analysis of multiple data sets from diverse mitochondrial and nuclear gene partitions in the pursuit of robust organismal phylogenies. The present study used separate, simultaneous, and conditional data combination methods to analyze 3,135 bp of data from four mitochondrial partitions and the seventh intron of the beta fibrinogen gene in the Asian pit viper genus, Trimeresurus sensu stricto. The phylogenetic utility and homogeneity of all partitions were estimated via a combination of homogeneity partition tests, homoplasy indices, and partitioned Bremer support. Despite the detection of significant heterogeneity of phylogenetic signal between the mitochondrial and nuclear partitions, the simultaneous analysis represented the best-supported topology of all the data. The relatively slow rate (approximately one quarter of the rate of mtDNA) and functionally unconstrained molecular evolution of the intron resulted in much lower levels of homoplasy compared with the mitochondrial partitions. This was further shown via partitioned Bremer support, which, when considered throughout hierarchical clade levels, highlighted the phylogenetic strength and limitations of the intron at deeper and shallower phylogenetic levels, respectively. The simultaneous analysis helped to resolve the phylogenetic relationships of taxa that were unresolved throughout all individual gene trees and tentatively supports the existence of morphologically and genetically distinct clades within the genus. Topological appraisals of the mitochondrial gene partitions suggest that the cytochrome b and the NADH subunit 4 gene partitions are better estimators of phylogenetic relationships than are the 12S and 16S ribosomal RNA partitions at the taxonomic levels under consideration.