Resolution of phylogenetic relationships among recently evolved species as a function of amount of DNA sequence: an empirical study based on woodpeckers (Aves: Picidae)

Synonymous substitutions in the 13 mitochondrial encoded protein genes form a large pool of characters that should approach the ideal for phylogenetic analysis of being independently and identically distributed. Pooling sequences from multiple mitochondrial protein-coding genes should result in statistically more powerful estimates of relationships among species that diverged sufficiently recently that most nucleotide substitutions are synonymous. Cytochrome oxidase I (COI) was sequenced for woodpecker species for which cytochrome b (cyt b) sequences were available. A pairing-design test based on the normal distribution indicated that cyt b evolves more rapidly than COI when all nucleotides are compared but their rates are equal for synonymous substitutions. Nearly all of the phylogenetically informative substitutions among woodpeckers are synonymous. Statistical support for relationships, as measured by bootstrap proportions, increased as the number of nucleotides increased from 1047 (cyt b) to 1512 (COI) to 2559 nucleotides (aggregate data set). Pseudo-bootstrap replicates showed the same trend and increasing the amount of sequence beyond the actual length of 2559 nucleotides to 5120 (2x) resulted in stronger bootstrap support, even though the amount of phylogenetic information was the same. However, the amount of sequence required to resolve an internode depends on the length of the internode and its depth in the phylogeny.     

A phylogenetic analysis of woodpeckers and their allies using 12S, Cyt b, and COI nucleotide sequences (class Aves; order Piciformes)

Although the woodpeckers have long been recognized as a natural, monophyletic taxon, morphological analyses of their intra- and intergeneric relationships have produced conflicting results. To clarify this issue, and as part of a larger study of piciform relationships, nucleotide sequences for the 12S ribosomal RNA (12S; 1123 bp), cytochrome b (Cyt b; 1022 bp), and cytochrome oxidase c subunit 1 (COI; 1512 bp) mitochondrial genes were obtained from 34 piciform species that included 16 of the 23 currently recognized woodpecker genera (subfamily Picinae), three piculets (subfamily Picumninae), a wryneck (subfamily Jynginae), a honeyguide (family Indicatoridae), and three barbets (infraorder Ramphastides). Analyses were conducted on the individual and combined 12S, Cyt b, and COI sequences with maximum parsimony, neighbor-joining, maximum likelihood, and Bayesian algorithms. Based on the strong, congruent support among the different data partitions and models of sequence evolution, a highly resolved consensus of the relationships among woodpeckers and their allies could be formed. The monophyly of Indicatoridae + Picidae (infraorder Picides), Picidae, Picinae + Picumninae, and Picinae was strongly supported in all analyses. However, the tribes Colaptini, Picini, Campephilini, and Campetherini were shown to be paraphyletic as were the genera of Colaptes and Piculus. A revision of the tribal-level classification of woodpeckers is proposed and the importance of plumage convergence among woodpeckers is discussed.     

Marmot phylogeny revisited: molecular evidence for a diphyletic origin of sociality

We established the phylogeny of 11 species of the genus Marmota based on the entire sequence of the mitochondrial cytochrome b ( cyt-b ) gene (1.1 kb) and a partial sequence of the NADH dehydrogenase subunit 4 ( ND4 ) gene (1.2 kb). In three species ( Marmota caligata , Marmota olympus , and Marmota bobac ) full-sized nuclear pseudogenes of the mitochondrial cyt-b were identified. The mitochondrial cyt-b genes and the three pseudogenes form separate clusters in the maximum parsimony dendrogram. This finding suggests that the pseudogenes originated from a single transfer to the nucleus that may have occurred prior to the radiation of the genus Marmota . Notably, compared with their functional mitochondrial equivalents the pseudogenes show a much lower substitution rate. In the dendrograms deduced from the mitochondrial sequences two distinct clusters become apparent: one cluster consists of the North-west American species, the other contains the Eurasian species together with the North American species Marmota monax . The position of M. monax as a member of the Eurasian clade is in accordance with the evolution of chromosome numbers. The results are of special interest with respect to the evolution of social systems in the genus that vary from solitary species ( M. monax ) to highly social species living in family groups (e.g. Marmota marmota ). The molecular phylogeny suggests a diphyletic origin of high sociality in the genus Marmota .     

Searching for evidence of selection in avian DNA barcodes

The barcode of life project has assembled a tremendous number of mitochondrial cytochrome c oxidase I (COI) sequences. Although these sequences were gathered to develop a DNA-based system for species identification, it has been suggested that further biological inferences may also be derived from this wealth of data. Recurrent selective sweeps have been invoked as an evolutionary mechanism to explain limited intraspecific COI diversity, particularly in birds, but this hypothesis has not been formally tested. In this study, I collated COI sequences from previous barcoding studies on birds and tested them for evidence of selection. Using this expanded data set, I re-examined the relationships between intraspecific diversity and interspecific divergence and sampling effort, respectively. I employed the McDonald-Kreitman test to test for neutrality in sequence evolution between closely related pairs of species. Because amino acid sequences were generally constrained between closely related pairs, I also included broader intra-order comparisons to quantify patterns of protein variation in avian COI sequences. Lastly, using 22 published whole mitochondrial genomes, I compared the evolutionary rate of COI against the other 12 protein-coding mitochondrial genes to assess intragenomic variability. I found no conclusive evidence of selective sweeps. Most evidence pointed to an overall trend of strong purifying selection and functional constraint. The COI protein did vary across the class Aves, but to a very limited extent. COI was the least variable gene in the mitochondrial genome, suggesting that other genes might be more informative for probing factors constraining mitochondrial variation within species.     

The amino acid sequence of cytochrome c553 from Microcystis aeruginosa

Cytochrome c553 is an electron donor to P700 in the photosynthetic electron transfer chain of cyanobacteria and eukaryotic algae. We have purified this cytochrome from the cyanobacterium Microcystis aeruginosa and determined its amino acid sequence. When the amino acid sequence of this protein is compared to sequences of cytochromes c553 from other organisms, one sees that the evolution of net charge is more pronounced than the evolution of overall structure, further documenting a pronounced shift in the isoelectric point of this protein during the evolution of cyanobacteria. Cyanobacteria and algae also contain cytochrome c550 (Mr 15,500) which is quite different from cytochrome c553 (Mr 10,500). When the amino acid sequence of cytochrome c553 is compared to that of cytochrome c550, two regions of similar sequence are recognized.     

Phylogeny of the genus Chironomus (Diptera) inferred from DNA sequences of mitochondrial cytochrome b and cytochrome oxidase I

Two mitochondrial genes, Cytochrome b (Cytb) and Cytochrome c oxidase subunit I (COI), have been used as phylogenetic markers in Chironomids. The nucleotide sequences of 685 bp from Cytb and 596 bp from COI have been determined for 36 Chironomus species from the Palearctic, or Holarctic, and Australasia. The concatenated sequence of 1281 bp from both genes was used to investigate the phylogenetic relationships among these species. The nucleotide sequence alignments were used for construction of phylogenetic trees based on maximum-parsimony and neighbor-joining methods. Both techniques produced similar phylogenies. Monophyly of the genus Chironomus is supported by a bootstrap value of 100% at the basal branch. Six clusters of species have been revealed with high bootstrap values supporting both monophyly of each cluster and the validity of the branching order within each cluster. Four species, C. circumdatus, C. nepeanensis, C. dorsalis, and C. crassiforceps, cannot be placed into any cluster. Cytological phylogenies were constructed using the same set of species, except for C. biwaprimus. These trees showed many similarities to that obtained from the mitochondrial (mt) sequence analysis, but also a number of significant differences. When compared with the tree constructed from the sequence of 23 species available for one of the globin genes, globin 2b (gb2b), there was better support for the mt tree than for the cytological trees. An intron, which varies in its occurrence and position in gb2b, was also investigated and the distribution of the introns supports the phylogenetic history of the genus Chironomus obtained with mt data. The differences observed in the cytological trees seem to be attributable more to the retention of the same chromosome banding sequence across several species, rather than convergent evolutionary events. An important question is the determination of the position of the subgenus Camptochironomus in relation to the representatives of the nominal subgenus Chironomus, since it has been suggested that this is a separate genus. The Camptochironomus species are internal to the trees and have arisen more recently than some of the species of the subgenus Chironomus, indicating that they are not sufficiently differentiated to be considered more than a subgenus.     

Cytochrome b(5) forms homomeric complexes in living cells

Cytochrome b(5) (cyt-b(5)) is a ubiquitous hemoprotein also associated with microsomal cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1). In the steroidogenic pathway CYP17A1 catalyses the metabolism of pregnenolone, yielding both glucocorticoid and androgen precursors. While not affecting the 17α-hydroxylation of pregnenolone, cyt-b(5) augments the 17,20 lyase reaction of 17-hydroxypregnenolone, catalyzing the formation of DHEA, through direct protein-protein interactions. In this study, multimeric complex formation of cyt-b(5) and the possible regulatory role of these complexes were investigated. Cyt-b(5) was isolated from ovine liver and used to raise anti-sheep cyt-b(5) immunoglobulins. Immunochemical studies revealed that, in vivo, cyt-b(5) is primarily found in the tetrameric form. Subsequent fluorescent resonance energy transfer (FRET) studies in COS-1 cells confirmed the formation of homomeric complexes by cyt-b(5) in live cells. Site-directed mutagenesis revealed that the C-terminal linker domain of cyt-b(5) is vital for complex formation. The 17,20-lyase activity of CYP17 was augmented by truncated cyt-b(5), which is unable to form complexes when co-expressed in COS-1 cells, thereby implicating the monomeric form of cyt-b(5) as the active species. This study has shown for the first time that cyt-b(5) forms homomeric complexes in vivo, implicating complex formation as a possible regulatory mechanism in steroidogenesis.     

Sequences from 14 mitochondrial genes provide a well-supported phylogeny of the Charadriiform birds congruent with the nuclear RAG-1 tree

Because of the difficulties of constructing a robust phylogeny for Charadriiform birds using morphological characters, recent studies have turned to DNA sequences to resolve the systematic uncertainties of family-level relationships in this group. However, trees constructed using nuclear genes or the mitochondrial Cytochrome b gene suggest deep-level relationships of shorebirds that differ from previous studies based on morphology or DNA-DNA hybridization distances. To test phylogenetic hypotheses based on nuclear genes (RAG-1, myoglobin intron-2) and single mitochondrial genes (Cytochrome b), approximately 13,000 bp of mitochondrial sequence was collected for one exemplar species of 17 families of Charadriiformes plus potential outgroups. Maximum likelihood and Bayesian analyses show that trees constructed from long mitochondrial sequences are congruent with the nuclear gene topologies [Chardrii (Lari, Scolopaci)]. Unlike short mitochondrial sequences (such as Cytochrome b alone), longer sequences yield a well-supported phylogeny for shorebirds across various taxonomic levels. Examination of substitution patterns among mitochondrial genes reveals specific genes (especially ND5, ND4, ND2, and COI) that are better suited for phylogenetic analyses among shorebird families because of their relatively homogeneous nucleotide composition among lineages, slower accumulation of substitutions at third codon positions, and phylogenetic utility in both closely and distantly related lineages. For systematic studies of birds in which family and generic levels are examined simultaneously, we recommend the use of both nuclear and mitochondrial sequences as the best strategy to recover relationships that most likely reflect the phylogenetic history of these lineages.     

Patterns of Protein Evolution in Cytochrome c Oxidase 1 (COI) from the Class Arachnida

Because sequence information is now available for the 648bp barcode region of cytochrome c oxidase 1 (COI) from more than 400,000 animal species, this gene segment can be used to probe patterns of mitochondrial evolution. The present study examines levels of amino acid substitution and the frequency of indels in COI from 4177 species of arachnids, including representatives from all 16 orders and 43% of its families (267/625). It examines divergences at three taxonomic levels—among members of each order to an outgroup, among families in each order and among BINs, a species proxy, in each family. Order Distances vary fourfold (0.10–0.39), while the mean of the Family Distances for the ten orders ranges fivefold (0.07–0.35). BIN Distances show great variation, ranging from 0.01 or less in 12 families to more than 0.25 in eight families. Patterns of amino acid substitution in COI are generally congruent with previously reported variation in nucleotide substitution rates in arachnids, but provide some new insights, such as clear rate acceleration in the Opiliones. By revealing a strong association between elevated rates of nucleotide and amino acid substitution, this study builds evidence for the selective importance of the rate variation among arachnid lineages. Moreover, it establishes that groups whose COI genes have elevated levels of amino acid substitution also regularly possess indels, a dramatic form of protein reconfiguration. Overall, this study suggests that the mitochondrial genome of some arachnid groups is dynamic with high rates of amino acid substitution and frequent indels, while it is ‘locked down’ in others. Dynamic genomes are most prevalent in arachnids with short generation times, but the possible impact of breeding system deserves investigation since many of the rapidly evolving lineages reproduce by haplodiploidy, a mode of reproduction absent in ‘locked down’ taxa.     

Structure and evolution of human α-fetoprotein deduced from partial sequence of cloned cDNA

The nucleotide sequence of a recombinant DNA clone, containing a partial mRNA sequence for human α-fetoprotein (AFP) in the plasmid vector pBR322, has been determined. Two regions of the cloned nucleotide sequence were found to agree with published amino acid sequences of two cyanogen bromide peptides derived from human AFP. Examination of the amino acid sequence, deduced from the cloned portion of the mRNA coding region, reveals extensive homology with the third domain of the human serum albumin molecule. A total of 44% ( ) amino acids and 54% ( ) nucleotides are identical in the two structures. The landmark cysteine residues are found in the same positions in both polypeptide chains, presumably forming the same disulfide bridges in AFP as those found in the albumin. The sequence homology reinforces the evidence that human AFP and albumin constitute a gene family, in analogy to the same family found in rodents. A comparison of the human and rodent sequence data suggests that the rate of molecular evolution has been faster for AFP than for albumin.     

Genomic structure of the luciferase gene and phylogenetic analysis in the Hotaria-group fireflies

The luminescent fireflies have species specific flash patterns, being recognized as sexual communication. The luciferase gene is the sole enzyme responsible for bioluminescence. We describe here the complete nucleotide sequence and the exon-intron structure of the luciferase gene of the Hotaria-group fireflies, H. unmunsana, H. papariensis and H. tsushimana. The luciferase gene of the Hotaria-group firefly including the known H. parvula spans 1950 bp and consisted of six introns and seven exons coding for 548 amino acid residues, suggesting highly conserved structure among the Hotaria-group fireflies. Although only one luciferase gene was cloned from H. papariensis, each of the two sequences of the gene was found in H. unmunsana (U1 and Uc) and H. tsushimana (T1 and T2). The amino acid sequence divergence among H. unmunsana, H. papariensis, and H. tsushimana only ranged from zero to three amino acid residues, but H. parvula differed by 10-11 amino acid residues from the other Hotaria-group fireflies, suggesting a divergent relationship of this species. Phylogenetic analysis using the deduced amino acid sequences of the luciferase gene resulted in a monophyletic group in the Hotaria excluding H. parvula, suggesting a close relationship among H. unmunsana, H. papariensis and H. tsushimana. Additionally, we also analyzed the mitochondrial cytochrome oxidase I (COI) gene of the Hotaria-group fireflies. The deduced amino acid sequence of the COI gene of H. unmunsana was identical to that of H. papariensis and H. tsushimana, but different by three positions from H. parvula. In terms of nucleotide sequences of the COI gene, intraspecific sequence divergence was sometimes larger than interspecies level, and phylogenetic analysis placed the three species into monophyletic groups unresolved among them, but excluded H. parvula. In conclusion, our results suggest that H. unmunsana, H. papariensis and H. tsushimana are very closely related or might be an identical species, at least based on the luciferase and COI genes.     

Mitochondrial COI sequences of brachiopods: genetic code shared with protostomes and limits of utility for phylogenetic reconstruction

A 1230-bp region of the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA of each of 16 brachiopod species, representing all five living orders, was amplified by polymerase chain reaction and sequenced. Pairwise comparisons of sequence differences plotted against divergence times estimated from the brachiopod fossil record revealed that, although there are considerable variations in the expected substitution rate among different lineages, amino acid substitutions of the COI sequences may largely become saturated in 100 Ma, due mostly to multiple substitutions at the same site. Coinciding with this result, phylogenetic analysis indicated low bootstrap values for nodes corresponding to divergence events that occurred before 100 Ma, suggesting that COI sequences are suitable only for inference of phylogenetic events subsequent to the Mesozoic. Examination of brachiopod codons corresponding to invariant amino acids in the COI of various other animals suggest the nonuniversal codon relationships UGA = Trp, AUA = Met, AAA/G = Lys, and AGA/G = Ser. These are identical to those in mollusks, annelids, and arthropods, consistent with the conclusion that brachiopods are protostomes, as indicated by previous molecular analyses.     

Molecular systematics of cytochrome oxidase I and 16S from Neochlamisus leaf beetles and the importance of sampling.

If a gene tree is to be judiciously used for inferring the histories of closely related taxa, (1) its topology must be sufficiently resolved and robust that noteworthy phylogenetic patterns can be confidently documented, and (2) sampling of species, populations, and pertinent biological variation must be sufficiently broad that otherwise misleading sources of genetic variation can be detected. These principles are illustrated by the complex gene tree of Neochlamisus leaf beetles that I reconstructed using 90,000 bp of cytochrome oxidase I (COI) and 16S mitochondrial DNA (mtDNA) sequences from over 100 specimens. Cytochrome oxidase I haplotypes varied up to 25.1% within Neochlamisus and up to 11.1% within the gibbosus species group, while exhibiting very low A + T bias for insect mtDNA (63%), low transition saturation, and conservative patterns of amino acid variation. 16S exhibited lower sequence divergences and greater A + T bias and transition saturation than COI, and substitutions were more constrained in stems than in loops. Comparisons with an earlier study of Ophraella leaf beetles highlighted conservative and labile elements of molecular evolution across genes and taxa. Cytochrome oxidase I parsimony and neighbor-joining analyses strongly supported a robust mtDNA genealogy that revealed the monophyly of Neochlamisus and of the gibbosus species group. Phylogeographic relationships suggested that the eastern U.S. gibbosus group derives from southwestern velutinus group ancestors. Haplotypes from individual velutinus group species clustered monophyletically, as expected. However, haplotypes from each of several gibbosus group taxa were polyphyletically distributed, appearing in divergent parts of the tree. 16S provided a less-resolved gibbosus group topology that was congruent with the COI tree and corroborated patterns of mitochondrial polyphyly. By subsampling haplotypes corresponding to particular species, populations, and ecological variants of gibbosus group taxa, I demonstrate that recovered topologies and genetic distances vary egregiously according to sampling regime. This study thus documents the potentially dire consequences of inadequate sampling when inferring the evolutionary history of closely related and mitochondrially polyphyletic taxa.     

Constraints on Viral Evolution during Chronic Hepatitis C Virus Infection Arising from a Common-Source Exposure

Extraordinary viral sequence diversity and rapid viral genetic evolution are hallmarks of hepatitis C virus (HCV) infection. Viral sequence evolution has previously been shown to mediate escape from cytotoxic T-lymphocyte (CTL) and neutralizing antibody responses in acute HCV infection. HCV evolution continues during chronic infection, but the pressures driving these changes are poorly defined. We analyzed plasma virus sequence evolution in 5.2-kb hemigenomes from multiple longitudinal time points isolated from individuals in the Irish anti-D cohort, who were infected with HCV from a common source in 1977 to 1978. We found phylogenetically distinct quasispecies populations at different plasma time points isolated late in chronic infection, suggesting ongoing viral evolution and quasispecies replacement over time. We saw evidence of early pressure driving net evolution away from a computationally reconstructed common ancestor, known as Bole1b, in predicted CTL epitopes and E1E2, with balanced evolution toward and away from the Bole1b amino acid sequence in the remainder of the genome. Late in chronic infection, the rate of evolution toward the Bole1b sequence increased, resulting in net neutral evolution relative to Bole1b across the entire 5.2-kb hemigenome. Surprisingly, even late in chronic infection, net amino acid evolution away from the infecting inoculum sequence still could be observed. These data suggest that, late in chronic infection, ongoing HCV evolution is not random genetic drift but rather the product of strong pressure toward a common ancestor and concurrent net ongoing evolution away from the inoculum virus sequence, likely balancing replicative fitness and ongoing immune escape.     

Restriction fragment length polymorphism of mitochondrial DNA and phylogenetic relationships among five species of Indian freshwater turtles

DNA-based identification of species for phylogenetic analysis as well as forensic identification is widely being carried out with the help of polymerase chain reaction (PCR). In this study, a successful effort has been made to identify 5 species of Indian freshwater turtles, including 3 hard-shell turtles (Geoemydidae), i.e. Kachuga dhongoka, K. kachuga and Geoclemys hamiltoni, and 2 species of soft-shell turtles (Trionychidae), i.e. Aspideretes gangeticus and Lissemys punctata punctata, by using a well-optimized PCR-RFLP method. The analysis of nucleotide sequence variations in the PCR-amplified mitochondrial cyt-b genes (encoding cytochrome b) from the 5 species revealed its usefulness in the taxonomic differentiation of these species. On the basis of cyt-b sequence data and the PCR-RFLP pattern, a phylogeny was developed to resolve the genetic relationships between these species, living in the same habitat type. In comparison, the PCR-RFLP of mitochondrial 16S rDNA genes appeared less decisive in analysing phylogenetic relationships or even in species differentiation. Further, the molecular method (PCR-RFLP) developed here is simple, rapid, reliable and reproducible; hence it can be routinely applied for species identification, essential for conservation and management of endangered chelonian species.     

Estimating the influence of selection on the variable amino acid sites of the cytochrome B protein functional domains

We evaluated the effects of selection on the molecular evolution of the functional domains of the mammalian cytochrome b gene as it relates to physicochemical properties shown to correlate with rates of amino acid replacement. Two groups of mammals were considered: pocket gophers of the rodent family Geomyidae, and cetaceans and ungulates of the monophyletic taxon Cetartopdactyla. Several characteristics of cytochrome b evolution were common to both mammal groups. The evolution of the matrix domain reflected the region's relative lack of function. Goodness of fit to neutral expectations indicated that external influences have had very little effect on the evolution of the matrix, although in some cases conservative and moderate changes have been favored. Although rates of synonymous nucleotide substitution have been relatively high, the transmembrane domain exhibited poor goodness of fit to neutral expectations. However, the evolution of the transmembrane domain has been constrained by negative selection, allowing a preponderance of conservative and moderate amino acid replacements. We hypothesize that a high rate of substitution is maintained in spite of negative selection because the codons of the transmembrane coding region are predisposed to conservative changes in all amino acid properties. The evolutionary patterns of the intermembrane domain in pocket gophers and cetartiodactyls, however, were very different. Changes inferred from the pocket gopher phylogenetic tree exhibited a significant fit to neutral expectations for each of the amino acid properties. Changes inferred from the cetartiodactyl tree exhibited significant fit to neutral expectations for polarity and isoelectric point, but not for composition, molecular volume, polar requirement, or hydropathy. In each case, lack of fit was due to selection that promoted conservative or moderate change, with the noteworthy exception of polar requirement. We detected an unexpectedly large change in polar requirement (from aspartic acid to threonine) in two separate lineages (Camelus bactrianus and all cetaceans) at amino acid position 159. This inferred change occurred in a region of the cyt-b protein that directly interacts with external surface proteins of the cytochrome bc(1) complex and resulted in a reversion to a more common character state in vertebrates.     

Barcoding Queensland Fruit Flies (Bactrocera tryoni): impediments and improvements

Identification of adult fruit flies primarily involves microscopic examination of diagnostic morphological characters, while immature stages, such as larvae, can be more problematic. One of the Australia’s most serious horticultural pests, the Queensland Fruit Fly (Bactrocera tryoni: Tephritidae), is of particular biosecurity/quarantine concern as the immature life stages occur within food produce and can be difficult to identify using morphological characteristics. DNA barcoding of the mitochondrial Cytochrome Oxidase I (COI) gene could be employed to increase the accuracy of fruit fly species identifications. In our study, we tested the utility of standard DNA barcoding techniques and found them to be problematic for Queensland Fruit Flies, which (i) possess a nuclear copy (a numt pseudogene) of the barcoding region of COI that can be co‐amplified; and (ii) as in previous COI phylogenetic analyses closely related B. tryoni complex species appear polyphyletic. We found that the presence of a large deletion in the numt copy of COI allowed an alternative primer to be designed to only amplify the mitochondrial COI locus in tephritid fruit flies. Comparisons of alternative commonly utilized mitochondrial genes, Cytochrome Oxidase II and Cytochrome b, revealed a similar level of variation to COI; however, COI is the most informative for DNA barcoding, given the large number of sequences from other tephritid fruit fly species available for comparison. Adopting DNA barcoding for the identification of problematic fly specimens provides a powerful tool to distinguish serious quarantine fruit fly pests (Tephritidae) from endemic fly species of lesser concern.     

Mitochondrial DNA Variation, Phylogenetic Relationships, and Evolution of Four Mediterranean Genera of Soles (Soleidae, Pleuronectiformes)

To increase knowledge about the systematics and evolution of Mediterranean soles, we assessed mitochondrial DNA variation, molecular phylogeny, and evolution in eight species from the genera Solea, Microchirus, Monochirus, and Buglossidium by large ribosomal subunit (16S) and cytochrome b (cytb) sequence analysis. Relevant molecular features are the great variation of base composition among species at the third codon in cytb and the heterogeneity of the nucleotide substitution rate. Phylogenies recovered using 16S nucleotide and cytb amino acid sequences agree with those based on morphology in assessing monophyly of Solea species and ancestry of Buglossidium luteum, but they are against the intergeneric differentiation of Microchirus and Monochirus. Conversely, phylogenetic trees based on cytb nucleotide sequences yielded relationships among taxa regardless of their evolutionary histories. The incongruities between morphological and molecular issues suggest the need for reassessing the systematic value of some morphological characters. Approximate estimates of the divergence time of Mediterranean soleid lineages range from 40 to 13 Mya (Oligocene–Miocene), indicating an ancient origin for the group. Received August 31, 1999; accepted December 17, 1999.     

Characteristics of pre-S2 region of hepatitis B virus

The nucleotide sequence of our cloned HBV DNA (subtype adw) has been determined. When the 165-nucleotide sequence of the pre-S2 region was compared with 7 other published sequences (subtypes adw, adr, ayw, and adyw), we found 38 nucleotide substitutions among different subtypes and 4 (adr) or 6 (adw and ayw) substitutions within the same subtype. Analysis of the predicted amino acid sequence from the known nucleotide sequences indicates that: there are 20 amino acid substitutions, the longest conserved amino acid sequence is located between amino acids 23 to 34, and the 54th amino acid is identical within the same subtype but varies in different subtypes.     

Phylogeny of the Heelwalkers (Insecta: Mantophasmatodea) based on mtDNA sequences, with evidence for additional taxa in South Africa

We examined the phylogeny of Mantophasmatodea from southern Africa (South Africa, Namibia) using approx. 1300 bp of mitochondrial DNA sequence data from the genes encoding COI and 16S. The taxon sample comprised multiple specimens from eight described species (Namaquaphasma ookiepense, Austrophasma rawsonvillense, A. caledonense, A. gansbaaiense, Lobatophasma redelinghuysense, Hemilobophasma montaguense, Karoophasma botterkloofense, K. biedouwense) and four undescribed species of Austrophasmatidae; three specimens of Sclerophasma paresisense (Mantophasmatidae); and two specimens of Praedatophasma maraisi and one of Tyrannophasma gladiator (not yet convincingly assigned to any family). For outgroup comparison a broad selection from hemimetabolous insect orders was included. Equally weighted parsimony analyses of the combined COI+16S data sets with gaps in 16S scored as a fifth character state supported Austrophasmatidae and all species and genera of Mantophasmatodea as being monophyletic. Most species were highly supported with 98-100% bootstrap/7-39 Bremer support (BS), but K. biedouwense had moderate support (87/4) and A. caledonense low support (70/1). Mantophasmatodea, Austrophasmatidae, and a clade Tyrannophasma gladiator+Praedatophasma maraisi were all strongly supported (99-100/12-25), while relationships among the two latter clades and Mantophasmatidae remain ambiguous. Concerning the relationships among genera of Austrophasmatidae, support values are moderately high for some nodes, but not significant for others. We additionally calculated the partitioned BS values of COI and 16S for all nodes in the strict consensus of the combined tree. COI and 16S are highly congruent at the species level as well as at the base of Mantophasmatodea, but congruence is poor for most intergeneric relationships. In forthcoming studies, deeper relationships in the order should be additionally explored by nuclear genes, such as 18S and 28S, for a reduced sample of specimens.