Overcoming deep roots, fast rates, and short internodes to resolve the ancient rapid radiation of eupolypod II ferns

Backbone relationships within the large eupolypod II clade, which includes nearly a third of extant fern species, have resisted elucidation by both molecular and morphological data. Earlier studies suggest that much of the phylogenetic intractability of this group is due to three factors: (i) a long root that reduces apparent levels of support in the ingroup; (ii) long ingroup branches subtended by a series of very short backbone internodes (the "ancient rapid radiation" model); and (iii) significantly heterogeneous lineage-specific rates of substitution. To resolve the eupolypod II phylogeny, with a particular emphasis on the backbone internodes, we assembled a data set of five plastid loci (atpA, atpB, matK, rbcL, and trnG-R) from a sample of 81 accessions selected to capture the deepest divergences in the clade. We then evaluated our phylogenetic hypothesis against potential confounding factors, including those induced by rooting, ancient rapid radiation, rate heterogeneity, and the Bayesian star-tree paradox artifact. While the strong support we inferred for the backbone relationships proved robust to these potential problems, their investigation revealed unexpected model-mediated impacts of outgroup composition, divergent effects of methods for countering the star-tree paradox artifact, and gave no support to concerns about the applicability of the unrooted model to data sets with heterogeneous lineage-specific rates of substitution. This study is among few to investigate these factors with empirical data, and the first to compare the performance of the two primary methods for overcoming the Bayesian star-tree paradox artifact. Among the significant phylogenetic results is the near-complete support along the eupolypod II backbone, the demonstrated paraphyly of Woodsiaceae as currently circumscribed, and the well-supported placement of the enigmatic genera Homalosorus, Diplaziopsis, and Woodsia.     

Mitochondrial gene fragments suggest paraphyly of the genus Panorpa (Mecoptera, Panorpidae)

Life history studies of scorpionfly species have been used to test predictions of evolutionary theory, but comparative analysis has been hampered by a lacking phylogeny of scorpionflies. We present a molecular phylogeny of selected panorpid scorpionflies inferred from mitochondrial 12S, 16S rRNA, and COI gene fragments, using parsimony and maximum-likelihood methods. Maximum-likelihood reconstructions depend on an explicit evolutionary substitution model; therefore, we estimated fit of substitution models to our data and used an optimal evolutionary substitution model in subsequent reconstructions. Both reconstruction methods converge on compatible trees with considerable statistical support for a majority of nodes. We performed parametric tests of most important phylogenetic conclusions employing the fitted GTR + %I + Gamma substitution model. Parametric bootstrapping allowed rejection of alternative explanations of the data set, where classical tests, like the KHY test, failed. Parametric bootstrapping confirmed that the association of Neopanorpa sp. with Asian Panorpa species is currently the superior explanation of the data set. Therefore, it is concluded that the genus Panorpa is most likely paraphyletic to the representative of the genus Neopanorpa. We conclude that the sequenced mitochondrial gene fragments appear to be well suited for future more comprehensive phylogenetic investigations of panorpid scorpionflies.     

Multiple lateral transfers of dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes

A large fragment of the dissimilatory sulfite reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G+C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with delta-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the delta-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the delta-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.     

Species Diversity,Phylogeny and Large Scale Biogeographic Patterns of the Genus Padina (Phaeophyceae,Dictyotales)

The brown algal genus Padina (Dictyotales, Phaeophyceae) is distributed worldwide in tropical and temperate seas. Global species diversity and distribution ranges, however, remain largely unknown. Species‐level diversity was reassessed using DNA‐based, algorithmic species delineation techniques based on cox3 and rbcL sequence data from 221 specimens collected worldwide. This resulted in estimates ranging from 39 to 61 putative species (ESUs), depending on the technique as well as the locus. We discuss the merits, potential pitfalls, and evolutionary and biogeographic significance of algorithmic species delineation. We unveil patterns whereby ESUs are in all but one case restricted to either the Atlantic or Indo‐Pacific Ocean. Within ocean basins we find evidence for the vast majority of ESUs to be confined to a single marine realm. Exceptions, whereby ESUs span up to three realms, are located in the Indo‐Pacific Ocean. Patterns of range‐restricted species likely arise by repeated founder events and subsequent peripatric speciation, hypothesized to dominate speciation mechanisms for coastal marine organisms in the Indo‐Pacific. Using a three‐gene (cox3, psaA and rbcL), relaxed molecular clock phylogenetic analysis we estimated divergence times, providing a historical framework to interpret biogeographic patterns.     

Estimation of the age of extant Ephedra using chloroplast rbcL sequence data

The distinctive gymnosperm genus Ephedra is sometimes considered to have originated over 200 million years (Myr) ago on the basis of "ephedroid" fossil pollen. In this article we estimate the age of extant Ephedra using chloroplast rbcL gene sequences. Relative rate tests fail to reject the null hypothesis of equal rates of nucleotide substitution of the rbcL sequences among three landmark lineages (Gnetales, Pinaceae, and Ginkgo). The most divergent sequences we have found in Ephedra differ by only 7 bp for an 1,110 bp region of rbcL sequence, whereas the differences among genera range from 92 to 107 bp in Gnetales and from 35 to 92 bp in Pinaceae. Using three landmark events, the age of extant Ephedra is estimated to be approximately 8-32 Myr. Our result is consistent with the current distribution of many Ephedra species in geologically recent habitats and points out difficulties in the identification of older ephedroid pollen fossils with the modern genus Ephedra.     

93 Progress in characterizing the symbiodinium sp. in soritid foraminifera

Our research seeks to clarify the phylogeny of the Caulerpales through analyses of rbcL (large subunit of ribulose 1,5 biphosphate carboxylase/oxygenase) gene sequences. In a review of caulerpalean taxonomy, Hillis-Colinvaux (1984) recognized two suborders (Bryopsidineae and Halimedineae) on the basis of anatomical, physiological, and habitat characteristics. The Bryopsidineae (including the genera Bryopsis, Derbesia , and Codium ) have cosmopolitan distributions, non-holocarpic reproduction, and homoplasty, while the Halimedineae (including Caulerpa, Halimeda, and Udotea) have tropical to subtropical distributions, holocarpic reproduction, and heteroplasty. Previous phylogenetic analyses based on 18S rRNA sequence data supported the hypothesis of two monophyletic suborders within the Caulerpales (Zechman et al 1990). However, cladistic analyses of morphological characters (Vroom 1998) suggested that only the Halimedineae was monophyletic. Preliminary maximum likelihood and Bayesian analyses suggest the Halimedineae and Bryopsidineae form separate monophyletic groups, with robust support (bootstrap and posterior probabilities) for the former and moderate to poor support for the latter. The families of the Halimedineae (Caulerpaceae, Udoteaceae) form monophyletic sister groups with robust support. The freshwater family Dichotomosiphonaceae was inferred to be basal to the marine Halimedineae clade. The families within the Bryopsidineae (Derbesiaceae, Bryopsidaceae, Codiaceae) each form distinct monophyletic groups. The Codiaceae forms a basal monophyletic group to the sister clade of Bryopsidaceae and Derbeseaceae. This research was partially supported from a NSF grant (DEB-0128977 to FWZ).     

Phylogeny and biogeography of the flowering plant genus Styrax (Styracaceae) based on chloroplast DNA restriction sites and DNA sequences of the internal transcribed spacer region

Phylogenetic relationships within the flowering plant genus Styrax were investigated with DNA sequence data from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) and with chloroplast DNA restriction site data from the genes trnK, rpoC1, and rpoC2. The data sets from each genome were analyzed separately and in combination with parsimony methods. The results strongly support the monophyly of each of the four series of the genus but provide little phylogenetic resolution among them. Reticulate evolution may at least partly explain discordance between the molecular phylogenetic estimates and a prior morphological estimate within series Cyrta. The historical biogeography of the genus was inferred with unweighted parsimony character optimization of trees recovered from a combined ITS and morphological data set, after a series of combinability tests for data set congruence was conducted. The results are consistent with the fossil record in supporting a Eurasian origin of Styrax. The nested phylogenetic position of the South American members of the genus within those from southern North America and Eurasia suggests that the boreotropics hypothesis best explains the amphi-Pacific tropical disjunct distribution occurring within section Valvatae. The pattern of relationship recovered among the species of section Styrax ((western North America + western Eurasia) (eastern North America + eastern Eurasia)) is rare among north-temperate Tertiary forest relicts. The monophyly of the group of species from western North America and western Eurasia provides qualified support for the Madrean-Tethyan hypothesis, which posits a Tertiary floristic connection among the semiarid regions in which these taxa occur. A single vicariance event between eastern Asia and eastern North America accounts for the pattern of relationship among intercontinental disjuncts in series Cyrta.     

The use of chloroplast DNA to resolve plant phylogenies: noncoding versus rbcL sequences

Direct sequencing of polymerase chain reaction products is now an expanding area of plant systematics and evolution. Within angiosperms the rbcL gene has been widely sequenced and used for inferring plant phylogenies at higher taxonomic levels. Unfortunately rbcL does not usually contain enough information to resolve relationships between closely related genera, such as Hordeum, Triticum, and Aegilops. One solution to this problem could be to analyze noncoding regions of chloroplast DNA, which are supposed to evolve more rapidly than coding regions. Here we present pairwise comparisons among dicots and monocots for rbcL and two noncoding sequences of cpDNA (the trnL (UAA) intron and the intergenic spacer between the trnL (UAA) 3' exon and the trnF (GAA) gene). It appears that these regions evolve faster (more than three times faster, on average) than rbcL, as previously reported, and that the trnL intron evolves at a rate that is the same as that of the intergenic spacer. By the analysis of these regions, the genera Hordeum, Triticum, and Aegilops clearly could be distinguished. A phylogeny using trnL (UAA) intron sequences is also inferred for some species of the genus Gentiana L., clearly illustrating the phylogenetic utility of these zones at the generic level. The advantages and the disadvantages of the use of these regions to resolve plant phylogenies are discussed, as well as the desirability of a preliminary study before every large-scale analysis.      

On the systematic position of the genus Coris (Primulaceae)

The systematic position of the aberrant primulaceous genus Coris was investigated by means of cladistic analysis of nucleotide sequence data from the chloroplast gene rbcL . One new sequence ( Coris ) was added to a data matrix composed of a set of previously published sequences. The notion that Coris is most closely related to the Lythraceae is rejected, and very strong support for its position within the Primulaceae was found. The clariñcation of this issue is important for future analyses of phylogenetic interrelationships in the Primulales.     

Evolutionary relationships within the Neotropical, eusporangiate fern genus Danaea (Marattiaceae)

Genera within the eusporangiate fern family Marattiaceae have long been neglected in taxonomic and systematic studies. Here we present the first phylogenetic hypothesis of relationships within the exclusively Neotropical genus Danaea based on a sampling of 60 specimens representing 31 species from various Neotropical sites. We used DNA sequence data from three plastid regions (atpB, rbcL, and trnL-F), morphological characters from both herbarium specimens and live plants observed in the field, and geographical and ecological information to examine evolutionary patterns. Eleven representatives of five other marattioid genera (Angiopteris, Archangiopteris, Christensenia, Macroglossum, and Marattia) were used to root the topology. We identified three well-supported clades within Danaea that are consistent with morphological characters: the "leprieurii" clade (containing species traditionally associated with the name D. elliptica), the "nodosa" clade (containing all species traditionally associated with the name D. nodosa), and the "alata" clade (containing all other species). All three clades are geographically and ecologically widely distributed, but subclades within them show various distribution patterns. Our phylogenetic hypothesis provides a robust framework within which broad questions related to the morphology, taxonomy, biogeography, evolution, and ecology of these ferns can be addressed.     

The evolution of the atpbeta-rbcL intergenic spacer in the epacrids (Ericales) and its systematic and evolutionary implications

Sequence data from the noncoding region separating the plastid genes atpbeta and rbcL were gathered for 27 epacrid taxa, representing all previously recognized infrafamilial groups, and four outgroup taxa (Ericaceae), to address several persistent phylogenetic questions in the group. Parsimony analyses were conducted on these data, as well as on a complementary rbcL sequence dataset assembled from the literature and the combined dataset. The atpbeta-rbcL spacer was notable for the high frequency of insertion-deletion mutations (indels); their distributions were coded as binary characters and included as a adjunct matrix in some of the analyses. The phylogenetic patterns derived from the spacer and rbcL data and the combined analyses, both including and excluding the indel data, concur in resolving seven major lineages corresponding to the tribes of Crayn et al. (1998, Aust. J. Bot. 46, 187-200), viz. Prionoteae, Archerieae, Oligarrheneae, Cosmelieae, Richeeae, Epacrideae, and Styphelieae. The relationships of the tribes and within Styphelieae, however, are not convincingly resolved. Minor conflicts in the positions of some taxa between the spacer and the rbcL trees are poorly supported. Among epacrids, the spacer region provided more cladistically informative characters than rbcL and resulted in trees with lower homoplasy. Further, the spacer data, when analyzed alone and when combined with rbcL, resolved several clades that could not be retrieved on rbcL data alone and provided increased support for many other relationships. The evolution of a putative three-base inversion associated with a hairpin secondary structure in the spacer region is discussed in the light of the inferred phylogeny.     

Molecular phylogeny, historical biogeography, and divergence time estimates for swallowtail butterflies of the genus Papilio (Lepidoptera: Papilionidae)

Swallowtail butterflies are recognized as model organisms in ecology, evolutionary biology, genetics, and conservation biology but present numerous unresolved phylogenetic problems. We inferred phylogenetic relationships for 51 of about 205 species of the genus Papilio (sensu lato) from 3.3-Kilobase (kb) sequences of mitochondrial and nuclear DNA (2.3 kb of cytochrome oxidases I and II and 1.0 kb of elongation factor 1 alpha). Congruent phylogenetic trees were recovered within Papilio from analyses of combined data using maximum likelihood, Bayesian analysis, and maximum parsimony bootstrap consensus. Several disagreements with the traditional classification of Papilio were found. Five major previously hypothesized subdivisions within Papilio were well supported: Heraclides, Pterourus, Chilasa, Papilio (sensu stricto), and Eleppone. Further studies are required to clarify relationships within traditional "Princeps," which was paraphyletic. Several biologically interesting characteristics of Papilio appear to have polyphyletic origins, including mimetic adults, larval host associations, and larval morphology. Early diversification within Papilio is estimated at 55-65 million years ago based on a combination of biogeographic time constraints rather than fossils. This divergence time suggests that Papilio has slower apparent substitution rates than do Drosophila and fig-pollinating wasps and/or divergences corrected using best-fit substitution models are still being consistently underestimated. The amount of sequence divergence between Papilio subdivisions is equivalent to divergences between genera in other tribes of the Papilionidae, and between genera of moths of the noctuid subfamily Heliothinae.     

Incongruence between primary sequence data and the distribution of a mitochondrial atp1 group II intron among ferns and horsetails

Using DNA sequence data from multiple genes (often from more than one genome compartment) to reconstruct phylogenetic relationships has become routine. Augmenting this approach with genomic structural characters (e.g., intron gain and loss, changes in gene order) as these data become available from comparative studies already has provided critical insight into some long-standing questions about the evolution of land plants. Here we report on the presence of a group II intron located in the mitochondrial atp1 gene of leptosporangiate and marattioid ferns. Primary sequence data for the atp1 gene are newly reported for 27 taxa, and results are presented from maximum likelihood-based phylogenetic analyses using Bayesian inference for 34 land plants in three data sets: (1) single-gene mitochondrial atp1 (exon+intron sequences); (2) five combined genes (mitochondrial atp1 [exon only]; plastid rbcL, atpB, rps4; nuclear SSU rDNA); and (3) same five combined genes plus morphology. All our phylogenetic analyses corroborate results from previous fern studies that used plastid and nuclear sequence data: the monophyly of euphyllophytes, as well as of monilophytes; whisk ferns (Psilotidae) sister to ophioglossoid ferns (Ophioglossidae); horsetails (Equisetopsida) sister to marattioid ferns (Marattiidae), which together are sister to the monophyletic leptosporangiate ferns. In contrast to the results from the primary sequence data, the genomic structural data (atp1 intron distribution pattern) would seem to suggest that leptosporangiate and marattioid ferns are monophyletic, and together they are the sister group to horsetails--a topology that is rarely reconstructed using primary sequence data.     

Shared nucleotide composition biases among species and their impact on phylogenetic reconstructions of the Drosophilidae

Compositional changes are a major feature of genome evolution. Overlooking nucleotide composition differences among sequences can seriously mislead phylogenetic reconstructions. Large compositional variation exists among the members of the family Drosophilidae. Until now, however, base composition differences have been largely neglected in the formulations of the nucleotide substitution process used to reconstruct the phylogeny of this important group of species. The present study adopts a maximum-likelihood framework of phylogenetic inference in order to analyze five nuclear gene regions and shows that (1) the pattern of compositional variation in the Drosophilidae does not match the phylogeny of the species; (2) accounting for the heterogeneous GC content with Galtier and Gouy's nucleotide substitution model leads to a tree that differs in significant aspects from the tree inferred when the nucleotide composition differences are ignored, even though both phylogenetic hypotheses attain strong nodal support in the bootstrap analyses; and (3) the LogDet distance correction cannot completely overcome the distorting effects of the compositional variation that exists among the species of the Drosophilidae. Our analyses confidently place the Chymomyza genus as an outgroup closer than the genus Scaptodrosophila to the Drosophila genus and conclusively support the monophyly of the Sophophora subgenus.     

Relationships within Cornales and circumscription of Cornaceae-matK and rbcL sequence data and effects of outgroups and long branches

Phylogenetic relationships in Cornales were assessed using sequences rbcL and matK. Various combinations of outgroups were assessed for their suitability and the effects of long branches and outgroups on tree topology were examined using RASA 2.4 prior to conducting phylogenetic analyses. RASA identified several potentially problematic taxa having long branches in individual data sets that may have obscured phylogenetic signal, but when data sets were combined RASA no longer detected long branch problems. t(RASA) provides a more conservative measurement for phylogenetic signal than the PTP and skewness tests. The separate matK and rbcL sequence data sets were measured as not containing phylogenetic signal by RASA, but PTP and skewness tests suggested the reverse [corrected]. Nonetheless, the matK and rbcL sequence data sets suggested relationships within Cornales largely congruent with those suggested by the combined matK-rbcL sequence data set that contains significant phylogenetic signal as measured by t(RASA), PTP, and skewness tests. Our analyses also showed that a taxon having a long branch on the tree may not be identified as a "long-branched" taxon by RASA. The long branches identified by RASA had little effect on the arrangement of other taxa in the tree, but the placements of the long-branched taxa themselves were often problematic. Removing the long-branched taxa from analyses generally increased bootstrap support, often substantially. Use of non-optimal outgroups (as identified by RASA) decreased phylogenetic resolution in parsimony analyses and suggested different relationships in maximum likelihood analyses, although usually weakly supported clades (less than 50% support) were impacted. Our results do not recommend using t(RASA) as a sole criterion to discard data or taxa in phylogenetic analyses, but t(RASA) and the taxon variance ratio obtained from RASA may be useful as a guide for improved phylogenetic analyses. Results of parsimony and ML analyses of the sequence data using optimal outgroups suggested by RASA revealed four major clades within Cornales: (1) Curtisia-Grubbia, (2) Cornus-Alangium, (3) Nyssa-Camptotheca-Davidia-Mastixia-Diplopanax, and (4) Hydrangeaceae-Loasaceae, with clades (2) and (3) forming a monophyletic group sister to clade (4) and clade (1) sister to the remainder of Cornales. However, there was not strong bootstrap support for relationships among the major clades. The placement of Hydrostachys could not be reliably determined, although most analyses place the genus within Hydrangeaceae; ML analyses, for example, placed the genus as the sister of Hydrangeeae. Our results supported a Cornales including the systematically problematic Hydrostachys, a Cornaceae consisting of Cornus and Alangium, a Nyssaceae consisting of Nyssa and Camptotheca, a monogeneric Davidiaceae, a Mastixiaceae consisting of Mastixia and Diplopanax, and an expanded Grubbiaceae consisting of Grubbia and Curtisia, and two larger families, Hydrangeaceae and Loasaceae.     

Noncoding sequences from the slowly evolving chloroplast inverted repeat in addition to rbcL data do not support gnetalean affinities of angiosperms

We developed PCR primers against highly conserved regions of the rRNA operon located within the inverted repeat of the chloroplast genome and used these to amplify the region spanning from the 3' terminus of the 23S rRNA gene to the 5' terminus of the 5S rRNA gene. The sequence of this roughly 500-bp region, which includes the 4.5S rRNA gene and two chloroplast intergenic transcribed spacer regions (cpITS2 and cpITS3), was determined from 20 angiosperms, 7 gymnosperms, and 16 ferns (21,700 bp). Sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL) from the same or confamilial genera were analyzed in both separate and combined data sets. Due to the low substitution rate in the inverted repeat region, noncoding sequences in the cpITS region are not saturated with substitutions, in contrast to synonymous sites in rbcL, which are shown to evolve roughly six times faster than noncoding cpITS sequences. Several length polymorphisms with very clear phylogenetic distributions were detected in the data set. Results of phylogenetic analyses provide very strong bootstrap support for monophyly of both spermatophytes and angiosperms. No support for a sister group relationship between Gnetales and angiosperms in either cpITS or rbcL data was found. Rather, weak bootstrap support for monophyly of gymnosperms studied and for a basal position for the aquatic angiosperm Nymphaea among angiosperms studied was observed. Noncoding sequences from the inverted repeat region of chloroplast DNA appear suitable for study of land plant evolution.      

Occurrence of matK in a trnK group II intron in charophyte green algae and phylogeny of the Characeae

A group II intron containing the matK gene, which encodes a splicing-associated maturase, was found in the trnK (lysine tRNA) exon in the chloroplast genome of the six extant genera of green algae in the family Characeae, which among green algae are the sister group to embryophytes (land plants). The characean trnK intron (～2.5 kilobases [kb]) and matK ORF (～1.5 kb) are comparable in size to the intron and ORF of land plants, in which they are similarly found inserted in the trnK exon. Domain X, a sequence of conserved amino acid residues within matK, occurs in the Characeae. Phylogenetic analysis using maximum likelihood (GTR + I + gamma likelihood model) and parsimony (branch and bound search) yielded one tree with high bootstrap support for all branches. The matK tree was congruent with the rbcL tree for the same taxa. The number and proportion of informative sites was higher in matK (501, 31% of matK sequence) compared to rbcL (122, 10%). Characeae branch lengths were on average more than five times longer for matK compared to rbcL and provided better resolution within the Characeae. These findings along with recent genomic analyses demonstrate that the intron and matK invaded the chloroplast genome of green algae prior to the evolution of land plants.     

Substitution Model of Sequence Evolution for the Human Immunodeficiency Virus Type 1 Subtype B gp120 Gene over the C2-V5 Region

Phylogenetic analyses frequently rely on models of sequence evolution that detail nucleotide substitution rates, nucleotide frequencies, and site-to-site rate heterogeneity. These models can influence hypothesis testing and can affect the accuracy of phylogenetic inferences. Maximum likelihood methods of simultaneously constructing phylogenetic tree topologies and estimating model parameters are computationally intensive, and are not feasible for sample sizes of 25 or greater using personal computers. Techniques that initially construct a tree topology and then use this non-maximized topology to estimate ML substitution rates, however, can quickly arrive at a model of sequence evolution. The accuracy of this two-step estimation technique was tested using simulated data sets with known model parameters. The results showed that for a star-like topology, as is often seen in human immunodeficiency virus type 1 (HIV-1) subtype B sequences, a random starting topology could produce nucleotide substitution rates that were not statistically different than the true rates. Samples were isolated from 100 HIV-1 subtype B infected individuals from the United States and a 620 nt region of the env gene was sequenced for each sample. The sequence data were used to obtain a substitution model of sequence evolution specific for HIV-1 subtype B env by estimating nucleotide substitution rates and the site-to-site heterogeneity in 100 individuals from the United States. The method of estimating the model should provide users of large data sets with a way to quickly compute a model of sequence evolution, while the nucleotide substitution model we identified should prove useful in the phylogenetic analysis of HIV-1 subtype B env sequences. Received: 4 October 2000 / Accepted: 1 March 2001     

Testing the phylogenetic position of a parasitic plant (Cuscuta, Convolvulaceae, asteridae): Bayesian inference and the parametric bootstrap on data drawn from three genomes

Previous findings on structural rearrangements in the chloroplast genome of Cuscuta (dodder), the only parasitic genus in the morning-glory family, Convolvulaceae, were attributed to its parasitic life style, but without proper comparison to related nonparasitic members of the family. Before molecular evolutionary questions regarding genome evolution can be answered, the phylogenetic problems within the family need to be resolved. However, the phylogenetic position of parasitic angiosperms and their precise relationship to nonparasitic relatives are difficult to infer. Problems are encountered with both morphological and molecular evidence. Molecular data have been used in numerous studies to elucidate relationships of parasitic taxa, despite accelerated rates of sequence evolution. To address the question of the position of the genus Cuscuta within Convolvulaceae, we generated a new molecular data set consisting of mitochondrial (atpA) and nuclear (RPB2) genes, and analyzed these data together with an existing chloroplast data matrix (rbcL, atpB, trnL-F, and psbE-J), to which an additional chloroplast gene (rpl2) was added. This data set was analyzed with an array of phylogenetic methods, including Bayesian analysis, maximum likelihood, and maximum parsimony. Further exploration of data was done by using methods of phylogeny hypothesis testing. At least two nonparasitic lineages are shown to diverge within the Convolvulaceae before Cuscuta. However, the exact sister group of Cuscuta could not be ascertained, even though many alternatives were rejected with confidence. Caution is therefore warranted when interpreting the causes of molecular evolution in Cuscuta. Detailed comparisons with nonparasitic Convolvulaceae are necessary before firm conclusions can be reached regarding the effects of the parasitic mode of life on patterns of molecular evolution in Cuscuta.