Levels Of Sequence Divergence Research Articles

Phylogeny and molecular evolution of the DMC1 gene within the StH genome species in Triticeae (Poaceae)

To estimate the phylogeny and molecular evolution of a single-copy nuclear disrupted meiotic cDNA (DMC1) gene within the StH genome species, two DMC1 homoeologous sequences were isolated from nearly all the sampled StH genome species and were analyzed with those from seven diploid taxa representing the St and H genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1) there is a close relationship among North American StH genome species; (2) the DMC1 gene sequences of the StH genome species from North America and Eurasia are evolutionarily distinct; (3) the StH genome polyploids have higher levels of sequence diversity in the St genome homoeolog than the H genome homoeolog; (4) the DMC1 sequence may evolve faster in the polyploid species than in the diploids; (5) high dN and dN/dS values in the St genome within polyploid species could be caused by low selective constraints or AT-biased mutation pressure. Our result provides some insight on evolutionary dynamics of duplicate DMC1 gene, the polyploidization events and phylogeny of the StH genome species.

PHYRN: a robust method for phylogenetic analysis of highly divergent sequences.

Both multiple sequence alignment and phylogenetic analysis are problematic in the “twilight zone” of sequence similarity (≤25% amino acid identity). Herein we explore the accuracy of phylogenetic inference at extreme sequence divergence using a variety of simulated data sets. We evaluate four leading multiple sequence alignment (MSA) methods (MAFFT, T-COFFEE, CLUSTAL, and MUSCLE) and six commonly used programs of tree estimation (Distance-based: Neighbor-Joining; Character-based: PhyML, RAxML, GARLI, Maximum Parsimony, and Bayesian) against a novel MSA-independent method (PHYRN) described here. Strikingly, at “midnight zone” genetic distances (∼7% pairwise identity and 4.0 gaps per position), PHYRN returns high-resolution phylogenies that outperform traditional approaches. We reason this is due to PHRYN's capability to amplify informative positions, even at the most extreme levels of sequence divergence. We also assess the applicability of the PHYRN algorithm for inferring deep evolutionary relationships in the divergent DANGER protein superfamily, for which PHYRN infers a more robust tree compared to MSA-based approaches. Taken together, these results demonstrate that PHYRN represents a powerful mechanism for mapping uncharted frontiers in highly divergent protein sequence data sets.

Phylogenetic relationships of the extinct Carolina Parakeet (Conuropsis carolinensis) inferred from DNA sequence data

We obtained the first DNA sequences from the extinct Carolina Parakeet (Conuropsis carolinensis) and used these data to infer the phylogenetic relationships of this iconic North American parrot. We compared our sequences of the mitochondrial COI and ND2 genes obtained from multiple Carolina Parakeet museum specimens to homologous sequences from individuals representing 43 species in 28 genera of Neotropical parrots (Tribe Arini), and four species from more distantly related Old World species of the Order Psittaciformes. Bayesian and maximum likelihood analyses place C.carolinensis on a long branch within a well-supported clade of parakeets that also includes Aratinga solstitialis, A. auricapillus, and Nandayus nenday. These species of Aratinga (but not N. nenday) closely resemble C.carolinensis in the presence of yellow and orange head plumage and blue feathers in the wings. Our data do not support a close relationship with the Monk Parakeet (Myiopsitta monachus), with which the Carolina Parakeet shares fully feathered ceres, a putative adaptation for cold tolerance that appears to have evolved independently in both species. Given the high level of sequence divergence from all sampled species, we recommend continued recognition of the monotypic genus Conuropsis. Taxonomic revision of the highly polyphyletic genus Aratinga is needed.

Contrasted Patterns of Molecular Evolution in Dominant and Recessive Self-Incompatibility Haplotypes in Arabidopsis

Self-incompatibility has been considered by geneticists a model system for reproductive biology and balancing selection, but our understanding of the genetic basis and evolution of this molecular lock-and-key system has remained limited by the extreme level of sequence divergence among haplotypes, resulting in a lack of appropriate genomic sequences. In this study, we report and analyze the full sequence of eleven distinct haplotypes of the self-incompatibility locus (S-locus) in two closely related Arabidopsis species, obtained from individual BAC libraries. We use this extensive dataset to highlight sharply contrasted patterns of molecular evolution of each of the two genes controlling self-incompatibility themselves, as well as of the genomic region surrounding them. We find strong collinearity of the flanking regions among haplotypes on each side of the S-locus together with high levels of sequence similarity. In contrast, the S-locus region itself shows spectacularly deep gene genealogies, high variability in size and gene organization, as well as complete absence of sequence similarity in intergenic sequences and striking accumulation of transposable elements. Of particular interest, we demonstrate that dominant and recessive S-haplotypes experience sharply contrasted patterns of molecular evolution. Indeed, dominant haplotypes exhibit larger size and a much higher density of transposable elements, being matched only by that in the centromere. Overall, these properties highlight that the S-locus presents many striking similarities with other regions involved in the determination of mating-types, such as sex chromosomes in animals or in plants, or the mating-type locus in fungi and green algae.

Utility of Genetic Markers and Morphology for Species Discrimination within the Order Tintinnida (Ciliophora, Spirotrichea)

We evaluated the small- and large-subunit rDNA (SSU and LSU, respectively) for their ability to discriminate morphospecies of tintinnid ciliates. Multiple individuals from 29 morphospecies were identified according to microscopically-observed characteristics of the lorica, and then sequenced for both loci (21 new species for SSU and all of them new for LSU). Sequences from public databases were included in our analyses, and two hypervariable SSU regions (V4 and V9) were separately examined. Of the four regions, LSU is the most useful as a potential barcoding tool. It showed a gap in distances within and between species, and discriminated the maximum number of phylotypes (86% at 1% cut-off). SSU and V4 were less consistent, sometimes lumping together very distinctive morphospecies, even at the 1% level of sequence divergence. V9 was the least reliable marker in delimitating morphospecies. The agreement in sequences and morphology suggests that the lorica is useful for species discrimination, even in agglomerated forms. However, the observation of both genetically constant yet polymorphic groups of species, as well as similar morphospecies with divergent sequences, indicates that previous taxonomic schemes are complementary to the emerging molecular database.

Rapid Evolution of Enormous, Multichromosomal Genomes in Flowering Plant Mitochondria with Exceptionally High Mutation Rates

Author SummaryA fundamental challenge in evolutionary biology is to explain why organisms exhibit dramatic variation in genome size and complexity. One hypothesis predicts that high rates of mutation in DNA sequence create selection against large and complex genomes, which are more susceptible to mutational disruption. Species of flowering plants in the genus Silene vary by approximately 100-fold in the rates of mutation in their mitochondrial DNA, providing an excellent opportunity to test the predicted effects of high mutation rates on genome evolution. Contrary to expectation, Silene species with elevated mutation rates have experienced dramatic expansions in mitochondrial genome size compared to their slowly evolving relatives, resulting in the largest known mitochondrial genomes. In addition to the increases in size and mutation rate, these genomes also reveal a history of rapid change in genome structure. They have been fragmented into dozens of chromosomes and appear to have experienced major reductions in recombination activity. All of these changes have occurred in just the past few million years. This mitochondrial genome diversity within the genus Silene provides a striking example of rapid genomic change and raises new hypotheses regarding the relationship between mutation rate and genome evolution.

Genetic diversity of ITS sequences of Bursaphelenchus xylophilus

The sequence variation of internal transcribed spacer (ITS) regions of ribosomal DNA has been routinely used for species identification and species-level phylogeny of the pinewood nematode, Bursaphelenchus xylophilus. In this study, the intraspecies ITS genetic diversity of B. xylophilus was evaluated. Three pinewood nematode isolates from the United States, Japan, and Portugal were used for polymerase chain reaction (PCR) ITS region amplification and sequencing. Multiple peaks were observed in sequencing chromatograms from ITS regions of American and Japanese isolates, suggesting the presence of more than one ribosomal sequence for each isolate. PCR products were further cloned and 10 clones of each isolate were subsequently sequenced. Additionally, the ITS regions of individual nematodes from each isolate were amplified, cloned and sequenced. Among the 3 B. xylophilus isolates analyzed, an intraspecific and intra-isolate molecular variability was found. The intra-isolate ITS molecular diversity in the American isolate was higher than that in the Japanese and Portuguese isolates. However, the level of sequence variation observed within isolates was about the same as that described among ITS repeats within individuals.

A comparative analysis of Y chromosome and mtDNA phylogenies of the Hylobates gibbons

BackgroundThe evolutionary relationships of closely related species have long been of interest to biologists since these species experienced different evolutionary processes in a relatively short period of time. Comparison of phylogenies inferred from DNA sequences with differing inheritance patterns, such as mitochondrial, autosomal, and X and Y chromosomal loci, can provide more comprehensive inferences of the evolutionary histories of species. Gibbons, especially the genus Hylobates, are particularly intriguing as they consist of multiple closely related species which emerged rapidly and live in close geographic proximity. Our current understanding of relationships among Hylobates species is largely based on data from the maternally-inherited mitochondrial DNAs (mtDNAs).ResultsTo infer the paternal histories of gibbon taxa, we sequenced multiple Y chromosomal loci from 26 gibbons representing 10 species. As expected, we find levels of sequence variation some five times lower than observed for the mitochondrial genome (mtgenome). Although our Y chromosome phylogenetic tree shows relatively low resolution compared to the mtgenome tree, our results are consistent with the monophyly of gibbon genera suggested by the mtgenome tree. In a comparison of the molecular dating of divergences and on the branching patterns of phylogeny trees between mtgenome and Y chromosome data, we found: 1) the inferred divergence estimates were more recent for the Y chromosome than for the mtgenome, 2) the species H. lar and H. pileatus are monophyletic in the mtgenome phylogeny, respectively, but a H. pileatus individual falls into the H. lar Y chromosome clade.ConclusionsBased on the ~6.4 kb of Y chromosomal DNA sequence data generated for each of the 26 individuals in this study, we provide molecular inferences on gibbon and particularly on Hylobates evolution complementary to those from mtDNA data. Overall, our results illustrate the utility of comparative studies of loci with different inheritance patterns for investigating potential sex specific processes on the evolutionary histories of closely related taxa, and emphasize the need for further sampling of gibbons of known provenance.

Genetic and phylogenetic analysis of South Korean sacbrood virus isolates from infected honey bees (Apis cerana)

Sacbrood virus (SBV) is one of the most destructive honey bee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Genetic analysis of SBV infected honey bees (Apis cerana) from five different provinces was carried out based on three nucleotide sequences; one partial structural protein coding sequence and two non-structural protein coding sequences. Sequences amplified by three specific primer pairs were aligned and compared with reference sequences deposited in the GenBank database. Sequence alignments revealed a low level of sequence variation among Korean isolates (≥98.6% nucleotide identity), regardless of the genome regions studied or the geographic origins of the strains. Multiple sequence comparisons indicated that Korean SBV isolates are genetically closely related to Chinese and other Asian strains. Interestingly, the Korean SBV isolates showed a number of unique nucleotides and amino acids that had not been observed in other published strains. Korean and other Asian isolates from the host A. cerana and the UK, European and Japanese strains from the host Apis mellifera showed differences in nucleotide and deduced amino acid identities. This suggests that host-specificity exists among SBV strains isolated from different species. Phylogenetic relatedness between compared sequences was analyzed by MEGA 4.1 software using the neighbor-joining (NJ) method with a boot-strap value of 1000 replicates. Obtained topologies were in agreement with previous studies, in which a distinct group of SBV was formed by UK and European genotypes and another group was comprised of Asian genotypes including strains that originated from China, Japan (japonica), India and Nepal. However, phylogeny based on a partial protein structural coding sequence grouped all Korean SBV isolates identified in A. cerana as a separate cluster. Our findings suggest that further study, including Korean SBV isolated from A. mellifera, is needed.

Oxygenation properties and oxidation rates of mouse hemoglobins that differ in reactive cysteine content

House mice (genus Mus) harbor extensive allelic variation at two tandemly duplicated genes that encode the β-chain subunits of adult hemoglobin (Hb). Alternative haplotypes differ in the level of sequence divergence between the two β-globin gene duplicates: the Hbb d and Hbb p haplotypes harbor two structurally distinct β-globin genes, whereas the Hbb s haplotype harbors two β-globin duplicates that are identical in sequence. One especially salient difference between the s-type Hbs relative to the d- and p-type Hbs relates to the number of reactive β-chain cysteine residues. In addition to the highly conserved cysteine residue at β93, the d- and p-type Hbs contain an additional reactive cysteine residue at β13. To assess the functional consequences of allelic variation in β-globin cysteine content, we measured O 2-binding properties and H 2O 2-induced oxidation rates of mono- and dicysteinyl β-Hbs from 4 different inbred strains of mice: C57BL/6J, BALB/cJ, MSM/Ms, and CAROLI/EiJ. The experiments revealed that purified Hbs from the various mouse strains did not exhibit substantial variation in O 2-binding properties, but s-type Hb (which contains a single reactive β-chain cysteine residue) was far more readily oxidized to Fe 3 + metHb by H 2O 2 than other mouse Hbs that contain two reactive β-chain cysteine residues. These results suggest that the possession of an additional reactive cysteine residue may protect against metHb formation under oxidizing conditions. The allelic differences in β-globin cysteine content could affect aspects of redox signaling and oxidative/nitrosative stress responses that are mediated by Hb-S-nitrosylation and Hb-S-glutathionylation pathways.

Detection of Human Rhinovirus C Viral Genome in Blood among Children with Severe Respiratory Infections in the Philippines

Human rhinovirus (HRV) C was recently identified as the third species of HRV using a molecular technique. Infections caused by previously identified HRVs (A and B) are thought to be limited to the respiratory tract; however, pathogenesis of HRVC is still largely unknown. A total of 816 nasopharyngeal swabs from hospitalized children with severe respiratory infections in the Philippines (May 2008–May 2009) were tested for HRV by reverse transcription polymerase chain reaction (RT-PCR), and 243 samples (29.8%) were positive for HRV. Among these patients, serum samples were also tested to determine whether specific HRV species were associated with viremia. Only 30 serum samples (12.3%) were positive for HRV. However, the HRV positive rates were different among HRV species, 3% (4/135) for HRVA, 0% (0/25) for HRVB, and 31% (26/83) for HRVC, and were the highest on 2 days after the onset of symptoms. These results suggest that HRVC may have a different pathogenicity and can more commonly cause viremia than HRVA and HRVB. Serum positive rates for HRV are affected by age, i.e., higher positive rates for those aged 1 year or more. HRVC that were detected from serum exhibited the same level of sequence diversity as those positive only for nasopharyngeal samples in phylogenetic analysis. However, all HRVA which were detected from serum were clustered in a monophyletic clade based on their 5′ non-coding region (NCR) sequences, which is closely related with a certain HRVC genotype (A2) in 5′-NCR. This finding suggests that the 5′NCR region may be associated with viremia.

Growing coffee: Psilanthus (Rubiaceae) subsumed on the basis of molecular and morphological data; implications for the size, morphology, distribution and evolutionary history of Coffea

Morphological and molecular phylogenetic studies show that there is a close relationship between Coffea and Psilanthus. In this study we reassess species relationships based on improved species sampling for Psilanthus, including P. melanocarpus, a species that shares morpho-taxonomic characters of both genera. Analyses are performed using parsimony and Bayesian inference, on sequence data from four plastid regions [trnL–F intron, trnL–F IGS, rpl16 intron and accD–psa1 intergenic spacer (IGS)] and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (ITS 1/5.8S/ITS 2). Several major lineages with geographical coherence, as identified in previous studies based on smaller and larger data sets, are supported. Our results also confirm previous studies showing that the level of sequence divergence between Coffea and Psilanthus species is negligible, particularly given the much longer branch lengths separating other genera of tribe Coffeeae. There are strong indications that neither Psilanthus nor Coffea is monophyletic. Psilanthus melanocarpus is nested with the Coffea–Psilanthus clade, which means that there is only one critical difference between Coffea and Psilanthus; the former has a long-emergent style and the latter a short, included style. Based on these new data, in addition to other systematically informative evidence from a broad range of studies, and especially morphology, Psilanthus is subsumed into Coffea. This decision increases the number of species in Coffea from 104 to 124, extends the distribution to tropical Asia and Australasia and broadens the morphological characterization of the genus. The implications for understanding the evolutionary history of Coffea are discussed. A group of closely related species is informally named the ‘Coffea liberica alliance’. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 357–377.

Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipesreveal birth-and-death evolution and strong purifying selection

BackgroundThe 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context.ResultsThe nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types.ConclusionsThese results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection.

Genomic Analysis of the Vaccinia Virus Strain Variants Found in Dryvax Vaccine

Smallpox was eradicated using variant forms of vaccinia virus-based vaccines. One of these was Dryvax, a calf lymph vaccine derived from the New York City Board of Health strain. We used genome-sequencing technology to examine the genetic diversity of the population of viruses present in a sample of Dryvax. These studies show that the conserved cores of these viruses exhibit a lower level of sequence variation than do the telomeres. However, even though the ends of orthopoxviruses are more genetically plastic than the cores, there are still many telomeric genes that are conserved as intact open reading frames in the 11 genomes that we, and 4 genomes that others, have sequenced. Most of these genes likely modulate inflammation. Our sequencing also detected an evolving pattern of mutation, with some genes being highly fragmented by randomly assorting mutations (e.g., M1L), while other genes are intact in most viruses but have been disrupted in individual strains (e.g., I4L in strain DPP17). Over 85% of insertion and deletion mutations are associated with repeats, and a rare new isolate bearing a large deletion in the right telomere was identified. All of these strains cluster in dendrograms consistent with their origin but which also surprisingly incorporate horsepox virus. However, these viruses also exhibit a "patchy" pattern of polymorphic sites characteristic of recombinants. There is more genetic diversity detected within a vial of Dryvax than between variola virus major and minor strains, and our study highlights how propagation methods affect the genetics of orthopoxvirus populations.

Nuclear 18S rDNA As A Species-Level Molecular Marker for Leucothoidae (Amphipoda)

Abstract Leucothoid amphipods were investigated using nuclear 18S ribosomal DNA sequences. Analysis of sequences from eight species in two genera supported the current morphological species designations and the separation of the family into two clades. Additionally, an anamorph-leucomorph connection has been confirmed with molecular sequence data that demonstrated both morphologies conclusively belonged to the same species, Anamixis vanga Thomas, 1997. Analyses suggest that morphology is reflecting the genetic evolutionary history of the family. Levels of sequence divergence are compared for 18S rDNA and COI mitochondrial DNA in Leucothoidae.

Transcriptome analysis and comparison reveal divergence between two invasive whitefly cryptic species

BackgroundInvasive species are valuable model systems for examining the evolutionary processes and molecular mechanisms associated with their specific characteristics by comparison with closely related species. Over the past 20 years, two species of the whitefly Bemisia tabaci species complex, Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED), have both spread from their origin Middle East/Mediterranean to many countries despite their apparent differences in many life history parameters. Previously, we have sequenced the transcriptome of MED. In this study, we sequenced the transcriptome of MEAM1 and took a comparative genomic approach to investigate the transcriptome evolution and the genetic factors underlying the differences between MEAM1 and MED.ResultsUsing Illumina sequencing technology, we generated 17 million sequencing reads for MEAM1. These reads were assembled into 57,741 unique sequences and 15,922 sequences were annotated with an E-value above 10-5. Compared with the MED transcriptome, we identified 3,585 pairs of high quality orthologous genes and inferred their sequence divergences. The average differences in coding, 5' untranslated and 3' untranslated region were 0.83%, 1.66% and 1.43%, respectively. The level of sequence divergence provides additional support to the proposition that MEAM1 and MED are two species. Based on the ratio of nonsynonymous and synonymous substitutions, we identified 24 sequences that have evolved in response to positive selection. Many of those genes are predicted to be involved in metabolism and insecticide resistance which might contribute to the divergence of the two whitefly species.ConclusionsOur data present a comprehensive sequence comparison between the two invasive whitefly species. This study will provide a road map for future investigations on the molecular mechanisms underlying their biological differences.

Phylogeographic patterns and cryptic speciation across oceanographic barriers in South African intertidal fishes

Biogeographic boundaries are the meeting zone of broadly distributed faunas, or the actual cause of a faunal break. In the latter case, closely related sister species should be found across such a boundary. To achieve such a situation, preliminary stages are expected, where phylogeographic breaks followed by genetic cryptic speciation would be observed. Biogeographic boundaries, in the Cape Point/Cape Agulhas region of southern Africa, offer an ideal system to test such predictions. Here, we studied two intertidal clinid fish species that are endemic to southern Africa, Clinus superciliosus (n = 127) and Muraenoclinus dorsalis (n = 114). Using mitochondrial control region, 16S rRNA, 12S rRNA and NADH2 genes and the nuclear rhodopsin and the first intron of the S7 ribosomal protein gene, we show both phylogeographic breaks and likely cryptic speciation in each species. Pairwise Φ(st) results suggest population genetic structuring for both species, with higher levels for M. dorsalis (Φ(st) = 0.34-0.93) than for C. superciliosus (Φ(st) = 0.1-0.74). Further, we recover two and three distinct lineages within M. dorsalis and C. superciliosus, respectively. Phylogenetic topologies, concordance between nuclear and mitochondrial markers and levels of sequence divergence, which are consistent with closely related sister species pairs, suggest the presence of cryptic species. Our results therefore meet the expectation for reduced gene flow at a biogeographic barrier, which translates into significant genetic breaks and cryptic sister species.

What does it take to resolve relationships and to identify species with molecular markers? An example from the epiphytic Rhipsalideae (Cactaceae)

The Cactaceae are a major New World plant family and popular in horticulture. Still, taxonomic units and species limits have been difficult to define, and molecular phylogenetic studies so far have yielded largely unresolved trees, so relationships within Cactaceae remain insufficiently understood. This study focuses on the predominantly epiphytic tribe Rhipsalideae and evaluates the utility of a spectrum of plastid genomic regions. • We present a phylogenetic study including 52 of the 53 Rhipsalideae species and all the infraspecific taxa. Seven regions (trnK intron, matK, rbcL, rps3-rpl16, rpl16 intron, psbA-trnH, trnQ-rps16), ca. 5600 nucleotides (nt) were sequenced per sample. The regions used were evaluated for their phylogenetic performance and performance in DNA-based species recognition based on operational taxonomic units (OTUs) defined beforehand. • The Rhipsalideae are monophyletic and contain five clades that correspond to the genera Rhipsalis, Lepismium, Schlumbergera, Hatiora, and Rhipsalidopsis. The species-level tree was well resolved and supported; the rpl16 and trnK introns yielded the best phylogenetic signal. Although the psbA-trnH and trnQ-rps16 spacers were the most successful individual regions for OTU identification, their success rate did not significantly exceed 70%. The highest OTU identification rate of 97% was found using the combination of psbA-trnH, rps3-rpl16, trnK intron, and trnQ-rps16 as a minimum possible marker length (ca. 1660 nt). • The phylogenetic performance of a marker is not determined by the level of sequence variability, and species discrimination power does not necessarily correlate with phylogenetic utility.

A set of plastid DNA-specific universal primers for flowering plants

MatKand rbcL are recommended as the official barcode loci for higher plants but there remains a need for additional universal markers. We generated a series of 84 new universal primers targeting 42 plastid loci that all yielded single amplicons when applied to DNA templates from 19 diverse higher plant families. Marker utility ultimately depends on sequence variability, with rapidly evolving loci being useful for barcoding or biogeographic applications and more conserved loci being better suited to deep phylogeny reconstruction. Whereas excessive size variation is undesirable for many applications, modest size variability caused by indels and the sequence variation frequently associated with indels are highly desirable. We therefore performed a quick screen of the markers for size and sequence variation using pooled DNA templates from 96 taxonomically diverse species. All markers produced little or no size variation (consistent with the presence of minor indels). The seven regions exhibiting most size variation in pooled (rpl23&rpl2.1, 16S, 23S, 4.5S&5S, petB&D, and rpl2, rpoCl and trnK introns) were then amplified for all species individually, confirming the pooled template results. When the most variable loci (introns of trnK and rpoC1) were sequenced for all 96 species, a high level of sequence variation (nucleotide substitutions and indels) was observed among congeneric species groups for both loci. Both markers therefore have potential as supplementary barcode markers.

Phylogenetics, Morphology, and Evolution of the Large GenusMyrcias.l. (Myrtaceae)

Phylogenetic relationships of Myrcia s.l., the most species-rich tree genus in the Brazilian cerrado and Atlantic forests, are reconstructed by analysis, with parsimony and Bayesian inference, of four plastid regions (psbA-trnH intergenic spacer, trnL intron, trnL-F intergenic spacer, and matK exon) and the external and internal transcribed spacer regions of nuclear ribosomal DNA. The resulting phylogenetic hypothesis is evaluated in conjunction with key morphological characters and abiotic factors suggested to have influenced the group’s current distribution. Some lineages identified by previous classifications are supported; others with morphological and/or ecological correspondence are recognized for the first time. Sequence divergence levels provide good resolution of relationships within but not between the main clades of Myrcia s.l. The previously recognized genera Calyptranthes and Gomidesia are shown to be monophyletic, whereas Marlierea is shown not to be; all are nested within paraphyletic Myrcia. A new subgeneric classification is required. An interpretation of the origins of Myrcia s.l. and potential causes of speciation in the group is made on the basis of recently published data and accepted geological events.