Parallel rate heterogeneity in chloroplast and mitochondrial genomes of Brazil nut trees (Lecythidaceae) is consistent with lineage effects

We investigated whether relative rates of divergence were correlated between the mitochondrial and chloroplast genomes as expected under lineage effects or were genome specific as expected with locus-specific effects. Five mitochondrial noncoding regions (nad1B_C, nad4exon1_2, nad7exon2_3, nad7exon3_4, and rps14-cob) for 21 samples from Lecythidaceae were sequenced. Three chloroplast regions (rpl20-5'rps12, trnS-trnG, and psbA-trnH) were sequenced to expand the taxa in an existing data set. Absolute rates of nucleotide and insertion and deletion (indel) changes were 13 times faster in the chloroplast genome than in the mitochondrial genome. Similar indel length frequency distributions for both organelles suggested that common mechanisms were responsible for generating indels. Molecular clock tests applied to phylogenetic trees estimated from mitochondrial and chloroplast sequences revealed global rate heterogeneity of nucleotide substitution. Maximum likelihood and Tajima's 1D relative rate tests show that Lecythis zabucajo exhibited a rate acceleration for both the mitochondrial and chloroplast sequences. Whereas Eschweilera romeu-cardosoi showed a significant rate slowdown for chloroplast sequences, the mitochondrial sequences for 3 Eschweilera taxa showed evidence for a rate slowdown only when compared with L. zabucajo. Significant rate heterogeneity was also observed for indel changes in the mitochondrial genome but not for the chloroplast. The lack of mitochondrial nucleotide changes for some taxa as well as chloroplast indel homoplasy may have limited the power of relative rate tests to detect rate variation. Relative ratio tests consistently indicated rate proportionality among branch lengths between the mitochondrial and chloroplast phylogenetic trees. The relative ratio tests showed that taxa possessing rate heterogeneity had parallel relative divergence rates in both mitochondrial and chloroplast sequences as expected under lineage effects. A neutral replication-dependent model of rate heterogeneity for both nucleotide and indel changes provides a simple explanation for common patterns of rate heterogeneity across the 2 organelle genomes in Lecythidaceae. The lineage effects observed here were uncoupled from annual/perennial habit because all the species from this study are perennial.     

Reevaluation of the cox1 group I intron in Araceae and angiosperms indicates a history dominated by loss rather than horizontal transfer

The origin and modes of transmission of introns remain matters of much debate. Previous studies of the group I intron in the angiosperm cox1 gene inferred frequent angiosperm-to-angiosperm horizontal transmission of the intron from apparent incongruence between intron phylogenies and angiosperm phylogenies, patchy distribution of the intron among angiosperms, and differences between cox1 exonic coconversion tracts (the first 22 nt downstream of where the intron inserted). We analyzed the cox1 gene in 179 angiosperms, 110 of them containing the intron (intron(+)) and 69 lacking it (intron(-)). Our taxon sampling in Araceae is especially dense to test hypotheses about vertical and horizontal intron transmission put forward by Cho and Palmer (1999. Multiple acquisitions via horizontal transfer of a group I intron in the mitochondrial coxl gene during evolution of the Araceae family. Mol Biol Evol. 16:1155-1165). Maximum likelihood trees of Araceae cox1 introns, and also of all angiosperm cox1 introns, are largely congruent with known phylogenetic relationships in these taxa. The exceptions can be explained by low signal in the intron and long-branch attraction among a few taxa with high mitochondrial substitution rates. Analysis of the 179 coconversion tracts reveals 20 types of tracts (11 of them only found in single species, all involving silent substitutions). The distribution of these tracts on the angiosperm phylogeny shows a common ancestral type, characterizing most intron(+) and some intron(-) angiosperms, and several derivative tract types arising from gradual back mutation of the coconverted nucleotides. Molecular clock dating of small intron(+) and intron(-) sister clades suggests that coconversion tracts have persisted for 70 Myr in Araceae, whose cox1 sequences evolve comparatively slowly. Sequence similarity among the 110 introns ranges from 91% to identical, whereas putative homologs from fungi are highly different, but sampling in fungi is still sparse. Together, these results suggest that the cox1 intron entered angiosperms once, has largely or entirely been transmitted vertically, and has been lost numerous times, with coconversion tract footprints providing unreliable signal of former intron presence.     

Molecular evolution and phylogenetic utility of the petD group II intron: a case study in basal angiosperms

Sequences of spacers and group I introns in plant chloroplast genomes have recently been shown to be very effective in phylogenetic reconstruction at higher taxonomic levels and not only for inferring relationships among species. Group II introns, being more frequent in those genomes than group I introns, may be further promising markers. Because group II introns are structurally constrained, we assumed that sequences of a group II intron should be alignable across seed plants. We designed universal amplification primers for the petD intron and sequenced this intron in a representative selection of 47 angiosperms and three gymnosperms. Our sampling of taxa is the most representative of major seed plant lineages to date for group II introns. Through differential analysis of structural partitions, we studied patterns of molecular evolution and their contribution to phylogenetic signal. Nonpairing stretches (loops, bulges, and interhelical nucleotides) were considerably more variable in both substitutions and indels than in helical elements. Differences among the domains are basically a function of their structural composition. After the exclusion of four mutational hotspots accounting for less than 18% of sequence length, which are located in loops of domains I and IV, all sequences could be aligned unambiguously across seed plants. Microstructural changes predominantly occurred in loop regions and are mostly simple sequence repeats. An indel matrix comprising 241 characters revealed microstructural changes to be of lower homoplasy than are substitutions. In showing Amborella first branching and providing support for a magnoliid clade through a synapomorphic indel, the petD data set proved effective in testing between alternative hypotheses on the basal nodes of the angiosperm tree. Within angiosperms, group II introns offer phylogenetic signal that is intermediate in information content between that of spacers and group I introns on the one hand and coding sequences on the other.     

Angiosperm phylogeny inferred from sequences of four mitochondrial genes

An angiosperm phylogeny was reconstructed in a maximum likelihood analysis of sequences of four mitochondrial genes, atpl, matR, had5, and rps3, from 380 species that represent 376 genera and 296 families of seed plants. It is largely congruent with the phylogeny of angiosperms reconstructed from chloroplast genes atpB, matK, and rbcL, and nuclear 18S rDNA. The basalmost lineage consists of Amborella and Nymphaeales (including Hydatellaceae). Austrobaileyales follow this clade and are sister to the mesangiosperms, which include Chloranthaceae, Ceratophyllum, magnoliids, monocots, and eudicots. With the exception of Chloranthaceae being sister to Ceratophyllum, relationships among these five lineages are not well supported. In eudicots, Ranunculales, Sabiales, Proteales, Trochodendrales, Buxales, Gunnerales, Saxifragales, Vitales, Berberidopsidales, and Dilleniales form a basal grade of lines that diverged before the diversification of rosids and asterids. Within rosids, the COM (Celastrales-Oxalidales-Malpighiales) clade is sister to malvids (or rosid Ⅱ), instead of to the nitrogen-fixing clade as found in all previous large-scale molecular analyses of angiosperms. Santalales and Caryophyllales are members of an expanded asterid clade. This study shows that the mitochondrial genes are informative markers for resolving relationships among genera, families, or higher rank taxa across angiosperms. The low substitution rates and low homoplasy levels of the mitochondrial genes relative to the chloroplast genes, as found in this study, make them particularly useful for reconstructing ancient phylogenetic relationships. A mitochondrial gene-based angiosperm phylogeny provides an independent and essential reference for comparison with hypotheses of angiosperm phylogeny based on chloroplast genes, nuclear genes, and non-molecular data to reconstruct the underlying organismal phylogeny.     

The pattern of insertion/deletion polymorphism in Arabidopsis thaliana

Little is known about variation of nucleotide insertion/deletions (indels) within species. In Arabidopsis thaliana, we investigated indel polymorphism patterns between two genome sequences and among 96 accessions at 1215 loci. Our study identified patterns in the variation of indel density, size, GC content and distribution, and a correlation between indels and substitutions. We found that the GC content in indel sequences was lower than that in non-indel sequences and that indels typically occur in regions with lower GC content. Patterns of indel frequency distribution among populations were more consistent with neutral expectation than substitution patterns. We also found that the local level of substitutions is positively correlated with indel density and negatively correlated with their distance to the closed indel, suggesting that indels play an important role in nucleotide variation.     

Is the rate of insertion and deletion mutation male biased?: Molecular evolutionary analysis of avian and primate sex chromosome sequences.

The rate of mutation for nucleotide substitution is generally higher among males than among females, likely owing to the larger number of DNA replications in spermatogenesis than in oogenesis. For insertion and deletion (indel) mutations, data from a few human genetic disease loci indicate that the two sexes may mutate at similar rates, possibly because such mutations arise in connection with meiotic crossing over. To address origin- and sex-specific rates of indel mutation we have conducted the first large-scale molecular evolutionary analysis of indels in noncoding DNA sequences from sex chromosomes. The rates are similar on the X and Y chromosomes of primates but about twice as high on the avian Z chromosome as on the W chromosome. The fact that indels are not uncommon on the nonrecombining Y and W chromosomes excludes meiotic crossing over as the main cause of indel mutation. On the other hand, the similar rates on X and Y indicate that the number of DNA replications (higher for Y than for X) is also not the main factor. Our observations are therefore consistent with a role of both DNA replication and recombination in the generation of short insertion and deletion mutations. A significant excess of deletion compared to insertion events is observed on the avian W chromosome, consistent with gradual DNA loss on a nonrecombining chromosome.     

Indel evolution of mammalian introns and the utility of non-coding nuclear markers in eutherian phylogenetics

Nuclear DNA intron sequences are increasingly used to investigate evolutionary relationships among closely related organisms. The phylogenetic usefulness of intron sequences at higher taxonomic levels has, however, not been firmly established and very few studies have used these markers to address evolutionary questions above the family level. In addition, the mechanisms driving intron evolution are not well understood. We compared DNA sequence data derived from three presumably independently segregating introns (THY, PRKC I and MGF) across 158 mammalian species. All currently recognized extant eutherian mammalian orders were included with the exception of Cingulata, Dermoptera and Scandentia. The total aligned length of the data was 6366 base pairs (bp); after the exclusion of autapomorphic insertions, 1511 bp were analyzed. In many instances the Bayesian and parsimony analyses were complementary and gave significant posterior probability and bootstrap support (>80) for the monophyly of Afrotheria, Euarchontoglires, Laurasiatheria and Boreoeutheria. Apart from finding congruent support when using these methods, the intron data also provided several indels longer than 3 bp that support, among others, the monophyly of Afrotheria, Paenungulata, Ferae and Boreoeutheria. A quantitative analysis of insertions and deletions suggested that there was a 75% bias towards deletions. The average insertion size in the mammalian data set was 16.49 bp +/- 57.70 while the average deletion was much smaller (4.47 bp +/- 14.17). The tendency towards large insertions and small deletions is highlighted by the observation that out of a total of 17 indels larger than 100 bp, 15 were insertions. The majority of indels (>60% of all events) were 1 or 2 bp changes. Although the average overall indel substitution rate of 0.00559 per site is comparable to that previously reported for rodents and primates, individual analyses among different evolutionary lineages provide evidence for differences in the formation rate of indels among the different mammalian groups.     

Polymorphism and structural variation of rps16 group-II intron in the Solanum species

Nucleotide sequences of the self-splicing group-II intron of rps16 have first been determined in nine species of the Solanum genus. It was found that the observed variations in the intron length (855–864 bp) was associated with indels of 1 to 9 bp. Altogether, five indels and 50 nucleotide substitutions were detected, which were used to identify six Solanum haplotypes. Although the intron sequence was in general fairly well conserved, the distribution of the described mutations among its structural elements corresponding to six pre-RNA domains was qualitatively and quantitatively nonuniform. The highest polymorphism levels were observed in domains I, II, and IV. The sequence of domain V was absolutely invariable, which is in agreement with its functional significance. The chloroplast rpS16 intron sequences have been characterized in nine Solanum species. The intron length ranged from 855 bp to 864 bp, which is associated with 1–9-nucleotide indels. In total five indels and 50 nucleotide substitutions have been detected and six Solanum haplotypes have been revealed. Solanum rpS16 introns has been characterized by mutation rate heterogeneity between structure regions of all six domains its pre-RNA. Intron domains I, II, IV are shown to be more variable. Sequences of the domain V are invariant, that agrees with its functional significance.     

Comparative evolution of the mitochondrial cytochrome b gene and nuclear beta-fibrinogen intron 7 in woodpeckers

Most molecular phylogenetic studies of vertebrates have been based on DNA sequences of mitochondrial-encoded genes. MtDNA evolves rapidly and is thus particularly useful for resolving relationships among recently evolved groups. However, it has the disadvantage that all of the mitochondrial genes are inherited as a single linkage group so that only one independent gene tree can be inferred regardless of the number of genes sequenced. Introns of nuclear genes are attractive candidates for independent sources of rapidly evolving DNA: they are pervasive, most of their nucleotides appear to be unconstrained by selection, and PCR primers can be designed for sequences in adjacent exons where nucleotide sequences are conserved. We sequenced intron 7 of the beta-fibrinogen gene (beta-fibint7) for a diversity of woodpeckers and compared the phylogenetic signal and nucleotide substitution properties of this DNA sequence with that of mitochondrial-encoded cytochrome b (cyt b) from a previous study. A few indels (insertions and deletions) were found in the beta-fibint7 sequences, but alignment was not difficult, and the indels were phylogentically informative. The beta-fibint7 and cyt b gene trees were nearly identical to each other but differed in significant ways from the traditional woodpecker classification. Cyt b evolves 2.8 times as fast as beta-fibint7 (14. 0 times as fast at third codon positions). Despite its relatively slow substitution rate, the phylogenetic signal in beta-fibint7 is comparable to that in cyt b for woodpeckers, because beta-fibint7 has less base composition bias and more uniform nucleotide substitution probabilities. As a consequence, compared with cyt b, beta-fibint7 nucleotide sites are expected to enter more distinct character states over the course of evolution and have fewer multiple substitutions and lower levels of homoplasy. Moreover, in contrast to cyt b, in which nearly two thirds of nucleotide sites rarely vary among closely related taxa, virtually all beta-fibint7 nucleotide sites appear free of selective constraints, which increases informative sites per unit sequenced. However, the estimated gamma distribution used to model rate variation among sites suggests constraints on some beta-fibint7 sites. This study suggests that introns will be useful for phylogenetic studies of recently evolved groups.     

Investigations of the coxII intron structure in the mitochondrial genes of angiosperms

The molecular structure of the intron of the mitochondrial gene coding for cytochrome oxidase susbunit II has been investigated by sequence analysis in eight angiosperm orders. Comparison of the overall primary structure of the intron, made with respect to Magnoliales, the ancestor group of all the angiosperms, reveals a high conservation among dicot plants. On the contrary, the introns of both ancient and advanced monocot orders show relevant divergences represented by insertions and deletions in conserved specific regions of the introns. As a consequence of some of these rearrangements, a structure similar to a transposable element is generated in advanced monocots. This specific structure, common in all the analyzed species of Poales, seems to be generated by a stepwise process and it is not dependent on an insertion event.     

Lineage-specific group II intron gains and losses of the mitochondrial rps3 gene in gymnosperms

According to PCR assays and sequencing, we now report the shared presence of two rps3 introns, namely the rps3i74 and the rps3i249, in the mitochondria of all the classes representing the surviving lineages of gymnosperms, and unveil several lineages experiencing intron loss.Interestingly, the rps3 intron gains and losses within the four groups of gymnosperms let us sort out the Pinaceae and the non-Pinaceae into intron (+)- and intron (?)-lineages, respectively. Worthy of mention is also the finding that only Gnetum within the Gnetales harbours both the rps3 introns.This intron distribution pattern is consistent with the hypothesis that the two rps3 introns were likely present in the common ancestor of the seed plants and, then, independently lost in the non-Pinaceae during gymnosperm evolution.The derived secondary structural model of the novel group IIA intron improves our understanding of the significance and origin of the extraordinary length polymorphisms observed among rps3i249 orthologs.Despite the remarkable structural plasticity to adopt and reject introns, the rps3 mRNAs undergo accurate processing by splicing and extensive editing in gymnosperm mitochondria.This study provides additional insights into the evolutionarily high dynamics of mitochondrial introns which may come and go in closely related plant species.The turnover of the mitochondrial rps3 group II introns seen among lineages of seed plants further suggests that these introns might be an additional signature to discriminate between particularly cryptical taxonomic groups for which there is a need of a further evaluation of their evolutionary affiliation.     

Evolution of the nad3-rps12 Gene Cluster in Angiosperm Mitochondria: Comparison of Edited and Unedited Sequences

We have analyzed the nad3-rps12 locus for eight angiosperms in order to compare the utility of mitochondrial DNA and edited mRNA sequences in phylogenetic reconstruction. The two coding regions, containing from 25 to 35 editing sites in the various plants, have been concatenated in order to increase the significance of the analysis. Differing from the corresponding chloroplast sequences, unedited mitochondrial DNA sequences seem to evolve under a quasi-neutral substitution process which undifferentiates the nucleotide substitution rates for the three codon positions. By using complete gene sequences (all codon positions) we found that genomic sequences provide a classical angiosperm phylogenetic tree with a clear-cut grouping of monocotyledons and dicotyledons with Magnoliidae at the basal branch of the tree. Conversely, owing to their low nucleotide substitution rates, edited mRNA sequences were found not to be suitable for studying phylogenetic relationships among angiosperms. Received: 24 January 1996 / Accepted: 5 June 1996     

Patterns and relative rates of nucleotide and insertion/deletion evolution at six chloroplast intergenic regions in new world species of the Lecythidaceae

Insertions and deletions (indels) in chloroplast noncoding regions are common genetic markers to estimate population structure and gene flow, although relatively little is known about indel evolution among recently diverged lineages such as within plant families. Because indel events tend to occur nonrandomly along DNA sequences, recurrent mutations may generate homoplasy for indel haplotypes. This is a potential problem for population studies, because indel haplotypes may be shared among populations after recurrent mutation as well as gene flow. Furthermore, indel haplotypes may differ in fitness and therefore be subject to natural selection detectable as rate heterogeneity among lineages. Such selection could contribute to the spatial patterning of cpDNA haplotypes, greatly complicating the interpretation of cpDNA population structure. This study examined both nucleotide and indel cpDNA variation and divergence at six noncoding regions (psbB-psbH, atpB-rbcL, trnL-trnH, rpl20-5'rps12, trnS-trnG, and trnH-psbA) in 16 individuals from eight species in the Lecythidaceae and a Sapotaceae outgroup. We described patterns of cpDNA changes, assessed the level of indel homoplasy, and tested for rate heterogeneity among lineages and regions. Although regression analysis of branch lengths suggested some degree of indel homoplasy among the most divergent lineages, there was little evidence for indel homoplasy within the Lecythidaceae. Likelihood ratio tests applied to the entire phylogenetic tree revealed a consistent pattern rejecting a molecular clock. Tajima's 1D and 2D tests revealed two taxa with consistent rate heterogeneity, one showing relatively more and one relatively fewer changes than other taxa. In general, nucleotide changes showed more evidence of rate heterogeneity than did indel changes. The rate of evolution was highly variable among the six cpDNA regions examined, with the trnS-trnG and trnH-psbA regions showing as much as 10% and 15% divergence within the Lecythidaceae. Deviations from rate homogeneity in the two taxa were constant across cpDNA regions, consistent with lineage-specific rates of evolution rather than cpDNA region-specific natural selection. There is no evidence that indels are more likely than nucleotide changes to experience homoplasy within the Lecythidaceae. These results support a neutral interpretation of cpDNA indel and nucleotide variation in population studies within species such as Corythophora alta.     

Constraints on the evolution of plastid introns: the group II intron in the gene encoding tRNA-Val(UAC).

The evolution of the group II intron in the plastid gene encoding tRNA(Val)UAC (trnV) from seven plant taxa was studied by aligning secondary and other structural features. Levels of evolutionary divergence between six angiosperms and a liverwort, Marchantia polymorpha, were compared for the six domains commonly demonstrated for group II introns and were shown to be statistically heterogeneous. Evolutionary rates varied substantially among various domains and other features. Domain II showed the highest evolutionary rate, approaching the synonymous substitution rate reported for cpDNA-encoded genes, while domain VI and the helix and loop region bearing EBS1 evolved at rates similar to those for nonsynonymous substitutions of a number of cpDNA-encoded genes. The minimum free-energy structure of domain I varied among the seven taxa, suggesting that possible protein-RNA or tertiary interactions are important for intron processing.     

Reticulate phylogenetics and phytogeographical structure of Heliosperma (Sileneae, Caryophyllaceae) inferred from chloroplast and nuclear DNA sequences

The Balkan Peninsula is known to be one of the most diverse and species-rich parts of Europe, but its biota has gained much less attention in phylogenetic and evolutionary studies compared to other southern European mountain systems. We used nuclear ribosomal internal transcribed spacer (ITS) sequences and intron sequences of the chloroplast gene rps16 to examine phylogenetic and biogeographical patterns within the genus Heliosperma (Sileneae, Caryophyllaceae). The ITS and rps16 intron sequences both support monophyly of Heliosperma, but the data are not conclusive with regard to its exact origin. Three strongly supported clades are found in both data sets, corresponding to Heliosperma alpestre, Heliosperma macranthum and the Heliosperma pusillum clade, including all other taxa. The interrelationships among these three differ between the nuclear and the plastid data sets. Hierarchical relationships within the H. pusillum clade are poorly resolved by the ITS data, but the rps16 intron sequences form two well-supported clades which are geographically, rather than taxonomically, correlated. A similar geographical structure is found in the ITS data, when analyzed with the NeighbourNet method. The apparent rate of change within Heliosperma is slightly higher for rps16 as compared to ITS. In contrast, in the Sileneae outgroup, ITS substitution rates are more than twice as high as those for rps16, a situation more in agreement with what has been found in other rate comparisons of noncoding cpDNA and ITS. Unlike most other Sileneae ITS sequences, the H. pusillum group sequences display extensive polymorphism. A possible explanation to these patterns is extensive hybridization and gene flow within Heliosperma, which together with concerted evolution may have eradicated the ancient divergence suggested by the rps16 data. The morphological differentiation into high elevation, mainly widely distributed taxa, and low elevation narrow endemics is not correlated with the molecular data, and is possibly a result of ecological differentiation.     

Regional Context in the Alignment of Biological Sequence Pairs

Sequence divergence derives from either point substitution or indel (insertion or deletion) processes. We investigated the rates of these two processes both in protein and non-protein coding DNA. We aligned sequence pairs using two pair-hidden Markov models (PHMMs) conjoined by one silent state. The two PHMMs had their own set of parameters to model rates in their respective regions. The aim was to test the hypothesis that the indel mutation rate mimics the point mutation rate. That is, indels are found less often in conserved regions (slow point substitution rate) and more often in non-conserved regions (fast point substitution rate). Both polypeptides and rRNA molecules in our data exhibited a clear distinction between slow and fast rates of the two processes. These two rates served as surrogates to conserved and non-conserved secondary structure components, respectively. With polypeptides we found both the fast indel rate and the fast replacement rate were co-located with hydrophilic residues. We also found that the average concordance, of our alignments with corresponding curated alignments, improves markedly when the model allows either of the two fast rates to colocate with hydrophilic residues. With rRNA molecules, our model did not detect colocation between the fast indel rate and the fast substitution rate. Nevertheless, coupling the indel rates with the point substitution rates across the two regions markedly increased model fit. This result suggests that rRNA pairwise alignments should be modeled after allowing for the two processes to vary simultaneously and independently in the two regions.     

Molecular evolution of insertions and deletion in the chloroplast genome of silene

Insertions, deletions, and inversions in the chloroplast genome of higher plants have been shown to be extremely useful for resolving phylogenetic relationships both between closely related taxa and among more basal lineages. Introns and intergenic spacers from the chloroplast genome are now increasingly used for phylogenetic and population genetic studies of populations from a single species, and it is therefore interesting to know whether indels can provide useful data and hence increase the power of intraspecific studies. Here, we show that indels in three cpDNA intergenic spacers and one cpDNA intron for two species of Silene evolve at slightly higher rates than base pair substitutions. Repeat indels appear to have the highest rate of evolution and are thus more prone to homoplasy. We show that coded indel data have high information content for phylogenetic analysis, and indels thus provide useful information to infer phylogenetic relationships at the intraspecific level.     

Optimal intron analyses in the trimeresurus radiation of Asian pitvipers

Nuclear introns are commonly used as phylogenetic markers, but a number of issues related to alignment strategies, indel treatments, and the incorporation of length-variant heterozygotes (LVHs) are not routinely addressed when generating phylogenetic hypotheses. Topological congruence in relation to an extensive mitochondrial DNA multigene phylogeny (derived from 2,423 bp of 12S, 16S, ND4, and CYTB genes) of the Asian pitviper Trimeresurus radiation was used to compare combinations of "by eye" and edited and unedited ClustalX 1.8 alignments of two nuclear introns. Indels were treated as missing data, fifth character states, and assigned simple and multistate codes. Upon recovery of the optimal alignment and indel treatment strategy, a total evidence approach was used to investigate the phylogenetic utility of the indels and test new generic arrangements within Trimeresurus. Approximately one third of the intron data partitions exhibited LVHs, suggesting that they are common in introns. Furthermore, a simple concatenation approach can facilitate the incorporation of LVHs into phylogenetic analyses to make use of all available data and investigate mechanisms of molecular evolution. Analyses of ClustalX 1.8-assisted alignments were generally more congruent than the "by eye" alignment and the analysis of a simple coded, edited ClustalX 1.8 (gap opening cost 5, gap extension cost 1) alignment revealed the most congruent tree. The total evidence approach supported the new arrangements within Trimeresurus, suggesting that the phylogeny should be considered as a working benchmark in Asian pitviper systematics. Finally, a critical appraisal of the diverse array of indels (56 to 57 per intron, ranging from 1 to 151 bp in length) suggested that they are a combination of Hennigian and homoplasious events unrelated to indel size or location within the intron. [Alignment; indels; intron analysis; length-variant heterozygotes; Trimeresurus.].     

Molecular phylogenetics of the lizard genus Microlophus (squamata:tropiduridae): aligning and retrieving indel signal from nuclear introns

We use a multigene data set (the mitochondrial locus and nine nuclear gene regions) to test phylogenetic relationships in the South American "lava lizards" (genus Microlophus) and describe a strategy for aligning noncoding sequences that accounts for differences in tempo and class of mutational events. We focus on seven nuclear introns that vary in size and frequency of multibase length mutations (i.e., indels) and present a manual alignment strategy that incorporates insertions and deletions (indels) for each intron. Our method is based on mechanistic explanations of intron evolution that does not require a guide tree. We also use a progressive alignment algorithm (Probabilistic Alignment Kit; PRANK) and distinguishes insertions from deletions and avoids the "gapcost" conundrum. We describe an approach to selecting a guide tree purged of ambiguously aligned regions and use this to refine PRANK performance. We show that although manual alignment is successful in finding repeat motifs and the most obvious indels, some regions can only be subjectively aligned, and there are limits to the size and complexity of a data matrix for which this approach can be taken. PRANK alignments identified more parsimony-informative indels while simultaneously increasing nucleotide identity in conserved sequence blocks flanking the indel regions. When comparing manual and PRANK with two widely used methods (CLUSTAL, MUSCLE) for the alignment of the most length-variable intron, only PRANK recovered a tree congruent at deeper nodes with the combined data tree inferred from all nuclear gene regions. We take this concordance as an objective function of alignment quality and present a strongly supported phylogenetic hypothesis for Microlophus relationships. From this hypothesis we show that (1) a coded indel data partition derived from the PRANK alignment contributed significantly to nodal support and (2) the indel data set permitted detection of significant conflict between mitochondrial and nuclear data partitions, which we hypothesize arose from secondary contact of distantly related taxa, followed by hybridization and mtDNA introgression.