Accurate Phylogenetic Reconstruction Research Articles

Three-Way Alignment Improves Multiple Sequence Alignment of Highly Diverged Sequences

The standard approach for constructing a phylogenetic tree from a set of sequences consists of two key stages. First, a multiple sequence alignment (MSA) of the sequences is computed. The aligned data are then used to reconstruct the phylogenetic tree. The accuracy of the resulting tree heavily relies on the quality of the MSA. The quality of the popularly used progressive sequence alignment depends on a guide tree, which determines the order of aligning sequences. Most MSA methods use pairwise comparisons to generate a distance matrix and reconstruct the guide tree. However, when dealing with highly diverged sequences, constructing a good guide tree is challenging. In this work, we propose an alternative approach using three-way dynamic programming alignment to generate the distance matrix and the guide tree. This three-way alignment incorporates information from additional sequences to compute evolutionary distances more accurately. Using simulated datasets on two symmetric and asymmetric trees, we compared MAFFT with its default guide tree with MAFFT with a guide tree produced using the three-way alignment. We found that (1) the three-way alignment can reconstruct better guide trees than those from the most accurate options of MAFFT, and (2) the better guide tree, on average, leads to more accurate phylogenetic reconstruction. However, the improvement over the L-INS-i option of MAFFT is small, attesting to the excellence of the alignment quality of MAFFT. Surprisingly, the two criteria for choosing the best MSA (phylogenetic accuracy and sum-of-pair score) conflict with each other.

Unraveling the Phylogenetic Relationships and Taxonomic Status of a Puzzling Freshwater Mussel Genus Inversidens (Bivalvia, Unionidae) through Multilocus Phylogeny and Mitochondrial Phylogenomics

Accurate phylogenetic reconstructions are crucial for comprehending the evolutionary histories, reproductive traits, and ecological habits of organisms. The subfamily Gonideinae of freshwater mussels is currently thought to include eight tribes. However, due to inadequate taxon sampling and molecular data, the assignment of the freshwater mussel genus Inversidens at the tribe level has been unstable. Additionally, the lack of phylogenetic data has hindered efforts to understand the basic biology and implementation of conservation efforts of Inversidens rentianensis, an endemic species to China. Here, we first present the complete mitochondrial genome of Inversidens rentianensis and offer a detailed description of its anatomical morphology. Based on DNA sequence data from five genes (COI, ND1, 16S rRNA, 18S rRNA, and 28S rRNA) and complete mitochondrial genomes, we investigated the phylogenetic position of Inversidens using various analytical methods. Both the concatenated five-gene and mitogenome datasets strongly supported that Inversidens classified to the tribe Gonideini in Gonideinae and formed a basal clade within the tribe Gonideini. Molecular dating analysis suggested that Inversidens originated during the mid-Cretaceous era (102.73 Mya, 95% highest posterior density HPD=72.22‐137.03 Mya) and underwent diversification in the Late Paleogene era (37.92 Mya, 95% HPD=20.39‐60.59 Mya). Moreover, based on the Quantitative Assessment of Species for Conservation (QASCP), Inversidens rentianensis is ranked as second priority, providing valuable insights for its management and conservation efforts. Taken together, this study provides a comprehensive understanding of the systematic position and evolutionary history of Inversidens within the currently accepted subfamily Gonideinae classification framework. These findings establish a solid foundation for future investigations on the ecology, reproductive behavior patterns, and conservation biology of this taxonomic group.

Abstract 213: Phylogenetic reconstruction across 303 metastatic tumor samples using Ultima whole-genome sequencing dramatically increases subclonal resolution

Abstract Comprehensive characterization of tumor evolution is essential for understanding the drivers of metastasis and treatment resistance, which are still largely unknown. Studying evolution and resistance requires large cohorts of patients; for each patient, a comprehensive phylogenetic tree is used to follow clones over time and space. Deep whole-genome sequencing (WGS) enables the creation of more robust phylogenies, since (i) each clone is characterized by far more clone-specific mutations (compared to whole-exome or panel sequencing); and (ii) WGS yields more accurate clonal and sub-clonal allelic copy-number estimates, enabling more precise estimation of the fraction of cancer cells that harbor each mutation. Due to recent development of more affordable WGS by Ultima Genomics, we set out to test our ability to construct phylogenetic trees based on Ultima WGS. Ultima sequencing has demonstrated high sensitivity and specificity for detecting germline polymorphisms, but its performance on somatic mutation calling has not yet been rigorously studied. First, we adjusted our somatic mutation detection pipeline to work with Ultima-generated WGS data and tested its performance using well-studied cell line tumor/normal pairs. We assembled a ground truth set of somatic mutations based on deep sequencing data from multiple platforms for the breast cancer cell line pairs (HCC1954/HCC1954-BL and HCC1143/HCC1143-BL) and the melanoma tumor/normal cell line pair (COLO829/COLO829-BL). We found that mutation detection performance was on-par with other platforms for single nucleotide variations. Next, we identified 32 patients from various cancer types (breast, lung, cholangiocarcinoma, and melanoma) treated with a variety of therapies. We collected between 4 to 14 samples per patient at autopsy (total of 303), and sequenced them with Ultima to an average coverage of 63x (range 48x-91x). We identified mutations using our Ultima-adjusted WGS pipeline and constructed phylogenetic trees using our PhylogicNDT suite of tools. We then compared the results to trees previously constructed from mostly whole-exomes (and a few whole-genomes) sequenced on Illumina. We demonstrate that the trees generated from Ultima WGS data are consistent with the previously generated trees, and proved to be much more detailed. At commercialization, we anticipate that WGS for projects like this will be performed at ~1/6th the cost of current NGS offerings (on par with current WES costs). Therefore, it will enable larger studies and more accurate phylogenetic reconstruction, which will advance the study of tumor evolution and resistance. Citation Format: Julian Hess, Elizabeth Martin, Ilya Soifer, Hila Benjamin, Mendy Miller, Carrie Cibulskis, Brian P. Danysh, Matthew Coole, Stacey Gabriel, Dejan Juric, Doron Lipson, Gad Getz. Phylogenetic reconstruction across 303 metastatic tumor samples using Ultima whole-genome sequencing dramatically increases subclonal resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 213.

Intra-genomic rRNA gene variability of Nassellaria and Spumellaria (Rhizaria, Radiolaria) assessed by Sanger, MinION and Illumina sequencing.

SummaryRibosomal RNA (rRNA) genes are known to be valuable markers for the barcoding of eukaryotic life and its phylogenetic classification at various taxonomic levels. The large‐scale exploration of environmental microbial diversity through metabarcoding approaches has been focused mainly on the V4 and V9 regions of the 18S rRNA gene. The accurate interpretation of such environmental surveys is hampered by technical (e.g. PCR and sequencing errors) and biological biases (e.g. intra‐genomic variability). Here we explored the intra‐genomic diversity of Nassellaria and Spumellaria specimens (Radiolaria) by comparing Sanger sequencing with Illumina and Oxford Nanopore Technologies (MinION). Our analysis determined that intra‐genomic variability of Nassellaria and Spumellaria is generally low, yet some Spumellaria specimens showed two different copies of the V4 with <97% similarity. Of the different sequencing methods, Illumina showed the highest number of contaminations (i.e. environmental DNA, cross‐contamination, tag‐jumping), revealed by its high sequencing depth; and MinION showed the highest sequencing rate error (~14%). Yet the long reads produced by MinION (~2900 bp) allowed accurate phylogenetic reconstruction studies. These results highlight the requirement for a careful interpretation of Illumina‐based metabarcoding studies, in particular regarding low abundant amplicons, and open future perspectives towards full‐length rDNA environmental metabarcoding surveys.

On the transformation of MinHash-based uncorrected distances into proper evolutionary distances for phylogenetic inference

Recently developed MinHash-based techniques were proven successful in quickly estimating the level of similarity between large nucleotide sequences. This article discusses their usage and limitations in practice to approximating uncorrected distances between genomes, and transforming these pairwise dissimilarities into proper evolutionary distances. It is notably shown that complex distance measures can be easily approximated using simple transformation formulae based on few parameters. MinHash-based techniques can therefore be very useful for implementing fast yet accurate alignment-free phylogenetic reconstruction procedures from large sets of genomes. This last point of view is assessed with a simulation study using a dedicated bioinformatics tool.

On the transformation of MinHash-based uncorrected distances into proper evolutionary distances for phylogenetic inference.

Recently developed MinHash-based techniques were proven successful in quickly estimating the level of similarity between large nucleotide sequences. This article discusses their usage and limitations in practice to approximating uncorrected distances between genomes, and transforming these pairwise dissimilarities into proper evolutionary distances.It is notably shown that complex distance measures can be easily approximated using simple transformation formulae based on few parameters.MinHash-based techniques can therefore be very useful for implementing fast yet accurate alignment-free phylogenetic reconstruction procedures from large sets of genomes.This last point of view is assessed with a simulation study using a dedicated bioinformatics tool.

Reconstruction of real and simulated phylogenies based on quartet plurality inference

BackgroundDeciphering the history of life on Earth has long been regarded as one of the most central tasks in biology. In past years, widespread discordance between the evolutionary histories of different groups of orthologous genes of prokaryotes have been revealed, primarily due to horizontal gene transfers (HGTs). Nonetheless, evidence that support a strong tree-like signal of evolution have been uncovered, despite the presence of HGT events. Therefore, a challenging task is to distill this tree-like signal from the noise induced by all sources of non-tree-like events.ResultsIn this work we tackle this question, using real and simulated data. We first tighten a recent related theoretical result in this field. In a simulation study, we infer individual quartet topologies, and then use the inferred quartets to reconstruct simulated species trees. We demonstrate that accurate tree reconstruction is feasible despite surprisingly high rates of HGT. In a real data study, we construct phylogenies of two sets of prokaryotes, and show that our tree reconstruction scheme is comparable with (and complementary better than) other commonly used methods.ConclusionsUsing a blend of theoretical and empirical investigations, our study proves the feasibility of accurate quartet-based phylogenetic reconstruction, the vast impact of HGT events notwithstanding.

Evaluation of phylogenetic reconstruction methods using bacterial whole genomes: a simulation based study.

Background: Phylogenetic reconstruction is a necessary first step in many analyses which use whole genome sequence data from bacterial populations. There are many available methods to infer phylogenies, and these have various advantages and disadvantages, but few unbiased comparisons of the range of approaches have been made. Methods: We simulated data from a defined "true tree" using a realistic evolutionary model. We built phylogenies from this data using a range of methods, and compared reconstructed trees to the true tree using two measures, noting the computational time needed for different phylogenetic reconstructions. We also used real data from Streptococcus pneumoniae alignments to compare individual core gene trees to a core genome tree. Results: We found that, as expected, maximum likelihood trees from good quality alignments were the most accurate, but also the most computationally intensive. Using less accurate phylogenetic reconstruction methods, we were able to obtain results of comparable accuracy; we found that approximate results can rapidly be obtained using genetic distance based methods. In real data we found that highly conserved core genes, such as those involved in translation, gave an inaccurate tree topology, whereas genes involved in recombination events gave inaccurate branch lengths. We also show a tree-of-trees, relating the results of different phylogenetic reconstructions to each other. Conclusions: We recommend three approaches, depending on requirements for accuracy and computational time. Quicker approaches that do not perform full maximum likelihood optimisation may be useful for many analyses requiring a phylogeny, as generating a high quality input alignment is likely to be the major limiting factor of accurate tree topology. We have publicly released our simulated data and code to enable further comparisons.

Evaluation of phylogenetic reconstruction methods using bacterial whole genomes: a simulation based study

Background: Phylogenetic reconstruction is a necessary first step in many analyses which use whole genome sequence data from bacterial populations. There are many available methods to infer phylogenies, and these have various advantages and disadvantages, but few unbiased comparisons of the range of approaches have been made. Methods: We simulated data from a defined 'true tree' using a realistic evolutionary model. We built phylogenies from this data using a range of methods, and compared reconstructed trees to the true tree using two measures, noting the computational time needed for different phylogenetic reconstructions. We also used real data from Streptococcus pneumoniae alignments to compare individual core gene trees to a core genome tree. Results: We found that, as expected, maximum likelihood trees from good quality alignments were the most accurate, but also the most computationally intensive. Using less accurate phylogenetic reconstruction methods, we were able to obtain results of comparable accuracy; we found that approximate results can rapidly be obtained using genetic distance based methods. In real data we found that highly conserved core genes, such as those involved in translation, gave an inaccurate tree topology, whereas genes involved in recombination events gave inaccurate branch lengths. We also show a tree-of-trees, relating the results of different phylogenetic reconstructions to each other. Conclusions: We recommend three approaches, depending on requirements for accuracy and computational time. For the most accurate tree, use of either RAxML or IQ-TREE with an alignment of variable sites produced by mapping to a reference genome is best. Quicker approaches that do not perform full maximum likelihood optimisation may be useful for many analyses requiring a phylogeny, as generating a high quality input alignment is likely to be the major limiting factor of accurate tree topology. We have publicly released our simulated data and code to enable further comparisons.

Evaluation of phylogenetic reconstruction methods using bacterial whole genomes: a simulation based study

Background: Phylogenetic reconstruction is a necessary first step in many analyses which use whole genome sequence data from bacterial populations. There are many available methods to infer phylogenies, and these have various advantages and disadvantages, but few unbiased comparisons of the range of approaches have been made. Methods: We simulated data from a defined “true tree” using a realistic evolutionary model. We built phylogenies from this data using a range of methods, and compared reconstructed trees to the true tree using two measures, noting the computational time needed for different phylogenetic reconstructions. We also used real data from Streptococcus pneumoniae alignments to compare individual core gene trees to a core genome tree. Results: We found that, as expected, maximum likelihood trees from good quality alignments were the most accurate, but also the most computationally intensive. Using less accurate phylogenetic reconstruction methods, we were able to obtain results of comparable accuracy; we found that approximate results can rapidly be obtained using genetic distance based methods. In real data we found that highly conserved core genes, such as those involved in translation, gave an inaccurate tree topology, whereas genes involved in recombination events gave inaccurate branch lengths. We also show a tree-of-trees, relating the results of different phylogenetic reconstructions to each other. Conclusions: We recommend three approaches, depending on requirements for accuracy and computational time. Quicker approaches that do not perform full maximum likelihood optimisation may be useful for many analyses requiring a phylogeny, as generating a high quality input alignment is likely to be the major limiting factor of accurate tree topology. We have publicly released our simulated data and code to enable further comparisons.

Molecular phylogenetics of Australian weevils (Coleoptera: Curculionoidea): exploring relationships in a hyperdiverse lineage through comparison of independent analyses

AbstractWith over 60 000 described species in approximately 5800 genera, weevils (Curculionoidea) represent one of the most diverse and species‐rich superfamilies of eukaryotes on the planet. Recent attempts to resolve the phylogeny of family‐group taxa in weevils using morphological, molecular or combined data sets have produced vastly different patterns of relationships, particularly within the largest family, Curculionidae. Here we present an estimation of the phylogeny of Australian weevils and of the divergence dates of the major lineages based on a multi‐gene data set (28S, 16S and COI) spanning ∼3.5 kbp of DNA sequence. We assess its topological similarities to, and differences from, other recently published phylogenetic trees, particularly in relation to taxon sampling and relative diversity of the lineages, and we discuss the implications for weevil systematics. Our results, derived from a different set of taxa that has different combination of loci and different fossil calibration points, recover a number of relationships and age estimates that are congruent with those obtained by other recent weevil phylogeny estimates, indicating that we are beginning to recognise the major monophyletic lineages and reconstruct the major diversification events within Curculionoidea. Resolution of natural groups within families (e.g. subfamilies and tribes) however remains poor, even for studies with the largest volume of sequence data (whole mitochondrial genome analyses), evidently largely due to deficient taxon sampling. Although taxon sampling is known to be one of the most critical determinants of accurate phylogenetic reconstruction, it is rarely addressed in phylogenetic assessments. Our case study of weevils highlights not only its importance, but also the fact that it is very difficult to achieve comprehensive representative sampling in hyperdiverse lineages because of their sheer taxic diversity. Doubts concerning the monophyly of many subordinate lineages exacerbate this problem, restricting the use of exemplar approaches to taxon sampling. The value of comparing different data sets and analyses for assessing systematic relationships is often overlooked, even despite the presence of many conflicting results in previous phylogenetic hypotheses. A summary of relationships recovered relative to taxon sampling and methodological differences between different phylogenetic reconstructions provides an important stepping‐stone in understanding the diversification of weevils.

TCS: a web server for multiple sequence alignment evaluation and phylogenetic reconstruction.

This article introduces the Transitive Consistency Score (TCS) web server; a service making it possible to estimate the local reliability of protein multiple sequence alignments (MSAs) using the TCS index. The evaluation can be used to identify the aligned positions most likely to contain structurally analogous residues and also most likely to support an accurate phylogenetic reconstruction. The TCS scoring scheme has been shown to be accurate predictor of structural alignment correctness among commonly used methods. It has also been shown to outperform common filtering schemes like Gblocks or trimAl when doing MSA post-processing prior to phylogenetic tree reconstruction. The web server is available from http://tcoffee.crg.cat/tcs.

Strepsiptera, phylogenomics and the long branch attraction problem.

Insect phylogeny has recently been the focus of renewed interest as advances in sequencing techniques make it possible to rapidly generate large amounts of genomic or transcriptomic data for a species of interest. However, large numbers of markers are not sufficient to guarantee accurate phylogenetic reconstruction, and the choice of the model of sequence evolution as well as adequate taxonomic sampling are as important for phylogenomic studies as they are for single-gene phylogenies. Recently, the sequence of the genome of a strepsipteran has been published and used to place Strepsiptera as sister group to Coleoptera. However, this conclusion relied on a data set that did not include representatives of Neuropterida or of coleopteran lineages formerly proposed to be related to Strepsiptera. Furthermore, it did not use models that are robust against the long branch attraction artifact. Here we have sequenced the transcriptomes of seven key species to complete a data set comprising 36 species to study the higher level phylogeny of insects, with a particular focus on Neuropteroidea (Coleoptera, Strepsiptera, Neuropterida), especially on coleopteran taxa considered as potential close relatives of Strepsiptera. Using models robust against the long branch attraction artifact we find a highly resolved phylogeny that confirms the position of Strepsiptera as a sister group to Coleoptera, rather than as an internal clade of Coleoptera, and sheds new light onto the phylogeny of Neuropteroidea.

Accurate phylogenetic tree reconstruction from quartets: a heuristic approach.

Supertree methods construct trees on a set of taxa (species) combining many smaller trees on the overlapping subsets of the entire set of taxa. A ‘quartet’ is an unrooted tree over taxa, hence the quartet-based supertree methods combine many -taxon unrooted trees into a single and coherent tree over the complete set of taxa. Quartet-based phylogeny reconstruction methods have been receiving considerable attentions in the recent years. An accurate and efficient quartet-based method might be competitive with the current best phylogenetic tree reconstruction methods (such as maximum likelihood or Bayesian MCMC analyses), without being as computationally intensive. In this paper, we present a novel and highly accurate quartet-based phylogenetic tree reconstruction method. We performed an extensive experimental study to evaluate the accuracy and scalability of our approach on both simulated and biological datasets.

Multilocus assessment of phylogenetic relationships in Alytes (Anura, Alytidae)

With the advent of large multilocus datasets, molecular systematics is experiencing very rapid progress, but important challenges remain regarding data analysis and interpretation. Midwife toads (genus Alytes) exemplify two of the most widespread problems for accurate phylogenetic reconstruction: discerning the causes of discordance between gene trees, and resolving short internodes produced during rapid, successive splitting events. The three species in subgenus Baleaphryne (A. maurus, A. dickhilleni and A. muletensis), the sister group to A. obstetricans, have disjunct and highly restricted geographical ranges, which are thought to result from old vicariant events affecting their common ancestor, but their phylogenetic relationships are still unresolved. In this study we re-address the phylogeny of Alytes with a special focus on the relationships in Baleaphryne with a multilocus dataset including >9000 base pairs of mitochondrial DNA and four nuclear markers (3142bp) in all recognized taxa, including all subspecies of A. obstetricans. Both concatenation and species tree analyses suggest that A. muletensis, endemic to the Balearic island of Mallorca, is the sister taxon to a clade comprising the southeastern Iberian endemic A. dickhilleni and the North African A. maurus. This scenario is consistent with palaeogeological evidence associated with the fragmentation of the Betic-Rifean Massif, followed by the opening of the Strait of Gibraltar. On the other hand, analyses of intraspecific variation in A. obstetricans are inconclusive regarding relationships between major clades and conflict with current subspecific taxonomy.

Phylogenetic quantification of intra-tumour heterogeneity.

Intra-tumour genetic heterogeneity is the result of ongoing evolutionary change within each cancer. The expansion of genetically distinct sub-clonal populations may explain the emergence of drug resistance, and if so, would have prognostic and predictive utility. However, methods for objectively quantifying tumour heterogeneity have been missing and are particularly difficult to establish in cancers where predominant copy number variation prevents accurate phylogenetic reconstruction owing to horizontal dependencies caused by long and cascading genomic rearrangements. To address these challenges, we present MEDICC, a method for phylogenetic reconstruction and heterogeneity quantification based on a Minimum Event Distance for Intra-tumour Copy-number Comparisons. Using a transducer-based pairwise comparison function, we determine optimal phasing of major and minor alleles, as well as evolutionary distances between samples, and are able to reconstruct ancestral genomes. Rigorous simulations and an extensive clinical study show the power of our method, which outperforms state-of-the-art competitors in reconstruction accuracy, and additionally allows unbiased numerical quantification of tumour heterogeneity. Accurate quantification and evolutionary inference are essential to understand the functional consequences of tumour heterogeneity. The MEDICC algorithms are independent of the experimental techniques used and are applicable to both next-generation sequencing and array CGH data.

High-throughput sequencing of Bacillus anthracis in France: investigating genome diversity and population structure using whole-genome SNP discovery.

BackgroundSingle nucleotide polymorphisms (SNPs) are ideal signatures for subtyping monomorphic pathogens such as Bacillus anthracis. Here we report the use of next-generation sequencing technology to investigate the historical, geographic and genetic diversity of Bacillus anthracis in France. 122 strains isolated over a 60-years period throughout the country were whole-genome sequenced and comparative analyses were carried out with a focus on SNPs discovery to discriminate regional sub-groups of strains.ResultsA total of 1581 chromosomal SNPs precisely establish the phylogenetic relationships existing between the French strains. Phylogeography patterns within the three canSNP sub-lineages present in France (i.e. B.Br.CNEVA, A.Br.011/009 and A.Br.001/002) were observed. One of the more remarkable findings was the identification of a variety of genotypes within the A.Br.011/009 sub-group that are persisting in the different regions of France. The 560 SNPs defining the A.Br.011/009- affiliated French strains split the Trans-Eurasian sub-group into six distinct branches without any intermediate nodes. Distinct sub-branches, with some geographic clustering, were resolved. The 345 SNPs defining the major B.Br CNEVA sub-lineage clustered three main phylogeographic clades, the Alps, the Pyrenees, and the Massif Central, with a small Saône-et-Loire sub-cluster nested within the latter group. The French strains affiliated to the minor A.Br.001/002 group were characterized by 226 SNPs. All recent isolates collected from the Doubs department were closely related. Identification of SNPs from whole-genome sequences facilitates high-resolution strain tracking and provides the level of discrimination required for outbreak investigations. Eight diagnostic SNPs, representative of the main French-specific phylogeographic clusters, were therefore selected and developed into high-resolution melting SNP discriminative assays.ConclusionsThis work has established one of the most accurate phylogenetic reconstruction of B. anthracis population structure in a country. An extensive next-generation sequencing (NGS) dataset of 122 French strains have been created that allowed the identification of novel diagnostic SNPs useful to rapidly determine the geographic origin of any strain found in France.

Untangling the influences of unmodeled evolutionary processes on phylogenetic signal in a forensically important HIV-1 transmission cluster

Stochastic models of sequence evolution have been developed to reflect many biologically important processes, allowing for accurate phylogenetic reconstruction when an appropriate model is selected. However, commonly used models do not incorporate several potentially important biological processes. Spurious phylogenetic inference may result if these processes play an important role in the evolution of a dataset yet are not incorporated into assumed models. Few studies have attempted to assess the relative importance of multiple processes in producing spurious inferences. The application of phylogenetic methods to infer the source of HIV-1 transmission clusters depends upon accurate phylogenetic results, yet there are several relevant unmodeled biological processes (e.g., recombination and convergence) that may cause complications. Here, through analyses of HIV-1 env sequences from a small, forensically important transmission cluster, we tease apart the impact of these processes and present evidence suggesting that convergent evolution and high rates of insertions and deletions (causing alignment uncertainty) led to spurious phylogenetic signal with forensic relevance. Previous analyses show paraphyly of HIV-1 lineages sampled from an individual who, based on non-phylogenetic evidence, had never acted as a source of infection for others in this transmission cluster. If true, this pattern calls into question assumptions underlying phylogenetic approaches to source and recipient identification. By systematically assessing the contribution of different unmodeled processes, we demonstrate that removal of sites likely influenced by strong positive selection both reduces the alignment-wide signal supporting paraphyly of viruses sampled from this individual and eliminates support for the effects of recombination. Additionally, the removal of ambiguously aligned sites alters strongly supported relationships among viruses sampled from different individuals. These observations highlight the need to jointly consider multiple unmodeled evolutionary processes and motivate a phylogenomic perspective when inferring viral transmission histories.

Pollinium development in Spiranthes sinensis (Pers.) Ames.and Cymbidium pendulum SW: a comparative study

Comparative studies on the early anther development, anther wall differentiation, pollinium development and pollen grains surface features in Spiranthes sinensis (Pers.) Ames. (a terrestrial orchid) and Cymbidium pendulum Sw. (an epiphytic orchid) using light and scanning electron microscopy were described. Anther primordium initiated as a homogenous mass of meristematic cells which developed two thecae, each with a group of archesporial cells of dense cytoplasmic contents. The sporogenous cells of the hypodermal layer formed the anther wall which was 4-5 layered in S. sinensis and 7-layered in C. pendulum. The rare feature of 2-layered endothecium in S. sinensis and a 2-layered tapetum in C. pendulum was reported. Papillate epidermis was observed in C. pendulum. A complete septum of sterile cells in S. sinensis and partial in C. pendulum was observed. Pollen grains were mono-aperturate, reticulate and shed as tetrads in S. sinensis while either monoaperturate or inaperturate, tectate pollen grains and dispersed as hard pollinia in C. pendulum. This is a see-through information for accurate classification and phylogenetic reconstructions of orchid species. DOI: http://dx.doi.org/10.3329/bjb.v42i2.18035 Bangladesh J. Bot. 42(2): 307-314, 2013 (December)

Multiple ITS Haplotypes in the Genome of the Lichenized Basidiomycete Cora inversa (Hygrophoraceae): Fact or Artifact?

The internal transcribed spacer region (ITS) of the nuclear rDNA cistron represents the barcoding locus for Fungi. Intragenomic variation of this multicopy gene can interfere with accurate phylogenetic reconstruction of biological entities. We investigated the amount and nature of this variation for the lichenized fungus Cora inversa in the Hygrophoraceae (Basidiomycota: Agaricales), analyzing base call and length variation in ITS1 454 pyrosequencing data of three samples of the target mycobiont, for a total of 16,665 reads obtained from three separate repeats of the same samples under different conditions. Using multiple fixed alignment methods (PaPaRa) and maximum likelihood phylogenetic analysis (RAxML), we assessed phylogenetic relationships of the obtained reads, together with Sanger ITS sequences from the same samples. Phylogenetic analysis showed that all ITS1 reads belonged to a single species, C. inversa. Pyrosequencing data showed 266 insertion sites in addition to the 325 sites expected from Sanger sequences, for a total of 15,654 insertions (0.94 insertions per read). An additional 3,279 substitutions relative to the Sanger sequences were detected in the dataset, out of 5,461,125 bases to be called. Up to 99.3% of the observed indels in the dataset could be interpreted as 454 pyrosequencing errors, approximately 65% corresponding to incorrectly recovered homopolymer segments, and 35% to carry-forward-incomplete-extension errors. Comparison of automated clustering and alignment-based phylogenetic analysis demonstrated that clustering of these reads produced a 35-fold overestimation of biological diversity in the dataset at the 95% similarity threshold level, whereas phylogenetic analysis using a maximum likelihood approach accurately recovered a single biological entity. We conclude that variation detected in 454 pyrosequencing data must be interpreted with great care and that a combination of a sufficiently large number of reads per taxon, a set of Sanger references for the same taxon, and at least two runs under different emulsion PCR and sequencing conditions, are necessary to reliably separate biological variation from 454 sequencing errors. Our study shows that clustering methods are highly sensitive to artifactual sequence variation and inadequate to properly recover biological diversity in a dataset, if sequencing errors are substantial and not removed prior to clustering analysis.