On the importance of sequence alignment inspections in plastid phylogenomics - an example from revisiting the relationships of the water-lilies.

The water-lily clade represents the second earliest-diverging branch of angiosperms. Most of its species belong to Nymphaeaceae, of which the "core Nymphaeaceae"-comprising the genera Euryale, Nymphaea and Victoria-is the most diverse clade. Despite previous molecular phylogenetic studies on the core Nymphaeaceae, various aspects of their evolutionary relationships have remained unresolved. The length-variable introns and intergenic spacers are known to contain most of the sequence variability within the water-lily plastomes. Despite the challenges with multiple sequence alignment, any new molecular phylogenetic investigation on the core Nymphaeaceae should focus on these noncoding plastome regions. For example, a new plastid phylogenomic study on the core Nymphaeaceae should generate DNA sequence alignments of all plastid introns and intergenic spacers based on the principle of conserved sequence motifs. In this investigation, we revisit the phylogenetic history of the core Nymphaeaceae by employing such an approach. Specifically, we use a plastid phylogenomic analysis strategy in which all coding and noncoding partitions are separated and then undergo software-driven DNA sequence alignment, followed by a motif-based alignment inspection and adjustment. This approach allows us to increase the reliability of the character base compared to the default practice of aligning complete plastomes through software algorithms alone. Our approach produces significantly different phylogenetic tree reconstructions for several of the plastome regions under study. The results of these reconstructions underscore that Nymphaea is paraphyletic in its current circumscription, that each of the five subgenera of Nymphaea is monophyletic, and that the subgenus Nymphaea is sister to all other subgenera of Nymphaea. Our results also clarify many evolutionary relationships within the Nymphaea subgenera Brachyceras, Hydrocallis and Nymphaea. In closing, we discuss whether the phylogenetic reconstructions obtained through our motif-based alignment adjustments are in line with morphological evidence on water-lily evolution.

airpg: automatically accessing the inverted repeats of archived plastid genomes.

BACKGROUND: In most flowering plants, the plastid genome exhibits a quadripartite genome structure, comprising a large and a small single copy as well as two inverted repeat regions. Thousands of plastid genomes have been sequenced and submitted to public sequence repositories in recent years. The quality of sequence annotations in many of these submissions is known to be problematic, especially regarding annotations that specify the length and location of the inverted repeats: such annotations are either missing or portray the length or location of the repeats incorrectly. However, many biological investigations employ publicly available plastid genomes at face value and implicitly assume the correctness of their sequence annotations. RESULTS: We introduce airpg, a Python package that automatically assesses the frequency of incomplete or incorrect annotations of the inverted repeats among publicly available plastid genomes. Specifically, the tool automatically retrieves plastid genomes from NCBI Nucleotide under variable search parameters, surveys them for length and location specifications of inverted repeats, and confirms any inverted repeat annotations through self-comparisons of the genome sequences. The package also includes functionality for automatic identification and removal of duplicate genome records and accounts for taxa that genuinely lack inverted repeats. A survey of the presence of inverted repeat annotations among all plastid genomes of flowering plants submitted to NCBI Nucleotide until the end of 2020 using airpg, followed by a statistical analysis of potential associations with record metadata, highlights that release year and publication status of the genome records have a significant effect on the frequency of complete and equal-length inverted repeat annotations. CONCLUSION: The number of plastid genomes on NCBI Nucleotide has increased dramatically in recent years, and many more genomes will likely be submitted over the next decade. airpg enables researchers to automatically access and evaluate the inverted repeats of these plastid genomes as well as their sequence annotations and, thus, contributes to increasing the reliability of publicly available plastid genomes. The software is freely available via the Python package index at http://pypi.python.org/pypi/airpg .

annonex2embl: automatic preparation of annotated DNA sequences for bulk submissions to ENA.

MOTIVATION: The submission of annotated sequence data to public sequence databases constitutes a central pillar in biological research. The surge of novel DNA sequences awaiting database submission due to the application of next-generation sequencing has increased the need for software tools that facilitate bulk submissions. This need has yet to be met with the concurrent development of tools to automate the preparatory work preceding such submissions. RESULTS: The author introduce annonex2embl, a Python package that automates the preparation of complete sequence flatfiles for large-scale sequence submissions to the European Nucleotide Archive. The tool enables the conversion of DNA sequence alignments that are co-supplied with sequence annotations and metadata to submission-ready flatfiles. Among other features, the software automatically accounts for length differences among the input sequences while maintaining correct annotations, automatically interlaces metadata to each record and displays a design suitable for easy integration into bioinformatic workflows. As proof of its utility, annonex2embl is employed in preparing a dataset of more than 1500 fungal DNA sequences for database submission. AVAILABILITY AND IMPLEMENTATION: annonex2embl is freely available via the Python package index at http://pypi.python.org/pypi/annonex2embl. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Plastid phylogenomics of the Gynoxoid group (Senecioneae, Asteraceae) highlights the importance of motif-based sequence alignment amid low genetic distances.

PREMISE: The genus Gynoxys and relatives form a species-rich lineage of Andean shrubs and trees with low genetic distances within the sunflower subtribe Tussilaginineae. Previous molecular phylogenetic investigations of the Tussilaginineae have included few, if any, representatives of this Gynoxoid group or reconstructed ambiguous patterns of relationships for it. METHODS: We sequenced complete plastid genomes of 21 species of the Gynoxoid group and related Tussilaginineae and conducted detailed comparisons of the phylogenetic relationships supported by the gene, intron, and intergenic spacer partitions of these genomes. We also evaluated the impact of manual, motif-based adjustments of automatic DNA sequence alignments on phylogenetic tree inference. RESULTS: Our results indicate that the inclusion of all plastid genome partitions is needed to infer well-supported phylogenetic trees of the Gynoxoid group. Whole plastome-based tree inference suggests that the genera Gynoxys and Nordenstamia are polyphyletic and form the core clade of the Gynoxoid group. This clade is sister to a clade of Aequatorium and Paragynoxys and also includes some but not all representatives of Paracalia. CONCLUSIONS: The concatenation and combined analysis of all plastid genome partitions and the construction of manually-curated, motif-based DNA sequence alignments are found to be instrumental in the recovery of well-supported relationships of the Gynoxoid group. We demonstrate that the correct assessment of homology in genome-level plastid sequence data sets is crucial for subsequent phylogeny reconstruction and that the manual post-processing of multiple sequence alignments improves the reliability of such reconstructions amid low genetic distances between taxa.

PACVr: plastome assembly coverage visualization in R.

BACKGROUND: Plastid genomes typically display a circular, quadripartite structure with two inverted repeat regions, which challenges automatic assembly procedures. The correct assembly of plastid genomes is a prerequisite for the validity of subsequent analyses on genome structure and evolution. The average coverage depth of a genome assembly is often used as an indicator of assembly quality. Visualizing coverage depth across a draft genome is a critical step, which allows users to inspect the quality of the assembly and, where applicable, identify regions of reduced assembly confidence. Despite the interplay between genome structure and assembly quality, no contemporary, user-friendly software tool can visualize the coverage depth of a plastid genome assembly while taking its quadripartite genome structure into account. A software tool is needed that fills this void. RESULTS: We introduce 'PACVr', an R package that visualizes the coverage depth of a plastid genome assembly in relation to the circular, quadripartite structure of the genome as well as the individual plastome genes. By using a variable window approach, the tool allows visualizations on different calculation scales. It also confirms sequence equality of, as well as visualizes gene synteny between, the inverted repeat regions of the input genome. As a tool for plastid genomics, PACVr provides the functionality to identify regions of coverage depth above or below user-defined threshold values and helps to identify non-identical IR regions. To allow easy integration into bioinformatic workflows, PACVr can be invoked from a Unix shell, facilitating its use in automated quality control. We illustrate the application of PACVr on four empirical datasets and compare visualizations generated by PACVr with those of alternative software tools. CONCLUSIONS: PACVr provides a user-friendly tool to visualize (a) the coverage depth of a plastid genome assembly on a circular, quadripartite plastome map and in relation to individual plastome genes, and (b) gene synteny across the inverted repeat regions. It contributes to optimizing plastid genome assemblies and increasing the reliability of publicly available plastome sequences. The software, example datasets, technical documentation, and a tutorial are available with the package at https://cran.r-project.org/package=PACVr.

Evolutionary diversification of the African achyranthoid clade (Amaranthaceae) in the context of sterile flower evolution and epizoochory.

Background and Aims: Many African genera of the Amaranthaceae exhibit unique inflorescences that include sterile flowers modified to hooks or spines. Considering that the abundance of large terrestrial herbivores increased on the African continent with the expansion of grassland and savannah ecosystems, modified sterile flowers could have been an innovation that boosted the diversification of an African achyranthoid clade of Amaranthaceae, with large animals serving dispersal. Methods: We generated an extensively sampled phylogeny comprising 26 of the 31 achyranthoid genera as well as representatives of all other lineages of Amaranthaceae. Phylogenetic tree inference employed four genomic regions, using parsimony, likelihood and Bayesian inference methods. We estimated divergence times, evaluated trait-dependant changes and species diversification rates using state-dependent speciation and extinction models, and reconstructed ancestral character states for modified sterile flowers. Key Results: The achyranthoids were found to be a major clade of the Amaranthaceae, comprising mostly African members. Phylogenetic relationships within this clade were well resolved and supported two main subclades. Several genera were found to be polyphyletic. Our results indicate that the achyranthoids started to diversify ~28 million years ago, and that modified sterile flowers evolved multiple times. An asymmetry in transition rates towards the gain of sterile flowers was observed, whereas no trait-dependent increase in species diversification rates was detected. Bayesian rate heterogeneity analyses indicated that the achyranthoids diversified without significant rate shifts. Conclusions: The accumulation of modified sterile flowers within achyranthoids appears to result from the higher transition rates in favour of modified sterile flowers. Multiple gains suggest an adaptive value for this trait. However, epizoochory does not appear to fuel species diversification, possibly due to extensive gene flow through regularly migrating mammals, which limits the possibility of speciation by isolation.

Phylogenetic analyses of Tolpis Adans. (Asteraceae) reveal patterns of adaptive radiation, multiple colonization and interspecific hybridization.

The plant genus Tolpis (Asteraceae) has been the subject of several investigations on the evolution of oceanic island plants. Its insular species were utilized in studies of artificial hybrid fertility, testing the validity of Baker's law, the application of DNA barcodes, and the phylogenetic utility of inter-simple sequence repeat markers. Despite this considerable interest in Tolpis, little is known about its phylogenetic history. Past investigations were unable to resolve most of the interspecific relationships, especially within the Canary Islands, where the genus is particularly diverse. Incomplete taxon sampling, the use of ambiguous outgroups and the limited utility of slowly evolving chloroplast DNA markers precluded detailed reconstructions. The present investigation presents a comprehensive molecular phylogeny of Tolpis. By utilizing highly variable nuclear DNA markers and a comprehensive taxon set, we have resolved the majority of interspecific relationships in the genus. Evaluations of competing tree topologies and ancestral area reconstructions complemented the analyses. Our results highlight the presence of three dominant mechanisms of island plant evolution-island colonization, adaptive radiation and interspecific hybridization-in Tolpis: (i) the extant distribution of the genus is the result of two independent colonization pathways, (ii) Tolpis has colonized at least one archipelago multiple times, (iii) the present insular diversity is the product of adaptive radiation, (iv) potential hybridization was detected between species now inhabiting different islands and archipelagoes, indicating sympatric historical distributions, and (v) several undescribed species await taxonomic recognition.

Software Choice and Sequencing Coverage Can Impact Plastid Genome Assembly-A Case Study in the Narrow Endemic Calligonum bakuense.

Most plastid genome sequences are assembled from short-read whole-genome sequencing data, yet the impact that sequencing coverage and the choice of assembly software can have on the accuracy of the resulting assemblies is poorly understood. In this study, we test the impact of both factors on plastid genome assembly in the threatened and rare endemic shrub Calligonum bakuense. We aim to characterize the differences across plastid genome assemblies generated by different assembly software tools and levels of sequencing coverage and to determine if these differences are large enough to affect the phylogenetic position inferred for C. bakuense compared to congeners. Four assembly software tools (FastPlast, GetOrganelle, IOGA, and NOVOPlasty) and seven levels of sequencing coverage across the plastid genome (original sequencing depth, 2,000x, 1,000x, 500x, 250x, 100x, and 50x) are compared in our analyses. The resulting assemblies are evaluated with regard to reproducibility, contig number, gene complement, inverted repeat length, and computation time; the impact of sequence differences on phylogenetic reconstruction is assessed. Our results show that software choice can have a considerable impact on the accuracy and reproducibility of plastid genome assembly and that GetOrganelle produces the most consistent assemblies for C. bakuense. Moreover, we demonstrate that a sequencing coverage between 500x and 100x can reduce both the sequence variability across assembly contigs and computation time. When comparing the most reliable plastid genome assemblies of C. bakuense, a sequence difference in only three nucleotide positions is detected, which is less than the difference potentially introduced through software choice.

Phylogeny of Barnadesioideae (Asteraceae) inferred from DNA sequence data and morphology.

Subfamily Barnadesioideae (Asteraceae) consists of nine genera and 91 species endemic to South America. They include annual and perennial herbs, arching shrubs and trees up to 30m tall. Presumed sister to all other Asteraceae, its intergeneric relationships are key to understanding the early evolution of the family. Results of the only molecular study on the subfamily conflict with relationships inferred from morphology. We investigate inter- and intrageneric relationships in Barnadesioideae with novel DNA sequence data and morphological characters using parsimony, likelihood and Bayesian inference. All results verify Barnadesioideae as monophyletic and sister to the rest of the family. A basal split within the subfamily is recognized, with Chuquiraga, Doniophyton and Duseniella in one clade, and Arnaldoa, Barnadesia, Dasyphyllum, Fulcaldea, Huarpea and possibly Schlechtendalia in another. The largest genus, Dasyphyllum, is revealed as biphyletic with the two clades separating along subgeneric and geographic lines. Schlechtendalia, suggested as the earliest diverging lineage of the subfamily by morphological studies and parsimony analyses, is found in a more derived position under model-based inference methods. Competing phylogenetic hypotheses, both previous and present, are evaluated using likelihood-based tests. Evolutionary trends within Barnadesioideae are inferred: hummingbird pollination has developed convergently at least three times. An early vicariance in the subfamily's distribution is revealed. X=9 is supported as the ancestral base chromosome number for both Barnadesioideae and the family as a whole.

EMBL2checklists: A Python package to facilitate the user-friendly submission of plant and fungal DNA barcoding sequences to ENA.

BACKGROUND: The submission of DNA sequences to public sequence databases is an essential, but insufficiently automated step in the process of generating and disseminating novel DNA sequence data. Despite the centrality of database submissions to biological research, the range of available software tools that facilitate the preparation of sequence data for database submissions is low, especially for sequences generated via plant and fungal DNA barcoding. Current submission procedures can be complex and prohibitively time expensive for any but a small number of input sequences. A user-friendly software tool is needed that streamlines the file preparation for database submissions of DNA sequences that are commonly generated in plant and fungal DNA barcoding. METHODS: A Python package was developed that converts DNA sequences from the common EMBL and GenBank flat file formats to submission-ready, tab-delimited spreadsheets (so-called 'checklists') for a subsequent upload to the annotated sequence section of the European Nucleotide Archive (ENA). The software tool, titled 'EMBL2checklists', automatically converts DNA sequences, their annotation features, and associated metadata into the idiosyncratic format of marker-specific ENA checklists and, thus, generates files that can be uploaded via the interactive Webin submission system of ENA. RESULTS: EMBL2checklists provides a simple, platform-independent tool that automates the conversion of common DNA barcoding sequences into easily editable spreadsheets that require no further processing but their upload to ENA via the interactive Webin submission system. The software is equipped with an intuitive graphical as well as an efficient command-line interface for its operation. The utility of the software is illustrated by its application in four recent investigations, including plant phylogenetic and fungal metagenomic studies. DISCUSSION: EMBL2checklists bridges the gap between common software suites for DNA sequence assembly and annotation and the interactive data submission process of ENA. It represents an easy-to-use solution for plant and fungal biologists without bioinformatics expertise to generate submission-ready checklists from common DNA sequence data. It allows the post-processing of checklists as well as work-sharing during the submission process and solves a critical bottleneck in the effort to increase participation in public data sharing.

Bioinformatic Workflows for Generating Complete Plastid Genome Sequences-An Example from Cabomba (Cabombaceae) in the Context of the Phylogenomic Analysis of the Water-Lily Clade.

The sequencing and comparison of plastid genomes are becoming a standard method in plant genomics, and many researchers are using this approach to infer plant phylogenetic relationships. Due to the widespread availability of next-generation sequencing, plastid genome sequences are being generated at breakneck pace. This trend towards massive sequencing of plastid genomes highlights the need for standardized bioinformatic workflows. In particular, documentation and dissemination of the details of genome assembly, annotation, alignment and phylogenetic tree inference are needed, as these processes are highly sensitive to the choice of software and the precise settings used. Here, we present the procedure and results of sequencing, assembling, annotating and quality-checking of three complete plastid genomes of the aquatic plant genus Cabomba as well as subsequent gene alignment and phylogenetic tree inference. We accompany our findings by a detailed description of the bioinformatic workflow employed. Importantly, we share a total of eleven software scripts for each of these bioinformatic processes, enabling other researchers to evaluate and replicate our analyses step by step. The results of our analyses illustrate that the plastid genomes of Cabomba are highly conserved in both structure and gene content.

WARACS: Wrappers to Automate the Reconstruction of Ancestral Character States.

PREMISE OF THE STUDY: Reconstructions of ancestral character states are among the most widely used analyses for evaluating the morphological, cytological, or ecological evolution of an organismic lineage. The software application Mesquite remains the most popular application for such reconstructions among plant scientists, even though its support for automating complex analyses is limited. A software tool is needed that automates the reconstruction and visualization of ancestral character states with Mesquite and similar applications. METHODS AND RESULTS: A set of command line-based Python scripts was developed that (a) communicates standardized input to and output from the software applications Mesquite, BayesTraits, and TreeGraph2; (b) automates the process of ancestral character state reconstruction; and (c) facilitates the visualization of reconstruction results. CONCLUSIONS: WARACS provides a simple tool that streamlines the reconstruction and visualization of ancestral character states over a wide array of parameters, including tree distribution, character state, and optimality criterion.

Community trees: Identifying codiversification in the Páramo dipteran community.

Groups of codistributed species that responded in a concerted manner to environmental events are expected to share patterns of evolutionary diversification. However, the identification of such groups has largely been based on qualitative, post hoc analyses. We develop here two methods (posterior predictive simulation [PPS], Kuhner-Felsenstein [K-F] analysis of variance [ANOVA]) for the analysis of codistributed species that, given a group of species with a shared pattern of diversification, allow empiricists to identify those taxa that do not codiversify (i.e., "outlier" species). The identification of outlier species makes it possible to jointly estimate the evolutionary history of co-diversifying taxa. To evaluate the approaches presented here, we collected data from Páramo dipterans, identified outlier species, and estimated a "community tree" from species that are identified as having codiversified. Our results demonstrate that dipteran communities from different Páramo habitats in the same mountain range are more closely related than communities in other ranges. We also conduct simulation testing to evaluate this approach. Results suggest that our approach provides a useful addition to comparative phylogeographic methods, while identifying aspects of the analysis that require careful interpretation. In particular, both the PPS and K-F ANOVA perform acceptably when there are one or two outlier species, but less so as the number of outliers increases. This is likely a function of the corresponding degradation of the signal of community divergence; without a strong signal from a codiversifying community, there is no dominant pattern from which to detect an outlier species. For this reason, both the magnitude of K-F distance distribution and outside knowledge about the phylogeographic history of each putative member of the community should be considered when interpreting the results.

Posterior predictive checks of coalescent models: P2C2M, an R package.

Bayesian inference operates under the assumption that the empirical data are a good statistical fit to the analytical model, but this assumption can be challenging to evaluate. Here, we introduce a novel r package that utilizes posterior predictive simulation to evaluate the fit of the multispecies coalescent model used to estimate species trees. We conduct a simulation study to evaluate the consistency of different summary statistics in comparing posterior and posterior predictive distributions, the use of simulation replication in reducing error rates and the utility of parallel process invocation towards improving computation times. We also test P2C2M on two empirical data sets in which hybridization and gene flow are suspected of contributing to shared polymorphism, which is in violation with the coalescent model: Tamias chipmunks and Myotis bats. Our results indicate that (i) probability-based summary statistics display the lowest error rates, (ii) the implementation of simulation replication decreases the rate of type II errors, and (iii) our r package displays improved statistical power compared to previous implementations of this approach. When probabilistic summary statistics are used, P2C2M corroborates the assumption that genealogies collected from Tamias and Myotis are not a good fit to the multispecies coalescent model. Taken as a whole, our findings argue that an assessment of the fit of the multispecies coalescent model should accompany any phylogenetic analysis that estimates a species tree.