Computational Reproducibility of Molecular Phylogenies.

Repeated runs of the same program can generate different molecular phylogenies from identical data sets under the same analytical conditions. This lack of reproducibility of inferred phylogenies casts a long shadow on downstream research employing these phylogenies in areas such as comparative genomics, systematics, and functional biology. We have assessed the relative accuracies and log-likelihoods of alternative phylogenies generated for computer-simulated and empirical data sets. Our findings indicate that these alternative phylogenies reconstruct evolutionary relationships with comparable accuracy. They also have similar log-likelihoods that are not inferior to the log-likelihoods of the true tree. We determined that the direct relationship between irreproducibility and inaccuracy is due to their common dependence on the amount of phylogenetic information in the data. While computational reproducibility can be enhanced through more extensive heuristic searches for the maximum likelihood tree, this does not lead to higher accuracy. We conclude that computational irreproducibility plays a minor role in molecular phylogenetics.

RNA or DNA? Revisiting the Chemical Nature of the Cenancestral Genome.

One of the central issues in the understanding of early cellular evolution is the characterisation of the cenancestor. This includes the description of the chemical nature of its genome. The disagreements on this question comprise several proposals, including the possibility that AlkB-mediated methylation repair of alkylated RNA molecules may be interpreted as evidence of a cenancestral RNA genome. We present here an evolutionary analysis of the cupin-like protein superfamily based on tertiary structure-based phylogenies that includes the oxygen-dependent AlkB and its homologs. Our results suggest that the repair of methylated RNA molecules is the outcome of the enzyme substrate ambiguity, and doesn´t necessarily indicates that the last common ancestor was endowed with an RNA genome.

SCONCE2: jointly inferring single cell copy number profiles and tumor evolutionary distances.

BACKGROUND: Single cell whole genome tumor sequencing can yield novel insights into the evolutionary history of somatic copy number alterations. Existing single cell copy number calling methods do not explicitly model the shared evolutionary process of multiple cells, and generally analyze cells independently. Additionally, existing methods for estimating tumor cell phylogenies using copy number profiles are sensitive to profile estimation errors. RESULTS: We present SCONCE2, a method for jointly calling copy number alterations and estimating pairwise distances for single cell sequencing data. Using simulations, we show that SCONCE2 has higher accuracy in copy number calling and phylogeny estimation than competing methods. We apply SCONCE2 to previously published single cell sequencing data to illustrate the utility of the method. CONCLUSIONS: SCONCE2 jointly estimates copy number profiles and a distance metric for inferring tumor phylogenies in single cell whole genome tumor sequencing across multiple cells, enabling deeper understandings of tumor evolution.

Genome-based reclassification of Azospirillum brasilense Az39 as the type strain of Azospirillum argentinense sp. nov.

Strain Az39T of Azospirillum is a diazotrophic plant growth-promoting bacterium isolated in 1982 from the roots of wheat plants growing in Marcos Juárez, Córdoba, Argentina. It produces indole-3-acetic acid in the presence of l-tryptophan as a precursor, grows at 20-38 °C (optimal 38 °C), and the cells are curved or spiral-shaped, with diameters ranging from 0.5-0.9 to 1.8-2.2 µm. They contain C16 : 0, C18 : 0 and C18 : 1 ω7c/ω6c as the main fatty acids. Phylogenetic analysis of its 16S rRNA gene sequence confirmed that this strain belongs to the genus Azospirillum, showing a close relationship with Azospirillum baldaniorum Sp245T, Azospirillum brasilense Sp7T and Azospirillum formosense CC-Nfb-7T. Housekeeping gene analysis revealed that Az39T, together with five strains of the genus (Az19, REC3, BR 11975, MTCC4035 and MTCC4036), form a cluster apart from A. baldaniorum Sp245T, A. brasilense Sp7T and A. formosense CC-Nfb-7T. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between Az39T and the aforementioned type strains revealed values below 96 %, the circumscription limit for the species delineation (ANI: 95.3, 94.1 and 94.0 %; dDDH: 62.9, 56.3 and 55.6 %). Furthermore, a phylogeny evaluation of the core proteome, including 809 common shared proteins, showed an independent grouping of Az39T, Az19, REC3, BR 11975, MTCC4035 and MTCC4036. The G+C content in the genomic DNA of these six strains varied from 68.3 to 68.5 %. Based on the combined phylogenetic, genomic and phenotypic characterization presented here, we consider that strain Az39T, along with strains Az19, REC3, BR 11975, MTCC4035 and MTCC4036, are members of a new Azospirillum species, for which the name Azospirillum argentinense sp. nov. is proposed. The type strain is Az39T (=LBPCV39T=BR 148428T=CCCT 22.01T).

Combining molecular and geographical data to infer the phylogeny of Lamiales and its dispersal patterns in and out of the tropics.

Lamiales is one of the most intractable orders of flowering plants, with several changes in family composition, and circumscription throughout history. The order is worldwide distributed, occurring in tropical forests and frozen habitats. In this study, a comprehensive phylogeny of Lamiales was reconstructed using DNA sequences. The tree was used to infer dispersal patterns, focusing on the tropics and extratropics. Molecular and species geographic data available from public repositories were combined to address both objectives. A total of 6,910 species, and 842 genera of Lamiales were sampled using the Python tool PyPHLAWD. The tree was inferred using RAxML, and recovered a monophyletic Lamiales. All 26 families were recovered as monophyletic with high support. The families Bignoniaceae, and Plantaginaceae are remarkable examples. The first emerged as monophyletic and included tribe Jacarandeae, while the later emerged as monophyletic in its sensu lato and included both the tribes Angelonieae, and Gratioleae. Distribution points for all species were retrieved from GBIF. After filtering, 1,136,425 records were retained. Species were coded as present in extratropical or tropical environments. The in and out of the tropics dispersal patterns were inferred using a maximum likelihood approach that identifies hidden rate changes. The model recovered higher rates of transition from extratropics to tropics, estimating two rates of state transitions. When ancestral states are considered, more discrete transitions from extratropics to tropics were observed. The extratropical state was also inferred for the crown node of Lamiales and old nested nodes, revealing a rare pattern of transitions to the tropics throughout the upper Cretaceous and Tertiary. A significant phylogenetic signal was recovered for the in and out of the tropics dispersal patterns, showing that state transitions are not frequent enough to erase the effect of tree structure on the data.

Ripe for reassessment: A synthesis of available molecular data for the speciose diatom family Bacillariaceae.

The Bacillariaceae is a very species-rich family of raphid diatoms and includes the large and taxonomically difficult genus Nitzschia, whose species are often small-celled and finely structured and have few discrete morphological characters visible in the light microscope. The classification of Nitzschia is still mostly based on one developed in the second half of the 19th century by Grunow, who separated the genus into a series of sections largely on cell shape and symmetry, the position of the raphe, transverse extension of the fibulae, and folding of the valve. We assembled and analysed single-gene and concatenated alignments of nSSU, nLSU, rbcL, psbC and cox1 to test Grunow's and subsequent classifications and to examine selected morphological characters for their potential to help define monophyletic groups. The maximum likelihood trees were equivocal as to monophyly of the family itself but showed good support for each of eight main clades of Bacillariaceae, three of which corresponded more or less to existing genera (Hantzschia, Cylindrotheca and Bacillaria). The other five main clades and some subclades comprised groups of Nitzschia species or assemblies of Nitzschia species with other genera (Pseudo-nitzschia, Fragilariopsis, Neodenticula, Tryblionella, Psammodictyon). Relationships between most of the eight main clades were not resolved robustly but all analyses recovered Nitzschia as non-monophyletic. The Grunowian classification of Nitzschia into sections was not supported, though in some respects (e.g. treatment of sigmoid species) it is better than subsequent reclassifications. Several of the main clades and subclades are cryptic (lacking morphological synapomorphies) and homoplasy is common in both light microscopical and ultrastructural characters (to the extent that organisms initially assigned to the same species sometimes prove to belong to a different main clade). Nevertheless, some characters, including the structure of the raphe canal and girdle, seem to be sufficiently conservative evolutionarily to give a provisional estimate of relationships if molecular data are unavailable. No new formal classifications are proposed but various options are explored and research needs identified.

Evolutionary management of coral-reef fisheries using phylogenies to predict density dependence.

Harvesting models are based upon the ideology that removing large, old individuals provides space for young, fast-growing counterparts that can maximize (fisheries) yields while maintaining population stability and ecosystem function. Yet, this compensatory density dependent response has rarely been examined in multispecies systems. We combined extensive data sets from coral-reef fisheries across a suite of Pacific islands and provided unique context to the universal assumptions of compensatory density dependence. We reported that size-and-age truncation only existed for 49% of target coral-reef fishes exposed to growing fishing pressure across a suite of Pacific islands. In contrast, most of the remaining species slowly disappeared from landings and reefs with limited change to their size structure (i.e., little to no compensation), often becoming replaced by smaller-bodied sister species. To understand these remarkable and disparate differences, we constructed phylogenies for dominant fish families and discovered that large patristic distances between sister species, or greater phylogenetic isolation, predicted size-and-age truncation. Isolated species appeared to have greater niche dominance or breadth, supported by their faster growth rates compared to species with similar sizes and within similar guilds, and many also have group foraging behavior. In contrast, closely related species may have more restricted, realized niches that led to their disappearance and replacement. We conclude that phylogenetic attributes offered novel guidance to proactively manage multispecies fisheries and improve our understanding of ecological niches and ecosystem stability.

Incongruent phylogenies and their implications for the study of diversification, taxonomy, and genome size evolution of Rhododendron.

PREMISE: Classification of taxa depends on the quality of inferred phylogenies. Rhododendron, a highly species-rich genus (>1156 species) of woody plants, has a highly debated infrageneric classification, due to its huge diversity, homoplasy in key characters, and incongruence among data sets. We provide a broad coverage of representative species to resolve Rhododendron infrageneric phylogeny and highlight the areas of incongruence. We further investigate the effect of polyploidy and genome size evolution on diversification of Rhododendron. METHODS: We generated two plastid and two nuclear loci for 260 Rhododendron species. We analyzed the loci separately as well as concatenated, utilizing both likelihood and Bayesian methods. We tested incongruence both among the data sets and with previous studies. We estimated genome sizes for 125 species through flow cytometry. RESULTS: Our results suggest stronger support for larger subgenera; however, the smaller subgenera pose several problems; for example, R. tomentosum (former genus Ledum) occupies incongruent positions based on different DNA regions. The main shift to higher diversification in the genus occurs in the Himalayan/Southeast Asian clade of R. subg. Hymenanthes. We found that polyploidy occurs in almost all subgenera but most frequently within R. subg. Rhododendron sections Rhododendron and Schistanthe. CONCLUSIONS: We endorse the recognition of five major clades at the subgeneric level, but a number of species cannot be confidently assigned to these clades due to incongruency. With regard to genome size evolution, results support previous reports that genome sizes of tropical plants are lower than those of colder and temperate regions and that genome downsizing promotes diversification.

Comparing copy-number profiles under multi-copy amplifications and deletions.

BACKGROUND: During cancer progression, malignant cells accumulate somatic mutations that can lead to genetic aberrations. In particular, evolutionary events akin to segmental duplications or deletions can alter the copy-number profile (CNP) of a set of genes in a genome. Our aim is to compute the evolutionary distance between two cells for which only CNPs are known. This asks for the minimum number of segmental amplifications and deletions to turn one CNP into another. This was recently formalized into a model where each event is assumed to alter a copy-number by 1 or -1, even though these events can affect large portions of a chromosome. RESULTS: We propose a general cost framework where an event can modify the copy-number of a gene by larger amounts. We show that any cost scheme that allows segmental deletions of arbitrary length makes computing the distance strongly NP-hard. We then devise a factor 2 approximation algorithm for the problem when copy-numbers are non-zero and provide an implementation called cnp2cnp. We evaluate our approach experimentally by reconstructing simulated cancer phylogenies from the pairwise distances inferred by cnp2cnp and compare it against two other alternatives, namely the MEDICC distance and the Euclidean distance. CONCLUSIONS: The experimental results show that our distance yields more accurate phylogenies on average than these alternatives if the given CNPs are error-free, but that the MEDICC distance is slightly more robust against error in the data. In all cases, our experiments show that either our approach or the MEDICC approach should preferred over the Euclidean distance.

Circulation of 2 Barmah Forest Virus Lineages in Military Training Areas, Australia.

During 2017-2018, Barmah Forest virus was recovered from mosquitoes trapped in military training areas in Australia and from a soldier infected at 1 of these areas. Phylogenies of the nucleotide sequences of the envelope glycoprotein gene E2 and the 3' untranslated region suggest that 2 lineages are circulating in eastern Australia.

Under the rug: Abandoning persistent misconceptions that obfuscate organelle evolution.

The advent and advance of next generation sequencing over the past two decades made it possible to accumulate large quantities of sequence reads that could be used to assemble complete or nearly complete organelle genomes (plastome or mitogenome). The result has been an explosive increase in the availability of organelle genome sequences with over 4000 different species of green plants currently available on GenBank. During the same time period, plant molecular biologists greatly enhanced the understanding of the structure, repair, replication, recombination, transcription and translation, and inheritance of organelle DNA. Unfortunately many plant evolutionary biologists are unaware of or have overlooked this knowledge, resulting in misrepresentation of several phenomena that are critical for phylogenetic and evolutionary studies using organelle genomes. We believe that confronting these misconceptions about organelle genome organization, composition, and inheritance will improve our understanding of the evolutionary processes that underly organelle evolution. Here we discuss four misconceptions that can limit evolutionary biology studies and lead to inaccurate phylogenies and incorrect structure of the organellar DNA used to infer organelle evolution.

Genome-based reclassification of Azospirillum brasilense Sp245 as the type strain of Azospirillum baldaniorum sp. nov.

Azospirillum sp. strain Sp245T, originally identified as belonging to Azospirillum brasilense, is recognized as a plant-growth-promoting rhizobacterium due to its ability to fix atmospheric nitrogen and to produce plant-beneficial compounds. Azospirillum sp. Sp245T and other related strains were isolated from the root surfaces of different plants in Brazil. Cells are Gram-negative, curved or slightly curved rods, and motile with polar and lateral flagella. Their growth temperature varies between 20 to 38 °C and their carbon source utilization is similar to other Azospirillum species. A preliminary 16S rRNA sequence analysis showed that the new species is closely related to A. brasilense Sp7T and A. formosense CC-Nfb-7T. Housekeeping genes revealed that Azospirillum sp. Sp245T, BR 12001 and Vi22 form a separate cluster from strain A. formosense CC-Nfb-7T, and a group of strains closely related to A. brasilense Sp7T. Overall genome relatedness index (OGRI) analyses estimated based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between Azospirillum sp. Sp245T and its close relatives to other Azospirillum species type strains, such as A. brasilense Sp7T and A. formosense CC-Nfb-7T , revealed values lower than the limit of species circumscription. Moreover, core-proteome phylogeny including 1079 common shared proteins showed the independent clusterization of A. brasilense Sp7T, A. formosense CC-Nfb-7T and Azospirillum sp. Sp245T, a finding that was corroborated by the genome clustering of OGRI values and housekeeping phylogenies. The DNA G+C content of the cluster of Sp245T was 68.4-68.6 %. Based on the phylogenetic, genomic, phenotypical and physiological analysis, we propose that strain Sp245T together with the strains Vi22 and BR12001 represent a novel species of the genus Azospirillum, for which the name Azospirillum baldaniorum sp. nov. is proposed. The type strain is Sp245T (=BR 11005T=IBPPM 219T) (GCF_007827915.1, GCF_000237365.1, and GCF_003119195.2).

Updating the taxonomy of Aspergillus in South Africa.

The taxonomy and nomenclature of the genus Aspergillus and its associated sexual (teleomorphic) genera have been greatly stabilised over the last decade. This was in large thanks to the accepted species list published in 2014 and associated metadata such as DNA reference sequences released at the time. It had a great impact on the community and it has never been easier to identify, publish and describe the missing Aspergillus diversity. To further stabilise its taxonomy, it is crucial to not only discover and publish new species but also to capture infraspecies variation in the form of DNA sequences. This data will help to better characterise and distinguish existing species and make future identifications more robust. South Africa has diverse fungal communities but remains largely unexplored in terms of Aspergillus with very few sequences available for local strains. In this paper, we re-identify Aspergillus previously accessioned in the PPRI and MRC culture collections using modern taxonomic approaches. In the process, we re-identify strains to 63 species, describe seven new species and release a large number of new DNA reference sequences.

Evolutionary dynamics of language systems.

Understanding how and why language subsystems differ in their evolutionary dynamics is a fundamental question for historical and comparative linguistics. One key dynamic is the rate of language change. While it is commonly thought that the rapid rate of change hampers the reconstruction of deep language relationships beyond 6,000-10,000 y, there are suggestions that grammatical structures might retain more signal over time than other subsystems, such as basic vocabulary. In this study, we use a Dirichlet process mixture model to infer the rates of change in lexical and grammatical data from 81 Austronesian languages. We show that, on average, most grammatical features actually change faster than items of basic vocabulary. The grammatical data show less schismogenesis, higher rates of homoplasy, and more bursts of contact-induced change than the basic vocabulary data. However, there is a core of grammatical and lexical features that are highly stable. These findings suggest that different subsystems of language have differing dynamics and that careful, nuanced models of language change will be needed to extract deeper signal from the noise of parallel evolution, areal readaptation, and contact.

[Sars-CoV-2 genomic and phylodynamic analyses]. / Analyses génomiques et phylodynamiques du Sars-CoV-2.

In line with the recent Ebola and Zika virus epidemics, the Covid-19 pandemic has led to an avalanche of genomic data. These data made it possible to better understand the origin of this virus, to date its emergence in China, but also in France, and to analyse the spread of the epidemic using techniques from the emerging field of phylodynamics.

HIV-1 molecular transmission clusters in nine European countries and Canada: association with demographic and clinical factors.

BACKGROUND: Knowledge of HIV-1 molecular transmission clusters (MTCs) is important, especially in large-scale datasets, for designing prevention programmes and public health intervention strategies. We used a large-scale HIV-1 sequence dataset from nine European HIV cohorts and one Canadian, to identify MTCs and investigate factors associated with the probability of belonging to MTCs. METHODS: To identify MTCs, we applied maximum likelihood inferences on partial pol sequences from 8955 HIV-positive individuals linked to demographic and clinical data. MTCs were defined using two different criteria: clusters with bootstrap support >75% (phylogenetic confidence criterion) and clusters consisting of sequences from a specific region at a proportion of >75% (geographic criterion) compared to the total number of sequences within the network. Multivariable logistic regression analysis was used to assess factors associated with MTC clustering. RESULTS: Although 3700 (41%) sequences belonged to MTCs, proportions differed substantially by country and subtype, ranging from 7% among UK subtype C sequences to 63% among German subtype B sequences. The probability of belonging to an MTC was independently less likely for women than men (OR = 0.66; P < 0.001), older individuals (OR = 0.79 per 10-year increase in age; P < 0.001) and people of non-white ethnicity (OR = 0.44; P < 0.001 and OR = 0.70; P = 0.002 for black and 'other' versus white, respectively). It was also more likely among men who have sex with men (MSM) than other risk groups (OR = 0.62; P < 0.001 and OR = 0.69; P = 0.002 for people who inject drugs, and sex between men and women, respectively), subtype B (ORs 0.36-0.70 for A, C, CRF01 and CRF02 versus B; all P < 0.05), having a well-estimated date of seroconversion (OR = 1.44; P < 0.001), a later calendar year of sampling (ORs 2.01-2.61 for all post-2002 periods versus pre-2002; all P < 0.01), and being naïve to antiretroviral therapy at sampling (OR = 1.19; P = 0.010). CONCLUSIONS: A high proportion (>40%) of individuals belonged to MTCs. Notably, the HIV epidemic dispersal appears to be driven by subtype B viruses spread within MSM networks. Expansion of regional epidemics seems mainly associated with recent MTCs, rather than the growth of older, established ones. This information is important for designing prevention and public health intervention strategies.

Distribution and asymptotic behavior of the phylogenetic transfer distance.

The transfer distance (TD) was introduced in the classification framework and studied in the context of phylogenetic tree matching. Recently, Lemoine et al. (Nature 556(7702):452-456, 2018. https://doi.org/10.1038/s41586-018-0043-0 ) showed that TD can be a powerful tool to assess the branch support on large phylogenies, thus providing a relevant alternative to Felsenstein's bootstrap. This distance allows a reference branch[Formula: see text] in a reference tree [Formula: see text] to be compared to a branch b from another tree T (typically a bootstrap tree), both on the same set of n taxa. The TD between these branches is the number of taxa that must be transferred from one side of b to the other in order to obtain [Formula: see text]. By taking the minimum TD from [Formula: see text] to all branches in T we define the transfer index, denoted by [Formula: see text], measuring the degree of agreement of T with [Formula: see text]. Let us consider a reference branch [Formula: see text] having p tips on its light side and define the transfer support (TS) as [Formula: see text]. Lemoine et al. (2018) used computer simulations to show that the TS defined in this manner is close to 0 for random "bootstrap" trees. In this paper, we demonstrate that result mathematically: when T is randomly drawn, TS converges in probability to 0 when n tends to [Formula: see text]. Moreover, we fully characterize the distribution of [Formula: see text] on caterpillar trees, indicating that the convergence is fast, and that even when n is small, moderate levels of branch support cannot appear by chance.

A comprehensive bioinformatic analysis of hepatitis D virus full-length genomes.

In association with hepatitis B virus (HBV), hepatitis delta virus (HDV) is a subviral agent that may promote severe acute and chronic forms of liver disease. Based on the percentage of nucleotide identity of the genome, HDV was initially classified into three genotypes. However, since 2006, the original classification has been further expanded into eight clades/genotypes. The intergenotype divergence may be as high as 35%-40% over the entire RNA genome, whereas sequence heterogeneity among the isolates of a given genotype is <20%; furthermore, HDV recombinants have been clearly demonstrated. The genetic diversity of HDV is related to the geographic origin of the isolates. This study shows the first comprehensive bioinformatic analysis of the complete available set of HDV sequences, using both nucleotide and protein phylogenies (based on an evolutionary model selection, gamma distribution estimation, tree inference and phylogenetic distance estimation), protein composition analysis and comparison (based on the presence of invariant residues, molecular signatures, amino acid frequencies and mono- and di-amino acid compositional distances), as well as amino acid changes in sequence evolution. Taking into account the congruent and consistent results of both nucleotide and amino acid analyses of GenBank available sequences (recorded as of January, 2017), we propose that the eight hepatitis D virus genotypes may be grouped into three large genogroups fully supported by their shared characteristics.

Recent radiation and dispersal of an ancient lineage: The case of Fouquieria (Fouquiericeae, Ericales) in North American deserts.

Arid biomes are particularly prominent in the Neotropics providing some of its most emblematic landscapes and a substantial part of its species diversity. To understand some of the evolutionary processes underlying the speciation of lineages in the Mexican Deserts, the diversification of Fouquieria is investigated, which includes eleven species, all endemic to the warm deserts and dry subtropical regions of North America. Using a phylogeny from plastid DNA sequences with samples of individuals from populations of all the species recognized in Fouquieria, we estimate divergence times, test for temporal diversification heterogeneity, test for geographical structure, and conduct ancestral area reconstruction. Fouquieria is an ancient lineage that diverged from Polemoniaceae ca. 75.54 Ma. A Mio-Pliocene diversification of Fouquieria with vicariance, associated with Neogene orogenesis underlying the early development of regional deserts is strongly supported. Test for temporal diversification heterogeneity indicates that during its evolutionary history, Fouquieria had a drastic diversification rate shift at ca.12.72 Ma, agreeing with hypotheses that some of the lineages in North American deserts diversified as early as the late Miocene to Pliocene, and not during the Pleistocene. Long-term diversification dynamics analyses suggest that extinction also played a significant role in Fouquieria's evolution, with a very high rate at the onset of the process. From the late Miocene onwards, Fouquieria underwent substantial diversification change, involving high speciation decreasing to the present and negligible extinction, which is congruent with its scant fossil record during this period. Geographic phylogenetic structure and the pattern of most sister species inhabiting different desert nucleus support that isolation by distance could be the main driver of speciation.

Can we build it? Yes we can, but should we use it? Assessing the quality and value of a very large phylogeny of campanulid angiosperms.

PREMISE OF THE STUDY: The study of very large and very old clades holds the promise of greater insights into evolution across the tree of life. However, there has been a fair amount of criticism regarding the interpretations and quality of studies to date, with some suggesting that detailed studies carried out on smaller, tractable scales should be preferred over the increasingly grand syntheses of these data. METHODS: We provided in detail our trials and tribulations of compiling a large, sparsely sampled matrix from GenBank data and inferring a well-supported, time-calibrated phylogeny of Campanulidae. We also used a simulation approach to assess tree quality and to study the value of using very large, comprehensive phylogenies in a comparative context. KEY RESULTS: A robust and well-supported phylogeny can be produced as long as automated procedures are supplemented with some human intervention. In the case of campanulids, the overall topology may be driven not only by particular genes, but also particular sequences for a gene. We also determined that estimates of divergence times should be fairly robust to issues related to clade-specific heterogeneity. Finally, we demonstrated how relying on results from smaller, younger clades are prone to produce biased interpretations of tropical to temperate evolution across campanulids as a whole. CONCLUSIONS: While we were both surprised and encouraged by the robust and fairly well-resolved, comprehensive phylogeny of campanulids, challenges still remain. Nevertheless, large phylogenies are inherently valuable in a comparative context if only to attenuate the issue of ascertainment bias.