Diversity and Molecular Evolution of Antimicrobial Peptides in Caecilian Amphibians.

Antimicrobial peptides (AMPs) are key molecules in the innate immune defence of vertebrates with rapid action, broad antimicrobial spectrum, and ability to evade pathogen resistance mechanisms. To date, amphibians are the major group of vertebrates from which most AMPs have been characterised, but most studies have focused on the bioactive skin secretions of anurans (frogs and toads). In this study, we have analysed the complete genomes and/or transcriptomes of eight species of caecilian amphibians (order Gymnophiona) and characterised the diversity, molecular evolution, and antimicrobial potential of the AMP repertoire of this order of amphibians. We have identified 477 candidate AMPs within the studied caecilian genome and transcriptome datasets. These candidates are grouped into 29 AMP families, with four corresponding to peptides primarily exhibiting antimicrobial activity and 25 potentially serving as AMPs in a secondary function, either in their entirety or after cleavage. In silico prediction methods were used to identify 62 of those AMPs as peptides with promising antimicrobial activity potential. Signatures of directional selection were detected for five candidate AMPs, which may indicate adaptation to the different selective pressures imposed by evolutionary arms races with specific pathogens. These findings provide encouraging support for the expectation that caecilians, being one of the least-studied groups of vertebrates, and with ~300 million years of separate evolution, are an underexplored resource of great pharmaceutical potential that could help to contest antibiotic resistance and contribute to biomedical advance.

Multiple Instances of Adaptive Evolution in Aquaporins of Amphibious Fishes.

Aquaporins (AQPs) are a highly diverse family of transmembrane proteins involved in osmotic regulation that played an important role in the conquest of land by tetrapods. However, little is known about their possible implication in the acquisition of an amphibious lifestyle in actinopterygian fishes. Herein, we investigated the molecular evolution of AQPs in 22 amphibious actinopterygian fishes by assembling a comprehensive dataset that was used to (1) catalogue AQP paralog members and classes; (2) determine the gene family birth and death process; (3) test for positive selection in a phylogenetic framework; and (4) reconstruct structural protein models. We found evidence of adaptive evolution in 21 AQPs belonging to 5 different classes. Almost half of the tree branches and protein sites that were under positive selection were found in the AQP11 class. The detected sequence changes indicate modifications in molecular function and/or structure, which could be related to adaptation to an amphibious lifestyle. AQP11 orthologues appear to be the most promising candidates to have facilitated the processes of the water-to-land transition in amphibious fishes. Additionally, the signature of positive selection found in the AQP11b stem branch of the Gobiidae clade suggests a possible case of exaptation in this clade.

Determining spin-orbit coupling in graphene by quasiparticle interference imaging.

Inducing and controlling spin-orbit coupling (SOC) in graphene is key to create topological states of matter, and for the realization of spintronic devices. Placing graphene onto a transition metal dichalcogenide is currently the most successful strategy to achieve this goal, but there is no consensus as to the nature and the magnitude of the induced SOC. Here, we show that the presence of backscattering in graphene-on-WSe2 heterostructures can be used to probe SOC and to determine its strength quantitatively, by imaging quasiparticle interference with a scanning tunneling microscope. A detailed theoretical analysis of the Fourier transform of quasiparticle interference images reveals that the induced SOC consists of a valley-Zeeman (λvZ ≈ 2 meV) and a Rashba (λR ≈ 15 meV) term, one order of magnitude larger than what theory predicts, but in excellent agreement with earlier transport experiments. The validity of our analysis is confirmed by measurements on a 30 degree twist angle heterostructure that exhibits no backscattering, as expected from symmetry considerations. Our results demonstrate a viable strategy to determine SOC quantitatively by imaging quasiparticle interference.

Light sources with bias tunable spectrum based on van der Waals interface transistors.

Light-emitting electronic devices are ubiquitous in key areas of current technology, such as data communications, solid-state lighting, displays, and optical interconnects. Controlling the spectrum of the emitted light electrically, by simply acting on the device bias conditions, is an important goal with potential technological repercussions. However, identifying a material platform enabling broad electrical tuning of the spectrum of electroluminescent devices remains challenging. Here, we propose light-emitting field-effect transistors based on van der Waals interfaces of atomically thin semiconductors as a promising class of devices to achieve this goal. We demonstrate that large spectral changes in room-temperature electroluminescence can be controlled both at the device assembly stage -by suitably selecting the material forming the interfaces- and on-chip, by changing the bias to modify the device operation point. Even though the precise relation between device bias and kinetics of the radiative transitions remains to be understood, our experiments show that the physical mechanism responsible for light emission is robust, making these devices compatible with simple large areas device production methods.

Occurrence records and metadata for sand flies (Diptera, Psychodidae, Phlebotominae) collected in the lands of indigenous people in the Brazilian Amazon

In order to contribute to knowledge of the epidemiology of American cutaneous leishmaniasis (ACL) among indigenous people living in sylvatic regions, we studied the sand fly fauna collected in areas of disease transmission in the Brazilian Amazon. Our two datasets reported here are comprised of occurrence data for sand flies from the Suruwaha Indigenous Land in the state of Amazonas collected between 2012-1013, and the Wajãpi Indigenous Land in the state of Amapá collected between 2013-2014. Sand flies were collected using unbaited CDC-like light traps at various sites within each study area and were identified to species-level by taxonomists with expertise in Amazonian fauna. A total of 4,646 records are reported: 1,428 from the Suruwaha and 3,218 from the Wajãpi. These records will contribute to a better understanding of ACL transmission dynamics, as well as the distribution of insect vectors, in these areas.

Genomic Fishing and Data Processing for Molecular Evolution Research.

Molecular evolution analyses, such as detection of adaptive/purifying selection or ancestral protein reconstruction, typically require three inputs for a target gene (or gene family) in a particular group of organisms: sequence alignment, model of evolution, and phylogenetic tree. While modern advances in high-throughput sequencing techniques have led to rapid accumulation of genomic-scale data in public repositories and databases, mining such vast amount of information often remains a challenging enterprise. Here, we describe a comprehensive, versatile workflow aimed at the preparation of genome-extracted datasets readily available for molecular evolution research. The workflow involves: (1) fishing (searching and capturing) specific gene sequences of interest from taxonomically diverse genomic data available in databases at variable levels of annotation, (2) processing and depuration of retrieved sequences, (3) production of a multiple sequence alignment, (4) selection of best-fit model of evolution, and (5) solid reconstruction of a phylogenetic tree.

Occurrence records and metadata for sand flies (Diptera, Psychodidae, Phlebotominae) collected in the lands of indigenous people in the Brazilian Amazon.

To contribute to knowledge of the epidemiology of American cutaneous leishmaniasis (ACL) among indigenous people living in sylvatic regions, we studied the sand fly fauna collected in areas of disease transmission in the Brazilian Amazon. Here we report two datasets comprising occurrence data for sand flies from the Suruwaha Indigenous Land in the state of Amazonas collected in 2012-2013, and the Wajãpi Indigenous Land in the state of Amapá collected in 2013-2014. Sand flies were collected using unbaited CDC-like light traps at various sites within each study area and were identified to either genus or species-level by taxonomists with expertise in Amazonian fauna. A total of 4,646 records are reported: 1,428 from the Suruwaha and 3,218 from the Wajãpi. These records will contribute to a better understanding of ACL transmission dynamics, as well as the distribution of insect vectors, in these areas.

Feeding specialization and longer generation time are associated with relatively larger brains in bees.

Despite their miniature brains, insects exhibit substantial variation in brain size. Although the functional significance of this variation is increasingly recognized, research on whether differences in insect brain sizes are mainly the result of constraints or selective pressures has hardly been performed. Here, we address this gap by combining prospective and retrospective phylogenetic-based analyses of brain size for a major insect group, bees (superfamily Apoidea). Using a brain dataset of 93 species from North America and Europe, we found that body size was the single best predictor of brain size in bees. However, the analyses also revealed that substantial variation in brain size remained even when adjusting for body size. We consequently asked whether such variation in relative brain size might be explained by adaptive hypotheses. We found that ecologically specialized species with single generations have larger brains-relative to their body size-than generalist or multi-generation species, but we did not find an effect of sociality on relative brain size. Phylogenetic reconstruction further supported the existence of different adaptive optima for relative brain size in lineages differing in feeding specialization and reproductive strategy. Our findings shed new light on the evolution of the insect brain, highlighting the importance of ecological pressures over social factors and suggesting that these pressures are different from those previously found to influence brain evolution in other taxa.

Insights into the skin of caecilian amphibians from gene expression profiles.

BACKGROUND: Gene expression profiles can provide insights into the molecular machinery behind tissue functions and, in turn, can further our understanding of environmental responses, and developmental and evolutionary processes. During vertebrate evolution, the skin has played a crucial role, displaying a wide diversity of essential functions. To unravel the molecular basis of skin specialisations and adaptations, we compared gene expression in the skin with eight other tissues in a phylogenetically and ecologically diverse species sample of one of the most neglected vertebrate groups, the caecilian amphibians (order Gymnophiona). RESULTS: The skin of the five studied caecilian species showed a distinct gene expression profile reflecting its developmental origin and showing similarities to other epithelial tissues. We identified 59 sequences with conserved enhanced expression in the skin that might be associated with caecilian dermal specialisations. Some of the up-regulated genes shared expression patterns with human skin and potentially are involved in skin functions across vertebrates. Variation trends in gene expression were detected between mid and posterior body skin suggesting different functions between body regions. Several candidate biologically active peptides were also annotated. CONCLUSIONS: Our study provides the first atlas of differentially expressed sequences in caecilian tissues and a baseline to explore the molecular basis of the skin functions in caecilian amphibians, and more broadly in vertebrates.

What lies beneath? Molecular evolution during the radiation of caecilian amphibians.

BACKGROUND: Evolution leaves an imprint in species through genetic change. At the molecular level, evolutionary changes can be explored by studying ratios of nucleotide substitutions. The interplay among molecular evolution, derived phenotypes, and ecological ranges can provide insights into adaptive radiations. Caecilians (order Gymnophiona), probably the least known of the major lineages of vertebrates, are limbless tropical amphibians, with adults of most species burrowing in soils (fossoriality). This enigmatic order of amphibians are very distinct phenotypically from other extant amphibians and likely from the ancestor of Lissamphibia, but little to nothing is known about the molecular changes underpinning their radiation. We hypothesised that colonization of various depths of tropical soils and of freshwater habitats presented new ecological opportunities to caecilians. RESULTS: A total of 8540 candidate groups of orthologous genes from transcriptomic data of five species of caecilian amphibians and the genome of the frog Xenopus tropicalis were analysed in order to investigate the genetic machinery behind caecilian diversification. We found a total of 168 protein-coding genes with signatures of positive selection at different evolutionary times during the radiation of caecilians. The majority of these genes were related to functional elements of the cell membrane and extracellular matrix with expression in several different tissues. The first colonization of the tropical soils was connected to the largest number of protein-coding genes under positive selection in our analysis. From the results of our study, we highlighted molecular changes in genes involved in perception, reduction-oxidation processes, and aging that likely were involved in the adaptation to different soil strata. CONCLUSIONS: The genes inferred to have been under positive selection provide valuable insights into caecilian evolution, potentially underpin adaptations of caecilians to their extreme environments, and contribute to a better understanding of fossorial adaptations and molecular evolution in vertebrates.

Inadvertent Paralog Inclusion Drives Artifactual Topologies and Timetree Estimates in Phylogenomics.

Increasingly, large phylogenomic data sets include transcriptomic data from nonmodel organisms. This not only has allowed controversial and unexplored evolutionary relationships in the tree of life to be addressed but also increases the risk of inadvertent inclusion of paralogs in the analysis. Although this may be expected to result in decreased phylogenetic support, it is not clear if it could also drive highly supported artifactual relationships. Many groups, including the hyperdiverse Lissamphibia, are especially susceptible to these issues due to ancient gene duplication events and small numbers of sequenced genomes and because transcriptomes are increasingly applied to resolve historically conflicting taxonomic hypotheses. We tested the potential impact of paralog inclusion on the topologies and timetree estimates of the Lissamphibia using published and de novo sequencing data including 18 amphibian species, from which 2,656 single-copy gene families were identified. A novel paralog filtering approach resulted in four differently curated data sets, which were used for phylogenetic reconstructions using Bayesian inference, maximum likelihood, and quartet-based supertrees. We found that paralogs drive strongly supported conflicting hypotheses within the Lissamphibia (Batrachia and Procera) and older divergence time estimates even within groups where no variation in topology was observed. All investigated methods, except Bayesian inference with the CAT-GTR model, were found to be sensitive to paralogs, but with filtering convergence to the same answer (Batrachia) was observed. This is the first large-scale study to address the impact of orthology selection using transcriptomic data and emphasizes the importance of quality over quantity particularly for understanding relationships of poorly sampled taxa.

Multi-tissue transcriptomes of caecilian amphibians highlight incomplete knowledge of vertebrate gene families.

RNA sequencing (RNA-seq) has become one of the most powerful tools to unravel the genomic basis of biological adaptation and diversity. Although challenging, RNA-seq is particularly promising for research on non-model, secretive species that cannot be observed in nature easily and therefore remain comparatively understudied. Among such animals, the caecilians (order Gymnophiona) likely constitute the least known group of vertebrates, despite being an old and remarkably distinct lineage of amphibians. Here, we characterize multi-tissue transcriptomes for five species of caecilians that represent a broad level of diversity across the order. We identified vertebrate homologous elements of caecilian functional genes of varying tissue specificity that reveal a great number of unclassified gene families, especially for the skin. We annotated several protein domains for those unknown candidate gene families to investigate their function. We also conducted supertree analyses of a phylogenomic dataset of 1,955 candidate orthologous genes among five caecilian species and other major lineages of vertebrates, with the inferred tree being in agreement with current views of vertebrate evolution and systematics. Our study provides insights into the evolution of vertebrate protein-coding genes, and a basis for future research on the molecular elements underlying the particular biology and adaptations of caecilian amphibians.

Scanning Tunneling Microscopy of an Air Sensitive Dichalcogenide Through an Encapsulating Layer.

Many atomically thin exfoliated two-dimensional (2D) materials degrade when exposed to ambient conditions. They can be protected and investigated by means of transport and optical measurements if they are encapsulated between chemically inert single layers in the controlled atmosphere of a glovebox. Here, we demonstrate that the same encapsulation procedure is also compatible with scanning tunneling microscopy (STM) and spectroscopy (STS). To this end, we report a systematic STM/STS investigation of a model system consisting of an exfoliated 2H-NbSe2 crystal capped with a protective 2H-MoS2 monolayer. We observe different electronic coupling between MoS2 and NbSe2 from a strong coupling when their lattices are aligned within a few degrees to essentially no coupling for 30° misaligned layers. We show that STM always probes intrinsic NbSe2 properties such as the superconducting gap and charge density wave at low temperature when setting the tunneling bias inside the MoS2 band gap, irrespective of the relative angle between the NbSe2 and MoS2 lattices. This study demonstrates that encapsulation is fully compatible with STM/STS investigations of 2D materials.

Müller glia reactivity follows retinal injury despite the absence of the glial fibrillary acidic protein gene in Xenopus.

Intermediate filament proteins are structural components of the cellular cytoskeleton with cell-type specific expression and function. Glial fibrillary acidic protein (GFAP) is a type III intermediate filament protein and is up-regulated in glia of the nervous system in response to injury and during neurodegenerative diseases. In the retina, GFAP levels are dramatically increased in Müller glia and are thought to play a role in the extensive structural changes resulting in Müller cell hypertrophy and glial scar formation. In spite of similar changes to the morphology of Xenopus Müller cells following injury, we found that Xenopus lack a gfap gene. Other type III intermediate filament proteins were, however, significantly induced following rod photoreceptor ablation and retinal ganglion cell axotomy. The recently available X. tropicalis and X. laevis genomes indicate a small deletion most likely resulted in the loss of the gfap gene during anuran evolution. Lastly, a survey of representative species from all three extant amphibian orders including the Anura (frogs, toads), Caudata (salamanders, newts), and Gymnophiona (caecilians) suggests that deletion of the gfap locus occurred in the ancestor of all Anura after its divergence from the Caudata ancestor around 290 million years ago. Our results demonstrate that extensive changes in Müller cell morphology following retinal injury do not require GFAP in Xenopus, and other type III intermediate filament proteins may be involved in the gliotic response.

Next-Generation Mitogenomics: A Comparison of Approaches Applied to Caecilian Amphibian Phylogeny.

Mitochondrial genome (mitogenome) sequences are being generated with increasing speed due to the advances of next-generation sequencing (NGS) technology and associated analytical tools. However, detailed comparisons to explore the utility of alternative NGS approaches applied to the same taxa have not been undertaken. We compared a 'traditional' Sanger sequencing method with two NGS approaches (shotgun sequencing and non-indexed, multiplex amplicon sequencing) on four different sequencing platforms (Illumina's HiSeq and MiSeq, Roche's 454 GS FLX, and Life Technologies' Ion Torrent) to produce seven (near-) complete mitogenomes from six species that form a small radiation of caecilian amphibians from the Seychelles. The fastest, most accurate method of obtaining mitogenome sequences that we tested was direct sequencing of genomic DNA (shotgun sequencing) using the MiSeq platform. Bayesian inference and maximum likelihood analyses using seven different partitioning strategies were unable to resolve compellingly all phylogenetic relationships among the Seychelles caecilian species, indicating the need for additional data in this case.

Life-history evolution and mitogenomic phylogeny of caecilian amphibians.

We analyze mitochondrial genomes to reconstruct a robust phylogenetic framework for caecilian amphibians and use this to investigate life-history evolution within the group. Our study comprises 45 caecilian mitochondrial genomes (19 of them newly reported), representing all families and 27 of 32 currently recognized genera, including some for which molecular data had never been reported. Support for all relationships in the inferred phylogenetic tree is high to maximal, and topology tests reject all investigated alternatives, indicating an exceptionally robust molecular phylogenetic framework of caecilian evolution consistent with current morphology-based supraspecific classification. We used the mitogenomic phylogenetic framework to infer ancestral character states and to assess correlation among three life-history traits (free-living larvae, viviparity, specialized pre-adult or vernal teeth), each of which occurs only in some caecilian species. Our results provide evidence that an ancestor of the Seychelles caecilians abandoned direct development and re-evolved a free-living larval stage. This study yields insights into the concurrent evolution of direct development and of vernal teeth in an ancestor of Teresomata that likely gave rise to skin-feeding (maternal dermatophagy) behavior and subsequently enabled evolution of viviparity, with skin feeding possibly a homologous precursor of oviduct feeding in viviparous caecilians.

Molecular phylogenetics of Gobioidei and phylogenetic placement of European gobies.

Gobioidei is one of the largest suborders of teleost fishes, with nearly 2000 extant species currently recognized. They have a worldwide distribution and show a spectacular variety in morphology, ecology, and behavior. Despite their importance, phylogenetic relationships among many groups of gobioids (including some of the major lineages) still remain poorly understood. In this study, we analyze sequence data of five molecular markers (two mitochondrial and three nuclear) averaging 6000 bp for 222 species of gobioids. Our study is the first to include both multiple nuclear and mitochondrial genes to reconstruct a comprehensive multilocus phylogeny of gobioids encompassing most major lineages representing the overall diversity of one of the most speciose vertebrate lineages. Two separate datasets are produced and used to specifically address the phylogenetic placement of Rhyacichthyidae and Odontobutidae, and the phylogenetic relationships among gobioid lineages. Our results strongly support that the initial split in the gobioid tree separated a clade containing Rhyacichthyidae+Odontobutidae as the sister group of all other lineages. The family Eleotrididae branches off the gobioid tree after the Rhyacichthyidae+Odontobutidae clade, followed by the Butidae as sister group to the Gobiidae. Additionally, several major monophyletic groups are confidently identified within the two major Gobiidae subclades, the gobiine-like gobiids and the gobionelline-like gobiids. Robustness of the phylogenetic trees inferred here is significantly higher than that of previous studies, hence our results provide the most compelling molecular phylogenetic hypothesis of Gobioidei thus far. For the first time, we provide a comprehensive sampling of European gobies that traditionally have been divided into "transverse" gobies and "sand gobies". We show that the European gobies cluster in three distinct lineages, the Pomatoschistus-, Aphia-, and Gobius-lineages. The former resolved within the gobionelline-like gobiids and the latter two within the gobiine-like gobiids. These findings have significant implications for our understanding of the phylogeographic origin of European gobies in the light of the closure of the Paratethys. A rogue taxon analysis identified Kraemeria as an unstable taxon decreasing support at the base of the gobiine-like gobiids. Removal of this rogue taxon significantly increased phylogenetic resolution in that part of the tree and revealed additional insights into early bursts of cladogenesis of the gobiine-like gobiids.

The origin of modern frogs (Neobatrachia) was accompanied by acceleration in mitochondrial and nuclear substitution rates.

BACKGROUND: Understanding the causes underlying heterogeneity of molecular evolutionary rates among lineages is a long-standing and central question in evolutionary biology. Although several earlier studies showed that modern frogs (Neobatrachia) experienced an acceleration of mitochondrial gene substitution rates compared to non-neobatrachian relatives, no further characterization of this phenomenon was attempted. To gain new insights on this topic, we sequenced the complete mitochondrial genomes and nine nuclear loci of one pelobatoid (Pelodytes punctatus) and five neobatrachians, Heleophryne regis (Heleophrynidae), Lechriodus melanopyga (Limnodynastidae), Calyptocephalella gayi (Calyptocephalellidae), Telmatobius bolivianus (Ceratophryidae), and Sooglossus thomasseti (Sooglossidae). These represent major clades not included in previous mitogenomic analyses, and most of them are remarkably species-poor compared to other neobatrachians. RESULTS: We reconstructed a fully resolved and robust phylogeny of extant frogs based on the new mitochondrial and nuclear sequence data, and dated major cladogenetic events. The reconstructed tree recovered Heleophryne as sister group to all other neobatrachians, the Australasian Lechriodus and the South American Calyptocephalella formed a clade that was the sister group to Nobleobatrachia, and the Seychellois Sooglossus was recovered as the sister group of Ranoides. We used relative-rate tests and direct comparison of branch lengths from mitochondrial and nuclear-based trees to demonstrate that both mitochondrial and nuclear evolutionary rates are significantly higher in all neobatrachians compared to their non-neobatrachian relatives, and that such rate acceleration started at the origin of Neobatrachia. CONCLUSIONS: Through the analysis of the selection coefficient (ω) in different branches of the tree, we found compelling evidence of relaxation of purifying selection in neobatrachians, which could (at least in part) explain the observed higher mitochondrial and nuclear substitution rates in this clade. Our analyses allowed us to discard that changes in substitution rates could be correlated with increased mitochondrial genome rearrangement or diversification rates observed in different lineages of neobatrachians.

Discovery of a new family of amphibians from northeast India with ancient links to Africa.

The limbless, primarily soil-dwelling and tropical caecilian amphibians (Gymnophiona) comprise the least known order of tetrapods. On the basis of unprecedented extensive fieldwork, we report the discovery of a previously overlooked, ancient lineage and radiation of caecilians from threatened habitats in the underexplored states of northeast India. Molecular phylogenetic analyses of mitogenomic and nuclear DNA sequences, and comparative cranial anatomy indicate an unexpected sister-group relationship with the exclusively African family Herpelidae. Relaxed molecular clock analyses indicate that these lineages diverged in the Early Cretaceous, about 140 Ma. The discovery adds a major branch to the amphibian tree of life and sheds light on both the evolution and biogeography of caecilians and the biotic history of northeast India-an area generally interpreted as a gateway between biodiversity hotspots rather than a distinct biogeographic unit with its own ancient endemics. Because of its distinctive morphology, inferred age and phylogenetic relationships, we recognize the newly discovered caecilian radiation as a new family of modern amphibians.

Experimental design in phylogenetics: testing predictions from expected information.

Taxon and character sampling are central to phylogenetic experimental design; yet, we lack general rules. Goldman introduced a method to construct efficient sampling designs in phylogenetics, based on the calculation of expected Fisher information given a probabilistic model of sequence evolution. The considerable potential of this approach remains largely unexplored. In an earlier study, we applied Goldman's method to a problem in the phylogenetics of caecilian amphibians and made an a priori evaluation and testable predictions of which taxon additions would increase information about a particular weakly supported branch of the caecilian phylogeny by the greatest amount. We have now gathered mitogenomic and rag1 sequences (some newly determined for this study) from additional caecilian species and studied how information (both expected and observed) and bootstrap support vary as each new taxon is individually added to our previous data set. This provides the first empirical test of specific predictions made using Goldman's method for phylogenetic experimental design. Our results empirically validate the top 3 (more intuitive) taxon addition predictions made in our previous study, but only information results validate unambiguously the 4th (less intuitive) prediction. This highlights a complex relationship between information and support, reflecting that each measures different things: Information is related to the ability to estimate branch length accurately and support to the ability to estimate the tree topology accurately. Thus, an increase in information may be correlated with but does not necessitate an increase in support. Our results also provide the first empirical validation of the widely held intuition that additional taxa that join the tree proximal to poorly supported internal branches are more informative and enhance support more than additional taxa that join the tree more distally. Our work supports the view that adding more data for a single (well chosen) taxon may increase phylogenetic resolution and support in weakly supported parts of the tree without adding more characters/genes. Altogether our results corroborate that, although still underexplored, Goldman's method offers a powerful tool for experimental design in molecular phylogenetic studies. However, there are still several drawbacks to overcome, and further assessment of the method is needed in order to make it better understood, more accessible, and able to assess the addition of multiple taxa.