Recent research clarified the genetic identity of the ten nominal species of Amazonian Bolitoglossa (subgenus Eladinea) and proposed a remarkable number of undescribed candidate species in this group of salamanders. Here, we provide a genetic analysis of Amazonian Bolitoglossa species using available GenBank data and new DNA sequences of the mitochondrial genes 16S rRNA and cytochrome b, and the nuclear-encoded genes RAG-1 and POMC. Justified by substantial genetic differentiation (uncorrected genetic distance to all other nominal species > 4% for 16S and >= 12% for COB), lack of haplotype sharing in nuclear genes, and morphological differences, we describe a candidate species occurring at various sites in Amazonian Peru as Bolitoglossa chrysothyma sp. nov. After about 50 years of zoological research at Panguana, the type locality of the new species, it was observed by researchers for the first time in 2007, suggesting a recent colonization of the area from the slopes of the adjacent Serran & iacute;a de Sira. When threatened, some individuals displayed a coiled antipredator posture which we speculate could represent a bird dropping mimesis. Aspects of species delimitation in Amazonian Bolitoglossa, their biogeography, possible temporal faunal changes at Panguana, and current threats are discussed. https://zoobank.org/urn:lsid:zoobank.org:pub:FE753BAF-437A-4F00-8FF2-E4BA5E6677DE

Missing data is a long-standing issue in phylogenetic inference, which often results in high levels of taxonomic instability, obscuring otherwise well-supported relationships. Multiple approaches have been developed to deal with the negative effects of ineffective overlap on tree resolution, often by identifying taxa for removal. Here, we repurpose a heuristic method developed to identify unstable taxa in morphological data matrices, concatabominations, and combine it with a novel gene-tree jackknifing on matrix representation of trees to identify candidates for targeted sequencing. Using a multilocus caecilian data set, we illustrate the method's capacity to identify candidate taxa and loci for additional sequencing, compare the results with those of the mathematics-based gene sampling sufficiency approach, and explore the terrace space associated with the multilocus data set. We show that our approach yields tractable numbers of loci/taxa for targeted sequencing that successfully mitigate topological instability due to ineffective overlap, even when modest amounts of data are added.

BACKGROUND:The microbiome is known to play key roles in health and disease, including host susceptibility to parasite infections. The freshwater snail Galba truncatula is the intermediate host for many trematode species, including the liver and rumen flukes Fasciola hepatica and Calicophoron daubneyi, respectively. The snail-parasite system has previously been investigated. However, the specific interaction between the snail-associated microbiota and intra-snail developmental stages of trematodes has yet to be explored. METHODS:Galba truncatula snails were collected from farms in Northern Ireland and trematode infection was diagnosed using PCR. High-throughput sequencing analysis of the bacterial 16S ribosomal DNA V3-V4 hypervariable regions was subsequently applied to characterise the microbiota of both uninfected and infected snails. RESULTS:We first showed that the snail harboured microbiota that was distinct for its environment. The microbiota of infected snails was found to differ significantly from that of uninfected snails. In particular, the bacterial genera Mycoplasma and Methylotenera were significantly more abundant in infected snails, while genera Sphingomonas and Nocardioides were predominantly associated with uninfected snails. CONCLUSION:These findings pave the way to future studies on the functional roles of bacteria in host-parasite relationships.

Amphibians represent a diverse group of tetrapods, marked by deep divergence times between their three systematic orders and families. Studying amphibian biology through the genomics lens increases our understanding of the features of this animal class and that of other terrestrial vertebrates. The need for amphibian genomic resources is more urgent than ever due to the increasing threats to this group. Amphibians are one of the most imperiled taxonomic groups, with approximately 41% of species threatened with extinction due to habitat loss, changes in land use patterns, disease, climate change, and their synergistic effects. Amphibian genomic resources have provided a better understanding of ontogenetic diversity, tissue regeneration, diverse life history and reproductive modes, antipredator strategies, and resilience and adaptive responses. They also serve as essential models for studying broad genomic traits, such as evolutionary genome expansions and contractions, as they exhibit the widest range of genome sizes among all animal taxa and possess multiple mechanisms of genetic sex determination. Despite these features, genome sequencing of amphibians has significantly lagged behind that of other vertebrates, primarily due to the challenges of assembling their large, repeat-rich genomes and the relative lack of societal support. The emergence of long-read sequencing technologies, combined with advanced molecular and computational techniques that improve scaffolding and reduce computational workloads, is now making it possible to address some of these challenges. To promote and accelerate the production and use of amphibian genomics research through international coordination and collaboration, we launched the Amphibian Genomics Consortium (AGC, https://mvs.unimelb.edu.au/amphibian-genomics-consortium) in early 2023. This burgeoning community already has more than 282 members from 41 countries. The AGC aims to leverage the diverse capabilities of its members to advance genomic resources for amphibians and bridge the implementation gap between biologists, bioinformaticians, and conservation practitioners. Here we evaluate the state of the field of amphibian genomics, highlight previous studies, present challenges to overcome, and call on the research and conservation communities to unite as part of the AGC to enable amphibian genomics research to "leap" to the next level.

A molecular level perspective on how novel phenotypes evolve is contingent on our understanding of how genomes evolve through time, and of particular interest is how novel elements emerge or are lost. Mechanisms of protein evolution such as gene duplication have been well established. Studies of gene fusion events show they often generate novel functions and adaptive benefits. Identifying gene fusion and fission events on a genome scale allows us to establish the mode and tempo of emergence of composite genes across the animal tree of life, and allows us to test the repeatability of evolution in terms of determining how often composite genes can arise independently. Here we show that ∼5% of all animal gene families are composite, and their phylogenetic distribution suggests an abrupt, rather than gradual, emergence during animal evolution. We find that gene fusion occurs at a higher rate than fission (73.3% vs 25.4%) in animal composite genes, but many gene fusions (79% of the 73.3%) have more complex patterns including subsequent fission or loss. We demonstrate that nodes such as Bilateria, Euteleostomi, and Eutheria, have significantly higher rates of accumulation of composite genes. We observe that in general deuterostomes have a greater amount of composite genes as compared to protostomes. Intriguingly, up to 41% of composite gene families have evolved independently in different clades showing that the same solutions to protein innovation have evolved time and again in animals. Significance statement New genes emerge and are lost from genomes over time. Mechanisms that can produce new genes include, but are not limited to, gene duplication, retrotransposition, de novo gene genesis, and gene fusion/fission. In this work, we show that new genes formed by fusing distinct homologous gene families together comprise a significant portion of the animal proteome. Their pattern of emergence through time is not gradual throughout the animal phylogeny - it is intensified on nodes of major transition in animal phylogeny. Interestingly, we see that evolution replays the tape frequently in these genes with 41% of gene fusion/fission events occurring independently throughout animal evolution.

There is conflicting evidence as to whether Porifera (sponges) or Ctenophora (comb jellies) comprise the root of the animal phylogeny. Support for either a Porifera-sister or Ctenophore-sister tree has been extensively examined in the context of model selection, taxon sampling, and outgroup selection. The influence of dataset construction is comparatively understudied. We re-examine five animal phylogeny datasets that have supported either root hypothesis using an approach designed to enrich orthologous signal in phylogenomic datasets. We find that many component orthogroups in animal datasets fail to recover major lineages as monophyletic with the exception of Ctenophora, regardless of the supported root. Enriching these datasets to retain orthogroups recovering ≥3 major lineages reduces dataset size by up to 50% while retaining underlying phylogenetic information and taxon sampling. Site-heterogeneous phylogenomic analysis of these enriched datasets recovers both Porifera-sister and Ctenophora-sister positions, even with additional constraints on outgroup sampling. Two datasets which previously supported Ctenophora-sister support Porifera-sister upon enrichment. All enriched datasets display improved model fitness under posterior predictive analysis. While not conclusively rooting animals at either Porifera or Ctenophora, we do see an increase in signal for Porifera-sister and a decrease in signal for Ctenophore-sister when data are filtered for orthologous signal. Our results indicate that dataset size and construction as well as model fit influence animal root inference.

Conflicting studies place a group of bilaterian invertebrates containing xenoturbellids and acoelomorphs, the Xenacoelomorpha, as either the primary emerging bilaterian phylum(1-6) or within Deuterostomia, sister to Am-bulacraria.(7-11) Although their placement as sister to the rest of Bilateria supports relatively simple morphology in the ancestral bilaterian, their alternative placement within Deuterostomia suggests a morphologically com-plex ancestral bilaterian along with extensive loss of major phenotypic traits in the Xenacoelomorpha. Recent studies have questioned whether Deuterostomia should be considered monophyletic at all.(10,12,13) Hidden pa-ralogy and poor phylogenetic signal present a major challenge for reconstructing species phylogenies.(14-18) Here, we assess whether these issues have contributed to the conflict over the placement of Xenacoelomor-pha. We reanalyzed published datasets, enriching for orthogroups whose gene trees support well-resolved clans elsewhere in the animal tree.(16) We find that most genes in previously published datasets violate incon-testable clans, suggesting that hidden paralogy and low phylogenetic signal affect the ability to reconstruct branching patterns at deep nodes in the animal tree. We demonstrate that removing orthogroups that cannot recapitulate incontestable relationships alters the final topology that is inferred, while simultaneously improving the fit of the model to the data. We discover increased, but ultimately not conclusive, support for the existence of Xenambulacraria in our set of filtered orthogroups. At a time when we are progressing toward sequencing all life on the planet, we argue that long-standing contentious issues in the tree of life will be resolved using smaller amounts of better quality data that can be modeled adequately.(19)

Dietary lipids increase energy density in dairy cow diets and in some cases can increase beneficial fatty acids (FA) in milk and dairy products. However, the degree of FA saturation may affect the rumen microbiome. The objective of this study was to determine the long-term effects of feeding saturated (hydrogenated vegetable oil; HVO) or unsaturated (olive oil; OO) fatty acid (FA) sources on the rumen microbiome of dairy cows. For 63 days, 15 mid-lactating cows were fed with either a basal diet (no fat supplement), or the basal diet supplemented with 3% dry matter (DM), either HVO or OO. Rumen contents were collected on days 21, 42 and 63 for 16S rRNA gene sequencing using the Illumina MiSeq platform. The results reveal dominance of the phyla Firmicutes (71.5%) and Bacteroidetes (26.2%), and their respective prevalent genera Succiniclasticum (19.4%) and Prevotella (16.6%). Succiniclasticum increased with both treatments at all time points. Prevotella was reduced on day 42 in both diets. Bacterial diversity alpha or beta were not affected by diets. Predicted bacterial functions by CowPI showed changes in energy and protein metabolism. Overall, 3% DM of lipid supplementation over 63 days can be used in dairy cow diets without major impacts on global bacterial community structure.

SummaryConflicting studies place a group of bilaterian invertebrates containing xenoturbellids and acoelomorphs, the Xenacoelomorpha, as either the primary emerging bilaterian phylum, or within Deuterostomia, sister to Ambulacraria. While their placement as sister to the rest of Bilateria supports relatively simple morphology in the ancestral bilaterian, their alternative placement within Deuterostomia suggests a morphologically complex ancestral Bilaterian along with extensive loss of major phenotypic traits in the Xenacoelomorpha. More recently, further studies have brought into question whether Deuterostomia should be considered monophyletic at all. Hidden paralogy presents a major challenge for reconstructing species phylogenies. Here we assess whether hidden paralogy has contributed to the conflict over the placement of Xenacoelomorpha. Our approach assesses previously published datasets, enriching for orthogroups whose gene trees support well resolved clans elsewhere in the animal tree of life. We find that the majority of constituent genes in previously published datasets violate incontestable clans, suggesting that hidden paralogy is rife at this depth. We demonstrate that enrichment for genes with orthologous signal alters the final topology that is inferred, whilst simultaneously improving fit of the model to the data. We discover increased, but ultimately not conclusive, support for the existence of Xenambulacraria in our orthology enriched set of genes. At a time when we are steadily progressing towards sequencing all of life on the planet, we argue that long-standing contentious issues in the tree of life will be resolved using smaller amounts of better quality data that can be modelled adequately.

BackgroundButyrivibrio and Pseudobutyrivibrio dominate in anaerobic gastrointestinal microbiomes, particularly the rumen, where they play a key role in harvesting energy from the diet. Within these genera, 5 rumen species have been classified (B. fibrisolvens, B. hungatei, B. proteoclasticus, P. ruminis and P. xylanivorans); nonetheless, the phylogeny and evolution of these genera is still unclear. Given the recent increase in available genomes, a re-investigation of the phylogenetic systematics and evolution of Butyrivibrio and Pseudobutyrivibrio is timely.ResultsWe show, using both a 16S rDNA and 40 gene marker phylogenetic tree, that 6 species, namely 1. P. ruminis, 2. P. xylanivorans, 3. B. fibrisolvens, 4. Butyrivibrio sp., 5. B. hungatei, and 6. B. proteclasticus likely exist. Pangenome analysis at 100% core definition showed a high abundance of accessory genes (91.50 to 99.34%) compared with core genes (0.66 to 8.50%), illustrating possession of very open genomes. Across the 71 genomes, 870 COGs (clusters of orthologous genes) were shared by all taxa, suggesting evolution through speciation from a common ancestor. Further analysis of Carbohydrate-Active Enzymes (CAZymes) genes show that most are within the accessory genome and orthologous in descent with numerous within-family CAZyme isoforms apparent, CAZyme family tree lineages show that these isoforms largely group according to the 6 species, suggesting extensive horizontal gene transfer within these families.ConclusionsWe show the extensive genomic variation found within Butyrivibrio, and to a lesser extent, Pseudobutyrivibrio. and demonstrate the existence of a new Butyrivibrio species. The Butyrivibrio and Pseudobutyrivibrio genomes are very open with very low % core genomes and high % accessory genomes., and possess a number of GH isoforms that we hypothesise facilitate metabolic plasticity and resilience under dietary perturbations. This study utilizes all currently available genomes and consequently provides a major advancement in our understanding of these important anaerobic bacteria.

The Rhinella spinulosa group is a Glade of toads that inhabit the Andes mountains from northern Ecuador to Patagonia. Its taxonomy was recently revised, and in its new arrangement comprises nine species, including Rhinella gallardoi, traditionally placed in a different intrageneric group. In this work we studied the larval external and internal morphology in this group, by describing for the first time tadpoles of R. achalensis, R. gallardoi, and R. vellardi, and then summarizing morphological data for R altiperuviana, R limensis, R. papillosa, R. spinulosa, and R. trifolium. Although we found no diagnostic larval features for the whole Glade, two distinct morphs were identified. Most tadpoles were highlypigmented and slender, and their oral discs showed a long gap in the second labial tooth row; conversely, tadpoles of R. limensis and R. vellardi shared a globose body and a very short gap. Buccal and musculoskeletal features were highly conserved within the group and regarding other Rhinella, and included four lingual papillae, nonkeratinized spurs, tripartite suprarostral cartilages, quadrato-orbital commissure, and in musculature, m. subarcualis rectus II-IV invading the branchial septum III and laryngeal muscles reduced or absent.

SMXL alignment. Trimmed alignment showing all conserved parts of the gene, not only those residues used for phylogenetic reconstruction. (FAS 335 kb)

We describe two new species of poison frog from central and southern Peru that have been referred to as Ameerega picta, A. hahneli, or A. altamazonica throughout the past thirty years. Our phylogenies generated with genomic data provide strong support that the two new species are successive sisters to two described taxa, A. rubriventris and A. altamazonica, and collectively comprise the Ameerega rubriventris complex. The first new taxon, Ameerega panguana sp. nov., can be distinguished from all other Ameerega by its combination of a unique white venter and an advertisement call of 1-2 notes per second. The second new taxon, Ameerega imasmari sp. nov., is the only cryptically colored Ameerega species that is disttributed across the Fitzcarrald Arch in Southern Peru which possesses a 'peep' advertisement call consisting of 3-4 notes per second and a dominant frequency of 4.3-4.5 kHz. Within the Ameerega rubriventris complex, we observed differences between species in their ventral coloration, tympanum diameter, and call, which suggest that these taxa are reproductively isolated from each other.

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 singlecopy 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.

Exploring and understanding the phylogeny of the Butyrivibrio group is imperative if we are ever to fully understand the consortium of ruminal microbial enzymes that are responsible for the catalysis of multifaceted reactions, such as biohydrogenation. At present, taxonomic classification of the Butyrivibrio group is based primarily on butyrate production. This approach has become antiquated with the development of sequencing technologies and downstream bioinformatics analysis. This study investigated the taxonomic relatedness and functional capacity of the ruminal Butyrivibrio group using 72 genomes. Seventy-one Butyrivibrio group genomes were obtained via JGI (the Hungate 1000 project), and one additional bacterial strain was sequenced by ourselves. A 40 marker phylogenetic tree was constructed and visualised with the interactive Tree Of Life (iTOL), and pangenome analysis conducted using Spine/ClustAGE. Orthologous gene affiliations were identified using OrthAgogue, and glycosyl hydrolase families were identified using dbCAN then aligned with Clustal Omega. Data obtained showed that three primary clades were observed, namely the genus Pseudobutyrivibrio, B. fibrisolvens, and the remaining Butyrivibrio species. Pangenome analysis and orthologous gene affiliations revealed greater diversity within Butyrivibrio than Pseudobutyrivibrio. Butyrivibrio clades consistently showed smaller core genome sizes in comparison to Pseudobutyrivibrio, with core genome percentages as low as 4 %, indicating high levels of variance. Glycosyl hydrolase alignment shows extensive sequence dissimilarity between genes on a nucleotide and amino acid level These findings suggest that the Butyrivibrio group are highly evolved to maintain competitiveness in the rumen and emphasises the need for further research into the biochemical capacity of the Butyrivibrio group.

Background Strigolactones (SLs) are an important class of carotenoid-derived signalling molecule in plants, which function both as exogenous signals in the rhizosphere and as endogenous plant hormones. In flowering plants, SLs are synthesized by a core pathway of four enzymes and are perceived by the DWARF14 (D14) receptor, leading to degradation of SMAX1-LIKE7 (SMXL7) target proteins in a manner dependent on the SCFMAX2 ubiquitin ligase. The evolutionary history of SLs is poorly understood, and it is not clear whether SL synthesis and signalling are present in all land plant lineages, nor when these traits evolved. Results We have utilized recently-generated genomic and transcriptomic sequences from across the land plant clade to resolve the origin of each known component of SL synthesis and signalling. We show that all enzymes in the core SL synthesis pathway originated at or before the base of land plants, consistent with the previously observed distribution of SLs themselves in land plant lineages. We also show that the late-acting enzyme LATERAL BRANCHING OXIDOREDUCTASE (LBO) may be considerably more ancient than previously thought. We perform a detailed phylogenetic analysis of SMXL proteins and show that specific SL target proteins only arose in flowering plants. We also assess diversity and protein structure in the SMXL family, identifying several previously unknown clades. Conclusions Overall, our results suggest that SL synthesis is much more ancient than canonical SL signalling, consistent with the idea that SLs first evolved as rhizosphere signals and were only recruited much later as hormonal signals.

Poison frogs secrete alkaloid toxins in their skin as defence mechanisms against predators. Numerous studies have shown that the origin of alkaloid toxins in the skin is through ‘sequestration from diet’, i.e. uptake and storage of toxins or their chemical precursors, mostly from consumed arthropods. There exists the intriguing possibility that the gut microbiome of these frogs may play a role in this process. We address this question by looking at the organism together with its associated microbial communities, an effective symbiotic relationship between host and microbiome that could have allowed phenotypic adaptation of the host to a toxic diet. We sequenced the Bacterial and Archaeal 16S rRNA regions of the gut microbiome of 7 Poison frog species and 9 outgroup frog species caught in the rainforest of Eastern Peru. Frog species were selected based on sharing similar microhabitats and comparable individual sizes. A comparative analysis of the microbiome composition across all our samples allowed us to identify a core group of abundant symbiotic microbes unique to poison frogs in spite of intrinsic variation within species. We speculate this group could be associated to their ability to sequester toxins and we carried further metagenomic sequencing to allow us to determine possible functions that may be involved in toxin processing in these frogs.

Metataxonomic 16S rDNA based studies are a commonplace and useful tool in the research of the microbiome, but they do not provide the full investigative power of metagenomics and metatranscriptomics for revealing the functional potential of microbial communities. However, the use of metagenomic and metatranscriptomic technologies is hindered by high costs and skills barrier necessary to generate and interpret the data. To address this, a tool for Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was developed for inferring the functional potential of an observed microbiome profile, based on 16S data. This allows functional inferences to be made from metataxonomic 16S rDNA studies with little extra work or cost, but its accuracy relies on the availability of completely sequenced genomes of representative organisms from the community being investigated. The rumen microbiome is an example of a community traditionally underrepresented in genome and sequence databases, but recent efforts by projects such as the Global Rumen Census and Hungate 1000 have resulted in a wide sampling of 16S rDNA profiles and almost 500 fully sequenced microbial genomes from this environment. Using this information, we have developed “CowPI,” a focused version of the PICRUSt tool provided for use by the wider scientific community in the study of the rumen microbiome. We evaluated the accuracy of CowPI and PICRUSt using two 16S datasets from the rumen microbiome: one generated from rDNA and the other from rRNA where corresponding metagenomic and metatranscriptomic data was also available. We show that the functional profiles predicted by CowPI better match estimates for both the meta-genomic and transcriptomic datasets than PICRUSt, and capture the higher degree of genetic variation and larger pangenomes of rumen organisms. Nonetheless, whilst being closer in terms of predictive power for the rumen microbiome, there were differences when compared to both the metagenomic and metatranscriptome data and so we recommend, where possible, functional inferences from 16S data should not replace metagenomic and metatranscriptomic approaches. The tool can be accessed at http://www.cowpi.org and is provided to the wider scientific community for use in the study of the rumen microbiome.