Reconstruction of ancient microbial genomes from the human gut.

Loss of gut microbial diversity1-6 in industrial populations is associated with chronic diseases7, underscoring the importance of studying our ancestral gut microbiome. However, relatively little is known about the composition of pre-industrial gut microbiomes. Here we performed a large-scale de novo assembly of microbial genomes from palaeofaeces. From eight authenticated human palaeofaeces samples (1,000-2,000 years old) with well-preserved DNA from southwestern USA and Mexico, we reconstructed 498 medium- and high-quality microbial genomes. Among the 181 genomes with the strongest evidence of being ancient and of human gut origin, 39% represent previously undescribed species-level genome bins. Tip dating suggests an approximate diversification timeline for the key human symbiont Methanobrevibacter smithii. In comparison to 789 present-day human gut microbiome samples from eight countries, the palaeofaeces samples are more similar to non-industrialized than industrialized human gut microbiomes. Functional profiling of the palaeofaeces samples reveals a markedly lower abundance of antibiotic-resistance and mucin-degrading genes, as well as enrichment of mobile genetic elements relative to industrial gut microbiomes. This study facilitates the discovery and characterization of previously undescribed gut microorganisms from ancient microbiomes and the investigation of the evolutionary history of the human gut microbiota through genome reconstruction from palaeofaeces.

Relaxation of Natural Selection in the Evolution of the Giant Lungfish Genomes.

Nonadaptive hypotheses on the evolution of eukaryotic genome size predict an expansion when the process of purifying selection becomes weak. Accordingly, species with huge genomes, such as lungfish, are expected to show a genome-wide relaxation signature of selection compared with other organisms. However, few studies have empirically tested this prediction using genomic data in a comparative framework. Here, we show that 1) the newly assembled transcriptome of the Australian lungfish, Neoceratodus forsteri, is characterized by an excess of pervasive transcription, or transcriptional leakage, possibly due to suboptimal transcriptional control, and 2) a significant relaxation signature in coding genes in lungfish species compared with other vertebrates. Based on these observations, we propose that the largest known animal genomes evolved in a nearly neutral scenario where genome expansion is less efficiently constrained.

Wood feeding and social living: Draft genome of the subterranean termite Reticulitermes lucifugus (Blattodea; Termitoidae).

Termites (Insecta, Blattodea, Termitoidae) are a widespread and diverse group of eusocial insects known for their ability to digest wood matter. Herein, we report the draft genome of the subterranean termite Reticulitermes lucifugus, an economically important species and among the most studied taxa with respect to eusocial organization and mating system. The final assembly (~813 Mb) covered up to 88% of the estimated genome size and, in agreement with the Asexual Queen Succession Mating System, it was found completely homozygous. We predicted 16,349 highly supported gene models and 42% of repetitive DNA content. Transposable elements of R. lucifugus show similar evolutionary dynamics compared to that of other termites, with two main peaks of activity localized at 25% and 8% of Kimura divergence driven by DNA, LINE and SINE elements. Gene family turnover analyses identified multiple instances of gene duplication associated with R. lucifugus diversification, with significant lineage-specific gene family expansions related to development, perception and nutrient metabolism pathways. Finally, we analysed P450 and odourant receptor gene repertoires in detail, highlighting the large diversity and dynamical evolutionary history of these proteins in the R. lucifugus genome. This newly assembled genome will provide a valuable resource for further understanding the molecular basis of termites biology as well as for pest control.

Drosophila Evolution over Space and Time (DEST): A New Population Genomics Resource.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome data sets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate data sets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in >20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This data set, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental metadata. A web-based genome browser and web portal provide easy access to the SNP data set. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan data set. Our resource will enable population geneticists to analyze spatiotemporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

Investigating the microbial community of Cacopsylla spp. as potential factor in vector competence of phytoplasma.

Phytoplasmas are obligatory intracellular bacteria that colonize the phloem of many plant species and cause hundreds of plant diseases worldwide. In nature, phytoplasmas are primarily transmitted by hemipteran vectors. While all phloem-feeding insects could in principle transmit phytoplasmas, only a limited number of species have been confirmed as vectors. Knowledge about factors that might determine the vector capacity is currently scarce. Here, we characterized the microbiomes of vector and non-vector species of apple proliferation (AP) phytoplasma 'Candidatus Phytoplasma mali' to investigate their potential role in the vector capacity of the host. We performed high-throughput 16S rRNA metabarcoding of the two principal AP-vectors Cacopsylla picta and Cacopsylla melanoneura and eight Cacopsylla species, which are not AP-vectors but co-occur in apple orchards. The microbiomes of all species are dominated by Carsonella, the primary endosymbiont of psyllids and a second uncharacterized Enterobacteriaceae endosymbiont. Each Cacopsylla species harboured a species-specific phylotype of both symbionts. Moreover, we investigated differences between the microbiomes of AP-vector versus non-vector species and identified the predominant endosymbionts but also Wolbachia and several minor taxa as potential indicator species. Our study highlights the importance of considering the microbiome in future investigations of potential factors influencing host vector competence. We investigated the potential role of symbiotic bacteria in the acquisition and transmission of phytoplasma. By comparing the two main psyillid vector species of Apple proliferation (AP) phytoplasma and eight co-occurring species, which are not able to vector AP-phytoplasma, we found differences in the microbial communities of AP-vector and non-vector species, which appear to be driven by the predominant symbionts in both vector species and Wolbachia and several minor taxa in the non-vector species. In contrast, infection with AP-phytoplasma did not affect microbiome composition in both vector species. Our study provides new insights into the endosymbiont diversity of Cacopsylla spp. and highlights the importance of considering the microbiome when investigating potential factors influencing host vector competence.

Viviparity and habitat restrictions may influence the evolution of male reproductive genes in tsetse fly (Glossina) species.

BACKGROUND: Glossina species (tsetse flies), the sole vectors of African trypanosomes, maintained along their long evolutionary history a unique reproductive strategy, adenotrophic viviparity. Viviparity reduces their reproductive rate and, as such, imposes strong selective pressures on males for reproductive success. These species live in sub-Saharan Africa, where the distributions of the main sub-genera Fusca, Morsitans, and Palpalis are restricted to forest, savannah, and riverine habitats, respectively. Here we aim at identifying the evolutionary patterns of the male reproductive genes of six species belonging to these three main sub-genera. We then interpreted the different patterns we found across the species in the light of viviparity and the specific habitat restrictions, which are known to shape reproductive behavior. RESULTS: We used a comparative genomic approach to build consensus evolutionary trees that portray the selective pressure acting on the male reproductive genes in these lineages. Such trees reflect the long and divergent demographic history that led to an allopatric distribution of the Fusca, Morsitans, and Palpalis species groups. A dataset of over 1700 male reproductive genes remained conserved over the long evolutionary time scale (estimated at 26.7 million years) across the genomes of the six species. We suggest that this conservation may result from strong functional selective pressure on the male imposed by viviparity. It is noteworthy that more than half of these conserved genes are novel sequences that are unique to the Glossina genus and are candidates for selection in the different lineages. CONCLUSIONS: Tsetse flies represent a model to interpret the evolution and differentiation of male reproductive biology under different, but complementary, perspectives. In the light of viviparity, we must take into account that these genes are constrained by a post-fertilization arena for genomic conflicts created by viviparity and absent in ovipositing species. This constraint implies a continuous antagonistic co-evolution between the parental genomes, thus accelerating inter-population post-zygotic isolation and, ultimately, favoring speciation. Ecological restrictions that affect reproductive behavior may further shape such antagonistic co-evolution.

Genomic Analysis of European Drosophila melanogaster Populations Reveals Longitudinal Structure, Continent-Wide Selection, and Previously Unknown DNA Viruses.

Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatiotemporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterize variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples.

Divergence and hybridization in sea turtles: Inferences from genome data show evidence of ancient gene flow between species.

Reconstructing past events of hybridization and population size changes are required to understand speciation mechanisms and current patterns of genetic diversity, and ultimately contribute to species' conservation. Sea turtles are ancient species currently facing anthropogenic threats including climate change, fisheries, and illegal hunting. Five of the seven extant sea turtle species are known to currently hybridize, especially along the Brazilian coast where some populations can have ~32%-42% of hybrids. Although frequently observed today, it is not clear what role hybridization plays in the evolutionary diversification of this group of reptiles. In this study, we generated whole genome resequencing data of the five globally distributed sea turtle species to estimate a calibrated phylogeny and the population size dynamics, and to understand the role of hybridization in shaping the genomes of these ancient species. Our results reveal discordant species divergence dates between mitochondrial and nuclear genomes, with a high frequency of conflicting trees throughout the nuclear genome suggesting that some sea turtle species frequently hybridized in the past. The reconstruction of the species' demography showed a general decline in effective population sizes with no signs of recovery, except for the leatherback sea turtle. Furthermore, we discuss the influence of reference bias in our estimates. We show long-lasting ancestral gene flow events within Chelonioidea that continued for millions of years after initial divergence. Speciation with gene flow is a common pattern in marine species, and it raises questions whether current hybridization events should be considered as a part of these species' evolutionary history or a conservation issue.

Age, tissue, genotype and virus infection regulate Wolbachia levels in Drosophila.

The bacterial symbiont Wolbachia can protect insects against viral pathogens, and the varying levels of antiviral protection are correlated with the endosymbiont load within the insects. To understand why Wolbachia strains differ in their antiviral effects, we investigated the factors controlling Wolbachia density in five closely related strains in their natural Drosophila hosts. We found that Wolbachia density varied greatly across different tissues and between flies of different ages, and these effects depended on the host-symbiont association. Some endosymbionts maintained largely stable densities as flies aged while others increased, and these effects in turn depended on the tissue being examined. Measuring Wolbachia rRNA levels in response to viral infection, we found that viral infection itself also altered Wolbachia levels, with Flock House virus causing substantial reductions in symbiont loads late in the infection. This effect, however, was virus-specific as Drosophila C virus had little impact on Wolbachia in all of the five host systems. Because viruses have strong tissue tropisms and antiviral protection is thought to be cell-autonomous, these effects are likely to affect the virus-blocking phenomenon. However, we were unable to find any evidence of a correlation between Wolbachia and viral titres within the same tissues. We conclude that Wolbachia levels within flies are regulated in a complex host-symbiont-virus-dependent manner and this trinity is likely to influence the antiviral effects of Wolbachia.

Survival and divergence in a small group: The extraordinary genomic history of the endangered Apennine brown bear stragglers.

About 100 km east of Rome, in the central Apennine Mountains, a critically endangered population of ∼50 brown bears live in complete isolation. Mating outside this population is prevented by several 100 km of bear-free territories. We exploited this natural experiment to better understand the gene and genomic consequences of surviving at extremely small population size. We found that brown bear populations in Europe lost connectivity since Neolithic times, when farming communities expanded and forest burning was used for land clearance. In central Italy, this resulted in a 40-fold population decline. The overall genomic impact of this decline included the complete loss of variation in the mitochondrial genome and along long stretches of the nuclear genome. Several private and deleterious amino acid changes were fixed by random drift; predicted effects include energy deficit, muscle weakness, anomalies in cranial and skeletal development, and reduced aggressiveness. Despite this extreme loss of diversity, Apennine bear genomes show nonrandom peaks of high variation, possibly maintained by balancing selection, at genomic regions significantly enriched for genes associated with immune and olfactory systems. Challenging the paradigm of increased extinction risk in small populations, we suggest that random fixation of deleterious alleles (i) can be an important driver of divergence in isolation, (ii) can be tolerated when balancing selection prevents random loss of variation at important genes, and (iii) is followed by or results directly in favorable behavioral changes.

Phylogenomic proof of Recurrent Demipolyploidization and Evolutionary Stalling of the "Triploid Bridge" in Arundo (Poaceae).

Polyploidization is a frequent phenomenon in plants, which entails the increase from one generation to the next by multiples of the haploid number of chromosomes. While tetraploidization is arguably the most common and stable outcome of polyploidization, over evolutionary time triploids often constitute only a transient phase, or a "triploid bridge", between diploid and tetraploid levels. In this study, we reconstructed in a robust phylogenomic and statistical framework the evolutionary history of polyploidization in Arundo, a small genus from the Poaceae family with promising biomass, bioenergy and phytoremediation species. Through the obtainment of 10 novel leaf transcriptomes for Arundo and outgroup species, our results prove that recurrent demiduplication has likely been a major driver of evolution in this species-poor genus. Molecular dating further demonstrates that the species originating by demiduplication stalled in the "triploid bridge" for evolutionary times in the order of millions of years without undergoing tetratploidization. Nevertheless, we found signatures of molecular evolution highlighting some of the processes that accompanied the genus radiation. Our results clarify the complex nature of Arundo evolution and are valuable for future gene functional validation as well as reverse and comparative genomics efforts in the Arundo genus and other Arundinoideae.

Genomic data do not support comb jellies as the sister group to all other animals.

Understanding how complex traits, such as epithelia, nervous systems, muscles, or guts, originated depends on a well-supported hypothesis about the phylogenetic relationships among major animal lineages. Traditionally, sponges (Porifera) have been interpreted as the sister group to the remaining animals, a hypothesis consistent with the conventional view that the last common animal ancestor was relatively simple and more complex body plans arose later in evolution. However, this premise has recently been challenged by analyses of the genomes of comb jellies (Ctenophora), which, instead, found ctenophores as the sister group to the remaining animals (the "Ctenophora-sister" hypothesis). Because ctenophores are morphologically complex predators with true epithelia, nervous systems, muscles, and guts, this scenario implies these traits were either present in the last common ancestor of all animals and were lost secondarily in sponges and placozoans (Trichoplax) or, alternatively, evolved convergently in comb jellies. Here, we analyze representative datasets from recent studies supporting Ctenophora-sister, including genome-scale alignments of concatenated protein sequences, as well as a genomic gene content dataset. We found no support for Ctenophora-sister and conclude it is an artifact resulting from inadequate methodology, especially the use of simplistic evolutionary models and inappropriate choice of species to root the metazoan tree. Our results reinforce a traditional scenario for the evolution of complexity in animals, and indicate that inferences about the evolution of Metazoa based on the Ctenophora-sister hypothesis are not supported by the currently available data.

TRPA5, an Ankyrin Subfamily Insect TRP Channel, is Expressed in Antennae of Cydia pomonella (Lepidoptera: Tortricidae) in Multiple Splice Variants.

Transient receptor potential (TRP) channels are an ancient family of cation channels, working as metabotropic triggers, which respond to physical and chemical environmental cues. Perception of chemical signals mediate reproductive behaviors and is therefore an important target for sustainable management tactics against the codling moth Cydia pomonella L. (Lepidoptera: Tortricidae). However, olfactory behavior strongly depends on diel periodicity and correlation of chemical with physical cues, like temperature, and physical cues thus essentially contribute to the generation of behavioral response. From an antennal transcriptome generated by next generation sequencing, we characterized five candidate TRPs in the codling moth. The coding DNA sequence of one of these was extended to full length, and phylogenetic investigation revealed it to be orthologous of the TRPA5 genes, reported in several insect genomes as members of the insect TRPA group with unknown function but closely related to the thermal sensor pyrexia Reverse transcription PCR revealed the existence of five alternate splice forms of CpTRPA5. Identification of a novel TRPA and its splice forms in codling moth antennae open for investigation of their possible sensory roles and implications in behavioral responses related to olfaction.

Interkingdom transfer of the acne-causing agent, Propionibacterium acnes, from human to grapevine.

Here, we report the surprising and, to our knowledge, unique example of horizontal interkingdom transfer of a human opportunistic pathogen (Propionibacterium acnes) to a crop plant (the domesticated grapevine Vitis vinifera L.). Humans, like most organisms, have established a long-lasting cohabitation with a variety of microbes, including pathogens and gut-associated bacteria. Studies which have investigated the dynamics of such associations revealed numerous cases of bacterial host switches from domestic animals to humans. Much less is, however, known about the exchange of microbial symbionts between humans and plants. Fluorescent in situ hybridization localized P. acnes in the bark, in xylem fibers, and, more interestingly, inside pith tissues. Phylogenetic and population genetic analyses suggest that the establishment of the grapevine-associated P. acnes as obligate endophyte is compatible with a recent transfer event, likely during the Neolithic, when grapevine was domesticated.

Loss of Drosophila pheromone reverses its role in sexual communication in Drosophila suzukii.

The Drosophila pheromone cis-11-octadecenyl acetate (cVA) is used as pheromone throughout the melanogaster group and fulfils a primary role in sexual and social behaviours. Here, we found that Drosophila suzukii, an invasive pest that oviposits in undamaged ripe fruit, does not produce cVA. In fact, its production site, the ejaculatory bulb, is atrophied. Despite loss of cVA production, its receptor, Or67d, and cognate sensillum, T1, which are essential in cVA-mediated behaviours, were fully functional. However, T1 expression was dramatically reduced in D. suzukii, and the corresponding antennal lobe glomerulus, DA1, minute. Behavioural responses to cVA depend on the input balance of Or67d neurons (driving cVA-mediated behaviours) and Or65a neurons (inhibiting cVA-mediated behaviours). Accordingly, the shifted input balance in D. suzukii has reversed cVA's role in sexual behaviour: perfuming D. suzukii males with Drosophila melanogaster equivalents of cVA strongly reduced mating rates. cVA has thus evolved from a generic sex pheromone to a heterospecific signal that disrupts mating in D. suzukii, a saltational shift, mediated through offsetting the input balance that is highly conserved in congeneric species. This study underlines that dramatic changes in a species' sensory preference can result from rather 'simple' numerical shifts in underlying neural circuits.

Serine codon-usage bias in deep phylogenomics: pancrustacean relationships as a case study.

Phylogenomic analyses of ancient relationships are usually performed using amino acid data, but it is unclear whether amino acids or nucleotides should be preferred. With the 2-fold aim of addressing this problem and clarifying pancrustacean relationships, we explored the signals in the 62 protein-coding genes carefully assembled by Regier et al. in 2010. With reference to the pancrustaceans, this data set infers a highly supported nucleotide tree that is substantially different to the corresponding, but poorly supported, amino acid one. We show that the discrepancy between the nucleotide-based and the amino acids-based trees is caused by substitutions within synonymous codon families (especially those of serine-TCN and AGY). We show that different arthropod lineages are differentially biased in their usage of serine, arginine, and leucine synonymous codons, and that the serine bias is correlated with the topology derived from the nucleotides, but not the amino acids. We suggest that a parallel, partially compositionally driven, synonymous codon-usage bias affects the nucleotide topology. As substitutions between serine codon families can proceed through threonine or cysteine intermediates, amino acid data sets might also be affected by the serine codon-usage bias. We suggest that a Dayhoff recoding strategy would partially ameliorate the effects of such bias. Although amino acids provide an alternative hypothesis of pancrustacean relationships, neither the nucleotides nor the amino acids version of this data set seems to bring enough genuine phylogenetic information to robustly resolve the relationships within group, which should still be considered unresolved.

MicroRNAs and phylogenomics resolve the relationships of Tardigrada and suggest that velvet worms are the sister group of Arthropoda.

Morphological data traditionally group Tardigrada (water bears), Onychophora (velvet worms), and Arthropoda (e.g., spiders, insects, and their allies) into a monophyletic group of invertebrates with walking appendages known as the Panarthropoda. However, molecular data generally do not support the inclusion of tardigrades within the Panarthropoda, but instead place them closer to Nematoda (roundworms). Here we present results from the analyses of two independent genomic datasets, expressed sequence tags (ESTs) and microRNAs (miRNAs), which congruently resolve the phylogenetic relationships of Tardigrada. Our EST analyses, based on 49,023 amino acid sites from 255 proteins, significantly support a monophyletic Panarthropoda including Tardigrada and suggest a sister group relationship between Arthropoda and Onychophora. Using careful experimental manipulations--comparisons of model fit, signal dissection, and taxonomic pruning--we show that support for a Tardigrada + Nematoda group derives from the phylogenetic artifact of long-branch attraction. Our small RNA libraries fully support our EST results; no miRNAs were found to link Tardigrada and Nematoda, whereas all panarthropods were found to share one unique miRNA (miR-276). In addition, Onychophora and Arthropoda were found to share a second miRNA (miR-305). Our study confirms the monophyly of the legged ecdysozoans, shows that past support for a Tardigrada + Nematoda group was due to long-branch attraction, and suggests that the velvet worms are the sister group to the arthropods.

A multidisciplinary approach to tackling invasive species: barcoding, morphology, and metataxonomy of the leafhopper Arboridia adanae.

The leafhopper genus Arboridia includes several species that feed on Vitis vinifera and cause leaf chlorosis. We report the first alien Arboridia infestation in Italy in 2021 in an Apulian vineyard. To confirm the taxonomic status of the species responsible for crop damage, and reconstruct its demographic history, we barcoded individuals from Apulia together with Arboridia spp. from Crete (Greece), A. adanae from Central Turkey and other specimens of the presumed sister species, A. dalmatina from Dalmatia (Croatia). Molecular phylogenies and barcoding gap analysis identified clades not associated with sampling locations. This result is incongruent with classical specimen assignment and is further supported by morphological analyses, which did not reveal significant differences among the populations. Therefore, we propose A. dalmatina as a junior synonym of A. adanae, which would become the only grapevine-related Arboridia species in the eastern Mediterranean. To further characterise A. adanae evolution, we performed a molecular clock analysis that suggested a radiation during the Pleistocene glaciations. Finally, to assess whether the Apulian individuals carried microorganisms of agricultural relevance, we sequenced their bacterial microbiota using 16S rRNA amplicon sequencing identifying three phytopathogens not generally associated with Arboridia activities as well as Wolbachia in one Apulian haplogroup. We discuss the agricultural implications of this infestation.

Discovering and exploring the hidden diversity of human gut viruses using highly enriched virome samples.

Viruses are an abundant and crucial component of the human microbiome, but accurately discovering them via metagenomics is still challenging. Currently, the available viral reference genomes poorly represent the diversity in microbiome samples, and expanding such a set of viral references is difficult. As a result, many viruses are still undetectable through metagenomics even when considering the power of de novo metagenomic assembly and binning, as viruses lack universal markers. Here, we describe a novel approach to catalog new viral members of the human gut microbiome and show how the resulting resource improves metagenomic analyses. We retrieved >3,000 viral-like particles (VLP) enriched metagenomic samples (viromes), evaluated the efficiency of the enrichment in each sample to leverage the viromes of highest purity, and applied multiple analysis steps involving assembly and comparison with hundreds of thousands of metagenome-assembled genomes to discover new viral genomes. We reported over 162,000 viral sequences passing quality control from thousands of gut metagenomes and viromes. The great majority of the retrieved viral sequences (~94.4%) were of unknown origin, most had a CRISPR spacer matching host bacteria, and four of them could be detected in >50% of a set of 18,756 gut metagenomes we surveyed. We included the obtained collection of sequences in a new MetaPhlAn 4.1 release, which can quantify reads within a metagenome matching the known and newly uncovered viral diversity. Additionally, we released the viral database for further virome and metagenomic studies of the human microbiome.