Mitochondrial metagenomics: letting the genes out of the bottle

‘Mitochondrial metagenomics’ (MMG) is a methodology for shotgun sequencing of total DNA from specimen mixtures and subsequent bioinformatic extraction of mitochondrial sequences. The approach can be applied to phylogenetic analysis of taxonomically selected taxa, as an economical alternative to mitogenome sequencing from individual species, or to environmental samples of mixed specimens, such as from mass trapping of invertebrates. The routine generation of mitochondrial genome sequences has great potential both for systematics and community phylogenetics. Mapping of reads from low-coverage shotgun sequencing of environmental samples also makes it possible to obtain data on spatial and temporal turnover in whole-community phylogenetic and species composition, even in complex ecosystems where species-level taxonomy and biodiversity patterns are poorly known. In addition, read mapping can produce information on species biomass, and potentially allows quantification of within-species genetic variation. The success of MMG relies on the formation of numerous mitochondrial genome contigs, achievable with standard genome assemblers, but various challenges for the efficiency of assembly remain, particularly in the face of variable relative species abundance and intra-specific genetic variation. Nevertheless, several studies have demonstrated the power of mitogenomes from MMG for accurate phylogenetic placement, evolutionary analysis of species traits, biodiversity discovery and the establishment of species distribution patterns; it offers a promising avenue for unifying the ecological and evolutionary understanding of species diversity

The geographic and phylogenetic structure of public DNA barcode databases: an assessment using Chrysomelidae (leaf beetles)

IntroductionDNA barcoding in insects has progressed rapidly, with the ultimate goal of a complete inventory of the world’s species. However, the barcoding effort to date has been driven by a few national campaigns and leaves much of the world unsampled. This study investigates to what degree the current barcode data cover the species diversity across the globe, using the leaf beetle family Chrysomelidae as an example.MethodsA recent version (June 2023) of the Barcode-of-Life database was subjected to test of sampling completeness using the barcode-to-BIN ratio and sampling coverage (SC) metric. All barcodes were placed in a phylogenetic tree of ~600 mitochondrial genomes, applying phylogenetic diversity (PD) and metrics of community phylogenetics to national barcode sets to test for sampling completeness at clade level and reveal the global structure of species diversity.ResultsThe database included 73342 barcodes, grouped into 5310 BINs (species proxies) from 101 countries. Costa Rica contributed nearly half of all barcode sequences, while nearly 50 countries were represented by less than ten barcodes. Only five countries, Costa Rica, Canada, South Africa, Germany, and Spain, had a high sampling completeness, although collectively the barcode database covers most major taxonomic and biogeographically confined lineages. PD showed moderate saturation as more species diversity is added in a country, and community phylogenetics indicated clustering of national faunas. However, at the species level the inventory remained incomplete even in the most intensely sampled countries, and the sampling was insufficient for assessment of global species richness patterns.DiscussionThe sequence-based inventory in Chrysomelidae needs to be greatly expanded to include more areas and deeper local sampling before reaching a knowledge base similar to the existing Linnaean taxonomy. However, placing the barcodes into a backbone phylogenetic tree from mitochondrial genomes, a taxonomically and biogeographically highly structured pattern of global diversity emerges into which all species can be integrated via their barcodes

Evolutionary dynamics of autosomal-heterosomal rearrangements in a multiple-X chromosome system of tiger beetles (Cicindelidae)

<p>Abstract</p> <p>Background</p> <p>Genetic systems involving multiple X chromosomes have arisen repeatedly in sexually reproducing animals. Tiger beetles (Cicindelidae) exhibit a phylogenetically ancient multiple-X system typically consisting of 2–4 X chromosomes and a single Y. Because recombination rates are suppressed in sex chromosomes, changes in their numbers and movement of genes between sex chromosomes and autosomes, could have important consequences for gene evolution and rates of speciation induced by these rearrangements. However, it remains unclear how frequent these rearrangements are and which genes are affected.</p> <p>Results</p> <p>Karyotype analyses were performed for a total of 26 North American species in the highly diverse genus <it>Cicindela</it>, tallying the number of X chromosomes and autosomes during mitosis and meiosis. The chromosomal location of the ribosomal rRNA gene cluster (rDNA) was used as an easily scored marker for genic turnover between sex chromosomes or autosomes. The findings were assessed in the light of a recent phylogenetic analysis of the group. While autosome numbers remained constant throughout the lineage, sex chromosome numbers varied. The predominant karyotype was n = 9+X₁X₂X₃Y which was also inferred to be the ancestral state, with several changes to X₁X₂Y and X₁X₂X₃X₄Y confined to phylogenetically isolated species. The total (haploid) numbers of rDNA clusters varied between two, three, and six (in one exceptional case), and clusters were localized either on the autosomes, the sex chromosomes, or both. Transitions in rDNA localization and in numbers of rDNA clusters varied independently of each other, and also independently of changes in sex chromosome numbers.</p> <p>Conclusion</p> <p>Changes of X chromosome numbers and transposition of the rDNA locus (and presumably other genes) between autosomes and sex chromosomes in <it>Cicindela </it>occur frequently, and are likely to be the result of fusions or fissions between X chromosomes, rather than between sex chromosomes and autosomes. Yet, translocations between sex chromosomes and autosomes appear to be common, as indicated by the patterns of rDNA localization. Rearranged karyotypes involving multiple sex chromosomes would reduce recombination, and hybrid dysgenesis selects against polymorphic populations. Hence, the high frequency of these rearrangements could be a cause of the great species diversity in <it>Cicindela</it>.</p

The SITE-100 Project: Site-Based Biodiversity Genomics for Species Discovery, Community Ecology, and a Global Tree-of-Life

Most insect communities are composed of evolutionarily diverse lineages, but detailed phylogenetic analyses of whole communities are lacking, in particular in species-rich tropical faunas. Likewise, our knowledge of the Tree-of-Life to document evolutionary diversity of organisms remains highly incomplete and especially requires the inclusion of unstudied lineages from species-rich ecosystems. Here we present the SITE-100 program, which is an attempt at building the Tree-of-Life from whole-community sampling of high-biodiversity sites around the globe. Combining the local site-based sets into a global tree produces an increasingly comprehensive estimate of organismal phylogeny, while also re-tracing evolutionary history of lineages constituting the local community. Local sets are collected in bulk in standardized passive traps and imaged with large-scale high-resolution cameras, which is followed by a parataxonomy step for the preliminary separation of morphospecies and selection of specimens for phylogenetic analysis. Selected specimens are used for individual DNA extraction and sequencing, usually to sequence mitochondrial genomes. All remaining specimens are bulk extracted and subjected to metabarcoding. Phylogenetic analysis on the mitogenomes produces a reference tree to which short barcode sequences are added in a secondary analysis using phylogenetic placement methods or backbone constrained tree searches. However, the approach may be hampered because (1) mitogenomes are limited in phylogenetic informativeness, and (2) site-based sampling may produce poor taxon coverage which causes challenges for phylogenetic inference. To mitigate these problems, we first assemble nuclear shotgun data from taxonomically chosen lineages to resolve the base of the tree, and add site-based mitogenome and DNA barcode data in three hierarchical steps. We posit that site-based sampling, though not meeting the criterion of “taxon-completeness,” has great merits given preliminary studies showing representativeness and evenness of taxa sampled. We therefore argue in favor of site-based sampling as an unorthodox but logistically efficient way to construct large phylogenetic trees.Copyright © 2022 Bian, Garner, Liu and Vogler. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. The attached file is the published version of the article

Recalibrated Tree of Leaf Beetles (Chrysomelidae) Indicates Independent Diversification of Angiosperms and Their Insect Herbivores

BACKGROUND: The great diversity of the “Phytophaga” (weevils, longhorn beetles and leaf beetles) has been attributed to their co-radiation with the angiosperms based on matching age estimates for both groups, but phylogenetic information and molecular clock calibrations remain insufficient for this conclusion. METHODOLOGY: A phylogenetic analysis of the leaf beetles (Chrysomelidae) was conducted based on three partial ribosomal gene markers (mitochondrial rrnL, nuclear small and large subunit rRNA) including over 3000 bp for 167 taxa representing most major chrysomelid lineages and outgroups. Molecular clock calibrations and confidence intervals were based on paleontological data from the oldest (K-T boundary) leaf beetle fossil, ancient feeding traces ascribed to hispoid Cassidinae, and the vicariant split of Nearctic and Palearctic members of the Timarchini. PRINCIPAL FINDINGS: The origin of the Chrysomelidae was dated to 73–79 Mya (confidence interval 63–86 Mya), and most subfamilies were post-Cretaceous, consistent with the ages of all confirmed body fossils. Two major monocot feeding chrysomelid lineages formed widely separated clades, demonstrating independent colonization of this ancient (early Cretaceous) angiosperm lineage. CONCLUSIONS: Previous calibrations proposing a much older origin of Chrysomelidae were not supported. Therefore, chrysomelid beetles likely radiated long after the origin of their host lineages and their diversification was driven by repeated radiaton on a pre-existing diverse resource, rather than ancient host associations

Rare failures of DNA bar codes to separate morphologically distinct species in a biodiversity survey of Iberian leaf beetles

During a survey of genetic and species diversity patterns of leaf beetle (Coleoptera: Chrysomelidae) assemblages across the Iberian Peninsula we found a broad congruence between morphologically delimited species and variation in the cytochrome oxidase (cox1) gene. However, one species pair each in the genera Longitarsus Berthold and Pachybrachis Chevrolat was inseparable using molecular methods, whereas diagnostic morphological characters (including male or female genitalia) unequivocally separated the named species. Parsimony haplotype networks and maximum likelihood trees built from cox1 showed high genetic structure within each species pair, but no correlation with the morphological types and neither with geographic distributions. This contrasted with all analysed congeneric species, which were recovered as monophyletic. A limited number of specimens were sequenced for the nuclear 18S rRNA gene, which showed no or very limited variation within the species pair and no separation of morphological types. These results suggest that processes of lineage sorting for either group are lagging behind the clear morphological and presumably reproductive separation. In the Iberian chrysomelids, incongruence between DNA-based and morphological delimitations is a rare exception, but the discovery of these species pairs may be useful as an evolutionary model for studying the process of speciation in this ecological and geographical setting. In addition, the study of biodiversity patterns based on DNA requires an evolutionary understanding of these incongruences and their potential causes.AB was funded by the Spanish Ministry of Science and Innovation (grant CGL2009-10111). CGR is funded by the Xunta de Galicia(postdoctoral fellowship POS-A/2012/052)S

Intraspecific genetic variation in complex assemblages from mitochondrial metagenomics: comparison with DNA barcodes

Metagenomic shotgun sequencing, using Illumina technology, and de novo genome assembly of mixed field-collected amples of invertebrates readily produce mitochondrial genome sequences, allowing rapid identification and quantification of species diversity. However, intraspecific genetic variability present in the specimen pools is lost during mitogenome assembly, which limits the utility of ‘mitochondrial metagenomics’ for studies of population diversity. 2. Using 10 natural communities (>2600 individuals) of leaf beetles (Chrysomelidae), DNA variation in the mitochondrial cox1-5’ ‘barcode’ was compared for Sanger sequenced individuals and Illumina shotgun sequenced specimen pools. 3. Generally, only a single mitochondrial contig was assembled per species, even in the presence of intraspecific variation. Ignoring ambiguity from the use of two different assemblers, the cox1 barcode regions from these assemblies were exact nucleotide matches of a Sanger sequenced barcode in 90.7% of cases, which dropped to 76.0% in assemblies from samples with large intra and interspecific variability. Nucleotide differences between barcodes from both data types were almost exclusively in synonymous 3rd codon position, although the number of affected sites was very low, and the greatest discrepancies were correlated with poor quality of Sanger sequences. 4. Unassembled shotgun reads were also used to score single nucleotide polymorphisms and to calculate intraspecific nucleotide diversity (pi) for all available populations at each site. These values correlated with Sanger sequenced cox1 variation but were significantly higher. 5. Overall, the assemblage-focused shotgun sequencing of pooled samples produced nucleotide variation data comparable to the well-established specimen-focused Sanger approach. The findings thus extend the application of mitochondrial metagenomics of complex biodiversity samples to the estimation of diversity below the species level

The limited spatial scale of dispersal in soil arthropods revealed with whole‐community haplotype‐level metabarcoding

Soil arthropod communities are highly diverse and critical for ecosystem functioning. However, our knowledge of spatial structure and the underlying processes of community assembly are scarce, hampered by limited empirical data on species diversity and turnover. We implement a high‐throughput sequencing approach to generate comparative data for thousands of arthropods at three hierarchical levels: genetic, species and supra‐specific lineages. A joint analysis of the spatial arrangement across these levels can reveal the predominant processes driving the variation in biological assemblages at the local scale. This multihierarchical approach was performed using haplotype‐level COI metabarcoding of entire communities of mites, springtails and beetles from three Iberian mountain regions. Tens of thousands of specimens were extracted from deep and superficial soil layers and produced comparative phylogeographic data for >1,000 codistributed species and nearly 3,000 haplotypes. Local assemblage composition differed greatly between grasslands and forests and, within each habitat, showed strong spatial structure and high endemicity. Distance decay was high at all levels, even at the scale of a few kilometres or less. The local distance decay patterns were self‐similar for the haplotypes and higher hierarchical entities, and this fractal structure was similar in all regions, suggesting that uniform processes of limited dispersal determine local‐scale community assembly. Our results from whole‐community metabarcoding provide insight into how dispersal limitations constrain mesofauna community structure within local spatial settings over evolutionary timescales. If generalized across wider areas, the high turnover and endemicity in the soil locally may indicate extremely high richness globally, challenging our current estimations of total arthropod diversity on Earth.This research was funded by Newton International Program, UK, to PA and the NHM Biodiversity Initiative to APV. PA was supported by postdoctoral grants from the Royal Society (Newton International Program, UK) and the Spanish Ministry of Economy and Competitiveness (MINECO, Spain) within the Juan de la Cierva Formación Program. CA was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Spain; CGL2015‐74178‐JIN MINECO/FEDER, UE).Peer reviewe

The evolutionary genetics of highly divergent alleles of the mimicry locus in Papilio dardanus

Background: The phylogenetic history of genes underlying phenotypic diversity can offer insight into the evolutionary origin of adaptive traits. This is especially true where single genes have large phenotypic effects, for example in determining polymorphic mimicry in butterflies. Here, we characterise the evolutionary history of two candidate genes for the mimicry switch in the polymorphic Batesian mimic Papilio dardanus coding for the transcription factors engrailed and invected. Results: We show that phased haplotypes associated with the dominant morphs f. poultoni and f. planemoides are phylogenetically highly divergent, in particular at non-synonymous sites. Some non-synonymous changes are shared between the divergent alleles suggesting either convergence or a shared ancestry. Gene trees for invected do not show this pattern. Despite their great divergence, all engrailed alleles of P. dardanus were monophyletic with respect to alleles of closely related species. Phylogenetic analyses therefore reveal no evidence for introgression from other species. A McDonald-Kreitman test conducted on a population sample from South Africa confirms a significant excess of intraspecific non-synonymous diversity in P. dardanus engrailed, suggesting long-term balanced polymorphism at this locus. Conclusions: The divergence between engrailed haplotypes suggests an evolutionary history distorted by selection with multiple changes reflecting recurrent selective sweeps. The high level of intraspecific polymorphism observed is characteristic of balancing selection on this locus, as expected if the gene engrailed is under phenotypic selection for the maintenance of multiple mimetic morphs. Non-synonymous changes in key functional portions of a major transcription factor are likely to be deleterious but if maintained in a dominant allele at low frequency, heterozygosity would reduce the associated genetic load

Bulk de novo mitogenome assembly from pooled total DNA elucidates the phylogeny of weevils (Coleoptera: Curculionoidea)

Complete mitochondrial genomes have been shown to be reliable markers for phylogeny reconstruction among diverse animal groups. However, the relative difficulty and high cost associated with obtaining de novo full mitogenomes have frequently led to conspicuously low taxon sampling in ensuing studies. Here, we report the successful use of an economical and accessible method for assembling complete or near-complete mitogenomes through shot-gun next-generation sequencing of a single library made from pooled total DNA extracts of numerous target species. To avoid the use of separate indexed libraries for each specimen, and an associated increase in cost, we incorporate standard polymerase chain reaction-based “bait” sequences to identify the assembled mitogenomes. The method was applied to study the higher level phylogenetic relationships in the weevils (Coleoptera: Curculionoidea), producing 92 newly assembled mitogenomes obtained in a single Illumina MiSeq run. The analysis supported a separate origin of wood-boring behavior by the subfamilies Scolytinae, Platypodinae, and Cossoninae. This finding contradicts morphological hypotheses proposing a close relationship between the first two of these but is congruent with previous molecular studies, reinforcing the utility of mitogenomes in phylogeny reconstruction. Our methodology provides a technically simple procedure for generating densely sampled trees from whole mitogenomes and is widely applicable to groups of animals for which bait sequences are the only required prior genome knowledge