Optimizing insect metabarcoding using replicated mock communities.

1: Metabarcoding (high-throughput sequencing of marker gene amplicons) has emerged as a promising and cost-effective method for characterizing insect community samples. Yet, the methodology varies greatly among studies and its performance has not been systematically evaluated to date. In particular, it is unclear how accurately metabarcoding can resolve species communities in terms of presence-absence, abundances, and biomass. 2: Here we use mock community experiments and a simple probabilistic model to evaluate the effect of different DNA extraction protocols on metabarcoding performance. Specifically, we ask four questions: (Q1) How consistent are the recovered community profiles across replicate mock communities?; (Q2) How does the choice of lysis buffer affect the recovery of the original community?; (Q3) How are community estimates affected by differing lysis times and homogenization?; and (Q4) Is it possible to obtain adequate species abundance estimates through the use of biological spike-ins? 3: We show that estimates are quite variable across community replicates. In general, a mild lysis protocol is better at reconstructing species lists and approximate counts, while homogenization is better at retrieving biomass composition. Small insects are more likely to be detected in lysates, while some tough species require homogenization to be detected. Results are less consistent across biological replicates for lysates than for homogenates. Some species are associated with strong PCR amplification bias, which complicates the reconstruction of species counts. Yet, with adequate spike-in data, species abundance can be determined with roughly 40% standard error for homogenates, and with roughly 50% standard error for lysates, under ideal conditions. In the latter case, however, this often requires species-specific reference data, while spike-in data generalizes better across species for homogenates. 4: We conclude that a non-destructive, mild lysis approach shows the highest promise for presence/absence description of the community, while also allowing future morphological or molecular work on the material. However, homogenization protocols perform better for characterizing community composition, in particular in terms of biomass.

FAVIS: Fast and versatile protocol for non-destructive metabarcoding of bulk insect samples.

Insects are diverse and sustain essential ecosystem functions, yet remain understudied. Recent reports about declines in insect abundance and diversity have highlighted a pressing need for comprehensive large-scale monitoring. Metabarcoding (high-throughput bulk sequencing of marker gene amplicons) offers a cost-effective and relatively fast method for characterizing insect community samples. However, the methodology applied varies greatly among studies, thus complicating the design of large-scale and repeatable monitoring schemes. Here we describe a non-destructive metabarcoding protocol that is optimized for high-throughput processing of Malaise trap samples and other bulk insect samples. The protocol details the process from obtaining bulk samples up to submitting libraries for sequencing. It is divided into four sections: 1) Laboratory workspace preparation; 2) Sample processing-decanting ethanol, measuring the wet-weight biomass and the concentration of the preservative ethanol, performing non-destructive lysis and preserving the insect material for future work; 3) DNA extraction and purification; and 4) Library preparation and sequencing. The protocol relies on readily available reagents and materials. For steps that require expensive infrastructure, such as the DNA purification robots, we suggest alternative low-cost solutions. The use of this protocol yields a comprehensive assessment of the number of species present in a given sample, their relative read abundances and the overall insect biomass. To date, we have successfully applied the protocol to more than 7000 Malaise trap samples obtained from Sweden and Madagascar. We demonstrate the data yield from the protocol using a small subset of these samples.

Emerging technologies revolutionise insect ecology and monitoring.

Insects are the most diverse group of animals on Earth, but their small size and high diversity have always made them challenging to study. Recent technological advances have the potential to revolutionise insect ecology and monitoring. We describe the state of the art of four technologies (computer vision, acoustic monitoring, radar, and molecular methods), and assess their advantages, current limitations, and future potential. We discuss how these technologies can adhere to modern standards of data curation and transparency, their implications for citizen science, and their potential for integration among different monitoring programmes and technologies. We argue that they provide unprecedented possibilities for insect ecology and monitoring, but it will be important to foster international standards via collaboration.

An Anthropocene map of genetic diversity.

The Anthropocene is witnessing a loss of biodiversity, with well-documented declines in the diversity of ecosystems and species. For intraspecific genetic diversity, however, we lack even basic knowledge on its global distribution. We georeferenced 92,801 mitochondrial sequences for >4500 species of terrestrial mammals and amphibians, and found that genetic diversity is 27% higher in the tropics than in nontropical regions. Overall, habitats that are more affected by humans hold less genetic diversity than wilder regions, although results for mammals are sensitive to choice of genetic locus. Our study associates geographic coordinates with publicly available genetic sequences at a massive scale, yielding an opportunity to investigate both the drivers of this component of biodiversity and the genetic consequences of the anthropogenic modification of nature.

Competitive release leads to range expansion and rampant speciation in malagasy dung beetles.

Competition is often thought to promote ecological diversification and thereby to facilitate the coexistence of competitors during evolutionary radiations. At large spatial scales, species may also coexist by having allopatric distributions, which raises the question about the role of range expansion in the proliferation of species during radiations. Here, we integrate a well-sampled (50 out of 74 species) and timed phylogeny of Nanos and Apotolamprus dung beetles (Canthonini) in Madagascar with data on species' geographical ranges, abundances, and body sizes. There is an overall decline in lineage accumulation through time since the colonization of northern Madagascar in the mid Miocene (24-13 Ma). A clade of 24 extant Nanos species (clade L) originating 6.0 Ma exhibits an increase in speciation rate, which is associated with a significant increase in body size and strikingly allopatric distributions of the species. Large body size typically confers a competitive advantage in dung beetles, which is here reflected by strong numerical dominance of clade L species in local communities. We suggest that the "key innovation" of large body size has allowed range expansion due to competitive release, which has created extensive opportunities for allopatric speciation and differentiation along environmental gradients. Most theories to explain diversification patterns in Madagascar rely on allopatric modes of speciation, but they fail to explain how ancestral species became widespread in the first place. The mechanism proposed here, involving range expansion following competitive release via a "key innovation", may have operated in other Malagasy taxa with large numbers of species with small geographic ranges. [biodiversity hotspot; competition; Madagascar; microendemism; radiation.].

Numts help to reconstruct the demographic history of the ocellated lizard (Lacerta lepida) in a secondary contact zone.

In northwestern Iberia, two largely allopatric Lacerta lepida mitochondrial lineages occur, L5 occurring to the south of Douro River and L3 to the north, with a zone of putative secondary contact in the region of the Douro River valley. Cytochrome b sequence chromatograms with polymorphisms at nucleotide sites diagnostic for the two lineages were detected in individuals in the region of the Douro River and further north within the range of L3. We show that these polymorphisms are caused by the presence of four different numts (I-IV) co-occurring with the L3 genome, together with low levels of heteroplasmy. Two of the numts (I and II) are similar to the mitochondrial genome of L5 but are quite divergent from the mitochondrial genome of L3 where they occur. We show that these numts are derived from the mitochondrial genome of L5 and were incorporated in L3 through hybridization at the time of secondary contact between the lineages. The additional incidence of these numts to the north of the putative contact zone is consistent with an earlier postglacial northward range expansion of L5, preceding that of L3. We show that genetic exchange between the lineages responsible for the origin of these numts in L3 after secondary contact occurred prior to, or coincident with, the northward expansion of L3. This study shows that, in the context of phylogeographic analysis, numts can provide evidence for past demographic events and can be useful tools for the reconstruction of complex evolutionary histories.

Phylogeography and demographic history of Lacerta lepida in the Iberian Peninsula: multiple refugia, range expansions and secondary contact zones.

BACKGROUND: The Iberian Peninsula is recognized as an important refugial area for species survival and diversification during the climatic cycles of the Quaternary. Recent phylogeographic studies have revealed Iberia as a complex of multiple refugia. However, most of these studies have focused either on species with narrow distributions within the region or species groups that, although widely distributed, generally have a genetic structure that relates to pre-Quaternary cladogenetic events. In this study we undertake a detailed phylogeographic analysis of the lizard species, Lacerta lepida, whose distribution encompasses the entire Iberian Peninsula. We attempt to identify refugial areas, recolonization routes, zones of secondary contact and date demographic events within this species. RESULTS: Results support the existence of 6 evolutionary lineages (phylogroups) with a strong association between genetic variation and geography, suggesting a history of allopatric divergence in different refugia. Diversification within phylogroups is concordant with the onset of the Pleistocene climatic oscillations. The southern regions of several phylogroups show a high incidence of ancestral alleles in contrast with high incidence of recently derived alleles in northern regions. All phylogroups show signs of recent demographic and spatial expansions. We have further identified several zones of secondary contact, with divergent mitochondrial haplotypes occurring in narrow zones of sympatry. CONCLUSIONS: The concordant patterns of spatial and demographic expansions detected within phylogroups, together with the high incidence of ancestral haplotypes in southern regions of several phylogroups, suggests a pattern of contraction of populations into southern refugia during adverse climatic conditions from which subsequent northern expansions occurred. This study supports the emergent pattern of multiple refugia within Iberia but adds to it by identifying a pattern of refugia coincident with the southern distribution limits of individual evolutionary lineages. These areas are important in terms of long-term species persistence and therefore important areas for conservation.

Origin and Diversification of Dung Beetles in Madagascar.

Madagascar has a rich fauna of dung beetles (Scarabaeinae and Aphodiinae) withalmost 300 species described to date. Like most other taxa in Madagascar, dung beetles exhibit an exceptionally high level of endemism (96% of the species). Here,we review the current knowledge of the origin and diversification of Malagasy dung beetles. Based on molecular phylogenies, the extant dung beetles originate from eight colonizations, of which four have given rise to extensive radiations. These radiations have occurred in wet forests, while the few extant species in the less successfulradiations occur in open and semi-open habitats. We discuss the likely mechanisms of speciation and the ecological characteristics of the extant communities, emphasizing the role of adaptation along environmental gradients and allopatric speciation in generating the exceptionally high beta diversity in Malagasy dung beetles. Phylogeographic analyses of selected species reveal complex patterns with evidence for genetic introgression between old taxa. The introduction of cattle to Madagascar 1500 years ago created a new abundant resource, onto which a few species haveshifted and thereby been able to greatly expand their geographical ranges.