Biology subjects

Weber, S.

Publications and source records attributed to Weber, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Spiders as natural DNA samplers recover arthropod community diversity and biotic interactions across space and time

Global arthropod decline demands effective biodiversity monitoring strategies. However, most current monitoring approaches do not provide an exhaustive picture of arthropod community structure. In particular, biotic interactions and temporal patterns of biodiversity change are still poorly understood. Here we explore the possibility of addressing these two shortfalls using spiders, one of the most important predators of terrestrial arthropods, as natural samplers for arthropod community DNA. We conducted several experiments comparing the recovered community composition between spider gut contents and traditional monitoring methods. Additionally, we used archived spiders from long-term collections to assess the preservation of prey DNA over time. Spiders proved to be highly efficient natural DNA samplers, with gut content metabarcoding revealing similar community composition and alpha- and beta-diversity like traditional methods. Unique arthropod taxa were detected by spider gut contents and traditional methods respectively, indicating that these two approaches can serve as valuable complements to each other. Well-preserved archived spiders effectively reconstructed historical diets up to 20 years back in time, showing that historical spider collections can act as time capsules of the wider diversity that occurred in the spider's habitat. Spiders as natural samplers overcome critical shortfalls in biodiversity monitoring and contribute to our future understanding of community assembly across space and time.

ecology↗

bioRxiv2026

Extraction workflow determines marker-specific recovery andreproducibility in leaf-litter eDNA metabarcoding

Forest-floor leaf litter is a dynamic and structurally complex ecological transition zone and thus a promising substrate for terrestrial eDNA metabarcoding. Yet, extraction workflows for this heterogeneous matrix remain poorly standardized, especially in tropical systems, making it largely impossible to compare ecological functions across space, time, and taxa. To guide workflow selection across a series of selection criteria, including biological target, research question and practical considerations, we compared DNA extraction workflows for leaf-litter eDNA collected from 42 biological samples across seven different forest sites on Oahu, Hawaii. We evaluated four DNA extraction workflows: (1) Two low-volume approaches, with DNA extracted directly from 200 mg of homogenized litter using (i) CTAB or (ii) DNeasy PowerSoil(R); and (2) two high-volume approaches using PBS wash-based from 10 g of litter followed by (i) Centrifugation or (ii) Filtration. Taxonomic recovery from each workflow was evaluated with two COI primer sets targeting arthropods (ANML and shorter NoPlant), and one ITS marker targeting fungi. Results show that eDNA workflows tested here recovered site-level differences among forest-floor communities, but biodiversity recovery depended strongly on extraction workflow and marker. For low volumes, PowerSoil recovered the highest fungal richness (with ITS marker), and produced the most reproducible PCR-replicate profiles across markers, and required the least hands-on time, while CTAB was less expensive but required handling hazardous chemicals. For high volumes workflow, Centrifugation recovered higher arthropod diversity with ANML primer. Differences in community composition were nonetheless recovered by each method. At the same time, sampling sites explained more ASV-level compositional variation than extraction workflow across markers, showing that all workflows retained site-level ecological signals. Together, these results support a workflow framework in which extraction choice depends on target organism group, DNA state, reproducibility needs, and practical constraints.

ecology↗

bioRxiv2026

Emerging Beetle-Pathogen Symbioses and Their Consequences for Forest Health: Lessons from Rapid 'Ohi'a Death in Hawai'i

Invasive ambrosia beetles and fungal pathogens threaten forest ecosystems worldwide, exemplified in Hawaii by the widespread loss of keystone species [o]hia (Metrosideros polymorpha), due to Rapid [O]hia Death (ROD). A unique occurrence of five ambrosia beetle species (one native, four introduced) that vary in their symbiotic relationships with two introduced fungal pathogens provide an opportunity to test hypotheses of how opportunistic symbioses facilitate disease dynamics involving dominant forest trees. ROD is caused by two novel Ceratocystis fungal pathogens whose spores can spread via association with ambrosia beetles as they bore into [o]hia trees. We examined beetle-pathogen interactions of all five ambrosia beetle species in three ROD-affected regions on Hawaii Island, and used quantitative PCR (qPCR) to provide the first molecular confirmation of the two ROD pathogens associated with the exterior, mycangia, and gut of each beetle species. Results from generalized linear models and correlation networks show that pathogen acquisition and transport, including the potential for consumption and the presence of the pathogens, are determined by beetle invasion status and mycangia morphology. A niche construction framework suggests that both varying symbioses and opportunism facilitate disease spread, with the three invasive Xyleborus species emerging as key disease vectors. Identifying the beetle species that are more likely to contribute to disease spread, and understanding their biology as vectors, can inform targeted conservation strategies for [o]hia and for insect-pathogen threats in forests worldwide, and illustrates the potential ecosystem-level impacts of novel and opportunistic symbioses between globally distributed invasive vectors and pathogens.

ecology↗

bioRxiv2026

Topography structures of arthropod communities revealed by leaf-derived environmental DNA on Oahu, Hawaii

Arthropod communities on oceanic islands are shaped by spatial isolation, environmental gradients, and biological invasions, yet their structure remains difficult to resolve due to incomplete taxonomic coverage. In particular, it remains unclear (i) how non-native arthropods can influence community composition and (ii) how they interact with native and non-native plants. To answer the first question, we used leaf-derived environmental DNA (eDNA) to characterize arthropod communities across elevational gradients on five ridges on Oahu, using the native tree Metrosideros polymorpha as a standardized plant. To understand the second question, we compared leaf-derived eDNA from Metrosideros polymorpha (Native), Acacia koa (native), and Psidium cattleianum (invasive), co-occurring in two ridges on Oahu. Additionally, to overcome limitations of reference databases, we applied NIClassify to infer native versus introduced status without requiring species-level identification. Across 96 leaf samples (with 851 Arthropod ASVs), we found arthropod richness increased with elevation, while the proportion of introduced taxa declined significantly. Community composition was primarily structured by ridge, with strong distance-decay relationships indicating high spatial turnover in both native and non-native assemblages. In contrast, plant species effects were context dependent and did not show a consistent native versus invasive signal. Threshold analyses identified a community transition (native vs introduced) near 500 m elevation. These results show that plant-derived eDNA can resolve spatial and environmental structuring of arthropod communities while capturing invasion dynamics under incomplete taxonomic knowledge. Classifier-based inference enables community-level ecological interpretation beyond reference-limited taxa, providing a scalable framework for biodiversity monitoring in data-poor systems.

ecology↗

bioRxiv2026

The Contextual Specificity of Pausing: Interpreting Electromyographic Partial Responses During Action Cancellation and Attentional Capture

A prominent model of action stopping theorises that the reactive cancellation of movement is underpinned by two dissociable processes: a rapid, involuntary "pause" that transiently suppresses motor output, and a slower, voluntary cancellation of movement. As a fundamental component of motor control, the pause process has been posited to generalise broadly to infrequent and salient stimuli (irrespective of whether they bear an imperative to stop) and to be observable as suppression in electromyographical (EMG) recordings. Over two experiments (N = 24 in each), participants completed standard stop signal and flanker tasks, and novel task variants, where flanking arrows occurred infrequently, with or without a delay (relative to the central go stimulus), or coincident with a stop stimulus. Relative to a standard flanker task, presenting flankers infrequently specifically increased slowing in incongruent trials after at least three preceding trials without flanking stimuli, with no effect in congruent or neutral trials, indicating involuntary delays to infrequent events are sensitive to both stimulus features and the recency of perceptually similar events. Presenting flanker arrows of any congruence alongside a stop stimulus resulted in faster EMG suppression than stop stimuli alone, indicating that additional infrequent stimuli can facilitate inhibitory responses. However, presenting flanker stimuli at an equivalent frequency and delay outside of a stopping task did not reliably elicit EMG suppression or behavioural delays. The current findings challenge current models by demonstrating that physiological signatures previously interpreted as manifestations of a generalisable pause process are instead context specific, requiring the anticipation of action cancellation.

neuroscience↗

bioRxiv2026

Stopping and Changing Expected and Unexpected Movements

The capacity to cancel or adapt planned actions in response to changing environmental demands is essential for navigating our complex world. While past research has shown that an individuals expectations of upcoming movement demands influence the speed of action initiation, the effect this has on subsequent cancellation or adaption of that movement remains unknown. 25 healthy adults completed stop signal tasks and stop change tasks in which biasing cues (e.g., "70% left") accurately indicated the probability that a left, or right button press would be required. As expected, responses that were congruent with the cue were faster than incongruent responses; however, biasing cues had no effect on behavioural or physiological (electromyographical) indices of stopping speed. Stopping latencies were found to be faster in the stop change task than the stop signal task, corroborating other recent work. However, a second experiment (25 healthy adults) which used the same stimuli for both tasks (varying only the instructions), revealed no difference - highlighting the sensitivity of the stop process to stimulus effects, and a common confound in the literature. We also observed that physiological indices of action reprogramming (following a stop) were faster in congruent than incongruent trials. Collectively, these results suggests that preparatory changes that accompany expected movements influence the enaction of movement both prior to, and after stopping, but the stop mechanism itself, remains independent of these preparations. These results inform how action cancellation and adaption are applied in real world environments, where expectations continually interface with our motor plans. HighlightsO_LI* Anticipating a movement increases the speed of its enaction but not subsequent cancellation C_LIO_LI* Expected movements can be reprogrammed more quickly than unexpected movements C_LIO_LI* The latency of action cancellation is highly sensitive to stimulus effects C_LI

neuroscience↗

bioRxiv2026

Comparison of environmental DNA and bulk DNA metabarcoding for assessing terrestrial arthropod diversity across three habitat types on Guam

DNA based methods offer a rapid and cost-effective way for detecting species occurrence and monitoring biodiversity; among them bulk DNA metabarcoding is well-established, and recently developed environmental DNA (eDNA)-based methods offer a non-destructive alternative. With a goal to develop suitable methods for assessing insect biodiversity in ecosystems for which DNA reference libraries are not well developed and incomplete, such as remote islands, we compared established bulk DNA metabarcoding methods with eDNA across three replicated terrestrial ecosystem types (limestone forest, degraded forest, and grassland) in Guam. Using two mitochondrial COI primer pairs, we performed bulk DNA metabarcoding of standard entomological collection methods (malaise traps, pan traps, vegetation beating), and compared the assessment of biodiversity with that from different eDNA sources (flowers, spider webs, leaves, tree trunks). In our samples, eDNA and bulk DNA metabarcoding both detected a large proportion of overall taxa (OTUs, 86.6% and 60.3%, respectively). Although DNA metabarcoding detected significantly more taxa, eDNA proved to be a reasonable non-destructive alternative. As expected, because of limitations in existing reference databases for remote habitats, species-level identification was achieved for only a few OTUs. Overall, the sampling approach was the dominant driver of arthropod diversity, explaining [~]17% of the observed variation, while habitat type accounted for [~]4%. Thus, each sampling approach captured some unique diversity signals and contributed to the complementary effect of maximizing detection. For rapid insect biodiversity surveys of terrestrial arthropods, we recommend an integrated metabarcoding approach, and in sensitive habitats where insect capture is undesirable, eDNA offers a powerful alternative to monitor diversity and community change.

ecology↗

bioRxiv2025

Light-induced conformational switching and magnetic sensitivity of Drosophila cryptochrome

Cryptochromes are flavoproteins with a number of established and proposed biological functions based on their sensitivity to light. Amongst the latter is the possibility that cryptochromes mediate the geomagnetic compass sense used by migratory birds as a navigational cue. This hypothesis rests on a magnetically sensitive photochemical reaction of the flavin chromophore in which a series of electron transfers within the protein scaffold ultimately generates a signal propagated within the central nervous system of the animal. Although there is a good understanding of the photochemistry and the electron transfer pathway, the protein-mediated mechanisms of signal transduction are still unclear. Here we have examined the response of Drosophila melanogaster cryptochrome - DmCRY, an archetypal cryptochrome - to photochemical activation by means of molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and cavity ring-down spectroscopy. We were able to measure the dynamics of DmCRY at near-residue level resolution, revealing a reversible, long-lived, blue-light induced conformational change in the C-terminal tail of the protein. This putative signalling state was validated using different illumination conditions, and by examining DmCRY variants in which the electron transfer chain was disrupted by point mutation. Our results show how the photochemical behaviour of the flavin chromophore generates a state of DmCRY that may act as a key primer for modulating downstream interactions.

biophysics↗

bioRxiv2025

When Stopping Requires Going; Physiological Similarities Between Action Cancellation and the Cancellation of Action Cancellation

The reactive cancellation of real-world actions typically requires complex combinations of both muscle contraction and/or muscle suppression. However, current experimental paradigms solely examine contexts in which action cancellation requires muscle suppression. To provide fundamental insights into inhibitory control mechanisms, we directly compared the latency of action cancellation in novel paradigms where stopping required either suppression of planned activation or reinstatement of ongoing activity. 20 healthy adults (mean age = 32.2 years) completed novel variants of the stop signal task (SST) in which each trial began with tonic force production to depress two buttons. When a go signal appeared, participants were required to release these buttons. On a subset of trials, a stop signal occurred after a brief delay, and participants were required to cancel the release of one of the buttons. Data in these variants were compared to conventional response-selective SSTs, in which the go signal required bilateral button presses and stop signals necessitated cancellation of one of these responses. Electromyographic (EMG) recordings allowed detailed comparison of the characteristics of muscle contraction and suppression (i.e., stopping speed) across these tasks. When physiological evidence of synchronous action cancellation in both hands was observed (supporting recent models of complex stopping), EMG measures of action cancellation speed did not differ (p = 0.863, BF01 = 8.49) between cancellation of releases and cancellation of presses conditions. This result suggests that response inhibition may broadly characterise reactive control to maintain a current physiological state rather than specific cancellation of a voluntary response.

physiology↗

bioRxiv2024

Harnessing Environmental DNA (eDNA) to Explore Frugivorous Interactions: A Case Study in Papaya (Carica papaya) and Pineapple (Ananas comosus)

Plant-animal interactions (PAIs) are critical in ecosystem function, mediating energy flow and species interactions. Traditional methods of tracking PAIs, such as morphological identification and camera trapping, are limited in speed and scalability, posing challenges for comprehensive biodiversity monitoring. Recently, environmental DNA (eDNA) metabarcoding has emerged as a promising technique for detecting species interactions non-destructively. This pilot study explores the application of eDNA metabarcoding to investigate interactions involving partially consumed and intact fruits of Carica papaya and Ananas comosus. eDNA metabarcoding were performed from of 36 partially consumed and 6 intact fruit samples. Metabarcoding of mitochondrial COI gene fragments revealed a diverse range of taxa, with Arthropoda, particularly insects, being the most abundant. Results indicated significant differences in taxonomic composition between pineapple and papaya samples, where both the fruit hold some unique as well as shared taxa. Furthermore, the diversity also differed between consumed and intact fruits, suggesting that partially consumed fruits serve as rich eDNA sources, capturing interactions with various frugivores and decomposers. Signal from various organisms detected through eDNA metabarcoding from consumed and damaged fruits allowed us to capture a wide array of taxa, revealing insights into species composition and ecological relationships. The unique ASVs associated with each fruit type suggest that certain taxa may showing preferences based on fruit characteristics such as sugar content, texture, or chemical profile. Present work highlighted the importance of eDNA based methods in unraveling the taxonomic composition of fruit-associated plant-animal interactions. This method needs limited taxonomic expertise, less labors, fast and effective, which can be implemented in monitoring ecological and economical species interactions.

ecology↗

bioRxiv2024

An exploration of complex action stopping across multiple datasets: Insights into the mechanisms of action cancellation and re-programming

A long history of psychological experiments has utilised stop signal paradigms to assess action inhibition. Recent studies have investigated complex stopping behaviours, such as response-selective stopping where only one component of a bimanual action requires cancellation. A current emphasis has been to utilise electromyographical (EMG) recordings to assess the temporal dynamics of action inhibition at the level of the muscle, beyond those based solely on observable behavioural events. Here, we combine EMG and behavioural data from 17 cohorts of healthy younger and older adults yielding over 42,000 response-selective stopping trials, providing unique insights into this emerging field. Expanding from past research in this area, our robust single-trial EMG analyses permit detection of cancelled (partial) and response-generating EMG bursts in both hands, revealing substantial overlaps in the distributions of timing of action cancellation and re-programming. These findings are consistent with recent experimental and modelling evidence, suggesting that response- selective stopping involves two independent processes: a discrete bimanual stop and initiation of a new unimanual response. This overlap appears incompatible with the recent pause-then-cancel model, and more consistent with a broader "pause-then-retune" account, where a slower process mediates any action updating, not just cancellation. Moreover, this independence means that cancellation can happen at any time during motor planning and execution, against the notion of an observable "point of no return" in motor actions. We also discuss best practices for the analysis of EMG data and indicate how methodological aspects, such as choosing appropriate reference time points, can influence the outcomes and their interpretation.

neuroscience↗

bioRxiv2024

Neural dynamics of visual working memory representation during sensory distraction

Recent studies have provided evidence for the concurrent encoding of sensory percepts and visual working memory contents (VWM) across visual areas; however, it has remained unclear how these two types of representations are concurrently present. Here, we reanalyzed an open-access fMRI dataset where participants memorized a sensory stimulus while simultaneously being presented with sensory distractors. First, we found that the VWM code in several visual regions did not fully generalize between different time points, suggesting a dynamic code. A more detailed analysis revealed that this was due to shifts in coding spaces across time. Second, we collapsed neural signals across time to assess the degree of interference between VWM contents and sensory distractors, specifically by testing the alignment of their encoding spaces. We find that VWM and feature-matching sensory distractors are encoded in coding spaces that do not fully overlap, but the separation decreases when distractors negatively impact behavioral performance in recalling the target. Together, these results indicate a role of dynamic coding and temporally stable coding spaces in helping multiplex perception and VWM within visual areas.

neuroscience↗

bioRxiv2024

Graphical pangenomics-enabled characterisation of structural variant impact on gene expression in Brassica napus

Structural variants (SVs, eg. insertions and deletions) are genomic variations > 50 bp that are known to be associated with a range of crop traits, from yield to flowering behaviour and stress responses. Recently, pangenome graphs have emerged as a powerful framework for analysing genomic data by encoding population- or species-level diversity in one data structure. Pangenome graphs have the potential to serve as unbiased references for downstream applications, including SV genotyping and pan-transcriptomic analyses. In this work, we hypothesized that extensive variation affects transcript quantification and expression quantitative trait locus (eQTL) analysis when relying on a single reference, and that using pangenome graphs can mitigate reference sequence bias. We combined long and short read whole genome sequencing data with expression profiling of Brassica napus (oilseed rape) to assess the impact of SVs on gene expression regulation and explored the utility of pangenome graphs for eQTL analysis. We demonstrate that pangenome graphs provides a superior framework for eQTL analysis by eliminating single reference bias in gene expression quantification. Combined with the graph-based genotyping of SVs, we identified 240 eQTL-SVs found in close proximity of target loci. These SVs affect expression of genes related to important traits, are often not in linkage with SNPs and represent diversity unaccounted for in classical SNP-based analyses. This study highlights the multiple advantages of graph-based approaches in population-scale studies and provides novel insight into gene expression regulation in an important crop.

plant biology↗

bioRxiv2024

Stopping Speed to Auditory and Visual Stop Signals Depends on Go Signal Modality

Past research has found that the speed of the action cancellation process is influenced by the sensory modality/modalities of the environmental change that triggers it. However, the effect on selective stopping processes (where participants must cancel only one component of a multi-component movement) remains unknown, despite these complex movements often being required as we navigate our busy modern world. Thirty healthy adults (mean age = 31.1 years, SD = 10.5) completed five response-selective stop signal tasks featuring different combinations of "go signal" modality (the environmental change baring an imperative to initiate movement; auditory or visual) and "stop signal" modality (the environmental change indicating that action cancellation is required; auditory, visual, or audiovisual). Electromyographical (EMG) recordings of effector muscles allowed detailed comparison of the characteristics of voluntary action and cancellation between tasks. Behavioural and physiological measures of stopping speed demonstrated that the modality of the go signal influenced how quickly participants cancelled movement in response to the stop signal: stopping was faster in two cross-modal experimental conditions (auditory go - visual stop; visual go - auditory stop), than in two conditions using the same modality for both signals. A separate condition testing for multisensory facilitation revealed that stopping was fastest when the stop signal consisted of a combined audiovisual stimulus, compared to all other go-stop stimulus combinations. These findings provide novel evidence regarding the role of attentional networks in action cancellation and suggest modality specific cognitive resources influence the latency of the stopping process.

neuroscience↗

bioRxiv2023

Structural determinants of pure antiestrogenicity

Inhibitory activities of antiestrogens on estrogen receptor alpha (ER) range from mixed antagonism/agonism (selective ER modulators; SERMs) to complete antiestrogenicity associated with accelerated ER turnover (selective ER degraders; SERDs). Here, we show using a panel of SERMs, SERDs and a PROTAC that efficient induction of ER SUMOylation better accounts for complete transcriptional repression than increased suppression of coactivator recruitment or accelerated ER degradation. Antiestrogen-induced ER SUMOylation depends on the hydrophobicity of N-terminal residues of ligand binding domain (LBD) helix 12 (H12). L536 mutations, including those occurring in endocrine therapy-resistant breast cancer, abolished SUMOylation with all antiestrogens. Structures of the L536S human ER LBD bound to fulvestrant analogs and molecular dynamics simulations predict dynamic side chain interactions with wild-type ER H12 in the coactivator-binding groove and model the impact of L536 mutations. Together, our results show that induced SUMOylation contributes to the complete suppression of ER activity and provide insights into its structural determinants.

molecular biology↗

bioRxiv2023

Selective Cancellation of Reactive and Anticipated Movement: Differences in Speed of Action Reprogramming, but not Stopping.

The ability to inhibit movements is an essential component of a healthy executive control system. Two distinct but commonly used tasks to assess motor inhibition are the stop signal task (SST) and the anticipated response inhibition (ARI) task. The SST and ARI tasks are similar in that they both require cancelation of a prepotent movement; however, the SST involves cancelation of a speeded reaction to a temporally unpredictable signal, while the ARI task involves cancelation of an anticipated response that the participant has prepared to enact at a wholly predictable time. 33 participants (mean age = 33.3 years, range = 18-55 years) completed variants of the SST and ARI task. In each task, the majority of trials required bimanual button presses, while on a subset of trials a stop signal indicated that one of the presses should be cancelled (i.e., motor selective inhibition). Additional variants of the tasks also included trials featuring signals which were to be ignored, allowing for insights into the attentional component of the inhibitory response. Electromyographic (EMG) recordings allowed detailed comparison of the characteristics of voluntary action and cancellation. The speed of the inhibitory process was not influenced by whether the enacted movement was reactive (SST) or anticipated (ARI task). However, the ongoing (non-cancelled) component of anticipated movements was more efficient than reactive movements, as a result of faster action reprogramming (i.e., faster ongoing actions following successful selective inhibition). Older age was associated with both slower inhibition and slower action reprogramming across all reactive and anticipated tasks.

neuroscience↗

bioRxiv2023

Structural characterization of two nanobodies targeting the ligand-binding pocket of human Arc

The activity-regulated cytoskeleton-associated protein (Arc) is a complex regulator of synaptic plasticity in glutamatergic neurons. Understanding its molecular function is key to elucidate the neurobiology of memory and learning, stress regulation, and multiple neurological and psychiatric diseases. The recent development of anti-Arc nanobodies has promoted the characterization of the molecular structure and function of Arc. This study aimed to validate two anti-Arc nanobodies, E5 and H11, as selective modulators of the human Arc N-lobe (Arc-NL), a domain that mediates several molecular functions of Arc through its peptide ligand binding site. The structural characteristics of recombinant Arc-NL-nanobody complexes were solved at atomic resolution using X-ray crystallography. The structures revealed that both anti-Arc nanobodies specifically bind to the multi-peptide binding site of Arc-NL. Isothermal titration calorimetry showed that the Arc-NL-nanobody interactions occur at nanomolar affinity, and that the nanobodies can displace a TARP{gamma}2-derived peptide from the Arc-NL binding site. Thus, both anti-Arc-NL nanobodies can be used in vitro as competitive inhibitors of endogenous Arc ligands. Differences in the CDR3 loops between the two nanobodies indicate that the spectrum of short linear motifs recognized by the Arc-NL should be expanded. We provide a robust biochemical background to support the use of anti-Arc nanobodies in attempts to target Arc-dependent synaptic plasticity. Function-blocking anti-Arc nanobodies could eventually help unravel the complex neurobiology of synaptic plasticity and help diagnose and treat Arc-associated synaptic disorders.

biochemistry↗

bioRxiv2023

Facilitating taxonomy and phylogenetics: An informative and cost-effective protocol integrating long amplicon PCRs and third-generation sequencing

Phylogenetic inference has become a standard technique in integrative taxonomy and systematics, as well as in biogeography and ecology. DNA barcodes are often used for phylogenetic inference, despite being strongly limited due to their low number of informative sites. Also, because current DNA barcodes are based on a fraction of a single, fast-evolving gene, they are highly unsuitable for resolving deeper phylogenetic relationships due to saturation. In recent years, methods that analyse hundreds and thousands of loci at once have improved the resolution of the Tree of Life, but these methods require resources, experience and molecular laboratories that most taxonomists do not have. This paper introduces a PCR-based protocol that produces long amplicons of both slow- and fast-evolving unlinked mitochondrial and nuclear gene regions, which can be sequenced by the affordable and portable ONT MinION platform with low infrastructure or funding requirements. As a proof of concept, we inferred a phylogeny of a sample of 63 spider species from 20 families using our proposed protocol. The results were overall consistent with the results from approaches based on hundreds and thousands of loci, while requiring just a fraction of the cost and labour of such approaches, making our protocol accessible to taxonomists worldwide. HighlightsO_LIDNA barcoding is an invaluable tool for fast and accurate taxonomic classification C_LIO_LIExisting DNA barcodes are still insufficient for obtaining well-supported phylogenies C_LIO_LIWe present a protocol that produces long amplicons of unlinked loci for spiders C_LIO_LIAmplicons are sequenced at very low cost per specimen with ONT MinION C_LIO_LIOur recovered phylogeny is largely consistent with that of high-cost approaches. C_LI

evolutionary biology↗