A CRISPR-based strategy for targeted sequencing in biodiversity science.

Many applications in molecular ecology require the ability to match specific DNA sequences from single- or mixed-species samples with a diagnostic reference library. Widely used methods for DNA barcoding and metabarcoding employ PCR and amplicon sequencing to identify taxa based on target sequences, but the target-specific enrichment capabilities of CRISPR-Cas systems may offer advantages in some applications. We identified 54,837 CRISPR-Cas guide RNAs that may be useful for enriching chloroplast DNA across phylogenetically diverse plant species. We tested a subset of 17 guide RNAs in vitro to enrich plant DNA strands ranging in size from diagnostic DNA barcodes of 1,428 bp to entire chloroplast genomes of 121,284 bp. We used an Oxford Nanopore sequencer to evaluate sequencing success based on both single- and mixed-species samples, which yielded mean chloroplast sequence lengths of 2,530-11,367 bp, depending on the experiment. In comparison to mixed-species experiments, single-species experiments yielded more on-target sequence reads and greater mean pairwise identity between contigs and the plant species' reference genomes. But nevertheless, these mixed-species experiments yielded sufficient data to provide ≥48-fold increase in sequence length and better estimates of relative abundance for a commercially prepared mixture of plant species compared to DNA metabarcoding based on the chloroplast trnL-P6 marker. Prior work developed CRISPR-based enrichment protocols for long-read sequencing and our experiments pioneered its use for plant DNA barcoding and chloroplast assemblies that may have advantages over workflows that require PCR and short-read sequencing. Future work would benefit from continuing to develop in vitro and in silico methods for CRISPR-based analyses of mixed-species samples, especially when the appropriate reference genomes for contig assembly cannot be known a priori.

Evidence-based strategies to navigate complexity in dietary DNA metabarcoding: A reply.

It is clearly beneficial to eliminate low-abundance sequences that arise in error during dietary DNA metabarcoding studies, but to purge all low-abundance sequences is to risk eliminating real sequences and complicating ecological analyses. Our prior literature review noted that DNA sequence relative read abundance (RRA) thresholds can help ameliorate false-positive taxon occurrences, but that historical emphasis on this utility has fostered uncertainty about the associated risk of inflating the false-negative rate (Littleford-Colquhoun et al., 2022). To address this, we combined a simulation study and an empirical data set to both illustrate the issue and provide blueprints for simulation studies and sensitivity analyses that can help investigators avoid overcorrecting and thereby bolster confidence in ecological inferences. Awareness of both the costs and the benefits of abundance-filtering is needed because accurately characterizing dietary distributions can be critically important for understanding animal diets, nutrition and trophic networks. Highlighting the need to raise awareness, a critique of our paper emphasized the misleading notion that "false positive interactions between species can present fundamentally incorrect network structures in network ecology, whereas false negatives will provide a correct but incomplete version of the network" (Tercel & Cuff, 2022). Asserting that the reliability of results will be eroded by false positives but resilient to the omission of true positives is risky and runs counter to evidence. Unfortunately, abundance-filtering methods can introduce false negatives at higher rates than they eliminate false positives and thereby undermine the analysis of otherwise reliable sequencing data. Overcorrecting can qualitatively alter and ultimately undermine ecological interpretations.

Selection in the city: Rapid and fine-scale evolution of urban eastern water dragons.

Oceanic archipelagos have long been treated as a Petri dish for studies of evolutionary and ecological processes. Like archipelagos, cities exhibit similar patterns and processes, such as the rapid phenotypic divergence of a species between urban and nonurban environments. However, on a local scale, cities can be highly heterogenous, where geographically close populations can experience dramatically different environmental conditions. Nevertheless, we are yet to understand the evolutionary and ecological implications for populations spread across a heterogenous cityscape. To address this, we compared neutral genetic divergence to quantitative trait divergence within three native riparian and four city park populations of an iconic urban adapter, the eastern water dragon. We demonstrated that selection is likely acting to drive divergence of snout-vent length and jaw width across native riparian populations that are geographically isolated and across city park populations that are geographically close yet isolated by urbanization. City park populations as close as 0.9 km exhibited signs of selection-driven divergence to the same extent as native riparian populations isolated by up to 114.5 km. These findings suggest that local adaptation may be occurring over exceptionally small geographic and temporal scales within a single metropolis, demonstrating that city parks can act as archipelagos for the study of rapid evolution.

The precautionary principle and dietary DNA metabarcoding: Commonly used abundance thresholds change ecological interpretation.

Dietary DNA metabarcoding enables researchers to identify and characterize trophic interactions with a high degree of taxonomic precision. It is also sensitive to sources of bias and contamination in the field and laboratory. One of the earliest and most common strategies for dealing with such sensitivities has been to remove all low-abundance sequences and conduct ecological analyses based on the presence or absence of food taxa. Although this step is now often perceived to be necessary, evidence of its sufficiency is lacking and more attention to the risk of introducing other errors is needed. Using computer simulations, we demonstrate that common strategies to remove low-abundance sequences can erroneously eliminate true dietary sequences in ways that impact downstream inferences. Using real data from well-studied wildlife populations in Yellowstone National Park, we further show how these strategies can markedly alter the composition of dietary profiles in ways that scale-up to obscure ecological interpretations about dietary generalism, specialism, and composition. Although the practice of removing low-abundance sequences may continue to be a useful strategy to address research questions that focus on a subset of relatively abundant foods, its continued widespread use risks generating misleading perceptions about the structure of trophic networks. Researchers working with dietary DNA metabarcoding data-or similar data such as environmental DNA, microbiomes, or pathobiomes-should be aware of drawbacks and consider alternative bioinformatic, experimental, and statistical solutions.

Cross-continental emergence of Nannizziopsis barbatae disease may threaten wild Australian lizards.

Members of the genus Nannizziopsis are emerging fungal pathogens of reptiles that have been documented as the cause of fatal mycoses in a wide range of reptiles in captivity. Cases of severe, proliferative dermatitis, debility and death have been detected in multiple free-living lizard species from locations across Australia, including a substantial outbreak among Eastern water dragons (Intellagama lesueurii) in Brisbane, Queensland. We investigated this disease in a subset of severely affected lizards and identified a clinically consistent syndrome characterized by hyperkeratosis, epidermal hyperplasia, dermal inflammation, necrosis, ulceration, and emaciation. Using a novel fungal isolation method, histopathology, and molecular techniques, we identified the etiologic agent as Nannizziopsis barbatae, a species reported only once previously from captive lizards in Australia. Here we report severe dermatomycosis caused by N. barbatae in five species of Australian lizard, representing the first cases of Nannizziopsis infection among free-living reptiles, globally. Further, we evaluate key pathogen and host characteristics that indicate N. barbatae-associated dermatomycosis may pose a concerning threat to Australian lizards.

City life alters the gut microbiome and stable isotope profiling of the eastern water dragon (Intellagama lesueurii).

Urbanisation is one of the most significant threats to biodiversity, due to the rapid and large-scale environmental alterations it imposes on the natural landscape. It is, therefore, imperative that we understand the consequences of and mechanisms by which, species can respond to it. In recent years, research has shown that plasticity of the gut microbiome may be an important mechanism by which animals can adapt to environmental change, yet empirical evidence of this in wild non-model species remains sparse. Using an empirical replicated study system, we show that city life alters the gut microbiome and stable isotope profiling of a wild native non-model species - the eastern water dragon (Intellagama lesueurii) in Queensland, Australia. City dragons exhibit a more diverse gut microbiome than their native habitat counterparts and show gut microbial signatures of a high fat and plant rich diet. Additionally, we also show that city dragons have elevated levels of the Nitrogen-15 isotope in their blood suggesting that a city diet, which incorporates novel anthropogenic food sources, may also be richer in protein. These results highlight the role that gut microbial plasticity plays in an animals' response to human-altered landscapes.

Fresh is best: Accurate SNP genotyping from koala scats.

Maintaining genetic diversity is a crucial component in conserving threatened species. For the iconic Australian koala, there is little genetic information on wild populations that is not either skewed by biased sampling methods (e.g., sampling effort skewed toward urban areas) or of limited usefulness due to low numbers of microsatellites used. The ability to genotype DNA extracted from koala scats using next-generation sequencing technology will not only help resolve location sample bias but also improve the accuracy and scope of genetic analyses (e.g., neutral vs. adaptive genetic diversity, inbreeding, and effective population size). Here, we present the successful SNP genotyping (1272 SNP loci) of koala DNA extracted from scat, using a proprietary DArTseq™ protocol. We compare genotype results from two-day-old scat DNA and 14-day-old scat DNA to a blood DNA template, to test accuracy of scat genotyping. We find that DNA from fresher scat results in fewer loci with missing information than DNA from older scat; however, 14-day-old scat can still provide useful genetic information, depending on the research question. We also find that a subset of 209 conserved loci can accurately identify individual koalas, even from older scat samples. In addition, we find that DNA sequences identified from scat samples through the DArTseq™ process can provide genetic identification of koala diet species, bacterial and viral pathogens, and parasitic organisms.

Archipelagos of the Anthropocene: rapid and extensive differentiation of native terrestrial vertebrates in a single metropolis.

Some of the best evidence for rapid evolutionary change comes from studies of archipelagos and oceanic islands. City parks are analogous systems as they create geographically isolated green spaces that differ in size, structure and complexity. Very little, however, is known about whether city parks within a single urban centre drive selection and result in the diversification of native species. Here, we provide evidence for the rapid genetic and morphological differentiation of a native lizard (Intellagama lesueurii) at four geographically close yet unconnected parks within one city. Year of establishment of each city park varied from 1855 (oldest) to 2001 (youngest) equating to a generation time range of 32 to three generations. Genetic divergence among city park populations was large despite the small pairwise geographic distances (<5 km) and found to be two to three times higher for microsatellites and three to 33 times higher for mtDNA relative to nonurban populations. Patterns of morphological differentiation were also found to be most extensive among the four city park populations. In contrast to nonurban populations, city park populations showed significant differentiation in relative body size, relative head and limb morphology and relative forelimb and hindlimb length. Crucially, we show that these patterns of differentiation are unlikely to have been caused by founder events and/or drift alone. Our results suggest that city park 'archipelagos' could represent theatres for rapid evolution that may, in time, favour adaptive diversification.

Population differentiation and hybridisation of Australian snubfin (Orcaella heinsohni) and Indo-Pacific humpback (Sousa chinensis) dolphins in north-western Australia.

Little is known about the Australian snubfin (Orcaella heinsohni) and Indo-Pacific humpback (Sousa chinensis) dolphins ('snubfin' and 'humpback dolphins', hereafter) of north-western Australia. While both species are listed as 'near threatened' by the IUCN, data deficiencies are impeding rigorous assessment of their conservation status across Australia. Understanding the genetic structure of populations, including levels of gene flow among populations, is important for the assessment of conservation status and the effective management of a species. Using nuclear and mitochondrial DNA markers, we assessed population genetic diversity and differentiation between snubfin dolphins from Cygnet (n = 32) and Roebuck Bays (n = 25), and humpback dolphins from the Dampier Archipelago (n = 19) and the North West Cape (n = 18). All sampling locations were separated by geographic distances >200 km. For each species, we found significant genetic differentiation between sampling locations based on 12 (for snubfin dolphins) and 13 (for humpback dolphins) microsatellite loci (FST = 0.05-0.09; P<0.001) and a 422 bp sequence of the mitochondrial control region (FST = 0.50-0.70; P<0.001). The estimated proportion of migrants in a population ranged from 0.01 (95% CI 0.00-0.06) to 0.13 (0.03-0.24). These are the first estimates of genetic diversity and differentiation for snubfin and humpback dolphins in Western Australia, providing valuable information towards the assessment of their conservation status in this rapidly developing region. Our results suggest that north-western Australian snubfin and humpback dolphins may exist as metapopulations of small, largely isolated population fragments, and should be managed accordingly. Management plans should seek to maintain effective population size and gene flow. Additionally, while interactions of a socio-sexual nature between these two species have been observed previously, here we provide strong evidence for the first documented case of hybridisation between a female snubfin dolphin and a male humpback dolphin.