Non-invasive genetic monitoring for the threatened valley elderberry longhorn beetle.

The valley elderberry longhorn beetle (VELB), Desmocerus californicus dimorphus (Coleoptera: Cerambycidae), is a federally threatened subspecies endemic to the Central Valley of California. The VELB range partially overlaps with that of its morphologically similar sister taxon, the California elderberry longhorn beetle (CELB), Desmocerus californicus californicus (Coleoptera: Cerambycidae). Current surveying methods are limited to visual identification of larval exit holes in the VELB/CELB host plant, elderberry (Sambucus spp.), into which larvae bore and excavate feeding galleries. Unbiased genetic approaches could provide a much-needed complementary approach that has more precision than relying on visual inspection of exit holes. In this study we developed a DNA sequencing-based method for indirect detection of VELB/CELB from frass (insect fecal matter), which can be easily and non-invasively collected from exit holes. Frass samples were collected from 37 locations and the 12S and 16S mitochondrial genes were partially sequenced using nested PCR amplification. Three frass-derived sequences showed 100% sequence identity to VELB/CELB barcode references from museum specimens sequenced for this study. Database queries of frass-derived sequences also revealed high similarity to common occupants of old VELB feeding galleries, including earwigs, flies, and other beetles. Overall, this non-invasive approach is a first step towards a genetic assay that could augment existing VELB monitoring and accurately discriminate between VELB, CELB, and other insects. Furthermore, a phylogenetic analysis of 12S and 16S data from museum specimens revealed evidence for the existence of a previously unrecognized, genetically distinct CELB subpopulation in southern California

Non-invasive genetic monitoring for the threatened valley elderberry longhorn beetle.

The valley elderberry longhorn beetle (VELB), Desmocerus californicus dimorphus (Coleoptera: Cerambycidae), is a federally threatened subspecies endemic to the Central Valley of California. The VELB range partially overlaps with that of its morphologically similar sister taxon, the California elderberry longhorn beetle (CELB), Desmocerus californicus californicus (Coleoptera: Cerambycidae). Current surveying methods are limited to visual identification of larval exit holes in the VELB/CELB host plant, elderberry (Sambucus spp.), into which larvae bore and excavate feeding galleries. Unbiased genetic approaches could provide a much-needed complementary approach that has more precision than relying on visual inspection of exit holes. In this study we developed a DNA sequencing-based method for indirect detection of VELB/CELB from frass (insect fecal matter), which can be easily and non-invasively collected from exit holes. Frass samples were collected from 37 locations and the 12S and 16S mitochondrial genes were partially sequenced using nested PCR amplification. Three frass-derived sequences showed 100% sequence identity to VELB/CELB barcode references from museum specimens sequenced for this study. Database queries of frass-derived sequences also revealed high similarity to common occupants of old VELB feeding galleries, including earwigs, flies, and other beetles. Overall, this non-invasive approach is a first step towards a genetic assay that could augment existing VELB monitoring and accurately discriminate between VELB, CELB, and other insects. Furthermore, a phylogenetic analysis of 12S and 16S data from museum specimens revealed evidence for the existence of a previously unrecognized, genetically distinct CELB subpopulation in southern California

Non-invasive genetic monitoring for the threatened valley elderberry longhorn beetle.

The valley elderberry longhorn beetle (VELB), Desmocerus californicus dimorphus (Coleoptera: Cerambycidae), is a federally threatened subspecies endemic to the Central Valley of California. The VELB range partially overlaps with that of its morphologically similar sister taxon, the California elderberry longhorn beetle (CELB), Desmocerus californicus californicus (Coleoptera: Cerambycidae). Current surveying methods are limited to visual identification of larval exit holes in the VELB/CELB host plant, elderberry (Sambucus spp.), into which larvae bore and excavate feeding galleries. Unbiased genetic approaches could provide a much-needed complementary approach that has more precision than relying on visual inspection of exit holes. In this study we developed a DNA sequencing-based method for indirect detection of VELB/CELB from frass (insect fecal matter), which can be easily and non-invasively collected from exit holes. Frass samples were collected from 37 locations and the 12S and 16S mitochondrial genes were partially sequenced using nested PCR amplification. Three frass-derived sequences showed 100% sequence identity to VELB/CELB barcode references from museum specimens sequenced for this study. Database queries of frass-derived sequences also revealed high similarity to common occupants of old VELB feeding galleries, including earwigs, flies, and other beetles. Overall, this non-invasive approach is a first step towards a genetic assay that could augment existing VELB monitoring and accurately discriminate between VELB, CELB, and other insects. Furthermore, a phylogenetic analysis of 12S and 16S data from museum specimens revealed evidence for the existence of a previously unrecognized, genetically distinct CELB subpopulation in southern California

Identifying hidden biocomplexity and genomic diversity in Chinook salmon, an imperiled species with a history of anthropogenic influence

Biocomplexity is an important mechanism for population resilience in changing environments. However, we are just beginning to understand how to identify biocomplexity so that species management efforts promote resilience and stability. Genomic techniques are emerging as an important method for identifying biocomplexity. Central Valley (CV) Chinook salmon are an example of a species at risk of extinction if better methods for identifying and protecting biocomplexity are not employed. To address this knowledge gap, we employed restriction site associated DNA sequencing to conduct the first genomic study of all major populations of CV Chinook salmon. We found greater population structure across the Central Valley than previously documented. Additionally, we show evidence for differentiation and adaptation within migratory phenotypes despite high levels of gene flow. We also determined that genomic data can vastly improve our ability to assign individuals to their natal populations, even as they mix during migration, a finding that will assist management practices. These results demonstrate how genomic study can greatly improve our ability to identify and conserve biocomplexity.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

The First Record of the Large-scale Loach Paramisgurnus dabryanus (Cobitidae) in the United States

Exotic species have been implicated as a major threat to native freshwater fish communities in the Unites States. The San Francisco Estuary watershed has been recognized as one of the most invaded systems where exotics often dominate the fish community. On October 6, 2014, members of the U.S. Fish and Wildlife Service detected a previously unknown exotic fish in a disconnected pool immediately upstream from the Chowchilla Bifurcation Structure in the San Joaquin River, a major tributary of the San Francisco Estuary. A member of the U.S. Fish and Wildlife Service initially identified the fish as an Oriental Weatherfish Misgurnus anguillicaudatus using external morphological characteristics. We conducted additional fish sampling near the Chowchilla Bifurcation Structure in November 2014 and collected a total of six additional specimens in disconnected pool habitats. Unexpectedly, genetic and meristic techniques revealed that these specimens were Large-Scale Loach Paramisgurnus dabryanus. To our knowledge this is the first confirmed occurrence of Large-Scale Loach in the United States and the suspected pathway of introduction is release from aquaria. Very little is known about the population in the San Joaquin River. We recommend further evaluation of the ecology, distribution, and abundance of Large-Scale Loach to better understand their potential impact on the fish communities of the San Joaquin River and the likelihood of establishment throughout the United States

Introduction of Bluefin Killifish Lucania goodei into the Sacramento-San Joaquin Delta

Biological invasion by non-native species has been identified as one of the major threats to native fish communities worldwide. The fish community of San Francisco Estuary is no exception, as the estuary has been recognized as one of the most invaded on the planet and the system has been impacted significantly by these invasions. Here, we summarize the introduction and probable establishment of a new species in the Sacramento–San Joaquin Delta, the Bluefin Killifish (Lucania goodei), as discovered by the US Fish and Wildlife Service Delta Juvenile Fish Monitoring Program (DJFMP). The DJFMP has conducted a large-scale beach seine survey since 1976, and it is the longest-running monitoring program in the San Francisco Estuary that extensively monitors the shallow-water nearshore habitat. Possibly introduced as discarded aquarium fish within the vicinity of the Delta Cross Channel, Bluefin Killifish is a close relative of the Rainwater Killifish (Lucania parva), another non-native fish species that has been present in the San Francisco Estuary system for decades. Studies in their native range suggest that Bluefin Killifish will fill a similar niche to Rainwater Killifish, albeit with a more freshwater distribution. The potential ecological impact of Bluefin Killifish remains unclear in the absence of additional studies. However, we have been able to track the spread of the species within the Sacramento–San Joaquin Delta through the existence of long-term monitoring programs. Our findings demonstrate the value of monitoring across various habitats for the early detection and proactive management of invasive species

Meek_et_al_EandE_Seq_Count_data

File contains the sequence counts for each individual in the RAD-seq study at each SNP locus, showing the counts for both allele 1 and allele 2, separated by a comma

Sequencing improves our ability to study threatened migratory species: Genetic population assignment in California's Central Valley Chinook salmon

Effective conservation and management of migratory species requires accurate identification of unique populations, even as they mix along their migratory corridors. While telemetry has historically been used to study migratory animal movement and habitat use patterns, genomic tools are emerging as a superior alternative in many ways, allowing large-scale application at reduced costs. Here, we demonstrate the usefulness of genomic resources for identifying single-nucleotide polymorphisms (SNPs) that allow fast and accurate identification of the imperiled Chinook salmon in the Great Central Valley of California. We show that 80 well-chosen loci, drawn from a pool of over 11,500 SNPs developed from restriction site-associated DNA sequencing, can accurately identify Chinook salmon runs and select populations within run. No other SNP panel for Central Valley Chinook salmon has been able to achieve the high accuracy of assignment we show here. This panel will greatly improve our ability to study and manage this ecologically, economically, and socially important species and demonstrates the great utility of using genomics to study migratory species

Rapid and accurate species identification for ecological studies and monitoring using CRISPR‐based SHERLOCK

One of the most fundamental aspects of ecological research and monitoring is accurate species identification, but cryptic speciation and observer error can confound phenotype-based identification. The CRISPR-Cas toolkit has facilitated remarkable advances in many scientific disciplines, but the fields of ecology and conservation biology have yet to fully embrace this powerful technology. The recently developed CRISPR-Cas13a platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) enables highly accurate taxonomic identification and has all the characteristics needed to transition to ecological and environmental disciplines. Here we conducted a series of "proof of principle" experiments to characterize SHERLOCK's ability to accurately, sensitively and rapidly distinguish three fish species of management interest co-occurring in the San Francisco Estuary that are easily misidentified in the field. We improved SHERLOCK's ease of field deployment by combining the previously demonstrated rapid isothermal amplification and CRISPR genetic identification with a minimally invasive and extraction-free DNA collection protocol, as well as the option of instrument-free lateral flow detection. This approach opens the door for redefining how, where and by whom genetic identifications occur in the future

Rapid CRISPR‐Cas13a genetic identification enables new opportunities for listed Chinook salmon management

Accurate taxonomic identification is foundational for effective species monitoring and management. When visual identifications are infeasible or inaccurate, genetic approaches provide a reliable alternative. However, these approaches are sometimes less viable (e.g., need for near real-time results, remote locations, funding concerns, molecular inexperience). In these situations, CRISPR-based genetic tools can fill an unoccupied niche between real-time, inexpensive, but error-prone visual identification and more expensive or time-consuming, but accurate genetic identification for taxonomic units that are difficult or impossible to visually identify. Herein, we use genomic data to develop CRISPR-based SHERLOCK assays capable of rapidly (<1 h), accurately (94%-98% concordance between phenotypic and genotypic assignments), and sensitively (detects 1-10 DNA copies/reaction) distinguishing ESA-listed Chinook salmon runs (winter- and spring-run) from each other and from unlisted runs (fall- and late fall-run) in California's Central Valley. The assays can be field deployable with minimally invasive mucus swabbing negating the need for DNA extraction (decreasing costs and labour), minimal and inexpensive equipment needs, and minimal training to conduct following assay development. This study provides a powerful genetic approach for a species of conservation concern that benefits from near real-time management decision-making but also serves as a precedent for transforming how conservation scientists and managers view genetic identification going forward. Once developed, CRISPR-based tools can provide accurate, sensitive, and rapid results, potentially without the prohibitive need for expensive specialty equipment or extensive molecular training. Further adoption of this technology will have widespread value for the monitoring and protection of our natural resources