A practical guide to sample preservation and pre-PCR processing of aquatic environmental DNA.

Environmental DNA (eDNA) is rapidly growing in popularity as a tool for community assessments and species detection. While eDNA approaches are now widely applied, there is not yet agreement on best practices for sample collection and processing. Investigators looking to integrate eDNA approaches into their research programme are required to examine a growing collection of disparate studies to make an often uncertain decision about which protocols best fit their needs. To promote the application of eDNA approaches and to encourage the generation of high-quality data, here we review the most common techniques for the collection, preservation and extraction of metazoan eDNA from water samples. Specifically, we focus on experimental studies that compare various methods and outline the numerous challenges associated with eDNA. While the diverse applications of eDNA do not lend themselves to a one-size-fits-all recommendation, in most cases, capture/concentration of eDNA on cellulose nitrate filters (with pore size determined by water turbidity), followed by storage of filters in Longmire's buffer and extraction with a DNeasy Blood & Tissue Kit (or similar) has been shown to provide sufficient, high-quality DNA. However, we also emphasize the importance of testing and optimizing protocols for the system of interest.

Marine environmental DNA: Approaches, applications, and opportunities.

Environmental DNA (eDNA) is increasingly being used to document species distributions and habitat use in marine systems, with much of the recent effort focused on leveraging advances in next-generation DNA sequencing to assess and track biodiversity across taxonomic groups. Environmental DNA offers a number of important advantages over traditional survey techniques, including non-invasive sampling, sampling where traditional approaches are impractical or inefficient (e.g. deep oceans), reduced cost, and increased detection sensitivity. However, eDNA applications are currently limited because of an insufficient understanding of the influence of sample source, analytical approach, and marker type on eDNA detections. Because approaches vary considerably among eDNA studies, we present a summary of the current state of the field and emerging best practices. The impact of observed variation in rates of eDNA production, persistence, and transport are also discussed and future research needs are highlighted with the goal of expanding eDNA applications, including the development of statistical models to improve the predictability of eDNA detection and quantification.

Dramatic response to climate change in the Southwest: Robert Whittaker's 1963 Arizona Mountain plant transect revisited.

Models analyzing how Southwestern plant communities will respond to climate change predict that increases in temperature will lead to upward elevational shifts of montane species. We tested this hypothesis by reexamining Robert Whittaker's 1963 plant transect in the Santa Catalina Mountains of southern Arizona, finding that this process is already well underway. Our survey, five decades after Whittaker's, reveals large changes in the elevational ranges of common montane plants, while mean annual rainfall has decreased over the past 20 years, and mean annual temperatures increased 0.25°C/decade from 1949 to 2011 in the Tucson Basin. Although elevational changes in species are individualistic, significant overall upward movement of the lower elevation boundaries, and elevational range contractions, have occurred. This is the first documentation of significant upward shifts of lower elevation range boundaries in Southwestern montane plant species over decadal time, confirming that previous hypotheses are correct in their prediction that mountain communities in the Southwest will be strongly impacted by warming, and that the Southwest is already experiencing a rapid vegetation change.

Not All Larvae Stay Close to Home: Insights into Marine Population Connectivity with a Focus on the Brown Surgeonfish (Acanthurus nigrofuscus).

Recent reports of localized larval recruitment in predominately small-range fishes are countered by studies that show high genetic connectivity across large oceanic distances. This discrepancy may result from the different timescales over which genetic and demographic processes operate or rather may indicate regular long-distance dispersal in some species. Here, we contribute an analysis of mtDNA cytochrome b diversity in the widely distributed Brown Surgeonfish (Acanthurus nigrofuscus; N = 560), which revealed significant genetic structure only at the extremes of the range (ΦCT = 0.452; P < .001). Collections from Hawaii to the Eastern Indian Ocean comprise one large, undifferentiated population. This pattern of limited genetic subdivision across reefs of the central Indo-Pacific has been observed in a number of large-range reef fishes. Conversely, small-range fishes are often deeply structured over the same area. These findings demonstrate population connectivity differences among species at biogeographic and evolutionary timescales, which likely translates into differences in dispersal ability at ecological and demographic timescales. While interspecific differences in population connectivity complicate the design of management strategies, the integration of multiscale connectivity patterns into marine resource planning will help ensure long-term ecosystem stability by preserving functionally diverse communities.

Escaping paradise: Larval export from Hawaii in an Indo-Pacific reef fish, the Yellow Tang (Zebrasoma flavescens).

The depauperate marine ecosystems of the Hawaiian Archipelago share a high proportion of species with the southern and western Pacific, indicating historical and/or ongoing connections across the large oceanic expanse separating Hawaii from its nearest neighbors. The rate and direction of these interactions are, however, unknown. While previous biogeographic studies have consistently described Hawaii as a diversity sink, prevailing currents likely offer opportunities for larval export. To assess interactions between the remote reefs of the Hawaiian Archipelago and the species rich communities of the Central and West Pacific, we surveyed 14 nuclear microsatellite loci (nDNA; n = 857) and a 614 bp segment of mitochondrial cytochrome b (mtDNA; n = 654) in the Yellow Tang (Zebrasoma flavescens). Concordant frequency shifts in both nDNA and mtDNA reveal significant population differentiation among three West Pacific sites and Hawaii (nDNA F' CT = 0.116, mtDNA ϕ CT = 0.098, P < 0.001). SAMOVA analyses of microsatellite data additionally indicate fine scale differentiation within the 2600 km Hawaiian Archipelago (F' SC = 0.026; P < 0.001), with implications for management of this heavily-exploited aquarium fish. Mismatch analyses indicate the oldest contemporary populations are in the Hawaiian Archipelago (circa 318,000 y), with younger populations in the West Pacific (91,000 - 175,000 y). Estimates of Yellow Tang historical demography contradict expectations of Hawaii as a population sink, and instead indicate asymmetrical gene flow, with Hawaii exporting rather than importing Yellow Tang larvae.

Defining Boundaries for Ecosystem-Based Management: A Multispecies Case Study of Marine Connectivity across the Hawaiian Archipelago.

Determining the geographic scale at which to apply ecosystem-based management (EBM) has proven to be an obstacle for many marine conservation programs. Generalizations based on geographic proximity, taxonomy, or life history characteristics provide little predictive power in determining overall patterns of connectivity, and therefore offer little in terms of delineating boundaries for marine spatial management areas. Here, we provide a case study of 27 taxonomically and ecologically diverse species (including reef fishes, marine mammals, gastropods, echinoderms, cnidarians, crustaceans, and an elasmobranch) that reveal four concordant barriers to dispersal within the Hawaiian Archipelago which are not detected in single-species exemplar studies. We contend that this multispecies approach to determine concordant patterns of connectivity is an objective and logical way in which to define the minimum number of management units and that EBM in the Hawaiian Archipelago requires at least five spatially managed regions.

Isolation and characterization of 23 microsatellite loci in the yellow tang, Zebrasoma flavescens (Pisces: Acanthuridae).

Twenty-three microsatellites were isolated from the yellow tang (Zebrasoma flavescens), an ecologically and commercially important reef fish. Genetic diversity was assessed in 90 adults collected from Honokohau, Hawaii. The number of alleles per locus varied from four to 29 (mean = 13.8) and observed and expected heterozygosities ranged from 0.15 to 0.94 (mean = 0.70) and from 0.29 to 0.93 (mean = 0.81), respectively. Eight loci exhibited significant departure from Hardy-Weinberg equilibrium due to the presence of null alleles. Exact tests showed no evidence of genotypic disequilibrium between loci. Overall, loci were well resolved, easy to score and highly polymorphic.